Neuroprotective Effect of Juniperus communis on Chlorpromazine

Research ArticleNeuroprotective Effect of Juniperus communis onChlorpromazine Induced Parkinson Disease in Animal Model

Souravh Bais1 N S Gill2 and Nitan Kumar1

1Department of Pharmacology Rayat Institute of Pharmacy Railmajra Shaheed Bhagat Singh Nagar Punjab 144533 India2Department of Pharmaceutical Chemistry Rayat Institute of Pharmacy Railmajra Shaheed Bhagat Singh NagarPunjab 144533 India

Correspondence should be addressed to Souravh Bais souravh2008123rediffmailcom

Received 28 September 2014 Revised 8 January 2015 Accepted 26 January 2015

Academic Editor Qiusheng Zheng

Copyright copy 2015 Souravh Bais et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

We evaluated anti-Parkinsonrsquos activity of methanolic extract of Juniperus communis (MEJC) leaves in chlorpromazine (CPZ)induced experimental animal model In this study effects of Juniperus communis (100 and 200mgkg ip) were studied usingvarious behavior parameters like catalepsy (bar test) muscle rigidity (rotarod test) and locomotor activity (actophotometer) andits effect on neurochemical parameters (TBARS GSH nitrite and total protein) in rats The experiment was designed by givingchlorpromazine (3mgkg ip) for 21 days to induce Parkinsonrsquos disease-like symptoms Chlorpromazine significantly inducedmotor dysfunctions (catalepsy muscle rigidity and hypolocomotion) in a period of 21 days The MEJC significantly (119875 lt 0001)reduced catalepsy and muscle rigidity and significantly (119875 lt 0001) increased locomotor activity in rats The maximum reductionwas observed on the 21st day at a dose of 200mgkg (ip) The MEJC extract also showed an increase in the level of reducedgutathione (GSH) (119875 lt 0001) and total protein (119875 lt 0001) and decreased the elevated levels of TBARS (119875 lt 0001) and nitrite(119875 lt 0001) preferably at a higher dose (200mgkg) as compared to chlorpromazine group Thus the present study showed theneuroprotective effect of MEJC against CPZ induced Parkinsonrsquos disease-like symptoms or anti-Parkinsonrsquos activity

1 Introduction

Parkinsonrsquos disease (PD) is a neurodegenerative brain disor-der It ismainly characterized by progressive loss of dopamin-ergic and other neurons present in the substantia nigra parscompacta resulting in malfunctioning of the cerebral neu-ronal systems Clinically the disease may cause slowness ofmovement muscle rigidity and rest tremor [1] Pathologi-cally Parkinson disease may cause depletion of dopaminein brain due to the presence of intracytoplasmic inclusionsknown as Lewy bodies It is not clear why Lewy body forma-tion causes neuronal cell death These pathological changesare also seen in the locus coeruleus and parasympathetic aswell as sympathetic postganglionic neurons pedunculopon-tine nucleus raphe nucleus and dorsal motor nucleus of thevagal nerve [2] The Parkinson disease can be treated withvarious drugs including levodopa carbidopa orphenadrinebenztropine selegiline pergola andmanymore which act byreversing the symptoms of Parkinsonrsquos disease but these drugs

possess various side effects like nausea and vomiting respi-ratory disturbances hallucinations orange discoloration ofsaliva and urine mania dyskinesia convulsions and anxietyarrhythmia mydriasis dry mouth sore throat and transientdizziness on long term use [3]

Oxidative stress also plays a major role in the pathophysi-ology of PD Oxidative stress may cause neurodegenerationof neuronal cells Oxidative stress may cause depletion ofenzymes such as catalase superoxide dismutase and glu-tathione [4]

J communis L is a shrub belonging to family Cupres-saceae Lmainly found inHimachal Pradesh (India) at an alti-tude of 1500ndash4000 Fruit is subspherical purplish-black andseed contained 2-3 layers of thin-walled cells The seeds andfruits of the plant contain camphene d-120572-pinene formic acidacetic acid wax gum cyclohexinol terpene ascorbic aciddihydrojunene cadinene juniper and camphor [5ndash7] The Jcommunis (berry) oil mainly contained monoterpene hydro-carbons such as 120572-pinene (514) 120573-pinene (50) sabinene

