Research ArticleAnti-Parkinson Activity of Petroleum Ether Extract ofFicus religiosa (L) Leaves

Jitendra O Bhangale and Sanjeev R Acharya

Institute of Pharmacy Nirma University Ahmedabad Gujarat 382 481 India

Correspondence should be addressed to Jitendra O Bhangale jitu2586gmailcom

Received 24 September 2015 Revised 11 December 2015 Accepted 17 December 2015

Academic Editor Antonio Ferrer-Montiel

Copyright copy 2016 J O Bhangale and S R Acharya This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

In the present study we evaluated anti-Parkinsonrsquos activity of petroleumether extract ofFicus religiosa (PEFRE) leaves in haloperidoland 6 hydroxydopamine (6-OHDA) induced experimental animal models In this study effects of Ficus religiosa (100 200 and400mgkg po) were studied using in vivo behavioral parameters like catalepsy muscle rigidity and locomotor activity and itseffects on neurochemical parameters (MDA CAT SOD and GSH) in rats The experiment was designed by giving haloperidolto induce catalepsy and 6-OHDA to induce Parkinsonrsquos disease-like symptoms The increased cataleptic scores (induced byhaloperidol) were significantly (119901 lt 0001) found to be reduced with the PEFRE at a dose of 200 and 400mgkg (po) 6-OHDAsignificantly induced motor dysfunction (muscle rigidity and hypolocomotion) 6-OHDA administration showed significantincrease in lipid peroxidation level and depleted superoxide dismutase catalase and reduced glutathione level Daily administrationof PEFRE (400mgkg) significantly improved motor performance and also significantly attenuated oxidative damage Thus thestudy proved that Ficus religiosa treatment significantly attenuated the motor defects and also protected the brain from oxidativestress

1 Introduction

Parkinsonrsquos disease (PD) is caused by the gradual and selec-tive loss of dopaminergic neurons in the substantia nigrapars compacta (SNpc) [1 2] PD produces bradykinesiamuscular rigidity rest tremor and loss of postural balancealong with some secondary manifestations like dementiasialorrhoea [3] soft speech and difficulty in swallowingdue to uncoordinated movements of mouth and throat [4]PD occurs due to inhibition of mitochondrial complex-1[5 6] different mechanisms of cell damage like excitotoxicitycalcium homeostasis inflammation apoptosis distressedenergy metabolism and protein aggregation [7] and inter-action between genetic and environmental factors [8]

Oxidative stress interferes with dopamine metabolismleading to Parkinsonrsquos disease This oxidative damage leadsto formation of reactive oxygen species (ROS) leading toneuronal death [9 10] This was evidenced by reduced levelof endogenous antioxidant compoundsThese findings intro-duced the requirement of using antioxidants as a therapeuticintervention in PD in addition to other protective agents

The current available drug treatments for PD possessvarious side effects Therefore herbal therapies should beconsidered as alternativecomplementarymedicines for ther-apeutic approach

Since ancient times plants have been an ideal source ofmedicine Plants have played a noteworthy role in main-taining human health and improving the quality of life forthousands of years and have served humans as well asvaluable components of medicines seasonings beveragescosmetics and dyes Inmodern times focus on plant researchhas increased all over the world and a large body of evidencehas been collected to demonstrate immense potential ofmedicinal plants used in various traditional systems Ficusreligiosa Linn (Moraceae) commonly known as ldquoPimpalardquoor ldquoPipalrdquo tree is a large widely deciduous tree heart-shapedwithout aerial roots from the branches with spreadingbranches and grey bark [11ndash13] The tree is held sacredby Hindus and Buddhists In India it is known by severalvernacular names the most commonly used ones beingAsvatthah (Sanskrit) Sacred fig (Bengali) Peepal (Hindi)

Hindawi Publishing CorporationAdvances in Pharmacological SciencesVolume 2016 Article ID 9436106 9 pageshttpdxdoiorg10115520169436106

2 Advances in Pharmacological Sciences

Arayal (Malayalam) Ravi (Telugu) and Arasu (Tamil)Leaves contain campesterol stigmasterol isofucosterol 120572-amyrin lupeol tannic acid arginine serine aspartic acidglycine threonine alanine proline tryptophan tyrosinemethionine valine isoleucine leucine n-nonacosane n-hentricontane hexacosanol and n-octacosane [14 15] Ficusreligiosa has been used in traditional medicine for a widerange of ailments Its bark fruits leaves roots latex andseeds are medicinally used in different forms sometimesin combination with other herbs [16] The whole parts ofthe plant exhibit wide spectrum of activities such as anti-cancer antioxidant antidiabetic antimicrobial anticonvul-sant anthelmintic antiulcer antiasthmatic and antiamnesicactivities Bark of the plant has been used as astringentcooling aphrodisiac and antibacterial against Staphylococcusaureus and Escherichia coli gonorrhea diarrhea dysenteryhemorrhoids and gastrohelcosis as anti-inflammatory andfor burnsThe leaves of the plant have been used for hemopt-ysis epistaxis hematuria menorrhagia blood dysentery andskin diseases Leaf juice has been used for asthma coughsexual disorders diarrhea hematuria toothache migraineeye troubles gastric problems and scabies Fruits of the plantwere used in asthma and as laxative and digestive Seeds ofthe plant were used as refrigerant and laxative and latex wasused in neuralgia inflammations and hemorrhages [17] AsF religiosa has been used traditionally in the treatment ofneurodegenerative disorders (including Parkinsonrsquos disease)and has also been reported to possess antioxidant activity thisplant may prove to be effective in the remedy of PD HenceF religiosa was evaluated for its anti-Parkinsonrsquos effect usingneurotoxin induced Parkinsonrsquos model in rats

2 Materials and Methods

21 Collection of Plant Material Fresh leaves of Ficus reli-giosa were collected from local area of Ahmedabad districtGujarat India during JulyndashSeptember This plant was iden-tified and authenticated by Dr A Benniamin Scientist DBotanical Survey of India Pune Voucher specimens numberBSIWCTech2015JOB-1 have been kept in Botanical Sur-vey of India Pune Maharashtra India

22 Animals Adult male Wistar rats weighing 180ndash220 gand albino mice of either sex weighing 25ndash30 g were usedand acclimatized to laboratory condition for one week Allanimals were housed in well-ventilated polypropylene cagesat 12 12 h lightdark schedule at 25 plusmn 2∘C and 55ndash65 RHThe rats were fed with commercial pelleted rats chow andwater ad libitum as a standard diet Institutional AnimalEthics Committee approved the experimental protocol inaccordance with the Committee for the Purpose of Controland Supervision of Experiments on Animals (CPCSEA)

23 Preparation of Leaf Extract The leaveswere collected anddried in shade and ground Coarsely powdered plantmaterial(1000 g) was weighed and extracted with 5 lit of solvents likepetroleum ether (60ndash80∘C) ethyl acetate and ethanol bysuccessive extraction in a Soxhlet apparatus for 72 h After

each extraction the solvent was distilled off and concentratedextract was transferred to previously weighed petri dish andevaporated to dryness at room temperature (45ndash50∘C) toobtain dried extracts The dried extract was weighed and thepercentage yield of the extracts was calculated as follows

of extractive yield (ww)

=Weight of dried extract

Weight of dried leaves powdertimes 100

(1)

The yield of petroleum ether ethyl acetate and ethanolextract was 182 106 and 268 (ww) respectively

24 Preliminary Phytochemical Studies Preliminary qualita-tive phytochemical screening was done for the presence ofdifferent group of chemicals that is alkaloids flavonoidssaponins tannins sterols carbohydrates and glycosides asdescribed by Harborne [18]

241 Test for Tannins and Phenols 5mL of extract wasadded to 2mL of 5 of alcoholic FeCl

3solution Blue-Black

precipitate indicated the presence of tannins and phenols

242 Test for Alkaloids To the dry extract (10ndash20mg) dilutehydrochloric acid (1-2mL) was added shaken well andfiltered With filtrate the following tests were performed

(1) Mayerrsquos Test To 2-3mL of filtrate 2-3 drops of Mayerrsquosreagent were added Appearance of precipitate indicatedpresence of alkaloids

(2) Wagnerrsquos Test To 2-3mL of filtrate Wagnerrsquos (3ndash5 drops)reagent was added Appearance of reddish-brown precipitateindicated presence of alkaloids

(3) Hagerrsquos Test To 2-3mL of filtrate 4-5 drops of Hagerrsquosreagent were added Appearance of yellow precipitate indi-cated presence of alkaloids

(4) Dragendorff rsquos Test To 2-3mL of filtrate 4-5 drops ofDragendorff rsquos reagent were added Appearance of orange-brown precipitate indicated presence of alkaloids

243 Test for Saponins About 1 g of dried powdered samplewas boiled with 10mL distilled water Frothing persistenceindicated the presence of saponins

244 Test for Terpenoids 5mL of extract was mixed with2mL of chloroform and few drops of concentrated H

2SO4

were carefully added to form a layer Red ring indicated thatthe terpenoids are present

245 Test for Steroids (Liebermann-BurchardReaction) 5mLof extract was mixed with 10mL CHCl

3and 1mL acetic

anhydride and few drops of concentrated H2SO4were added

Green ring indicated the presence of steroids

Advances in Pharmacological Sciences 3

246 Test for Flavonoids (Shinoda Test) To dry extract (10ndash20mg) 5mLof ethanol (95) 2-3 drops of hydrochloric acidand 05 gmagnesium turningswere added Change of color ofsolution to pink indicated presence of flavonoids

247 Test for Carbohydrates (Molischrsquos Test) Few drops (2-3) of 120572-naphthol solution in alcohol were added to 2-3mLof solution of extract and shaken for few minutes and then05mL of conc sulfuric acid was added from the side oftest tube The formation of violet ring at the junction of twosolutions indicated presence of carbohydrates

248 Test for Glycosides

(1) Legalrsquos Test To the extract 1mL of pyridine and 1mL ofsodium nitroprusside were added Change in color to pink orred indicated presence of cardiac glycosides

(2) Keller-Kiliani Test Glacial acetic acid (3ndash5 drops) onedrop of 5 ferric chloride and concentrated sulfuric acid wasadded to the test tube containing 2mL of solution of extractAppearance of reddish-brown color at the junction of twolayers and bluish green in the upper layer indicated presenceof cardiac glycosides

(3) Borntragerrsquos Test Dilute sulfuric acid was added to 2mLof solution of extract boiled for few minutes and filtered Tothe filtrate 2mL of benzene or chloroform was added andshaken well The organic layer was separated and ammoniawas added The change in color of ammoniacal layer to pinkred indicated presence of anthraquinone glycosides

249 Test for Phlobatannins About 2mL of extract wasboiled with 2mL 1 HCl Deposition of red color indicatedthe presence of phlobatannins

2410 Test for Amino Acid (Ninhydrin Test) 5 to 6 drops ofNinhydrin reagent were added in 5mL of extract and heatedover boiling water bath for 5min Purple coloration indicatedthe presence of amino acid

2411 Test for Proteins (Biuret Test) 5-6 drops of 5 NaOHand 5ndash7 drops of 1 CuSO

4were added in 2mL of extract

Violet color indicated the presence of protein

25 Acute Oral Toxicity of the Extract Themice were dividedinto 5 groups of 10 animals each The mice were fastedfor 6 h and had access to only water ad libitum beforeexperimental study Group I received only vehicle (distilledwater) Groups II III IV and V received different doses ofpet ether extract of F religiosa (PEFRE) that is 1000 20003000 and 4000mgkg respectively All the doses and vehiclewere administered orallyThe animals were observed for 72 hfor mortality [19]

26 Haloperidol Induced Catalepsy Haloperidol causes dys-functioning of various neurotransmitters such as acetyl-choline GABA and serotonin Pathology of haloperidolinduced catalepsy underlying increased oxidative stressHaloperidol an antipsychotic drug blocks central dopaminereceptor in striatum It also produces a behavioral state inanimals like mice and rats in which they fail to correctexternally imposed postures (called catalepsy) thus keepingthe above fact in mind the haloperidol induced catalepsymodel was selected The method described by Elliott andClose in 1990 [20] was followed for the anticataleptic activityThe animals were divided into five groups (119899 = 6) GroupI served as vehicle control Group II served as standardLevodopa (6mgkg po) and Groups IIIndashV served as testgroup treated with PEFRE (100 200 and 400mgkg po)respectively Standard bar test was used to measure thecatalepsy Catalepsy was induced by haloperidol (1mgkgip) and examined at every 30min interval for 210min Theduration for which the rat retains the forepaws extended andresting on the elevated bar was considered as cataleptic scoreA cut-off time of 5min was applied

27 Induction of Parkinsonism by 6-OHDA The rats wereanesthetized with an intraperitoneal injection of 50mgkgof sodium pentobarbital and were fixed in a stereotaxicapparatus [21 22] A stainless steel needle (028mm od) wasinserted unilaterally into the substantia nigra with the follow-ing coordinates anteriorposterior minus48mm mediallateralminus22mm ventraldorsal minus72mmndash35mm from bregma andinjection of 6-OHDA (20 120583g of 6-OHDA hydrobromide in4 120583L 09 saline with 002120583gmL ascorbic acid) was thenmade over 5min and the needle was left in place for afurther 5min Then the skull was secured with stainlessmetallic screws and the wound area was covered by dentalcement Each rat was housed individually following thesurgical procedure Sham operated animals were also treatedin the same manner but they received equivalent volumes ofnormal saline instead of 6-OHDA

28 Experimental Design Animals were divided into sixgroups of 6 rats in each group Group I served as shamoperated animals and received normal saline (10mLkg po)Groups II toVIwere inducedwith parkinsonismby 6-OHDAas follows Group II served as a 6-OHDAcontrol and receivednormal saline (10mLkg) Group III served as a L DOPA(6mgkg po) and Groups IV to VI served as a test drugPEFRE (100 200 and 400mgkg po resp) The treatmentof animals was started after 48 h of induction with 6-OHDAaccording to their respective group once a day for 55 days

29 Behavioral Assessment All behavioral assessment wasperformedby an observer blinded to the groupDifferent testswere performed at different time points after lesion

291 Locomotor Activity The spontaneous locomotor activ-ity was monitored using digital actophotometer (Hiconinstrument India) equipped with infrared sensitive photo-cellsThe apparatus was placed in a darkened light and sound

4 Advances in Pharmacological Sciences

attenuated and ventilated testing room Each interruption ofa beamon the119909 or119910 axis generated an electric impulse whichwas presented on a digital counter Each animal was observedover a period of 5min on days 15 20 25 30 35 40 45 50and 55 following 6-OHDA administration and values wereexpressed as counts per 5min [23]

292 Rotarod Activity All animals were evaluated for gripstrength by using the rotarod The rotarod test is widelyused in rodents to assess their ldquominimal neurological deficitrdquosuch as motor function and coordination Each rat was givena prior training session before initialization of therapy toacclimatize them on a rotarod apparatus (EIE instrumentIndia) Animal was placed on the rotating rodwith a diameterof 7 cm (speed 25 rpm) Each rat was subjected to threeseparate trials at 5min interval on days 15 20 25 30 35 4045 50 and 55 following 6-OHDA administration and cut-offtime (180 s) was maintained throughout the experiment Theaverage results were recorded as fall of time [24]

210 Dissection and Homogenization After the treatmentperiod animals were scarified by decapitation under mildanesthesia The brains were immediately removed forebrainwas dissected out and cerebellum was discarded Brainswere put on ice and rinsed in ice-cold isotonic saline toremove blood A 10 (wv) tissue homogenate was preparedin 01M phosphate buffer (pH 74)The homogenate was cen-trifuged at 10000 g for 15minutes and aliquots of supernatantobtained were used for biochemical estimation

211 Biochemical Estimation

2111 Malondialdehyde (MDA) Level The amount of malon-dialdehyde was used as an indirect measure of lipid peroxida-tion and was determined by reaction with thiobarbituric acid(TBA) [25] Briefly 1mL of aliquots of supernatantwas placedin test tubes and added to 3mL of TBA reagent TBA 038(ww) 025M hydrochloric acid (HCl) and trichloroaceticacid (TCA 15) The solution was shaken and placed for15min followed by cooling in an ice bath After coolingsolution was centrifuged to 3500 g for 10min The upperlayer was collected and assessed with a spectrophotometer at532 nm All determinations were made in triplicate Resultswere expressed as nanomoles per mg of proteinThe concen-tration of MDA was calculated using the formula

Conc of MDA =Abs532times 100 times 119881

119879

(156 times 105

) times 119882119879times 119881119880

(2)

where Abs532

is absorbance 119881119879is total volume of mixture

(4mL) 156 times 105 is molar extinction coefficient119882119879is weight

of dissected brain (1 g) and 119881119880is aliquot volume (1mL)

2112 Superoxide Dismutase (SOD) Level SOD activity wasdetermined according to the method described by Beyer andFridovich in 1987 [26] 01mL of supernatant was mixed with01mL EDTA (1 times 10minus4M) 05mL of carbonate buffer (pH97) and 1mL of epinephrine (1mM) The optical density

of formed adrenochrome was read at 480 nm for 3minon spectrophotometer The enzyme activity was expressedin terms of Uminmg One unit of enzyme activity isdefined as the concentration required for the inhibition ofthe chromogen production by 50 in one minute under thedefined assay conditions

2113 Catalase (CAT) Level The catalase activity wasassessed by themethod ofAebi in 1974 [27]The assaymixtureconsists of 005mL of supernatant of tissue homogenate(10) and 195mL of 50mM phosphate buffer (pH 70) in3mL cuvette 1mL of 30mM hydrogen peroxide (H

2O2)

was added and changes in absorbance were followed for30 s at 240 nm at 15 s intervals The catalase activity wascalculated using the millimolar extinction coefficient ofH2O2(0071mmol cmminus1) and the activity was expressed as

micromoles of H2O2oxidized per minute per milligram

protein

CAT activity

=120575OD

119864 times Vol of Sample (mL) timesmg of protein

(3)

where 120575OD is change in absorbanceminute 119864 is extinctioncoefficient of hydrogen peroxide (0071mmol cmminus1)

2114 GSH Level (Reduced Glutathione) For the estimationof reduced glutathione the 1mL of tissue homogenate wasprecipitated with 1mL of 10 TCA To an aliquot of thesupernatant 4mL of phosphate solution and 05mL of 551015840-dithiobis-(2-nitrobenzoic acid) (DTNB) reagent were addedand absorbance was taken at 412 nm [28] The values wereexpressed as nM of reduced glutathione per mg of protein

GSH level = 119884 minus 00031400314

times119863119865

119861119879times 119881119880

(4)

where 119884 is Abs412

of tissue homogenate119863119865is dilution factor

(1) 119861119879is brain tissue homogenate (1mL) and 119881

119880is aliquot

volume (1mL)

212 Histopathological Studies The brains from control andexperimental groups were fixed with 10 formalin andembedded in paraffinwax and cut into longitudinal section of5 120583m thickness The sections were stained with hemotoxylinand eosin dye for histopathological observation

213 Statistical Analysis All the values were expressed asmean plusmn SEM Statistical evaluation of the data was done byone-way ANOVA (between control and drug treatments) fol-lowed by Dunnettrsquos 119905-test for multiple comparisons and two-way ANOVA followed by Bonferronirsquos multiple comparisontest with the level of significance chosen at 119901 lt 0001 usingGraph-Pad Prism 5 (San Diego CA) software

3 Results

31 Phytochemical Screening Table 1 showed the phytochem-ical screening of the different extract of F religiosa

Advances in Pharmacological Sciences 5

Table 1 Phytochemical investigation of F religiosa Linn leaves

Sr number Name of test Pet ether Ethyl acetate Ethanol1 Tannins and phenols minusve minusve +ve2 Alkaloids +ve +ve +ve3 Saponins minusve minusve minusve4 Terpenoids minusve +ve +ve5 Steroids minusve minusve minusve6 Flavonoids minusve +ve +ve7 Carbohydrates minusve minusve minusve8 Glycosides minusve minusve +ve9 Phlobatannins minusve minusve minusve10 Amino acid minusve minusve minusve11 Protein minusve minusve minusve

32 Acute Toxicity The PEFRE was found to be safe at all thedoses used and there was nomortality found up to the dose of4000mgkg of PEFRE when administered orally Thereforewe have taken 400mgkg as the therapeutic dose and madevariations by taking 100mgkg as lower dose and 400mgkgas higher dose

33 The Effects of PEFRE on Haloperidol Induced CatalepsyChronic oral administration of higher doses of PEFRE (200and 400mgkg) showed significant (119901 lt 0001) reduction incataleptic score from 60min to 210min as compared to vehi-cle treated animals Administration of PEFRE (100mgkg)did not show significant activity Treatment with Levodopa(6mgkg) significantly (119901 lt 0001) reduced duration ofcatalepsy as compared to vehicle treated group (Figure 1)

34 The Effects of PEFRE on 6-OHDAInduced Parkinsonrsquos Disease

341 The Effects of PEFRE on 6-OHDA Induced ParkinsonrsquosDisease in the Locomotor Activity Total locomotor activity ofrats in 6-OHDA treated group was significantly (119901 lt 0001)reduced as compared to vehicle treated group Oral admin-istration of PEFRE of different doses (200 and 400mgkg)showed significant (119901 lt 0001) increase in the locomotoractivity from day 20 to 55 as compared to 6-OHDA treatedcontrol animals Administration of PEFRE (100mgkg) didnot show significant activity Levodopa (6mgkg) signifi-cantly (119901 lt 0001) increased locomotor activity (Figure 2)

342 The Effects of PEFRE on 6-OHDA Induced ParkinsonrsquosDisease in the Rotarod Performance Treatment with 6-OHDA significantly decreased the fall of time when com-pared to the vehicle control animals Chronic oral admin-istration of PEFRE (200 and 400mgkg) significantly (119901 lt0001) increased the fall of time when compared to 6-OHDAgroup fromday 15 to day 55 Levodopa (6mgkg) significantly(119901 lt 0001) increased the fall of time as compared to 6-OHDA group Administration of PEFRE (100mgkg) did notshow significant activity (Figure 3)

IIIIII

IVV

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0

50

100

150

200

Cata

lept

ic sc

ore

9030 60 180 210120 150Time (min)

Figure 1 Effect of PEFRE on haloperidol induced catalepsy in ratsGroup I vehicle control group Group II Levodopa + haloperidoltreated group Group III PEFRE (100mgkg) + haloperidol treatedgroup Group IV PEFRE (200mgkg) + haloperidol treated groupGroup V PEFRE (400mgkg) + haloperidol treated group lowastlowastlowast119901 lt0001 as compared to Vehicle treated control group

343 The Effects of PEFRE on 6-OHDA Induced ParkinsonrsquosDisease in MDA CAT SOD and GSH Level Administrationof 6-OHDA resulted in significant changes in biochemicalparameters when compared to the vehicle control animalsThe inoculation of 6-OHDA induced oxidative stress asindicated by increased MDA level and decreased CAT SODand GSH levels when compared to vehicle control animalsin brain levels The treatment with pet ether extract ofFRE (400mgkg po) showed significant (119901 lt 0001)decrease in MDA level compared to OHDA rats Similarlydaily administration of PEFRE (400mgkg) attenuated theincrease in SOD and CAT activity with 6-OHDA treatedgroup Pretreatment with PEFRE (400mgkg) significantly(119901 lt 0001) increased GSH levels in the brain as comparedto 6-OHDA treated animals thus preventing the reductionin GSH induced by 6-OHDA (Table 2)

344 Effect of PEFRE onHistopathological Changes in the Brainof Normal and 6-OHDA Treated Animals The histopatho-logical study showed that neurotoxins that is 6-OHDAcaused marked hypertrophic changes increased intracellularspace infiltration of neutrophils decreased density of cellsalterations of architecture hemorrhage and neuronal dam-age and even cell death Furthermore many neurons wereshrunken pyknotic and darkly stained with small nuclei(Figure 4(b)) compared with normal vehicle treated rats(Figure 4(a))There is significant reversal of neuronal damageor neuronal alterations observed in Levodopa (6mgkg)treated rats (Figure 4(c)) and PEFRE treated rats at dosesof 200 (Figure 4(e)) and 400mgkg (Figure 4(f)) Treatmentwith PEFRE (100mgkg) did not show significant recovery ofneuronal damage (Figure 4(d))

6 Advances in Pharmacological Sciences

Table 2 Effect of PEFRE on the levels of lipid peroxidation (MDA) catalase (CAT) superoxide dismutase (SOD) and reduced glutathione(GSH) in the brain of 6-OHDA treated animals

Group MDA (nMmg of protein) CAT (120583moles of H2

O2

usedminmg protein) SOD (unitsmg protein) GSH (nMmg of protein)

Vehicle control 1311 plusmn 009315 5788 plusmn 0046 3185 plusmn 01852 4371 plusmn 0075766-OHDA control 2616 plusmn 01602 3463 plusmn 0035 206 plusmn 01068 3985 plusmn 0020

Levodopa 1659 plusmn 003551lowastlowast 6525 plusmn 020lowastlowastlowast 9667 plusmn 08333lowastlowastlowast 7023 plusmn 06013lowastlowastlowast

PEFRE (100) 2 plusmn 01558 3544 plusmn 015 2419 plusmn 08732 4182 plusmn 001312PEFRE (200) 1975 plusmn 02153 4234 plusmn 011 2801 plusmn 05034 4614 plusmn 01312PEFRE (400) 1347 plusmn 02501lowastlowastlowast 622 plusmn 031lowastlowastlowast 8833 plusmn 08333lowastlowastlowast 8455 plusmn 003936lowastlowastlowast

Values are expressed as mean plusmn SEM lowast119901 lt 005 lowastlowast119901 lt 001 and lowastlowastlowast119901 lt 0001 as compared to 6-OHDA treated control group (Group II) [Groups III to VIwere compared with Group II] 119901 lt 0001 as compared to vehicle treated group (Group I) [Group II was compared with Group I]

IIIIII

IVVVI

lowastlowast

lowastlowastlowast

lowastlowastlowast

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20 25 30 35 40 45 50 5515Days

Loco

mot

or ac

tivity

(cou

nts5

min

)

0

50

100

150

200

Figure 2 The effects of PEFRE on 6-OHDA induced Parkinsonrsquosdisease in the Locomotor activity Group I Vehicle control groupGroup II 6-OHDA treated group Group III Levodopa + 6-OHDAtreated group Group IV PEFRE (100mgkg) + 6-OHDA treatedgroup Group V PEFRE (200mgkg) + 6-OHDA treated groupGroup VI PEFRE (400mgkg) + 6-OHDA treated group lowastlowastlowast119901 lt0001 as compared to 6-OHDA treated negative control group(Group II) 119901 lt 0001 in 6-OHDA treated negative control group(Group II) compared to vehicle treated control group (Group I)

4 Discussion

Parkinsonrsquos disease is a chronic neurodegenerative disordercharacterized by loss of dopamine neurons of the SNpcThe pathogenesis of PD includes oxidative stress proteinaccumulation like a-synuclein mitochondrial dysfunctionapoptosis and neuronal excitotoxicity Among all oxidativestress is a crucial pathological mechanism for PD SNpc ismore vulnerable to reactive oxygen species as it containsmoreamount of dopamine

In the present study we evaluated the effect of pet etherextract of F religiosa in neurotoxins (haloperidol and 6-OHDA) induced Parkinson disease in experimental animals

Haloperidol induced catalepsy is a widely accepted ani-mal model of PD Haloperidol (nonselective D

2dopamine

IIIIII

IVVVI

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20 25 30 35 40 45 50 5515Days

0

50

100

150

Mea

n fa

ll of

tim

e (s)

