Research Article Ameliorative Effect of Chronic ...by aniline hydrochloride. At the end of treatment...

Research ArticleAmeliorative Effect of Chronic Supplementation ofProtocatechuic Acid Alone and in Combination with AscorbicAcid in Aniline Hydrochloride Induced Spleen Toxicity in Rats

Upasana Khairnar Aman Upaganlawar and Chandrashekhar Upasani

SNJBrsquos SSDJ College of Pharmacy Neminagar Chandwad 42310 India

Correspondence should be addressed to Aman Upaganlawar amanrxygmailcom

Received 28 December 2015 Revised 28 April 2016 Accepted 24 May 2016

Academic Editor Roland Bitsch

Copyright copy 2016 Upasana Khairnar 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

Background Present study was designed to evaluate the protective effects of protocatechuic acid alone and in combinationwith ascorbic acid in aniline hydrochloride induced spleen toxicity in rats Materials and Methods Male Wistar rats of eithersex (200ndash250 g) were used and divided into different groups Spleen toxicity was induced by aniline hydrochloride (100 ppm)in drinking water for a period of 28 days Treatment group received protocatechuic acid (40mgkgday po) ascorbic acid(40mgkgday po) and combination of protocatechuic acid (20mgkgday po) and ascorbic acid (20mgkgday po) followedby aniline hydrochloride At the end of treatment period serum and tissue parameters were evaluated Result Rats supplementedwith aniline hydrochloride showed a significant alteration in body weight spleen weight feed consumption water intakehematological parameters (haemoglobin content red blood cells white blood cells and total iron content) tissue parameters (lipidperoxidation reduced glutathione and nitric oxide content) and membrane bound phosphatase (ATPase) compared to controlgroupHistopathology of aniline hydrochloride induced spleen showed significant damage compared to control rats Treatmentwithprotocatechuic acid along with ascorbic acid showed better protection as compared to protocatechuic acid or ascorbic acid alonein aniline hydrochloride induced spleen toxicity Conclusion Treatment with protocatechuic acid and ascorbic acid in combinationshowed significant protection in aniline hydrochloride induced splenic toxicity in rats

1 Introduction

Spleen is the largest lymphoid tissue bean shaped organ forfiltering blood It plays an important role in the body such asformation of blood and removal of the old and ineffective cellsand allows only young active cells to pass into circulationIt is also involved in the iron metabolism and reacts againstinfection [1 2] Aniline a toxic aromatic amine is widely usedin industry for the manufacturing of dyes resins varnishesperfumes pesticides explosives isocyanates hydroquinoneand rubber chemicals [3] Various studies reported thatthe chronic exposure to aniline leads to the developmentof splenomegaly increased erythropoietic activity increasedpigmentation production of free radical hyperplasia andformation of malignant tumours [4 5] Clinical symptomssuch as cyanosis weakness dizziness headache stupor lossof coordination and coma occur commonly after exposure

to or contact with aniline [6] Earlier studies have shownthat aniline hydrochloride (AH) exposure leads to the for-mation of oxidative and nitrosative stress which are dueto iron overload and induction of lipid peroxidation AHenhance the production of reactive oxygennitrogen species(ROSRNS) which attacks proteins and nucleic acid leadingto the structural and functional changes in the spleen [7]Protection against free radicals can be enhanced by ampleintake of dietary antioxidants Substantial evidence indicatesthat foods containing antioxidant nutrients may be of majorimportance in disease prevention There is growing consen-sus among scientists that the combination of antioxidantrather than single entities may be more effective over thelong term Antioxidants may be of great benefit in improvingthe quality of life by preventing the onset of degenerativediseases In addition they have a potential for substantialsaving in the cost of healthcare delivery [8] Protocatechuic

Hindawi Publishing CorporationScientificaVolume 2016 Article ID 4306984 9 pageshttpdxdoiorg10115520164306984

2 Scientifica

acid (PCA) is a polyphenolic compound chemically it is34-dihydroxybenzoic acid and available mainly in the fruitsand vegetables [9] It is reported to possess antioxidant [10]antibacterial [11] anticancer [12] antiulcer [13] antidiabetic[14] antiaging [15] antifibrotic [16] antiviral [17] anti-inflammatory [18] antiatherosclerotic [19] cardioprotective[20] hepatoprotective [21] nephroprotective [22] and neu-roprotective [23] activities and have good effect on reproduc-tive system [24] Ascorbic acid (AA) is also known as vitaminC which is the enolic form of 3-keto L glucofuranolactoneit plays an active role in tissue metabolism and is connectedwith numerous electron transport processes where it behavesas a strong reducing agent [25] AA is an effective antioxidantand is involved in the biosynthesis of carnitine [26] Com-binations of various antioxidants are reported to producesynergistic activity Literature showed that there are no workscarried out to explore the protective effects of protocatechuicacid (polyphenolic antioxidant) and ascorbic acid (vitaminand antioxidant) alone and in combination in AH inducedspleen toxicity so the present study was initiated

2 Materials and Methods

21 Animals In the present study male Wistar rats (200ndash250 g) were used The rats were procured from registeredbreeder (Lachmi Biofarms Pune India) and kept separatelyin polypropylene cages (four rats per cage)with paddy husk asbeddingThe rats weremaintained under standard laboratoryconditions at temperature 23 plusmn 1∘C relative humidity 45ndash55 and 12 h light and 12 h dark cycles throughout theexperiments The experimental protocol was approved byInstitutional Animal Ethics Committee (IAEC) of SSDJ Col-lege of Pharmacy Neminagar Chandwad (approval numberSSDJIAEC2015028)

22 Drugs and Chemicals PCA was procured from Spec-trochem Pvt Ltd Mumbai India with certificate of anal-ysis AH 551015840-dithiobis-(2-nitrobenzoic acid) and N-(1-naphthyl) ethylenediamine dihydrochloride were purchasedfrom HiMedia Lab Pvt Ltd Mumbai AA was purchasedfrom Sigma Aldrich USA All the other chemicals used in thestudy were of analytical grade and procured from standardsupplier

Dose Selection for PCA and AA A preliminary study wascarried out using different doses that is 10 20 30 40and 50mgkgpo of both PCA as well as AA in AHinduced spleen toxicity At the end of treatment periodthe haemoglobin level was observed It was found that40mgkg dose was more effective in maintaining the levelof haemoglobin near to control value as compared to AHinduced rats So 40mgkg dose of PCA acid and AA wasselected for the study

Combination was selected based on combination index(CI index) as suggested by Chou and Talalay [27]

CI =(119863comb)1(119863alone)1

+(119863comb)2(119863alone)2

CI = 2040+20

40

CI = 05 + 05

CI = 1

CI = 1 indicates summation(1)

