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Pharmacological Reports xxx (2014) xxx–xxx

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Original research article

Effect of trimethylgallic acid esters against chronic stress-inducedanxiety-like behavior and oxidative stress in mice

Mamta Sachdeva Dhingra a, Sameer Dhingra b, Rachna Kumria c, Renu Chadha a,Tejvir Singh d, Anil Kumar a, Maninder Karan a,*a University Institute of Pharmaceutical Sciences, UGC Center of Advanced Study (UGC-CAS) in Pharmaceutical Sciences, Panjab University, Chandigarh, Indiab School of Pharmacy, Faculty of Medical Sciences, The University of the West Indies, St. Augustine, Trinidad and Tobagoc Swift School of Pharmacy, Swift Group of Colleges, Rajpura, Indiad Department of Chemistry, UGC Center of Advanced Study (UGC-CAS) in Chemistry, Panjab University, Chandigarh, India

A R T I C L E I N F O

Article history:

Received 16 June 2013

Received in revised form 20 December 2013

Accepted 13 January 2014

Available online xxx

Keywords:

Trimethylgallic acid esters

Chronic stress

Chronic fatigue syndrome

Antianxiety

Neuroinflammation

TNF-a

A B S T R A C T

Background: Many studies have shown that the levels of oxidative stress (increased lipid peroxidation,

decreased glutathione levels and endogenous antioxidant enzyme activities) and proinflammatory

cytokines (e.g., TNF-a) are increased in patients with chronic fatigue syndrome. Gallic acid and other

phenolic compounds are potent antioxidants and inhibitor of cytokine production. The present study

was designed to investigate the effect of newly synthesized conjugated esters of trimethylgallic acid in

an experimental model of chronic stress.

Methods: The animals were forced to swim individually for a period of 6 min every day for 15 days to

induce chronic stress. The locomotor activity, anxiety-like behavior, and memory retention were

evaluated in chronically stressed animals, followed by biochemical estimations and neuroinflammatory

surge in the brain.

Results: Chronic treatment with trimethylgallic acid esters for 15 days significantly reversed the chronic

stress-induced behavioral (impaired locomotor activity, anxiety-like behavior, and decreased

percentage of memory retention), biochemical (increased lipid peroxidation and nitrite levels;

decreased glutathione levels, superoxide dismutase and catalase activities), and inflammation surge

(serum TNF-a) in stressed mice.

Conclusions: The study revealed that trimethylgallic acid esters could ameliorate chronic stress-induced

various behavioral and biochemical alterations in mice, showing protective effects against chronic stress.

� 2014 Published by Elsevier Urban & Partner Sp. z o.o. on behalf of Institute of Pharmacology,

Polish Academy of Sciences.

Contents lists available at ScienceDirect

Pharmacological Reports

jou r nal h o mep ag e: w ww .e lsev ier . co m / loc ate /p h arep

1819202122

Introduction

Chronic fatigue stress (CFS) has been reported to producedetrimental effects on human health. Currently, CFS treatment isbased on the symptoms and is more of symptomatic nature rather

232425262728293031323334

Abbreviations: CFS, chronic fatigue stress; IL-1, interleukin-1; COX-2, cyclooxygen-

ase-2; ELISA, enzyme-linked immunosorbent assay; FST, forced swim test; GA, gallic

acid; IFN-g, interferon gamma; IL-6, interleukin 6; iNOS, inducible nitric oxide

synthase; MDA, malondialdehyde; MGG, 20-methoxyphenyl-3,4,5-trimethylgallate;

MRG, 30-hydroxyphenyl-3,4,5-trimethylgallate; MSG, 30 ,40-(methylenedioxy)phe-

nyl-3,4,5-trimethylgallate; MUG, 20-oxo-20H-chromene-70-yl-3,4,5-trimethylgallate;

NF-kb, nuclear factor kappa beta; nNOS, neuronal nitric oxide synthase; nNOS,

neuronal nitric oxide synthase; NO, nitric oxide; ROS, reactive oxygen species; TBARS,

thiobarbituric acid-reactive substances; TL, transfer latency; TMGA, trimethylgallic

acid; TNF-a, tumor necrosis factor-a.

* Corresponding author.

E-mail address: maninderkaran@hotmail.com (M. Karan).