Figure 3 The effects of PEFRE on 6-OHDA induced Parkinsonrsquosdisease in the rotarod performance Group I vehicle control groupGroup II 6-OHDA treated group Group III Levodopa + 6-OHDAtreated group Group IV PEFRE (100mgkg) + 6-OHDA treatedgroup Group V PEFRE (200mgkg) + 6-OHDA treated groupGroup VI PEFRE (400mgkg) + 6-OHDA treated group lowastlowastlowast119901 lt0001 as compared to 6-OHDA treated negative control group(Group II) 119901 lt 0001 6-OHDA treated negative control group(Group II) compared to vehicle treated control group (Group I)

antagonists) provides a pharmacological model of parkin-sonism by interfering with the storage of catecholaminersquosintracellularly resulting in dopamine depletion in nerveendings In the present study haloperidol (1mgkg ip)induced significant catalepsy in rats as evidenced by asignificant increase in the time spent on the block as com-pared to vehicle treated animals Treatment with F religiosasignificantly reduced the catalepsy in haloperidol treated ratsin dose dependent manner The PEFRE at doses of 200and 400mgkg showed protective effect against haloperidolinduced catalepsy indicated that this plant has an ability toprotect dopaminergic neurotransmission in striatum

There are well-known pharmacological PD models inmammalian systems including the classical and highly selec-tive neurotoxin 6-hydroxydopamine (6-OHDA) as well as 1-methyl-4-phenyl-1236-tetrahydropyridine (MPTP) and its

Advances in Pharmacological Sciences 7

(a) (b)

(c) (d)

(e) (f)

Figure 4 Effect of PEFRE on histopathological changes in the brain of normal and 6-OHDA treated animals (HampE staining originalmagnification 40x) (a) Normal control showing normal brain architecture (b) Rats treated with 6-OHDA showing degeneration of neurons(c) Rats treated with 6-OHDA and Levodopa (6mgkg) showing minimal changes in neuronal cell integrity and architecture (d) Rats treatedwith 6-OHDA and PEFRE (100mgkg) showing mild decrease in neurons and cellular hypertrophy (e) PEFRE (200mgkg) and (f) PEFRE(400mgkg) treated rats showing minimal changes in neuronal cell populations

metaboliteMPP+ (1-methyl-4-phenylpyridinium ion)Thesetoxins cause decreased ATP production increased ROSproduction and increased apoptosis of DAergic cells [29]

The efficacy of F religiosa in 6-OHDA induced PDhas notbeen well established In the present study 6-OHDA admin-istration to rats caused a significant decrease in locomotoractivity and muscle activity Lack of motor coordinationand maintenance of normal limb posture has been reportedin PD condition The evaluated data suggested damage tothe dopaminergic neurons and progression of Parkinsonrsquosdisease like behavioral abnormalities in rats exposed to 6-OHDA Pretreatment of rats with PEFRE at the doses of 200and 400mgkg exhibited significant increase in locomotoractivity and increase in muscle activity and thus could beproved with possible action on CNS

Oxidative stress generated as a result of mitochondrialdysfunction particularly mitochondrial complex-1 impair-ment plays an important role in the pathogenesis of PDThe oxidative stress was measured through determinationof levels of malondialdehyde catalase superoxide dismutaseand reduced glutathione in the brain tissue

6-OHDA generates an increase in the production ofhydrogen peroxide and free radicals [30 31] These reactiveoxygen species are generated through the nonenzymaticbreakdown of 6-OHDA or direct inhibition of complex-I and complex-IV of the mitochondrial electron transportchain [31ndash33] The resulting ROS production from 6-OHDAbreakdown leads to lipid peroxidation protein denaturationand increases in glutathione which are found in PD patients[34]

8 Advances in Pharmacological Sciences

Lipid peroxidation a sensitive marker of oxidative stresswas estimated by measuring the levels of thiobarbituricacid Lipid peroxidation occurs due to attack by radicalson double bond of unsaturated fatty acid and arachidonicacid which generate lipid peroxyl radicals and that initiatea chain reaction of further attacks on other unsaturatedfatty acid As we know lipid peroxidation is the process ofoxidative degradation of polyunsaturated fatty acids and itsoccurrence in biological membranes causes impaired mem-brane function impaired structural integrity [35] decreasedfluidity and inactivation of number of membrane boundenzymes Increased levels of the lipid peroxidation producthave been found in the substantia nigra of PD patient Inthe present investigation similar results were observed in thebrain homogenate of 6-OHDA treated control animals

Catalase is an antioxidant which helps in neutralizingthe toxic effects of hydrogen peroxide Hydrogen peroxide isconverted by the catalase enzyme to form water and nonre-active oxygen species thus preventing the accumulation ofprecursor to free radical biosynthesis Oxidative stress resultsin decrease in catalase level 6-OHDA inoculation in ratsinduced oxidative stress as indicated by a decrease in thecatalase levels

Superoxide dismutase (known as SOD) is an enzymewhich acts as a catalyst in the process of dismutation ofsuperoxide into nonreactive oxygen species and hydrogenperoxide It is therefore a critical antioxidant defense whichis present in nearly all cells which are exposed to oxygen[36 37] Superoxide dismutase helps in neutralizing the toxiceffects of free radicals [38 39] 6-OHDA treated control groupshowed a decrease in the level of SOD in the brain of animalsthus indicative of production of oxidative stress

GSH potent enzymes are an important factor in etiologyof PD [40] The depletion of reduced glutathione in thesubstantia nigra in Parkinsonrsquos disease could be the result ofneuronal loss As a matter of fact the positive correlation hasbeen found to exist between the extent of neuronal loss anddepletion of glutathione [41] A decrease in the availabilityof reduced glutathione would impair the capacity of neuronsto detoxify hydrogen peroxide and increase the risk of freeradical formation and lipid peroxidation A reduction inGSHlevels was evident in 6-OHDA treated control animals

Thus the 6-OHDA per se group showed a significantincrease in the levels of thiobarbituric acid (which is anindication of extent of lipid peroxidation) and decrease in thelevels of SODandGSH in the brain as compared to the vehicletreated control animals All these indicate an increase in theoxidative stress in the brain of animals treated with 6-OHDAPretreatmentwith higher dose of petroleumether extract ofFreligiosa (400mgkg) resulted in a decrease inMDA level andincrease in the levels of SOD catalase andGSH indicating itsantioxidant effect in the brain of 6-OHDA treated animals

Histopathological findings showed that pet ether extractof Ficus religiosa treated animals had decreased infiltrationof neutrophils reduced intracellular space increased densityof cells and regained normal architecture and moderatenecrosis in striatum region of brain

5 Conclusion

In view of the above facts we are concluding that petroleumether extract of Ficus religiosa plant showed to be an antiox-idant and showed a promising effect in animals with Parkin-sonrsquos disease And we appreciate further detailed molecularstudies with this drug in anti-Parkinsonrsquos pharmacology andtoxicology and also characterization of active constituentsresponsible for neuroprotective effect

Conflict of Interests

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

References

[1] M F Beal ldquoMitochondrial dysfunction in neurodegenerativediseasesrdquo Biochimica et Biophysica Acta (BBA)mdashBioenergeticsvol 1366 no 1-2 pp 211ndash223 1998

[2] P Jenner and C W Olanow ldquoThe pathogenesis of cell death inParkinsonrsquos diseaserdquo Neurology vol 66 no 10 supplement 4pp S24ndashS36 2006

[3] A E Lang and A M Lozano ldquoParkinsonrsquos disease second oftwo partsrdquo The New England Journal of Medicine vol 339 no16 pp 1130ndash1143 1998

[4] L Scott ldquoIdentifying poor symptom control in Parkinsonrsquosdiseaserdquo Nursing Times vol 102 no 12 pp 30ndash32 2006

[5] Y Mizuno S Ohta M Tanaka et al ldquoDeficiencies in complex-I subunits of the respiratory chain in Parkinsonrsquos diseaserdquoBiochemical and Biophysical Research Communications vol 163no 3 pp 1450ndash1455 1989

[6] W J Schmidt and M Alam ldquoControversies on new animalmodels of Parkinsonrsquos disease pro and con the rotenone modelof Parkinsonrsquos Disease (PD)rdquo Journal of Neural Transmissionvol 70 pp 273ndash276 2006

[7] T J Collier and C E Sortwell ldquoTherapeutic potential of nervegrowth factors in Parkinsonrsquos diseaserdquo Drugs and Aging vol 14no 4 pp 261ndash287 1999

[8] T B Sherer R Betarbet and J T Greenamyre ldquoEnvironmentmitochondria and Parkinsonrsquos diseaserdquo Neuroscientist vol 8no 3 pp 192ndash197 2002

[9] L Chen Y Ding B Cagniard et al ldquoUnregulated cytosolicdopamine causes neurodegeneration associated with oxidativestress in micerdquo The Journal of Neuroscience vol 28 no 2 pp425ndash433 2008

[10] F Zoccarato P Toscano andA Alexandre ldquoDopamine-deriveddopaminochrome promotes H

2

O2

release at mitochondriacomplex-1rdquo Journal of Biological Chemistry vol 280 no 16 pp15587ndash15594 2005

[11] A Ghani Medicinal Plants of Bangladesh with ChemicalConstituents and Uses Asiatic Society of Bangladesh DhakaBangladesh 1998

[12] D Singh andRKGoel ldquoAnticonvulsant effect of Ficus religiosarole of serotonergic pathwaysrdquo Journal of Ethnopharmacologyvol 123 no 2 pp 330ndash334 2009

[13] P V Prasad P K Subhaktha A Narayana and M M RaoldquoMedico-historical study of lsquoasvattharsquo (sacred fig tree)rdquo Bulletinof the Indian Institute of History of Medicine (Hyderabad) vol36 no 1 pp 1ndash20 2006

Advances in Pharmacological Sciences 9

[14] S K Panda N C Panda and B K Sahue ldquoPhytochemistryand pharmacological properties of Ficus religiosa an overviewrdquoIndian Veterinary Journal vol 60 pp 660ndash664 1976

[15] R S Verma and K S Bhatia ldquoChromatographic study of aminoacids of the leaf protein concentrates of Ficus religiosa Linn andMimusops elengi Linnrdquo Indian Journal of Pharmacy Practice vol23 pp 231ndash232 1986

[16] O A Aiyegoro and A I Okoh ldquoUse of bioactive plantproducts in combinationwith standard antibiotics implicationsin antimicrobial chemotherapyrdquo Journal of Medicinal PlantsResearch vol 3 no 13 pp 1147ndash1152 2009

[17] P K Warrier Indian Medicinal PlantsmdashA Compendium of 500Species vol 3 Orient Longman Chennai India 1996

[18] J B Harborne Phytochemical Methods Chapman and HallLondon UK 3rd edition 1998

[19] V Ravichandran B SureshMN Sathishkumar K Elango andR Srinivasan ldquoAntifertility activity of hydroalcoholic extract ofAilanthus excelsa (Roxb) an ethnomedicines used by tribals ofNilgiris region in Tamilnadurdquo Journal of Ethnopharmacologyvol 112 no 1 pp 189ndash191 2007

[20] P J Elliott and S P Close ldquoNeuroleptic-induced catalepsyas a model of Parkinsonrsquos disease I Effect of dopaminergicagentsrdquo Journal of Neural Transmission Parkinsonrsquos Disease andDementia Section vol 2 no 2 pp 79ndash89 1990

[21] R Deumens A Blokland and J Prickaerts ldquoModeling Parkin-sonrsquos disease in rats an evaluation of 6-OHDA lesions of thenigrostriatal pathwayrdquo Experimental Neurology vol 175 no 2pp 303ndash317 2002

[22] S Wang L-F Hu Y Yang J-H Ding and G Hu ldquoStudies ofATP-sensitive potassium channels on 6-hydroxydopamine andhaloperidol rat models of Parkinsonrsquos disease implications fortreating Parkinsonrsquos diseaserdquo Neuropharmacology vol 48 no7 pp 984ndash992 2005

[23] D S Reddy and S K Kulkarni ldquoPossible role of nitric oxidein the nootropic and antiamnesic effects of neurosteroids onaging- and dizocilpine-induced learning impairmentrdquo BrainResearch vol 799 no 2 pp 215ndash229 1998

[24] S RajaSankar T Manivasagam V Sankar et al ldquoWithaniasomnifera root extract improves catecholamines and physiolog-ical abnormalities seen in a Parkinsonrsquos disease model mouserdquoJournal of Ethnopharmacology vol 125 no 3 pp 369ndash373 2009

[25] H Ohkawa N Ohishi and K Yagi ldquoAssay for lipid peroxidesin animal tissues by thiobarbituric acid reactionrdquo AnalyticalBiochemistry vol 95 no 2 pp 351ndash358 1979

[26] W F Beyer Jr and I Fridovich ldquoAssaying for superoxidedismutase activity some large consequences of minor changesin conditionsrdquo Analytical Biochemistry vol 161 no 2 pp 559ndash566 1987

[27] H Aebi ldquoCatalase in vitrordquo inMethods in Enzymology vol 105pp 121ndash126 Academic Press New York NY USA 1984

[28] S K Srivastava and E Beutler ldquoAccurate measurement ofoxidized glutathione content of human rabbit and rat redblood cells and tissuesrdquoAnalytical Biochemistry vol 25 pp 70ndash76 1968

[29] F Blandini and M-T Armentero ldquoAnimal models of Parkin-sonrsquos diseaserdquo FEBS Journal vol 279 no 7 pp 1156ndash1166 2012

[30] G Cohen and R E Heikkila ldquoThe generation of hydrogenperoxide superoxide radical and hydroxyl radical by 6-hydroxydopamine dialuric acid and related cytotoxic agentsrdquoThe Journal of Biological Chemistry vol 249 no 8 pp 2447ndash2452 1974

[31] D G Graham S M Tiffany W R Bell Jr andW F GutknechtldquoAutoxidation versus covalent binding of quinones as themechanism of toxicity of dopamine 6-hydroxydopamine andrelated compounds toward C1300 neuroblastoma cells in vitrordquoMolecular Pharmacology vol 14 no 4 pp 644ndash653 1978

[32] Y Y Glinka andM B H Youdim ldquoInhibition of mitochondrialcomplexes I and IV by 6-hydroxydopaminerdquo European Journalof Pharmacology vol 292 no 3-4 pp 329ndash332 1995

[33] Y Glinka M Gassen and M B H Youdim ldquoMechanism of 6-hydroxydopamine neurotoxicityrdquo Journal of Neural Transmis-sion Supplement no 50 pp 55ndash66 1997

[34] P Jenner and C W Olanow ldquoUnderstanding cell death inParkinsonrsquos diseaserdquoAnnals of Neurology vol 44 no 3 pp S72ndashS84 1998

[35] B Halliwell and J M C Gutteridge ldquoOxygen radicals and thenervous systemrdquoTrends inNeurosciences vol 8 pp 22ndash26 1985

[36] L SMonk K V Fagerstedt and RM Crawford ldquoOxygen toxi-city and superoxide dismutase as an antioxidant in physiologicalstressrdquo Physiologia Plantarum vol 76 no 3 pp 456ndash459 1989

[37] C Bowler M Van Montagu and D Inze ldquoSuperoxide dismu-tase and stress tolerancerdquoAnnual Review of Plant Physiology andPlant Molecular Biology vol 43 no 1 pp 83ndash116 1992

[38] J G Scandalios ldquoResponse of plant antioxidant defense genesto environmental stressrdquo Advances in Genetics vol 28 pp 1ndash411990

[39] E BGralla andD J Kosman ldquoMolecular genetics of superoxidedismutases in yeasts and related fungirdquo Advances in Geneticsvol 30 pp 251ndash319 1992

[40] P Jenner ldquoOxidative mechanisms in nigral cell death in Parkin-sonrsquos diseaserdquoMovementDisorders vol 13 no 1 pp 24ndash34 1998

[41] P Riederer E Sofic W-D Rausch et al ldquoTransition metalsferritin glutathione and ascorbic acid in Parkinsonian brainsrdquoJournal of Neurochemistry vol 52 no 2 pp 515ndash520 1989

Submit your manuscripts athttpwwwhindawicom

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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

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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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

2 Advances in Pharmacological Sciences

Arayal (Malayalam) Ravi (Telugu) and Arasu (Tamil)Leaves contain campesterol stigmasterol isofucosterol 120572-amyrin lupeol tannic acid arginine serine aspartic acidglycine threonine alanine proline tryptophan tyrosinemethionine valine isoleucine leucine n-nonacosane n-hentricontane hexacosanol and n-octacosane [14 15] Ficusreligiosa has been used in traditional medicine for a widerange of ailments Its bark fruits leaves roots latex andseeds are medicinally used in different forms sometimesin combination with other herbs [16] The whole parts ofthe plant exhibit wide spectrum of activities such as anti-cancer antioxidant antidiabetic antimicrobial anticonvul-sant anthelmintic antiulcer antiasthmatic and antiamnesicactivities Bark of the plant has been used as astringentcooling aphrodisiac and antibacterial against Staphylococcusaureus and Escherichia coli gonorrhea diarrhea dysenteryhemorrhoids and gastrohelcosis as anti-inflammatory andfor burnsThe leaves of the plant have been used for hemopt-ysis epistaxis hematuria menorrhagia blood dysentery andskin diseases Leaf juice has been used for asthma coughsexual disorders diarrhea hematuria toothache migraineeye troubles gastric problems and scabies Fruits of the plantwere used in asthma and as laxative and digestive Seeds ofthe plant were used as refrigerant and laxative and latex wasused in neuralgia inflammations and hemorrhages [17] AsF religiosa has been used traditionally in the treatment ofneurodegenerative disorders (including Parkinsonrsquos disease)and has also been reported to possess antioxidant activity thisplant may prove to be effective in the remedy of PD HenceF religiosa was evaluated for its anti-Parkinsonrsquos effect usingneurotoxin induced Parkinsonrsquos model in rats

2 Materials and Methods

21 Collection of Plant Material Fresh leaves of Ficus reli-giosa were collected from local area of Ahmedabad districtGujarat India during JulyndashSeptember This plant was iden-tified and authenticated by Dr A Benniamin Scientist DBotanical Survey of India Pune Voucher specimens numberBSIWCTech2015JOB-1 have been kept in Botanical Sur-vey of India Pune Maharashtra India

22 Animals Adult male Wistar rats weighing 180ndash220 gand albino mice of either sex weighing 25ndash30 g were usedand acclimatized to laboratory condition for one week Allanimals were housed in well-ventilated polypropylene cagesat 12 12 h lightdark schedule at 25 plusmn 2∘C and 55ndash65 RHThe rats were fed with commercial pelleted rats chow andwater ad libitum as a standard diet Institutional AnimalEthics Committee approved the experimental protocol inaccordance with the Committee for the Purpose of Controland Supervision of Experiments on Animals (CPCSEA)

23 Preparation of Leaf Extract The leaveswere collected anddried in shade and ground Coarsely powdered plantmaterial(1000 g) was weighed and extracted with 5 lit of solvents likepetroleum ether (60ndash80∘C) ethyl acetate and ethanol bysuccessive extraction in a Soxhlet apparatus for 72 h After

each extraction the solvent was distilled off and concentratedextract was transferred to previously weighed petri dish andevaporated to dryness at room temperature (45ndash50∘C) toobtain dried extracts The dried extract was weighed and thepercentage yield of the extracts was calculated as follows

of extractive yield (ww)

=Weight of dried extract

Weight of dried leaves powdertimes 100

(1)

The yield of petroleum ether ethyl acetate and ethanolextract was 182 106 and 268 (ww) respectively

24 Preliminary Phytochemical Studies Preliminary qualita-tive phytochemical screening was done for the presence ofdifferent group of chemicals that is alkaloids flavonoidssaponins tannins sterols carbohydrates and glycosides asdescribed by Harborne [18]

241 Test for Tannins and Phenols 5mL of extract wasadded to 2mL of 5 of alcoholic FeCl

3solution Blue-Black

precipitate indicated the presence of tannins and phenols

242 Test for Alkaloids To the dry extract (10ndash20mg) dilutehydrochloric acid (1-2mL) was added shaken well andfiltered With filtrate the following tests were performed

(1) Mayerrsquos Test To 2-3mL of filtrate 2-3 drops of Mayerrsquosreagent were added Appearance of precipitate indicatedpresence of alkaloids

(2) Wagnerrsquos Test To 2-3mL of filtrate Wagnerrsquos (3ndash5 drops)reagent was added Appearance of reddish-brown precipitateindicated presence of alkaloids

(3) Hagerrsquos Test To 2-3mL of filtrate 4-5 drops of Hagerrsquosreagent were added Appearance of yellow precipitate indi-cated presence of alkaloids

(4) Dragendorff rsquos Test To 2-3mL of filtrate 4-5 drops ofDragendorff rsquos reagent were added Appearance of orange-brown precipitate indicated presence of alkaloids

243 Test for Saponins About 1 g of dried powdered samplewas boiled with 10mL distilled water Frothing persistenceindicated the presence of saponins

244 Test for Terpenoids 5mL of extract was mixed with2mL of chloroform and few drops of concentrated H

2SO4

were carefully added to form a layer Red ring indicated thatthe terpenoids are present

245 Test for Steroids (Liebermann-BurchardReaction) 5mLof extract was mixed with 10mL CHCl

3and 1mL acetic

anhydride and few drops of concentrated H2SO4were added

Green ring indicated the presence of steroids

Advances in Pharmacological Sciences 3

246 Test for Flavonoids (Shinoda Test) To dry extract (10ndash20mg) 5mLof ethanol (95) 2-3 drops of hydrochloric acidand 05 gmagnesium turningswere added Change of color ofsolution to pink indicated presence of flavonoids

247 Test for Carbohydrates (Molischrsquos Test) Few drops (2-3) of 120572-naphthol solution in alcohol were added to 2-3mLof solution of extract and shaken for few minutes and then05mL of conc sulfuric acid was added from the side oftest tube The formation of violet ring at the junction of twosolutions indicated presence of carbohydrates

248 Test for Glycosides

(1) Legalrsquos Test To the extract 1mL of pyridine and 1mL ofsodium nitroprusside were added Change in color to pink orred indicated presence of cardiac glycosides

(2) Keller-Kiliani Test Glacial acetic acid (3ndash5 drops) onedrop of 5 ferric chloride and concentrated sulfuric acid wasadded to the test tube containing 2mL of solution of extractAppearance of reddish-brown color at the junction of twolayers and bluish green in the upper layer indicated presenceof cardiac glycosides

(3) Borntragerrsquos Test Dilute sulfuric acid was added to 2mLof solution of extract boiled for few minutes and filtered Tothe filtrate 2mL of benzene or chloroform was added andshaken well The organic layer was separated and ammoniawas added The change in color of ammoniacal layer to pinkred indicated presence of anthraquinone glycosides

249 Test for Phlobatannins About 2mL of extract wasboiled with 2mL 1 HCl Deposition of red color indicatedthe presence of phlobatannins

2410 Test for Amino Acid (Ninhydrin Test) 5 to 6 drops ofNinhydrin reagent were added in 5mL of extract and heatedover boiling water bath for 5min Purple coloration indicatedthe presence of amino acid

2411 Test for Proteins (Biuret Test) 5-6 drops of 5 NaOHand 5ndash7 drops of 1 CuSO

4were added in 2mL of extract

Violet color indicated the presence of protein

25 Acute Oral Toxicity of the Extract Themice were dividedinto 5 groups of 10 animals each The mice were fastedfor 6 h and had access to only water ad libitum beforeexperimental study Group I received only vehicle (distilledwater) Groups II III IV and V received different doses ofpet ether extract of F religiosa (PEFRE) that is 1000 20003000 and 4000mgkg respectively All the doses and vehiclewere administered orallyThe animals were observed for 72 hfor mortality [19]

26 Haloperidol Induced Catalepsy Haloperidol causes dys-functioning of various neurotransmitters such as acetyl-choline GABA and serotonin Pathology of haloperidolinduced catalepsy underlying increased oxidative stressHaloperidol an antipsychotic drug blocks central dopaminereceptor in striatum It also produces a behavioral state inanimals like mice and rats in which they fail to correctexternally imposed postures (called catalepsy) thus keepingthe above fact in mind the haloperidol induced catalepsymodel was selected The method described by Elliott andClose in 1990 [20] was followed for the anticataleptic activityThe animals were divided into five groups (119899 = 6) GroupI served as vehicle control Group II served as standardLevodopa (6mgkg po) and Groups IIIndashV served as testgroup treated with PEFRE (100 200 and 400mgkg po)respectively Standard bar test was used to measure thecatalepsy Catalepsy was induced by haloperidol (1mgkgip) and examined at every 30min interval for 210min Theduration for which the rat retains the forepaws extended andresting on the elevated bar was considered as cataleptic scoreA cut-off time of 5min was applied

27 Induction of Parkinsonism by 6-OHDA The rats wereanesthetized with an intraperitoneal injection of 50mgkgof sodium pentobarbital and were fixed in a stereotaxicapparatus [21 22] A stainless steel needle (028mm od) wasinserted unilaterally into the substantia nigra with the follow-ing coordinates anteriorposterior minus48mm mediallateralminus22mm ventraldorsal minus72mmndash35mm from bregma andinjection of 6-OHDA (20 120583g of 6-OHDA hydrobromide in4 120583L 09 saline with 002120583gmL ascorbic acid) was thenmade over 5min and the needle was left in place for afurther 5min Then the skull was secured with stainlessmetallic screws and the wound area was covered by dentalcement Each rat was housed individually following thesurgical procedure Sham operated animals were also treatedin the same manner but they received equivalent volumes ofnormal saline instead of 6-OHDA

28 Experimental Design Animals were divided into sixgroups of 6 rats in each group Group I served as shamoperated animals and received normal saline (10mLkg po)Groups II toVIwere inducedwith parkinsonismby 6-OHDAas follows Group II served as a 6-OHDAcontrol and receivednormal saline (10mLkg) Group III served as a L DOPA(6mgkg po) and Groups IV to VI served as a test drugPEFRE (100 200 and 400mgkg po resp) The treatmentof animals was started after 48 h of induction with 6-OHDAaccording to their respective group once a day for 55 days

29 Behavioral Assessment All behavioral assessment wasperformedby an observer blinded to the groupDifferent testswere performed at different time points after lesion

291 Locomotor Activity The spontaneous locomotor activ-ity was monitored using digital actophotometer (Hiconinstrument India) equipped with infrared sensitive photo-cellsThe apparatus was placed in a darkened light and sound

4 Advances in Pharmacological Sciences

attenuated and ventilated testing room Each interruption ofa beamon the119909 or119910 axis generated an electric impulse whichwas presented on a digital counter Each animal was observedover a period of 5min on days 15 20 25 30 35 40 45 50and 55 following 6-OHDA administration and values wereexpressed as counts per 5min [23]

292 Rotarod Activity All animals were evaluated for gripstrength by using the rotarod The rotarod test is widelyused in rodents to assess their ldquominimal neurological deficitrdquosuch as motor function and coordination Each rat was givena prior training session before initialization of therapy toacclimatize them on a rotarod apparatus (EIE instrumentIndia) Animal was placed on the rotating rodwith a diameterof 7 cm (speed 25 rpm) Each rat was subjected to threeseparate trials at 5min interval on days 15 20 25 30 35 4045 50 and 55 following 6-OHDA administration and cut-offtime (180 s) was maintained throughout the experiment Theaverage results were recorded as fall of time [24]

210 Dissection and Homogenization After the treatmentperiod animals were scarified by decapitation under mildanesthesia The brains were immediately removed forebrainwas dissected out and cerebellum was discarded Brainswere put on ice and rinsed in ice-cold isotonic saline toremove blood A 10 (wv) tissue homogenate was preparedin 01M phosphate buffer (pH 74)The homogenate was cen-trifuged at 10000 g for 15minutes and aliquots of supernatantobtained were used for biochemical estimation

211 Biochemical Estimation

2111 Malondialdehyde (MDA) Level The amount of malon-dialdehyde was used as an indirect measure of lipid peroxida-tion and was determined by reaction with thiobarbituric acid(TBA) [25] Briefly 1mL of aliquots of supernatantwas placedin test tubes and added to 3mL of TBA reagent TBA 038(ww) 025M hydrochloric acid (HCl) and trichloroaceticacid (TCA 15) The solution was shaken and placed for15min followed by cooling in an ice bath After coolingsolution was centrifuged to 3500 g for 10min The upperlayer was collected and assessed with a spectrophotometer at532 nm All determinations were made in triplicate Resultswere expressed as nanomoles per mg of proteinThe concen-tration of MDA was calculated using the formula

