Anti-fatigue Effects of Polysaccharides from Morchella esculenta

Fuwei Yin

1 a

and Lantao Liu

2,* b

School of Physical Education, Hunan Normal University, Changsha 410012, China

Department of Physical Education, Central South University, Changsha 410083, China

Keywords:

Polysaccharides, M. esculenta, Anti-Fatigue, Mechanism, Forced Swimming Test.

Abstract:

Fatigue has caused indirect damage to the body or a direct cause of disease, which has led to the research

and development of anti-fatigue natural medicines and supplements that have become a hot spot at home

and abroad. The aim of present work was to evaluate the anti-fatigue effects of polysaccharides from M.

esculenta (PMe) by using a swimming exercise animal model. The mice were assigned into a normal control

group and three PMe treatment groups. The treatment groups received different doses of PMe (100, 200,

and 400 mg/kg) through gastric gavage once per day for 4 weeks, and normal control group received

distilled water. On the last day of the experiment, the forced swimming test was performed, and fatigue-

related biochemical parameters were analyzed. The results showed that PMe significantly prolonged

(p<0.05) the swimming time to exhaustion, reduced (p<0.05) the levels of lactate, urea nitrogen and

malondialdehyde in serum, increased (p<0.05) the levels of non-esterfied fatty acid in serum, as well as the

glycogen levels in liver and muscle. In conclusion, PMe has the anti-fatigue effects and its mechanisms

might be related to the fact that PMe could reduce the production of metabolites or delay the accumulation

of metabolites, attenuate protein and amino acid metabolism, and enhance fat metabolism, reduce oxidative

stress and protect oxidative damage induced by exercise, and improve the energy substance storage or

reduce energy substance consumption.

1 INTRODUCTION

Morchella esculenta (L.) Pers. (M. esculenta)

belongs to the family of Ascomycota of the order

Discomycetes, which grows in temperate regions of

Asia, Europe and Americas. It is a class of rare

edible and medicinal mushrooms with rich nutrition

and delicious taste (Nitha, Fijesh, Janardhanan,

2013). As early as 2000 years ago, M. esculenta was

used in Traditional Chinese medicine (TCM) for the

treatment of spleen and stomach weakness,

indigestion, phlegm and shortness of breath,

dizziness, insomnia and other diseases (Cui, Chen,

Wang, Kai, Fang, 2011). Various bioactive

ingredients from M. esculenta have been isolated

and reported, such as polysaccharides, tocopherol,

carotenoids, organic acids and polyphenols (Yang,

Yin, Zhang, 2015). Modern pharmacological studies

have demonstrated that polysaccharides is one of the

most important bioactive ingredients of M. esculenta

https://orcid.org/0000-0001-6749-4912

https://orcid.org/0000-0003-1498-3864

and have a variety of pharmacological actions,

including immunomodulatory, antioxidation,

antibacterial, anti-viral, antimicrobial, antitumor,

anti-proliferation, anti-inflammatory,

hepatoprotective, and many other effects (Liu, Pan,

2016). However, little is known about anti-fatigue

activity of polysaccharides from M. esculenta

(PMe). The aim of present work was to evaluate the

effects of PMe on physical fatigue by using a

swimming exercise animal model. Further, its

possible mechanism of anti-fatigue effects also will

be investigated, which will provide a scientific basis

for the use of this active ingredient

2 MATERIALS AND METHODS

2.1 Materials and Chemicals

Dry M. esculenta mycelium was obtained from

ZhongZhiKang Mushroom Science & Technology

Development Co., Ltd. (Hangzhou, China). Assay

kits for determination of lactate (LA) and glycogen

132

Yin, F. and Liu, L.

Anti-fatigue Effects of Polysaccharides from Morchella esculenta.

DOI: 10.5220/0011191100003443

In Proceedings of the 4th International Conference on Biomedical Engineering and Bioinformatics (ICBEB 2022), pages 132-138

ISBN: 978-989-758-595-1

were provided by the LeiGen Biotechnology Co.

(Beijing, China). Assay kits for determination of

urea nitrogen (UN) and non-esterfied fatty acid

(NEFA) were provided by the HuiLi Biochemical

Reagents Co. (Changchun, China). Assay kits for

determination of malondialdehyde (MDA) were

provided by the JianCheng Biotechnology Co.

