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Behavioural Brain Research 205 (2009) 414–420

Contents lists available at ScienceDirect

Behavioural Brain Research

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pigallocatechin gallate ameliorates chronic fatigue syndrome in mice:ehavioral and biochemical evidence

nand Kamal Sachdeva, Anurag Kuhad, Vinod Tiwari, Kanwaljit Chopra ∗

harmacology Research Laboratory, University Institute of Pharmaceutical Sciences, UGC Centre of Advanced Study, Punjab University, Chandigarh 160 014 India

r t i c l e i n f o

rticle history:eceived 19 June 2009eceived in revised form 20 July 2009ccepted 21 July 2009vailable online 28 July 2009

eywords:hronic fatigue syndrome

a b s t r a c t

Three decades after the coining of the term chronic fatigue syndrome, the diagnosis of this illnessis still symptom based and the aetiology remains elusive. Chronic fatigue syndrome pathogene-sis seems to be multifactorial and the possible involvement of immune system is supported. Thepresent study was designed to evaluate the effects of the epigallocatechin gallate in a mouse modelof immunologically induced chronic fatigue. On 19th day, after lipopolysaccharide/Brucella abor-tus administration, the mice showed significant increase in immobility period, post swim fatigueand thermal hyperalgesia. Behavioral deficits were coupled with enhanced oxidative–nitrosative

pigallocatechin gallateipopolysacchariderucella abortusumor necrosis factor-�

stress as evident by increased lipid peroxidation, nitrite levels and decreased endogenous antioxi-dant enzymes (superoxide dismutase, reduced glutathione and catalase) and inflammation (increasedlevels of tumor necrosis factor-� and tissue growth factor-�). Chronic treatment with epigallocate-chin gallate restored these behavioral and biochemical alterations in mice. The present study pointsout towards the beneficial effect of epigallocatechin gallate in the amelioration of chronic fatiguesyndrome and thus may provide a new, effective and powerful strategy to treat chronic fatiguesyndrome.

. Introduction

Chronic fatigue syndrome is a specific clinical condition thatharacterizes unexplained disabling fatigue and a combinationf non-specific accompanying symptoms for at least 6 months.he estimated world-wide prevalence of chronic fatigue syn-rome is 0.4–1%. The pathophysiology of CFS is unclear and theain hypotheses include altered chronic fatigue syndrome func-

ioning resulting from an abnormal immune response against aommon antigen, a neuroendocrine disturbance. Various studiesave sought evidence for a disturbance in immunity in peopleith chronic fatigue syndrome. Abnormal activation of the T lym-hocyte subsets and high levels of pro-inflammatory cytokinesxplains many manifestations of disease process. Gene expres-ion studies have demonstrated that genes crucial for T-cellctivation and other immunological functions are differentiallyxpressed in chronic fatigue syndrome patients [12,25,35]. The

ro-inflammatory cytokines trigger the formation of reactivexygen species, experimental evidences showed that oxidativetress and, more specifically, lipid peroxidation contribute tohe progression of chronic fatigue [14,16,37,41]. Evidences of

∗ Corresponding author. Tel.: +91 172 2534105; fax: +91 172 2541142.E-mail address: dr chopra k@yahoo.com (K. Chopra).

166-4328/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.bbr.2009.07.020

© 2009 Elsevier B.V. All rights reserved.

oxidative damage of DNA and lipids in the vastus lateralis mus-cles of chronic fatigue syndrome patients have been observed[32,33].

Antioxidants have also been shown to be effective in endotoxin-induced behaviors, they trap free radicals and may delay theonset of lipid peroxidation [17]. Various in vitro and in vivostudies showed the ability of melatonin is a strong antioxidantto reverse the rise in the lipopolysaccharide-induced pro-inflammatory cytokine levels and attenuate endotoxin-inducedsickness behavior [44]. We have recently shown that curcuminattenuated lipopolysaccharide-induced sickness behavior by itsantioxidant activity as well as by inhibiting lipopolysaccharide-induced cytokine production [16].