Hindawi Publishing CorporationChinese Journal of BiologyVolume 2015 Article ID 542542 7 pageshttpdxdoiorg1011552015542542

2 Chinese Journal of Biology

(58) andmyrcene (83) [8] J communis can be used (tra-ditionally) for renal suppression acute and chronic cystitiscatarrh of the bladder albuminuria leucorrhoea and amen-orrhoea [9] J communis fruit can be used as being antisepticstimulant and styptic It can also be used in the treatmentof migraine infantile tuberculosis rheumatic and painfulswellings chronic Brightrsquos disease piles and nephroticdropsy of children [6] The plant was reported to have anal-gesic activity [10] antibacterial activity [11] hepatoprotectiveactivity [12] antihypercholesterolemic activity [13] antiin-flammatory activity [14] antidiabetic and antihyperlipidemicactivity [15] anticataleptic activity [16] and antimicrobial[17] activity Previous study indicates that Parkinsonrsquos diseaseoccurs due to the increased oxidative stress [18] and severalplants with antioxidant activity have been found to beeffective in the treatment of disease [8]Thus the study aimedat screening of the plant for its anti-Parkinsonrsquos activity

2 Materials and Methods

21 Animals Experiments were carried out on Wistar rats(150ndash200 g) of either sex and were obtained from the LokanThermometer (Ambala India) They were housed understandard lightdark cycle and fedwith standard pellet diet andwater ad libitum The experimental protocols were approvedby the Institutional Animal Ethics Committee and conductedaccording to the guidelines of Committee for the Purposeof Control and Supervision of Experiments on Animals(CPCSEA) New Delhi India

22 Procurement of Plant Material Standardized dry meth-anolic extract of leaves was obtained from Amsar Pvt LtdIndore (M P)The extract dose was prepared using 2 CMC(suspending agents) prior to administration

23 Drugs and Chemicals Chlorpromazine levodopa andcarbidopa were procured as gift sample from Sun pharma-ceuticals (Baddi India) All chemicals are of analytical gradeand are purchased from Spruce Enterprises Ambala Cantt(Punjab India)

24 Phytochemical Evaluation The methanolic extract of Jcommunis was screened by different chemical tests to test thepresence of the active chemical constituents such as flavon-oids alkaloids steroids terpenoids glycosides and phenoliccompounds These tests were performed to get a preliminaryidea of the chemical constituents [19ndash21]

25 Determination of LD50 LD50

of the extract was deter-mined following the Organizations for Economic Cooper-ation and Development (OECD) guidelines Acute toxicityclass method (OECD guideline no 423) and revised up-and-down method (OECD guideline no 425) were followed forthe testing of chemicals Animals were observed for 24 hoursfor toxic symptoms such as behavioral changes convulsionslocomotion and mortality If one animal showed mortalitythen the same dose was repeated again to confirm the toxicdose If mortality was not observed then the same procedurewas repeated for further higher doses [22 23]

26 Methodology Rats were randomly allocated into fourgroups each containing 6 animals Animals of Group Iwere administered with 1 gum acacia as a vehicle andserved as control group Group II animals were administeredwith chlorpromazine 3mgkg ip (dissolved with 1 gumacacia in distilled water suspension) daily for a period of 21days and served as the negative control group Group IIIanimals received the combination of carbidopa + levodopa(1 10 ratio) (10mgkg ip) and served as standard groupGroup IVa and Group IVb animals were administered withmethanolic extract of J communis (MEJC) 100mgkg ipand 200mgkg ip respectively for a period of 21 daysChlorpromazine was given 30 minutes prior to standard andtest drug Body weight changes and behavioral assessmentswere carried out before the start of the treatment Vari-ous parameters like catalepsy (bar test) locomotor activity(actophotometer test) andmuscle activity (rotarod test) weremeasured in all animals [24ndash26] After the 21 days animalswere sacrificed and their brains were removed and weighedA 10 tissue homogenate was prepared in 01M phosphatebuffer (pH 8) for TBARS GSH nitrites and total protein