Please cite this article in press as: Dhingra MS, et al. Effect of trimebehavior and oxidative stress in mice. Pharmacol Rep (2014), http:/

http://dx.doi.org/10.1016/j.pharep.2014.01.004

1734-1140/� 2014 Published by Elsevier Urban & Partner Sp. z o.o. on behalf of Instit

than a cure [46]. Therapies such as cognitive behavior therapy,graded exercise therapy, and pharmacological interventions (e.g.antidepressants and corticosteroids) have been tried, but withlimited success. Although these drugs could attenuate the stress inpatients [56], the exact mechanism of how these therapiesameliorate fatigue and other related problems have not yet beenunderstood. However, some evidences indicate that CFS isaccompanied with signs of increased oxidative stress andinflammation in the peripheral blood, suggesting the involvementof inducible NO synthase (iNOS), inflammation, oxidative andnitrosative stress in the pathogenesis of CFS [43,45]. Further, CFSresults in intracellular inflammation, with increased productionsof tumor necrosis factor-alpha (TNF-a), interleukin-1 (IL-1)nuclear factor kappa beta (NF-kb), and iNOS. Thus, membranefatty acids and functional proteins are damaged, due to inflamma-tion, and oxidative and nitrosative stress [44]. A number of factorscan trigger the inflammatory, oxidative and nitrosative stress

thylgallic acid esters against chronic stress-induced anxiety-like/dx.doi.org/10.1016/j.pharep.2014.01.004

ute of Pharmacology, Polish Academy of Sciences.

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thways, including psychological stress, strenuous exercise, viralfections, etc. The ‘psychosomatic’ symptoms of CFS are caused bytracellular inflammation (ache and pain, muscular tension,tigue, irritability, sadness, and the subjective feeling of infection),mage caused by inflammatory and oxidative and nitrosative

ress (ache and pain, muscular tension and fatigue), and gut-rived inflammation (complaints of irritable bowel) [45,46].flammatory pathways (monocytic activation) are also detected somatizing disorder [46]. Recent studies demonstrate thatidative stress is involved in the pathophysiology of CFS andnificantly contributes to the clinical symptoms [18,46,58]. Theidative stress mechanisms need to be understood in order tosign therapeutic strategies. Chronic stress impairs the biosyn-esis of n-3 and n-6 long-chain polyunsaturated fatty acids byhibiting the delta-6 desaturation of the precursor essential fattyids, alpha-linolenic acid and linoleic acid. In turn, this influencese proper functions of the cell membrane, including cell signalingd has an adverse effect on the biosynthesis of eicosanoids frome long-chain polyunsaturated fatty acids, dihomo-a-linolenicid, arachidonic acid and eicosapentaenoic acid. Most of theseanges are associated with the increase of the inflammatorysponse and generation of free radicals [17,18].

During the past few decades, a large number of naturallycurring phenolic compounds such as resorcinol, guaiacol,genol, thymol, vanillin, isovanillin, sesamol, umbelliferone,d menthol have been identified as antioxidants, which are

ewed as promising therapeutic agents for treating free radicalediated inflammatory diseases. Several reviews have addressede anti-inflammatory activity of phenols, attributing theiroperty not only to the antioxidant capacity, but also toflammatory mediators’ modulation, namely cytokines and pro-flammatory proteins, such as inducible nitric oxide synthase andX-2 [12]. Gallic acid (GA) is another polyphenolic compound,

idely distributed in the plant kingdom and represents a largemily of plant secondary polyphenolic metabolites. GA and itsrivatives are present in the form of either methylated GAs

yringic acid) or galloyl conjugates of catechin derivatives, i.e.,van-3-ols, or polygalloyl esters of glucose, quinic acid or glycerol9]. GA and its natural derivatives have been widely reported forrious biological and pharmacological activities including anti-flammatory and neuroprotective effects [8,13,31,38]. Thesetivities are possibly linked with their antioxidant potentiale to their ability to prevent damage from free radicals or toevent the generation of these free radicals [55].Based on these observations and the previous studies reportedm our laboratory [41,64], it has been proposed that there aretential advantages in giving these naturally occurring phenolicmpounds with complimentary pharmacological activities in therm of a single chemical or drug entity. Such agents are designedith improved physicochemical and pharmacokinetic propertieshich release the parent drugs at the site of action [3,37,70]. Hence

our recent study [14], we reported the synthesis and evaluation trimethylgallic acid (TMGA) esters as potent anti-inflammatoryents with gastroprotective effects. The compounds with goodti-inflammatory activity and gastroprotective effect wererther considered studying their role in an animal model ofronic stress. Thus, in the present paper, we have reported thefects of these ester derivatives in chronic fatigue stress-inducedhavioral and biochemical alterations.

aterials and methods

imals

Male albino mice (LACA strain; weighing 20–30 g) were bred inntral Animal House facility of the Panjab University, Chandigarh.