23 Experimental Protocol The rats were divided into thefollowing groups (119899 = 6)

Group Ι served as normal control and received nor-mal saline as vehicleGroup ΙΙ rats received AH (100 ppm) in drinkingwater for 28 daysGroup ΙII rats received AH (100 ppm) via drinkingwater and PCA (40mgkgpo) for 28 daysGroup ΙV rats received AH (100 ppm) via drinkingwater and AA (40mgkgpo) for 28 daysGroup V rats received AH (100 ppm) via drinkingwater and PCA (20mgkgpo) in combination withAA both (20mgkgpo) for 28 days

3 Assessment of Spleen Toxicity

31 Estimation of General Parameters and Biochemical Eval-uation At the end of treatment period body weight spleenweight spleen hypertrophy index water intake and feed con-sumption were noted Blood was withdrawn from retroor-bital plexus using glass capillary and serum was separatedusing high speed centrifuge Blood was used for the estima-tion of haemoglobin contents (Sahlirsquos haemometer method)red blood cell (RBC) count and white blood cell (WBC)count using haemocytometer [28]

Protein Content Was Estimated Using Span Diagnostic Kit001mL of serum was mixed with 1mL of working reagent(cupric sulphate 7mmolL potassium iodide 6mmolL tar-trate 20mmolL surfactant 005wv and stabilizer) Theassay mixture was incubated for 5 minutes at 37∘C Aftercompletion of incubation period absorbance was measuredagainst standard and blank (standard concentration 6ndash8 gdL)

The Iron Content in the Serum Was Estimated by RamsayMethod Equal volumes of serum 01M sodium sulphite and221015840-dipyridyl reagent were mixed in glass stopper centrifugetubesThe tubes were heated in a boiling water bath for 5minThe content was cooled and 12mL of chloroform was addedin each tube The tube was mixed vigorously for 30 secondsand centrifuged for 5min at 1000 rpm The color intensitywas measured at 520 nm Standard iron solution 498mg offerrous sulphate was dissolved in distilled water and 10mLof conc H

2SO4was added and the final volume was made up

to 1 L (5ndash20mL of the standard iron) [29]

32 Assessment of Markers of Oxidative Stress The ani-mals were euthanasiously sacrificed and isolated spleen wasquickly transferred to ice-cold tris hydrochloric buffered

Scientifica 3

Table 1 Effect of PCA alone and in combination with AA on body weight spleen weight water intake and feed consumption

Parameters Control AH AH+PCA AH+AA AH+PCA+AABody weight (g) 2625 plusmn 4433 1780 plusmn 2129lowastlowastlowast 1947 plusmn 2362 1993 plusmn 2305 2287 plusmn 4063a

Spleen weight (g) 0701 plusmn 0027 1331 plusmn 0036lowastlowastlowast 0896 plusmn 0030 0924 plusmn 0030 0858 plusmn 0032b

Spleen hypertrophy 000267 000747lowastlowastlowast 000460 000463 000375ab

Water intake (mL) 37 plusmn 0966 1833 plusmn 0557lowastlowastlowast 22 plusmn 0730 2877 plusmn 0432 3921 plusmn 0747ab

Feed consumption (g) 1831 plusmn 021 1204 plusmn 0220lowastlowastlowast 1349 plusmn 0358 1373 plusmn 0338 1797 plusmn 0312ab

Values are expressed as mean plusmn SEM Level of significance is considered as lowastp lt 005 lowastlowast119901 lt 001 and lowastlowastlowast119901 lt 0001 compared to control group 119901 lt 005119901 lt 001 and

119901 lt 0001 compared to AH-treated group a119901 lt 005 compared to AH+PCA and b119901 lt 005 compared to AH+AA group

saline (pH 74) It was blotted free of blood and tissue fluidsweighed on electronic balance WENSAR (Model PGB200)The spleen was cross-chopped with surgical scalpel into fineslices suspended in chilled 025M sucrose solution andquickly blotted on a filter paper The tissue was then mincedandhomogenised in chilled tris hydrochloride buffer (10mMpH 74) to a concentration of 10wv The homogenate wascentrifuged at 10000 rpm at 0∘C for 15 minutes using RemiC-24 high speed cooling centrifuge The clear supernatantwas used for the determination of lipid peroxidation reducedglutathione and nitric oxide and the sediment was used forthe estimation of membrane bound phosphatase

321 Assay of Reduced Glutathione (GSH) GSH was deter-mined as follows equal volumes of tissue homogenate(supernatant) and 20 trichloroacetic acid were mixedThe precipitated fraction was centrifuged and to 025mLof supernatant 2mL of 551015840-dithiobis (2-nitrobenzoic acid)reagent was added The final volume was made up to 3mLwith phosphate buffer The color developed was read at412 nm against reagent blank Different concentrations (10ndash50120583g) of standard glutathione were taken and processed asabove for standard graphThe amount of reduced glutathionewas expressed as 120583g of GSHmg protein [30]

322 Assay of Lipid Peroxidation (MDA Content) It wasestimated using the method described by Slater and Sawyer[31] 20mL of the tissue homogenate was added to 20mL offreshly prepared 10wv trichloroacetic acid and themixturewas allowed to stand in an ice bath for 15 minutes After 15minutes the precipitate was separated by centrifugation and20mL of clear supernatant solution was mixed with 20mLof freshly prepared thiobarbituric acidThe resulting solutionwas heated in a boiling water bath for 10 minutes It was thenimmediately cooled in an ice bath for 5 minutes The colourdeveloped was measured at 532 nm against reagent blankDifferent concentrations (0-23 nM) of standardmalondialde-hyde were taken and processed as above for standard graphThe values were expressed as nM of MDAmg protein

323 Assay of Nitric Oxide To 1mL of tissue homogenateadd 1mL of Griess reagent and incubate it for 15min at 37∘CRead the absorbance at 540 nmagainst aGriess reagent blankDifferent concentration (0ndash25120583g) of sodium nitrite solutionwas used as the standardThe amount of nitrite present in thesamples was estimated from the standard curves obtained

33 Assessment of Membrane Bound Phosphatases (Na+K+ATPase Ca++ ATPase and Mg++ ATPase) It was estimatedthat the membrane fraction remains after centrifugation ofthe tissue homogenatesThe activities ofNa+K+ ATPase [32]Ca++ ATPase [33] and Mg++ ATPase [34] were determinedThe phosphorus content of the supernatant was estimated asdescribed by Fiske and Subbarow [35] The enzyme activitywas expressed as 120583M of inorganic phosphorus liberatedmgproteinmin Potassium dihydrogen orthophosphate at var-ious concentrations (4 to 20120583gmL) was used as standardphosphorus Regression coefficient was found to be 09965