27 Biochemical Estimation

271 Lipid Peroxidation Assay (TBARS) Thiobarbituric acidreactive substances (TBARS) measurement is an index oflipid peroxidation in brain For the estimation of TBARSten percent (wv) tissue homogenate was mixed with sodiumdodecyl sulfate acetate buffer (pH 35) and aqueous solutionof thiobarbituric acid After heating at 95∘C for 60minthe red pigment produced was extracted with n-butanol-pyridine mixture and estimated by the absorbance at 532 nmAs an external standard tetramethoxypropane was usedand lipid peroxide level was expressed in terms of nmolmalondialdehyde [27]

272 Estimation of Reduced Glutathione (GSH) For the esti-mation of reduced glutathione the 1ml of tissue homogenatewas precipitated with 1ml of 10 trichloroacetic acid (TCA)To an aliquot of the supernatant 4ml of phosphate solutionand 05ml of 551015840-dithio-bis-(2-nitrobenzoic acid) (DTNB)reagent were added and absorbance was taken at 412 nm Astandard curve of reduced glutathione was prepared and theconcentration of GSH in the supernatant was determinedfrom the standard curve [28]

273 Estimation of Nitrite The production of nitric oxide(NO) in the brainmay occur due to oxidative stress and it canbe determined by estimation of nitrite level The nitrite levelwas determined spectrophotometrically with Griess reagent(01 N-1-naphthyl ethylene amine dihydrochloride 1 sul-phanilamide and 25 phosphoric acid) Brain homogenateand Griess reagent were mixed equally and this mixture wasincubated for 10min and the absorbance was measured at546 nm The standard curve of sodium nitrite was preparedand the concentration of nitrite in the supernatant wasdetermined from standard curve [29]

274 Estimation of Protein For the estimation of proteincontent of brain Lowry method was used Standard curvewas determined using bovine serum albumin [30]

Chinese Journal of Biology 3

28 Statistical Analysis Data were analyzed with one-wayANOVAs followed by Tukeyrsquos post hoc tests with significanceset to 119875 lt 0001 All analyses were performed using SigmaStat (100) Results obtained were expressed as mean plusmn SEM

3 Results

31 Acute Toxicity (LD50) The Juniperus communis did not

produce any mortality up to a dose level of 2000mgkg bodyweight Juniperus communis extract showed less solubilityso further increment of doses was not possible (accordingto OECD-423) So the two dose ranges 100mgkg and200mgkg were selected

32 Phytochemical Screening of Methanolic Extract of Junipe-rus communis Leaves Phytochemical screening of methano-lic extract of Juniperus communis leaves showed the presenceof alkaloids flavonoids glycosides steroids and tannins

33 Effect of MEJC on Chlorpromazine Induced Catalepsy inRats All the animals were evaluated for catalepsy using bartest for 2ndash4 sec during weekly observation and at last on the21st day of treatment The control animals were evaluated fortheir catalepsy score (21ndash23 sec) during the entire observa-tion period

Animals of Group II were found to retain themselves fora longer duration as compared to the control Group (GroupI) The cataleptic behavior (inability to correct abnormalposture) of CPZ treated animals (Group II) was found toincrease significantly every weak (increase in mean retentiontime from 7 sec on the 14th day to 11 sec on the 21st day)and also on the 21st day treatment (15 sec 119875 lt 0001) whencompared to vehicle treated control group (Group I) animals

The animals treated with levodopa and carbidopa(10mgkg ip) group significantly (119875 lt 0001) preventedthe increase in catalepsy when compared to CPZ treatedanimals (Group II) on the 21st day The retention time ofanimals (Group III) on bar was found to reduce significantly(529 sec 119875 lt 0001) on the 21st day as compared to 229 sec(NS) 321 sec (119875 lt 0001) and 445 sec (119875 lt 0001) onthe 0th the 7th the 14th and the 21st days of treatment incomparison to CPZ treated negative control group (GroupII) The animals pretreated with the different doses of MEJC(100 and 200mgkg ip) showed a significant decrease incatalepsy score when compared to CPZ treated animals TheMEJC at a dose (200mgkg ip) was found to be significantlyon the 21st day (52 sec 119875 lt 0001) as compared to 23 (NS)337 (119875 lt 001) and 501 (119875 lt 0001) on the 0th the 7th the14th and the 21st days of treatment in comparison to CPZtreated negative control group (Group II) on respective days(Figure 1)

