Author for correspondence: Kanwaljit Chopra, Associate Professor of Pharmacology, Pharmacology Research Laboratory, University Institute of Pharmaceutical Sciences, UGC Centre of Advanced Study, Panjab University, Chandigarh-160 014, India (fax +91 172 2541142, e-mail email@example.com).
Abstract: Chronic fatigue syndrome (CFS) is a specific clinical condition that characterizes unexplained disabling fatigue. In the present study, chronic fatigue was produced in mice by subjecting them to forced swim inside a rectangular jar of specific dimensions for 6 min. daily for 15 days. Epigallocatechin gallate (EGCG; 25, 50 and 100 mg/kg, p.o.) was administered daily 30 min. before forced swim session. Immobility period and post-swim fatigue was assessed on alternate days. On the 16th day, after assessment of various behavioural parameters, mice were killed to harvest the brain, spleen and thymus. There was significant increase in oxidative–nitrosative stress and tumour necrosis factor-α levels in the brain of mice subjected to water-immersion stress as compared with naive group. These behavioural and biochemical alterations were restored after chronic treatment with EGCG. The present study points out that EGCG could be of therapeutic potential in the treatment of chronic fatigue.
Exposure to a stressful stimulus or chronic fatigue is perceived as a threat to the organism’s homoeostasis and elicits a variety of symptoms encompassing behavioural, biochemical and neurochemical aspects . Chronic fatigue syndrome (CFS) is an incapacitating illness defined by disabling chronic fatigue and characteristic accompanying signs  and its symptoms include reduction in daily activity >50% for at least 6 months . No physical signs are specific to either CFS or chronic fatigue, and there are no diagnostic tests to identify these syndromes which are generally based on symptom complaints, and may be characterized as heterogeneous with multiple aetiologies . In fact, chronic fatigue includes fewer symptoms than CFS . Chronic stress has been known to associate with other behavioural dysfunctions such as cognitive deficits, anxiety, neurochemical alterations and oxidative damage . Stress-related neuroendocrine mechanisms are also proposed to be involved in the pathogenesis of affective disorders. As there is no known cause of CFS, current treatment remains symptomatic with a focus on management rather than cure [6,7].
Oxidative stress has been suggested to be involved in the pathogenesis of the CFS [8–10]. Oxidative stress affects physical and mental function through various redox-sensitive signalling systems . CFS has also been found to be associated with increased immune activation and inflammatory cytokine levels . Hypofunctioning of the hypothalamic-pituitary-adrenal axis could lead to an exaggerated stress response and subsequently an excessive release of pro-inflammatory cytokines. Long-term stress increases levels of glucocorticoids and catecholamines, which suppresses immune function  and it has also been reported that during an immune response certain cytokines like interleukin (IL)-1, IL-6 and tumour necrosis factor-α (TNF-α) can signal the brain which triggers the activation of both the central nervous system and hypothalamic–pituitaryadrenal axis . Stress not only induces inflammatory reactions with an increased production of pro-inflammatory cytokines but it also induces pro-oxidant state and increases lipid peroxidation .
For the past decade, tea polyphenols have been picked up by the scientific community for its diverse biological activities . Epigallocatechin gallate (EGCG), the principal active constituent of green tea, is known to have anti-inflammatory, anti-carcinogenic and free radical-scavenging properties [15–17]. It also inhibits COX-2 without affecting COX-1 expression . EGCG possesses neuroprotective effects against a variety of toxic insults and inflammatory neuronal injuries [19–22]. EGCG also decreases the lipopolysaccharide (LPS)-induced nitrosative stress by down-regulating activity of nitric oxide synthase . Thus, the present study was carried out to assess the effect of EGCG in a murine water-immersion stress model of chronic fatigue.
Material and Methods
Animals. Albino Laca mice (20–30 g) bred in the Central Animal House facility of Panjab University, Chandigarh, India, were used for the study. The animals had free access to standard rodent food pellets and water. They were acclimatized to the laboratory conditions before the experiment. All the experiments were conducted between 9.00 a.m. and 5.00 p.m. The experimental protocol was approved by the Institutional Animal Ethics Committee, Panjab University, Chandigarh, and conducted according to the National Science Academy Guidelines for the use and care of animals.
Drugs. Epigallocatechin gallate was obtained as a gift sample from DSM Nutritional Products Ltd., Switzerland, and dissolved in distilled water. EGCG (25, 50 and 100 mg/kg) was administered by oral gavage daily, 30 min. before the forced swim test. All other chemicals were of analytical grade.
