www.elsevier.com/locate/jmcplaJournal of Medical Colleges of PLA 23(2008)88–93

Anti-fatigue effects of salidroside in mice

Ma Li1*, Cai Donglian2, Li Huaixing3, Tong Bende1, Song Lihua4, Wang Ying2

1Department of Clinical Nutrition, Changzheng Hospital, Second Military Medical University, Shanghai 200003, China

2Department of Clinical Nutrition, Changhai Hospital, Second Military Medical University, Shanghai 200433, China

3Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China

4Agriculture and Biological College, Shanghai Jiaotong University, Shanghai 200030, China Received 29 November 2007; accepted 09 January 2008

Abstract

Objective: To study the anti-fatigue effects of salidroside in mice. Methods: Totally 120 normal male Kunming mice were randomized into 5 groups (4 salidroside intervention groups and the control group) based on body weight. The control group was given distilled water and the 4 intervention groups were given various doses of salidroside (60, 180, 360, 720 mg/kg) for 15 consecutive days, respectively. The levels of lactate, serum urea nitrogen, muscle and liver glycogen, the longest swimming time and hemoglobin were determined before and after swimming test. Results: Different doses of salidroside significantly lengthened the swimming time and increased the contents of hemoglobin and muscle and liver glycogen, while reducing that of lactate in blood significantly compared with control group, especially in the 180 mg/kg salidroside group. Conclusion: Salidroside has noticeable anti-fatigue effect on mice. These effects were dose-dependent, and the strongest effect on most biomarkers was seen with an intermediate dose.

Keywords: Salidroside; Anti-fatigue

1. Introduction

Rhodiola rosea (“golden root”) is widely distributed at high altitudes in Arctic and mountainous regions throughout Europe and Asia and has been categorized as an adaptogen by Russian researchers due to its ability to increase resistance to a variety of chemical, biological, and physical stressors [1]. Its claimed benefits include

Supported by the Foundation of the Gym Sport Bureau of Shanghai (04JT017)

* Corresponding author. E-mail address: mali106@126.com (Ma L.)

anti-depressant, anticancer, anti-stress, anti- oxidation and improving immune function [2].

The chemical compositions of Rhodiola species are very complicated. The roots of Rhodiola rosea contain a range of biologically active substances including organic acids, flavonoids, tannins, and phenolic glycosides. Although Rhodiola rosea has been widely investigated as an adaptogen, its mechanism of action is not elucidated quite well. With the improvement of separation technique, many compounds have been isolated from the roots of Rhodiola rosea, such as phenylpropyl alcohol, benzene alcohol, flavanol, monoterpene, triterpene, phenolic acid and so on. This provides a chance to

Ma Li et al./Journal of Medical Colleges of PLA 23(2008)88–93 89

investigate Rhodiola rosea.Salidroside is thought to be one of the most

critical constituents in Rhodiola rosea that have therapeutic activity. But the anti-fatigue effects of salidroside and the dose-dependent relation haven’t been reported. The present study was designed to investigate the anti-fatigue effects of salidroside and its effective dose.

2. Materials and methods

2.1. Salidroside

Salidroside (>95%) was purchased from East China University of Science and Technology.

2.2. Animals and grouping

Kunming mice were purchased from the Center of Laboratory Animal of the Second Military Medical University (Shanghai, China). The mice were weighed between 15 and 21 g at the time of experiment. They were given fed water ad libitum and fed with standard mice pellets food, for one week before drug administration in the following environment: under 23±1 , with (50±5)% relative humidity, and ventilation at 15 air renewal cycles every hour and in a 24-hour light-dark cycle for one-week adaptation before drug administration. We obtained permission for performing the research protocols and all animal experiments, conducted during the present study got prior permission and followed the guidelines of the ethics committee of the Second Military Medical University. The mice were randomized into 5 groups equally based on body weight after one week adoption: low, medium, intermediate-high, high-dose salidroside intervene- tion groups and control group. The control group was given distilled water and the 4 intervention groups were given different doses of salidroside (60, 180, 360, 720 mg/kg) for 15 consecutive days, respectively. The salidroside solution used in intervention groups were prepared through dissolving salidroside in distilled water.

2.3. Determination of body weight, hemoglobin, lactic acid, serum blood urea nitrogen, hepatic and muscle glycogen

Body weights were measured by electronic balance before and after the experiment. Forty Kunming mice were dropped individually into water tank (50 cm in length, 40 cm in width) containing 40 cm of water maintained at 30±1 30 min after intra-gastric administration. All mice were forced to swim with a load (5% body weight) attached to the tail; then the longest swimming time was measured.

