Research Article Prophylactic Antioxidant Potential of Gallic Acid...

Research ArticleProphylactic Antioxidant Potential of Gallic Acid inMurine Model of Sepsis

Harikesh Maurya1 Vaishali Mangal1 Sanjay Gandhi2

Kathiresan Prabhu3 and Kathiresan Ponnudurai3

1 Siddhartha Institute of Pharmacy Near IT Park Sahastradhara Road Dehradun India2 City Heart Centre 19 Cross Road Dehradun 248001 India3 Nova College Pharmacy Vegavaram Jangareddigudem Andhra Pradesh 534447 India

Correspondence should be addressed to Harikesh Maurya mauryaharikesh2gmailcom

Received 12 February 2014 Revised 11 May 2014 Accepted 12 May 2014 Published 11 June 2014

Academic Editor Madhav Bhatia

Copyright copy 2014 Harikesh Maurya et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Present study is to investigate the effect of Gallic acid pretreatment on survival of septic animals and oxidative stress in differentorgans like lungs liver kidney spleen and vascular dysfunction of mice Sepsis was induced by cecal ligation and puncture (CLP)in healthy adult male albino mice (25ndash30 g) and was divided into 3 groups each consisting of 6 animals that is sham-operated(SO group (Group I) SO + sepsis (Group II) and Gallic acid + sepsis (Group III)) Group III animals were pretreated with Gallicacid at the dose rate of 20mgkg body weight for 2 days before induction of sepsis Animals were sacrificed on 8th day and thetissue samples were obtained for further investigation on lipid peroxidation (LPO)malondialdehyde (MDA) superoxide dismutase(SOD) catalase (CAT) and glutathione reductase (GSH) Gallic acid pretreatment significant (119875 lt 005) reduces kidney spleenliver and lungsrsquo malondialdehyde level in septic mice However it fails to improve reduced glutathione level in all given organswhile Gallic acid pretreated mice showed significant improvement in SOD activity of kidney and spleen when compared to septicmice Finally the beneficial effects of Gallic acid pretreatment in sepsis are evident from the observations that Gallic acid partiallyrestored SOD and catalase activity and completely reversed lipid peroxidation Further studies are required to find out the possiblemechanisms underlying the beneficial effects of Gallic acid on large population

1 Introduction

Sepsis is multiple organ dysfunction syndromes a leadingcause for morbidity and mortality worldwide [1] Its basicpathogenesis involves free radicals intervention these rad-icals can react with cell component leading to lipoper-oxidation with loss of membrane fluidity creating a stateof multiple organ failure [2ndash5] Immunosuppressive andantimicrobial resistance patients are more prone to developthe sepsis [6] Uncontrolled hyperinflammatory response andinappropriate cytokine response lead to early stages of sepsisHence it is necessary to control inflammation during earlystages of sepsis and to avoid the incidences of organ injurywhich may lead to organ failure and death Further it isreported that the activated leukocytes can produce reactive

oxygen species (ROS) [7 8] and disproportionate activationof respiratory burst involved in many diseases like asthmadiabetes Parkinsonrsquos and Alzheimerrsquos disease [9ndash11] may beresponsible for sepsis

Both gram negative (Pseudomonas aeruginosa Klebsiellapneumoniae etc) and gram positive (Escherichia coli Staphy-lococcus aureus etc) organism can cause sepsis [12 13] Fur-ther neuroimmunopathogenesis of sepsis involves peripheralrelease of proinflammatory cytokines (TNF-120572 IL-1120572 IL-1120573and IL-6) increasing blood brain barrier (BBB) permeabilitygenerating neuroinflammation and sepsis associated sicknessbehaviour [14ndash16]

In recent decades the reported incidence of sepsis hasincreased considerably due to the advancing age of thepopulation an increased number of invasive procedures

Hindawi Publishing CorporationInternational Journal of InflammationVolume 2014 Article ID 580320 7 pageshttpdxdoiorg1011552014580320

2 International Journal of Inflammation

performed along with immunosuppressive therapy [17]Approximately 750000 cases of sepsis occur each year outof which almost 225000 cases are fatal in United States alone[18 19] Gallic acid is related to phytochemical compoundsfound widely distributed in many plant tissues both as a freecompound and as a component of the plant polymer tanninGallic acid and its Catechin derived from its polymer tanninare present as main phenolic compounds of both black andgreen tea This phenolic compound is possessing antioxidantproperty and appears to have antimicrobial and anticanceractivities [20]

The present study was designed to decipher the role ofGallic acid pretreatment on survival of septic animals whichmay reduce inflammatory processes organ injury and deathduring early sepsis induced by cecal ligation and puncture(CLP) CLP shows progressive systemic inflammatory syn-drome followed by septic shock and multiorgan injury inmice [21] CLP model shows a cytokine profile similar tothat in human sepsis and development of fulminant multipleorgan failure [22]

2 Materials and Methods

21 Procurement of Gallic Acid and Reagents Gallic acidwas obtained as a gift sample from Natural Remedies PvtLtd Bangalore India All other chemicals and biochemicalreagents were of LR and AR grade respectively

22 Experimental Animals Healthy adult male albino mice(25ndash30 g) were obtained from Siddhartha Institute of Phar-macy Dehradun India They were housed in groups ofsix under standard laboratory conditions of temperature(25 plusmn 2∘C) and 1212 h lightdark cycle Animals had freeaccess to standard pellet diet and water ad libitum Thedistribution of animals in the groups the sequence of trialsand the treatment allotted to each group were randomizedthroughout the experiment Laboratory animal handling andexperimental procedures were performed in accordance withthe guidelines of Committee for the Purpose of Controland Supervision of Experiments on Animals (CPCSEA) andexperimental protocol was approved by Institutional AnimalEthical Committee