The animals were divided into four groups, consisting of 5–6 animals in each group. Group one received vehicle (water) while the second, third and fourth groups received EGCG 25, 50 and 100 mg/kg, p.o. respectively. Various behavioural parameters were assessed in mice 24 hr after the last chronic forced swim test.
Behavioural assessment. Assessment of immobility period. The mice were individually forced to swim inside a rectangular glass jar (25 × 12 × 25 cm3) containing 15 cm of water maintained at 22 ± 3°C; the total duration of immobility during a 6-min. test was recorded with the help of stop watch. The animal was judged to be immobile when it ceased struggling and remained floating motionless in water, making only movements necessary to keep its head above water. The animals were forced to swim 6 min. every day for a total of 15 days, and the recording of immobility period and post-swim fatigue was performed on alternate days.
Assessment of post-swim fatigue. Post-swim activity was measured immediately after the forced swim test. To assess post-exercise fatigue, we measured the time elapsed before the mice-initiated grooming (licking and rubbing of the skin/fur) after a 6-min. swim in water at 22 ± 3°C. Each mouse was removed from the forced swim test and excess water was allowed to drain. The mouse was then placed in a clear observation chamber. The time to grooming was recorded in seconds after each mouse was removed from the water .
Assessment of locomotor activity. Locomotor activity (ambulations) was measured by a computerized actophotometer (IMCORP; Ambala, Haryana, India) for 5 min. Mice were individually placed in a transparent plastic cage (30 × 23 × 22 cm3) and were allowed to acclimatize to the observation chamber for a period of 2 min. The locomotion was expressed in terms of total counts per 5 min. per animal .
Assessment of rota-rod test. Mice were subjected to motor function evaluation by placing them individually on rota rod, which was adjusted to the speed of 25 r.p.m. The fall-off time was recorded for each mouse and the longest period any animal was kept on the rod was 300 sec. .
Assessment of anxiety in the mirror chamber. The anxiety behaviour was measured using the mirror chamber. During the 5-min. test session, the following parameters were noted: (i) latency to enter the mirror chamber; (ii) the total time spent in mirror chamber; and (iii) the number of entries the animal made into the mirror chamber. Animals were placed individually at the distal corner of the mirror chamber at the beginning of the test. An anxiogenic response was defined as decreased number of entries and time spent in the mirror chamber .
Assessment of cognitive behaviour using plus-maze test. Cognitive behaviour was noted by elevated plus-maze learning task . Transfer latency, i.e. the time spent by the animal to move from the open arm to enclosed arm, was considered as an index of learned task (memory retention). The elevated plus maze consisted of two open arms (16 × 5 cm) and two closed arms (16 × 5 × 12 cm) with an open roof. The maze was elevated to a height of 25 cm from the floor. The animal was placed individually at the end of either of the open arms and the initial transfer latency was noted on the first day. If the animal did not enter an enclosed arm within 90 sec., it was gently pushed into the enclosed arm and the transfer latency was assigned as 90 sec. To become acclimatized with the maze, the animal was allowed to explore the plus maze for 20 sec. after reaching the closed arm and then returned to its home cage. Retention of the learned task was assessed 24 hr after the 1st day trial and expressed as per cent of initial transfer latency.
Assessment of stress-induced hyperalgesia. Stress-induced hyperalgesia was assessed by tail-immersion test. Each mouse was placed individually in restrainer leaving the tail hanging out freely. The terminal 1 cm part of the tail was immersed in a water bath maintained at 52.5 ± 0.5°C. The withdrawal latency was defined as the time for the animal to withdraw its tail from the water. A cut-off time of 10 sec. was used to prevent damage to the tail .
Biochemical estimations. Preparation of brain homogenate. On the 16th day of the study, the animals were killed by cervical dislocation. The brains were immediately removed, rinsed in ice-cold saline and weighed. A 10% w/v tissue homogenate was prepared in 0.1 M phosphate buffer (pH 7.4) which was further used for estimating lipid peroxidation, nitrite, reduced glutathione, superoxide dismutase (SOD) and catalase assay.
