International Journal of Biological Macromolecules 50 (2012) 59–62
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Anti-fatigue activity of polysaccharides extract from Radix Rehmanniae Preparata Wei Tan a,b , Ke-qiang Yu c , Yan-yan Liu a,b , Ming-zi Ouyang a,b , Mei-hua Yan a,b , Ren Luo a,b,∗ , Xiao-shan Zhao a,b,∗ a b c
Department of Traditional Chinese Medicine, Southern Hospital, Southern Medical University, Guangzhou, Guangdong 510515, China School of Traditional Chinese Medicine, Southern Medical University, Guangzhou, Guangdong 510515, China Department of Science and Technology, Southern Medical University, Guangzhou, Guangdong 510515, China
a r t i c l e
i n f o
Article history: Received 9 August 2011 Received in revised form 17 September 2011 Accepted 24 September 2011 Available online 1 October 2011 Keywords: Radix Rehmanniae Preparata Polysaccharides Anti-fatigue
a b s t r a c t The anti-fatigue effects of the Radix Rehmanniae Preparata polysaccharides (RRPP) were studied in mice. The RRPP were orally administered at doses of 50, 100 and 200 mg/kg for 4 weeks and the anti-fatigue activity was evaluated using a weight-loaded swimming test, along with the determination of serum urea nitrogen (SUN), hepatic glycogen and blood lactic acid (BLA) contents. The results showed that there was no significant difference in the body weight of mice in the three RRPP groups compared with the negative control group during initial, intermediate and terminal stages in the experiment (p > 0.05). The ratio of exhausting swimming time was obviously increased 31.48% (p < 0.05) and 61.51% (p < 0.01) in the middle-dose group and the high-dose RRPP group, respectively. The BLA and SUN levels were decreased in middle-dose and high-dose RRPP groups (p < 0.01). Hepatic glycogen level was increased in three RRPP treated groups (p < 0.01). Therefore, RRPP may be responsible for the pharmacological effect of anti-fatigue of Radix Rehmanniae Preparata. The mechanism was related to the increase of the storage of hepatic glycogen and the decrease of the accumulation of SUN and BLA. Crown Copyright © 2011 Published by Elsevier B.V. All rights reserved.
1. Introduction Radix Rehmanniae Preparata (Shu Dihuang) is prepared root of Rehmannia glutinosa Libosch. (Dihuang), which is in the family of Scrophulariaceae. It is a traditional Chinese medicinal herb, and it is widely used in China, Japan, Korea and other Asian countries. Recorded in Chinese medical classics Shennong’s Herba, it is considered as a top grade herb in China [1]. Radix Rehmanniae Preparata is on the list of herbs which can be used as health food (approved by ministry of health of China, 2002). It is basically accepted as a drug for nourishing Yin and tonifying the kidney that has the functions of storing essence, dominating growth, development and reproduction and regulating water metabolism in the body by traditional Chinese medicine (TCM) theory. It can be used to treat lumbar debility, blood deficiency and sallow complexion, metrorrhagia and metrostaxis, kidney and liver Yin deficiency, palpitations, abnormal menstruation et cetera [2]. Some famous Chinese traditional formulas have anti-fatigue activity, such as Zhibai Dihuang pills [3], Liuwei Dihuang decoction [4] and Qiju Dihuang pills [5]. In those formulas, Radix Rehmanniae Preparata (Shu Dihuang) is one
of the most important compositions. Radix Rehmanniae Preparata contains various compounds, such as polysaccharides, iridoid glycoside, phenol glycoside ionone, flavonoid, amino acid, inorganic ions and microelement [6]. Researches have showed that the contents of Radix Rehmanniae Preparata polysaccharides (RRPP) in different habitats were between 0.98% and 5.09% [7]. Modern researches have also indicated that polysaccharides are the main chemical components related to the bioactivities and pharmacological properties of Radix Rehmanniae Preparata. Research showed that there were two acidic polysaccharides, called rehmannan SA and rehmannan SB in RRPP. They were commonly composed of l-arabinose:d-galactose:l-rhamnose:d-galacturonic acid in the molar ratios of 10:10:1:1 (rehmannan SA) and 14:7:3:8 (rehmannan SB) [8]. According to reports, RRPP can stimulate hemopoiesis [9,10]; it has anti-tumor [11–15], immune enhancement [11,12], and anti-diabetes [16] effect. In this study, we have focused on the anti-fatigue activity of RRPP in order to understand part of the underlying mechanism of Radix Rehmanniae Preparata’s tonifying property. 2. Materials and methods
∗ Corresponding authors at: Department of Traditional Chinese Medicine, Southern Hospital, Southern Medical University, 1838 North Guangzhou Avenue, Guangzhou 510515, China. Tel.: +86 20 61641671. E-mail addresses:
[email protected] (R. Luo),
[email protected] (X.-s. Zhao).