34 Histopathology of Spleen After decapitation spleen wasrapidly dissected out and washed immediately with normalsaline and fixed in 10 buffered formalin Small section oftissue was cut stained with haematoxylin and eosin (HampE)for general morphological evaluation It was carried out fromRane Pathology Laboratory Pune India

Statistical Analysis All the values are presented as mean plusmnSEM Statistical significance between more than two groupswas tested using one-way analysis of variance (ANOVA)followed by Dunnettrsquos test as appropriate using computer-based fitting program (Prism 5) Differences were consideredto be statistically significant when 119901 lt 005

4 Results

41 Effect of PCAAlone and inCombinationwithAAon SpleenHypertrophy Index Water Intake and Feed Intake At theend of treatment period body weight spleen weight spleenhypertrophy index water intake and feed consumption fromall the groups were monitored It was found that rats treatedwith AH showed a significant reduction in water intakeand feed consumption whereas spleen hypertrophy indexwas increased significantly compared to normal control ratsChronic treatment with PCA AA and PCA+AA showed asignificant recovery in alteration of water intake feed con-sumption spleen weight body weight and spleen hypertro-phy index as compared to AH-treated rats Combination ofPCA+AA (20mgkg resp) showed better result as comparedto antioxidants alone (Table 1)

42 Effect of PCA Alone and in Combination with AAon RBC WBC and Haemoglobin Level RBCs count andhaemoglobin level were significantly (119901 lt 001) decreasedand WBC count was significantly (119901 lt 001) increased

4 Scientifica

ControlAH

0

5

10

15

20

AH+PCA+AAAH+AA

AH+PCA

lowastlowastlowast

lowastlowastlowast

RBCs

and

WBC

s (times10

6c

ell)

WBCs (times106cell)RBCs (times106cell)

a b

a b

Figure 1 Effect of PCA alone and in combination with AA on RBCsandWBCs in AH-treated rats Values are expressed as mean plusmn SEMLevel of significance is considered as lowast119901 lt 005 lowastlowast119901 lt 001 andlowastlowastlowast119901 lt 0001 compared to control group 119901 lt 005 119901 lt 001 and119901 lt 0001 compared to AH-treated group a119901 lt 005 comparedto AH+PCA and b119901 lt 005 compared to AH+AA group

in AH-treated rats as compared to control rats Treatmentwith PCA (40mgkg day po) and AA (40mgkgday po)showed a significant (119901 lt 001) increase in RBC count andhaemoglobin level and a significant (119901 lt 001) decrease inWBC count as compared to AH-treated rats Combinationof PCA and AA (20mgkg day po each) in AH-treated ratsshowed significant improvement in RBC haemoglobin andWBC count as compared to AH-treated group Combinationwas found to bemore effective as compared to PCA alone andAA treated groups (Figures 1 and 2)

43 Effect of PCA Alone and in Combination with AA onSerum Total Protein and Iron Contents Total protein andserum iron content were monitored at the end of treatmentperiod and are shown in Figures 3 and 4 respectively Asignificant (119901 lt 0001) decrease in the level of serum proteinand a significant (119901 lt 0001) increase in serum iron contentwere observed in AH-treated group compared to controlTreatment with PCA in combination with AA (20mgkgdaypo each) showed a significant (119901 lt 0001) increase intotal protein and significant (119901 lt 0001) decrease in ironcontent as compared to AH-treated rats The combinationshowed additive effects in maintaining protein and iron levelas compared to alone antioxidants (Figures 3 and 4)

44 Effect of PCA Alone and in Combination with AA onTissue Lipid Peroxidation Reduced Glutathione Content andSerum NO Levels The level of endogenous antioxidantssuch as LPO GSH and NO was measured in spleen tissuehomogenate LPO and NO levels were found to be signifi-cantly (119901 lt 0001) increased and GSH level was significantlydecreased in spleen of AH-treated rats as compared to control

0

5

10

15

Hb

(gd

L)

ControlAH

lowastlowastlowast

AH+PCA+AAAH+AA

AH+PCA

a b

Figure 2 Effect of PCA alone and in combination with AA onhaemoglobin in AH-treated rats Values are expressed as mean plusmnSEM Level of significance is considered as lowast119901 lt 005 lowastlowast119901 lt 001and lowastlowastlowast119901 lt 0001 compared to control group 119901 lt 005 119901 lt

001 and 119901 lt 0001 compared to AH-treated group a119901 lt 005compared to AH+PCA and b119901 lt 005 compared to AH+AA group

a

0

2

4

6

8

10

Tota

l pro

tein

(gd

L)

ControlAH

lowastlowastlowast

AH+PCA+AAAH+AA

AH+PCA

Figure 3 Effect of PCA alone and in combination with AA on totalprotein in AH-treated rats Values are expressed as mean plusmn SEMLevel of significance is considered as lowast119901 lt 005 lowastlowast119901 lt 001 andlowastlowastlowast119901 lt 0001 compared to control group 119901 lt 005 119901 lt 001 and119901 lt 0001 compared to AH-treated group a119901 lt 005 comparedto AH+PCA and b119901 lt 005 compared to AH+AA group

group Chronic treatment with PCA AA (40mgkgdaypo) and PCA along with AA (20mgkgday po each)showed a significant (119901 lt 0001) decrease in LPO andNO level (Figures 6 and 7) and a significant (119901 lt 0001)increase in GSH level as compared to AH-treated group(Figure 5)The combinationwas found to bemore effective in

Scientifica 5

0

200

400

600

800

1000

ControlAH

lowastlowastlowast

a b

AH+PCA+AAAH+AA

AH+PCA

Tota

l iro

n (120583

gdL

)

Figure 4 Effect of PCA alone and in combination with AA on totaliron content in AH-treated rats Values are expressed as mean plusmnSEM Level of significance is considered aslowast119901 lt 005 lowastlowast119901 lt 001and lowastlowastlowast119901 lt 0001 compared to control group 119901 lt 005 119901 lt


0

1

2

3

4

ControlAH

lowastlowastlowastGSH

(120583g

g)

AH+PCA+AAAH+AA

AH+PCA

a b

Figure 5 Effect of PCA alone and in combination with AA onreduced glutathione in AH-treated rats Values are expressed asmean plusmn SEM Level of significance is considered as lowast119901 lt 005lowastlowast119901 lt 001 and lowastlowastlowast119901 lt 0001 compared to control group 119901 lt 005119901 lt 001 and 119901 lt 0001 compared to AH-treated groupa119901 lt 005 compared to AH+PCA and b119901 lt 005 compared toAH+AA group

maintaining markers of oxidative stress as compared to PCAalone and AA treated groups