Measurement of oxidative stress. The malondialdehyde content, a measure of lipid peroxidation, was assayed in the form of thiobarbituric acid-reactive substances by the method of Wills et al. . Thiobarbituric acid-reactive substances were quantified using an extinction coefficient of 1.56 × 105/M/cm and expressed as nmol of malondialdehyde per mg protein. Tissue protein was estimated using the Biuret method and the brain malondialdehyde content expressed as nmol of malondialdehyde per mg of protein. Non-protein thiols were assayed by the method of Jollow et al. . The result was expressed as nmol of non-protein sulfhydryl per mg protein. Cytosolic SOD activity was assayed by the method of Kono et al. . Catalase activity was assayed by the method of Claiborne et al. .
Measurement of nitrosative stress. Nitric oxide (nitrate–nitrite) by product in brain tissue was determined using the standard total nitric oxide assay kit (Assay Design Inc., Ann Arbor, MI, USA). Nitrate was reduced to nitrite by 3-hr incubation with nitrate reductase in the presence of nicotinamide adenine dinucleotide 3-phosphate (NADPH). Nitrite was converted to a deep purple azo compound by the addition of Griess reagent. Total nitrite/nitrate concentration was calculated using sodium nitrate as standard. Results were expressed as μmol/mg protein.
Tumour necrosis factor-α estimation. The quantification of TNF-α was conducted by the help and instructions provided by R&D Systems Quantikine Mouse TNF-α immunoassay kits (R&D Systems Inc., McKinley Place NE, MN, USA), which is a 4.5-hr solid phase ELISA designed to measure mouse TNF-α levels. The assays employ the sandwich enzyme immunoassay technique. A monoclonal antibody specific for mouse TNF-α has been pre-coated in the microplate. Standards, control and samples are pipetted into the wells and any mouse TNF-α present is bound by the immobilized antibody. After washing away any unbound substance an enzyme-linked polyclonal antibody specific for mouse TNF-α is added to the wells. Following a wash to remove any unbound antibody–enzyme reagent, a substrate solution is added to the wells. The enzyme reaction yields a blue product that turns yellow when the stop solution is added. The intensity of the colour measured is in proportion to the amount of mouse TNF-α bound in the initial steps. The sample values are then read off the standard curve.
Statistical analysis. The results were measured in seconds and expressed as mean ± S.E.M. The intergroup variation was measured by one-way anova followed by Tukey’s test. Statistical significance was considered at p < 0.05. Immobility time and post-swimming fatigue were analysed by using repeated measure anova. The statistical analysis was performed using the Jandel Sigma Stat statistical Software (JANDEL SIGMA STAT, San Jose, CA, USA) and spss Statistical Software version 16 (SPSS South Asia Pvt. Ltd, Bangalore, India).
Effect of epigallocatechin gallate on body weight, food and water consumption.
The chronic fatigue group showed significant reduction in the overall body weight, food and water consumption of the mice. EGCG administration in higher doses (50 and 100 mg/kg) significantly prevented this reduction in body weight [F(5,24) = 24.697 (p < 0.001)], food [F(5,24) = 9.156 (p < 0.001)] and water consumption [F(5,24) = 13.27 (p < 0.001)] (table 1).
Table 1. Effect of EGCG on food (A) and water (B) intake, Body weight, thymus weight and spleen weight in mouse model of chronic fatigue syndrome.
*p < 0.05 as compared with control group; #p < 0.05 as compared with chronic fatigue group; $p < 0.05 different from one another.
31.2 ± 0.490
60.4 ± 1.21
112.8 ± 1.39
5.8 ± 0.37
6.2 ± 0.37
24 ± 0.632*
22 ± 0.32*
170.4 ± 1.32*
3 ± 0.31*
3.2 ± 0.2*
CFS + EGCG 25
25.2 ± 0.490
30 ± 0.45#$
153.2 ± 1.52#$
4 ± 0.44
4.2 ± 0.37
CFS + EGCG 50
27.6 ± 0.748#
38.8 ± 0.86#$
142 ± 1.14#$
4.4 ± 0.4
4.8 ± 0.37#
CFS + EGCG 100
30.4 ± 0.748#$
50.4 ± 0.93#$
127.8 ± 1.15#$
5.2 ± 0.2#
5.6 ± 0.24#
Effect of epigallocatechin gallate on spleen and thymus gland weight.
The chronic fatigue group resulted in hypertrophy of spleen and hypotrophy of thymus glands of mice. After 15 days of forced swimming fatigue, the organ weight index of the spleen was increased and thymus weight was decreased. Chronic treatment of EGCG significantly restored both the spleen [F(5,24) = 288.05 (p < 0.001)] and thymus weight [F(5,24) = 351.73 (p < 0.001)] (table 1).