2.1. Materials Radix Rehmanniae Preparata was purchased from Guangdong Tianchen Decoction Pieces of Traditional Chinese Medicine Co. Ltd.
0141-8130/$ – see front matter. Crown Copyright © 2011 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.ijbiomac.2011.09.019
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Table 1 Effects of RRPP on body weight in BALB/c mice. Group
N
Dose (mg/kg)
CG RRPP-LG RRPP-MG RRPP-HG
10 10 10 10
0 50 100 200
Body weight (g) Initial
(lot No. 100305, Guangdong, China). Reagent kits for the determination of blood lactic acid (BLA, lot No. 20100410), serum blood urea nitrogen (BUN, lot No. 20100430) and liver glycogen (lot No. 20100516) were purchased from Jiancheng Biotechnology Co. (Nanjing, China).
2.2. Preparation of Radix Rehmanniae Preparata polysaccharides The powdered dry Radix Rehmanniae Preparata (200 g) was homogenized and extracted two times with 1600 ml of distilled water for 4 h at 98 ∼ 100 ◦ C by consulting Cui’s method [17]. The whole extract was filtered and centrifuged at 1000 × g for 30 min at 4 ◦ C. The supernatant was concentrated to 100 ml and precipitated by the addition of 95% ethanol in 1:4.3 ratio (v/v) at room temperature. After 24 h precipitation, the sample was centrifuged as described above, and the precipitate was dissolved in 100 ml of distilled water. This process was repeated three times. The final precipitate was then washed with sevage reagent (isoamyl alcohol and chloroform in 1:4 ratio) [18] and freeze-dried, which yielded the crude polysaccharide from Radix Rehmanniae Preparata (RRPP). The crude RRPP from starting crude materials was approximately 21.13% (w/w). Total sugar in the crude RRPP was 25.82% (by the phenol–sulfuric acid method using glucose as standard solution).
2.3. Experimental animals Male BALB/c mice (8 weeks old, 17 ∼ 20 g) were obtained from Medical Laboratory Animal Center of Guangdong province (Approval No. SCXK (Yue) 2008-0002), Foshan, China. The mice were housed at a room temperature of 23 ◦ C ± 1 ◦ C with a 12h-light and12h-dark cycle (lights on from 6: 00 am to 6: 00 pm). Food and water were available ad libitum. Mice were treated in compliance with the current law and the Guiding Principles for the Care and Use of Laboratory Animals approved by the Animal Ethics Committee of China.
2.4. Experimental design Mice were trained to accustom themselves to swimming twice (10 min per time) in the first week. During the period, the mice which could not learn to swim were screened out. Then 80 mice were chosen and randomly divided into four groups, with 20 mice in each group. RRPP was given to the mice at doses of 0, 50, 100 and 200 mg/kg and the four groups were accordingly named as the negative control group (CG), the low-dose group (RRPP-LG), the middle-dose group (RRPP-MG) and the high-dose group (RRPPHG). The same volume of distilled water was given to mice in CG. Samples were orally administered (8: 00 am) into mice using a feeding atraumatic needle once per day for 4 weeks. Changes of the body weight of the mice were observed during initial, intermediate and terminal stages of the test along with the swimming capacity and corresponding biochemical parameters including serum urea nitrogen (SUN), blood lactic acid (BLA) and hepatic glycogen.