23 Surgical Details Experimental mice were fastedovernight before the induction of sepsis They wereanesthetized by an intraperitoneal injection of xylazine10 120583gg and ketamine 80 120583gg body weights Animals wereallowed to breath spontaneously during the surgery Aheating lamp was used to preserve the body temperatureat approximately 37∘C Peritonitis was induced by CLP asdescribed previously by Wichterman et al (1980) [23 24]

A 2 cm ventral midline incision was performed on theanimals under anaesthesia and the cecum was exposed Adistended portion of the cecum just distal to the ileocecalvalve was isolated filled with fecal content and tied with a3-0 silk suture in a manner that would not disrupt bowelcontinuity The ligated portion of the cecum was puncturedtwice with a 21-gauge needle to avoid intestinal obstruction

and a small amount of stool was expelled from the puncturesto ensure leakage of the intestinal content The cecum wasreplaced in its original position within the abdomen theabdomen was closed with a 3-0 suture in 2 layers andthe animals were allowed to recover and normal saline(1mLmouse)was given subcutaneously to allmice to preventdehydration The SO group mice had undergone the samesurgical procedure except cecal ligation and puncture [25ndash27]

After 24 hours of CLP the abdomen was reopened andthe ligated cecum was excised Samples were taken by swabsfor verification of the induced peritonitis Peritoneal lavagewas further continued with 40mL of warm sterile salineTheleft colon was transected from 3 to 4 cm above the peritonealreflection an end-to-end anastomosis was performed byusing 1 layer of interrupted 7-0 sutures and then the abdomenwas closed primarily in all animals [28]

24 Study Groups Animals were divided into 3 groups withsix animals in each group In Group I (SO group) ceacumwas ligated without puncture In Group II (SO + sepsis)ceacum was ligated with puncture and then sutured Surgerywas performed by CLP After the CLP surgery of group firstand group second animals they were kept for observationGroup III (Gallic acid + sepsis) animals were pretreated withGallic acid at the dose rate of 20mgkg body weight for 2 daysbefore induction of sepsis Animals were fed with standardmice chow and water postoperatively [29]

25 Survival Study Sepsis was induced through CLP withSO group animals serving as controls In preliminary exper-iments we observed a hyperdynamic physiological statefollowed by a hypodynamic physiological state similar tohemodynamic alterations in septic patients It was thusdecided to initiate Gallic acid therapy 2 days before sepsisinduction by CLP Mice were treated with Gallic acid orplacebo The degree of sepsis induced by CLP in micewas assessed by the presence of conjunctivitis absenceof grooming activities with resulting ruffled fur no oraluptake of food or water and lethargy Survival curve andmean survival time for all the groups were performed byKaplan-Meier survival curve and analyzed using log rank test[24]

26 Tissue Preparation261 Preparation of Liver Homogenate A 200mg of livertissue was weighed and taken in 2mL of ice-cold PBS (pH74) Another 200mg of sample was weighed and taken in2mL of 002M ethylene diamine tetra acetic acid (EDTA)solution for reduced glutathione (GSH) estimation Thehomogenate (10) prepared with IKA homogenizer (Ger-many) under ice-cold condition was centrifuged for 10min at3000 rpm The supernatant was stored at 20∘C until assay ofdifferent oxidative stress-related biochemical parameters Adouble beamUV-VIS Spectrophotometer (UV 5704SS ECILIndia) was used for recording the absorbance of test samples[30]

International Journal of Inflammation 3

262 Preparation of Kidney Homogenate A small portion ofkidney tissue were immediately removed weighed mincedand homogenized (10 wv) separately in ice-cold 115KCl-001M sodium potassium phosphate buffer (pH 74) ina Potter-Elvehjem type homogenizer The homogenate wascentrifuged at 18000timesg for 20min at 4∘C and the resultantsupernatant was used for subsequent biochemical analyses[31]

263 Preparation of Lungs and Spleen Homogenate Lungsand spleen were quickly removed and washed in ice-coldphosphate buffer saline (PBS) Washed tissues were immedi-ately immersed in liquid nitrogen and stored at minus70∘C untilbiochemical analysis On the day of use frozen tissue sampleswere quickly weighed and homogenized 1 10 in ice-coldPBSThe homogenates were then centrifuged at 14000timesg for15min at 4∘C The supernatants were separated and used forenzyme activities [32]

27 Biochemical Estimation

271 Lipid Peroxidation (LPO) inDifferent Tissues Theextentof lipid peroxidation was evaluated in terms of MDA (mal-ondialdehyde) production determined by the thiobarbituricacid (TBA) method [33]

272 Assessment of Antioxidant Eminence

(a) Superoxide Dismutase Superoxide dismutase (SOD) wasestimated as per the method described by Madesh and Bal-asubramanian [34] It involves the generation of superoxideby pyrogallol autooxidation and inhibition of superoxidedependent reduction of the tetrazolium dye MTT [3-(4-5dimethylthiazol 2-yl) 25-diphenyltetrazoliumbromide] to itsformazan which is measured at 570 nm The reaction wasended by the addition of dimethyl sulfoxide (DMSO) whichhelps to solubilize the formazan formed The colour evolvedwas stable for many hrs and was expressed as SOD units (oneunit of SOD is the amount of (120583g) protein required to inhibitthe MTT reduction by 50) [35 36]