The levels of hemoglobin (Hb) were determined according to the recommended procedures provided by the kits purchased from Nanjing Jiancheng Bioengineering Institute. The blood samples were collected from eye sockets of 40 Kunming mice 30 min after intragastric administration and 30 min after weight loading swimming (2% body weight), respectively. Then blood lactic acid was tested according to the recommended procedures provided by the kit purchased from Nanjing Jiancheng Bioengineering Institute. Forty Kunming mice were forced to swim for 90 min without a load. After an hour’s resting, the mice were killed to collect liver, gastrocnemiusmuscle, and plasma samples for enzyme activity assays. Levels of serum blood urea nitrogen (BUN) were determined by an auto-analyzer. The contents of hepatic and muscle glycogen were tested according to the recommended procedures provided by the kits purchased from Nanjing Jiancheng Bioengineering Institute.

2.4. Statistical analysis

The data were analyzed with SPSS 10.0 software. ANOVA was used to determine the effects of salidroside on anti-fatigue. The values were expressed as mean±SD. The test differences were considered statistically significant when a Pvalue was less than 0.05.

90 Ma Li et al./Journal of Medical Colleges of PLA 23(2008)88–93

3. Results

3.1. Effects of salidroside on body weight

As shown in Table 1, the body weight of mice in 4 intervention groups was not different significantly from that in the control group before and after experiment (P>0.05), which means the salidroside has no effect on body weight.

3.2. Effect of salidroside on the longest swimming time

We investigated the anti-fatigue effects of salidroside by swimming test. As shown in Fig. 1, the longest swimming time of mice in 180, 360, 720 mg/kg salidroside intervention groups was significantly prolonged compared with that in the control group (P<0.05), which is 4.2, 3.3 and 1.75 times longer that in the control group, respectively.

3.3. Effect of salidroside on Hb

The Hb levels in 60 mg/kg sal idroside intervention group (156.8±35.6 g/L) and 180 mg/kg salidroside intervention group(153.7±23.2 g/L) were much higher than that in control group (144±17.2 g/L) (P<0.05). Although the Hb levels

Table 1 Effects of salidroside on body weight of mice of different groups (mean ± SD, n=24, g)

Groups Before

experiment After

experiment A (Control) 25.3±2.2 35.3±3.1 B (Low dose) 25.4±1.8 33.6±2.3 C (Intermediate dose) 25.4±1.2 34.4±2.5 D (Intermediate-high dose)

25.1±1.8 36.8±2.1

E (High dose) 25.6±2.6 34.4±4.1

Dose of salidroside, B: 60 mg/kg; C: 180 mg/kg; D: 360 mg/kg; E: 720 mg/kg.

Fig.1. Effects of salidroside on the longest swimming time of the mice. Dose of salidroside, A: Control; B: 60 mg/kg, C: 180 mg/kg, D: 360 mg/kg, E: 720 mg/kg.

in 360 salidroside group (148.3±15.7 g/L) and 720 mg/kg salidroside group(151.4±29.1 g/L) were also increased, no significant difference was observed (P>0.05). Therefore low and medium dose salidroside could significantly increase the Hb of mice (Table 2).

3.4. Effect of salidroside on serum BUN

The serum BUN of the low, intermediate and intermediate-high dose intervention groups were 9.08±0.96, 8.90±0.77, 8.98±1.19 mmol/L, which were lower than that of the control group (9.94±1.59 mmol/L), but there was not significant difference (P>0.05). See Table 2.

Table 2 Effects of salidroside on Hb and serum BUN of mice of different groups (mean ± SD, n=8)

Groups Hemoglobin

g/LBUN

(mmol/L ) A (Control) 144.0±17.2 9.94±1.59

B (Low dose) 156.8±35.6a 9.08±0.96

C (Intermediate dose) 153.7±23.2a 8.90±0.77 D (Intermediate-high dose) 148.3±15.7 8.98±1.19

E (High dose) 151.4±29.1 10.38±1.03 aP <0.05 vs group A Dose of salidroside, B: 60 mg/kg; C: 180 mg/kg; D:

360 mg/kg; E: 720 mg/kg.

0

1000

2000

3000

4000

A B C D E

Long

est s

wim

min

g tim

es

Ma Li et al./Journal of Medical Colleges of PLA 23(2008)88–93 91

3.5. Effect of salidroside on blood lactic acid

As shown in Table 3, there was no significant difference in the levels of blood lactic acid between 4 intervention groups and the control group before swimming (P>0.05). After swimming, the level of blood lactic acid of intermediate, intermediate-high and high dose salidroside intervention groups were significantly lower than that of control group (P<0.05). Particularly, the blood lactic acid level of intermediate group was lower than that of other three salidroside intervention groups (P<0.05). These results hinted that salidroside can prevent the increase of blood lactic acid of mice after swimming.