Please cite this article in press as: Dhingra MS, et al. Effect of trimebehavior and oxidative stress in mice. Pharmacol Rep (2014), http

The animals were housed under standard laboratory conditionsunder a 12-h light/dark cycle, with food and water ad libitum. Theanimals were acclimatized to the laboratory conditions 2 h prior toexperiment, and thereafter they were kept in the laboratory everyday in a similar way till the end of the experiment. All theexperiments were carried out between 9:00 and 16:00 in order tominimize the influences of circadian changes and inter-groupvariations. The experimental protocol was approved by theInstitutional Animal Ethics Committee (IAEC/98-112 dated28.03.11) and was conducted strictly according to the guidelinesof Committee for the Purpose of Control and Supervision ofExperiments on Animals (CPCSEA), New Delhi, India.

Drugs and treatment

The test compounds – 30-hydroxyphenyl-3,4,5-trimethylgallate(MRG), 20-methoxyphenyl-3,4,5-trimethylgallate (MGG), 30,40-(methylenedioxy)phenyl-3,4,5-trimethylgallate (MSG), and 20-oxo-20H-chromene-70-yl-3,4,5-trimethylgallate (MUG) were suspendedin 0.25% w/v carboxymethylcellulose (CMC). Doses of testcompounds were selected according to the previous studyreported from our laboratory [14] and administered orally30 min before the swimming session for 15 consecutive days.The animals were randomized into 6 groups ( n = 10 in eachgroup), including the naıve group, in which the mice received onlythe vehicle for 15 days without forced swimming session; thecontrol (chronically stressed) group, in which the mice receivedthe vehicle; the MRG (28.71 mg/kg, po) group; the MGG(30.03 mg/kg, po) group; the MSG (31.35 mg/kg, po) group; andthe MUG (33.62 mg/kg, po) group.

After 15 days, various behavioral assessments followed bybiochemical estimations were conducted, on the subsequent day16. Besides, there was a sufficient time gap (at least 1 h) betweenthe behavioral tests. Locomotor activity was assessed first,followed by elevated plus maze and mirror chamber test.

Measurement of immobility period

The animals were forced to swim individually in a glass jar(25 cm � 12 cm � 25 cm) at 25 � 2 8C for 6 min every day for 15consecutive days using forced swim test (FST). The water was 15 cmin height. After an initial period of vigorous activity, each animalattained a typical immobile posture. The duration of immobility wasmeasured. The animal was considered to be immobile when it ceasedto struggle, and the limbs seldom moved to keep the head above thewater [56]. This test session was repeated for 15 days.

Behavioral assessments

Various behavioral parameters were assessed in mice on day 16,24 h after the last forced swim challenge.

Measurement of locomotor activity

The locomotor activity was recorded for 5 min using actophot-ometer. Each animal was observed in a closed square(30 cm � 30 cm) arena equipped with infrared light sensitivephotocells using digital actophotometer. Locomotor activity wasexpressed in terms of total photobeam counts for 5 min per animal[27]. The apparatus was placed in a darkened, light and soundattenuated and ventilated testing room.

Measurement of anxiety (mirror chamber test)

The mirror chamber consisted of a wooden chamber with amirror cube open on one side, and the mirror cube was placed into

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M.S. Dhingra et al. / Pharmacological Reports xxx (2014) xxx–xxx 3

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a square plexiglass box. The box (40 cm � 40 cm � 30.5 cm) had awhite floor and opaque black walls. The mirrored cube(30 cm � 30 cm � 30 cm) was made up of 5 pieces of mirroredglass with one mirrored side and an opposite side painted darkbrown. In the standard configuration, the mirrored surfaces (3 sidepanes, a top pane and the floor pane) face the interior of the cube.Placement of the mirrored cube into the center of the containerforms a 5 cm corridor completely surrounding the mirroredchamber. The animal was placed individually at the distal cornerof the mirror chamber at the beginning of the test. During the5-min test, the following parameters were evaluated, includingthe latency to enter the mirror chamber, the number of entriesin mirror chamber, and the total time spent in mirror chamber.An anxiogenic response was identified when the numberof entries and time spent in the mirror chamber were decreased[32].