(b) Catalase Catalase was assayed by spectrophotometricmethod as described byAebi [37] 10 tissue homogenatewasused for estimation of catalase activity

(c) Reduced Glutathione (GSH) GSH was determined by esti-mating free-SH groups using 5-51015840 dithiobis 2-nitrobenzoicacid (DTNB) method of Sedlak and Lindsay [38] 10 tissuehomogenate was made in 002M EDTA for estimation ofGSH level

273 Estimation of Gallic Acid Pretreatment in Ach InducedRelaxations in Endothelium Intact Mouse Aorta Thoracicaorta was cut into rings of 3-4mm length These aorticrings were mounted between two hooks made from 37 gaugestainless steel wire and were kept under a resting tension of10 g in a thermostatically controlled (370plusmn01∘C) organ bath(UGO Basile Italy) of 10mL capacity containing ModifiedKrebs-Henseleit solution (MKHS) and were continuously

bubbled with medical gas (74 N2+ 21 O

2+ 5 CO

2)

The aortic rings were equilibrated for 60ndash80min in organbath filled with MKHS before recording tension Duringequilibration period the bath fluid was repeatedly changedonce in every 15min The change in tension was measuredby a high-sensitivity isometric force transducer and recordedin a PC using Lab Chart V613 Pro software programme(Powerlab AD Instruments Australia)

After equilibration aortic ringswere contractedwith highK+ (80mM) depolarizing solution On attaining contraction-plateau high potassium solution was replaced by normalMKHS The preparations were washed with normal MKHSto restore baseline resting tension [39] Following a lapse of30min and 2-3 washes with normal MKHS aortic rings wereagain contracted with phenylephrine and concentration-response curves to Ach were elicited by its cumulativeaddition to the bath solution at an increment of 05 log unitat plateau phase of agonist-induced contraction

28 Statistical Analysis Relaxation responses were expressedas the percentage reversal of the phenylephrine contractionBoth 119864max (the maximal response) and EC

50(the con-

centration producing 50 of the maximal response) weredetermined by nonlinear regression analysis (sigmoidal dose-response with variable slope) using Graph Pad Prism version500 (San Diego California USA) Sensitivitypotency wasexpressed as pD

2

= minus log EC50 Results were expressed as

mean plusmn SE with 119899 equal to number of animals Data wereanalyzed by Student-Newman-Keuls method for multiplegroup analysis Survival percent was estimated by Kaplan-Meiermethod and compared by log rank test Concentration-dependent agonist response data were analyzed by two-wayANOVA followed by Bonferroni post hoc test Differences invalues were considered statistically significant at 119875 lt 005(Snedecor and Cochran)

3 Results

31 Assessment of Gallic Acid Pretreatment in Sepsis onSurvival Time in Mice The results denote mean survivaltime for sepsis (treated with vehicle) and pretreatment withGallic acid (20mgkg BW orally) was 27 10 plusmn 1 26 h and38 08 plusmn 1 80 h respectively (Figure 1) Thus Gallic acidshows significant (119875 lt 005) effect on the survival timein comparison to the septic mice (group II) All the sham-operatedmice (group I) survived during 72 00 h observationperiod

32 Effect of Gallic Acid Pretreatment in Sepsis on LipidPeroxidation Level (nmolmg) in Different Organs of MiceThe extent of lipid peroxidation was evaluated in termsof MDA (malondialdehyde) production determined by thethiobarbituric acid (TBA) method (Table 1)

Kidney Tissue The effect of Gallic acid pretreatment in sepsison kidneyMDA level was found to be 587plusmn082 nmolmg insham-operated (SO group) treatment group In comparisonto this the sepsis treatment group shows the significant


Table 1 Effect of Gallic acid pretreatment in sepsis on lipid peroxidation level (nmolmg) in different organs of mice

Sr number Number of groups Kidney tissue Spleen tissue Liver tissue Lungs tissue1 Group I 0587 plusmn 082 0750 plusmn 080 1268 plusmn 092 0422 plusmn 075

2 Group II 0898 plusmn 079lowast

1100 plusmn 048lowast



3 Group III 0706 plusmn 078

0800 plusmn 085

1896 plusmn 088

0621 plusmn 068

lowast

119875 lt 005 shows statistically significant difference in comparison to first group119875 lt 005 shows statistically significant difference in comparison to second group

Table 2 Effect of Gallic acid pre-treatment in sepsis on superoxide dismutase (SOD unitsmg) activity in different organs of mice







1321 plusmn 155 3086 plusmn 180 5133 plusmn 154

lowast


125

100

75

50

25

0

0 10 20 30 40 50 60 70

Time

Sham

Surv

ival

()

Survival study

SepsisGA

Figure 1 Effect ofGallic acid pretreatment in sepsis on survival timein mice

(119875 lt 005) increased level of MDA to 898 plusmn 079 nmolmgHowever Gallic acid pretreatment significantly (119875 lt 005)reduced kidney MDA level to 706 plusmn 078 nmolmg in septicmice

Spleen Tissue The effect of Gallic acid pretreatment in sepsison spleen MDA level was found to be 750 plusmn 080 nmolmgin SO treatment group In comparison to this the sepsistreatment group shows the significant (119875 lt 005) increasedlevel of MDA to 1100 plusmn 048 nmolmg However Gallic acidpretreatment significantly (119875 lt 005) reduced spleen MDAlevel to 800 plusmn 085 nmolmg in septic mice