(Nanjing, China). All the other chemicals and

reagents used in this study were of analytical grade

and were provided by the commercial channels. All

solutions were prepared with deionized water to

eliminate metal ion contamination.

2.2 Polysaccharides from M. Esculenta

Preparation

PMe was extracted according to the pre-described

method by published literature and subjected to

small modification (Cui, Chen, Wang, Kai, Fang,

2011). The dry M. esculenta mycelium were further

dried to constant weight at 45°C, and ground into

fine powder (100 mesh) using a shredder. Then, the

powder was extracted by refluxing in 80% ethanol

for 7 h to remove the ethanol-soluble materials,

including colored substances, small molecule

substances, monosaccharides, and oligosaccharides.

The ethanol was volatilized and the pretreaed dry

powder was obtained. Pretreated powder was

extracted with 10 volumes of distilled water at 90°C

for 3 h. After centrifugation (2432 × g, 15 min), the

residue was extracted for another 3 h at 90ºC. All

supernatants were combined and concentrated in a

rotary evaporator under low pressure. The

concentrated supernatants were mixed with 3

volumes of 95% ethanol at 4°C for 24 h. After

centrifugation (2432 × g, 15 min), the precipitate

was washed twice with 100% ethanol and acetone,

subsequently re-dissolved in distilled water, and

again precipitated with 95% ethanol. The resulting

precipitate was collected by centrifugation (2432 ×

g, 15 min) and the proteins were removed by Sevag

method. Finally, the supernatant was collected and

lyophilized to obtain PMe.

2.3 Experimental Animals and Care

Conditions

Healthy male Kunming mice weighting between 18

and 22g were housed in standard conditions with

maintained relative humidity (50 ± 10%), controlled

temperature (22 ± 1°C), and an artificial 12-h

light/dark cycle (lights on 07:30-19:30 h). The mice

were allowed free access to standard food particles

(unless otherwise stated) and tap water ad libitum

throughout the experimental period. All animals are

subjected to humanitarian care in compliance with

the "Measures for the Administration of Laboratory

Animals in Hunan Province", which strictly comply

with the orders issued by the Ministry of Science

and Technology of China in 1988. The experimental

protocol was approved by the Ethics Committee of

Central South University

2.4 Experimental Design

After 7 days of adaptation to the feeding

environment, the mice were randomly assigned into

the following four groups (10 mice per group).

Group 1: normal control (NC) group, the mice were

treated with distilled water. Group 2: low dose PMe

treatment (LPT) group, the mice were treated with

100 mg/kg of PMe. Group 3: middle dose PMe

treatment (MPT) group, the mice were treated with

200 mg/kg of PMe. Group 4: high dose PMe

treatment (HPT) group, the mice were treated with

400 mg/kg of PMe. PMe was dissolved in distilled

water. The treatment groups received different doses

of PMe through gastric gavage once per day for 4

weeks, and NC group received the same volume of

distilled water.

On the last day of the experiment, 30 min after

the last treatment, the forced swimming test was

performed as previously reported. The apparatus

used was a plastic water tank (length: 65 cm, width:

50 cm, depth: 50 cm) filled with water of 30 cm

deep at room temperature (25 ± 1°C). Each mouse

tail tied a wire bundle (equivalent to 10% body

weight) to swim in order to shorten the test time.

Mice are considered exhausted when the animal sink

into the water and can not float on the water surface

within 10 s (Zhang, 2015), and their swimming time

to exhaustion was immediately recorded.

2.5 Biochemical Analysis

After the forced swimming test, the mice were

anesthetized by intraperitoneal injection of 10%

(w/v) chloral hydrate (350 mg/kg body weight) and

sacrificed via decapitation. Blood samples were

collected and serum were prepared by centrifugation

(1800 × g, 15 min) at 4°C for the estimations of

levels of LA, UN, NFFA, and MDA. Then the liver

and quadriceps femoris muscle were quickly

resected, washed with physiological saline, and

stored in liquid nitrogen at -80°C for the estimations

of glycogen contents. All biochemical parameters

were measured using the corresponding commercial

Anti-fatigue Effects of Polysaccharides from Morchella esculenta

133

assay kits according to the manufacturer's

recommended instructions.

2.6 Statistical Analysis

Values are presented as Mean ± standard deviation

(SD). Statistical analysis was performed using SPSS

data analysis software (version 18.0, Chicago,

USA). Statistical significance was done using one

way analysis of variance (ANOVA) test and then by

Dunnett's test. p<0.05 was considered significant.