Epigallocatechin gallate, the principal active constituent ofgreen tea, is known to have anti-inflammatory, anticarcinogenic,and free radical-scavenging properties [31,20,48]. It also inhibitsCOX-2 without affecting COX-1 expression [21]. Epigallocatechingallate possesses neuroprotective effects against a variety of toxicinsults and inflammatory neuronal injuries [4,8,36,57]. Epigallocat-echin gallate also blocks the induction of nitric oxide synthase by

down-regulating lipopolysaccharide-induced activity [31].

In the current study, we examined the effects of the polyphe-nol, epigallocatechin gallate, in a mouse model of immunologicallyinduced fatigue, wherein purified lipopolysaccharide and Brucellaabortus antigens were used as immunogens.

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. Material and methods

.1. Animals

Albino Laca mice (20–30 g) bred in the Central Animal House facility of Punjabniversity, Chandigarh, India were used for the study. The animals had free access

o standard rodent food pellets and water. They were acclimatized to the laboratoryonditions before the experiment. All the experiments were conducted between9.00 and 17.00. The experimental protocol was approved by the Institutional Ani-al Ethics Committee, Punjab University, Chandigarh and conducted according to

he National Science Academy Guidelines for the use and care of animals.

.2. Drugs

Escherichia coli lipopolysaccharide serotype 0111:B4 containing not less than00,000 EU/mg was (purchased from Sigma, USA), dissolved in sterile saline (0.9%aCl); Brucella abortus containing not less than 4 × 108 particles/ml was procured

rom the Central Research Institute, Kasauali, Himachal Pradesh, India. Epigallo-atechin gallate was obtained as gift sample from DSM Nutritional Products Ltd.,witzerland. Lipopolysaccharide and Brucella abortus were administered as singlentra-peritoneal injection. Epigallocatechin gallate was dissolved in distilled waternd administered by oral gavage. All other chemicals were of analytical grade.

.3. Treatment protocol and experimental procedure

Animals were divided in six groups comprising of six animals in each group.roup I comprised the control group, where animals received distilled water

vehicle) for 21 days. Group II control animals received epigallocatechin gallate100 mg/kg; oral gavage) for 21 days. Group III animals were challenged with sin-le intra-peritoneal injection of lipopolysaccharide (1 mg/kg), followed by distilledater for 21 days. Group IV included animals challenged with Brucella abor-

us (0.2 ml/mouse/i.p.) followed by administration of distilled water for 21 days.roups V and VI comprised the epigallocatechin gallate-treated group, where twoubgroups (each for lipopolysaccharide and Brucella abortus) of animals receivedifferent doses of epigallocatechin gallate (50, 100 mg/kg; oral gavage) 30 min before

ipopolysaccharide/Brucella abortus administration and continued for 21 days afteripopolysaccharide/Brucella abortus challenge.

Epigallocatechin gallate was administered 30 min before the lipopolysaccha-ide/Brucella abortus challenge on day 1 followed by the recording of the immobilityeriod, post-swim fatigue and tail withdrawal latency (for the measurement oftress-induced hyperalgesia). The animals were subjected to water-immersion testor 10 min daily and immobility was recorded on days 1, 7, 14, 19 and 21. The tailithdrawal latency was also measured on the same days. The post-swim fatigue waseasured daily for 21 days. Body weight as well as food and water intake was mon-

tored weekly. On day 22, blood was collected through tail vein and then animalsere sacrificed to harvest the brains, spleens and thymus.

.4. Assessment of fatigue and related parameters

.4.1. Water-immersion stress testMice were forced to swim individually in glass jar (25 cm × 12 cm × 25 cm) con-

aining 15 cm-deep water at room temperature (22 ± 3 ◦C). After an initial period ofigorous activity each animal assumed a typical immobile posture. The mice wereonsidered to be immobile when they ceased to struggle and made minimal limbovements to keep their head above the water level. Animals were forced to swim

aily for 10 min. The immobility period was noted for a period of initial 6 min in aotal period of 10 min duration on days 1, 7, 14, 19 and 21.

.4.2. Post-swim fatigue assessmentPost-swim activity was measured immediately after the forced swim test. To

ssess post exercise fatigue, we measured the time elapsed before mice initiatedrooming (licking and rubbing of the skin/fur) after a 10-min swim in water at2 ± 3 ◦C. Each mouse was removed from the forced swim test and placed in clearbservation chamber. The time to grooming was recorded in seconds after eachouse was removed from the water [7].