Measurement of memory (plus maze test)

Elevated plus maze was used to evaluate the spatial long-termmemory [27,31]. Briefly, the apparatus consisted of 2 open arms(16 cm � 5 cm) and 2 enclosed arms (16 cm � 5 cm � 12 cm). Thearm extended from a central platform (5 cm � 5 cm). The mice wereplaced individually at the end of one of the open arms facing awayfrom the central platform. The time that the mice spent moving fromthe open arm to either of the enclosed arms was recorded. Transferlatency (TL) indicated the elapse between the placement of theanimal on the open arm and its full entry (all the 4 paws) in theenclosed arm. On the first day, the mice were allowed to explorethe plus maze for 20 s after the measurement of TL. The micereturned to their home cages after the first trial. Retention wasexamined 24 h later. Each animal was again placed into the maze,and TL was recorded. Percentage of the retention of memory wascalculated as follows:

memory retention ¼ final transfer latency � initial transfer latency

initial transfer latency

� 100%

Biochemical estimations

Dissection and homogenization

On day 16, animals were sacrificed by decapitation. The wholebrain was removed and homogenized (10%, w/v) in 0.1 mol/Lphosphate buffer (pH 7.4). Homogenates were centrifuged for20 min at 15,000 � g. The supernatant was collected for theestimation of lipid peroxidation and reduced glutathione levels.The post nuclear fractions for the catalase assay were obtained bycentrifugation of the homogenate at 1000 � g for 20 min at 4 8C.For other enzyme assays, the homogenate was centrifuged at12,000 � g for 60 min at 4 8C.

Estimation of lipid peroxidation

The malondialdehyde (MDA) content, a measure of lipidperoxidation, was assayed in the form of thiobarbituric acid-reactive substances (TBARS) by the method of Wills [74]. In brief,0.5 ml of postmitochondrial supernatant and 0.5 ml of Tris–hydrochloric acid were incubated at 37 8C for 2 h. After incubation,1 ml of 10% trichloroacetic acid was added and centrifuged at1000 � g for 10 min. To 1 ml of supernatant, 1 ml of 0.67%thiobarbituric acid was added, and the tubes were kept in boilingwater for 10 min. After cooling, 1 ml of double distilled water wasadded, and absorbance was measured at 532 nm. TBARS werequantified using an extinction coefficient of 1.56 � 105 M�1 cm�1

and expressed as nanomole of MDA per milligram of protein.

Please cite this article in press as: Dhingra MS, et al. Effect of trimebehavior and oxidative stress in mice. Pharmacol Rep (2014), http:/

Tissue protein was estimated using the Biuret method, and thebrain MDA content expressed as nanomole of MDA per milligramof protein.

Estimation of superoxide dismutase activity

Cytosolic superoxide dismutase activity was assayed by themethod of Kono [29]. The assay system consisted of 0.1 mmethylenediaminetetracetic acid, 50 mM sodium carbonate, and96 mM of nitro blue tetrazolium. In the cuvette, 2 ml of the abovemixture was taken, and to it, 0.05 ml of postmitochondrialsupernatant and 0.05 ml of hydroxylamine hydrochloride (adjust-ed to pH 6 with NaOH) were added. The auto-oxidation ofhydroxylamine was observed by measuring the change in opticaldensity at 560 nm for 2 min at 30/60 s intervals. The superoxidedismutase activity was expressed as units per milligram protein.

Estimation of reduced glutathione

Reduced glutathione was assayed by the method of Jollow et al.[26]. In brief, 1 ml of postmitochondrial supernatant (10%) wasprecipitated with 1 ml of sulphosalicylic acid (4%). The samples werekept at 4 8C for at least 1 h and then subjected to centrifugation at1200 � g for 15 min at 4 8C. The assay mixture contained 0.1 mlsupernatant, 2.7 ml phosphate buffer (0.1 M, pH 7.4), and 0.2 ml 5,5,dithiobis-(2-nitro benzoic acid) (Ellman’s reagent, 0.1 mM, pH 8) in atotal volume of 3 ml. The yellow color developed was readimmediately at 412 nm and glutathione levels were calculatedusing molar extinction coefficient of 1.36 � 104 M�1 cm�1 andexpressed as micromole per milligram protein.