Conc of MDA =Abs532times 100 times 119881

119879

(156 times 105

) times 119882119879times 119881119880

(2)

where Abs532

is absorbance 119881119879is total volume of mixture

(4mL) 156 times 105 is molar extinction coefficient119882119879is weight

of dissected brain (1 g) and 119881119880is aliquot volume (1mL)

2112 Superoxide Dismutase (SOD) Level SOD activity wasdetermined according to the method described by Beyer andFridovich in 1987 [26] 01mL of supernatant was mixed with01mL EDTA (1 times 10minus4M) 05mL of carbonate buffer (pH97) and 1mL of epinephrine (1mM) The optical density

of formed adrenochrome was read at 480 nm for 3minon spectrophotometer The enzyme activity was expressedin terms of Uminmg One unit of enzyme activity isdefined as the concentration required for the inhibition ofthe chromogen production by 50 in one minute under thedefined assay conditions

2113 Catalase (CAT) Level The catalase activity wasassessed by themethod ofAebi in 1974 [27]The assaymixtureconsists of 005mL of supernatant of tissue homogenate(10) and 195mL of 50mM phosphate buffer (pH 70) in3mL cuvette 1mL of 30mM hydrogen peroxide (H

2O2)

was added and changes in absorbance were followed for30 s at 240 nm at 15 s intervals The catalase activity wascalculated using the millimolar extinction coefficient ofH2O2(0071mmol cmminus1) and the activity was expressed as

micromoles of H2O2oxidized per minute per milligram

protein

CAT activity

=120575OD

119864 times Vol of Sample (mL) timesmg of protein

(3)

where 120575OD is change in absorbanceminute 119864 is extinctioncoefficient of hydrogen peroxide (0071mmol cmminus1)

2114 GSH Level (Reduced Glutathione) For the estimationof reduced glutathione the 1mL of tissue homogenate wasprecipitated with 1mL of 10 TCA To an aliquot of thesupernatant 4mL of phosphate solution and 05mL of 551015840-dithiobis-(2-nitrobenzoic acid) (DTNB) reagent were addedand absorbance was taken at 412 nm [28] The values wereexpressed as nM of reduced glutathione per mg of protein

GSH level = 119884 minus 00031400314

times119863119865

119861119879times 119881119880

(4)

where 119884 is Abs412

of tissue homogenate119863119865is dilution factor

(1) 119861119879is brain tissue homogenate (1mL) and 119881

119880is aliquot

volume (1mL)

212 Histopathological Studies The brains from control andexperimental groups were fixed with 10 formalin andembedded in paraffinwax and cut into longitudinal section of5 120583m thickness The sections were stained with hemotoxylinand eosin dye for histopathological observation

213 Statistical Analysis All the values were expressed asmean plusmn SEM Statistical evaluation of the data was done byone-way ANOVA (between control and drug treatments) fol-lowed by Dunnettrsquos 119905-test for multiple comparisons and two-way ANOVA followed by Bonferronirsquos multiple comparisontest with the level of significance chosen at 119901 lt 0001 usingGraph-Pad Prism 5 (San Diego CA) software

3 Results

31 Phytochemical Screening Table 1 showed the phytochem-ical screening of the different extract of F religiosa

Advances in Pharmacological Sciences 5

Table 1 Phytochemical investigation of F religiosa Linn leaves

Sr number Name of test Pet ether Ethyl acetate Ethanol1 Tannins and phenols minusve minusve +ve2 Alkaloids +ve +ve +ve3 Saponins minusve minusve minusve4 Terpenoids minusve +ve +ve5 Steroids minusve minusve minusve6 Flavonoids minusve +ve +ve7 Carbohydrates minusve minusve minusve8 Glycosides minusve minusve +ve9 Phlobatannins minusve minusve minusve10 Amino acid minusve minusve minusve11 Protein minusve minusve minusve

32 Acute Toxicity The PEFRE was found to be safe at all thedoses used and there was nomortality found up to the dose of4000mgkg of PEFRE when administered orally Thereforewe have taken 400mgkg as the therapeutic dose and madevariations by taking 100mgkg as lower dose and 400mgkgas higher dose

33 The Effects of PEFRE on Haloperidol Induced CatalepsyChronic oral administration of higher doses of PEFRE (200and 400mgkg) showed significant (119901 lt 0001) reduction incataleptic score from 60min to 210min as compared to vehi-cle treated animals Administration of PEFRE (100mgkg)did not show significant activity Treatment with Levodopa(6mgkg) significantly (119901 lt 0001) reduced duration ofcatalepsy as compared to vehicle treated group (Figure 1)

34 The Effects of PEFRE on 6-OHDAInduced Parkinsonrsquos Disease

341 The Effects of PEFRE on 6-OHDA Induced ParkinsonrsquosDisease in the Locomotor Activity Total locomotor activity ofrats in 6-OHDA treated group was significantly (119901 lt 0001)reduced as compared to vehicle treated group Oral admin-istration of PEFRE of different doses (200 and 400mgkg)showed significant (119901 lt 0001) increase in the locomotoractivity from day 20 to 55 as compared to 6-OHDA treatedcontrol animals Administration of PEFRE (100mgkg) didnot show significant activity Levodopa (6mgkg) signifi-cantly (119901 lt 0001) increased locomotor activity (Figure 2)

342 The Effects of PEFRE on 6-OHDA Induced ParkinsonrsquosDisease in the Rotarod Performance Treatment with 6-OHDA significantly decreased the fall of time when com-pared to the vehicle control animals Chronic oral admin-istration of PEFRE (200 and 400mgkg) significantly (119901 lt0001) increased the fall of time when compared to 6-OHDAgroup fromday 15 to day 55 Levodopa (6mgkg) significantly(119901 lt 0001) increased the fall of time as compared to 6-OHDA group Administration of PEFRE (100mgkg) did notshow significant activity (Figure 3)

IIIIII

IVV

lowastlowast

lowast

lowastlowast

lowastlowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowastlowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowastlowastlowast

lowast

0

50

100

150

200

Cata

lept

ic sc

ore

9030 60 180 210120 150Time (min)

Figure 1 Effect of PEFRE on haloperidol induced catalepsy in ratsGroup I vehicle control group Group II Levodopa + haloperidoltreated group Group III PEFRE (100mgkg) + haloperidol treatedgroup Group IV PEFRE (200mgkg) + haloperidol treated groupGroup V PEFRE (400mgkg) + haloperidol treated group lowastlowastlowast119901 lt0001 as compared to Vehicle treated control group

343 The Effects of PEFRE on 6-OHDA Induced ParkinsonrsquosDisease in MDA CAT SOD and GSH Level Administrationof 6-OHDA resulted in significant changes in biochemicalparameters when compared to the vehicle control animalsThe inoculation of 6-OHDA induced oxidative stress asindicated by increased MDA level and decreased CAT SODand GSH levels when compared to vehicle control animalsin brain levels The treatment with pet ether extract ofFRE (400mgkg po) showed significant (119901 lt 0001)decrease in MDA level compared to OHDA rats Similarlydaily administration of PEFRE (400mgkg) attenuated theincrease in SOD and CAT activity with 6-OHDA treatedgroup Pretreatment with PEFRE (400mgkg) significantly(119901 lt 0001) increased GSH levels in the brain as comparedto 6-OHDA treated animals thus preventing the reductionin GSH induced by 6-OHDA (Table 2)

344 Effect of PEFRE onHistopathological Changes in the Brainof Normal and 6-OHDA Treated Animals The histopatho-logical study showed that neurotoxins that is 6-OHDAcaused marked hypertrophic changes increased intracellularspace infiltration of neutrophils decreased density of cellsalterations of architecture hemorrhage and neuronal dam-age and even cell death Furthermore many neurons wereshrunken pyknotic and darkly stained with small nuclei(Figure 4(b)) compared with normal vehicle treated rats(Figure 4(a))There is significant reversal of neuronal damageor neuronal alterations observed in Levodopa (6mgkg)treated rats (Figure 4(c)) and PEFRE treated rats at dosesof 200 (Figure 4(e)) and 400mgkg (Figure 4(f)) Treatmentwith PEFRE (100mgkg) did not show significant recovery ofneuronal damage (Figure 4(d))

6 Advances in Pharmacological Sciences

Table 2 Effect of PEFRE on the levels of lipid peroxidation (MDA) catalase (CAT) superoxide dismutase (SOD) and reduced glutathione(GSH) in the brain of 6-OHDA treated animals

Group MDA (nMmg of protein) CAT (120583moles of H2

O2

usedminmg protein) SOD (unitsmg protein) GSH (nMmg of protein)

Vehicle control 1311 plusmn 009315 5788 plusmn 0046 3185 plusmn 01852 4371 plusmn 0075766-OHDA control 2616 plusmn 01602 3463 plusmn 0035 206 plusmn 01068 3985 plusmn 0020

Levodopa 1659 plusmn 003551lowastlowast 6525 plusmn 020lowastlowastlowast 9667 plusmn 08333lowastlowastlowast 7023 plusmn 06013lowastlowastlowast

PEFRE (100) 2 plusmn 01558 3544 plusmn 015 2419 plusmn 08732 4182 plusmn 001312PEFRE (200) 1975 plusmn 02153 4234 plusmn 011 2801 plusmn 05034 4614 plusmn 01312PEFRE (400) 1347 plusmn 02501lowastlowastlowast 622 plusmn 031lowastlowastlowast 8833 plusmn 08333lowastlowastlowast 8455 plusmn 003936lowastlowastlowast

Values are expressed as mean plusmn SEM lowast119901 lt 005 lowastlowast119901 lt 001 and lowastlowastlowast119901 lt 0001 as compared to 6-OHDA treated control group (Group II) [Groups III to VIwere compared with Group II] 119901 lt 0001 as compared to vehicle treated group (Group I) [Group II was compared with Group I]

IIIIII

IVVVI

lowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

lowastlowast

lowastlowastlowast

lowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

20 25 30 35 40 45 50 5515Days

Loco

mot

or ac

tivity

(cou

nts5

min

)

0

50

100

150

200

Figure 2 The effects of PEFRE on 6-OHDA induced Parkinsonrsquosdisease in the Locomotor activity Group I Vehicle control groupGroup II 6-OHDA treated group Group III Levodopa + 6-OHDAtreated group Group IV PEFRE (100mgkg) + 6-OHDA treatedgroup Group V PEFRE (200mgkg) + 6-OHDA treated groupGroup VI PEFRE (400mgkg) + 6-OHDA treated group lowastlowastlowast119901 lt0001 as compared to 6-OHDA treated negative control group(Group II) 119901 lt 0001 in 6-OHDA treated negative control group(Group II) compared to vehicle treated control group (Group I)

4 Discussion

Parkinsonrsquos disease is a chronic neurodegenerative disordercharacterized by loss of dopamine neurons of the SNpcThe pathogenesis of PD includes oxidative stress proteinaccumulation like a-synuclein mitochondrial dysfunctionapoptosis and neuronal excitotoxicity Among all oxidativestress is a crucial pathological mechanism for PD SNpc ismore vulnerable to reactive oxygen species as it containsmoreamount of dopamine

In the present study we evaluated the effect of pet etherextract of F religiosa in neurotoxins (haloperidol and 6-OHDA) induced Parkinson disease in experimental animals

Haloperidol induced catalepsy is a widely accepted ani-mal model of PD Haloperidol (nonselective D

2dopamine

IIIIII

IVVVI

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowastlowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowastlowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowastlowastlowast

lowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

20 25 30 35 40 45 50 5515Days

0

50

100

150

Mea

n fa

ll of

tim

e (s)

Figure 3 The effects of PEFRE on 6-OHDA induced Parkinsonrsquosdisease in the rotarod performance Group I vehicle control groupGroup II 6-OHDA treated group Group III Levodopa + 6-OHDAtreated group Group IV PEFRE (100mgkg) + 6-OHDA treatedgroup Group V PEFRE (200mgkg) + 6-OHDA treated groupGroup VI PEFRE (400mgkg) + 6-OHDA treated group lowastlowastlowast119901 lt0001 as compared to 6-OHDA treated negative control group(Group II) 119901 lt 0001 6-OHDA treated negative control group(Group II) compared to vehicle treated control group (Group I)

antagonists) provides a pharmacological model of parkin-sonism by interfering with the storage of catecholaminersquosintracellularly resulting in dopamine depletion in nerveendings In the present study haloperidol (1mgkg ip)induced significant catalepsy in rats as evidenced by asignificant increase in the time spent on the block as com-pared to vehicle treated animals Treatment with F religiosasignificantly reduced the catalepsy in haloperidol treated ratsin dose dependent manner The PEFRE at doses of 200and 400mgkg showed protective effect against haloperidolinduced catalepsy indicated that this plant has an ability toprotect dopaminergic neurotransmission in striatum

There are well-known pharmacological PD models inmammalian systems including the classical and highly selec-tive neurotoxin 6-hydroxydopamine (6-OHDA) as well as 1-methyl-4-phenyl-1236-tetrahydropyridine (MPTP) and its

Advances in Pharmacological Sciences 7

(a) (b)

(c) (d)

(e) (f)

Figure 4 Effect of PEFRE on histopathological changes in the brain of normal and 6-OHDA treated animals (HampE staining originalmagnification 40x) (a) Normal control showing normal brain architecture (b) Rats treated with 6-OHDA showing degeneration of neurons(c) Rats treated with 6-OHDA and Levodopa (6mgkg) showing minimal changes in neuronal cell integrity and architecture (d) Rats treatedwith 6-OHDA and PEFRE (100mgkg) showing mild decrease in neurons and cellular hypertrophy (e) PEFRE (200mgkg) and (f) PEFRE(400mgkg) treated rats showing minimal changes in neuronal cell populations

metaboliteMPP+ (1-methyl-4-phenylpyridinium ion)Thesetoxins cause decreased ATP production increased ROSproduction and increased apoptosis of DAergic cells [29]

The efficacy of F religiosa in 6-OHDA induced PDhas notbeen well established In the present study 6-OHDA admin-istration to rats caused a significant decrease in locomotoractivity and muscle activity Lack of motor coordinationand maintenance of normal limb posture has been reportedin PD condition The evaluated data suggested damage tothe dopaminergic neurons and progression of Parkinsonrsquosdisease like behavioral abnormalities in rats exposed to 6-OHDA Pretreatment of rats with PEFRE at the doses of 200and 400mgkg exhibited significant increase in locomotoractivity and increase in muscle activity and thus could beproved with possible action on CNS

Oxidative stress generated as a result of mitochondrialdysfunction particularly mitochondrial complex-1 impair-ment plays an important role in the pathogenesis of PDThe oxidative stress was measured through determinationof levels of malondialdehyde catalase superoxide dismutaseand reduced glutathione in the brain tissue

6-OHDA generates an increase in the production ofhydrogen peroxide and free radicals [30 31] These reactiveoxygen species are generated through the nonenzymaticbreakdown of 6-OHDA or direct inhibition of complex-I and complex-IV of the mitochondrial electron transportchain [31ndash33] The resulting ROS production from 6-OHDAbreakdown leads to lipid peroxidation protein denaturationand increases in glutathione which are found in PD patients[34]

8 Advances in Pharmacological Sciences

Lipid peroxidation a sensitive marker of oxidative stresswas estimated by measuring the levels of thiobarbituricacid Lipid peroxidation occurs due to attack by radicalson double bond of unsaturated fatty acid and arachidonicacid which generate lipid peroxyl radicals and that initiatea chain reaction of further attacks on other unsaturatedfatty acid As we know lipid peroxidation is the process ofoxidative degradation of polyunsaturated fatty acids and itsoccurrence in biological membranes causes impaired mem-brane function impaired structural integrity [35] decreasedfluidity and inactivation of number of membrane boundenzymes Increased levels of the lipid peroxidation producthave been found in the substantia nigra of PD patient Inthe present investigation similar results were observed in thebrain homogenate of 6-OHDA treated control animals

Catalase is an antioxidant which helps in neutralizingthe toxic effects of hydrogen peroxide Hydrogen peroxide isconverted by the catalase enzyme to form water and nonre-active oxygen species thus preventing the accumulation ofprecursor to free radical biosynthesis Oxidative stress resultsin decrease in catalase level 6-OHDA inoculation in ratsinduced oxidative stress as indicated by a decrease in thecatalase levels

Superoxide dismutase (known as SOD) is an enzymewhich acts as a catalyst in the process of dismutation ofsuperoxide into nonreactive oxygen species and hydrogenperoxide It is therefore a critical antioxidant defense whichis present in nearly all cells which are exposed to oxygen[36 37] Superoxide dismutase helps in neutralizing the toxiceffects of free radicals [38 39] 6-OHDA treated control groupshowed a decrease in the level of SOD in the brain of animalsthus indicative of production of oxidative stress

GSH potent enzymes are an important factor in etiologyof PD [40] The depletion of reduced glutathione in thesubstantia nigra in Parkinsonrsquos disease could be the result ofneuronal loss As a matter of fact the positive correlation hasbeen found to exist between the extent of neuronal loss anddepletion of glutathione [41] A decrease in the availabilityof reduced glutathione would impair the capacity of neuronsto detoxify hydrogen peroxide and increase the risk of freeradical formation and lipid peroxidation A reduction inGSHlevels was evident in 6-OHDA treated control animals

Thus the 6-OHDA per se group showed a significantincrease in the levels of thiobarbituric acid (which is anindication of extent of lipid peroxidation) and decrease in thelevels of SODandGSH in the brain as compared to the vehicletreated control animals All these indicate an increase in theoxidative stress in the brain of animals treated with 6-OHDAPretreatmentwith higher dose of petroleumether extract ofFreligiosa (400mgkg) resulted in a decrease inMDA level andincrease in the levels of SOD catalase andGSH indicating itsantioxidant effect in the brain of 6-OHDA treated animals

Histopathological findings showed that pet ether extractof Ficus religiosa treated animals had decreased infiltrationof neutrophils reduced intracellular space increased densityof cells and regained normal architecture and moderatenecrosis in striatum region of brain

5 Conclusion

In view of the above facts we are concluding that petroleumether extract of Ficus religiosa plant showed to be an antiox-idant and showed a promising effect in animals with Parkin-sonrsquos disease And we appreciate further detailed molecularstudies with this drug in anti-Parkinsonrsquos pharmacology andtoxicology and also characterization of active constituentsresponsible for neuroprotective effect

Conflict of Interests

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

References

[1] M F Beal ldquoMitochondrial dysfunction in neurodegenerativediseasesrdquo Biochimica et Biophysica Acta (BBA)mdashBioenergeticsvol 1366 no 1-2 pp 211ndash223 1998

[2] P Jenner and C W Olanow ldquoThe pathogenesis of cell death inParkinsonrsquos diseaserdquo Neurology vol 66 no 10 supplement 4pp S24ndashS36 2006

[3] A E Lang and A M Lozano ldquoParkinsonrsquos disease second oftwo partsrdquo The New England Journal of Medicine vol 339 no16 pp 1130ndash1143 1998

[4] L Scott ldquoIdentifying poor symptom control in Parkinsonrsquosdiseaserdquo Nursing Times vol 102 no 12 pp 30ndash32 2006

[5] Y Mizuno S Ohta M Tanaka et al ldquoDeficiencies in complex-I subunits of the respiratory chain in Parkinsonrsquos diseaserdquoBiochemical and Biophysical Research Communications vol 163no 3 pp 1450ndash1455 1989

[6] W J Schmidt and M Alam ldquoControversies on new animalmodels of Parkinsonrsquos disease pro and con the rotenone modelof Parkinsonrsquos Disease (PD)rdquo Journal of Neural Transmissionvol 70 pp 273ndash276 2006

[7] T J Collier and C E Sortwell ldquoTherapeutic potential of nervegrowth factors in Parkinsonrsquos diseaserdquo Drugs and Aging vol 14no 4 pp 261ndash287 1999

[8] T B Sherer R Betarbet and J T Greenamyre ldquoEnvironmentmitochondria and Parkinsonrsquos diseaserdquo Neuroscientist vol 8no 3 pp 192ndash197 2002

[9] L Chen Y Ding B Cagniard et al ldquoUnregulated cytosolicdopamine causes neurodegeneration associated with oxidativestress in micerdquo The Journal of Neuroscience vol 28 no 2 pp425ndash433 2008

[10] F Zoccarato P Toscano andA Alexandre ldquoDopamine-deriveddopaminochrome promotes H

2

O2

release at mitochondriacomplex-1rdquo Journal of Biological Chemistry vol 280 no 16 pp15587ndash15594 2005

[11] A Ghani Medicinal Plants of Bangladesh with ChemicalConstituents and Uses Asiatic Society of Bangladesh DhakaBangladesh 1998

[12] D Singh andRKGoel ldquoAnticonvulsant effect of Ficus religiosarole of serotonergic pathwaysrdquo Journal of Ethnopharmacologyvol 123 no 2 pp 330ndash334 2009

[13] P V Prasad P K Subhaktha A Narayana and M M RaoldquoMedico-historical study of lsquoasvattharsquo (sacred fig tree)rdquo Bulletinof the Indian Institute of History of Medicine (Hyderabad) vol36 no 1 pp 1ndash20 2006

Advances in Pharmacological Sciences 9

[14] S K Panda N C Panda and B K Sahue ldquoPhytochemistryand pharmacological properties of Ficus religiosa an overviewrdquoIndian Veterinary Journal vol 60 pp 660ndash664 1976

[15] R S Verma and K S Bhatia ldquoChromatographic study of aminoacids of the leaf protein concentrates of Ficus religiosa Linn andMimusops elengi Linnrdquo Indian Journal of Pharmacy Practice vol23 pp 231ndash232 1986

[16] O A Aiyegoro and A I Okoh ldquoUse of bioactive plantproducts in combinationwith standard antibiotics implicationsin antimicrobial chemotherapyrdquo Journal of Medicinal PlantsResearch vol 3 no 13 pp 1147ndash1152 2009

[17] P K Warrier Indian Medicinal PlantsmdashA Compendium of 500Species vol 3 Orient Longman Chennai India 1996

[18] J B Harborne Phytochemical Methods Chapman and HallLondon UK 3rd edition 1998

[19] V Ravichandran B SureshMN Sathishkumar K Elango andR Srinivasan ldquoAntifertility activity of hydroalcoholic extract ofAilanthus excelsa (Roxb) an ethnomedicines used by tribals ofNilgiris region in Tamilnadurdquo Journal of Ethnopharmacologyvol 112 no 1 pp 189ndash191 2007

[20] P J Elliott and S P Close ldquoNeuroleptic-induced catalepsyas a model of Parkinsonrsquos disease I Effect of dopaminergicagentsrdquo Journal of Neural Transmission Parkinsonrsquos Disease andDementia Section vol 2 no 2 pp 79ndash89 1990

[21] R Deumens A Blokland and J Prickaerts ldquoModeling Parkin-sonrsquos disease in rats an evaluation of 6-OHDA lesions of thenigrostriatal pathwayrdquo Experimental Neurology vol 175 no 2pp 303ndash317 2002

[22] S Wang L-F Hu Y Yang J-H Ding and G Hu ldquoStudies ofATP-sensitive potassium channels on 6-hydroxydopamine andhaloperidol rat models of Parkinsonrsquos disease implications fortreating Parkinsonrsquos diseaserdquo Neuropharmacology vol 48 no7 pp 984ndash992 2005

[23] D S Reddy and S K Kulkarni ldquoPossible role of nitric oxidein the nootropic and antiamnesic effects of neurosteroids onaging- and dizocilpine-induced learning impairmentrdquo BrainResearch vol 799 no 2 pp 215ndash229 1998

[24] S RajaSankar T Manivasagam V Sankar et al ldquoWithaniasomnifera root extract improves catecholamines and physiolog-ical abnormalities seen in a Parkinsonrsquos disease model mouserdquoJournal of Ethnopharmacology vol 125 no 3 pp 369ndash373 2009

[25] H Ohkawa N Ohishi and K Yagi ldquoAssay for lipid peroxidesin animal tissues by thiobarbituric acid reactionrdquo AnalyticalBiochemistry vol 95 no 2 pp 351ndash358 1979

[26] W F Beyer Jr and I Fridovich ldquoAssaying for superoxidedismutase activity some large consequences of minor changesin conditionsrdquo Analytical Biochemistry vol 161 no 2 pp 559ndash566 1987

[27] H Aebi ldquoCatalase in vitrordquo inMethods in Enzymology vol 105pp 121ndash126 Academic Press New York NY USA 1984

[28] S K Srivastava and E Beutler ldquoAccurate measurement ofoxidized glutathione content of human rabbit and rat redblood cells and tissuesrdquoAnalytical Biochemistry vol 25 pp 70ndash76 1968

[29] F Blandini and M-T Armentero ldquoAnimal models of Parkin-sonrsquos diseaserdquo FEBS Journal vol 279 no 7 pp 1156ndash1166 2012

[30] G Cohen and R E Heikkila ldquoThe generation of hydrogenperoxide superoxide radical and hydroxyl radical by 6-hydroxydopamine dialuric acid and related cytotoxic agentsrdquoThe Journal of Biological Chemistry vol 249 no 8 pp 2447ndash2452 1974

[31] D G Graham S M Tiffany W R Bell Jr andW F GutknechtldquoAutoxidation versus covalent binding of quinones as themechanism of toxicity of dopamine 6-hydroxydopamine andrelated compounds toward C1300 neuroblastoma cells in vitrordquoMolecular Pharmacology vol 14 no 4 pp 644ndash653 1978

[32] Y Y Glinka andM B H Youdim ldquoInhibition of mitochondrialcomplexes I and IV by 6-hydroxydopaminerdquo European Journalof Pharmacology vol 292 no 3-4 pp 329ndash332 1995

[33] Y Glinka M Gassen and M B H Youdim ldquoMechanism of 6-hydroxydopamine neurotoxicityrdquo Journal of Neural Transmis-sion Supplement no 50 pp 55ndash66 1997

[34] P Jenner and C W Olanow ldquoUnderstanding cell death inParkinsonrsquos diseaserdquoAnnals of Neurology vol 44 no 3 pp S72ndashS84 1998

[35] B Halliwell and J M C Gutteridge ldquoOxygen radicals and thenervous systemrdquoTrends inNeurosciences vol 8 pp 22ndash26 1985

[36] L SMonk K V Fagerstedt and RM Crawford ldquoOxygen toxi-city and superoxide dismutase as an antioxidant in physiologicalstressrdquo Physiologia Plantarum vol 76 no 3 pp 456ndash459 1989

[37] C Bowler M Van Montagu and D Inze ldquoSuperoxide dismu-tase and stress tolerancerdquoAnnual Review of Plant Physiology andPlant Molecular Biology vol 43 no 1 pp 83ndash116 1992

[38] J G Scandalios ldquoResponse of plant antioxidant defense genesto environmental stressrdquo Advances in Genetics vol 28 pp 1ndash411990

[39] E BGralla andD J Kosman ldquoMolecular genetics of superoxidedismutases in yeasts and related fungirdquo Advances in Geneticsvol 30 pp 251ndash319 1992

[40] P Jenner ldquoOxidative mechanisms in nigral cell death in Parkin-sonrsquos diseaserdquoMovementDisorders vol 13 no 1 pp 24ndash34 1998

[41] P Riederer E Sofic W-D Rausch et al ldquoTransition metalsferritin glutathione and ascorbic acid in Parkinsonian brainsrdquoJournal of Neurochemistry vol 52 no 2 pp 515ndash520 1989

Submit your manuscripts athttpwwwhindawicom

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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

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Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

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AntibioticsInternational Journal of

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StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Advances in Pharmacological Sciences 3

246 Test for Flavonoids (Shinoda Test) To dry extract (10ndash20mg) 5mLof ethanol (95) 2-3 drops of hydrochloric acidand 05 gmagnesium turningswere added Change of color ofsolution to pink indicated presence of flavonoids

247 Test for Carbohydrates (Molischrsquos Test) Few drops (2-3) of 120572-naphthol solution in alcohol were added to 2-3mLof solution of extract and shaken for few minutes and then05mL of conc sulfuric acid was added from the side oftest tube The formation of violet ring at the junction of twosolutions indicated presence of carbohydrates