.4.3. Stress-induced hyperalgesiaStress-induced hyperalgesia was assessed by tail immersion test. Mice were held

ndividually and their tails were immersed in hot water (52.5 ± 0.5 ◦C) for not morehan 10 s (cut off time) and withdrawal latency was observed on the same days ashe immobility was observed [24].

.5. Biochemical studies

.5.1. Preparation of brain homogenateOn the 22nd day of the study, the animals were sacrificed by cervical dislocation.

he 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) whichas further used for estimating lipid peroxidation, nitrite, reduced glutathione,

uperoxide dismutase and catalase assay.

Research 205 (2009) 414–420 415

2.5.2. Assessment of oxidative stressThe malondialdehyde content, a measure of lipid peroxidation, was assayed in

the form of thiobarbituric acid-reactive substances by the method of Wills [53]. Thio-barbituric acid-reactive substances were quantified using an extinction coefficientof 1.56 × 105 M−1cm−1 and expressed as nmol of malondialdehyde per mg protein.Tissue protein was estimated using the Biuret method and the brain malondialde-hyde content expressed as nanomoles of malondialdehyde per milligram of protein.Non-protein thiols were assayed by the method of Jollow et al. [23]. The result wasexpressed as nmol of NPSH per mg protein. Cytosolic superoxide dismutase activitywas assayed by the method of Kono et al. [26]. Catalase activity was assayed by themethod of Claiborne [9].

2.5.3. Assessment of nitrosative stressNitric oxide (nitrate–nitrite) by product in brain tissue was determined using

the standard total nitric oxide assay kit (Assay Design Inc., USA). Nitrate was reducedto nitrite by 3 h incubation with nitrate reductase in the presence of nicotinamideadenine dinucleotide 3-phosphate (NADPH). Nitrite was converted to a deep purpleazo compound by the addition of Griess reagent. Total nitrite–nitrate concentrationwas calculated by using the standard of sodium nitrate. Results were expressed asmicromoles/mg protein.

2.5.4. TNF-˛ and TGF-ˇ estimationsThe quantification of TNF-� and TGF-� was done by the help and instruc-

tions provided by R&D Systems Quantikine Mouse TNF-� and TGF-� immunoassaykits. The Quantikine Mouse TNF-� and TGF-� immunoassays are 4.5 h solid phaseELISA designed to measure mouse TNF-� and TGF-� levels. The assays employthe sandwich enzyme immunoassay technique. A monoclonal antibody specificfor mouse TNF-�/TGF-� has been pre-coated in the microplate. Standards, con-trol and samples are pipetted into the wells and any mouse TNF-�/TGF-� presentis bound by the immobilized antibody. After washing away any unbound sub-stance an enzyme linked polyclonal antibody specific for mouse TNF-�/TGF-� isadded to the wells. Following a wash to remove any unbound antibody-enzymereagent, a substrate solution is added to the wells. The enzyme reaction yieldsa blue product that turns yellow when the stop solution is added. The inten-sity of the color measured is in proportion to the amount of mouse TNF-�/TGF-�bound in the initial steps. The sample values are then read off the standardcurve.

2.6. Statistical analysis

The results were measured in seconds and expressed as mean ± SEM. Theintergroup variation was measured by one-way analysis of variance (ANOVA)followed by Dunnett’s test. Statistical significance was considered at p < 0.05.The statistical analysis was done using the Jandel Sigma Stat statisticalSoftware.

3. Results

3.1. Effect of epigallocatechin gallate on body weight, food andwater consumption

Lipopolysaccharide/Brucella abortus challenge produced a sig-nificant reduction in the overall body weight, food and waterconsumption by the mice. Epigallocatechin gallate administra-tion significantly prevented this reduction in body weight, foodand water consumption (Fig. 1). However epigallocatechin gallate(100 mg/kg) alone group did not show any effect.

3.2. Effect of epigallocatechin gallate on immobility time,post-swim fatigue and hyperalgesia

Chronic water-immersion stress for 10 min session per dayfor 21 days after lipopolysaccharide/Brucella challenge producedsignificant increase in immobility time, post-swim fatigue alongwith decrease in tail withdrawal latency in mice as comparedto the control group indicating severe fatigue as noted on days1, 7, 14, 19 and 21. However, the peak effects were seen onday 19. Therefore, the effects of epigallocatechin gallate were

tested on the 19th day after lipopolysaccharide/Brucella chal-lenge.