Liver Tissue The effect of Gallic acid pretreatment in sepsison liver MDA level was found to be 1268 plusmn 092 nmolmgin SO treatment group In comparison to this the sepsistreatment group shows the significant (119875 lt 005) increasedlevel of MDA to 3602 plusmn 062 nmolmg However Gallic acid

pretreatment significantly (119875 lt 005) reduced liver MDAlevel to 1896 plusmn 088 nmolmg in septic mice

Lungs Tissue The effect of Gallic acid pretreatment in sepsison lungs MDA level was found to be 422 plusmn 075 nmolmgin SO treatment group In comparison to this the sepsistreatment group shows the significant (119875 lt 005) increasedlevel of MDA to 906 plusmn 080 nmolmg However Gallic acidpretreatment significantly (119875 lt 005) reduced lungs MDAlevel to 621 plusmn 068 nmolmg in septic mice

33 Effect of Gallic Acid Pretreatment in Sepsis on SuperoxideDismutase (SOD Unitsmg) Activity in Different Organs ofMice The SOD level was evaluated in different tissues asmentioned in Table 2

Kidney Tissue The SOD activity in kidney tissue showssignificant reduction in sepsis (Group II) which was 2380 plusmn206unitsmg as in comparison to SO treatment group that is3368plusmn 108 unitsmg Gallic acid pretreated mice (group III)showed significant improvement in SOD activity 5746plusmn138unitsmg as compared to septic mice

Spleen Tissue The level of SOD activity in septic mice (GroupII) 1022 plusmn 178 unitsmg was significantly lower than SOtreatment group that is 19 plusmn 16 unitsmg Gallic acidpretreated mice (Group III) did not show significant changein SOD activity (1321plusmn155 unitsmg) as compared to groupII

Liver Tissue Significant reduction in SOD activity found inseptic mice was 2501 plusmn 162 unitsmg as compared to SOtreatment group that is 5812 plusmn 200 unitsmg Gallic acidpretreatment was not able to reverse the SOD activity (2501plusmn162 unitsmg)

Lungs Tissue There was significant decrease in SOD activity(2048 plusmn 170 unitsmg) in group II as compared to SODactivity (4263 plusmn 166 unitsmg) with group I Gallic acidpretreatment reversed the SOD activity to 5133 plusmn 154unitsmg


Table 3 Effect of Gallic acid pretreatment in sepsis on catalase (CAT) activity (mmol H2O2minmg) in different organs of mice



027 plusmn 001 046 plusmn 005lowast

038 plusmn 002


042 plusmn 004

035 plusmn 003 055 plusmn 002

lowast


34 Effect of Gallic Acid Pretreatment in Sepsis on Catalase(CAT) Activity (mmol H

2O2minmg) in Different Organs of

Mice Thecatalase activitywas performed ondifferent organsafter Gallic acid pretreatment The results are mentioned inTable 3

Kidney Tissue The change of catalase activity in sepsis treat-ment group was 033 plusmn 003mMH

2O2minmg as compared

to SO treatment group that is 072plusmn004mMH2O2minmg

Gallic acid pretreatment did not show any significant changein catalase activity 054 plusmn 003mM H

2O2minmg as com-

pared to septic mice

Spleen Tissue Group II animals did not show significantchange of catalase activity 027 plusmn 001mM H

2O2minmg

in comparison to group I treatment animals 032 plusmn002mM H

2O2minmg while Gallic acid pretreatment

shows significant increase in catalase activity 042 plusmn 004mMH2O2minmg in comparison to group I animals

Liver Tissue In this there was no significant differenceobserved in catalase activity of group II animals 046 plusmn005mM H

2O2minmg as compared to group I animals

066 plusmn 004mM H2O2minmg Gallic acid pretreated mice

also did not show any significant change in catalase activity035 plusmn 003mMH

2O2minmg as compared to septic mice

Lungs Tissue Septic mice did not show significant decrease incatalase activity 038 plusmn 002mMH


to SO mice 052 plusmn 005mM H2O2minmg Gallic acid

pretreatment did not reverse significant change in catalaseactivity 055 plusmn 002mMH

2O2minmg as compared to septic

mice

35 Effect of Gallic Acid Pretreatment in Sepsis on ReducedGlutathione Level (mM GSHg Wet Tissue) in Liver of MiceThe effect of Gallic acid pretreatment in sepsis on reducedGSH level in liver tissuewas determined inTable 4 Sepsis sig-nificantly decreased the reduced GSH level (046 plusmn 004mMGSHg) of wet tissue as compared to SOmice 085plusmn006mMGSHgwet tissue However Gallic acid pretreatment in sepsisfailed to improve reduced glutathione level (065 plusmn 005mMGSHg wet tissue)

36 Effect of Gallic Acid Pretreatment in Sepsis on AchInduced Relaxations in Endothelium Intact Mouse Aorta PE(1 120583M) precontracted endothelium intact rings from aortaof SO mice exhibited concentration-dependent relaxationsto Ach (01 Nmndash10 120583M) with pD

2and 119864max of 0696 plusmn 007

Table 4 Effect of Gallic acid pretreatment in sepsis on reducedglutathione level (mMGSHgwet tissue) in liver of mice

Sr number Number of groups Liver tissue1 Group I 085 plusmn 006



lowast

119875 lt 005 shows statistically significant difference in comparison to SOgroup119875 lt 005 shows statistically significant difference in comparison to sepsis