34 Effect of MEJC on Chlorpromazine Induced Muscle Rigid-ity in Rats Muscle rigidity of animals was evaluated byusing the rotarod apparatusThemean fall-off time of vehicletreated control group (Group I) animals from the rotarodwasfound to be 1162ndash1165 seconds during weekly observation ofthe treatment

Days

Cata

leps

y (s

)

0th day 7th day 14th day 21st day0

5

10

15

20

IIIIII

IVaIVb

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowast


Figure 1 Effect of MEJC on chlorpromazine induced catalepsy inrats lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 as compared to vehicletreated control group (Group I) 119875 lt 005 119875 lt 001 and

119875 lt

0001 as compared to chlorpromazine treated negative control group(Group II)

CPZ treated groups (Group II) showed a nonsignificantdifference in muscle rigidity on the 0th day (1039 plusmn 2730)which then significantly reduced every week The rotarodreadings (muscle rigidity) decreased to 835 plusmn 3940 on the7th day to 6029 plusmn 1720 on the 14th day and finally to 385 plusmn1730 on the 21st day Then there was a significant decrease(119875 lt 0001) in the muscle activity of CPZ treated group whencompared to control group

The animals treated with standard drug (10mgkg ip)also showed a nonsignificant increase in muscle activity onthe 0th day (1118 plusmn 3990) which then significantly increased(119875 lt 0001) on the 7th day (10974 plusmn 4730) the 14th day(995 plusmn 2050) and the 21st day (975 plusmn 2110) when comparedto CPZ treated (Group II) animals on the same day whereaspretreatment with MEJC (Group IV) at a dose of 100mgkgip showed a rotarod reading 1063 plusmn 1930 on the 0th dayand a significant increase (119875 lt 005) in muscle activity on the7th day of treatment as compared to Group II A significantincreased (119875 lt 001) activity was observed on the 14th day(715 plusmn 1890) and on the 21st day (652 plusmn 3760)

The dose of MEJC (200mgkg ip) also showed a signif-icant activity (119875 lt 001) on the 7th day (1045 plusmn 4150) ascompared to Group II A significant activity (119875 lt 0001) wasobserved on the 14th day (895 plusmn 2340) and on the 21st day(7573plusmn 5320) as compared to negative control group (GroupII) (Figure 2)

35 Effect of MEJC on Chlorpromazine Induced Hypolocomo-tion in Rats Locomotor activity of animals was evaluatedusing actophotometer Locomotor activity of vehicle treatedcontrol group (Group I) was found to be 71ndash73 counts5minfor all the four weeks of treatment

CPZ treatment of animals of Group II showed a non-significant difference in locomotion on the 0th day (7193 plusmn1930) which then significantly reduced every week Theactophotometer readings decreased to 575 plusmn 1980 on the 7thday to 425 plusmn 2870 (119875 lt 0001) on the 14th day and finally


Days

Mea

n fa

ll-off

tim

e (s)


50

100

150

IIIIII

IVaIVb

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowastlowast lowastlowast

lowastlowastlowastlowastlowast

lowastlowastlowast

Figure 2 Effect of MEJC on chlorpromazine induced musclerigidity in rats lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 ascompared to vehicle treated control group (Group I)

119875 lt 005119875 lt 001 and

119875 lt 0001 as compared to chlorpromazine treatednegative control group (Group II)

to 335 plusmn 1230 on the 21st day Then there was a significantdecrease (119875 lt 0001) in the locomotor activity of CPZtreated negative control animals (Group II) when comparedto control group (Group I)

Animals treated with standard drug (10mgkg ip)showed a nonsignificant variance in locomotor activity on the0th day (6998 plusmn 2730) which then significantly increased inlocomotor activity on the 7th day (7143 plusmn 2820) on the 14thday (635 plusmn 1770 119875 lt 0001) and on the 21st day (6221 plusmn1230 119875 lt 0001) Thus there was a significant increase (119875 lt0001) in the locomotor activity of Group III when comparedto Group I whereas pretreatment with MEJC (Group IV)showed significant (119875 lt 0001) changes in locomotor activityAnimals treated with the 1st dose (100mgkg ip) showed anonsignificant difference in the 0th day (7132 plusmn 2070) andthen a gradual increase in locomotor activity on the 7th day(655 plusmn 1120 119875 lt 005) on the 14th day (525 plusmn 2430 119875 lt005) and finally on the 21st day (5723 plusmn 1830 119875 lt 0001)