Lipopolysaccharide/Brucella treatment produced significantincrease in mean immobility time, mean post-swim fatigue timeas well as significant decrease in mean tail withdrawal latency in

416 A.K. Sachdeva et al. / Behavioural Brain

Fig. 1. Effect of epigallocatechin gallate on body weight (A), food (B) and water (C)intake in mouse model of chronic fatigue syndrome. EGCG 50 mg = epigallocatechingg#

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dently improved by the treatment with epigallocatechin gallatein brain of lipopolysaccharide/Brucella treated mice. Serum TNF-�and TGF-� levels were markedly increased in lipopolysaccha-ride/Brucella challenged mice. Epigallocatechin gallate (100 mg/kg)

allate 50 mg/kg oral gavage; EGCG 100 mg = epigallocatechin gallate 100 mg/kg oralavage; CF = chronic fatigue (LPS/BA treated). *p < 0.05 as compared to control group,p < 0.05 as compared to chronic fatigue group.

ice as compared with the vehicle group (Fig. 2). Oral admin-stration of epigallocatechin gallate (50, 100 mg/kg) producedignificant and dose-dependent decrease in immobility time, post-wim fatigue along with increased tail withdrawal latency in mices compared with the challenged group (Fig. 2). However epi-allocatechin gallate (100 mg/kg) alone group did not show anyffect.

.3. Effect of epigallocatechin gallate on spleen and thymus glandeight

The lipopolysaccharide/Brucella challenge resulted in hypertro-hy of spleen and hypotrophy of thymus glands of mice. After

ipopolysaccharide and Brucella challenge, the organ weight index

f the spleen was increased by 104% and 106% and thymus weightas decreased by 57% and 52% respectively. Chronic treatment of

pigallocatechin gallate significantly restored spleen and thymuseight (Fig. 3). However epigallocatechin gallate (100 mg/kg) alone

roup did not show any effect.

Research 205 (2009) 414–420

3.4. Effect of epigallocatechin gallate on biochemical alterations

Malonaldehyde and nitrite levels were significantly increasedin the brain of lipopolysaccharide/Brucella abortus treated mice ascompared to control group. Chronic treatment with epigallocate-chin gallate produced a significant (p < 0.05) and dose-dependentreduction in malonaldehyde and nitrite levels in brain oflipopolysaccharide/Brucella treated mice (Fig. 4). Reduced glu-tathione levels and enzyme activity of superoxide dismutaseand catalase significantly decreased in the brains of lipopolysac-charide/Brucella treated mice as compared to control groupmice (Fig. 4). This reduction was significantly and dose depen-

Fig. 2. Effect of epigallocatechin gallate on immobility (A), post-swimfatigue (B) and hyperalgesia (C) in mouse model of chronic fatigue syn-drome. EGCG 50 mg = epigallocatechin gallate 50 mg/kg oral gavage; EGCG100 mg = epigallocatechin gallate 100 mg/kg oral gavage; CF = chronic fatigue(LPS/BA treated). *p < 0.05 as compared to control group, #p < 0.05 as compared tochronic fatigue group.

A.K. Sachdeva et al. / Behavioural Brain

Fig. 3. Effect of epigallocatechin gallate on thymus (A) and spleen (B) weight inm5a#

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ouse model of chronic fatigue syndrome. EGCG 50 mg = epigallocatechin gallate0 mg/kg oral gavage; EGCG 100 mg = epigallocatechin gallate 100 mg/kg oral gav-ge; CF = chronic fatigue (LPS/BA treated). *p < 0.05 as compared to control group,p < 0.05 as compared to chronic fatigue group.

ignificantly decreased TNF-� and TGF-� levels as compared withipopolysaccharide/Brucella challenged mice (Table 1). Howeverpigallocatechin gallate (100 mg/kg) alone group did not show anyffect on these parameters.