Table 5 Effect of Gallic acid pretreatment in sepsis on Ach inducedrelaxations in endothelium intact mouse aorta

Sr number Number of groups pD2 119864max ()1 Group I 0696 plusmn 007 9355 plusmn 284

2 Group II 0609 plusmn 019 7272 plusmn 567lowast

3 Group III 0739 plusmn 043 7297 plusmn 859lowast

lowast

119875 lt 001 shows statistically significant difference as comparison to SOgroup

and 9355 plusmn 284 respectively as given in Table 5 Sepsissignificantly (119875 lt 005) reduced the concentration dependentrelaxation to Ach with pD

2and 119864max of 0609 plusmn 019 and

7272 plusmn 567 respectively and also did not uphold therelaxation to Ach (119864max and pD

2) in sepsis

4 Discussion and Conclusion

Sepsis inflammation and particularly septic shock areassociated with global and local hypoperfusion ischemia-reperfusion events endothelial injury with an associatedprocoagulant state and monocyte-macrophage system acti-vation They also induce the production of large amountsof free radicals in a nonregulated fashion associated withhigh-oxidative potential damage In fact several sources ofreactive oxygen species (ROS) have been detected in sepsisand septic shock including the mitochondrial respiratoryelectron transport chain immune cell and xanthine oxidaseactivation as a result of ischemia and reperfusion and therespiratory burst associated with NADPH oxidase Severalstudies have shown the presence of oxidative stress in sep-sis Early production of reactive oxygen species (ROS) hasbeen demonstrated in experimental studies in sepsis andexcessive release of superoxide anion has been shown tocontribute to postreperfusion oxidative damage in severalischemic organs During sepsis activation of proinflamma-tory pathways leads to dysfunction ofmitochondria and cells


which contributes to multiorgan failure and poor outcomesProinflammatory cytokines like TNF-120572 increase cytosoliclevels of Ca2+ via inositol-145-triphosphate-mediated (IP

3-

mediated) pathway from the ER The increase in cytosolicCa2+ is followed by a rapid increase in mitochondrial Ca2+leading to a rise in mitochondrial ROS generation fromcomplex III of the electron transport chain Higher level ofROS results in the release of cytochrome c and cell death

Gallic acid and its structurally related compounds arefound widely distributed in fruits and plants Studies utilisingthese compounds have found them to possess many potentialtherapeutic properties including anti-cancer and antimicro-bial properties In addition to direct antioxidant activityGallic acid and its derivatives may function indirectly as anantioxidant by enhancing antioxidant enzymes such as hemeoxygenase glutathione peroxidase glutathione reductaseand catalase and phase-2 enzymes such as glutathione Stransferase and quinone reductase In Gallic acid pretreat-ment group we studied the role of Gallic acid against sepsisinduced oxidative stress in different organs of albino miceGallic acid showed protective role by decreasing the LPOlevel in animals exposed to CLP It worked as an antioxidantand increased the enzymatic antioxidants CAT and SOD inanimals exposed to CLP The organ specific effect may befurther studied to reach at any conclusion The reduction ofoxidative stress in the test animalsmay be by scavenging ROSprotecting the antioxidant enzymes from being denaturedand reducing the oxidative stress marker LPO A study hasreported the improvements of haemodynamics and vascularresponse by Gallic acid pre- and posttreatment during endo-toxemia (LPSmodel) in which they have attributed to allevia-tion of oxidative stress by reducing aortic-derived superoxideproduction suppression of lipid peroxidation and proteinoxidation and decrease in urinary nitric oxide metaboliteswith preservation of the ratio of glutathioneglutathionedisulfide Similar to their observation current study showedsuppression of lipid peroxidation in all the tissues under studyin CLP model of polymicrobial sepsis In contrast to theirstudy we did not find significant improvement in reducedglutathione content in liver and SOD activity in spleen andliver [40]

In the present study the elevated level of LPO in CLPmodel of mice may be due to oxidative stress and enhancedreactive oxygen species (ROS) production by the inflam-matory process In accordance with the results of previousstudy we have found increased MDA production in all thetissues after 18 hrs of sepsis which is also supportive of theclinical study [41 42] In an older study the authors havereported the increased thiobarbituric acid reactive substances(TBARSs) which are a marker of lipoperoxidation in criti-cally ill patients in association withmultiorgan failure (MOF)development [43]

In conclusion the beneficial effects of Gallic acid pre-treatment in sepsis are evident from the observations thatGallic acid partially restored SOD and catalase activity andcompletely reversed lipid peroxidation Further studies arerequired to find out the possible mechanisms underlyingthe beneficial effects of Gallic acid It is integrated that theGallic acid can be exploited in the treatment of inflammatory

disease (sepsis) on the basis of results and outcomes ofpresent research work

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] C C Winterbourn H Buss T P Chan L D Plank M AClark and J A Windsor ldquoProtein carbonyl measurementsshow evidence of early oxidative stress in critically ill patientsrdquoCritical Care Medicine vol 28 no 1 pp 143ndash149 2000

[2] M Andrades C Ritter J C F Moreira and F Dal-PizzolldquoOxidative parameters differences during non-lethal and lethalsepsis developmentrdquo Journal of Surgical Research vol 125 no 1pp 68ndash72 2005

[3] T Barichello J J Fortunato A M Vitali et al ldquoOxidativevariables in the rat brain after sepsis induced by cecal ligationand perforationrdquo Critical Care Medicine vol 34 no 3 pp 886ndash889 2006