21.17 21.23 21.21 21.17
Intermediate ± ± ± ±
1.12 1.17 1.10 1.11
25.37 25.27 25.55 25.18
± ± ± ±
1.06 1.04 1.14 1.18
Final 28.65 28.35 28.74 28.46
± ± ± ±
0.84 0.98 0.83 1.01
2.5. Weight-loaded swimming test After 28 days, 10 mice were taken out from each group for weight-loaded swimming test. The procedure used in this experiment was similar to that described by Porsolt et al. [19]. Briefly, 30 min after the last intragastric administration, the mice were placed individually in a swimming pool (30 cm high, 25 cm in diameter) in which the mice could only support themselves by touching the bottom with their feet (at 25 ◦ C ± 1 ◦ C). A tin wire (7% of body weight) was loaded on the tail root of each mouse. The swimming period was regarded as the time spent by the mouse floating in the water with struggling and making necessary movements until exhausting its strength. The mice were assessed to be exhausted when they failed to rise to the surface of water to breathe within a 10 s period. At the end of the session, the mice were removed from the water, dried with a paper towel, and placed back in their home cages. Water in the container was drained after each session. The swimming time to exhaustion was used as the index of the forced swimming capacity. The ratio of average exhausted swimming time was equal to the average swimming time of mice in each group/the average swimming time of mice in the control group. 2.6. Determination of hepatic glycogen, SUN and BLA After 28 days, the other 10 mice were taken out from each group for analyses of hepatic glycogen and blood biochemical parameters. 30 min after the last intragastric administration of RRPP, the mice were forced to swim in the swimming pool (weight-unloaded) for 90 min. Rested for 60 min, the mice were anesthetized with pentobarbital sodium. The blood samples of the mice were respectively collected in heparinized tubes and tubes without anticoagulant by removing the left eyeball. Blood plasma was prepared by centrifugation at 1000 × g, 4 ◦ C for 10 min. Serum was prepared by centrifugation at 1000 × g, 4 ◦ C for 15 min. The blood plasma was tested to determine the concentration of BLA and the serum for SUN. After the blood was collected, the livers of the mice were immediately dissected, frozen in liquid nitrogen, and kept at −80 ◦ C until analysis of glycogen concentration was performed. The concentration of BLA, SUN and hepatic glycogen were tested following the recommended procedures provided by the kits. 2.7. Statistical analysis All data were expressed as the mean ± standard error (SE) in the tables and indicated by vertical bars in the figures. Differences between groups were determined by analysis of variance and Student’s t-test. Probability value p less than 0.05 was considered significant. 3. Results and discussion 3.1. Effect of RRPP on body weights in BALB/c mice Change of body weight during the experimental period was shown in Table 1. Body weight was recorded before experiment (initial), after 14 days (intermediate) and after 28 days (final). There
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such as the RRPP in this study, the carbohydrate may accumulate in the liver as an energy metabolism mass. Therefore, the rate of conversion of protein into urea may slow down. Table 2 showed that the group of RRPP-HG and RRPP-MG reduced the SUN level in BALB/c mice compared with CG (p < 0.01). The result indicated that reduce the level of SUN may be one of the pathways of RRPP’s anti-fatigue effect. 3.4. RRPP decreased lactic acid in the blood
Fig. 1. Effects of RRPP on the ratio of exhausted swimming time in BALB/c mice (a, p < 0.05 compared with that in the CG; b, p < 0.01 compared with that in the CG).
was no significant difference in the body weight of mice in the three RRPP groups compared with that in the CG during initial, intermediate and terminal stages in the experiment (p > 0.05). In the present study, RRPP had no significant effect on the body weight. 3.2. RRPP prolonged the exhaustive swimming time The present study demonstrated an anti-fatigue activity of RRPP in the weight-loaded forced swimming test, and a valid animal model for screening anti-fatigue agents [20]. The length of the swimming time to exhaustion indicated the degree of fatigue. The improvement of exercise endurance was the most powerful representation of anti-fatigue effect. The exhausting swimming time of the mice was measured to investigate the effect of RRPP inhibiting fatigue. The swimming capacity was increased after administration of RRPP for 28 days compared with that of the CG (Figs. 1 and 2). Significant increases were observed in RRPP-MG (100 mg/kg, p < 0.05) and RRPP-HG (200 mg/kg, p < 0.01). Fig. 1 showed that the increased ratio of exhausting swimming time of each treatment group (RRPPLG, RRPP-MG and RRPP-HG) were 10.07%, 31.48% and 61.51%, respectively. Fig. 2 showed that the exhausting swimming time in RRPP-LG, RRPP-MG, RRPP-HG and CG were 406.6 s, 485.7 s, 596.6 s and 369.4 s, respectively. These results indicated that RRPP had significant effects on the endurance of mice in this experiment.