. Discussion

The central nervous system is extremely susceptible to immuno-ogical reactions which occur during the disease and injury30,39]. It is postulated that an infection may modulate the cen-ral nervous system endocrine-immune interactions via enhanced

xidative–nitrosative stress and cytokine release. Studies havestablished that cytokines induced by lipopolysaccharide can acti-ate the hypothalamus–pituitary–adrenal axis [46], induce fever11], prolong slow wave sleep [27], reduce food [38] and waterntake [6] and decrease motility [10]. It is suggested that an

able 1ffect epigallocatechin gallate on serum TNF-� and TGF-� levels in mouse model ofhronic fatigue syndrome.

Treatment group Serum TNF-�level (pg/ml)

Serum TGF �level (pg/ml)

Control 25.13 ± 1.3 50 ± 1.43Lipopolysaccharide 243.20 ± 4.71* 90.33 ± 1.8*

Brucella abortus 208.5 ± 2.75* 88.83 ± 1.35*

LPS + EGCG 100 80.41 ± 2.69# 58.33 ± 2.42#

BA + EGCG 100 69.79 ± 1.91# 62.66 ± 2.01#

PS = lipopolysaccharide, BA = Brucella abortus, and EGCG 100 = epigallocatechinallate 100 mg/kg.

* p < 0.05 as compared to control group.# p < 0.05 as compared to LPS/BA treated group.

Research 205 (2009) 414–420 417

infection and/or reactivation of a latent virus may have a contribu-tory role in a subset of chronic fatigue syndrome cases [7]. Severalcytokines produced during the immune response to infection aredescribed to be mediators of some symptoms including fever, som-nolence, lymphadenopathy and appetite loss [52]. TNF-� may beassociated with central nervous system pathology because it hasbeen associated with demyelination and may also lead to loss ofappetite [5,54]. Patarca et al. [42] had found elevations in serumlevels of TNF-� and TNF-� level in CFS patients.

In the present study, mice were immunologically challengedwith lipopolysaccharide/Brucella and were forced to swim for10 min daily so as to cause severe fatigue. Administration oflipopolysaccharide as well as killed Brucella abortus producedrobust increase in the immobility as well as post-swim fatigueduration, which reached peak levels on the 19th day [16]. Fewother workers have used lipopolysaccharide to produce immobil-ity; however, they have observed animals only for 24 h. By thattime, the activity tends to return to normal [22]. We observedthe animals for 21 days after immunologic challenge coupled withdaily forced swimming. This model elucidates long-term behav-ioral and pathophysiological consequences of immune activation.The results of the present study demonstrated that administrationof immune challenge induces several fatigue symptoms such asincreased immobility period, post-swim fatigue, and hyperalgesia.Administration of epigallocatechin gallate significantly decreasedimmobility time, post-swim fatigue and hyperalgesia. Quercetinand green tea extract have attenuated lipopolysaccharide-inducedcentral and peripheral hyperalgesia [3,24].

Lipopolysaccharide treated mice displayed reduced bodyweight along with decreased food and water consumption. Chronictreatment with epigallocatechin gallate significantly improvedbody weight, food and water consumption in lipopolysaccharide-treated mice. These findings are in concurrence with few previousstudies which demonstrated that activation of the immune systemby lipopolysaccharide, as well as other immune challenges, inducesa reduction in appetite and body weight, suppression of locomotor,exploratory and social activity, fatigue and malaise, impairmentin cognitive abilities, reduced libido and anhedonia [28]. Further,IL-1 has been shown to reduce food intake at doses within thepathophysiologic range and is recognized along with TNF-� as aprincipal mediator of anorexia. Previous work has established thatIL-1-induced feeding suppression is mediated by IL-1R1 receptorswithin the brain [29]. TNF-� is also known to depress food intakeby a centrally mediated effect leading to body weight loss [13]. Thiseffect might be due to a direct action of peripheral cytokines or viaprostaglandin release on glucose sensitive neurons in hypothala-mic nuclei such as the lateral hypothalamic arc. Chronic treatmentwith green tea extract successfully ameliorated these effects whichmay be ascribed to potent inhibitory effects of green tea extracton cytokines such as IL-6 and TNF-� [19,55]. Recently it has beenshown that epigallocatechin gallate which is one of the active con-stituents of green tea extract may be a therapeutically effectiveinhibitor of IL-1�-induced inflammatory effects [1].