45 Effect of PCA Alone and in Combination with AA onMembrane Bound Phosphatases (Na+K+ Ca++ and Mg++ATPase) The activities of membrane bound phosphatasesuch as Na+K+ ATPase Ca++ ATPase and Mg++ ATPase in

0

1

2

3

4

5

MD

Ag

tiss

ue (n

mol

L)

ControlAH

lowastlowastlowast

a b

AH+PCA+AAAH+AA

AH+PCA

Figure 6 Effect of PCA alone and in combination with AA on lipidperoxidation in AH-treated rats Values are expressed as mean plusmnSEM Level of significance is considered as lowast119901 lt 005 lowastlowast119901 lt 001and lowastlowastlowast119901 lt 0001 compared to control group 119901 lt 005 119901 lt


lowastlowastlowast

ControlAniline hydrochloride

0

10

20

30

40

NO

(nm

olL

)

AH+PCA+AAAH+AA

AH+PCA

a b

Figure 7 Effect of PCA alone and in combination with AA onnitric oxide inAH-treated rats Values are expressed asmeanplusmn SEMLevel of significance is considered as lowast119901 lt 005 lowastlowast119901 lt 001 andlowastlowastlowast119901 lt 0001 compared to control group 119901 lt 005 119901 lt 001 and119901 lt 0001 compared to AH-treated group a119901 lt 005 comparedto AH+PCA and b119901 lt 005 compared to AH+AA group

the spleen were estimated The level of Na+K+ Ca++ andMg++ ATPase was significantly (119901 lt 0001) decreased in AH-treated rats compared to control group Treatment with PCAAA (40mgkgday po) and PCA+AA (20mgkgday poeach) for 28 days showed significant (119901 lt 0001) increase inthe level of Na+K+ Ca++ and Mg++ ATPase as compared to

6 Scientifica

0

50

100

150

200

250

nm o

f ino

rgan

ic p

hosp

horu

slib

erat

edg

m ti

ssue

Control AH AH+PCA+AA

AH+AAAH+PCA

Na+K+ ATPasesCa++ ATPases

Mg++ ATPases

a b

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

Figure 8 Effect of PCA alone and in combination with AA onNa+K+ ATPase Ca++ ATPase and Mg++ ATPase in AH-treatedrats Values are expressed as mean plusmn SEM Level of significance isconsidered as lowast119901 lt 005 lowastlowast119901 lt 001 and lowastlowastlowast119901 lt 0001 compared tocontrol group 119901 lt 005 119901 lt 001 and 119901 lt 0001 compared toAH-treated group a119901 lt 005 compared to AH+PCA and b119901 lt 005

compared to AH+AA group

AH-treated group Combination of both antioxidants did notshow any significant changes compared to antioxidants aloneexcept Ca++ ATPase (Figure 8)

46 Effect of PCA Alone and in Combination with AA onHistoarchitecture of Spleen The section of control rat(Figure 9(a)) showed the normal red pulp of the spleenAH-treated group (100 ppm in drinking water) showedmultiple areas of sinusoidal congestion and accumulationof red blood cells called the ldquoBanti spleenrdquo (Figure 9(b))(arrow) The section of PCA and AA treated rats spleenshowed decrease in sinusoidal congestion and decreasein the accumulation of damaged red blood cells (Figures9(c) and 9(d)) Combination of PCA and AA showedcomparatively more protection as compared to drug aloneand AH-treated groups (Figure 9(e))

5 Discussion

Aniline exposure leads to the development of splenic toxicityin rats Previous studies show that exposure to anilineproduces increases in total iron content and oxidative stressin rats and it leads to enlargement of spleen (splenomegaly)due to excess deposition of damaged RBC [4 5 36] Thepresent study shows the splenoprotective effect of PCAalone and in combination with AA Splenic toxicity in ratswas induced by chronic supplementation of AH (100 ppm)via drinking water Toxicity of spleen was confirmed byevaluating the haemoglobin level and RBC count on 28thdayThe haemoglobin level and RBC count were significantlydecreased indicating the development of spleen toxicitySignificant decrease in body weight feed consumption andwater intake inAH-treated ratsmight be due to toxicity ofAHwhich decreased the food consumption and can be directlycorrelated to reduced body weight One important feature ofthis studywas increase in theweight of spleen (splenomegaly)

and spleen hypertrophy ratio in AH-treated rats indicated thedeposition of damaged RBCs in the spleen [4 5]

PCA is reported to play a major role in the treatmentof various conditions due to its strong antioxidant propertyPCA and AA alone and in combination were reported toexhibit antioxidant activity which can modify serum lipidlevel In the present study PCA and AA alone and incombination with treatments reverse the changes in bodyweight feed consumption water intake and spleen weightin AH-treated animals The alteration of general parameterssuggested the positive effect of PCA and AA alone and incombination in AH toxicity

In the present study AH exposure in rats showed signif-icant rise in the level of haemoglobin RBC and WBC whencompared to normal control ratsThese changesmight be dueto the excessive generation of oxidative and nitrosative stress[37 38] Treatment with PCA showed significant alteration ofhaemoglobin level and RBC andWBC count whichmight bedue to the strong antioxidantfree radical scavenging activityof PCA [9 15 23] Aniline administered rats showed a signif-icant increase in iron load and decrease in protein contentsIron plays a significant role as a mediator of aniline-inducedsplenotoxicity [6 7] AH toxicity causes accumulation of ironwhich may catalyze excessive formation of reactive oxygenspecies and damage proteins nucleic acids and lipids [39]AH exposure is reported to increase lipid peroxidation inthe tissue which might be due to increased iron contentLipid peroxidation and protein oxidation are at least twoimportant early biochemical events in AH induced splenictoxicity In the present study AH induced group showed asignificant increase in lipid peroxidation andNO content anda significant decrease inGSH level in spleenThese alterationsin oxidative stress markers produced structural modificationof native proteins and their function which might lead tosplenic toxicity [40]

In the present study membrane bound phosphatasessuchas Na+K+ ATPase Ca++ ATPase and Mg++ ATPase wereestimated Na+K+ Ca++ ATPase and Mg++ ATPase play asignificant role in the contraction and relaxation of muscle[32] These enzymes are located in the outer cell membraneand could have been affected by the excessive productionof free radical induced by AH and due to toxicity transportof electron may be affected thereby altering the energyproduction [33 34]