248 Test for Glycosides

(1) Legalrsquos Test To the extract 1mL of pyridine and 1mL ofsodium nitroprusside were added Change in color to pink orred indicated presence of cardiac glycosides

(2) Keller-Kiliani Test Glacial acetic acid (3ndash5 drops) onedrop of 5 ferric chloride and concentrated sulfuric acid wasadded to the test tube containing 2mL of solution of extractAppearance of reddish-brown color at the junction of twolayers and bluish green in the upper layer indicated presenceof cardiac glycosides

(3) Borntragerrsquos Test Dilute sulfuric acid was added to 2mLof solution of extract boiled for few minutes and filtered Tothe filtrate 2mL of benzene or chloroform was added andshaken well The organic layer was separated and ammoniawas added The change in color of ammoniacal layer to pinkred indicated presence of anthraquinone glycosides

249 Test for Phlobatannins About 2mL of extract wasboiled with 2mL 1 HCl Deposition of red color indicatedthe presence of phlobatannins

2410 Test for Amino Acid (Ninhydrin Test) 5 to 6 drops ofNinhydrin reagent were added in 5mL of extract and heatedover boiling water bath for 5min Purple coloration indicatedthe presence of amino acid

2411 Test for Proteins (Biuret Test) 5-6 drops of 5 NaOHand 5ndash7 drops of 1 CuSO

4were added in 2mL of extract

Violet color indicated the presence of protein

25 Acute Oral Toxicity of the Extract Themice were dividedinto 5 groups of 10 animals each The mice were fastedfor 6 h and had access to only water ad libitum beforeexperimental study Group I received only vehicle (distilledwater) Groups II III IV and V received different doses ofpet ether extract of F religiosa (PEFRE) that is 1000 20003000 and 4000mgkg respectively All the doses and vehiclewere administered orallyThe animals were observed for 72 hfor mortality [19]

26 Haloperidol Induced Catalepsy Haloperidol causes dys-functioning of various neurotransmitters such as acetyl-choline GABA and serotonin Pathology of haloperidolinduced catalepsy underlying increased oxidative stressHaloperidol an antipsychotic drug blocks central dopaminereceptor in striatum It also produces a behavioral state inanimals like mice and rats in which they fail to correctexternally imposed postures (called catalepsy) thus keepingthe above fact in mind the haloperidol induced catalepsymodel was selected The method described by Elliott andClose in 1990 [20] was followed for the anticataleptic activityThe animals were divided into five groups (119899 = 6) GroupI served as vehicle control Group II served as standardLevodopa (6mgkg po) and Groups IIIndashV served as testgroup treated with PEFRE (100 200 and 400mgkg po)respectively Standard bar test was used to measure thecatalepsy Catalepsy was induced by haloperidol (1mgkgip) and examined at every 30min interval for 210min Theduration for which the rat retains the forepaws extended andresting on the elevated bar was considered as cataleptic scoreA cut-off time of 5min was applied

27 Induction of Parkinsonism by 6-OHDA The rats wereanesthetized with an intraperitoneal injection of 50mgkgof sodium pentobarbital and were fixed in a stereotaxicapparatus [21 22] A stainless steel needle (028mm od) wasinserted unilaterally into the substantia nigra with the follow-ing coordinates anteriorposterior minus48mm mediallateralminus22mm ventraldorsal minus72mmndash35mm from bregma andinjection of 6-OHDA (20 120583g of 6-OHDA hydrobromide in4 120583L 09 saline with 002120583gmL ascorbic acid) was thenmade over 5min and the needle was left in place for afurther 5min Then the skull was secured with stainlessmetallic screws and the wound area was covered by dentalcement Each rat was housed individually following thesurgical procedure Sham operated animals were also treatedin the same manner but they received equivalent volumes ofnormal saline instead of 6-OHDA

28 Experimental Design Animals were divided into sixgroups of 6 rats in each group Group I served as shamoperated animals and received normal saline (10mLkg po)Groups II toVIwere inducedwith parkinsonismby 6-OHDAas follows Group II served as a 6-OHDAcontrol and receivednormal saline (10mLkg) Group III served as a L DOPA(6mgkg po) and Groups IV to VI served as a test drugPEFRE (100 200 and 400mgkg po resp) The treatmentof animals was started after 48 h of induction with 6-OHDAaccording to their respective group once a day for 55 days

29 Behavioral Assessment All behavioral assessment wasperformedby an observer blinded to the groupDifferent testswere performed at different time points after lesion

291 Locomotor Activity The spontaneous locomotor activ-ity was monitored using digital actophotometer (Hiconinstrument India) equipped with infrared sensitive photo-cellsThe apparatus was placed in a darkened light and sound

4 Advances in Pharmacological Sciences

attenuated and ventilated testing room Each interruption ofa beamon the119909 or119910 axis generated an electric impulse whichwas presented on a digital counter Each animal was observedover a period of 5min on days 15 20 25 30 35 40 45 50and 55 following 6-OHDA administration and values wereexpressed as counts per 5min [23]

292 Rotarod Activity All animals were evaluated for gripstrength by using the rotarod The rotarod test is widelyused in rodents to assess their ldquominimal neurological deficitrdquosuch as motor function and coordination Each rat was givena prior training session before initialization of therapy toacclimatize them on a rotarod apparatus (EIE instrumentIndia) Animal was placed on the rotating rodwith a diameterof 7 cm (speed 25 rpm) Each rat was subjected to threeseparate trials at 5min interval on days 15 20 25 30 35 4045 50 and 55 following 6-OHDA administration and cut-offtime (180 s) was maintained throughout the experiment Theaverage results were recorded as fall of time [24]

210 Dissection and Homogenization After the treatmentperiod animals were scarified by decapitation under mildanesthesia The brains were immediately removed forebrainwas dissected out and cerebellum was discarded Brainswere put on ice and rinsed in ice-cold isotonic saline toremove blood A 10 (wv) tissue homogenate was preparedin 01M phosphate buffer (pH 74)The homogenate was cen-trifuged at 10000 g for 15minutes and aliquots of supernatantobtained were used for biochemical estimation

211 Biochemical Estimation

2111 Malondialdehyde (MDA) Level The amount of malon-dialdehyde was used as an indirect measure of lipid peroxida-tion and was determined by reaction with thiobarbituric acid(TBA) [25] Briefly 1mL of aliquots of supernatantwas placedin test tubes and added to 3mL of TBA reagent TBA 038(ww) 025M hydrochloric acid (HCl) and trichloroaceticacid (TCA 15) The solution was shaken and placed for15min followed by cooling in an ice bath After coolingsolution was centrifuged to 3500 g for 10min The upperlayer was collected and assessed with a spectrophotometer at532 nm All determinations were made in triplicate Resultswere expressed as nanomoles per mg of proteinThe concen-tration of MDA was calculated using the formula

Conc of MDA =Abs532times 100 times 119881

119879

(156 times 105

) times 119882119879times 119881119880

(2)

where Abs532

is absorbance 119881119879is total volume of mixture

(4mL) 156 times 105 is molar extinction coefficient119882119879is weight

of dissected brain (1 g) and 119881119880is aliquot volume (1mL)

2112 Superoxide Dismutase (SOD) Level SOD activity wasdetermined according to the method described by Beyer andFridovich in 1987 [26] 01mL of supernatant was mixed with01mL EDTA (1 times 10minus4M) 05mL of carbonate buffer (pH97) and 1mL of epinephrine (1mM) The optical density

of formed adrenochrome was read at 480 nm for 3minon spectrophotometer The enzyme activity was expressedin terms of Uminmg One unit of enzyme activity isdefined as the concentration required for the inhibition ofthe chromogen production by 50 in one minute under thedefined assay conditions

2113 Catalase (CAT) Level The catalase activity wasassessed by themethod ofAebi in 1974 [27]The assaymixtureconsists of 005mL of supernatant of tissue homogenate(10) and 195mL of 50mM phosphate buffer (pH 70) in3mL cuvette 1mL of 30mM hydrogen peroxide (H

2O2)

was added and changes in absorbance were followed for30 s at 240 nm at 15 s intervals The catalase activity wascalculated using the millimolar extinction coefficient ofH2O2(0071mmol cmminus1) and the activity was expressed as

micromoles of H2O2oxidized per minute per milligram

protein

CAT activity

=120575OD

119864 times Vol of Sample (mL) timesmg of protein

(3)

where 120575OD is change in absorbanceminute 119864 is extinctioncoefficient of hydrogen peroxide (0071mmol cmminus1)

2114 GSH Level (Reduced Glutathione) For the estimationof reduced glutathione the 1mL of tissue homogenate wasprecipitated with 1mL of 10 TCA To an aliquot of thesupernatant 4mL of phosphate solution and 05mL of 551015840-dithiobis-(2-nitrobenzoic acid) (DTNB) reagent were addedand absorbance was taken at 412 nm [28] The values wereexpressed as nM of reduced glutathione per mg of protein

GSH level = 119884 minus 00031400314

times119863119865

119861119879times 119881119880

(4)

where 119884 is Abs412

of tissue homogenate119863119865is dilution factor

(1) 119861119879is brain tissue homogenate (1mL) and 119881

119880is aliquot

volume (1mL)

212 Histopathological Studies The brains from control andexperimental groups were fixed with 10 formalin andembedded in paraffinwax and cut into longitudinal section of5 120583m thickness The sections were stained with hemotoxylinand eosin dye for histopathological observation

213 Statistical Analysis All the values were expressed asmean plusmn SEM Statistical evaluation of the data was done byone-way ANOVA (between control and drug treatments) fol-lowed by Dunnettrsquos 119905-test for multiple comparisons and two-way ANOVA followed by Bonferronirsquos multiple comparisontest with the level of significance chosen at 119901 lt 0001 usingGraph-Pad Prism 5 (San Diego CA) software

3 Results

31 Phytochemical Screening Table 1 showed the phytochem-ical screening of the different extract of F religiosa

Advances in Pharmacological Sciences 5

Table 1 Phytochemical investigation of F religiosa Linn leaves

Sr number Name of test Pet ether Ethyl acetate Ethanol1 Tannins and phenols minusve minusve +ve2 Alkaloids +ve +ve +ve3 Saponins minusve minusve minusve4 Terpenoids minusve +ve +ve5 Steroids minusve minusve minusve6 Flavonoids minusve +ve +ve7 Carbohydrates minusve minusve minusve8 Glycosides minusve minusve +ve9 Phlobatannins minusve minusve minusve10 Amino acid minusve minusve minusve11 Protein minusve minusve minusve

32 Acute Toxicity The PEFRE was found to be safe at all thedoses used and there was nomortality found up to the dose of4000mgkg of PEFRE when administered orally Thereforewe have taken 400mgkg as the therapeutic dose and madevariations by taking 100mgkg as lower dose and 400mgkgas higher dose

33 The Effects of PEFRE on Haloperidol Induced CatalepsyChronic oral administration of higher doses of PEFRE (200and 400mgkg) showed significant (119901 lt 0001) reduction incataleptic score from 60min to 210min as compared to vehi-cle treated animals Administration of PEFRE (100mgkg)did not show significant activity Treatment with Levodopa(6mgkg) significantly (119901 lt 0001) reduced duration ofcatalepsy as compared to vehicle treated group (Figure 1)

34 The Effects of PEFRE on 6-OHDAInduced Parkinsonrsquos Disease

341 The Effects of PEFRE on 6-OHDA Induced ParkinsonrsquosDisease in the Locomotor Activity Total locomotor activity ofrats in 6-OHDA treated group was significantly (119901 lt 0001)reduced as compared to vehicle treated group Oral admin-istration of PEFRE of different doses (200 and 400mgkg)showed significant (119901 lt 0001) increase in the locomotoractivity from day 20 to 55 as compared to 6-OHDA treatedcontrol animals Administration of PEFRE (100mgkg) didnot show significant activity Levodopa (6mgkg) signifi-cantly (119901 lt 0001) increased locomotor activity (Figure 2)

342 The Effects of PEFRE on 6-OHDA Induced ParkinsonrsquosDisease in the Rotarod Performance Treatment with 6-OHDA significantly decreased the fall of time when com-pared to the vehicle control animals Chronic oral admin-istration of PEFRE (200 and 400mgkg) significantly (119901 lt0001) increased the fall of time when compared to 6-OHDAgroup fromday 15 to day 55 Levodopa (6mgkg) significantly(119901 lt 0001) increased the fall of time as compared to 6-OHDA group Administration of PEFRE (100mgkg) did notshow significant activity (Figure 3)

IIIIII

IVV

lowastlowast

lowast

lowastlowast

lowastlowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowastlowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowastlowastlowast

lowast

0

50

100

150

200

Cata

lept

ic sc

ore

9030 60 180 210120 150Time (min)

Figure 1 Effect of PEFRE on haloperidol induced catalepsy in ratsGroup I vehicle control group Group II Levodopa + haloperidoltreated group Group III PEFRE (100mgkg) + haloperidol treatedgroup Group IV PEFRE (200mgkg) + haloperidol treated groupGroup V PEFRE (400mgkg) + haloperidol treated group lowastlowastlowast119901 lt0001 as compared to Vehicle treated control group

343 The Effects of PEFRE on 6-OHDA Induced ParkinsonrsquosDisease in MDA CAT SOD and GSH Level Administrationof 6-OHDA resulted in significant changes in biochemicalparameters when compared to the vehicle control animalsThe inoculation of 6-OHDA induced oxidative stress asindicated by increased MDA level and decreased CAT SODand GSH levels when compared to vehicle control animalsin brain levels The treatment with pet ether extract ofFRE (400mgkg po) showed significant (119901 lt 0001)decrease in MDA level compared to OHDA rats Similarlydaily administration of PEFRE (400mgkg) attenuated theincrease in SOD and CAT activity with 6-OHDA treatedgroup Pretreatment with PEFRE (400mgkg) significantly(119901 lt 0001) increased GSH levels in the brain as comparedto 6-OHDA treated animals thus preventing the reductionin GSH induced by 6-OHDA (Table 2)

344 Effect of PEFRE onHistopathological Changes in the Brainof Normal and 6-OHDA Treated Animals The histopatho-logical study showed that neurotoxins that is 6-OHDAcaused marked hypertrophic changes increased intracellularspace infiltration of neutrophils decreased density of cellsalterations of architecture hemorrhage and neuronal dam-age and even cell death Furthermore many neurons wereshrunken pyknotic and darkly stained with small nuclei(Figure 4(b)) compared with normal vehicle treated rats(Figure 4(a))There is significant reversal of neuronal damageor neuronal alterations observed in Levodopa (6mgkg)treated rats (Figure 4(c)) and PEFRE treated rats at dosesof 200 (Figure 4(e)) and 400mgkg (Figure 4(f)) Treatmentwith PEFRE (100mgkg) did not show significant recovery ofneuronal damage (Figure 4(d))

6 Advances in Pharmacological Sciences

Table 2 Effect of PEFRE on the levels of lipid peroxidation (MDA) catalase (CAT) superoxide dismutase (SOD) and reduced glutathione(GSH) in the brain of 6-OHDA treated animals

Group MDA (nMmg of protein) CAT (120583moles of H2

O2

usedminmg protein) SOD (unitsmg protein) GSH (nMmg of protein)

Vehicle control 1311 plusmn 009315 5788 plusmn 0046 3185 plusmn 01852 4371 plusmn 0075766-OHDA control 2616 plusmn 01602 3463 plusmn 0035 206 plusmn 01068 3985 plusmn 0020

Levodopa 1659 plusmn 003551lowastlowast 6525 plusmn 020lowastlowastlowast 9667 plusmn 08333lowastlowastlowast 7023 plusmn 06013lowastlowastlowast

PEFRE (100) 2 plusmn 01558 3544 plusmn 015 2419 plusmn 08732 4182 plusmn 001312PEFRE (200) 1975 plusmn 02153 4234 plusmn 011 2801 plusmn 05034 4614 plusmn 01312PEFRE (400) 1347 plusmn 02501lowastlowastlowast 622 plusmn 031lowastlowastlowast 8833 plusmn 08333lowastlowastlowast 8455 plusmn 003936lowastlowastlowast

Values are expressed as mean plusmn SEM lowast119901 lt 005 lowastlowast119901 lt 001 and lowastlowastlowast119901 lt 0001 as compared to 6-OHDA treated control group (Group II) [Groups III to VIwere compared with Group II] 119901 lt 0001 as compared to vehicle treated group (Group I) [Group II was compared with Group I]

IIIIII

IVVVI

lowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

lowastlowast

lowastlowastlowast

lowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

20 25 30 35 40 45 50 5515Days

Loco

mot

or ac

tivity

(cou

nts5

min

)

0

50

100

150

200

Figure 2 The effects of PEFRE on 6-OHDA induced Parkinsonrsquosdisease in the Locomotor activity Group I Vehicle control groupGroup II 6-OHDA treated group Group III Levodopa + 6-OHDAtreated group Group IV PEFRE (100mgkg) + 6-OHDA treatedgroup Group V PEFRE (200mgkg) + 6-OHDA treated groupGroup VI PEFRE (400mgkg) + 6-OHDA treated group lowastlowastlowast119901 lt0001 as compared to 6-OHDA treated negative control group(Group II) 119901 lt 0001 in 6-OHDA treated negative control group(Group II) compared to vehicle treated control group (Group I)

4 Discussion

Parkinsonrsquos disease is a chronic neurodegenerative disordercharacterized by loss of dopamine neurons of the SNpcThe pathogenesis of PD includes oxidative stress proteinaccumulation like a-synuclein mitochondrial dysfunctionapoptosis and neuronal excitotoxicity Among all oxidativestress is a crucial pathological mechanism for PD SNpc ismore vulnerable to reactive oxygen species as it containsmoreamount of dopamine

In the present study we evaluated the effect of pet etherextract of F religiosa in neurotoxins (haloperidol and 6-OHDA) induced Parkinson disease in experimental animals

Haloperidol induced catalepsy is a widely accepted ani-mal model of PD Haloperidol (nonselective D

2dopamine

IIIIII

IVVVI

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowastlowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowastlowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowastlowastlowast

lowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

20 25 30 35 40 45 50 5515Days

0

50

100

150

Mea

n fa

ll of

tim

e (s)

Figure 3 The effects of PEFRE on 6-OHDA induced Parkinsonrsquosdisease in the rotarod performance Group I vehicle control groupGroup II 6-OHDA treated group Group III Levodopa + 6-OHDAtreated group Group IV PEFRE (100mgkg) + 6-OHDA treatedgroup Group V PEFRE (200mgkg) + 6-OHDA treated groupGroup VI PEFRE (400mgkg) + 6-OHDA treated group lowastlowastlowast119901 lt0001 as compared to 6-OHDA treated negative control group(Group II) 119901 lt 0001 6-OHDA treated negative control group(Group II) compared to vehicle treated control group (Group I)

antagonists) provides a pharmacological model of parkin-sonism by interfering with the storage of catecholaminersquosintracellularly resulting in dopamine depletion in nerveendings In the present study haloperidol (1mgkg ip)induced significant catalepsy in rats as evidenced by asignificant increase in the time spent on the block as com-pared to vehicle treated animals Treatment with F religiosasignificantly reduced the catalepsy in haloperidol treated ratsin dose dependent manner The PEFRE at doses of 200and 400mgkg showed protective effect against haloperidolinduced catalepsy indicated that this plant has an ability toprotect dopaminergic neurotransmission in striatum

There are well-known pharmacological PD models inmammalian systems including the classical and highly selec-tive neurotoxin 6-hydroxydopamine (6-OHDA) as well as 1-methyl-4-phenyl-1236-tetrahydropyridine (MPTP) and its

Advances in Pharmacological Sciences 7

(a) (b)

(c) (d)

(e) (f)

Figure 4 Effect of PEFRE on histopathological changes in the brain of normal and 6-OHDA treated animals (HampE staining originalmagnification 40x) (a) Normal control showing normal brain architecture (b) Rats treated with 6-OHDA showing degeneration of neurons(c) Rats treated with 6-OHDA and Levodopa (6mgkg) showing minimal changes in neuronal cell integrity and architecture (d) Rats treatedwith 6-OHDA and PEFRE (100mgkg) showing mild decrease in neurons and cellular hypertrophy (e) PEFRE (200mgkg) and (f) PEFRE(400mgkg) treated rats showing minimal changes in neuronal cell populations

metaboliteMPP+ (1-methyl-4-phenylpyridinium ion)Thesetoxins cause decreased ATP production increased ROSproduction and increased apoptosis of DAergic cells [29]

The efficacy of F religiosa in 6-OHDA induced PDhas notbeen well established In the present study 6-OHDA admin-istration to rats caused a significant decrease in locomotoractivity and muscle activity Lack of motor coordinationand maintenance of normal limb posture has been reportedin PD condition The evaluated data suggested damage tothe dopaminergic neurons and progression of Parkinsonrsquosdisease like behavioral abnormalities in rats exposed to 6-OHDA Pretreatment of rats with PEFRE at the doses of 200and 400mgkg exhibited significant increase in locomotoractivity and increase in muscle activity and thus could beproved with possible action on CNS

Oxidative stress generated as a result of mitochondrialdysfunction particularly mitochondrial complex-1 impair-ment plays an important role in the pathogenesis of PDThe oxidative stress was measured through determinationof levels of malondialdehyde catalase superoxide dismutaseand reduced glutathione in the brain tissue

6-OHDA generates an increase in the production ofhydrogen peroxide and free radicals [30 31] These reactiveoxygen species are generated through the nonenzymaticbreakdown of 6-OHDA or direct inhibition of complex-I and complex-IV of the mitochondrial electron transportchain [31ndash33] The resulting ROS production from 6-OHDAbreakdown leads to lipid peroxidation protein denaturationand increases in glutathione which are found in PD patients[34]

8 Advances in Pharmacological Sciences

Lipid peroxidation a sensitive marker of oxidative stresswas estimated by measuring the levels of thiobarbituricacid Lipid peroxidation occurs due to attack by radicalson double bond of unsaturated fatty acid and arachidonicacid which generate lipid peroxyl radicals and that initiatea chain reaction of further attacks on other unsaturatedfatty acid As we know lipid peroxidation is the process ofoxidative degradation of polyunsaturated fatty acids and itsoccurrence in biological membranes causes impaired mem-brane function impaired structural integrity [35] decreasedfluidity and inactivation of number of membrane boundenzymes Increased levels of the lipid peroxidation producthave been found in the substantia nigra of PD patient Inthe present investigation similar results were observed in thebrain homogenate of 6-OHDA treated control animals

Catalase is an antioxidant which helps in neutralizingthe toxic effects of hydrogen peroxide Hydrogen peroxide isconverted by the catalase enzyme to form water and nonre-active oxygen species thus preventing the accumulation ofprecursor to free radical biosynthesis Oxidative stress resultsin decrease in catalase level 6-OHDA inoculation in ratsinduced oxidative stress as indicated by a decrease in thecatalase levels

Superoxide dismutase (known as SOD) is an enzymewhich acts as a catalyst in the process of dismutation ofsuperoxide into nonreactive oxygen species and hydrogenperoxide It is therefore a critical antioxidant defense whichis present in nearly all cells which are exposed to oxygen[36 37] Superoxide dismutase helps in neutralizing the toxiceffects of free radicals [38 39] 6-OHDA treated control groupshowed a decrease in the level of SOD in the brain of animalsthus indicative of production of oxidative stress

GSH potent enzymes are an important factor in etiologyof PD [40] The depletion of reduced glutathione in thesubstantia nigra in Parkinsonrsquos disease could be the result ofneuronal loss As a matter of fact the positive correlation hasbeen found to exist between the extent of neuronal loss anddepletion of glutathione [41] A decrease in the availabilityof reduced glutathione would impair the capacity of neuronsto detoxify hydrogen peroxide and increase the risk of freeradical formation and lipid peroxidation A reduction inGSHlevels was evident in 6-OHDA treated control animals

Thus the 6-OHDA per se group showed a significantincrease in the levels of thiobarbituric acid (which is anindication of extent of lipid peroxidation) and decrease in thelevels of SODandGSH in the brain as compared to the vehicletreated control animals All these indicate an increase in theoxidative stress in the brain of animals treated with 6-OHDAPretreatmentwith higher dose of petroleumether extract ofFreligiosa (400mgkg) resulted in a decrease inMDA level andincrease in the levels of SOD catalase andGSH indicating itsantioxidant effect in the brain of 6-OHDA treated animals

Histopathological findings showed that pet ether extractof Ficus religiosa treated animals had decreased infiltrationof neutrophils reduced intracellular space increased densityof cells and regained normal architecture and moderatenecrosis in striatum region of brain

5 Conclusion

In view of the above facts we are concluding that petroleumether extract of Ficus religiosa plant showed to be an antiox-idant and showed a promising effect in animals with Parkin-sonrsquos disease And we appreciate further detailed molecularstudies with this drug in anti-Parkinsonrsquos pharmacology andtoxicology and also characterization of active constituentsresponsible for neuroprotective effect

Conflict of Interests

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

References

[1] M F Beal ldquoMitochondrial dysfunction in neurodegenerativediseasesrdquo Biochimica et Biophysica Acta (BBA)mdashBioenergeticsvol 1366 no 1-2 pp 211ndash223 1998

[2] P Jenner and C W Olanow ldquoThe pathogenesis of cell death inParkinsonrsquos diseaserdquo Neurology vol 66 no 10 supplement 4pp S24ndashS36 2006

[3] A E Lang and A M Lozano ldquoParkinsonrsquos disease second oftwo partsrdquo The New England Journal of Medicine vol 339 no16 pp 1130ndash1143 1998

[4] L Scott ldquoIdentifying poor symptom control in Parkinsonrsquosdiseaserdquo Nursing Times vol 102 no 12 pp 30ndash32 2006

[5] Y Mizuno S Ohta M Tanaka et al ldquoDeficiencies in complex-I subunits of the respiratory chain in Parkinsonrsquos diseaserdquoBiochemical and Biophysical Research Communications vol 163no 3 pp 1450ndash1455 1989

[6] W J Schmidt and M Alam ldquoControversies on new animalmodels of Parkinsonrsquos disease pro and con the rotenone modelof Parkinsonrsquos Disease (PD)rdquo Journal of Neural Transmissionvol 70 pp 273ndash276 2006

[7] T J Collier and C E Sortwell ldquoTherapeutic potential of nervegrowth factors in Parkinsonrsquos diseaserdquo Drugs and Aging vol 14no 4 pp 261ndash287 1999

[8] T B Sherer R Betarbet and J T Greenamyre ldquoEnvironmentmitochondria and Parkinsonrsquos diseaserdquo Neuroscientist vol 8no 3 pp 192ndash197 2002

[9] L Chen Y Ding B Cagniard et al ldquoUnregulated cytosolicdopamine causes neurodegeneration associated with oxidativestress in micerdquo The Journal of Neuroscience vol 28 no 2 pp425ndash433 2008

[10] F Zoccarato P Toscano andA Alexandre ldquoDopamine-deriveddopaminochrome promotes H

2

O2

release at mitochondriacomplex-1rdquo Journal of Biological Chemistry vol 280 no 16 pp15587ndash15594 2005

[11] A Ghani Medicinal Plants of Bangladesh with ChemicalConstituents and Uses Asiatic Society of Bangladesh DhakaBangladesh 1998

[12] D Singh andRKGoel ldquoAnticonvulsant effect of Ficus religiosarole of serotonergic pathwaysrdquo Journal of Ethnopharmacologyvol 123 no 2 pp 330ndash334 2009

[13] P V Prasad P K Subhaktha A Narayana and M M RaoldquoMedico-historical study of lsquoasvattharsquo (sacred fig tree)rdquo Bulletinof the Indian Institute of History of Medicine (Hyderabad) vol36 no 1 pp 1ndash20 2006

Advances in Pharmacological Sciences 9

[14] S K Panda N C Panda and B K Sahue ldquoPhytochemistryand pharmacological properties of Ficus religiosa an overviewrdquoIndian Veterinary Journal vol 60 pp 660ndash664 1976

[15] R S Verma and K S Bhatia ldquoChromatographic study of aminoacids of the leaf protein concentrates of Ficus religiosa Linn andMimusops elengi Linnrdquo Indian Journal of Pharmacy Practice vol23 pp 231ndash232 1986