In this study, lipopolysaccharide/Brucella abortus challengemarkedly increased the serum TNF-� and TGF-� levels in miceand this rise in serum TNF-� and TGF-� levels were signif-icantly reduced by epigallocatechin gallate administration. Asignificant elevation in serum levels of IL-1, TNF-� and TGF-� in the patients with chronic fatigue syndrome has beenreported [43,51]. Epigallocatechin gallate has been reported todecrease lipopolysaccharide-induced TNF-� production in a dose-

dependent manner in the murine macrophage cell line [55]. In onestudy, epigallocatechin gallate inhibits TNF-� induced MCP-1 pro-duction [2]. Epigallocatechin gallate significantly suppressed theheat shock protein 27 induction stimulated by TGF-� in a dose-dependent manner between 10 and 30 �M without affecting the

418 A.K. Sachdeva et al. / Behavioural Brain Research 205 (2009) 414–420

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eat shock protein 70 levels [18]. It was also found that epigal-ocatechin gallate interrupts TGF-� signaling in activated hepatictellate cells by suppressing gene expression of types I and II TGF-�eceptors [56]. Results of various studies further suggest that epi-allocatechin gallate may be a therapeutically effective inhibitor ofL-1�-induced inflammatory effects that are dependent on NF-�Bctivation in human osteoarthritis chondrocytes [47].

Treatment with the Brucella abortus antigen was known tonduce changes in cytokine gene expression in lymphocytes of

ice [50]. IFN-� deficient mice have demonstrated that at 3 weeksost infection with Brucella abortus, these animals had twice thepleen weights, a three-fold increase in the total number of splenic

eukocytes, and a higher number of bacteria than the control mice40]. Lipopolysaccharide accumulates in tissues rich in cells ofhe reticulo-endothelial system such as liver and spleen [49]. Athe same time, lipopolysaccharide induces a marked decrease inhe thymus weight. In the present study, the spleen weight was

duced glutathione (B), nitrite levels (C), superoxide dismutase (D) and catalase (E)g/kg oral gavage; EGCG 100 mg = epigallocatechin gallate 100 mg/kg oral gavage;

ompared to chronic fatigue group.

increased and thymus weight was decreased and these alterationswere restored by epigallocatechin gallate.

Lipopolysaccharide stimulates the production of reactive oxy-gen species intermediates by macrophages either directly or byinduction of IL-1 or TNF-� [45]. Lipopolysaccharide induces NF-�� that activates production of pro-inflammatory cytokines andchemokines that selectively induce the inflammatory enzymessuch as inducible nitric oxide synthase, COX-2 and adhesionmolecules [15,34]. In this study, we also observed an increasedlevel of various markers of oxidative–nitrosative stress. Bothlipopolysaccharide and Brucella abortus increased the levels ofthiobarbituric acid reactive substances in the brain, which is a

marker of lipid peroxidation in the brain along with enhancednitrite levels. Further, the levels of various endogenous antioxi-dants were decreased by chronic stress after immune activation.This oxidative damage is more likely a contributor to the biochem-ical as well as behavioral changes in experimental model of chronic

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atigue syndrome. Chronic treatment with epigallocatechin gal-ate attenuated enhanced oxidative–nitrosative stress and restoredndogenous antioxidant profile. Epigallocatechin gallate, the mostctive ingredient in green tea, has 25 times more potency thanitamin E in protecting the brain against free radical attack [2].pigallocatechin gallate decreased the activity and protein levelsf inducible nitric oxide synthase by reducing the expression ofnducible nitric oxide synthase mRNA [31].

In conclusion, epigallocatechin gallate, a polyphenolic antiox-dant, ameliorates the development of chronic fatigue syndromend thus may provide a new, effective and powerful strategy toreat chronic fatigue syndrome.

cknowledgements

The research grant sanctioned to Dr. (Ms) Kanwaljit Chopray Central Council for Research in Ayurveda and Siddha (CCRAS),ew Delhi is gratefully acknowledged. Mr. Anurag Kuhad andr. Vinod Tiwari are Indian Council of Medical Research (ICMR)-

enior Research Fellows. Mr. Anand Kamal Sachdeva is UGC Junioresearch Fellow. Authors are thankful to DSM Nutritional Prod-cts Ltd., Switzerland for providing gift sample of epigallocatechinallate.

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