Effect of epigallocatechin gallate on immobility time, post-swim fatigue. Chronic water-immersion stress for a 6-min. session daily for 15 days produced a significant increase in immobility time, post-swim fatigue as compared with the control group indicating severe fatigue as noted on days 1, 3, 5, 7, 9, 11, 13 and 15. However, the peak effects were observed on day 15. Oral administration of EGCG (25, 50, 100 mg/kg, p.o.) produced significant and dose-dependent decrease in immobility time [F(5,24) = 858.861 (EGCG-25), 620.61 (EGCG-50), 169.11 (EGCG-100); (p < 0.001)], post-swim fatigue [F(5,24) = 78.287 (EGCG-25), 40.558 (EGCG-50), 67.189 (EGCG-100); (p < 0.001)] in mice as compared with the challenged group (fig. 1).
Locomotor activity. Mice exposed to chronic forced swimming showed an increased in the locomotor activity as compared with unstressed mice; however, chronic treatment with EGCG (25, 50 and 100 mg/kg, p.o. for 15 days) significantly decreased the ambulatory scores [F(5,24) = 264.37 (p < 0.001)] in the chronic forced swim test (fig. 2A).
Rota rod test. Similarly, chronically stressed mice showed a significant decrease in the fall-off time as compared with unstressed mice, thus displaying muscle in-coordination. Daily treatment with EGCG (25, 50 and 100 mg/kg, p.o. for 15 days) before the exposure increased the mean fall-off time [F(5,24) = 899.07 (p < 0.001)] as compared with chronic water-immersion stress group (fig. 2B).
Hyperalgesia. Animals chronically subjected to water-immersion stress showed a significant decrease in tail withdrawal latency indicating hyperalgesia as compared with unstressed mice. Chronic treatment with EGCG (25, 50 and 100 mg/kg, p.o. for 15 days) significantly attenuated the development of the hyperalgesia [F(5,24) = 32.58 (p < 0.001)] in chronically stressed animals (fig. 2C).
Plus maze. Similarly, chronic fatigue significantly increased per cent initial transfer latency [F(5,24) = 11.06 (p < 0.001)] in mice as compared with unstressed mice which was reversed by chronic administration of EGCG (25, 50 and 100 mg/kg, p.o. for 15 days) (fig. 3A).
Mirror chamber. Chronic fatigue produced anxiety response in mice as the latency to enter the mirror chamber was significantly increased [F(5,24) = 253.88 (p < 0.001)] (fig. 3B), decreased the number of entries [F(5,24) = 21.03 (p < 0.001)] (fig. 3C), and also decreased the mean time spent in the mirror chamber [F(5,24) = 81.75 (p < 0.001)] (fig. 3D) as compared with unstressed mice. Daily treatment with EGCG (25, 50 and 100 mg/kg, p.o. for 15 days) reversed these responses in chronically exposed mice.
Effect of epigallocatechin gallate on biochemical alterations. Malonaldehyde (MDA) and nitrite levels were significantly increased in the brain of chronic fatigue mice as compared with the control group. Chronic treatment with EGCG produced significant (p <0.05) and dose-dependent reduction in MDA [F(5,24) = 73.99 (p < 0.001)] and nitrite levels [F(5,24) = 163.185 (p < 0.001)] in brain of chronic forced swimming mice. Reduced glutathione levels [F(5,24) = 41.65 (p < 0.001)] and enzyme activity of SOD [F(5,24) = 60.99 (p < 0.001)] and catalase significantly [F(5,24) = 52.92 (p < 0.001)] decreased in the brains of chronic forced swimming mice as compared with control group of mice (table 2). This reduction was significantly and dose-dependently restored by the treatment with EGCG in the brain of chronic forced swimming mice. Serum TNF-α level was markedly increased in chronic fatigue mice. EGCG (100 mg/kg) significantly decreased TNF-α level as compared with chronic fatigue group (table 3).
Table 2. Effect of EGCG on biochemical alterations LPO, nitrite, GSH, SOD and Catalase in mouse model of chronic fatigue syndrome
*p < 0.05 as compared with control group; #p < 0.05 as compared with chronic fatigue group.