[16] O A Aiyegoro and A I Okoh ldquoUse of bioactive plantproducts in combinationwith standard antibiotics implicationsin antimicrobial chemotherapyrdquo Journal of Medicinal PlantsResearch vol 3 no 13 pp 1147ndash1152 2009

[17] P K Warrier Indian Medicinal PlantsmdashA Compendium of 500Species vol 3 Orient Longman Chennai India 1996

[18] J B Harborne Phytochemical Methods Chapman and HallLondon UK 3rd edition 1998

[19] V Ravichandran B SureshMN Sathishkumar K Elango andR Srinivasan ldquoAntifertility activity of hydroalcoholic extract ofAilanthus excelsa (Roxb) an ethnomedicines used by tribals ofNilgiris region in Tamilnadurdquo Journal of Ethnopharmacologyvol 112 no 1 pp 189ndash191 2007

[20] P J Elliott and S P Close ldquoNeuroleptic-induced catalepsyas a model of Parkinsonrsquos disease I Effect of dopaminergicagentsrdquo Journal of Neural Transmission Parkinsonrsquos Disease andDementia Section vol 2 no 2 pp 79ndash89 1990

[21] R Deumens A Blokland and J Prickaerts ldquoModeling Parkin-sonrsquos disease in rats an evaluation of 6-OHDA lesions of thenigrostriatal pathwayrdquo Experimental Neurology vol 175 no 2pp 303ndash317 2002

[22] S Wang L-F Hu Y Yang J-H Ding and G Hu ldquoStudies ofATP-sensitive potassium channels on 6-hydroxydopamine andhaloperidol rat models of Parkinsonrsquos disease implications fortreating Parkinsonrsquos diseaserdquo Neuropharmacology vol 48 no7 pp 984ndash992 2005

[23] D S Reddy and S K Kulkarni ldquoPossible role of nitric oxidein the nootropic and antiamnesic effects of neurosteroids onaging- and dizocilpine-induced learning impairmentrdquo BrainResearch vol 799 no 2 pp 215ndash229 1998

[24] S RajaSankar T Manivasagam V Sankar et al ldquoWithaniasomnifera root extract improves catecholamines and physiolog-ical abnormalities seen in a Parkinsonrsquos disease model mouserdquoJournal of Ethnopharmacology vol 125 no 3 pp 369ndash373 2009

[25] H Ohkawa N Ohishi and K Yagi ldquoAssay for lipid peroxidesin animal tissues by thiobarbituric acid reactionrdquo AnalyticalBiochemistry vol 95 no 2 pp 351ndash358 1979

[26] W F Beyer Jr and I Fridovich ldquoAssaying for superoxidedismutase activity some large consequences of minor changesin conditionsrdquo Analytical Biochemistry vol 161 no 2 pp 559ndash566 1987

[27] H Aebi ldquoCatalase in vitrordquo inMethods in Enzymology vol 105pp 121ndash126 Academic Press New York NY USA 1984

[28] S K Srivastava and E Beutler ldquoAccurate measurement ofoxidized glutathione content of human rabbit and rat redblood cells and tissuesrdquoAnalytical Biochemistry vol 25 pp 70ndash76 1968

[29] F Blandini and M-T Armentero ldquoAnimal models of Parkin-sonrsquos diseaserdquo FEBS Journal vol 279 no 7 pp 1156ndash1166 2012

[30] G Cohen and R E Heikkila ldquoThe generation of hydrogenperoxide superoxide radical and hydroxyl radical by 6-hydroxydopamine dialuric acid and related cytotoxic agentsrdquoThe Journal of Biological Chemistry vol 249 no 8 pp 2447ndash2452 1974

[31] D G Graham S M Tiffany W R Bell Jr andW F GutknechtldquoAutoxidation versus covalent binding of quinones as themechanism of toxicity of dopamine 6-hydroxydopamine andrelated compounds toward C1300 neuroblastoma cells in vitrordquoMolecular Pharmacology vol 14 no 4 pp 644ndash653 1978

[32] Y Y Glinka andM B H Youdim ldquoInhibition of mitochondrialcomplexes I and IV by 6-hydroxydopaminerdquo European Journalof Pharmacology vol 292 no 3-4 pp 329ndash332 1995

[33] Y Glinka M Gassen and M B H Youdim ldquoMechanism of 6-hydroxydopamine neurotoxicityrdquo Journal of Neural Transmis-sion Supplement no 50 pp 55ndash66 1997

[34] P Jenner and C W Olanow ldquoUnderstanding cell death inParkinsonrsquos diseaserdquoAnnals of Neurology vol 44 no 3 pp S72ndashS84 1998

[35] B Halliwell and J M C Gutteridge ldquoOxygen radicals and thenervous systemrdquoTrends inNeurosciences vol 8 pp 22ndash26 1985

[36] L SMonk K V Fagerstedt and RM Crawford ldquoOxygen toxi-city and superoxide dismutase as an antioxidant in physiologicalstressrdquo Physiologia Plantarum vol 76 no 3 pp 456ndash459 1989

[37] C Bowler M Van Montagu and D Inze ldquoSuperoxide dismu-tase and stress tolerancerdquoAnnual Review of Plant Physiology andPlant Molecular Biology vol 43 no 1 pp 83ndash116 1992

[38] J G Scandalios ldquoResponse of plant antioxidant defense genesto environmental stressrdquo Advances in Genetics vol 28 pp 1ndash411990

[39] E BGralla andD J Kosman ldquoMolecular genetics of superoxidedismutases in yeasts and related fungirdquo Advances in Geneticsvol 30 pp 251ndash319 1992

[40] P Jenner ldquoOxidative mechanisms in nigral cell death in Parkin-sonrsquos diseaserdquoMovementDisorders vol 13 no 1 pp 24ndash34 1998

[41] P Riederer E Sofic W-D Rausch et al ldquoTransition metalsferritin glutathione and ascorbic acid in Parkinsonian brainsrdquoJournal of Neurochemistry vol 52 no 2 pp 515ndash520 1989

Submit your manuscripts athttpwwwhindawicom

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MEDIATORSINFLAMMATION

of

4 Advances in Pharmacological Sciences

attenuated and ventilated testing room Each interruption ofa beamon the119909 or119910 axis generated an electric impulse whichwas presented on a digital counter Each animal was observedover a period of 5min on days 15 20 25 30 35 40 45 50and 55 following 6-OHDA administration and values wereexpressed as counts per 5min [23]

292 Rotarod Activity All animals were evaluated for gripstrength by using the rotarod The rotarod test is widelyused in rodents to assess their ldquominimal neurological deficitrdquosuch as motor function and coordination Each rat was givena prior training session before initialization of therapy toacclimatize them on a rotarod apparatus (EIE instrumentIndia) Animal was placed on the rotating rodwith a diameterof 7 cm (speed 25 rpm) Each rat was subjected to threeseparate trials at 5min interval on days 15 20 25 30 35 4045 50 and 55 following 6-OHDA administration and cut-offtime (180 s) was maintained throughout the experiment Theaverage results were recorded as fall of time [24]

210 Dissection and Homogenization After the treatmentperiod animals were scarified by decapitation under mildanesthesia The brains were immediately removed forebrainwas dissected out and cerebellum was discarded Brainswere put on ice and rinsed in ice-cold isotonic saline toremove blood A 10 (wv) tissue homogenate was preparedin 01M phosphate buffer (pH 74)The homogenate was cen-trifuged at 10000 g for 15minutes and aliquots of supernatantobtained were used for biochemical estimation

211 Biochemical Estimation

2111 Malondialdehyde (MDA) Level The amount of malon-dialdehyde was used as an indirect measure of lipid peroxida-tion and was determined by reaction with thiobarbituric acid(TBA) [25] Briefly 1mL of aliquots of supernatantwas placedin test tubes and added to 3mL of TBA reagent TBA 038(ww) 025M hydrochloric acid (HCl) and trichloroaceticacid (TCA 15) The solution was shaken and placed for15min followed by cooling in an ice bath After coolingsolution was centrifuged to 3500 g for 10min The upperlayer was collected and assessed with a spectrophotometer at532 nm All determinations were made in triplicate Resultswere expressed as nanomoles per mg of proteinThe concen-tration of MDA was calculated using the formula

Conc of MDA =Abs532times 100 times 119881

119879

(156 times 105

) times 119882119879times 119881119880

(2)

where Abs532

is absorbance 119881119879is total volume of mixture

(4mL) 156 times 105 is molar extinction coefficient119882119879is weight

of dissected brain (1 g) and 119881119880is aliquot volume (1mL)

2112 Superoxide Dismutase (SOD) Level SOD activity wasdetermined according to the method described by Beyer andFridovich in 1987 [26] 01mL of supernatant was mixed with01mL EDTA (1 times 10minus4M) 05mL of carbonate buffer (pH97) and 1mL of epinephrine (1mM) The optical density

of formed adrenochrome was read at 480 nm for 3minon spectrophotometer The enzyme activity was expressedin terms of Uminmg One unit of enzyme activity isdefined as the concentration required for the inhibition ofthe chromogen production by 50 in one minute under thedefined assay conditions

2113 Catalase (CAT) Level The catalase activity wasassessed by themethod ofAebi in 1974 [27]The assaymixtureconsists of 005mL of supernatant of tissue homogenate(10) and 195mL of 50mM phosphate buffer (pH 70) in3mL cuvette 1mL of 30mM hydrogen peroxide (H

2O2)

was added and changes in absorbance were followed for30 s at 240 nm at 15 s intervals The catalase activity wascalculated using the millimolar extinction coefficient ofH2O2(0071mmol cmminus1) and the activity was expressed as

micromoles of H2O2oxidized per minute per milligram

protein

CAT activity

=120575OD

119864 times Vol of Sample (mL) timesmg of protein

(3)

where 120575OD is change in absorbanceminute 119864 is extinctioncoefficient of hydrogen peroxide (0071mmol cmminus1)

2114 GSH Level (Reduced Glutathione) For the estimationof reduced glutathione the 1mL of tissue homogenate wasprecipitated with 1mL of 10 TCA To an aliquot of thesupernatant 4mL of phosphate solution and 05mL of 551015840-dithiobis-(2-nitrobenzoic acid) (DTNB) reagent were addedand absorbance was taken at 412 nm [28] The values wereexpressed as nM of reduced glutathione per mg of protein

GSH level = 119884 minus 00031400314

times119863119865

119861119879times 119881119880

(4)

where 119884 is Abs412

of tissue homogenate119863119865is dilution factor

(1) 119861119879is brain tissue homogenate (1mL) and 119881

119880is aliquot

volume (1mL)

212 Histopathological Studies The brains from control andexperimental groups were fixed with 10 formalin andembedded in paraffinwax and cut into longitudinal section of5 120583m thickness The sections were stained with hemotoxylinand eosin dye for histopathological observation

213 Statistical Analysis All the values were expressed asmean plusmn SEM Statistical evaluation of the data was done byone-way ANOVA (between control and drug treatments) fol-lowed by Dunnettrsquos 119905-test for multiple comparisons and two-way ANOVA followed by Bonferronirsquos multiple comparisontest with the level of significance chosen at 119901 lt 0001 usingGraph-Pad Prism 5 (San Diego CA) software

3 Results

31 Phytochemical Screening Table 1 showed the phytochem-ical screening of the different extract of F religiosa

Advances in Pharmacological Sciences 5

Table 1 Phytochemical investigation of F religiosa Linn leaves

Sr number Name of test Pet ether Ethyl acetate Ethanol1 Tannins and phenols minusve minusve +ve2 Alkaloids +ve +ve +ve3 Saponins minusve minusve minusve4 Terpenoids minusve +ve +ve5 Steroids minusve minusve minusve6 Flavonoids minusve +ve +ve7 Carbohydrates minusve minusve minusve8 Glycosides minusve minusve +ve9 Phlobatannins minusve minusve minusve10 Amino acid minusve minusve minusve11 Protein minusve minusve minusve

32 Acute Toxicity The PEFRE was found to be safe at all thedoses used and there was nomortality found up to the dose of4000mgkg of PEFRE when administered orally Thereforewe have taken 400mgkg as the therapeutic dose and madevariations by taking 100mgkg as lower dose and 400mgkgas higher dose

33 The Effects of PEFRE on Haloperidol Induced CatalepsyChronic oral administration of higher doses of PEFRE (200and 400mgkg) showed significant (119901 lt 0001) reduction incataleptic score from 60min to 210min as compared to vehi-cle treated animals Administration of PEFRE (100mgkg)did not show significant activity Treatment with Levodopa(6mgkg) significantly (119901 lt 0001) reduced duration ofcatalepsy as compared to vehicle treated group (Figure 1)

34 The Effects of PEFRE on 6-OHDAInduced Parkinsonrsquos Disease

341 The Effects of PEFRE on 6-OHDA Induced ParkinsonrsquosDisease in the Locomotor Activity Total locomotor activity ofrats in 6-OHDA treated group was significantly (119901 lt 0001)reduced as compared to vehicle treated group Oral admin-istration of PEFRE of different doses (200 and 400mgkg)showed significant (119901 lt 0001) increase in the locomotoractivity from day 20 to 55 as compared to 6-OHDA treatedcontrol animals Administration of PEFRE (100mgkg) didnot show significant activity Levodopa (6mgkg) signifi-cantly (119901 lt 0001) increased locomotor activity (Figure 2)

342 The Effects of PEFRE on 6-OHDA Induced ParkinsonrsquosDisease in the Rotarod Performance Treatment with 6-OHDA significantly decreased the fall of time when com-pared to the vehicle control animals Chronic oral admin-istration of PEFRE (200 and 400mgkg) significantly (119901 lt0001) increased the fall of time when compared to 6-OHDAgroup fromday 15 to day 55 Levodopa (6mgkg) significantly(119901 lt 0001) increased the fall of time as compared to 6-OHDA group Administration of PEFRE (100mgkg) did notshow significant activity (Figure 3)

IIIIII

IVV

lowastlowast

lowast

lowastlowast

lowastlowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowastlowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowastlowastlowast

lowast

0

50

100

150

200

Cata

lept

ic sc

ore

9030 60 180 210120 150Time (min)

Figure 1 Effect of PEFRE on haloperidol induced catalepsy in ratsGroup I vehicle control group Group II Levodopa + haloperidoltreated group Group III PEFRE (100mgkg) + haloperidol treatedgroup Group IV PEFRE (200mgkg) + haloperidol treated groupGroup V PEFRE (400mgkg) + haloperidol treated group lowastlowastlowast119901 lt0001 as compared to Vehicle treated control group

343 The Effects of PEFRE on 6-OHDA Induced ParkinsonrsquosDisease in MDA CAT SOD and GSH Level Administrationof 6-OHDA resulted in significant changes in biochemicalparameters when compared to the vehicle control animalsThe inoculation of 6-OHDA induced oxidative stress asindicated by increased MDA level and decreased CAT SODand GSH levels when compared to vehicle control animalsin brain levels The treatment with pet ether extract ofFRE (400mgkg po) showed significant (119901 lt 0001)decrease in MDA level compared to OHDA rats Similarlydaily administration of PEFRE (400mgkg) attenuated theincrease in SOD and CAT activity with 6-OHDA treatedgroup Pretreatment with PEFRE (400mgkg) significantly(119901 lt 0001) increased GSH levels in the brain as comparedto 6-OHDA treated animals thus preventing the reductionin GSH induced by 6-OHDA (Table 2)

344 Effect of PEFRE onHistopathological Changes in the Brainof Normal and 6-OHDA Treated Animals The histopatho-logical study showed that neurotoxins that is 6-OHDAcaused marked hypertrophic changes increased intracellularspace infiltration of neutrophils decreased density of cellsalterations of architecture hemorrhage and neuronal dam-age and even cell death Furthermore many neurons wereshrunken pyknotic and darkly stained with small nuclei(Figure 4(b)) compared with normal vehicle treated rats(Figure 4(a))There is significant reversal of neuronal damageor neuronal alterations observed in Levodopa (6mgkg)treated rats (Figure 4(c)) and PEFRE treated rats at dosesof 200 (Figure 4(e)) and 400mgkg (Figure 4(f)) Treatmentwith PEFRE (100mgkg) did not show significant recovery ofneuronal damage (Figure 4(d))

6 Advances in Pharmacological Sciences

Table 2 Effect of PEFRE on the levels of lipid peroxidation (MDA) catalase (CAT) superoxide dismutase (SOD) and reduced glutathione(GSH) in the brain of 6-OHDA treated animals

Group MDA (nMmg of protein) CAT (120583moles of H2

O2

usedminmg protein) SOD (unitsmg protein) GSH (nMmg of protein)

Vehicle control 1311 plusmn 009315 5788 plusmn 0046 3185 plusmn 01852 4371 plusmn 0075766-OHDA control 2616 plusmn 01602 3463 plusmn 0035 206 plusmn 01068 3985 plusmn 0020

Levodopa 1659 plusmn 003551lowastlowast 6525 plusmn 020lowastlowastlowast 9667 plusmn 08333lowastlowastlowast 7023 plusmn 06013lowastlowastlowast

PEFRE (100) 2 plusmn 01558 3544 plusmn 015 2419 plusmn 08732 4182 plusmn 001312PEFRE (200) 1975 plusmn 02153 4234 plusmn 011 2801 plusmn 05034 4614 plusmn 01312PEFRE (400) 1347 plusmn 02501lowastlowastlowast 622 plusmn 031lowastlowastlowast 8833 plusmn 08333lowastlowastlowast 8455 plusmn 003936lowastlowastlowast

Values are expressed as mean plusmn SEM lowast119901 lt 005 lowastlowast119901 lt 001 and lowastlowastlowast119901 lt 0001 as compared to 6-OHDA treated control group (Group II) [Groups III to VIwere compared with Group II] 119901 lt 0001 as compared to vehicle treated group (Group I) [Group II was compared with Group I]

IIIIII

IVVVI

lowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

lowastlowast

lowastlowastlowast

lowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

20 25 30 35 40 45 50 5515Days

Loco

mot

or ac

tivity

(cou

nts5

min

)

0

50

100

150

200

Figure 2 The effects of PEFRE on 6-OHDA induced Parkinsonrsquosdisease in the Locomotor activity Group I Vehicle control groupGroup II 6-OHDA treated group Group III Levodopa + 6-OHDAtreated group Group IV PEFRE (100mgkg) + 6-OHDA treatedgroup Group V PEFRE (200mgkg) + 6-OHDA treated groupGroup VI PEFRE (400mgkg) + 6-OHDA treated group lowastlowastlowast119901 lt0001 as compared to 6-OHDA treated negative control group(Group II) 119901 lt 0001 in 6-OHDA treated negative control group(Group II) compared to vehicle treated control group (Group I)

4 Discussion

Parkinsonrsquos disease is a chronic neurodegenerative disordercharacterized by loss of dopamine neurons of the SNpcThe pathogenesis of PD includes oxidative stress proteinaccumulation like a-synuclein mitochondrial dysfunctionapoptosis and neuronal excitotoxicity Among all oxidativestress is a crucial pathological mechanism for PD SNpc ismore vulnerable to reactive oxygen species as it containsmoreamount of dopamine

In the present study we evaluated the effect of pet etherextract of F religiosa in neurotoxins (haloperidol and 6-OHDA) induced Parkinson disease in experimental animals

Haloperidol induced catalepsy is a widely accepted ani-mal model of PD Haloperidol (nonselective D

2dopamine

IIIIII

IVVVI

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowastlowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowastlowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowastlowastlowast

lowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

20 25 30 35 40 45 50 5515Days

0

50

100

150

Mea

n fa

ll of

tim

e (s)

Figure 3 The effects of PEFRE on 6-OHDA induced Parkinsonrsquosdisease in the rotarod performance Group I vehicle control groupGroup II 6-OHDA treated group Group III Levodopa + 6-OHDAtreated group Group IV PEFRE (100mgkg) + 6-OHDA treatedgroup Group V PEFRE (200mgkg) + 6-OHDA treated groupGroup VI PEFRE (400mgkg) + 6-OHDA treated group lowastlowastlowast119901 lt0001 as compared to 6-OHDA treated negative control group(Group II) 119901 lt 0001 6-OHDA treated negative control group(Group II) compared to vehicle treated control group (Group I)

antagonists) provides a pharmacological model of parkin-sonism by interfering with the storage of catecholaminersquosintracellularly resulting in dopamine depletion in nerveendings In the present study haloperidol (1mgkg ip)induced significant catalepsy in rats as evidenced by asignificant increase in the time spent on the block as com-pared to vehicle treated animals Treatment with F religiosasignificantly reduced the catalepsy in haloperidol treated ratsin dose dependent manner The PEFRE at doses of 200and 400mgkg showed protective effect against haloperidolinduced catalepsy indicated that this plant has an ability toprotect dopaminergic neurotransmission in striatum

There are well-known pharmacological PD models inmammalian systems including the classical and highly selec-tive neurotoxin 6-hydroxydopamine (6-OHDA) as well as 1-methyl-4-phenyl-1236-tetrahydropyridine (MPTP) and its

Advances in Pharmacological Sciences 7

(a) (b)

(c) (d)

(e) (f)

Figure 4 Effect of PEFRE on histopathological changes in the brain of normal and 6-OHDA treated animals (HampE staining originalmagnification 40x) (a) Normal control showing normal brain architecture (b) Rats treated with 6-OHDA showing degeneration of neurons(c) Rats treated with 6-OHDA and Levodopa (6mgkg) showing minimal changes in neuronal cell integrity and architecture (d) Rats treatedwith 6-OHDA and PEFRE (100mgkg) showing mild decrease in neurons and cellular hypertrophy (e) PEFRE (200mgkg) and (f) PEFRE(400mgkg) treated rats showing minimal changes in neuronal cell populations

metaboliteMPP+ (1-methyl-4-phenylpyridinium ion)Thesetoxins cause decreased ATP production increased ROSproduction and increased apoptosis of DAergic cells [29]

The efficacy of F religiosa in 6-OHDA induced PDhas notbeen well established In the present study 6-OHDA admin-istration to rats caused a significant decrease in locomotoractivity and muscle activity Lack of motor coordinationand maintenance of normal limb posture has been reportedin PD condition The evaluated data suggested damage tothe dopaminergic neurons and progression of Parkinsonrsquosdisease like behavioral abnormalities in rats exposed to 6-OHDA Pretreatment of rats with PEFRE at the doses of 200and 400mgkg exhibited significant increase in locomotoractivity and increase in muscle activity and thus could beproved with possible action on CNS

Oxidative stress generated as a result of mitochondrialdysfunction particularly mitochondrial complex-1 impair-ment plays an important role in the pathogenesis of PDThe oxidative stress was measured through determinationof levels of malondialdehyde catalase superoxide dismutaseand reduced glutathione in the brain tissue

6-OHDA generates an increase in the production ofhydrogen peroxide and free radicals [30 31] These reactiveoxygen species are generated through the nonenzymaticbreakdown of 6-OHDA or direct inhibition of complex-I and complex-IV of the mitochondrial electron transportchain [31ndash33] The resulting ROS production from 6-OHDAbreakdown leads to lipid peroxidation protein denaturationand increases in glutathione which are found in PD patients[34]

8 Advances in Pharmacological Sciences

Lipid peroxidation a sensitive marker of oxidative stresswas estimated by measuring the levels of thiobarbituricacid Lipid peroxidation occurs due to attack by radicalson double bond of unsaturated fatty acid and arachidonicacid which generate lipid peroxyl radicals and that initiatea chain reaction of further attacks on other unsaturatedfatty acid As we know lipid peroxidation is the process ofoxidative degradation of polyunsaturated fatty acids and itsoccurrence in biological membranes causes impaired mem-brane function impaired structural integrity [35] decreasedfluidity and inactivation of number of membrane boundenzymes Increased levels of the lipid peroxidation producthave been found in the substantia nigra of PD patient Inthe present investigation similar results were observed in thebrain homogenate of 6-OHDA treated control animals

Catalase is an antioxidant which helps in neutralizingthe toxic effects of hydrogen peroxide Hydrogen peroxide isconverted by the catalase enzyme to form water and nonre-active oxygen species thus preventing the accumulation ofprecursor to free radical biosynthesis Oxidative stress resultsin decrease in catalase level 6-OHDA inoculation in ratsinduced oxidative stress as indicated by a decrease in thecatalase levels

Superoxide dismutase (known as SOD) is an enzymewhich acts as a catalyst in the process of dismutation ofsuperoxide into nonreactive oxygen species and hydrogenperoxide It is therefore a critical antioxidant defense whichis present in nearly all cells which are exposed to oxygen[36 37] Superoxide dismutase helps in neutralizing the toxiceffects of free radicals [38 39] 6-OHDA treated control groupshowed a decrease in the level of SOD in the brain of animalsthus indicative of production of oxidative stress

GSH potent enzymes are an important factor in etiologyof PD [40] The depletion of reduced glutathione in thesubstantia nigra in Parkinsonrsquos disease could be the result ofneuronal loss As a matter of fact the positive correlation hasbeen found to exist between the extent of neuronal loss anddepletion of glutathione [41] A decrease in the availabilityof reduced glutathione would impair the capacity of neuronsto detoxify hydrogen peroxide and increase the risk of freeradical formation and lipid peroxidation A reduction inGSHlevels was evident in 6-OHDA treated control animals

Thus the 6-OHDA per se group showed a significantincrease in the levels of thiobarbituric acid (which is anindication of extent of lipid peroxidation) and decrease in thelevels of SODandGSH in the brain as compared to the vehicletreated control animals All these indicate an increase in theoxidative stress in the brain of animals treated with 6-OHDAPretreatmentwith higher dose of petroleumether extract ofFreligiosa (400mgkg) resulted in a decrease inMDA level andincrease in the levels of SOD catalase andGSH indicating itsantioxidant effect in the brain of 6-OHDA treated animals

Histopathological findings showed that pet ether extractof Ficus religiosa treated animals had decreased infiltrationof neutrophils reduced intracellular space increased densityof cells and regained normal architecture and moderatenecrosis in striatum region of brain

5 Conclusion

In view of the above facts we are concluding that petroleumether extract of Ficus religiosa plant showed to be an antiox-idant and showed a promising effect in animals with Parkin-sonrsquos disease And we appreciate further detailed molecularstudies with this drug in anti-Parkinsonrsquos pharmacology andtoxicology and also characterization of active constituentsresponsible for neuroprotective effect

Conflict of Interests

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

References

[1] M F Beal ldquoMitochondrial dysfunction in neurodegenerativediseasesrdquo Biochimica et Biophysica Acta (BBA)mdashBioenergeticsvol 1366 no 1-2 pp 211ndash223 1998

[2] P Jenner and C W Olanow ldquoThe pathogenesis of cell death inParkinsonrsquos diseaserdquo Neurology vol 66 no 10 supplement 4pp S24ndashS36 2006

[3] A E Lang and A M Lozano ldquoParkinsonrsquos disease second oftwo partsrdquo The New England Journal of Medicine vol 339 no16 pp 1130ndash1143 1998

[4] L Scott ldquoIdentifying poor symptom control in Parkinsonrsquosdiseaserdquo Nursing Times vol 102 no 12 pp 30ndash32 2006

[5] Y Mizuno S Ohta M Tanaka et al ldquoDeficiencies in complex-I subunits of the respiratory chain in Parkinsonrsquos diseaserdquoBiochemical and Biophysical Research Communications vol 163no 3 pp 1450ndash1455 1989

[6] W J Schmidt and M Alam ldquoControversies on new animalmodels of Parkinsonrsquos disease pro and con the rotenone modelof Parkinsonrsquos Disease (PD)rdquo Journal of Neural Transmissionvol 70 pp 273ndash276 2006

[7] T J Collier and C E Sortwell ldquoTherapeutic potential of nervegrowth factors in Parkinsonrsquos diseaserdquo Drugs and Aging vol 14no 4 pp 261ndash287 1999

[8] T B Sherer R Betarbet and J T Greenamyre ldquoEnvironmentmitochondria and Parkinsonrsquos diseaserdquo Neuroscientist vol 8no 3 pp 192ndash197 2002