Catalase estimation

Catalase activity was assayed by the method of Claiborne [9]. Inbrief, the assay mixture consisted of 1.95 ml phosphate buffer(0.05 M, pH 7), 1 ml hydrogen peroxide (0.019 M), and 0.05 mlpostmitochondrial supernatant (10%) in a final volume of 3 ml.Changes in absorbance were recorded at 240 nm. Catalase activitywas quantified using the millimolar extinction coefficient ofhydrogen peroxide (0.07 mM) and expressed as micromolesof hydrogen peroxide decomposed per minute per milligramprotein.

Nitrite estimation

Nitrite was estimated using the Greiss reagent and served as anindicator of nitric oxide production. A measure of 500 ml of Greissreagent (1:1 solution of 1% sulfanilamide in 5% phosphoric acid and0.1% napthylaminediaminedihydrochloric acid in water) wasadded to 100 ml of postmitochondrial supernatant, and absorbancewas measured at 546 nm [21]. Nitrite concentration was calculatedusing a standard curve for sodium nitrite. Nitrite levels wereexpressed as microgram per milliliter.

Estimation of TNF-a levels

TNF-a was estimated using TNF-a kit (R&D systems). It is asolid-phase sandwich enzyme-linked immunosorbent assay(ELISA) using a microtiter plate reader at 450 nm. Concentrations(picogram per milliliter) of TNF-a were calculated from plottedstandard curve. TNF-a levels were expressed as mean � SEM.

Statistical analysis

Statistical analysis was carried out using one-way analysis ofvariance (ANOVA). In all cases, post hoc comparisons of the meansof individual groups were performed using Dunnett’s test. Asignificance level of p < 0.05 denoted the significance in all cases.

thylgallic acid esters against chronic stress-induced anxiety-like/dx.doi.org/10.1016/j.pharep.2014.01.004

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Table 1Antianxiety effect of test compounds in mirror chamber test in chronically fatigue

mice.

Treatment

groups

(mg/kg)

Latency to enter

mirror chamber

(s)

Number of entries

in mirror chamber

Time spent in

mirror chamber

(s)

Naıve 52 � 1.64 6.8 � 0.59 43.08 � 2.1

Control 138.26 � 9.45a 2.1 � 0.28a 24.68 � 1.9a

MRG (28.71) 88.6 � 6.05b 4.0 � 0.37b 34.2 � 2.3b

MGG (30.03) 75.25 � 5.78b 3.6 � 0.42b 38.29 � 3.19b

MSG (31.35) 72.24 � 5.38b 5.0 � 0.33b 36.0 � 3.8b

MUG (33.62) 83.4 � 6.15b 4.0 � 0.62b 34.87 � 3.15b

Values are expressed as mean � SEM, n = 10 in each group.a p < 0.05 vs. naıve group.b p < 0.05 vs. control group mice.

M.S. Dhingra et al. / Pharmacological Reports xxx (2014) xxx–xxx4

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PHAREP 102 1–7

sults

havioral observations

fects of test compounds on immobility period in FST

As shown in Fig. 1, exposure to 15-day forced swimming (6 minch day) significantly increased the immobility period asmpared to the naıve group (p < 0.05). However, pretreatmentith MRG (28.71 mg/kg), MGG (30.03 mg/kg), MSG (31.35 mg/kg),d MUG (33.62 mg/kg) significantly reversed the increase inmobility period as compared to control (stressed mice).

fects of test compounds on locomotor activity

The locomotor activity was assessed in the actophotometerst. There was a significant decrease in locomotor activity inronic stressed mice as compared to that in naıve groupithout chronically forced swimming). However, pretreatment

ith MRG, MGG, MSG, and MUG at the tested doses significantlyproved locomotor activity as compared to chronic stressed

ice (Fig. 2).

fects of test compounds on anxiety

As shown in Table 1, there was a significant delay in the latency enter the mirror chamber in stressed mice after 15-day forcedimming test session. Meanwhile, there was also decreased inth the number of entries and time spent in the mirror chamber

compared to those in naıve group. However, pretreatment withl the test compounds significantly shortened the latency period

enter the mirror chamber, increased the number of entries ande spent in the mirror chamber as compared with the control

oup (Table 1).