PCA and AA alone and in combination with treatmentshowed the attenuation of splenic toxicity induced by AHwhich might be due to its potential of reactive oxygen speciesas well as potent free radical scavenging activity The bettereffects of the combination suggest that they can cooperate inpreserving the physiological integrity of cell exposed to freeradical [41 42] This effect could occur because AA rapidlyreduced the phenoxyl radicals formed by wheat peroxidaseback to the initial phenol avoiding the formation of ferulatedimers until it was completely oxidized to dehydroascorbicacid [43] In our study we have also observed that thecombination of PCA and AA offered better protection tothe spleen when compared with individual treatment ofPCA as well as AA Present study suggests that a possiblemechanism for the observed better effect could be due to

Scientifica 7

Control(a)

AH(b)

PCA(c)

AA(d)

PCA+AA(e)

Figure 9 Histopathology of spleen stained with hematoxylin-eosin staining

the AA quenching the radical itself and thereby protectingthe PCA AA also preserves the intracellular LPO NO andGSH level GSH may react with nitric oxide to form S-nitroglutathione that is farmore potent than nitric oxide itself [43]

The morphological changes are always supported withhistopathological alterationThehistopathological changes inthe AH-treated rat spleen include vascular congestion andincreased red blood cells [44] These changes are closely

associated with increased iron deposition in the red pulpof the spleen The vascular congestion and marked irondeposition in the spleen with the increasing AH exposureare consistent with scavenging of damaged red blood cellsin the red pulp This in conjunction with the accumulationof aniline metabolites within the spleen could lead to thetransformation of mesenchymal cells of the spleen [38] Thepresent study is associatedwith increased iron deposition and

8 Scientifica

development of fibrotic lesions in the AH-treated rats [45]due to iron-mediated production of ROS which might act asa stimulus for increased collagen production in splenic tissueleading to fibrosis An increase in collagen gene transcriptionand collagen production occurred when cultured humanfibroblasts were subjected to iron-induced lipid peroxidationor exposed tomalondialdehyde [46]The histoarchitecture ofthe spleen supports the biochemical findings in the presentstudy Free radicals damage RBCs which might be the reasonfor observed changes in spleen histology Treatment withPCA andor AA showed better protection compared toAH-treated rats spleen which might be due to the strongantioxidant property of the drugs and their combinations

6 Conclusion

In conclusion our study reveals that the combination of PCAand AA showed better protection in 50 reduced dose ascompared to antioxidant alone by preventing the oxidativeand nitrosative stress in AH-treated spleen toxicity in rats

Abbreviations

AH Aniline hydrochlorideAA Ascorbic acidPCA Protocatechuic acid

Competing Interests

The authors declare that they have no competing interests

References

[1] B Steiniger and P Barth ldquoMicroanatomy and function of thespleenrdquoAdvances in Anatomy Embryology andCell Biology vol151 pp 1ndash101 2000

[2] C C Chatterji ldquoHuman physiologyrdquoMedical Allied Agency vol1 pp 199ndash202 2004

[3] ldquoFacts and figuresrdquo Chemical amp Engineering News vol 75 pp40ndash46 1997

[4] M F Khan P J Boor Y Gu N W Alcock and G A S AnsarildquoOxidative stress in the splenotoxicity of anilinerdquo Fundamentaland Applied Toxicology vol 35 no 1 pp 22ndash30 1997

[5] M F Khan X Wu P J Boor and G A S Ansari ldquoOxidativemodification of lipids and proteins in aniline-induced splenictoxicityrdquo Toxicological Sciences vol 48 no 1 pp 134ndash140 1999

[6] M F Khan S Kannan and JWang ldquoActivation of transcriptionfactor AP-1 and mitogen-activated protein kinases in aniline-induced splenic toxicityrdquo Toxicology and Applied Pharmacologyvol 210 no 1-2 pp 86ndash93 2006

[7] M F Khan X Wu G A S Ansari and P J Boor ldquoMalon-dialdehyde-protein adducts in the spleens of aniline-treatedrats immunochemical detection and localizationrdquo Journal ofToxicology and Environmental Health Part A Current Issuesvol 66 no 1 pp 93ndash102 2003

[8] S Chanda and R Dave ldquoIn vitro models for antioxidant activ-ity evaluation and some medicinal plants possessing antioxi-dant properties an overviewrdquo African Journal of MicrobiologyResearch vol 3 no 13 pp 981ndash996 2009

[9] G Williamson and C Manach ldquoBioavailability and bio efficacyof polyphenols in humans II Review of 93 interventionstudiesrdquoThe American Journal of Clinical Nutrition vol 81 no1 pp 234Sndash255S 2005

[10] S Guan B Jiang Y M Bao and L J An ldquoProtocatechuicacid suppresses MPP+-induced mitochondrial dysfunction andapoptotic cell death in PC12 cellsrdquo Food and Chemical Toxicol-ogy vol 44 no 10 pp 1659ndash1666 2006

[11] C-Y Chao andM-C Yin ldquoAntibacterial effects of roselle calyxextracts andprotocatechuic acid in groundbeef and apple juicerdquoFoodborne Pathogens and Disease vol 6 no 2 pp 201ndash2062009

[12] T Tanaka T Tanaka and M Tanaka ldquoPotential cancer chemo-preventive activity of protocatechuic acidrdquo Journal of Experi-mental and Clinical Medicine vol 3 no 1 pp 27ndash33 2011

[13] K J Kore P P Bramhkule R M Rachhadiya et al ldquoEvaluationof antiulcer activity of protocatechuic acid ethyl ester in ratsrdquoInternational Journal of Pharmacy Life Sciences vol 2 no 7 p909 2011

[14] B Scazzocchio R Varı C Filesi et al ldquoCyanidin-3-O-120573-glucoside and protocatechuic acid exert insulin-like effects byupregulating PPAR120574 activity in human omental adipocytesrdquoDiabetes vol 60 no 9 pp 2234ndash2244 2011

[15] G-F Shi L-J An B Jiang S Guan and Y-M Bao ldquoAlpiniaprotocatechuic acid protects against oxidative damage in vitroand reduces oxidative stress in vivordquo Neuroscience Letters vol403 no 3 pp 206ndash210 2006

[16] C Li W Jiang H Zhu and J Hou ldquoAntifibrotic effects ofprotocatechuic aldehyde on experimental liver fibrosisrdquo Phar-maceutical Biology vol 50 no 4 pp 413ndash419 2012

[17] Z Zhou Y Zhang X-R Ding et al ldquoProtocatechuic aldehydeinhibits hepatitis B virus replication both in vitro and in vivordquoAntiviral Research vol 74 no 1 pp 59ndash64 2007