[9] L Chen Y Ding B Cagniard et al ldquoUnregulated cytosolicdopamine causes neurodegeneration associated with oxidativestress in micerdquo The Journal of Neuroscience vol 28 no 2 pp425ndash433 2008

[10] F Zoccarato P Toscano andA Alexandre ldquoDopamine-deriveddopaminochrome promotes H

2

O2

release at mitochondriacomplex-1rdquo Journal of Biological Chemistry vol 280 no 16 pp15587ndash15594 2005

[11] A Ghani Medicinal Plants of Bangladesh with ChemicalConstituents and Uses Asiatic Society of Bangladesh DhakaBangladesh 1998

[12] D Singh andRKGoel ldquoAnticonvulsant effect of Ficus religiosarole of serotonergic pathwaysrdquo Journal of Ethnopharmacologyvol 123 no 2 pp 330ndash334 2009

[13] P V Prasad P K Subhaktha A Narayana and M M RaoldquoMedico-historical study of lsquoasvattharsquo (sacred fig tree)rdquo Bulletinof the Indian Institute of History of Medicine (Hyderabad) vol36 no 1 pp 1ndash20 2006

Advances in Pharmacological Sciences 9

[14] S K Panda N C Panda and B K Sahue ldquoPhytochemistryand pharmacological properties of Ficus religiosa an overviewrdquoIndian Veterinary Journal vol 60 pp 660ndash664 1976

[15] R S Verma and K S Bhatia ldquoChromatographic study of aminoacids of the leaf protein concentrates of Ficus religiosa Linn andMimusops elengi Linnrdquo Indian Journal of Pharmacy Practice vol23 pp 231ndash232 1986

[16] O A Aiyegoro and A I Okoh ldquoUse of bioactive plantproducts in combinationwith standard antibiotics implicationsin antimicrobial chemotherapyrdquo Journal of Medicinal PlantsResearch vol 3 no 13 pp 1147ndash1152 2009

[17] P K Warrier Indian Medicinal PlantsmdashA Compendium of 500Species vol 3 Orient Longman Chennai India 1996

[18] J B Harborne Phytochemical Methods Chapman and HallLondon UK 3rd edition 1998

[19] V Ravichandran B SureshMN Sathishkumar K Elango andR Srinivasan ldquoAntifertility activity of hydroalcoholic extract ofAilanthus excelsa (Roxb) an ethnomedicines used by tribals ofNilgiris region in Tamilnadurdquo Journal of Ethnopharmacologyvol 112 no 1 pp 189ndash191 2007

[20] P J Elliott and S P Close ldquoNeuroleptic-induced catalepsyas a model of Parkinsonrsquos disease I Effect of dopaminergicagentsrdquo Journal of Neural Transmission Parkinsonrsquos Disease andDementia Section vol 2 no 2 pp 79ndash89 1990

[21] R Deumens A Blokland and J Prickaerts ldquoModeling Parkin-sonrsquos disease in rats an evaluation of 6-OHDA lesions of thenigrostriatal pathwayrdquo Experimental Neurology vol 175 no 2pp 303ndash317 2002

[22] S Wang L-F Hu Y Yang J-H Ding and G Hu ldquoStudies ofATP-sensitive potassium channels on 6-hydroxydopamine andhaloperidol rat models of Parkinsonrsquos disease implications fortreating Parkinsonrsquos diseaserdquo Neuropharmacology vol 48 no7 pp 984ndash992 2005

[23] D S Reddy and S K Kulkarni ldquoPossible role of nitric oxidein the nootropic and antiamnesic effects of neurosteroids onaging- and dizocilpine-induced learning impairmentrdquo BrainResearch vol 799 no 2 pp 215ndash229 1998

[24] S RajaSankar T Manivasagam V Sankar et al ldquoWithaniasomnifera root extract improves catecholamines and physiolog-ical abnormalities seen in a Parkinsonrsquos disease model mouserdquoJournal of Ethnopharmacology vol 125 no 3 pp 369ndash373 2009

[25] H Ohkawa N Ohishi and K Yagi ldquoAssay for lipid peroxidesin animal tissues by thiobarbituric acid reactionrdquo AnalyticalBiochemistry vol 95 no 2 pp 351ndash358 1979

[26] W F Beyer Jr and I Fridovich ldquoAssaying for superoxidedismutase activity some large consequences of minor changesin conditionsrdquo Analytical Biochemistry vol 161 no 2 pp 559ndash566 1987

[27] H Aebi ldquoCatalase in vitrordquo inMethods in Enzymology vol 105pp 121ndash126 Academic Press New York NY USA 1984

[28] S K Srivastava and E Beutler ldquoAccurate measurement ofoxidized glutathione content of human rabbit and rat redblood cells and tissuesrdquoAnalytical Biochemistry vol 25 pp 70ndash76 1968

[29] F Blandini and M-T Armentero ldquoAnimal models of Parkin-sonrsquos diseaserdquo FEBS Journal vol 279 no 7 pp 1156ndash1166 2012

[30] G Cohen and R E Heikkila ldquoThe generation of hydrogenperoxide superoxide radical and hydroxyl radical by 6-hydroxydopamine dialuric acid and related cytotoxic agentsrdquoThe Journal of Biological Chemistry vol 249 no 8 pp 2447ndash2452 1974

[31] D G Graham S M Tiffany W R Bell Jr andW F GutknechtldquoAutoxidation versus covalent binding of quinones as themechanism of toxicity of dopamine 6-hydroxydopamine andrelated compounds toward C1300 neuroblastoma cells in vitrordquoMolecular Pharmacology vol 14 no 4 pp 644ndash653 1978

[32] Y Y Glinka andM B H Youdim ldquoInhibition of mitochondrialcomplexes I and IV by 6-hydroxydopaminerdquo European Journalof Pharmacology vol 292 no 3-4 pp 329ndash332 1995

[33] Y Glinka M Gassen and M B H Youdim ldquoMechanism of 6-hydroxydopamine neurotoxicityrdquo Journal of Neural Transmis-sion Supplement no 50 pp 55ndash66 1997

[34] P Jenner and C W Olanow ldquoUnderstanding cell death inParkinsonrsquos diseaserdquoAnnals of Neurology vol 44 no 3 pp S72ndashS84 1998

[35] B Halliwell and J M C Gutteridge ldquoOxygen radicals and thenervous systemrdquoTrends inNeurosciences vol 8 pp 22ndash26 1985

[36] L SMonk K V Fagerstedt and RM Crawford ldquoOxygen toxi-city and superoxide dismutase as an antioxidant in physiologicalstressrdquo Physiologia Plantarum vol 76 no 3 pp 456ndash459 1989

[37] C Bowler M Van Montagu and D Inze ldquoSuperoxide dismu-tase and stress tolerancerdquoAnnual Review of Plant Physiology andPlant Molecular Biology vol 43 no 1 pp 83ndash116 1992

[38] J G Scandalios ldquoResponse of plant antioxidant defense genesto environmental stressrdquo Advances in Genetics vol 28 pp 1ndash411990

[39] E BGralla andD J Kosman ldquoMolecular genetics of superoxidedismutases in yeasts and related fungirdquo Advances in Geneticsvol 30 pp 251ndash319 1992

[40] P Jenner ldquoOxidative mechanisms in nigral cell death in Parkin-sonrsquos diseaserdquoMovementDisorders vol 13 no 1 pp 24ndash34 1998

[41] P Riederer E Sofic W-D Rausch et al ldquoTransition metalsferritin glutathione and ascorbic acid in Parkinsonian brainsrdquoJournal of Neurochemistry vol 52 no 2 pp 515ndash520 1989

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Advances in Pharmacological Sciences 5

Table 1 Phytochemical investigation of F religiosa Linn leaves

Sr number Name of test Pet ether Ethyl acetate Ethanol1 Tannins and phenols minusve minusve +ve2 Alkaloids +ve +ve +ve3 Saponins minusve minusve minusve4 Terpenoids minusve +ve +ve5 Steroids minusve minusve minusve6 Flavonoids minusve +ve +ve7 Carbohydrates minusve minusve minusve8 Glycosides minusve minusve +ve9 Phlobatannins minusve minusve minusve10 Amino acid minusve minusve minusve11 Protein minusve minusve minusve

32 Acute Toxicity The PEFRE was found to be safe at all thedoses used and there was nomortality found up to the dose of4000mgkg of PEFRE when administered orally Thereforewe have taken 400mgkg as the therapeutic dose and madevariations by taking 100mgkg as lower dose and 400mgkgas higher dose

33 The Effects of PEFRE on Haloperidol Induced CatalepsyChronic oral administration of higher doses of PEFRE (200and 400mgkg) showed significant (119901 lt 0001) reduction incataleptic score from 60min to 210min as compared to vehi-cle treated animals Administration of PEFRE (100mgkg)did not show significant activity Treatment with Levodopa(6mgkg) significantly (119901 lt 0001) reduced duration ofcatalepsy as compared to vehicle treated group (Figure 1)

34 The Effects of PEFRE on 6-OHDAInduced Parkinsonrsquos Disease

341 The Effects of PEFRE on 6-OHDA Induced ParkinsonrsquosDisease in the Locomotor Activity Total locomotor activity ofrats in 6-OHDA treated group was significantly (119901 lt 0001)reduced as compared to vehicle treated group Oral admin-istration of PEFRE of different doses (200 and 400mgkg)showed significant (119901 lt 0001) increase in the locomotoractivity from day 20 to 55 as compared to 6-OHDA treatedcontrol animals Administration of PEFRE (100mgkg) didnot show significant activity Levodopa (6mgkg) signifi-cantly (119901 lt 0001) increased locomotor activity (Figure 2)

342 The Effects of PEFRE on 6-OHDA Induced ParkinsonrsquosDisease in the Rotarod Performance Treatment with 6-OHDA significantly decreased the fall of time when com-pared to the vehicle control animals Chronic oral admin-istration of PEFRE (200 and 400mgkg) significantly (119901 lt0001) increased the fall of time when compared to 6-OHDAgroup fromday 15 to day 55 Levodopa (6mgkg) significantly(119901 lt 0001) increased the fall of time as compared to 6-OHDA group Administration of PEFRE (100mgkg) did notshow significant activity (Figure 3)

IIIIII

IVV

lowastlowast

lowast

lowastlowast

lowastlowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowastlowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowastlowastlowast

lowast

0

50

100

150

200

Cata

lept

ic sc

ore

9030 60 180 210120 150Time (min)

Figure 1 Effect of PEFRE on haloperidol induced catalepsy in ratsGroup I vehicle control group Group II Levodopa + haloperidoltreated group Group III PEFRE (100mgkg) + haloperidol treatedgroup Group IV PEFRE (200mgkg) + haloperidol treated groupGroup V PEFRE (400mgkg) + haloperidol treated group lowastlowastlowast119901 lt0001 as compared to Vehicle treated control group

343 The Effects of PEFRE on 6-OHDA Induced ParkinsonrsquosDisease in MDA CAT SOD and GSH Level Administrationof 6-OHDA resulted in significant changes in biochemicalparameters when compared to the vehicle control animalsThe inoculation of 6-OHDA induced oxidative stress asindicated by increased MDA level and decreased CAT SODand GSH levels when compared to vehicle control animalsin brain levels The treatment with pet ether extract ofFRE (400mgkg po) showed significant (119901 lt 0001)decrease in MDA level compared to OHDA rats Similarlydaily administration of PEFRE (400mgkg) attenuated theincrease in SOD and CAT activity with 6-OHDA treatedgroup Pretreatment with PEFRE (400mgkg) significantly(119901 lt 0001) increased GSH levels in the brain as comparedto 6-OHDA treated animals thus preventing the reductionin GSH induced by 6-OHDA (Table 2)

344 Effect of PEFRE onHistopathological Changes in the Brainof Normal and 6-OHDA Treated Animals The histopatho-logical study showed that neurotoxins that is 6-OHDAcaused marked hypertrophic changes increased intracellularspace infiltration of neutrophils decreased density of cellsalterations of architecture hemorrhage and neuronal dam-age and even cell death Furthermore many neurons wereshrunken pyknotic and darkly stained with small nuclei(Figure 4(b)) compared with normal vehicle treated rats(Figure 4(a))There is significant reversal of neuronal damageor neuronal alterations observed in Levodopa (6mgkg)treated rats (Figure 4(c)) and PEFRE treated rats at dosesof 200 (Figure 4(e)) and 400mgkg (Figure 4(f)) Treatmentwith PEFRE (100mgkg) did not show significant recovery ofneuronal damage (Figure 4(d))

6 Advances in Pharmacological Sciences

Table 2 Effect of PEFRE on the levels of lipid peroxidation (MDA) catalase (CAT) superoxide dismutase (SOD) and reduced glutathione(GSH) in the brain of 6-OHDA treated animals

Group MDA (nMmg of protein) CAT (120583moles of H2

O2

usedminmg protein) SOD (unitsmg protein) GSH (nMmg of protein)

Vehicle control 1311 plusmn 009315 5788 plusmn 0046 3185 plusmn 01852 4371 plusmn 0075766-OHDA control 2616 plusmn 01602 3463 plusmn 0035 206 plusmn 01068 3985 plusmn 0020

Levodopa 1659 plusmn 003551lowastlowast 6525 plusmn 020lowastlowastlowast 9667 plusmn 08333lowastlowastlowast 7023 plusmn 06013lowastlowastlowast

PEFRE (100) 2 plusmn 01558 3544 plusmn 015 2419 plusmn 08732 4182 plusmn 001312PEFRE (200) 1975 plusmn 02153 4234 plusmn 011 2801 plusmn 05034 4614 plusmn 01312PEFRE (400) 1347 plusmn 02501lowastlowastlowast 622 plusmn 031lowastlowastlowast 8833 plusmn 08333lowastlowastlowast 8455 plusmn 003936lowastlowastlowast

Values are expressed as mean plusmn SEM lowast119901 lt 005 lowastlowast119901 lt 001 and lowastlowastlowast119901 lt 0001 as compared to 6-OHDA treated control group (Group II) [Groups III to VIwere compared with Group II] 119901 lt 0001 as compared to vehicle treated group (Group I) [Group II was compared with Group I]

IIIIII

IVVVI

lowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

lowastlowast

lowastlowastlowast

lowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

20 25 30 35 40 45 50 5515Days

Loco

mot

or ac

tivity

(cou

nts5

min

)

0

50

100

150

200

Figure 2 The effects of PEFRE on 6-OHDA induced Parkinsonrsquosdisease in the Locomotor activity Group I Vehicle control groupGroup II 6-OHDA treated group Group III Levodopa + 6-OHDAtreated group Group IV PEFRE (100mgkg) + 6-OHDA treatedgroup Group V PEFRE (200mgkg) + 6-OHDA treated groupGroup VI PEFRE (400mgkg) + 6-OHDA treated group lowastlowastlowast119901 lt0001 as compared to 6-OHDA treated negative control group(Group II) 119901 lt 0001 in 6-OHDA treated negative control group(Group II) compared to vehicle treated control group (Group I)

4 Discussion

Parkinsonrsquos disease is a chronic neurodegenerative disordercharacterized by loss of dopamine neurons of the SNpcThe pathogenesis of PD includes oxidative stress proteinaccumulation like a-synuclein mitochondrial dysfunctionapoptosis and neuronal excitotoxicity Among all oxidativestress is a crucial pathological mechanism for PD SNpc ismore vulnerable to reactive oxygen species as it containsmoreamount of dopamine

In the present study we evaluated the effect of pet etherextract of F religiosa in neurotoxins (haloperidol and 6-OHDA) induced Parkinson disease in experimental animals

Haloperidol induced catalepsy is a widely accepted ani-mal model of PD Haloperidol (nonselective D

2dopamine

IIIIII

IVVVI

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowastlowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowastlowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowastlowastlowast

lowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

20 25 30 35 40 45 50 5515Days

0

50

100

150

Mea

n fa

ll of

tim

e (s)

Figure 3 The effects of PEFRE on 6-OHDA induced Parkinsonrsquosdisease in the rotarod performance Group I vehicle control groupGroup II 6-OHDA treated group Group III Levodopa + 6-OHDAtreated group Group IV PEFRE (100mgkg) + 6-OHDA treatedgroup Group V PEFRE (200mgkg) + 6-OHDA treated groupGroup VI PEFRE (400mgkg) + 6-OHDA treated group lowastlowastlowast119901 lt0001 as compared to 6-OHDA treated negative control group(Group II) 119901 lt 0001 6-OHDA treated negative control group(Group II) compared to vehicle treated control group (Group I)

antagonists) provides a pharmacological model of parkin-sonism by interfering with the storage of catecholaminersquosintracellularly resulting in dopamine depletion in nerveendings In the present study haloperidol (1mgkg ip)induced significant catalepsy in rats as evidenced by asignificant increase in the time spent on the block as com-pared to vehicle treated animals Treatment with F religiosasignificantly reduced the catalepsy in haloperidol treated ratsin dose dependent manner The PEFRE at doses of 200and 400mgkg showed protective effect against haloperidolinduced catalepsy indicated that this plant has an ability toprotect dopaminergic neurotransmission in striatum

There are well-known pharmacological PD models inmammalian systems including the classical and highly selec-tive neurotoxin 6-hydroxydopamine (6-OHDA) as well as 1-methyl-4-phenyl-1236-tetrahydropyridine (MPTP) and its

Advances in Pharmacological Sciences 7

(a) (b)

(c) (d)

(e) (f)

Figure 4 Effect of PEFRE on histopathological changes in the brain of normal and 6-OHDA treated animals (HampE staining originalmagnification 40x) (a) Normal control showing normal brain architecture (b) Rats treated with 6-OHDA showing degeneration of neurons(c) Rats treated with 6-OHDA and Levodopa (6mgkg) showing minimal changes in neuronal cell integrity and architecture (d) Rats treatedwith 6-OHDA and PEFRE (100mgkg) showing mild decrease in neurons and cellular hypertrophy (e) PEFRE (200mgkg) and (f) PEFRE(400mgkg) treated rats showing minimal changes in neuronal cell populations

metaboliteMPP+ (1-methyl-4-phenylpyridinium ion)Thesetoxins cause decreased ATP production increased ROSproduction and increased apoptosis of DAergic cells [29]

The efficacy of F religiosa in 6-OHDA induced PDhas notbeen well established In the present study 6-OHDA admin-istration to rats caused a significant decrease in locomotoractivity and muscle activity Lack of motor coordinationand maintenance of normal limb posture has been reportedin PD condition The evaluated data suggested damage tothe dopaminergic neurons and progression of Parkinsonrsquosdisease like behavioral abnormalities in rats exposed to 6-OHDA Pretreatment of rats with PEFRE at the doses of 200and 400mgkg exhibited significant increase in locomotoractivity and increase in muscle activity and thus could beproved with possible action on CNS

Oxidative stress generated as a result of mitochondrialdysfunction particularly mitochondrial complex-1 impair-ment plays an important role in the pathogenesis of PDThe oxidative stress was measured through determinationof levels of malondialdehyde catalase superoxide dismutaseand reduced glutathione in the brain tissue

6-OHDA generates an increase in the production ofhydrogen peroxide and free radicals [30 31] These reactiveoxygen species are generated through the nonenzymaticbreakdown of 6-OHDA or direct inhibition of complex-I and complex-IV of the mitochondrial electron transportchain [31ndash33] The resulting ROS production from 6-OHDAbreakdown leads to lipid peroxidation protein denaturationand increases in glutathione which are found in PD patients[34]

8 Advances in Pharmacological Sciences

Lipid peroxidation a sensitive marker of oxidative stresswas estimated by measuring the levels of thiobarbituricacid Lipid peroxidation occurs due to attack by radicalson double bond of unsaturated fatty acid and arachidonicacid which generate lipid peroxyl radicals and that initiatea chain reaction of further attacks on other unsaturatedfatty acid As we know lipid peroxidation is the process ofoxidative degradation of polyunsaturated fatty acids and itsoccurrence in biological membranes causes impaired mem-brane function impaired structural integrity [35] decreasedfluidity and inactivation of number of membrane boundenzymes Increased levels of the lipid peroxidation producthave been found in the substantia nigra of PD patient Inthe present investigation similar results were observed in thebrain homogenate of 6-OHDA treated control animals

Catalase is an antioxidant which helps in neutralizingthe toxic effects of hydrogen peroxide Hydrogen peroxide isconverted by the catalase enzyme to form water and nonre-active oxygen species thus preventing the accumulation ofprecursor to free radical biosynthesis Oxidative stress resultsin decrease in catalase level 6-OHDA inoculation in ratsinduced oxidative stress as indicated by a decrease in thecatalase levels

Superoxide dismutase (known as SOD) is an enzymewhich acts as a catalyst in the process of dismutation ofsuperoxide into nonreactive oxygen species and hydrogenperoxide It is therefore a critical antioxidant defense whichis present in nearly all cells which are exposed to oxygen[36 37] Superoxide dismutase helps in neutralizing the toxiceffects of free radicals [38 39] 6-OHDA treated control groupshowed a decrease in the level of SOD in the brain of animalsthus indicative of production of oxidative stress

GSH potent enzymes are an important factor in etiologyof PD [40] The depletion of reduced glutathione in thesubstantia nigra in Parkinsonrsquos disease could be the result ofneuronal loss As a matter of fact the positive correlation hasbeen found to exist between the extent of neuronal loss anddepletion of glutathione [41] A decrease in the availabilityof reduced glutathione would impair the capacity of neuronsto detoxify hydrogen peroxide and increase the risk of freeradical formation and lipid peroxidation A reduction inGSHlevels was evident in 6-OHDA treated control animals

Thus the 6-OHDA per se group showed a significantincrease in the levels of thiobarbituric acid (which is anindication of extent of lipid peroxidation) and decrease in thelevels of SODandGSH in the brain as compared to the vehicletreated control animals All these indicate an increase in theoxidative stress in the brain of animals treated with 6-OHDAPretreatmentwith higher dose of petroleumether extract ofFreligiosa (400mgkg) resulted in a decrease inMDA level andincrease in the levels of SOD catalase andGSH indicating itsantioxidant effect in the brain of 6-OHDA treated animals

Histopathological findings showed that pet ether extractof Ficus religiosa treated animals had decreased infiltrationof neutrophils reduced intracellular space increased densityof cells and regained normal architecture and moderatenecrosis in striatum region of brain

5 Conclusion

In view of the above facts we are concluding that petroleumether extract of Ficus religiosa plant showed to be an antiox-idant and showed a promising effect in animals with Parkin-sonrsquos disease And we appreciate further detailed molecularstudies with this drug in anti-Parkinsonrsquos pharmacology andtoxicology and also characterization of active constituentsresponsible for neuroprotective effect

Conflict of Interests

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

References

[1] M F Beal ldquoMitochondrial dysfunction in neurodegenerativediseasesrdquo Biochimica et Biophysica Acta (BBA)mdashBioenergeticsvol 1366 no 1-2 pp 211ndash223 1998

[2] P Jenner and C W Olanow ldquoThe pathogenesis of cell death inParkinsonrsquos diseaserdquo Neurology vol 66 no 10 supplement 4pp S24ndashS36 2006

[3] A E Lang and A M Lozano ldquoParkinsonrsquos disease second oftwo partsrdquo The New England Journal of Medicine vol 339 no16 pp 1130ndash1143 1998

[4] L Scott ldquoIdentifying poor symptom control in Parkinsonrsquosdiseaserdquo Nursing Times vol 102 no 12 pp 30ndash32 2006

[5] Y Mizuno S Ohta M Tanaka et al ldquoDeficiencies in complex-I subunits of the respiratory chain in Parkinsonrsquos diseaserdquoBiochemical and Biophysical Research Communications vol 163no 3 pp 1450ndash1455 1989

[6] W J Schmidt and M Alam ldquoControversies on new animalmodels of Parkinsonrsquos disease pro and con the rotenone modelof Parkinsonrsquos Disease (PD)rdquo Journal of Neural Transmissionvol 70 pp 273ndash276 2006

[7] T J Collier and C E Sortwell ldquoTherapeutic potential of nervegrowth factors in Parkinsonrsquos diseaserdquo Drugs and Aging vol 14no 4 pp 261ndash287 1999

[8] T B Sherer R Betarbet and J T Greenamyre ldquoEnvironmentmitochondria and Parkinsonrsquos diseaserdquo Neuroscientist vol 8no 3 pp 192ndash197 2002

[9] L Chen Y Ding B Cagniard et al ldquoUnregulated cytosolicdopamine causes neurodegeneration associated with oxidativestress in micerdquo The Journal of Neuroscience vol 28 no 2 pp425ndash433 2008

[10] F Zoccarato P Toscano andA Alexandre ldquoDopamine-deriveddopaminochrome promotes H

2

O2

release at mitochondriacomplex-1rdquo Journal of Biological Chemistry vol 280 no 16 pp15587ndash15594 2005

[11] A Ghani Medicinal Plants of Bangladesh with ChemicalConstituents and Uses Asiatic Society of Bangladesh DhakaBangladesh 1998

[12] D Singh andRKGoel ldquoAnticonvulsant effect of Ficus religiosarole of serotonergic pathwaysrdquo Journal of Ethnopharmacologyvol 123 no 2 pp 330ndash334 2009

[13] P V Prasad P K Subhaktha A Narayana and M M RaoldquoMedico-historical study of lsquoasvattharsquo (sacred fig tree)rdquo Bulletinof the Indian Institute of History of Medicine (Hyderabad) vol36 no 1 pp 1ndash20 2006

Advances in Pharmacological Sciences 9

[14] S K Panda N C Panda and B K Sahue ldquoPhytochemistryand pharmacological properties of Ficus religiosa an overviewrdquoIndian Veterinary Journal vol 60 pp 660ndash664 1976

[15] R S Verma and K S Bhatia ldquoChromatographic study of aminoacids of the leaf protein concentrates of Ficus religiosa Linn andMimusops elengi Linnrdquo Indian Journal of Pharmacy Practice vol23 pp 231ndash232 1986

[16] O A Aiyegoro and A I Okoh ldquoUse of bioactive plantproducts in combinationwith standard antibiotics implicationsin antimicrobial chemotherapyrdquo Journal of Medicinal PlantsResearch vol 3 no 13 pp 1147ndash1152 2009

[17] P K Warrier Indian Medicinal PlantsmdashA Compendium of 500Species vol 3 Orient Longman Chennai India 1996

[18] J B Harborne Phytochemical Methods Chapman and HallLondon UK 3rd edition 1998

[19] V Ravichandran B SureshMN Sathishkumar K Elango andR Srinivasan ldquoAntifertility activity of hydroalcoholic extract ofAilanthus excelsa (Roxb) an ethnomedicines used by tribals ofNilgiris region in Tamilnadurdquo Journal of Ethnopharmacologyvol 112 no 1 pp 189ndash191 2007

[20] P J Elliott and S P Close ldquoNeuroleptic-induced catalepsyas a model of Parkinsonrsquos disease I Effect of dopaminergicagentsrdquo Journal of Neural Transmission Parkinsonrsquos Disease andDementia Section vol 2 no 2 pp 79ndash89 1990

[21] R Deumens A Blokland and J Prickaerts ldquoModeling Parkin-sonrsquos disease in rats an evaluation of 6-OHDA lesions of thenigrostriatal pathwayrdquo Experimental Neurology vol 175 no 2pp 303ndash317 2002

[22] S Wang L-F Hu Y Yang J-H Ding and G Hu ldquoStudies ofATP-sensitive potassium channels on 6-hydroxydopamine andhaloperidol rat models of Parkinsonrsquos disease implications fortreating Parkinsonrsquos diseaserdquo Neuropharmacology vol 48 no7 pp 984ndash992 2005

[23] D S Reddy and S K Kulkarni ldquoPossible role of nitric oxidein the nootropic and antiamnesic effects of neurosteroids onaging- and dizocilpine-induced learning impairmentrdquo BrainResearch vol 799 no 2 pp 215ndash229 1998

[24] S RajaSankar T Manivasagam V Sankar et al ldquoWithaniasomnifera root extract improves catecholamines and physiolog-ical abnormalities seen in a Parkinsonrsquos disease model mouserdquoJournal of Ethnopharmacology vol 125 no 3 pp 369ndash373 2009