25.13 ± 1.3
162 ± 2.12*
CFS + EGCG 25 mg/kg
139.2 ± 2.7#
CFS + EGCG 50 mg/kg
71.4 ± 5.1#
CFS + EGCG 100 mg/kg
47.4 ± 3.66#
In the present study, we have employed the forced swimming activity to induce a state of chronic fatigue in animals. Animals were to swim daily for 6 min. over a total period of 15 days . The results of the present study demonstrate that water immersion induces several fatigue symptoms such as increased immobility period, post-swim fatigue, hyperalgesia, anxiety along with week grip strength and impaired memory in mice. Various stress conditions induce hyperalgesia to thermal, chemical and mechanical stimuli . Patients with chronic fatigue often have difficulties with concentration and memory . Chronically stressed mice showed enhanced sensitivity to amphetamine-induced hyperlocomotion [36,37]. Usually, the patients with fatigue syndrome suffer from the co-morbid mood or anxiety syndrome [38,39]. The locomotor activity in mice was increased 24 hr after the last episode of forced swim test, which may be as a result of the fact that animals are more anxious 24 hr after the last forced swim session. Chronic treatment with EGCG significantly restored all the above-stated behavioural deficits including anxiety and hyperalgesia in the chronic fatigued mice in a dose-dependent manner. Vignes et al.  suggested that EGCG exerts anxiolytic effect by interacting with GABA receptors, and Kaur et al.  also found that green tea extract attenuates LPS-induced central and peripheral hyperalgesia by selective inhibition of cyclooxygenase-2 enzyme. In our study, we also found a significant impairment in memory of fatigued mice in elevated plus maze and this finding is supported by the results of Haiq-Ferquson et al. , who also found a significant deficit in memory and attention of children with CFS.
Fifteen days forced swimming increased lipid peroxidation, nitrite activity and depletion of reduced glutathione, SOD and catalase activity in the brains of fatigued mice suggesting involvement of oxidative–nitrosative in mediation of CFS. Chronic treatment with EGCG attenuated these biochemical alterations in mice brain and these effects were attributed to its strong antioxidant potential. EGCG is a hydrogen-donating antioxidant and free radical scavenger of reactive oxygen and nitrogen species [42,43]. In addition, it elevates the activity of oxygen radical species-metabolizing enzymes, namely SOD, glutathione peroxidase and catalase [44,45]. Furthermore, it also possesses metal-chelating properties and neutralizes ferric iron to form redox-inactive iron, thereby protecting cells against oxidative damage [46,47]. In a recent study by Lyle et al. , Nadostachys jatamansi extract alleviates symptoms of CFS by attenuating the enhanced oxido-nitrosative stress.
Experimental studies have reported the role of pro-inflammatory cytokines in CFS [49–51]. A hyper secretion of pro-inflammatory cytokines during stress caused by a hypofunctional neuroendocrine counter regulation could serve as a possible explanation of exercise- or stress-induced exacerbation of fatigue experienced by patients with CFS . In this study, water-immersion stress markedly increased the serum TNF-α levels in mice and this rise in serum TNF-α levels was significantly reduced by EGCG administration. TNF-α is also known to depress food intake by a centrally mediated effect leading to body weight loss [53,54]. In the present study, we also found that mice subjected to water-immersion stress showed reduced body weight along with decreased food and water consumption. Chronic treatment with EGCG significantly improved body weight, food and water consumption in murine model of CFS and this effect might be as a result of inhibition of TNF-α. Shin et al.  also suggest that treatment with EGCG inhibits secretion of TNF-α, IL-6 and IL-8 through the attenuation of extracellular signal-regulated kinase and nuclear factor kappa-B in human mast cell line-1 cells.
The core finding of the present study is that EGCG attenuates various behavioural and biochemical alterations because of chronic fatigue caused by daily exposure to forced swimming via inhibition of oxidative–nitrosative stress and inflammation and implicates its suitability as a therapeutic option in the treatment of patients suffering from CFS.
The research grant sanctioned to Dr (Ms) Kanwaljit Chopra by Central Council for Research in Ayurveda and Siddha (CCRAS), New Delhi is gratefully acknowledged. Mr Anurag Kuhad and Mr Vinod Tiwari are Indian Council of Medical Research (ICMR)-Senior Research Fellows. Mr Anand Kamal Sachdeva is UGC Junior Research Fellow. Authors are thankful to DSM Nutritional Products Ltd., Switzerland for providing the gift sample of epigallocatechin gallate.