[18] A B Lende A D Kshirsagar A D Deshpande et al ldquoAnti-inflammatory and analgesic activity of protocatechuic acid inrats and micerdquo Inflammopharmacology vol 19 no 5 pp 255ndash263 2011

[19] A R Borate A A Suralkar S S Birje P V Malusare and PA Bangale ldquoAntihyperlipidemic effect of protocatechuic acid infructose induced hyperlipidemia in ratsrdquo International Journalof Pharma and Bio Sciences vol 2 no 4 pp 456ndash460 2011

[20] R Zhou L-F He Y-J Li Y Shen R-B Chao and J-RDu ldquoCardioprotective effect of water and ethanol extract ofSalvia miltiorrhiza in an experimental model of myocardialinfarctionrdquo Journal of Ethnopharmacology vol 139 no 2 pp440ndash446 2012

[21] C-L Liu J-MWang C-Y ChuM-T Cheng and T-H TsengldquoIn vivo protective effect of protocatechuic acid on tert-butylhydroperoxide-induced rat hepatotoxicityrdquo Food and ChemicalToxicology vol 40 no 5 pp 635ndash641 2002

[22] J-H Lee H-J Lee H-J Lee et al ldquoRhus verniciflua Stokesprevents cisplatin-induced cytotoxicity and reactive oxygenspecies production in MDCK-I renal cells and intact micerdquoPhytomedicine vol 16 no 2-3 pp 188ndash197 2009

[23] S G Guan Y-M Bao B J Jiang and L-J An ldquoProtective effectof protocatechuic acid from Alpinia oxyphylla on hydrogenperoxide-induced oxidative PC12 cell deathrdquo European Journalof Pharmacology vol 538 no 1ndash3 pp 73ndash79 2006

[24] A Beytur O Ciftci M Aydin O Cakir N Timurkaan andF Yilmaz ldquoProtocatechuic acid prevents reproductive damagecaused by 2378-tetrachlorodibenzo-p-dioxin (TCDD) inmaleratsrdquo Andrologia vol 44 supplement 1 pp 454ndash461 2012

Scientifica 9

[25] A P Odin ldquoVitamins as antimutagens advantages and somepossible mechanisms of antimutagenic actionrdquo MutationResearchReviews in Mutation Research vol 386 no 1 pp39ndash67 1997

[26] S Kojo ldquoVitamin C basic metabolism and its function as anindex of oxidative stressrdquo Current Medicinal Chemistry vol 11no 8 pp 1041ndash1064 2004

[27] T C Chou and P Talalay ldquoA simple generalized equation forthe analysis of multiple inhibitions ofMichaelis-Menten kineticsystemsrdquoThe Journal of Biological Chemistry vol 252 no 18 pp6438ndash6442 1977

[28] P B Godkar and D P Godkar Determination of HemoglobinText Book of Medical Laboratory Technology Balani PublishingHouse Mumbai India 2nd edition 2008

[29] W N M Ramsay ldquoThe determination of the total iron-bindingcapacity of serumrdquo Clinica Chimica Acta vol 2 no 3 pp 221ndash226 1957

[30] M S Moron J W Depierre and B Mannervik ldquoLevels of glu-tathione glutathione reductase and glutathione S-transferaseactivities in rat lung and liverrdquo Biochimica et Biophysica Acta(BBA)mdashGeneral Subjects vol 582 no 1 pp 67ndash78 1979

[31] T F Slater and B C Sawyer ldquoThe stimulatory effect of carbontetrachloride and other halogenalkane or peroxidative reactionin the rat liver functions in vitrordquo Biochemistry Journal vol 123pp 805ndash815 1971

[32] S L Bonting ldquoPresence of enzyme system in mammaliantissuesrdquo in Membrane and Ion Transport pp 257ndash263 Wiley-Interscience 1970

[33] S Hjerten andH Pan ldquoPurification and characterization of twoforms of a low-affinity Ca2+-ATPase from erythrocyte mem-branesrdquo Biochimica et Biophysica Acta (BBA)mdashBiomembranesvol 728 no 2 pp 281ndash288 1983

[34] T Ohinishi Y Suzuki and K A Ozawa ldquoComparative study ofplasma membrane magnesium ion ATPase activities in normalregenerating and malignant cellsrdquo Biochimica et BiophysicaActa vol 684 pp 64ndash67 1982

[35] C H Fiske and Y Subbarow ldquoThe colorimetric determinationof phosphorusrdquoThe Journal of Biological Chemistry vol 66 pp375ndash400 1925

[36] E J Gralla J S Bus F Reno et al ldquoStudies of aniline HCL inratsrdquoToxicology andApplied Pharmacology vol 48 p A97 1979

[37] R Khan A B Upaganlawar and C Upasani ldquoProtective effectsofDioscorea alata L in aniline exposure-induced spleen toxicityin rats a biochemical studyrdquoToxicology International vol 21 no3 pp 294ndash299 2014

[38] J S Bus and J A Popp ldquoPerspectives on the mechanism ofaction of the splenic toxicity of aniline and structurally-relatedcompoundsrdquo Food and Chemical Toxicology vol 25 no 8 pp619ndash626 1987

[39] M F Khan SM Green G A S Ansari and P J Boor ldquoPhenyl-hydroxylamine role in aniline-associated splenic oxidativestress and induction of subendocardial necrosisrdquo ToxicologicalSciences vol 42 no 1 pp 64ndash71 1998

[40] FMKhan XWu and JWang ldquoUp-regulation of transforminggrowth factor-1205731 in the spleen of aniline-treated ratsrdquo Toxicol-ogy and Applied Pharmacology vol 187 no 1 pp 22ndash28 2003

[41] R L Prior X Wu and K Schaich ldquoStandardized methodsfor the determination of antioxidant capacity and phenolicsin foods and dietary supplementsrdquo Journal of Agricultural andFood Chemistry vol 53 no 10 pp 4290ndash4302 2005

[42] V L Kinnula and J D Crapo ldquoSuperoxide dismutases in thelung and human lung diseasesrdquoAmerican Journal of Respiratoryand Critical Care Medicine vol 167 no 12 pp 1600ndash1619 2003

[43] S K Yogeeta A Gnanapragasam S Senthilkumar R Sub-hashini and T Devaki ldquoSynergistic salubrious effect of Ferulicacid and ascorbic acid on membrane-bound phosphatases andlysosomal hydrolases during experimental myocardial infarc-tion in ratsrdquo Life Sciences vol 80 no 3 pp 258ndash263 2006

[44] M Omer B U Aman and C D Upasani ldquoDL-120572-lipoic acidattenuates acute aniline induced splenic toxicity in rats abiological and histoarchitecture studyrdquo Asian Journal of Phar-macology and Toxicology vol 3 no 8 pp 4ndash7 2005