[25] H Ohkawa N Ohishi and K Yagi ldquoAssay for lipid peroxidesin animal tissues by thiobarbituric acid reactionrdquo AnalyticalBiochemistry vol 95 no 2 pp 351ndash358 1979

[26] W F Beyer Jr and I Fridovich ldquoAssaying for superoxidedismutase activity some large consequences of minor changesin conditionsrdquo Analytical Biochemistry vol 161 no 2 pp 559ndash566 1987

[27] H Aebi ldquoCatalase in vitrordquo inMethods in Enzymology vol 105pp 121ndash126 Academic Press New York NY USA 1984

[28] S K Srivastava and E Beutler ldquoAccurate measurement ofoxidized glutathione content of human rabbit and rat redblood cells and tissuesrdquoAnalytical Biochemistry vol 25 pp 70ndash76 1968

[29] F Blandini and M-T Armentero ldquoAnimal models of Parkin-sonrsquos diseaserdquo FEBS Journal vol 279 no 7 pp 1156ndash1166 2012

[30] G Cohen and R E Heikkila ldquoThe generation of hydrogenperoxide superoxide radical and hydroxyl radical by 6-hydroxydopamine dialuric acid and related cytotoxic agentsrdquoThe Journal of Biological Chemistry vol 249 no 8 pp 2447ndash2452 1974

[31] D G Graham S M Tiffany W R Bell Jr andW F GutknechtldquoAutoxidation versus covalent binding of quinones as themechanism of toxicity of dopamine 6-hydroxydopamine andrelated compounds toward C1300 neuroblastoma cells in vitrordquoMolecular Pharmacology vol 14 no 4 pp 644ndash653 1978

[32] Y Y Glinka andM B H Youdim ldquoInhibition of mitochondrialcomplexes I and IV by 6-hydroxydopaminerdquo European Journalof Pharmacology vol 292 no 3-4 pp 329ndash332 1995

[33] Y Glinka M Gassen and M B H Youdim ldquoMechanism of 6-hydroxydopamine neurotoxicityrdquo Journal of Neural Transmis-sion Supplement no 50 pp 55ndash66 1997

[34] P Jenner and C W Olanow ldquoUnderstanding cell death inParkinsonrsquos diseaserdquoAnnals of Neurology vol 44 no 3 pp S72ndashS84 1998

[35] B Halliwell and J M C Gutteridge ldquoOxygen radicals and thenervous systemrdquoTrends inNeurosciences vol 8 pp 22ndash26 1985

[36] L SMonk K V Fagerstedt and RM Crawford ldquoOxygen toxi-city and superoxide dismutase as an antioxidant in physiologicalstressrdquo Physiologia Plantarum vol 76 no 3 pp 456ndash459 1989

[37] C Bowler M Van Montagu and D Inze ldquoSuperoxide dismu-tase and stress tolerancerdquoAnnual Review of Plant Physiology andPlant Molecular Biology vol 43 no 1 pp 83ndash116 1992

[38] J G Scandalios ldquoResponse of plant antioxidant defense genesto environmental stressrdquo Advances in Genetics vol 28 pp 1ndash411990

[39] E BGralla andD J Kosman ldquoMolecular genetics of superoxidedismutases in yeasts and related fungirdquo Advances in Geneticsvol 30 pp 251ndash319 1992

[40] P Jenner ldquoOxidative mechanisms in nigral cell death in Parkin-sonrsquos diseaserdquoMovementDisorders vol 13 no 1 pp 24ndash34 1998

[41] P Riederer E Sofic W-D Rausch et al ldquoTransition metalsferritin glutathione and ascorbic acid in Parkinsonian brainsrdquoJournal of Neurochemistry vol 52 no 2 pp 515ndash520 1989

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

6 Advances in Pharmacological Sciences

Table 2 Effect of PEFRE on the levels of lipid peroxidation (MDA) catalase (CAT) superoxide dismutase (SOD) and reduced glutathione(GSH) in the brain of 6-OHDA treated animals

Group MDA (nMmg of protein) CAT (120583moles of H2

O2

usedminmg protein) SOD (unitsmg protein) GSH (nMmg of protein)

Vehicle control 1311 plusmn 009315 5788 plusmn 0046 3185 plusmn 01852 4371 plusmn 0075766-OHDA control 2616 plusmn 01602 3463 plusmn 0035 206 plusmn 01068 3985 plusmn 0020

Levodopa 1659 plusmn 003551lowastlowast 6525 plusmn 020lowastlowastlowast 9667 plusmn 08333lowastlowastlowast 7023 plusmn 06013lowastlowastlowast

PEFRE (100) 2 plusmn 01558 3544 plusmn 015 2419 plusmn 08732 4182 plusmn 001312PEFRE (200) 1975 plusmn 02153 4234 plusmn 011 2801 plusmn 05034 4614 plusmn 01312PEFRE (400) 1347 plusmn 02501lowastlowastlowast 622 plusmn 031lowastlowastlowast 8833 plusmn 08333lowastlowastlowast 8455 plusmn 003936lowastlowastlowast

Values are expressed as mean plusmn SEM lowast119901 lt 005 lowastlowast119901 lt 001 and lowastlowastlowast119901 lt 0001 as compared to 6-OHDA treated control group (Group II) [Groups III to VIwere compared with Group II] 119901 lt 0001 as compared to vehicle treated group (Group I) [Group II was compared with Group I]

IIIIII

IVVVI

lowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

lowastlowast

lowastlowastlowast

lowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

20 25 30 35 40 45 50 5515Days

Loco

mot

or ac

tivity

(cou

nts5

min

)

0

50

100

150

200

Figure 2 The effects of PEFRE on 6-OHDA induced Parkinsonrsquosdisease in the Locomotor activity Group I Vehicle control groupGroup II 6-OHDA treated group Group III Levodopa + 6-OHDAtreated group Group IV PEFRE (100mgkg) + 6-OHDA treatedgroup Group V PEFRE (200mgkg) + 6-OHDA treated groupGroup VI PEFRE (400mgkg) + 6-OHDA treated group lowastlowastlowast119901 lt0001 as compared to 6-OHDA treated negative control group(Group II) 119901 lt 0001 in 6-OHDA treated negative control group(Group II) compared to vehicle treated control group (Group I)

4 Discussion

Parkinsonrsquos disease is a chronic neurodegenerative disordercharacterized by loss of dopamine neurons of the SNpcThe pathogenesis of PD includes oxidative stress proteinaccumulation like a-synuclein mitochondrial dysfunctionapoptosis and neuronal excitotoxicity Among all oxidativestress is a crucial pathological mechanism for PD SNpc ismore vulnerable to reactive oxygen species as it containsmoreamount of dopamine

In the present study we evaluated the effect of pet etherextract of F religiosa in neurotoxins (haloperidol and 6-OHDA) induced Parkinson disease in experimental animals

Haloperidol induced catalepsy is a widely accepted ani-mal model of PD Haloperidol (nonselective D

2dopamine

IIIIII

IVVVI

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowastlowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowastlowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowastlowastlowast

lowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

20 25 30 35 40 45 50 5515Days

0

50

100

150

Mea

n fa

ll of

tim

e (s)

Figure 3 The effects of PEFRE on 6-OHDA induced Parkinsonrsquosdisease in the rotarod performance Group I vehicle control groupGroup II 6-OHDA treated group Group III Levodopa + 6-OHDAtreated group Group IV PEFRE (100mgkg) + 6-OHDA treatedgroup Group V PEFRE (200mgkg) + 6-OHDA treated groupGroup VI PEFRE (400mgkg) + 6-OHDA treated group lowastlowastlowast119901 lt0001 as compared to 6-OHDA treated negative control group(Group II) 119901 lt 0001 6-OHDA treated negative control group(Group II) compared to vehicle treated control group (Group I)

antagonists) provides a pharmacological model of parkin-sonism by interfering with the storage of catecholaminersquosintracellularly resulting in dopamine depletion in nerveendings In the present study haloperidol (1mgkg ip)induced significant catalepsy in rats as evidenced by asignificant increase in the time spent on the block as com-pared to vehicle treated animals Treatment with F religiosasignificantly reduced the catalepsy in haloperidol treated ratsin dose dependent manner The PEFRE at doses of 200and 400mgkg showed protective effect against haloperidolinduced catalepsy indicated that this plant has an ability toprotect dopaminergic neurotransmission in striatum

There are well-known pharmacological PD models inmammalian systems including the classical and highly selec-tive neurotoxin 6-hydroxydopamine (6-OHDA) as well as 1-methyl-4-phenyl-1236-tetrahydropyridine (MPTP) and its

Advances in Pharmacological Sciences 7

(a) (b)

(c) (d)

(e) (f)

Figure 4 Effect of PEFRE on histopathological changes in the brain of normal and 6-OHDA treated animals (HampE staining originalmagnification 40x) (a) Normal control showing normal brain architecture (b) Rats treated with 6-OHDA showing degeneration of neurons(c) Rats treated with 6-OHDA and Levodopa (6mgkg) showing minimal changes in neuronal cell integrity and architecture (d) Rats treatedwith 6-OHDA and PEFRE (100mgkg) showing mild decrease in neurons and cellular hypertrophy (e) PEFRE (200mgkg) and (f) PEFRE(400mgkg) treated rats showing minimal changes in neuronal cell populations

metaboliteMPP+ (1-methyl-4-phenylpyridinium ion)Thesetoxins cause decreased ATP production increased ROSproduction and increased apoptosis of DAergic cells [29]

The efficacy of F religiosa in 6-OHDA induced PDhas notbeen well established In the present study 6-OHDA admin-istration to rats caused a significant decrease in locomotoractivity and muscle activity Lack of motor coordinationand maintenance of normal limb posture has been reportedin PD condition The evaluated data suggested damage tothe dopaminergic neurons and progression of Parkinsonrsquosdisease like behavioral abnormalities in rats exposed to 6-OHDA Pretreatment of rats with PEFRE at the doses of 200and 400mgkg exhibited significant increase in locomotoractivity and increase in muscle activity and thus could beproved with possible action on CNS

Oxidative stress generated as a result of mitochondrialdysfunction particularly mitochondrial complex-1 impair-ment plays an important role in the pathogenesis of PDThe oxidative stress was measured through determinationof levels of malondialdehyde catalase superoxide dismutaseand reduced glutathione in the brain tissue

6-OHDA generates an increase in the production ofhydrogen peroxide and free radicals [30 31] These reactiveoxygen species are generated through the nonenzymaticbreakdown of 6-OHDA or direct inhibition of complex-I and complex-IV of the mitochondrial electron transportchain [31ndash33] The resulting ROS production from 6-OHDAbreakdown leads to lipid peroxidation protein denaturationand increases in glutathione which are found in PD patients[34]

8 Advances in Pharmacological Sciences

Lipid peroxidation a sensitive marker of oxidative stresswas estimated by measuring the levels of thiobarbituricacid Lipid peroxidation occurs due to attack by radicalson double bond of unsaturated fatty acid and arachidonicacid which generate lipid peroxyl radicals and that initiatea chain reaction of further attacks on other unsaturatedfatty acid As we know lipid peroxidation is the process ofoxidative degradation of polyunsaturated fatty acids and itsoccurrence in biological membranes causes impaired mem-brane function impaired structural integrity [35] decreasedfluidity and inactivation of number of membrane boundenzymes Increased levels of the lipid peroxidation producthave been found in the substantia nigra of PD patient Inthe present investigation similar results were observed in thebrain homogenate of 6-OHDA treated control animals

Catalase is an antioxidant which helps in neutralizingthe toxic effects of hydrogen peroxide Hydrogen peroxide isconverted by the catalase enzyme to form water and nonre-active oxygen species thus preventing the accumulation ofprecursor to free radical biosynthesis Oxidative stress resultsin decrease in catalase level 6-OHDA inoculation in ratsinduced oxidative stress as indicated by a decrease in thecatalase levels

Superoxide dismutase (known as SOD) is an enzymewhich acts as a catalyst in the process of dismutation ofsuperoxide into nonreactive oxygen species and hydrogenperoxide It is therefore a critical antioxidant defense whichis present in nearly all cells which are exposed to oxygen[36 37] Superoxide dismutase helps in neutralizing the toxiceffects of free radicals [38 39] 6-OHDA treated control groupshowed a decrease in the level of SOD in the brain of animalsthus indicative of production of oxidative stress

GSH potent enzymes are an important factor in etiologyof PD [40] The depletion of reduced glutathione in thesubstantia nigra in Parkinsonrsquos disease could be the result ofneuronal loss As a matter of fact the positive correlation hasbeen found to exist between the extent of neuronal loss anddepletion of glutathione [41] A decrease in the availabilityof reduced glutathione would impair the capacity of neuronsto detoxify hydrogen peroxide and increase the risk of freeradical formation and lipid peroxidation A reduction inGSHlevels was evident in 6-OHDA treated control animals

Thus the 6-OHDA per se group showed a significantincrease in the levels of thiobarbituric acid (which is anindication of extent of lipid peroxidation) and decrease in thelevels of SODandGSH in the brain as compared to the vehicletreated control animals All these indicate an increase in theoxidative stress in the brain of animals treated with 6-OHDAPretreatmentwith higher dose of petroleumether extract ofFreligiosa (400mgkg) resulted in a decrease inMDA level andincrease in the levels of SOD catalase andGSH indicating itsantioxidant effect in the brain of 6-OHDA treated animals

Histopathological findings showed that pet ether extractof Ficus religiosa treated animals had decreased infiltrationof neutrophils reduced intracellular space increased densityof cells and regained normal architecture and moderatenecrosis in striatum region of brain

5 Conclusion

In view of the above facts we are concluding that petroleumether extract of Ficus religiosa plant showed to be an antiox-idant and showed a promising effect in animals with Parkin-sonrsquos disease And we appreciate further detailed molecularstudies with this drug in anti-Parkinsonrsquos pharmacology andtoxicology and also characterization of active constituentsresponsible for neuroprotective effect

Conflict of Interests

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

References

[1] M F Beal ldquoMitochondrial dysfunction in neurodegenerativediseasesrdquo Biochimica et Biophysica Acta (BBA)mdashBioenergeticsvol 1366 no 1-2 pp 211ndash223 1998

[2] P Jenner and C W Olanow ldquoThe pathogenesis of cell death inParkinsonrsquos diseaserdquo Neurology vol 66 no 10 supplement 4pp S24ndashS36 2006

[3] A E Lang and A M Lozano ldquoParkinsonrsquos disease second oftwo partsrdquo The New England Journal of Medicine vol 339 no16 pp 1130ndash1143 1998

[4] L Scott ldquoIdentifying poor symptom control in Parkinsonrsquosdiseaserdquo Nursing Times vol 102 no 12 pp 30ndash32 2006

[5] Y Mizuno S Ohta M Tanaka et al ldquoDeficiencies in complex-I subunits of the respiratory chain in Parkinsonrsquos diseaserdquoBiochemical and Biophysical Research Communications vol 163no 3 pp 1450ndash1455 1989

[6] W J Schmidt and M Alam ldquoControversies on new animalmodels of Parkinsonrsquos disease pro and con the rotenone modelof Parkinsonrsquos Disease (PD)rdquo Journal of Neural Transmissionvol 70 pp 273ndash276 2006

[7] T J Collier and C E Sortwell ldquoTherapeutic potential of nervegrowth factors in Parkinsonrsquos diseaserdquo Drugs and Aging vol 14no 4 pp 261ndash287 1999

[8] T B Sherer R Betarbet and J T Greenamyre ldquoEnvironmentmitochondria and Parkinsonrsquos diseaserdquo Neuroscientist vol 8no 3 pp 192ndash197 2002

[9] L Chen Y Ding B Cagniard et al ldquoUnregulated cytosolicdopamine causes neurodegeneration associated with oxidativestress in micerdquo The Journal of Neuroscience vol 28 no 2 pp425ndash433 2008

[10] F Zoccarato P Toscano andA Alexandre ldquoDopamine-deriveddopaminochrome promotes H

2

O2

release at mitochondriacomplex-1rdquo Journal of Biological Chemistry vol 280 no 16 pp15587ndash15594 2005

[11] A Ghani Medicinal Plants of Bangladesh with ChemicalConstituents and Uses Asiatic Society of Bangladesh DhakaBangladesh 1998

[12] D Singh andRKGoel ldquoAnticonvulsant effect of Ficus religiosarole of serotonergic pathwaysrdquo Journal of Ethnopharmacologyvol 123 no 2 pp 330ndash334 2009

[13] P V Prasad P K Subhaktha A Narayana and M M RaoldquoMedico-historical study of lsquoasvattharsquo (sacred fig tree)rdquo Bulletinof the Indian Institute of History of Medicine (Hyderabad) vol36 no 1 pp 1ndash20 2006

Advances in Pharmacological Sciences 9

[14] S K Panda N C Panda and B K Sahue ldquoPhytochemistryand pharmacological properties of Ficus religiosa an overviewrdquoIndian Veterinary Journal vol 60 pp 660ndash664 1976

[15] R S Verma and K S Bhatia ldquoChromatographic study of aminoacids of the leaf protein concentrates of Ficus religiosa Linn andMimusops elengi Linnrdquo Indian Journal of Pharmacy Practice vol23 pp 231ndash232 1986

[16] O A Aiyegoro and A I Okoh ldquoUse of bioactive plantproducts in combinationwith standard antibiotics implicationsin antimicrobial chemotherapyrdquo Journal of Medicinal PlantsResearch vol 3 no 13 pp 1147ndash1152 2009

[17] P K Warrier Indian Medicinal PlantsmdashA Compendium of 500Species vol 3 Orient Longman Chennai India 1996

[18] J B Harborne Phytochemical Methods Chapman and HallLondon UK 3rd edition 1998

[19] V Ravichandran B SureshMN Sathishkumar K Elango andR Srinivasan ldquoAntifertility activity of hydroalcoholic extract ofAilanthus excelsa (Roxb) an ethnomedicines used by tribals ofNilgiris region in Tamilnadurdquo Journal of Ethnopharmacologyvol 112 no 1 pp 189ndash191 2007

[20] P J Elliott and S P Close ldquoNeuroleptic-induced catalepsyas a model of Parkinsonrsquos disease I Effect of dopaminergicagentsrdquo Journal of Neural Transmission Parkinsonrsquos Disease andDementia Section vol 2 no 2 pp 79ndash89 1990

[21] R Deumens A Blokland and J Prickaerts ldquoModeling Parkin-sonrsquos disease in rats an evaluation of 6-OHDA lesions of thenigrostriatal pathwayrdquo Experimental Neurology vol 175 no 2pp 303ndash317 2002

[22] S Wang L-F Hu Y Yang J-H Ding and G Hu ldquoStudies ofATP-sensitive potassium channels on 6-hydroxydopamine andhaloperidol rat models of Parkinsonrsquos disease implications fortreating Parkinsonrsquos diseaserdquo Neuropharmacology vol 48 no7 pp 984ndash992 2005

[23] D S Reddy and S K Kulkarni ldquoPossible role of nitric oxidein the nootropic and antiamnesic effects of neurosteroids onaging- and dizocilpine-induced learning impairmentrdquo BrainResearch vol 799 no 2 pp 215ndash229 1998

[24] S RajaSankar T Manivasagam V Sankar et al ldquoWithaniasomnifera root extract improves catecholamines and physiolog-ical abnormalities seen in a Parkinsonrsquos disease model mouserdquoJournal of Ethnopharmacology vol 125 no 3 pp 369ndash373 2009

[25] H Ohkawa N Ohishi and K Yagi ldquoAssay for lipid peroxidesin animal tissues by thiobarbituric acid reactionrdquo AnalyticalBiochemistry vol 95 no 2 pp 351ndash358 1979

[26] W F Beyer Jr and I Fridovich ldquoAssaying for superoxidedismutase activity some large consequences of minor changesin conditionsrdquo Analytical Biochemistry vol 161 no 2 pp 559ndash566 1987

[27] H Aebi ldquoCatalase in vitrordquo inMethods in Enzymology vol 105pp 121ndash126 Academic Press New York NY USA 1984

[28] S K Srivastava and E Beutler ldquoAccurate measurement ofoxidized glutathione content of human rabbit and rat redblood cells and tissuesrdquoAnalytical Biochemistry vol 25 pp 70ndash76 1968

[29] F Blandini and M-T Armentero ldquoAnimal models of Parkin-sonrsquos diseaserdquo FEBS Journal vol 279 no 7 pp 1156ndash1166 2012

[30] G Cohen and R E Heikkila ldquoThe generation of hydrogenperoxide superoxide radical and hydroxyl radical by 6-hydroxydopamine dialuric acid and related cytotoxic agentsrdquoThe Journal of Biological Chemistry vol 249 no 8 pp 2447ndash2452 1974

[31] D G Graham S M Tiffany W R Bell Jr andW F GutknechtldquoAutoxidation versus covalent binding of quinones as themechanism of toxicity of dopamine 6-hydroxydopamine andrelated compounds toward C1300 neuroblastoma cells in vitrordquoMolecular Pharmacology vol 14 no 4 pp 644ndash653 1978

[32] Y Y Glinka andM B H Youdim ldquoInhibition of mitochondrialcomplexes I and IV by 6-hydroxydopaminerdquo European Journalof Pharmacology vol 292 no 3-4 pp 329ndash332 1995

[33] Y Glinka M Gassen and M B H Youdim ldquoMechanism of 6-hydroxydopamine neurotoxicityrdquo Journal of Neural Transmis-sion Supplement no 50 pp 55ndash66 1997

[34] P Jenner and C W Olanow ldquoUnderstanding cell death inParkinsonrsquos diseaserdquoAnnals of Neurology vol 44 no 3 pp S72ndashS84 1998

[35] B Halliwell and J M C Gutteridge ldquoOxygen radicals and thenervous systemrdquoTrends inNeurosciences vol 8 pp 22ndash26 1985

[36] L SMonk K V Fagerstedt and RM Crawford ldquoOxygen toxi-city and superoxide dismutase as an antioxidant in physiologicalstressrdquo Physiologia Plantarum vol 76 no 3 pp 456ndash459 1989

[37] C Bowler M Van Montagu and D Inze ldquoSuperoxide dismu-tase and stress tolerancerdquoAnnual Review of Plant Physiology andPlant Molecular Biology vol 43 no 1 pp 83ndash116 1992

[38] J G Scandalios ldquoResponse of plant antioxidant defense genesto environmental stressrdquo Advances in Genetics vol 28 pp 1ndash411990

[39] E BGralla andD J Kosman ldquoMolecular genetics of superoxidedismutases in yeasts and related fungirdquo Advances in Geneticsvol 30 pp 251ndash319 1992

[40] P Jenner ldquoOxidative mechanisms in nigral cell death in Parkin-sonrsquos diseaserdquoMovementDisorders vol 13 no 1 pp 24ndash34 1998

[41] P Riederer E Sofic W-D Rausch et al ldquoTransition metalsferritin glutathione and ascorbic acid in Parkinsonian brainsrdquoJournal of Neurochemistry vol 52 no 2 pp 515ndash520 1989

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Advances in Pharmacological Sciences 7

(a) (b)

(c) (d)

(e) (f)

Figure 4 Effect of PEFRE on histopathological changes in the brain of normal and 6-OHDA treated animals (HampE staining originalmagnification 40x) (a) Normal control showing normal brain architecture (b) Rats treated with 6-OHDA showing degeneration of neurons(c) Rats treated with 6-OHDA and Levodopa (6mgkg) showing minimal changes in neuronal cell integrity and architecture (d) Rats treatedwith 6-OHDA and PEFRE (100mgkg) showing mild decrease in neurons and cellular hypertrophy (e) PEFRE (200mgkg) and (f) PEFRE(400mgkg) treated rats showing minimal changes in neuronal cell populations

metaboliteMPP+ (1-methyl-4-phenylpyridinium ion)Thesetoxins cause decreased ATP production increased ROSproduction and increased apoptosis of DAergic cells [29]

The efficacy of F religiosa in 6-OHDA induced PDhas notbeen well established In the present study 6-OHDA admin-istration to rats caused a significant decrease in locomotoractivity and muscle activity Lack of motor coordinationand maintenance of normal limb posture has been reportedin PD condition The evaluated data suggested damage tothe dopaminergic neurons and progression of Parkinsonrsquosdisease like behavioral abnormalities in rats exposed to 6-OHDA Pretreatment of rats with PEFRE at the doses of 200and 400mgkg exhibited significant increase in locomotoractivity and increase in muscle activity and thus could beproved with possible action on CNS

Oxidative stress generated as a result of mitochondrialdysfunction particularly mitochondrial complex-1 impair-ment plays an important role in the pathogenesis of PDThe oxidative stress was measured through determinationof levels of malondialdehyde catalase superoxide dismutaseand reduced glutathione in the brain tissue

6-OHDA generates an increase in the production ofhydrogen peroxide and free radicals [30 31] These reactiveoxygen species are generated through the nonenzymaticbreakdown of 6-OHDA or direct inhibition of complex-I and complex-IV of the mitochondrial electron transportchain [31ndash33] The resulting ROS production from 6-OHDAbreakdown leads to lipid peroxidation protein denaturationand increases in glutathione which are found in PD patients[34]

8 Advances in Pharmacological Sciences

Lipid peroxidation a sensitive marker of oxidative stresswas estimated by measuring the levels of thiobarbituricacid Lipid peroxidation occurs due to attack by radicalson double bond of unsaturated fatty acid and arachidonicacid which generate lipid peroxyl radicals and that initiatea chain reaction of further attacks on other unsaturatedfatty acid As we know lipid peroxidation is the process ofoxidative degradation of polyunsaturated fatty acids and itsoccurrence in biological membranes causes impaired mem-brane function impaired structural integrity [35] decreasedfluidity and inactivation of number of membrane boundenzymes Increased levels of the lipid peroxidation producthave been found in the substantia nigra of PD patient Inthe present investigation similar results were observed in thebrain homogenate of 6-OHDA treated control animals

Catalase is an antioxidant which helps in neutralizingthe toxic effects of hydrogen peroxide Hydrogen peroxide isconverted by the catalase enzyme to form water and nonre-active oxygen species thus preventing the accumulation ofprecursor to free radical biosynthesis Oxidative stress resultsin decrease in catalase level 6-OHDA inoculation in ratsinduced oxidative stress as indicated by a decrease in thecatalase levels

Superoxide dismutase (known as SOD) is an enzymewhich acts as a catalyst in the process of dismutation ofsuperoxide into nonreactive oxygen species and hydrogenperoxide It is therefore a critical antioxidant defense whichis present in nearly all cells which are exposed to oxygen[36 37] Superoxide dismutase helps in neutralizing the toxiceffects of free radicals [38 39] 6-OHDA treated control groupshowed a decrease in the level of SOD in the brain of animalsthus indicative of production of oxidative stress

GSH potent enzymes are an important factor in etiologyof PD [40] The depletion of reduced glutathione in thesubstantia nigra in Parkinsonrsquos disease could be the result ofneuronal loss As a matter of fact the positive correlation hasbeen found to exist between the extent of neuronal loss anddepletion of glutathione [41] A decrease in the availabilityof reduced glutathione would impair the capacity of neuronsto detoxify hydrogen peroxide and increase the risk of freeradical formation and lipid peroxidation A reduction inGSHlevels was evident in 6-OHDA treated control animals

Thus the 6-OHDA per se group showed a significantincrease in the levels of thiobarbituric acid (which is anindication of extent of lipid peroxidation) and decrease in thelevels of SODandGSH in the brain as compared to the vehicletreated control animals All these indicate an increase in theoxidative stress in the brain of animals treated with 6-OHDAPretreatmentwith higher dose of petroleumether extract ofFreligiosa (400mgkg) resulted in a decrease inMDA level andincrease in the levels of SOD catalase andGSH indicating itsantioxidant effect in the brain of 6-OHDA treated animals

Histopathological findings showed that pet ether extractof Ficus religiosa treated animals had decreased infiltrationof neutrophils reduced intracellular space increased densityof cells and regained normal architecture and moderatenecrosis in striatum region of brain