3.6. Effects of salidroside on contents of hepatic and muscle glycogen

After swimming, the content of hepatic glycogen of salidroside groups, especially intermediate, intermediate-high and high-dose groups, were higher than that of control group (P<0.05). The content of muscle glycogen of salidroside groups especially intermediate and intermediate-high dose groups were higher than that of the control group (P<0.05). These data indicated that salidroside can significantly increase the

content of hepatic and muscle glycogen of mice after swimming. See Table 4.

4. Discussion

Salidroside is one of the most critical constituents isolated from the roots of Rhodiola rosea. Its chemical name is (4-hydroxy-phenethyl)-

-D-glucopyranoside and molecular weight is 300.3. Previous study showed that salidroside has many biology effects include hepatoprotective effect [3], inhibiting leukemic cell growth [4], promoting the glucose uptaking and suppressing the differentiation of 3T3-L1 adipocyte [5]. Its claimed benefits also include anti-oxygen [6], anti-hypoxia [7–8] and anti-aging [9] and so on. But the anti-fatigue effects of salidroside and its dose-effect relation haven’t been reported until now.

The forced-swimming test is commonly used in anti-fatigue and endurance tests [10–11]. Our results showed that different doses of salidroside especially intermediate one could significantly lengthen the longest swimming time, which indicated that salidroside can elevate the exercise tolerance of mice.

Energy for exercise is derived initially from the breakdown of glycogen and, later , from circulating glucose released by the liver [12]. So

Table 3 Effects of salidroside on blood lactic acid concentrations in mice of different groups (mean ± SD, n=8, mmol/L)

Groups Beforeswimming

After swimming

A (Control) 4.31±0.58 11.61±1.79

B (Low dose) 4.45±0.32 10.54±1.13c

C (Intermediate dose) 4.14±0.77 9.06±1.61b

D (Intermediate-high dose) 4.21±0.73 9.81±1.65ac

E (High dose) 4.50±0.69 9.76±1.82ac

a <0.05,b <0.01 vs group A; c <0.05 vs group C Dose of salidroside, B: 60 mg/kg; C: 180 mg/kg; D: 360 mg/kg; E: 720 mg/kg.

Table 4 Effects of salidroside on hepatic and muscle glycogen of the mice after swimming (mean ± SD, n=8, mg/g)

Groups Hepaticglycogen

Muscle glycogen

A (Control) 7.61±3.25 1.23±0.31

B (Low dose) 13.83±4.09a 1.87±0.67a

C (Intermediate dose) 18.42±3.96b 2.01±0.54b

D (Intermediate-high dose) 19.02±5.37b 1.93±0.36b

E (High dose) 17.10±3.53b 1.88±0.43a

a <0.05, b <0.01 vs group A Dose of salidroside, B: 60 mg/kg; C: 180 mg/kg; D: 360 mg/kg; E: 720 mg/kg.

92 Ma Li et al./Journal of Medical Colleges of PLA 23(2008)88–93

liver and muscle glycogen are sensitive parameters related to fatigue. The data mentioned above revealed that the content of liver and muscle glycogen of mice in salidroside intervention groups were higher than that of control group after swimming. However its detailed mechanism isn’t clear. The possible reason is that salidroside may increase the content of liver and muscle glycogen of mice postexercise by improving glycogen reserve, or by reducing the consume of glycogen during exercise, or both. It still needs the further studies.

Serum urea nitrogen and blood lactic acid [13] are important blood biochemical parameters related to fatigue. In our experiment, the blood lactic acid of intermediate, intermediate-high and high dose of salidroside intervention groups were lower than that of control group after swimming. The results suggest that salidroside can inhibit the production of blood lactic acid during exercise. The content of serum BUN raise with the exercise load increasing. Our results showed the trend that serum BUN of the low, intermediate and intermediate-high dose intervention groups were lower than control group. In addition, our study also showed that low dose salidroside can increase hemoglobin of mice.

In conclusion, our results suggest that salidroside had significant anti-fatigue effects on mice and these effects were dose-dependent, and the strongest effect on most biomarkers was seen with an intermediate dose. The detailed mechanism of anti-fatigue properties of salidroside might be mediated through regulating central nervous system,antioxygen and improving substance metabolism and aerobic capacity. Studies showed that low and mediate dose of Rhodiola rosea could excitate central nervous system, while high dose of Rhodiola

rosea had sedative effects on central nervous system [2]. Our result that the intermediate dose of salidroside is the optimum dose for anti-fatigue may be concerned with this property of Rhodiola rosea.

However further studies to clarify the detailed mechanisms involved in the anti-fatigue properties of salidroside are necessary.

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(Editor Li Danyang)