Animals treated with second dose (200mgkg ip) alsoshowed a highly significant activity on the 21st day (6002 plusmn2830 119875 lt 0001) when compared to negative controlgroup (Group II) The present study showed that the MEJChas significant protection in CPZ induced hypolocomotion(Figure 3)

36 Effect of MEJC on the TBARS Level in Rat Brain TheTBARS level in control group (Group I) animals was found tobe 1736 plusmn 08320 nMmg proteins and that of animals treatedwith levodopa and carbidopa (Group II) was found to be1876 nMmg proteinsThen there was a significant difference(119875 lt 001) found between these two groups

The animals treated with CPZ (Group II) showed asignificant increase in TBARS level in brain ((4675 plusmn0035) nMmg protein 119875 lt 0001) of CPZ treated animalsas compared to control group (Group I) Pretreatment withMEJC (Group IVa and Group IVb) showed a significantreduction in level of TBARS in brain (IVa-2377 plusmn 01240 andIVb-193 plusmn 09432 119875 lt 001) when compared to CPZ treatedanimals (Group II) respectively (Table 1)

Days0th day 7th day 14th day 21st day

0

20

40

60

80

100

IIIIII

IVaIVb

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast lowastlowastlowastlowastlowast lowastlowastlowast

lowast

Loco

mot

or ac

tivity

(cou

nts5

min

)

Figure 3 Effect of MEJC on chlorpromazine induced hypolocomo-tion in rats lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 as comparedto vehicle treated control group (Group I) 119875 lt 005 119875 lt 001and 119875 lt 0001 as compared to chlorpromazine treated negative

control group (Group II)

37 Effect of MEJC on the GSH Level in Rat Brain GSHlevel in control group (Group I) animals was found to be5826plusmn 06670120583Mmg proteinThe animals treatedwith CPZ(Group II) showed a significant decrease in GSH levels inbrain (1920 plusmn 00435119875 lt 001) as compared to control group(Group I) animals

The GSH level in Group III treated with standard drugwas increased up to (4654 plusmn 0720) as compared to CPZtreated animals (Group II) The pretreatment with MEJCshowed a significant increase in level of GSH in brain (IVa-4092 plusmn 05350 119875 lt 0005 and IVb 52874 plusmn 0572 119875 lt0001) when compared to CPZ treated animals (Group II)respectively (Table 1)

38 Effect of MEJC on the Nitrite Level in Rat Brain Nitritelevel in control group (Group I) animals was found to be2941 plusmn 04250 nMmg proteins Chronic CPZ treatment torats for 21 days induced lipid peroxidation indicated by asignificant rise in nitrite level in brain (6143 plusmn 05210) 119875 lt0001 as compared with control group animals (Group I)

The animals treated with standard drug (Group III) hadsignificantly reduced the nitrite level to (3375 plusmn 06260) 119875 lt0001 as compared to negative control group (Group II) Theanimals pretreated with two doses of MEJC also showeda significant reduction in nitrite level (Group IVa-3442 plusmn07370 119875 lt 005 Group IVb-3102 plusmn 04600 119875 lt 0001)respectively as compared to negative control animals (GroupII) (Table 1)

39 Effect of MEJC on the Total Protein Level in Rat BrainProtein level in control group (Group I) was found to be11143 plusmn 05160 and level was significantly decreased (119875 lt0001) in chronic treatment with CPZ group (Group II)(6143 plusmn 05210) as compared to control or vehicle treatedgroup (Group I) animals The animals treated with lev-odopa+carbidopa (Group III) showed a significant increasein protein level (9953 plusmn 07120mgmL 119875 lt 0001) in brain ascompared with negative group (Group II)


Table 1 Effect of MEJCon the different parameters in rat brain

GroupsGSH level

(nMmg protein)mean plusmn SEM

Nitrite level(nMmg of protein)

mean plusmn SEM

Total protein level(mgmL)

mean plusmn SEM

TBARS level(nMmg protein)mean plusmn SEM

I (control group) 5826 plusmn 06670 2941 plusmn 04250 11143 plusmn 05160 1736 plusmn 008320II (negative control) 1920 plusmn 00435a 6776 plusmn 05260a 6143 plusmn 05210a 4675 plusmn 00350a