[4] M Geoghegan D McAuley S Eaton and J Powell-TuckldquoSelenium in critical illnessrdquo Current Opinion in Critical Carevol 12 no 2 pp 136ndash141 2006

[5] J U Becker C Theodosis S T Jacob C R Wira and N EGroce ldquoSurviving sepsis in low-income and middle-incomecountries new directions for care and researchrdquo The LancetInfectious Diseases vol 9 no 9 pp 577ndash582 2009

[6] R P Wenzel ldquoTreating sepsisrdquo The New England Journal ofMedicine vol 347 no 13 pp 966ndash967 2002

[7] R B Johnston Jr B B Keele H P Misra et al ldquoThe role ofsuperoxide anion generation in phagocytic bactericidal activityStudies with normal and chronic granulomatous disease leuko-cytesrdquo The Journal of Clinical Investigation vol 55 no 6 pp1357ndash1372 1975

[8] R B Johnston Jr ldquoOxygen metabolism and the microbicidalactivity of macrophagesrdquo Federation Proceedings vol 37 no 13pp 2759ndash2764 1978

[9] J Monteseirin M J Camacho I Bonilla et al ldquoRespira-tory burst in neutrophils from asthmatic patientsrdquo Journal ofAsthma vol 39 no 7 pp 619ndash624 2002

[10] W L Hand D L Hand and Y Vasquez ldquoIncreased poly-morphonuclear leukocyte respiratory burst function in type 2diabetesrdquo Diabetes Research and Clinical Practice vol 76 no 1pp 44ndash50 2007

[11] Neuroinflammation Working Group ldquoInflamation andAlzheimerrsquos diseaserdquo Neurobiology of Aging vol 21 no 3 pp383ndash387 2000

[12] J Cohen ldquoThe immunopathogenesis of sepsisrdquoNature vol 420no 6917 pp 885ndash891 2002

[13] V Kumar and A Sharma ldquoInnate immunity in sepsis patho-genesis and its modulation new immunomodulatory targetsrevealedrdquo Journal of Chemotherapy vol 20 no 6 pp 672ndash6832008

[14] M Ebersoldt T Sharshar and D Annane ldquoSepsis-associateddeliriumrdquo Intensive Care Medicine vol 33 no 6 pp 941ndash9502007


[15] CWoiciechowsky B SchoningW R Lanksch H-D Volk andW-D Docke ldquoMechanisms of brain-mediated systemic anti-inflammatory syndrome causing immunodepressionrdquo Journalof Molecular Medicine vol 77 no 11 pp 769ndash780 1999

[16] R Dantzer J C OrsquoConnor G G Freund R W Johnson andK W Kelley ldquoFrom inflammation to sickness and depressionwhen the immune system subjugates the brainrdquoNature ReviewsNeuroscience vol 9 no 1 pp 46ndash56 2008

[17] A Padkin C Goldfrad A R Brady D Young N Black andK Rowan ldquoEpidemiology of severe sepsis occurring in the first24 hrs in intensive care units in England Wales and NorthernIrelandrdquo Critical Care Medicine vol 31 no 9 pp 2332ndash23382003

[18] R A Balk and R A Balk ldquoSevere sepsis and septic shockDefinitions epidemiology and clinical manifestationsrdquo CriticalCare Clinics vol 16 no 2 pp 179ndash192 2000

[19] G S Martin D M Mannino S Eaton and M Moss ldquoTheepidemiology of sepsis in the United States from 1979 through2000rdquoTheNew England Journal of Medicine vol 348 no 16 pp1546ndash1554 2003

[20] Y-Y Ow and I Stupans ldquoGallic acid and gallic acid deriva-tives effects on drug metabolizing enzymesrdquo Current DrugMetabolism vol 4 no 3 pp 241ndash248 2003

[21] T Miyaji X Hu P S T Yuen et al ldquoEthyl pyruvate decreasessepsis-induced acute renal failure and multiple organ damagein agedmicerdquoKidney International vol 64 no 5 pp 1620ndash16312003

[22] D G Remick D E Newcomb G L Bolgos and D R CallldquoComparison of the mortality and inflammatory response oftwo models of sepsis lipopolysaccharide vs cecal ligation andpuncturerdquo Shock vol 13 no 2 pp 110ndash116 2000

[23] K A Wichterman A Baue and I H Chaudry ldquoSepsis andseptic shock a review of laboratory models and a proposalrdquoJournal of Surgical Research vol 29 no 2 pp 189ndash201 1980

[24] M W Merx E A Liehn U Janssens et al ldquoHMG-CoAreductase inhibitor simvastatin profoundly improves survival inamurinemodel of sepsisrdquoCirculation vol 109 no 21 pp 2560ndash2565 2004

[25] M M Reijnen J F Meis V A Postma and H van GoorldquoPrevention of intra-abdominal abscesses and adhesions usinga hyaluronic acid solution in a rat peritonitis modelrdquo Archivesof Surgery vol 134 no 9 pp 997ndash1001 1999

[26] I H de Hingh B M de Man R M Lomme H van Goorand T Hendriks ldquoColonic anastomotic strength and matrixmetalloproteinase activity in an experimentalmodel of bacterialperitonitisrdquoBritish Journal of Surgery vol 90 no 8 pp 981ndash9882003

[27] M M Reijnen B M de Man T Hendriks V A Postma JF Meis and H van Goor ldquoHyaluronic acid-based agents donot affect anastomotic strength in the rat colon in either thepresence or absence of bacterial peritonitisrdquo British Journal ofSurgery vol 87 no 9 pp 1222ndash1228 2000