3 RESULTS

3.1 Effect of PMe on the Swimming

Time to Exhaustion of Mice

As shown in Fig. 1, the swimming time to

exhaustion of mice in the LPT, MPT, and HPT

groups (7.94 ± 1.04, 8.21 ± 0.86, and 8.98 ± 1.15

min, respectively) were significantly longer

(p<0.05) than that in the NC group (6.87 ± 0.97

min).

Figure 1: Effect of PMe on the swimming time to exhaustion of mice. Values are presented as Mean ±SD. ap<0.05

compared to the NC group.

3.2 Effect of PMe on the LA and UN in

Serum of Mice

As shown in Fig. 2, the serum LA levels of mice in

the LPT, MPT and HPT groups (13.49 ± 1.84, 12.16

± 2.23, and 10.83 ± 1.78 mmol/L, respectively) were

significantly lower (p<0.05) than that in the NC

group (15.21 ± 2.16 mmol/L). Meanwhile, the

serum UN levels of mice in the MPT and HPT

groups (8.74 ± 0.75 and 8.21 ± 0.97 mmol/L) were

significantly lower (p<0.05) than that in the NC

group (9.85 ± 1.16 mmol/L).

Figure 2: Effect of PMe on the LA and UN in serum of mice. Values are presented as Mean ±SD. ap<0.05 compared to the

NC group.

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3.3 Effect of PMe on the Serum NEFA

of Mice

As shown in Fig. 3, the serum NEFA levels of mice

in the MPT and HPT groups (39.47 ± 2.06 and

46.23 ± 6.12 umol/L) were significantly higher

(p<0.05) than that in the NC group (34.56 ± 5.13

umol/L).

Figure 3: Effect of PMe on the serum NEFA of mice. Values are presented as Mean ±SD. ap<0.05 compared to the NC

group.

3.4 Effect of PMe on the Serum MDA

of Mice

As shown in Fig. 4, the serum MDA levels of mice

in the MPT and HPT groups (10.34 ± 1.40 and 8.15

± 1.03 nmol/mL) were significantly lower (p<0.05)

than that in the NC group (12.51 ± 1.47 nmol/mL).

Figure 4: Effect of PMe on the serum MDA of mice. Values are presented as Mean ±SD. ap<0.05 compared to the NC

group.

3.5 Effect of PMe on the Glycogen in

Liver and Muscle of Mice

As shown in fig. 6, the liver glycogen levels of mice

in the LPT, MPT and HPT groups (8.86 ± 0.94, 9.47

± 1.16, and 11.32 ± 1.03 mg/g, respectively) were

significantly higher (p<0.05) than that in the NC

group (7.69 ± 0.89 mg/g). Similarly, the muscle

glycogen levels of mice in the MPT and HPT groups

(1.75 ± 0.27 and 1.94 ± 0.22 mg/g) were

significantly higher (p<0.05) than that in the NC

group (1.43 ± 0.21 mg/g).

Anti-fatigue Effects of Polysaccharides from Morchella esculenta

135

Figure 5: Effect of PMe on the glycogen in liver and muscle of mice. Values are presented as Mean ±SD. ap<0.05

compared to the NC group.

4 DISCUSSIONS

Exercise tolerance is the most direct and important

indicators to reflect the physical fatigue. Enhanced

exercise tolerance in an exercise test means a

lessening of fatigue (Xu, Zhang, 2013). The animal

model for evaluating exercise tolerance mainly

includes forced wheel running test, forced treadmill

running test, forced climbing test and forced

swimming test and so on. Forced swimming test

rather than other forced exercise tests in this study

was chosen as an animal model because it can cause

minimal damage to animals, and has a high

reproducibility (Jin, Wei, 2011). The lengths of

swimming time to exhaustion can reflect the degree

of exercise tolerance. The data obtained from this

study show that different dose of PMe significantly

prolonged the swimming time to exhaustion of

mice, which indicated that PMe could improve

exercise tolerance and had the anti-fatigue effects.