309310311

312313314315316

317318319320321322323324

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Naïve Con trol MRG MGG MSG MUG

Imm

obili

ty T

ime

a

b

b bb

. 1. Effect of test compounds on immobility period Values are expressed as

an � SEM, n = 10 in each group, ap < 0.05 vs. naıve group, bp < 0.05 vs. control

up mice.

0

50

100

150

200

250

Naïve Con trol MRG MGG MSG MUG

Loc

omot

or A

ctiv

ity

a

b

b b

b

. 2. Effect of test compounds on locomotor activity Values are expressed as

an � SEM, n = 10 in each group, ap < 0.05 vs. naıve group, bp < 0.05 vs. control

up mice.

Please cite this article in press as: Dhingra MS, et al. Effect of trimebehavior and oxidative stress in mice. Pharmacol Rep (2014), http

Effects of test compounds on memory (plus maze performance task)

As shown in Fig. 3, there was a significant loss of memory inchronic fatigue mice as indicated by a significant decrease in thepercentage of retention of memory as compared to that in naivegroup (p < 0.05). However, chronic administration of synthesizedtrimethylgallic acid esters significantly improved the memoryretention as compared to control group.

Biochemical observations

Effect of test compounds on chronic stress-induced changes in lipid

peroxidation

Thiobarbituric acid-reactive substance (TBARS) levels wereincreased significantly in the brain of chronically stressed miceafter 15 days of forced swimming as compared to naıve group ofanimals. Chronic administration of test compounds produced asignificant reduction in TBARS levels in the brain of stressed miceas compared to the control group (p < 0.05) (Table 2).

Effect of test compounds on chronic stress-induced nitrosative stress

Nitrite concentrations were significantly elevated in brains ofstressed mice as compared to naıve group. Chronic administrationof TMGA esters significantly attenuated nitrite concentration ascompared to the control group (p < 0.05) (Table 2).

Effect of test compounds on chronic stress-induced changes in the

antioxidant profile

Reduced glutathione levels and enzymatic activities of super-oxide dismutase and catalase significantly decreased in brains ofstressed mice as compared to control mice (Table 2). The reductionwas significantly improved by the chronic administration ofsynthesized compounds in brains of stressed mice as compared tothe control group (p < 0.05) (Table 2).

0

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Naïve Con trol MRG MGG MSG MUG

Perc

enta

ge o

f mem

ory

rete

ntio

n (%

)

a

b

bb

b

Fig. 3. Effect of test compounds on memory Values are expressed as mean � SEM,

n = 10 in each group, ap < 0.05 vs. naıve group, bp < 0.05 vs. control group mice.

thylgallic acid esters against chronic stress-induced anxiety-like://dx.doi.org/10.1016/j.pharep.2014.01.004

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Naïve Cont rol MRG MGG MSG MUG

TN

F-α

(pg/

mL

)

a

b

bb

b

Fig. 4. Effect of test compounds on TNF-a release Values are expressed as

mean � SEM, n = 10 in each group, ap < 0.05 vs. naıve group, bp < 0.05 vs. control

group mice.

Table 2Effect of test compounds on lipid peroxidation, reduced glutathione, superoxide dismutase, catalase, and nitrite levels.

Treatment groups

(mg/kg)

Lipid peroxidation

(nmol/mg protein)

Reduced glutathione

(mmoles/mg protein)

Superoxide dismutase

(units/mg protein)

Catalase (mmol of H2O2

decomposed/min/mg

protein)

Brain nitrite (mg/ml)

Naıve 2.25 � 0.11 18.23 � 0.64 5.23 � 0.2 8.51 � 0.33 267 � 16.2

Control 3.18 � 0.13a 10.51 � 0.49a 2.16 � 0.19a 4.01 � 0.26a 435 � 18.78a

MRG (28.71) 2.88 � 0.09b 13.24 � 0.53b 4.08 � 0.16b 6.12 � 0.32b 328 � 15.25b

MGG (30.03) 2.69 � 0.12b 14.39 � 0.51b 4.37 � 0.15b 6.43 � 0.31b 345 � 13.24b

MSG (31.35) 2.40 � 0.1b 15.28 � 0.46b 4.67 � 0.13b 6.98 � 0.28b 376 � 12.68b

MUG (33.62) 2.72 � 0.14b 13.21 � 0.38b 4.28 � 0.17b 6.36 � 0.21b 323 � 16.85b

Values are expressed as mean � SEM, n = 10 in each group.a p < 0.05 vs. naıve group.b p < 0.05 vs. control group mice.