[28] F R Dijkstra M Nieuwenhuijzen M M Reijnen and Hvan Goor ldquoRecent clinical developments in pathophysiologyepidemiology diagnosis and treatment of intra-abdominaladhesionsrdquo Scandinavian Journal of Gastroenterology Supple-ment vol 35 no 232 pp 52ndash59 2000

[29] D Rittirsch M S Huber-Lang M A Flierl and P A WardldquoImmunodesign of experimental sepsis by cecal ligation andpuncturerdquo Nature Protocols vol 4 no 1 pp 31ndash36 2009

[30] C-J Li S Kahl D Carbaugh and T H Elsasser ldquoTemporalresponse of liver signal transduction elements during in vivo

endotoxin challenge in cattle effects of growth hormone treat-mentrdquoDomestic Animal Endocrinology vol 32 no 2 pp 79ndash922007

[31] F El-Demerdash Y Dewer R H ElMazoudy and A AAttia ldquoKidney antioxidant status biochemical parameters andhistopathological changes induced bymethomyl in CD-1 micerdquoExperimental and Toxicologic Pathology vol 65 no 6 pp 897ndash901 2013

[32] M Jafari M Salehi A Asgari et al ldquoEffects of paraoxon onserum biochemical parameters and oxidative stress inductionin various tissues of Wistar and Norway ratsrdquo EnvironmentalToxicology and Pharmacology vol 34 no 3 pp 876ndash887 2012

[33] S Shafiq-ur-Rehman S Rehman O Chandra andM AbdullaldquoEvaluation of malondialdehyde as an index of lead damage inrat brain homogenatesrdquo BioMetals vol 8 no 4 pp 275ndash2791995

[34] M Madesh and K A Balasubramanian ldquoMicrotiter plate assayfor superoxide dismutase usingMTT reduction by superoxiderdquoIndian Journal of Biochemistry amp Biophysics vol 35 no 3 pp184ndash188 1998

[35] D C Angus W T Linde-Zwirble J Lidicker G Clermont JCarcillo and M R Pinsky ldquoEpidemiology of severe sepsis intheUnited States analysis of incidence outcome and associatedcosts of carerdquoCritical CareMedicine vol 29 no 7 pp 1303ndash13102001

[36] O Arunlakshana H O Schild and D H Jenkinson ldquoSomequantitative uses of drug antagonistsrdquo British Journal of Phar-macology vol 120 supplement 1 pp 148ndash150 1997

[37] H Aebi ldquoCatalase in vitrordquoMethods in Enzymology vol 105 pp121ndash126 1984

[38] J Sedlak and R H Lindsay ldquoEstimation of total protein-bound andnonprotein sulfhydryl groups in tissuewith Ellmanrsquosreagentrdquo Analytical Biochemistry vol 25 pp 192ndash205 1968

[39] H Babaei O Sadeghpour L Nahar et al ldquoAntioxidant andvasorelaxant activities of flavonoids from Amygdalus lycioidesvar horridrdquo Turkish Journal of Biological Sciences vol 32 pp203ndash208 2008

[40] K Sompamit U Kukongviriyapan S Nakmareong P Pan-nangpetch and V Kukongviriyapan ldquoCurcumin improvesvascular function and alleviates oxidative stress in non-lethallipopolysaccharide-induced endotoxaemia in micerdquo EuropeanJournal of Pharmacology vol 616 no 1ndash3 pp 192ndash199 2009

[41] K Kapoor S Basu B K Das and B D Bhatia ldquoLipidperoxidation and antioxidants in neonatal septicemiardquo Journalof Tropical Pediatrics vol 52 no 5 pp 372ndash375 2006

[42] D Muhl G Woth L Drenkovics et al ldquoComparison ofoxidative stress amp leukocyte activation in patients with severesepsis amp burn injuryrdquo Indian Journal of Medical ResearchSupplement vol 134 no 1 pp 69ndash78 2011

[43] M Andresen T Regueira A Bruhn et al ldquoLipoperoxidationand protein oxidative damage exhibit different kinetics duringseptic shockrdquo Mediators of Inflammation vol 2008 Article ID168652 8 pages 2008

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

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Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


performed along with immunosuppressive therapy [17]Approximately 750000 cases of sepsis occur each year outof which almost 225000 cases are fatal in United States alone[18 19] Gallic acid is related to phytochemical compoundsfound widely distributed in many plant tissues both as a freecompound and as a component of the plant polymer tanninGallic acid and its Catechin derived from its polymer tanninare present as main phenolic compounds of both black andgreen tea This phenolic compound is possessing antioxidantproperty and appears to have antimicrobial and anticanceractivities [20]

The present study was designed to decipher the role ofGallic acid pretreatment on survival of septic animals whichmay reduce inflammatory processes organ injury and deathduring early sepsis induced by cecal ligation and puncture(CLP) CLP shows progressive systemic inflammatory syn-drome followed by septic shock and multiorgan injury inmice [21] CLP model shows a cytokine profile similar tothat in human sepsis and development of fulminant multipleorgan failure [22]

2 Materials and Methods

21 Procurement of Gallic Acid and Reagents Gallic acidwas obtained as a gift sample from Natural Remedies PvtLtd Bangalore India All other chemicals and biochemicalreagents were of LR and AR grade respectively