Energy supply from the glycolysis is the main

energy source of strenuous exercise (Yan, Hao,

2016). The increase in muscle oxygen consumption

would lead to hypoxia of body, resulting in

accelerated glycolysis reaction, and produce a lot of

LA during strenuous exercise. The accumulation of

serum LA could cause the cell pH value to decrease,

leading to a series of biochemical changes, which

finally lead to fatigue. Therefore, the accumulation

of serum LA could show the speed and extent of

fatigue development. UN is a product of protein and

amino acid catabolic metabolism. During strenuous

exercise, protein and amino acid catabolic

metabolism would be strengthened when for a long

time body cannot get enough energy by means of

sugar and fat catabolic metabolism (Lin, Liu, 2014).

Meanwhile, nucleotides metabolism would also be

quickened. These two metabolic pathways

eventually form UN. Less produced serum UN

indicated stronger exercise tolerance and bearing

capability of body (Liu, Ji, Li, 2013). So, serum UN

is another sensitive indicator of fatigue. In this

study, middle and high dose PMe significantly

decreased the LA and UN levels in serum of mice,

which indicated that PMe could reduce the

production of serum LA or delay the accumulation

of serum LA, decrease serum UN levels by

attenuating protein and amino acid metabolism,

thereby delaying fatigue.

Enhancing the proportion of energy supply from

fat catabolic metabolism during strenuous exercise

could save the glycogen consumption of body,

keeping the blood glucose in a physiological range

in order to meet the needs of the brain central

nervous system (Xu, 2012). This could improve

exercise tolerance and delay the occurrence of

fatigue. It is reported that the increased availability

of NEFA results in greater fat metabolism in the

muscle. In this study, middle and high dose PMe

significantly increased the serum NEFA levels of

mice, which indicated that PMe could improve

exercise tolerance might due to enhanced fat

metabolism by increasing availability of NEFA.

Strenuous exercise increases the production of

free radicals and ROS, which thus attacks the

membrane lipid and causes the lipid peroxidation

product to form. In turn, the formation and

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accumulation of lipid peroxidation will damage the

cells, especially membrane structure and genetic

material changes, to further cause the body's

oxidative damage, accelerating the development of

fatigue (Yan, Hao, 2016). MDA, one of the

degradation products from lipid peroxidation, is

known to be the most sensitive parameter reflecting

oxidative damage (Chen, Li, Wang, Zhang, 2013).

In this study, middle and high dose PMe

significantly decreased the serum MDA levels of

mice, which indicated that the anti-fatigue effects of

PMe might be due to protecting oxidative damage

induced by strenuous exercise through reducing

lipid peroxidation.

Exercise energy is originally derived from the

decomposition of glycogen, which can supplement

blood glucose consumption, and maintain blood

glucose levels stable in the physiological range (Yu,

Huang, 2012). The increase in muscle glycogen

consumption in strenuous exercise will promote the

liver glycogen decomposition of glucose to speed up

to maintain blood glucose levels stable. Glycogen

storage directly affects exercise endurance. Thus,

the glycogen is another important indicator related

to fatigue. In this study, middle and high dose PMe

significantly increased the glycogen levels in liver

and muscle of mice, which indicated that anti-

fatigue effects of PMe might be due, at least in part,

to improving glycogen storage, or reducing

glycogen consumption during strenuous exercise.

In recent years, a series of mechanisms on

physical fatigue have been explored, such as free

radical theory, exhaustion theory, metabolic matter

accumulation theory, internal environmental

imbalance theory, mutation theory, protective

inhibition theory and so on (Wang, Xing, 2014). In

this study, we reveal the anti-fatigue mechanisms of

PMe from three aspects of energy metabolism and

storages, metabolite accumulation, and free radical

induced oxidative stress.

5 CONCLUSION

Based on the above tests and analysis, it can be

concluded that PMe has the anti-fatigue effects as

evidenced by prolonging the swimming time to

exhaustion of mice, reducing the levels of LA, UN

and MDA in serum, and increasing the levels of

NEFA in serum, as well as the glycogen levels in

liver and muscle. The anti-fatigue mechanisms of

PMe might be through the following pathways.

(1) PMe could reduce the production of

metabolites or delay the accumulation of

metabolites.

(2) PMe could attenuate protein and amino acid

metabolism, and enhance fat metabolism.

(3) PMe could reduce oxidative stress, and

protect oxidative damage induced by exercise.

(4) PMe could improve the energy substance

storage or reduce energy substance consumption.

Further research is needed to clarify the detailed

mechanism of PMe's anti-fatigue effects.

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