M.S. Dhingra et al. / Pharmacological Reports xxx (2014) xxx–xxx 5

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Effect of test compounds on TNF-aSerum TNF-a levels significantly increased in stressed mice as

compared to naıve group. Chronic administration of test compoundsfor 15 days resulted in a significant decrease in serum TNF-a levelsas compared to control mice (Fig. 4).

Discussion

CFS is characterized by motor impairment, cognitive dys-function and anxiety like behavior, which is not substantiallyalleviated by rest [68]. However, the etiology of chronic stressremains still unclear. Studies in patients with CFS have revealedalterations of the hypothalamic –pituitary–adrenal (HPA) axis[2]. Here, we investigated the protective effects of newlysynthesized conjugated TMGA esters (MRG, MGG, MSG, andMUG) on chronic fatigue-induced stress. Animals were exposedchronically (15 days) to FST so as to induce CFS. FST is based onthe premises that the animal develops an immobile posturewhen placed in an inescapable cylinder filled with water. In thisparadigm, immobility is interpreted as a passive stress copingstrategy ( behavioral despair) [56]. Exposure of animals to achronic FST indicates an onset of symptoms similar to CFS asindicated by a significant increase in immobility time (Fig. 1).Consistent with other published reports including few from ourlaboratory [10,28,33–36], 15-days forced swimming couldinduce CFS-like symptoms as revealed by increases in despair(increased immobility period) and anxiety-like behaviors, andimpaired locomotor activity [69]. Here, repetition of the 6-minsession for 15 days caused significant chronic fatigue-likeconditions in mice. It is also reported that chronic stressinduces depressive behavior (increase in immobility time) [35]and significantly influences exercise and physical activity [66].However, the underlying mechanisms are not well understood.These behavioral changes might be due to the accumulation ofstress since chronic stress has been well documented to cause

Please cite this article in press as: Dhingra MS, et al. Effect of trimebehavior and oxidative stress in mice. Pharmacol Rep (2014), http:/

anxiety like behaviors, reduce locomotor activity and lead tostress induced depression [10,20,35]. The impairment of motoractivity could be due to the stress-induced depression [22,51]. Itis clear that oxidative stress plays a significant role in thepathogenesis of motor activity. Hypoactivity of CNS has alsobeen strongly implicated in the pathophysiology of anxiety.Besides, immobilization stress has been reported to induce a 2–3-fold elevation of plasma cortisol level [51], which is correlatedwith anxiety-like behavior and painful response in human[6,16]. Moreover, acute stress has been reported to influence thebehavioral activities, such as motor activity, anxiety-like activityand depression [15,20]. In the present study, administration ofTMGA esters (MRG, MGG, MSG, and MUG) at experimental dosessignificantly reversed the impairment in locomotor activity andproduced a remarkable neuroprotective effect against chronicstress in mice. Previous studies [8,13,60–62] also report thatgallic acid and other polyphenolic compounds of the same origincould reverse stress-induced immobility period. In otherneurodegenerative disorders, the beneficial role of GA deriva-tives has also been suggested against stress-related conditions[39,47]. Here, chronic administration of newly synthesizedTMGA esters attenuated stress-induced decrease in locomotoractivity, suggesting their therapeutic potential against chronicstress-induced alterations. Consistent with the previous report[11], here we also found that chronic stress could cause anxiety-like behavior and cognitive dysfunctions. When exposed to themirror chamber, the chronic stressed animals exhibited anxietyas revealed by a decrease in the number of entries and timespent in mirror chamber. This indicates that chronic stress couldsignificantly induce anxiety-like behavior in animals. Studies inhuman and animals have shown that stress hormones gluco-corticoids influence cognition [11,59]. Glucocorticoids have 2types of receptors, both of which are abundantly expressed inthe hippocampus, a region involved in learning and memory andbeing one of the most vulnerable regions to stress in the brain[59]. In the hippocampus, chronic stress causes atrophy in theCA3 region. It has also been reported that exposure to chronicstress causes subsequent impairment of hippocampus-depen-dent memory in both human and animals [49]. Furthermore, thehippocampus also regulates the stress response and inhibits theresponse of the HPA-axis to stress. Chronic stress causes theatrophy of dendrites of pyramidal neurons in the CA3 region ofthe hippocampus through a mechanism involving both gluco-corticoids and excitatory amino acid neurotransmitters releasedduring and after stress [19]. In the present study, chronic stresscaused poor retention of memory, which could be amelioratedby pretreatment of TMGA esters. It has been well documentedthat high levels of stress and fear commonly cause memory lossand cognition disturbance [7]. Stress also stimulates numerouspathways leading to an increased production of free radicalsthat lead to the release of inflammatory substances whichaccount for the accumulation of oxidative mediators in brain,