[45] M Khan Firoze B S Kaphalia P J Boor and G A S AnsarildquoSubchronic toxicity of aniline hydrochloride in ratsrdquo Archivesof Environmental Contamination and Toxicology vol 24 no 3pp 368ndash374 1993

[46] M Chojkier K Houglum J Solis-Herruzo and D A BrennerldquoStimulation of collagen gene expression by ascorbic acid incultured human fibroblasts A role for lipid peroxidationrdquoTheJournal of Biological Chemistry vol 264 no 28 pp 16957ndash16962 1989

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

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StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of



Advances in Pharmacological Sciences

Tropical MedicineJournal of


Medicinal ChemistryInternational Journal of


AddictionJournal of



BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Autoimmune Diseases


Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of


Pharmaceutics


MEDIATORSINFLAMMATION

of

2 Scientifica

acid (PCA) is a polyphenolic compound chemically it is34-dihydroxybenzoic acid and available mainly in the fruitsand vegetables [9] It is reported to possess antioxidant [10]antibacterial [11] anticancer [12] antiulcer [13] antidiabetic[14] antiaging [15] antifibrotic [16] antiviral [17] anti-inflammatory [18] antiatherosclerotic [19] cardioprotective[20] hepatoprotective [21] nephroprotective [22] and neu-roprotective [23] activities and have good effect on reproduc-tive system [24] Ascorbic acid (AA) is also known as vitaminC which is the enolic form of 3-keto L glucofuranolactoneit plays an active role in tissue metabolism and is connectedwith numerous electron transport processes where it behavesas a strong reducing agent [25] AA is an effective antioxidantand is involved in the biosynthesis of carnitine [26] Com-binations of various antioxidants are reported to producesynergistic activity Literature showed that there are no workscarried out to explore the protective effects of protocatechuicacid (polyphenolic antioxidant) and ascorbic acid (vitaminand antioxidant) alone and in combination in AH inducedspleen toxicity so the present study was initiated

2 Materials and Methods

21 Animals In the present study male Wistar rats (200ndash250 g) were used The rats were procured from registeredbreeder (Lachmi Biofarms Pune India) and kept separatelyin polypropylene cages (four rats per cage)with paddy husk asbeddingThe rats weremaintained under standard laboratoryconditions at temperature 23 plusmn 1∘C relative humidity 45ndash55 and 12 h light and 12 h dark cycles throughout theexperiments The experimental protocol was approved byInstitutional Animal Ethics Committee (IAEC) of SSDJ Col-lege of Pharmacy Neminagar Chandwad (approval numberSSDJIAEC2015028)

22 Drugs and Chemicals PCA was procured from Spec-trochem Pvt Ltd Mumbai India with certificate of anal-ysis AH 551015840-dithiobis-(2-nitrobenzoic acid) and N-(1-naphthyl) ethylenediamine dihydrochloride were purchasedfrom HiMedia Lab Pvt Ltd Mumbai AA was purchasedfrom Sigma Aldrich USA All the other chemicals used in thestudy were of analytical grade and procured from standardsupplier

Dose Selection for PCA and AA A preliminary study wascarried out using different doses that is 10 20 30 40and 50mgkgpo of both PCA as well as AA in AHinduced spleen toxicity At the end of treatment periodthe haemoglobin level was observed It was found that40mgkg dose was more effective in maintaining the levelof haemoglobin near to control value as compared to AHinduced rats So 40mgkg dose of PCA acid and AA wasselected for the study

Combination was selected based on combination index(CI index) as suggested by Chou and Talalay [27]

CI =(119863comb)1(119863alone)1

+(119863comb)2(119863alone)2

CI = 2040+20

40

CI = 05 + 05

CI = 1

CI = 1 indicates summation(1)

23 Experimental Protocol The rats were divided into thefollowing groups (119899 = 6)

Group Ι served as normal control and received nor-mal saline as vehicleGroup ΙΙ rats received AH (100 ppm) in drinkingwater for 28 daysGroup ΙII rats received AH (100 ppm) via drinkingwater and PCA (40mgkgpo) for 28 daysGroup ΙV rats received AH (100 ppm) via drinkingwater and AA (40mgkgpo) for 28 daysGroup V rats received AH (100 ppm) via drinkingwater and PCA (20mgkgpo) in combination withAA both (20mgkgpo) for 28 days

3 Assessment of Spleen Toxicity

31 Estimation of General Parameters and Biochemical Eval-uation At the end of treatment period body weight spleenweight spleen hypertrophy index water intake and feed con-sumption were noted Blood was withdrawn from retroor-bital plexus using glass capillary and serum was separatedusing high speed centrifuge Blood was used for the estima-tion of haemoglobin contents (Sahlirsquos haemometer method)red blood cell (RBC) count and white blood cell (WBC)count using haemocytometer [28]

Protein Content Was Estimated Using Span Diagnostic Kit001mL of serum was mixed with 1mL of working reagent(cupric sulphate 7mmolL potassium iodide 6mmolL tar-trate 20mmolL surfactant 005wv and stabilizer) Theassay mixture was incubated for 5 minutes at 37∘C Aftercompletion of incubation period absorbance was measuredagainst standard and blank (standard concentration 6ndash8 gdL)

The Iron Content in the Serum Was Estimated by RamsayMethod Equal volumes of serum 01M sodium sulphite and221015840-dipyridyl reagent were mixed in glass stopper centrifugetubesThe tubes were heated in a boiling water bath for 5minThe content was cooled and 12mL of chloroform was addedin each tube The tube was mixed vigorously for 30 secondsand centrifuged for 5min at 1000 rpm The color intensitywas measured at 520 nm Standard iron solution 498mg offerrous sulphate was dissolved in distilled water and 10mLof conc H

2SO4was added and the final volume was made up

to 1 L (5ndash20mL of the standard iron) [29]

32 Assessment of Markers of Oxidative Stress The ani-mals were euthanasiously sacrificed and isolated spleen wasquickly transferred to ice-cold tris hydrochloric buffered

Scientifica 3
















4 Results



4 Scientifica

ControlAH

0

5

10

15

20

AH+PCA+AAAH+AA

AH+PCA

lowastlowastlowast

lowastlowastlowast

RBCs

and

WBC

s (times10

6c

ell)


a b

a b





0

5

10

15

Hb

(gd

L)

ControlAH

lowastlowastlowast

AH+PCA+AAAH+AA

AH+PCA

a b



a

0

2

4

6

8

10

Tota

l pro

tein

(gd

L)