5 Conclusion

In view of the above facts we are concluding that petroleumether extract of Ficus religiosa plant showed to be an antiox-idant and showed a promising effect in animals with Parkin-sonrsquos disease And we appreciate further detailed molecularstudies with this drug in anti-Parkinsonrsquos pharmacology andtoxicology and also characterization of active constituentsresponsible for neuroprotective effect

Conflict of Interests

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

References

[1] M F Beal ldquoMitochondrial dysfunction in neurodegenerativediseasesrdquo Biochimica et Biophysica Acta (BBA)mdashBioenergeticsvol 1366 no 1-2 pp 211ndash223 1998

[2] P Jenner and C W Olanow ldquoThe pathogenesis of cell death inParkinsonrsquos diseaserdquo Neurology vol 66 no 10 supplement 4pp S24ndashS36 2006

[3] A E Lang and A M Lozano ldquoParkinsonrsquos disease second oftwo partsrdquo The New England Journal of Medicine vol 339 no16 pp 1130ndash1143 1998

[4] L Scott ldquoIdentifying poor symptom control in Parkinsonrsquosdiseaserdquo Nursing Times vol 102 no 12 pp 30ndash32 2006

[5] Y Mizuno S Ohta M Tanaka et al ldquoDeficiencies in complex-I subunits of the respiratory chain in Parkinsonrsquos diseaserdquoBiochemical and Biophysical Research Communications vol 163no 3 pp 1450ndash1455 1989

[6] W J Schmidt and M Alam ldquoControversies on new animalmodels of Parkinsonrsquos disease pro and con the rotenone modelof Parkinsonrsquos Disease (PD)rdquo Journal of Neural Transmissionvol 70 pp 273ndash276 2006

[7] T J Collier and C E Sortwell ldquoTherapeutic potential of nervegrowth factors in Parkinsonrsquos diseaserdquo Drugs and Aging vol 14no 4 pp 261ndash287 1999

[8] T B Sherer R Betarbet and J T Greenamyre ldquoEnvironmentmitochondria and Parkinsonrsquos diseaserdquo Neuroscientist vol 8no 3 pp 192ndash197 2002

[9] L Chen Y Ding B Cagniard et al ldquoUnregulated cytosolicdopamine causes neurodegeneration associated with oxidativestress in micerdquo The Journal of Neuroscience vol 28 no 2 pp425ndash433 2008

[10] F Zoccarato P Toscano andA Alexandre ldquoDopamine-deriveddopaminochrome promotes H

2

O2

release at mitochondriacomplex-1rdquo Journal of Biological Chemistry vol 280 no 16 pp15587ndash15594 2005

[11] A Ghani Medicinal Plants of Bangladesh with ChemicalConstituents and Uses Asiatic Society of Bangladesh DhakaBangladesh 1998

[12] D Singh andRKGoel ldquoAnticonvulsant effect of Ficus religiosarole of serotonergic pathwaysrdquo Journal of Ethnopharmacologyvol 123 no 2 pp 330ndash334 2009

[13] P V Prasad P K Subhaktha A Narayana and M M RaoldquoMedico-historical study of lsquoasvattharsquo (sacred fig tree)rdquo Bulletinof the Indian Institute of History of Medicine (Hyderabad) vol36 no 1 pp 1ndash20 2006

Advances in Pharmacological Sciences 9

[14] S K Panda N C Panda and B K Sahue ldquoPhytochemistryand pharmacological properties of Ficus religiosa an overviewrdquoIndian Veterinary Journal vol 60 pp 660ndash664 1976

[15] R S Verma and K S Bhatia ldquoChromatographic study of aminoacids of the leaf protein concentrates of Ficus religiosa Linn andMimusops elengi Linnrdquo Indian Journal of Pharmacy Practice vol23 pp 231ndash232 1986

[16] O A Aiyegoro and A I Okoh ldquoUse of bioactive plantproducts in combinationwith standard antibiotics implicationsin antimicrobial chemotherapyrdquo Journal of Medicinal PlantsResearch vol 3 no 13 pp 1147ndash1152 2009

[17] P K Warrier Indian Medicinal PlantsmdashA Compendium of 500Species vol 3 Orient Longman Chennai India 1996

[18] J B Harborne Phytochemical Methods Chapman and HallLondon UK 3rd edition 1998

[19] V Ravichandran B SureshMN Sathishkumar K Elango andR Srinivasan ldquoAntifertility activity of hydroalcoholic extract ofAilanthus excelsa (Roxb) an ethnomedicines used by tribals ofNilgiris region in Tamilnadurdquo Journal of Ethnopharmacologyvol 112 no 1 pp 189ndash191 2007

[20] P J Elliott and S P Close ldquoNeuroleptic-induced catalepsyas a model of Parkinsonrsquos disease I Effect of dopaminergicagentsrdquo Journal of Neural Transmission Parkinsonrsquos Disease andDementia Section vol 2 no 2 pp 79ndash89 1990

[21] R Deumens A Blokland and J Prickaerts ldquoModeling Parkin-sonrsquos disease in rats an evaluation of 6-OHDA lesions of thenigrostriatal pathwayrdquo Experimental Neurology vol 175 no 2pp 303ndash317 2002

[22] S Wang L-F Hu Y Yang J-H Ding and G Hu ldquoStudies ofATP-sensitive potassium channels on 6-hydroxydopamine andhaloperidol rat models of Parkinsonrsquos disease implications fortreating Parkinsonrsquos diseaserdquo Neuropharmacology vol 48 no7 pp 984ndash992 2005

[23] D S Reddy and S K Kulkarni ldquoPossible role of nitric oxidein the nootropic and antiamnesic effects of neurosteroids onaging- and dizocilpine-induced learning impairmentrdquo BrainResearch vol 799 no 2 pp 215ndash229 1998

[24] S RajaSankar T Manivasagam V Sankar et al ldquoWithaniasomnifera root extract improves catecholamines and physiolog-ical abnormalities seen in a Parkinsonrsquos disease model mouserdquoJournal of Ethnopharmacology vol 125 no 3 pp 369ndash373 2009

[25] H Ohkawa N Ohishi and K Yagi ldquoAssay for lipid peroxidesin animal tissues by thiobarbituric acid reactionrdquo AnalyticalBiochemistry vol 95 no 2 pp 351ndash358 1979

[26] W F Beyer Jr and I Fridovich ldquoAssaying for superoxidedismutase activity some large consequences of minor changesin conditionsrdquo Analytical Biochemistry vol 161 no 2 pp 559ndash566 1987

[27] H Aebi ldquoCatalase in vitrordquo inMethods in Enzymology vol 105pp 121ndash126 Academic Press New York NY USA 1984

[28] S K Srivastava and E Beutler ldquoAccurate measurement ofoxidized glutathione content of human rabbit and rat redblood cells and tissuesrdquoAnalytical Biochemistry vol 25 pp 70ndash76 1968

[29] F Blandini and M-T Armentero ldquoAnimal models of Parkin-sonrsquos diseaserdquo FEBS Journal vol 279 no 7 pp 1156ndash1166 2012

[30] G Cohen and R E Heikkila ldquoThe generation of hydrogenperoxide superoxide radical and hydroxyl radical by 6-hydroxydopamine dialuric acid and related cytotoxic agentsrdquoThe Journal of Biological Chemistry vol 249 no 8 pp 2447ndash2452 1974

[31] D G Graham S M Tiffany W R Bell Jr andW F GutknechtldquoAutoxidation versus covalent binding of quinones as themechanism of toxicity of dopamine 6-hydroxydopamine andrelated compounds toward C1300 neuroblastoma cells in vitrordquoMolecular Pharmacology vol 14 no 4 pp 644ndash653 1978

[32] Y Y Glinka andM B H Youdim ldquoInhibition of mitochondrialcomplexes I and IV by 6-hydroxydopaminerdquo European Journalof Pharmacology vol 292 no 3-4 pp 329ndash332 1995

[33] Y Glinka M Gassen and M B H Youdim ldquoMechanism of 6-hydroxydopamine neurotoxicityrdquo Journal of Neural Transmis-sion Supplement no 50 pp 55ndash66 1997

[34] P Jenner and C W Olanow ldquoUnderstanding cell death inParkinsonrsquos diseaserdquoAnnals of Neurology vol 44 no 3 pp S72ndashS84 1998

[35] B Halliwell and J M C Gutteridge ldquoOxygen radicals and thenervous systemrdquoTrends inNeurosciences vol 8 pp 22ndash26 1985

[36] L SMonk K V Fagerstedt and RM Crawford ldquoOxygen toxi-city and superoxide dismutase as an antioxidant in physiologicalstressrdquo Physiologia Plantarum vol 76 no 3 pp 456ndash459 1989

[37] C Bowler M Van Montagu and D Inze ldquoSuperoxide dismu-tase and stress tolerancerdquoAnnual Review of Plant Physiology andPlant Molecular Biology vol 43 no 1 pp 83ndash116 1992

[38] J G Scandalios ldquoResponse of plant antioxidant defense genesto environmental stressrdquo Advances in Genetics vol 28 pp 1ndash411990

[39] E BGralla andD J Kosman ldquoMolecular genetics of superoxidedismutases in yeasts and related fungirdquo Advances in Geneticsvol 30 pp 251ndash319 1992

[40] P Jenner ldquoOxidative mechanisms in nigral cell death in Parkin-sonrsquos diseaserdquoMovementDisorders vol 13 no 1 pp 24ndash34 1998

[41] P Riederer E Sofic W-D Rausch et al ldquoTransition metalsferritin glutathione and ascorbic acid in Parkinsonian brainsrdquoJournal of Neurochemistry vol 52 no 2 pp 515ndash520 1989

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

8 Advances in Pharmacological Sciences

Lipid peroxidation a sensitive marker of oxidative stresswas estimated by measuring the levels of thiobarbituricacid Lipid peroxidation occurs due to attack by radicalson double bond of unsaturated fatty acid and arachidonicacid which generate lipid peroxyl radicals and that initiatea chain reaction of further attacks on other unsaturatedfatty acid As we know lipid peroxidation is the process ofoxidative degradation of polyunsaturated fatty acids and itsoccurrence in biological membranes causes impaired mem-brane function impaired structural integrity [35] decreasedfluidity and inactivation of number of membrane boundenzymes Increased levels of the lipid peroxidation producthave been found in the substantia nigra of PD patient Inthe present investigation similar results were observed in thebrain homogenate of 6-OHDA treated control animals

Catalase is an antioxidant which helps in neutralizingthe toxic effects of hydrogen peroxide Hydrogen peroxide isconverted by the catalase enzyme to form water and nonre-active oxygen species thus preventing the accumulation ofprecursor to free radical biosynthesis Oxidative stress resultsin decrease in catalase level 6-OHDA inoculation in ratsinduced oxidative stress as indicated by a decrease in thecatalase levels

Superoxide dismutase (known as SOD) is an enzymewhich acts as a catalyst in the process of dismutation ofsuperoxide into nonreactive oxygen species and hydrogenperoxide It is therefore a critical antioxidant defense whichis present in nearly all cells which are exposed to oxygen[36 37] Superoxide dismutase helps in neutralizing the toxiceffects of free radicals [38 39] 6-OHDA treated control groupshowed a decrease in the level of SOD in the brain of animalsthus indicative of production of oxidative stress

GSH potent enzymes are an important factor in etiologyof PD [40] The depletion of reduced glutathione in thesubstantia nigra in Parkinsonrsquos disease could be the result ofneuronal loss As a matter of fact the positive correlation hasbeen found to exist between the extent of neuronal loss anddepletion of glutathione [41] A decrease in the availabilityof reduced glutathione would impair the capacity of neuronsto detoxify hydrogen peroxide and increase the risk of freeradical formation and lipid peroxidation A reduction inGSHlevels was evident in 6-OHDA treated control animals

Thus the 6-OHDA per se group showed a significantincrease in the levels of thiobarbituric acid (which is anindication of extent of lipid peroxidation) and decrease in thelevels of SODandGSH in the brain as compared to the vehicletreated control animals All these indicate an increase in theoxidative stress in the brain of animals treated with 6-OHDAPretreatmentwith higher dose of petroleumether extract ofFreligiosa (400mgkg) resulted in a decrease inMDA level andincrease in the levels of SOD catalase andGSH indicating itsantioxidant effect in the brain of 6-OHDA treated animals

Histopathological findings showed that pet ether extractof Ficus religiosa treated animals had decreased infiltrationof neutrophils reduced intracellular space increased densityof cells and regained normal architecture and moderatenecrosis in striatum region of brain

5 Conclusion

In view of the above facts we are concluding that petroleumether extract of Ficus religiosa plant showed to be an antiox-idant and showed a promising effect in animals with Parkin-sonrsquos disease And we appreciate further detailed molecularstudies with this drug in anti-Parkinsonrsquos pharmacology andtoxicology and also characterization of active constituentsresponsible for neuroprotective effect

Conflict of Interests

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

References

[1] M F Beal ldquoMitochondrial dysfunction in neurodegenerativediseasesrdquo Biochimica et Biophysica Acta (BBA)mdashBioenergeticsvol 1366 no 1-2 pp 211ndash223 1998

[2] P Jenner and C W Olanow ldquoThe pathogenesis of cell death inParkinsonrsquos diseaserdquo Neurology vol 66 no 10 supplement 4pp S24ndashS36 2006

[3] A E Lang and A M Lozano ldquoParkinsonrsquos disease second oftwo partsrdquo The New England Journal of Medicine vol 339 no16 pp 1130ndash1143 1998

[4] L Scott ldquoIdentifying poor symptom control in Parkinsonrsquosdiseaserdquo Nursing Times vol 102 no 12 pp 30ndash32 2006

[5] Y Mizuno S Ohta M Tanaka et al ldquoDeficiencies in complex-I subunits of the respiratory chain in Parkinsonrsquos diseaserdquoBiochemical and Biophysical Research Communications vol 163no 3 pp 1450ndash1455 1989

[6] W J Schmidt and M Alam ldquoControversies on new animalmodels of Parkinsonrsquos disease pro and con the rotenone modelof Parkinsonrsquos Disease (PD)rdquo Journal of Neural Transmissionvol 70 pp 273ndash276 2006

[7] T J Collier and C E Sortwell ldquoTherapeutic potential of nervegrowth factors in Parkinsonrsquos diseaserdquo Drugs and Aging vol 14no 4 pp 261ndash287 1999

[8] T B Sherer R Betarbet and J T Greenamyre ldquoEnvironmentmitochondria and Parkinsonrsquos diseaserdquo Neuroscientist vol 8no 3 pp 192ndash197 2002

[9] L Chen Y Ding B Cagniard et al ldquoUnregulated cytosolicdopamine causes neurodegeneration associated with oxidativestress in micerdquo The Journal of Neuroscience vol 28 no 2 pp425ndash433 2008

[10] F Zoccarato P Toscano andA Alexandre ldquoDopamine-deriveddopaminochrome promotes H

2

O2

release at mitochondriacomplex-1rdquo Journal of Biological Chemistry vol 280 no 16 pp15587ndash15594 2005

[11] A Ghani Medicinal Plants of Bangladesh with ChemicalConstituents and Uses Asiatic Society of Bangladesh DhakaBangladesh 1998

[12] D Singh andRKGoel ldquoAnticonvulsant effect of Ficus religiosarole of serotonergic pathwaysrdquo Journal of Ethnopharmacologyvol 123 no 2 pp 330ndash334 2009

[13] P V Prasad P K Subhaktha A Narayana and M M RaoldquoMedico-historical study of lsquoasvattharsquo (sacred fig tree)rdquo Bulletinof the Indian Institute of History of Medicine (Hyderabad) vol36 no 1 pp 1ndash20 2006

Advances in Pharmacological Sciences 9

[14] S K Panda N C Panda and B K Sahue ldquoPhytochemistryand pharmacological properties of Ficus religiosa an overviewrdquoIndian Veterinary Journal vol 60 pp 660ndash664 1976

[15] R S Verma and K S Bhatia ldquoChromatographic study of aminoacids of the leaf protein concentrates of Ficus religiosa Linn andMimusops elengi Linnrdquo Indian Journal of Pharmacy Practice vol23 pp 231ndash232 1986

[16] O A Aiyegoro and A I Okoh ldquoUse of bioactive plantproducts in combinationwith standard antibiotics implicationsin antimicrobial chemotherapyrdquo Journal of Medicinal PlantsResearch vol 3 no 13 pp 1147ndash1152 2009

[17] P K Warrier Indian Medicinal PlantsmdashA Compendium of 500Species vol 3 Orient Longman Chennai India 1996

[18] J B Harborne Phytochemical Methods Chapman and HallLondon UK 3rd edition 1998

[19] V Ravichandran B SureshMN Sathishkumar K Elango andR Srinivasan ldquoAntifertility activity of hydroalcoholic extract ofAilanthus excelsa (Roxb) an ethnomedicines used by tribals ofNilgiris region in Tamilnadurdquo Journal of Ethnopharmacologyvol 112 no 1 pp 189ndash191 2007

[20] P J Elliott and S P Close ldquoNeuroleptic-induced catalepsyas a model of Parkinsonrsquos disease I Effect of dopaminergicagentsrdquo Journal of Neural Transmission Parkinsonrsquos Disease andDementia Section vol 2 no 2 pp 79ndash89 1990

[21] R Deumens A Blokland and J Prickaerts ldquoModeling Parkin-sonrsquos disease in rats an evaluation of 6-OHDA lesions of thenigrostriatal pathwayrdquo Experimental Neurology vol 175 no 2pp 303ndash317 2002

[22] S Wang L-F Hu Y Yang J-H Ding and G Hu ldquoStudies ofATP-sensitive potassium channels on 6-hydroxydopamine andhaloperidol rat models of Parkinsonrsquos disease implications fortreating Parkinsonrsquos diseaserdquo Neuropharmacology vol 48 no7 pp 984ndash992 2005

[23] D S Reddy and S K Kulkarni ldquoPossible role of nitric oxidein the nootropic and antiamnesic effects of neurosteroids onaging- and dizocilpine-induced learning impairmentrdquo BrainResearch vol 799 no 2 pp 215ndash229 1998

[24] S RajaSankar T Manivasagam V Sankar et al ldquoWithaniasomnifera root extract improves catecholamines and physiolog-ical abnormalities seen in a Parkinsonrsquos disease model mouserdquoJournal of Ethnopharmacology vol 125 no 3 pp 369ndash373 2009

[25] H Ohkawa N Ohishi and K Yagi ldquoAssay for lipid peroxidesin animal tissues by thiobarbituric acid reactionrdquo AnalyticalBiochemistry vol 95 no 2 pp 351ndash358 1979

[26] W F Beyer Jr and I Fridovich ldquoAssaying for superoxidedismutase activity some large consequences of minor changesin conditionsrdquo Analytical Biochemistry vol 161 no 2 pp 559ndash566 1987

[27] H Aebi ldquoCatalase in vitrordquo inMethods in Enzymology vol 105pp 121ndash126 Academic Press New York NY USA 1984

[28] S K Srivastava and E Beutler ldquoAccurate measurement ofoxidized glutathione content of human rabbit and rat redblood cells and tissuesrdquoAnalytical Biochemistry vol 25 pp 70ndash76 1968

[29] F Blandini and M-T Armentero ldquoAnimal models of Parkin-sonrsquos diseaserdquo FEBS Journal vol 279 no 7 pp 1156ndash1166 2012

[30] G Cohen and R E Heikkila ldquoThe generation of hydrogenperoxide superoxide radical and hydroxyl radical by 6-hydroxydopamine dialuric acid and related cytotoxic agentsrdquoThe Journal of Biological Chemistry vol 249 no 8 pp 2447ndash2452 1974

[31] D G Graham S M Tiffany W R Bell Jr andW F GutknechtldquoAutoxidation versus covalent binding of quinones as themechanism of toxicity of dopamine 6-hydroxydopamine andrelated compounds toward C1300 neuroblastoma cells in vitrordquoMolecular Pharmacology vol 14 no 4 pp 644ndash653 1978

[32] Y Y Glinka andM B H Youdim ldquoInhibition of mitochondrialcomplexes I and IV by 6-hydroxydopaminerdquo European Journalof Pharmacology vol 292 no 3-4 pp 329ndash332 1995

[33] Y Glinka M Gassen and M B H Youdim ldquoMechanism of 6-hydroxydopamine neurotoxicityrdquo Journal of Neural Transmis-sion Supplement no 50 pp 55ndash66 1997

[34] P Jenner and C W Olanow ldquoUnderstanding cell death inParkinsonrsquos diseaserdquoAnnals of Neurology vol 44 no 3 pp S72ndashS84 1998

[35] B Halliwell and J M C Gutteridge ldquoOxygen radicals and thenervous systemrdquoTrends inNeurosciences vol 8 pp 22ndash26 1985

[36] L SMonk K V Fagerstedt and RM Crawford ldquoOxygen toxi-city and superoxide dismutase as an antioxidant in physiologicalstressrdquo Physiologia Plantarum vol 76 no 3 pp 456ndash459 1989

[37] C Bowler M Van Montagu and D Inze ldquoSuperoxide dismu-tase and stress tolerancerdquoAnnual Review of Plant Physiology andPlant Molecular Biology vol 43 no 1 pp 83ndash116 1992

[38] J G Scandalios ldquoResponse of plant antioxidant defense genesto environmental stressrdquo Advances in Genetics vol 28 pp 1ndash411990

[39] E BGralla andD J Kosman ldquoMolecular genetics of superoxidedismutases in yeasts and related fungirdquo Advances in Geneticsvol 30 pp 251ndash319 1992

[40] P Jenner ldquoOxidative mechanisms in nigral cell death in Parkin-sonrsquos diseaserdquoMovementDisorders vol 13 no 1 pp 24ndash34 1998

[41] P Riederer E Sofic W-D Rausch et al ldquoTransition metalsferritin glutathione and ascorbic acid in Parkinsonian brainsrdquoJournal of Neurochemistry vol 52 no 2 pp 515ndash520 1989

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Advances in Pharmacological Sciences 9

[14] S K Panda N C Panda and B K Sahue ldquoPhytochemistryand pharmacological properties of Ficus religiosa an overviewrdquoIndian Veterinary Journal vol 60 pp 660ndash664 1976

[15] R S Verma and K S Bhatia ldquoChromatographic study of aminoacids of the leaf protein concentrates of Ficus religiosa Linn andMimusops elengi Linnrdquo Indian Journal of Pharmacy Practice vol23 pp 231ndash232 1986

[16] O A Aiyegoro and A I Okoh ldquoUse of bioactive plantproducts in combinationwith standard antibiotics implicationsin antimicrobial chemotherapyrdquo Journal of Medicinal PlantsResearch vol 3 no 13 pp 1147ndash1152 2009

[17] P K Warrier Indian Medicinal PlantsmdashA Compendium of 500Species vol 3 Orient Longman Chennai India 1996

[18] J B Harborne Phytochemical Methods Chapman and HallLondon UK 3rd edition 1998

[19] V Ravichandran B SureshMN Sathishkumar K Elango andR Srinivasan ldquoAntifertility activity of hydroalcoholic extract ofAilanthus excelsa (Roxb) an ethnomedicines used by tribals ofNilgiris region in Tamilnadurdquo Journal of Ethnopharmacologyvol 112 no 1 pp 189ndash191 2007

[20] P J Elliott and S P Close ldquoNeuroleptic-induced catalepsyas a model of Parkinsonrsquos disease I Effect of dopaminergicagentsrdquo Journal of Neural Transmission Parkinsonrsquos Disease andDementia Section vol 2 no 2 pp 79ndash89 1990

[21] R Deumens A Blokland and J Prickaerts ldquoModeling Parkin-sonrsquos disease in rats an evaluation of 6-OHDA lesions of thenigrostriatal pathwayrdquo Experimental Neurology vol 175 no 2pp 303ndash317 2002

[22] S Wang L-F Hu Y Yang J-H Ding and G Hu ldquoStudies ofATP-sensitive potassium channels on 6-hydroxydopamine andhaloperidol rat models of Parkinsonrsquos disease implications fortreating Parkinsonrsquos diseaserdquo Neuropharmacology vol 48 no7 pp 984ndash992 2005

[23] D S Reddy and S K Kulkarni ldquoPossible role of nitric oxidein the nootropic and antiamnesic effects of neurosteroids onaging- and dizocilpine-induced learning impairmentrdquo BrainResearch vol 799 no 2 pp 215ndash229 1998

[24] S RajaSankar T Manivasagam V Sankar et al ldquoWithaniasomnifera root extract improves catecholamines and physiolog-ical abnormalities seen in a Parkinsonrsquos disease model mouserdquoJournal of Ethnopharmacology vol 125 no 3 pp 369ndash373 2009

[25] H Ohkawa N Ohishi and K Yagi ldquoAssay for lipid peroxidesin animal tissues by thiobarbituric acid reactionrdquo AnalyticalBiochemistry vol 95 no 2 pp 351ndash358 1979

[26] W F Beyer Jr and I Fridovich ldquoAssaying for superoxidedismutase activity some large consequences of minor changesin conditionsrdquo Analytical Biochemistry vol 161 no 2 pp 559ndash566 1987

[27] H Aebi ldquoCatalase in vitrordquo inMethods in Enzymology vol 105pp 121ndash126 Academic Press New York NY USA 1984

[28] S K Srivastava and E Beutler ldquoAccurate measurement ofoxidized glutathione content of human rabbit and rat redblood cells and tissuesrdquoAnalytical Biochemistry vol 25 pp 70ndash76 1968

[29] F Blandini and M-T Armentero ldquoAnimal models of Parkin-sonrsquos diseaserdquo FEBS Journal vol 279 no 7 pp 1156ndash1166 2012

[30] G Cohen and R E Heikkila ldquoThe generation of hydrogenperoxide superoxide radical and hydroxyl radical by 6-hydroxydopamine dialuric acid and related cytotoxic agentsrdquoThe Journal of Biological Chemistry vol 249 no 8 pp 2447ndash2452 1974

[31] D G Graham S M Tiffany W R Bell Jr andW F GutknechtldquoAutoxidation versus covalent binding of quinones as themechanism of toxicity of dopamine 6-hydroxydopamine andrelated compounds toward C1300 neuroblastoma cells in vitrordquoMolecular Pharmacology vol 14 no 4 pp 644ndash653 1978

[32] Y Y Glinka andM B H Youdim ldquoInhibition of mitochondrialcomplexes I and IV by 6-hydroxydopaminerdquo European Journalof Pharmacology vol 292 no 3-4 pp 329ndash332 1995

[33] Y Glinka M Gassen and M B H Youdim ldquoMechanism of 6-hydroxydopamine neurotoxicityrdquo Journal of Neural Transmis-sion Supplement no 50 pp 55ndash66 1997

[34] P Jenner and C W Olanow ldquoUnderstanding cell death inParkinsonrsquos diseaserdquoAnnals of Neurology vol 44 no 3 pp S72ndashS84 1998

[35] B Halliwell and J M C Gutteridge ldquoOxygen radicals and thenervous systemrdquoTrends inNeurosciences vol 8 pp 22ndash26 1985

[36] L SMonk K V Fagerstedt and RM Crawford ldquoOxygen toxi-city and superoxide dismutase as an antioxidant in physiologicalstressrdquo Physiologia Plantarum vol 76 no 3 pp 456ndash459 1989

[37] C Bowler M Van Montagu and D Inze ldquoSuperoxide dismu-tase and stress tolerancerdquoAnnual Review of Plant Physiology andPlant Molecular Biology vol 43 no 1 pp 83ndash116 1992

[38] J G Scandalios ldquoResponse of plant antioxidant defense genesto environmental stressrdquo Advances in Genetics vol 28 pp 1ndash411990

[39] E BGralla andD J Kosman ldquoMolecular genetics of superoxidedismutases in yeasts and related fungirdquo Advances in Geneticsvol 30 pp 251ndash319 1992

[40] P Jenner ldquoOxidative mechanisms in nigral cell death in Parkin-sonrsquos diseaserdquoMovementDisorders vol 13 no 1 pp 24ndash34 1998

[41] P Riederer E Sofic W-D Rausch et al ldquoTransition metalsferritin glutathione and ascorbic acid in Parkinsonian brainsrdquoJournal of Neurochemistry vol 52 no 2 pp 515ndash520 1989

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of