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M.S. Dhingra et al. / Pharmacological Reports xxx (2014) xxx–xxx6

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itochondrial inhibition [40], increased excitatory amino acidlease [42], activation of second messenger systems [49] andcreased efficiency of antioxidant defense mechanisms [57]. CFSuses increased productions of NF-kappa beta, COX-2 and iNOS andduces damage to membrane fatty acids and functional proteinsrough ROS and RNS [50]. Clinical reports also document that CFStients show significantly lower levels of transforming growth

ctor-beta1 (TGF-b1) and natural killer (NK) cell production asmpared to the normal control [46,72]. These factors are implicated

the increase of lipid peroxidation level in stress. In addition, forcedimming for 15 days significantly raised lipid peroxidation leveld nitrite concentration, and inhibited the levels of reduced

utathione, superoxide dismutase, and catalase enzyme activity,ereby inducing oxidative damage. The role of oxidative stress andlated free radical generation in the pathogenesis of CFS has beenell documented [38,63]. ROS generated by a severe stressornificantly compromise the in vivo antioxidant defense capability

hen animals are subjected to CFS [58]. Accordingly, chronicidative damage alters mitochondrial function, calcium homeo-

asis, energy pathways, neuronal precursors, neurogenesis, and cellath [54]. A larger number of experimental works have indicatedat many polyphenols have in vitro and in vivo antioxidanttivities and anti-fatigue effects. They can scavenge free radicalsd increase the activities of antioxidant enzymes [75]. In theesent study, pretreatment with TMGA esters could reduce thevels of lipid peroxidation and nitrite concentration suggestingeir neuroprotective and antioxidant like effect against chronicress. An initial formation of large amounts of oxygen and nitrogenactive species during stress may also initiate lipid peroxidation [1],

has been demonstrated in brain, liver and heart [5]. A positiverrelation between nitrite and TBARS levels has been demonstrated

many reports, revealing that nitric oxide acts as a free radical inress, and stress results in the expressions of iNOS and nNOS3,53]. However, some evidence indicates that CFS is accompanied

increased oxidative stress and that NO pathways are involved in pathogenesis [43,48]. Moreover, free radical damage by ROS hasen suggested to play a critical role in the pathophysiology of CFSd stress-induced depression [4,73]. Many depressed patientsowed increased levels of proinflammatory cytokines, e.g., IL-1 andF-a [24,65]. Maes et al. [45] were the first to show that the

ychological stress induces an inflammatory response withcreased production of proinflammatory cytokines, such as TNF-and IFN-g in humans. Many studies have reported that the

creased levels of proinflammatory cytokines are associated withressful situations [67,71]. In experimental animals, it has beenown that psychological stressors increase cytokine levels such as-6 and TNF-a in the blood and in various brain regions [25,52]. Inr study, chronic challenge to forced swimming resulted in

evated TNF-a levels and inflammation. Chronic administration ofst compounds significantly reduced TNF-a level in chronictigued mice. Polyphenols such as epigallocatechin have shownarked inhibition of cytokines in laboratory animal models chronictigue syndrome [60,61].

In conclusion, TMGA esters could ameliorate chronic stress-duced various behavioral and biochemical alterations in mice,owing protective effects against chronic stress.

nflict of interest

The authors state no conflict of interest.

nding

The research grant provided by the University Grants Com-ission to Mamta Sachdeva Dhingra is duly acknowledged.

Please cite this article in press as: Dhingra MS, et al. Effect of trimebehavior and oxidative stress in mice. Pharmacol Rep (2014), http

Uncited reference Q4

[30].

Acknowledgments

Authors pay homage to Late Professor Pritam Dev Sharma, oneof the investigators. This work could not have been possiblewithout his guidance.

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