ControlAH

lowastlowastlowast

AH+PCA+AAAH+AA

AH+PCA



Scientifica 5

0

200

400

600

800

1000

ControlAH

lowastlowastlowast

a b

AH+PCA+AAAH+AA

AH+PCA

Tota

l iro

n (120583

gdL

)



0

1

2

3

4

ControlAH


(120583g

g)

AH+PCA+AAAH+AA

AH+PCA

a b




0

1

2

3

4

5

MD

Ag

tiss

ue (n

mol

L)

ControlAH

lowastlowastlowast

a b

AH+PCA+AAAH+AA

AH+PCA



lowastlowastlowast


0

10

20

30

40

NO

(nm

olL

)

AH+PCA+AAAH+AA

AH+PCA

a b



6 Scientifica

0

50

100

150

200

250

nm o

f ino

rgan

ic p

hosp

horu

slib

erat

edg

m ti

ssue


AH+AAAH+PCA


Mg++ ATPases

a b

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast





5 Discussion







Scientifica 7

Control(a)

AH(b)

PCA(c)

AA(d)

PCA+AA(e)





8 Scientifica


6 Conclusion


Abbreviations


Competing Interests


References

























Scientifica 9



























Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

Scientifica 3
















4 Results



4 Scientifica

ControlAH

0

5

10

15

20

AH+PCA+AAAH+AA

AH+PCA

lowastlowastlowast

lowastlowastlowast

RBCs

and

WBC

s (times10

6c

ell)


a b

a b





0

5

10

15

Hb

(gd

L)

ControlAH

lowastlowastlowast

AH+PCA+AAAH+AA

AH+PCA

a b



a

0

2

4

6

8

10

Tota

l pro

tein

(gd

L)

ControlAH

lowastlowastlowast

AH+PCA+AAAH+AA

AH+PCA



Scientifica 5

0

200

400

600

800

1000

ControlAH

lowastlowastlowast

a b

AH+PCA+AAAH+AA

AH+PCA

Tota

l iro

n (120583

gdL

)



0

1

2

3

4

ControlAH


(120583g

g)

AH+PCA+AAAH+AA

AH+PCA

a b




0

1

2

3

4

5

MD

Ag

tiss

ue (n

mol

L)

ControlAH

lowastlowastlowast

a b

AH+PCA+AAAH+AA

AH+PCA



lowastlowastlowast


0

10

20

30

40

NO

(nm

olL

)

AH+PCA+AAAH+AA

AH+PCA

a b



6 Scientifica

0

50

100

150

200

250

nm o

f ino

rgan

ic p

hosp

horu

slib

erat

edg

m ti

ssue


AH+AAAH+PCA


Mg++ ATPases

a b

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast





5 Discussion







Scientifica 7

Control(a)

AH(b)

PCA(c)

AA(d)

PCA+AA(e)





8 Scientifica


6 Conclusion


Abbreviations


Competing Interests


References

























Scientifica 9



























Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

4 Scientifica

ControlAH

0

5

10

15

20

AH+PCA+AAAH+AA

AH+PCA

lowastlowastlowast

lowastlowastlowast

RBCs

and

WBC

s (times10

6c

ell)


a b

a b





0

5

10

15

Hb

(gd

L)

ControlAH

lowastlowastlowast

AH+PCA+AAAH+AA

AH+PCA

a b



a

0

2

4

6

8

10

Tota

l pro

tein

(gd

L)

ControlAH

lowastlowastlowast

AH+PCA+AAAH+AA

AH+PCA



Scientifica 5

0

200

400

600

800

1000

ControlAH

lowastlowastlowast

a b

AH+PCA+AAAH+AA

AH+PCA

Tota

l iro

n (120583

gdL

)



0

1

2

3

4

ControlAH


(120583g

g)

AH+PCA+AAAH+AA

AH+PCA

a b




0

1

2

3

4

5

MD

Ag

tiss

ue (n

mol

L)

ControlAH

lowastlowastlowast

a b

AH+PCA+AAAH+AA

AH+PCA



lowastlowastlowast


0

10

20

30

40

NO

(nm

olL

)

AH+PCA+AAAH+AA

AH+PCA

a b



6 Scientifica

0

50

100

150

200

250

nm o

f ino

rgan

ic p

hosp

horu

slib

erat

edg

m ti

ssue


AH+AAAH+PCA


Mg++ ATPases

a b

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast





5 Discussion







Scientifica 7

Control(a)

AH(b)

PCA(c)

AA(d)

PCA+AA(e)





8 Scientifica


6 Conclusion


Abbreviations


Competing Interests


References

























Scientifica 9



























Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

Scientifica 5

0

200

400

600

800

1000

ControlAH

lowastlowastlowast

a b

AH+PCA+AAAH+AA

AH+PCA

Tota

l iro

n (120583

gdL

)



0

1

2

3

4

ControlAH


(120583g

g)

AH+PCA+AAAH+AA

AH+PCA

a b




0

1

2

3

4

5

MD

Ag

tiss

ue (n

mol

L)

ControlAH

lowastlowastlowast

a b

AH+PCA+AAAH+AA

AH+PCA



lowastlowastlowast


0

10

20

30

40

NO

(nm

olL

)

AH+PCA+AAAH+AA

AH+PCA

a b



6 Scientifica

0

50

100

150

200

250

nm o

f ino

rgan

ic p

hosp

horu

slib

erat

edg

m ti

ssue


AH+AAAH+PCA


Mg++ ATPases

a b

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast





5 Discussion







Scientifica 7

Control(a)

AH(b)

PCA(c)

AA(d)

PCA+AA(e)





8 Scientifica


6 Conclusion


Abbreviations


Competing Interests


References

























Scientifica 9



























Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

6 Scientifica

0

50

100

150

200

250

nm o

f ino

rgan

ic p

hosp

horu

slib

erat

edg

m ti

ssue


AH+AAAH+PCA


Mg++ ATPases

a b

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast





5 Discussion







Scientifica 7

Control(a)

AH(b)

PCA(c)

AA(d)

PCA+AA(e)





8 Scientifica


6 Conclusion


Abbreviations


Competing Interests


References

























Scientifica 9



























Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

Scientifica 7

Control(a)

AH(b)

PCA(c)

AA(d)

PCA+AA(e)





8 Scientifica


6 Conclusion


Abbreviations


Competing Interests


References

























Scientifica 9



























Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

8 Scientifica


6 Conclusion


Abbreviations


Competing Interests


References

























Scientifica 9



























Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

Scientifica 9



























Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of





Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

Research Article Ameliorative Effect of Chronic ...by aniline hydrochloride. At the end of treatment...

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Transcript of Research Article Ameliorative Effect of Chronic ...by aniline hydrochloride. At the end of treatment...