Generally, the muscle produces plenty of lactic acid when it obtains enough energy from anaerobic glycolysis almost at the same time when doing high-intensity exercise. One of the main pathways to remove excess lactic acid is the conversion of lactate to glucose via gluconeogenesis, and excess glucose will then be saved as hepatic glycogen (HG) [22]. Besides, lactic acid could also be used for replenishing muscle glycogen stores. The increased level of lactic acid will bring about a reduction of pH in muscle tissue and blood, and also induce many side effects of various biochemical and physiological processes, that were harmful to the body performance. So, blood lactic acid (BLA) was measured as an index of anaerobic glucose metabolism. Table 2 showed that the group of RRPP-HG and RRPP-MG reduced the BLA level in BALB/c mice compared with CG (p < 0.01). The result indicated that reduce the level of BLA may be another pathway of RRPP’s anti-fatigue effect. 3.5. RRPP increased hepatic glycogen Liver is the direct tissue for energy conservation and utilization. The liver converts lactate back to glycogen and releases glycogen into the blood. Energy for exercise is derived initially from the breakdown of glycogen, and later from circulation glycogen released by the liver and from non-esterified fatty acids [23]. So increasing the HG storage conduces to enhancing the endurance capacity and locomotory capacity. Table 2 showed that HG level of all three RRPP groups obviously increased (p < 0.01), compared with that in the CG. The decreased BLA level and the enhanced liver glycogen storage in this experiment signified the occurrence of liver gluconeogenesis during intense swimming exercise. So, the increased level of HG all so may be one of the pathways of RRPP’s anti-fatigue effect.
3.3. RRPP decreased serum urea nitrogen in the blood 3.6. Fatigue and polysaccharides Serum urea nitrogen (SUN) is one of blood biochemical parameters related to fatigue. SUN, the metabolic outcome of protein and amino acid, was a sensitive index to evaluate the bearing capability when body suffered from a physical load. In other words, the worse the body is adapted for exercise tolerance, the more significantly the SUN level increases [21]. Urea is formed in the liver as the endproduct of protein metabolism. When excess carbohydrate is taken,
Fig. 2. Effects of RRPP on the weight-loaded swimming time in BALB/c mice (a, p < 0.05 compared with that in the CG; b, p < 0.01 compared with that in the CG).
Fatigue is one of the most frequent physiological reactions. It often occurred in aging, cancer, depression, HIV infection, multiple sclerosis and Parkinson’s disease [24]. However, there were very few pharmacological drugs or therapies available for the treatment of fatigue [25]. Natural products not only could improve athletic ability, postpone fatigue and accelerate the elimination of fatigue in human beings, but also had few side effects [26]. Most research shows that the polysaccharides which were extracted from traditional Chinese medicine herb have anti-fatigue activity. Polysaccharides from Morinda officinalis [27], Panax ginseng Meyer [28], shiitake [29], Dimocarpus longan Lour. seed [30], Ganoderma lucidum [31], had been reported for their anti-fatigue activities. The anti-fatigue activity of the RRPP may partially explain the tonifying property in traditional Chinese medicine, which provided scientific evidence for traditional medicine and further development of medicinal products for prevention and treatment of diseases related to fatigue. In conclusion, RRPP enhanced the swimming capacity of mice by decreasing the accumulation of SUN, delaying the accumulation of lactic acid, and by improving the energy storage. However, further study is needed to find out which part of the polysaccharides act the most important anti-fatigue effect and to elucidate the more
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Table 2 Effects of RRPP on serum urea nitrogen, blood lactic acid and hepatic glycogen levels in BALB/c mice. Group
N
Dose (g/kg)
SUN (mmol/L)
CG RRPP-LG RRPP-MG RRPP-HG
10 10 10 10
– 50 100 200
9.40 8.99 8.60 8.19
a
± ± ± ±
0.52 0.34 0.31a 0.30a
BLA (mg/100 mL) 21.69 20.51 18.24 15.82
± ± ± ±
1.42 1.18 1.37a 1.52a
HG (mg/g) 9.47 12.01 17.13 17.38
± ± ± ±
1.33 1.81a 1.55a 1.36a
p < 0.01 compared with CG.
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