III (standard group) 4654 plusmn 00720NSy 3375 plusmn 06260NSx 9953 plusmn 07120NSx 1876 plusmn 00270y

IVa (Test 1) 4092 plusmn 05350NSz 3442 plusmn 07370NSy 7571 plusmn 003730az 2377 plusmn 01240NSy

IVb (Test 2) 52874 plusmn 05720NSx 3102 plusmn 04600NSx 898 plusmn 04230cy 193 plusmn 09432NSy

a = lowast lowast lowast (119875 lt 0001) b = lowastlowast (119875 lt 001) and c = lowast (119875 lt 005) when compared with control groupx = (119875 lt 0001) y = (119875 lt 001) and z = (119875 lt 005) when compared with negative control group

The animals pretreated with MEJC (100mgkg and200mgkg ip) also showed a significant increase in proteinlevel (IVa-7571 plusmn 003730 119875 lt 005 IVb-898 plusmn 04230119875 lt 001) in brain as compared to negative group (GroupII) animals (Table 1)

4 Discussion

Parkinson is a brain disorder which is clinically characterizedby bradykinesia resting tremor and postural instability InParkinson disease the depletion of dopamine (DA) mayoccur Parkinson disease occurs due to the degeneration ofdopaminergic neurons in the substantia nigra pars compacta[31] Various plants have been used for the treatment of neu-rodegenerative disease like Parkinson disease Various animalmodels have been developed for the evaluation of Parkinsonrsquosdisease and are generated through the administration oftoxins The purpose of the present study was to evaluate theantiparkinson activity of J communis leaves in rats treatedwith chlorpromazine

Chlorpromazine is one of the antipsychotic drugs listed asessential drugs by WHO in 2003 [32ndash34] to treat both acutepsychosis and chronic psychosis It has been associated withside effects such as antidopaminergic extra pyramidal syn-dromes [35] drymouth blurred vision andurinary retention(anticholinergic) [36] narcoleptic dysphoria blood pressuredisturbances temperature and muscle control diminishedlibido erectile impotence and ejaculation inhibition in malepatients [37] Chronic treatments with CPZ increase thedopamine receptor binding site in neostriatum and inmesolimbic region which could account for dopaminehypersensitivity that induced tardive dyskinesia [38] Chlor-promazine induced Parkinsonism by interfering with thestorage of catecholamines in intracellular granules whichmaycause monoamine depletion in nerve terminals and in theinduction of hypolocomotion and muscular rigidity [39ndash42] thus CPZ was produced by the Parkinson disease-likesymptoms [43] followed by chronic treatments of rats for 21days It results in an increase level of oxidative stress that maycause the reduction in antioxidant enzymes which were alsoseen with the atypical agentrsquos ziprasidone risperidone andolanzapine Except olanzapine both typical and atypicalagents increase lipid peroxidation after chronic dosing [36]There was a significant increase in catalepsy a decreasein movements and a decrease in body weight following

chlorpromazine administration to rats The present data sug-gested that chlorpromazine developed Parkinsonrsquos disease-like behavioral symptoms in rats The oxidative stress wasmeasured throughdetermination of levels of TBARS reducedglutathione and nitrite level in brain tissue Lipid peroxida-tion is a sensitive marker of oxidative stress Lipid peroxida-tion occurs due to attack by radicals on double bond ofunsaturated fatty acid and arachidonic acid which generatelipid peroxyl radicals These radicals have further attacks onother unsaturated fatty acids [44] Increased levels of the lipidperoxidation product have been found in the substantia nigraof PD patients [40] In the present study the same resultwas observed in the brain homogenates of chlorpromazinetreated negative control animals Brain protects againstoxidative stress by SOD catalase and glutathione peroxidaseand thus these antioxidant enzymes protect brain from neu-rodegeneration Glutathione peroxidase protects brain fromneurodegeneration by scavengingH