22 Experimental Animals Healthy adult male albino mice(25ndash30 g) were obtained from Siddhartha Institute of Phar-macy Dehradun India They were housed in groups ofsix under standard laboratory conditions of temperature(25 plusmn 2∘C) and 1212 h lightdark cycle Animals had freeaccess to standard pellet diet and water ad libitum Thedistribution of animals in the groups the sequence of trialsand the treatment allotted to each group were randomizedthroughout the experiment Laboratory animal handling andexperimental procedures were performed in accordance withthe guidelines of Committee for the Purpose of Controland Supervision of Experiments on Animals (CPCSEA) andexperimental protocol was approved by Institutional AnimalEthical Committee

23 Surgical Details Experimental mice were fastedovernight before the induction of sepsis They wereanesthetized by an intraperitoneal injection of xylazine10 120583gg and ketamine 80 120583gg body weights Animals wereallowed to breath spontaneously during the surgery Aheating lamp was used to preserve the body temperatureat approximately 37∘C Peritonitis was induced by CLP asdescribed previously by Wichterman et al (1980) [23 24]

A 2 cm ventral midline incision was performed on theanimals under anaesthesia and the cecum was exposed Adistended portion of the cecum just distal to the ileocecalvalve was isolated filled with fecal content and tied with a3-0 silk suture in a manner that would not disrupt bowelcontinuity The ligated portion of the cecum was puncturedtwice with a 21-gauge needle to avoid intestinal obstruction

and a small amount of stool was expelled from the puncturesto ensure leakage of the intestinal content The cecum wasreplaced in its original position within the abdomen theabdomen was closed with a 3-0 suture in 2 layers andthe animals were allowed to recover and normal saline(1mLmouse)was given subcutaneously to allmice to preventdehydration The SO group mice had undergone the samesurgical procedure except cecal ligation and puncture [25ndash27]

After 24 hours of CLP the abdomen was reopened andthe ligated cecum was excised Samples were taken by swabsfor verification of the induced peritonitis Peritoneal lavagewas further continued with 40mL of warm sterile salineTheleft colon was transected from 3 to 4 cm above the peritonealreflection an end-to-end anastomosis was performed byusing 1 layer of interrupted 7-0 sutures and then the abdomenwas closed primarily in all animals [28]

24 Study Groups Animals were divided into 3 groups withsix animals in each group In Group I (SO group) ceacumwas ligated without puncture In Group II (SO + sepsis)ceacum was ligated with puncture and then sutured Surgerywas performed by CLP After the CLP surgery of group firstand group second animals they were kept for observationGroup III (Gallic acid + sepsis) animals were pretreated withGallic acid at the dose rate of 20mgkg body weight for 2 daysbefore induction of sepsis Animals were fed with standardmice chow and water postoperatively [29]

25 Survival Study Sepsis was induced through CLP withSO group animals serving as controls In preliminary exper-iments we observed a hyperdynamic physiological statefollowed by a hypodynamic physiological state similar tohemodynamic alterations in septic patients It was thusdecided to initiate Gallic acid therapy 2 days before sepsisinduction by CLP Mice were treated with Gallic acid orplacebo The degree of sepsis induced by CLP in micewas assessed by the presence of conjunctivitis absenceof grooming activities with resulting ruffled fur no oraluptake of food or water and lethargy Survival curve andmean survival time for all the groups were performed byKaplan-Meier survival curve and analyzed using log rank test[24]

26 Tissue Preparation261 Preparation of Liver Homogenate A 200mg of livertissue was weighed and taken in 2mL of ice-cold PBS (pH74) Another 200mg of sample was weighed and taken in2mL of 002M ethylene diamine tetra acetic acid (EDTA)solution for reduced glutathione (GSH) estimation Thehomogenate (10) prepared with IKA homogenizer (Ger-many) under ice-cold condition was centrifuged for 10min at3000 rpm The supernatant was stored at 20∘C until assay ofdifferent oxidative stress-related biochemical parameters Adouble beamUV-VIS Spectrophotometer (UV 5704SS ECILIndia) was used for recording the absorbance of test samples[30]












2+ 5 CO

2)




50(the con-


2



3 Results












0800 plusmn 085

1896 plusmn 088

0621 plusmn 068

lowast










lowast


125

100

75

50

25

0

0 10 20 30 40 50 60 70

Time

Sham

Surv

ival

()

Survival study

SepsisGA

















038 plusmn 002


042 plusmn 004


lowast









2O2minmg as com-



2O2minmg














mice








lowast






lowast





2) in sepsis





3-








References



















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



























2+ 5 CO

2)




50(the con-


2



3 Results












0800 plusmn 085

1896 plusmn 088

0621 plusmn 068

lowast










lowast


125

100

75

50

25

0

0 10 20 30 40 50 60 70

Time

Sham

Surv

ival

()

Survival study

SepsisGA

















038 plusmn 002


042 plusmn 004


lowast









2O2minmg as com-



2O2minmg














mice








lowast






lowast





2) in sepsis





3-








References



















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























0800 plusmn 085

1896 plusmn 088

0621 plusmn 068

lowast










lowast


125

100

75

50

25

0

0 10 20 30 40 50 60 70

Time

Sham

Surv

ival

()

Survival study

SepsisGA

















038 plusmn 002


042 plusmn 004


lowast









2O2minmg as com-



2O2minmg














mice








lowast






lowast





2) in sepsis





3-








References



















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















038 plusmn 002


042 plusmn 004


lowast









2O2minmg as com-



2O2minmg














mice








lowast






lowast





2) in sepsis





3-








References



















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















3-








References



















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Research Article Prophylactic Antioxidant Potential of Gallic Acid...

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