2O2generated by cellular

metabolism and balance formation and decomposition ofH2O2in normal condition It is obvious that reduced glu-

tathione is the limiting factor in the removal of H2O2 Neu-

ronal cell loss may cause the depletion of reduced glutathionein the substantia nigra in Parkinson disease [45] Nitricoxide production can be determined by nitrite determina-tions in biological material We detected nitrite in the ratbrain homogenates by the Griess method [46] Nitric oxidehas been involved in the cytotoxicities by activation ofmacro-phages or excess stimulation of neurons by glutamate Infurther study on glutamate stimulation causes neurotoxicityin primary cultures of rat fetal cortical striatal and hip-pocampal neurons [29] CPZ group showed a significantincrease in the level of TBARS and gradual decrease in GSHlevels in brain as compared control group All observationsshowed that chlorpromazine increases the oxidative stressin the brain of animals Two doses of methanolic extractof J communis (100 and 200mgkg ip) were used inthe chlorpromazine model in rats both doses were foundto be significant in reducing the catalepsy increasing thelocomotor activity (actophotometer) and increasing themuscle activity (rotarod test) in a chlorpromazine model ofParkinson in rats which indicates J communis has potentialeffects against Parkinsonrsquos disease-like symptoms producedin various experimental modelsThe antioxidative propertiesof J communis reduced the duration of the catalepsy thatdecreased the elevated levels of lipid peroxidation in thechlorpromazine treated animals


5 Conclusion

Juniperus communis earlier proved to be an antioxidantand showed a promising effect in animals with Parkinsonrsquosdisease The above findings suggest that Juniperus communismay offer a safer therapeutic approach to the treatment ofParkinsonrsquos disease

Conflict of Interests

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

References

[1] K L Leenders and W H Oertel ldquoParkinsonrsquos disease clinicalsigns and symptoms neural mechanisms positron emissiontomography and therapeutic interventionsrdquo Neural Plasticityvol 8 no 1-2 pp 99ndash110 2001

[2] H Braak U Rub W P Gai and K Del Tredici ldquoIdiopathicParkinsonrsquos disease possible routes by which vulnerable neu-ronal types may be subject to neuroinvasion by an unknownpathogenrdquo Journal of Neural Transmission vol 110 no 5 pp517ndash536 2003

[3] A M Karch Focus on Nursing Pharmacology LippincottWilliams ampWilkins Philadelphia Pa USA 5th edition 2009

[4] S Nikam PNikam S K Ahaley andAV Sontakke ldquoOxidativestress in Parkinsonrsquos diseaserdquo Indian Journal of Clinical Biochem-istry vol 24 no 1 pp 98ndash101 2009

[5] S J Clifton L KWard and D S Ranner ldquoThe status of juniperJuniperus communis L In North-East Englandrdquo Biological Con-servation vol 79 no 1 pp 67ndash77 1997

[6] C Kailash B G Chaudhari B P Dhar et al Database onMedicinal Plants Used in Ayurveda vol 8 Central Council forResearch in Ayurveda amp Siddha 2007

[7] S Bais N S Gill N Rana and S Shandil ldquoA phytopharma-cological review on a medicinal plant Juniperus communisrdquoInternational Scholarly Research Notices vol 2014 Article ID634723 6 pages 2014

[8] M Hoferl I Stoilova E Schmidt et al ldquoChemical compo-sition and antioxidant properties of juniper berry (Juniperuscommunis L) essential oil Action of the essential oil onthe antioxidant protection of Saccharomyces cerevisiae modelorganismrdquo Antioxidants vol 3 no 1 pp 81ndash98 2014

[9] C P Khare Indian Medicinal Plants Springer New York NYUSA 2007

[10] S Banerjee A Mukherjee and T K Chatterjee ldquoEvaluationof analgesic activities of methanolic extract of medicinal plantJuniperus communis linnrdquo International Journal of Pharmacyand Pharmaceutical Sciences vol 4 no 5 pp 547ndash550 2012

[11] S C Sati and S Joshi ldquoAntibacterial potential of leaf extractsof Juniperus communis L from Kumaun Himalayardquo AfricanJournal of Microbiology Research vol 4 no 12 pp 1291ndash12942010

[12] G G P Manvi ldquoScreening and evaluation of pharmacognosticphytochemical and hepatoprotective activity of linn stemsrdquoInternational Journal of Pharma and Bio Sciences vol 1 no 32010

[13] M Akdogan A Koyu M Ciris and K Yildiz ldquoAnti-hyper-cholesterolemic activity of Oil in rats a biochemical and

histopathological investigationrdquo Biomedical Research vol 23no 3 pp 321ndash328 2012

[14] H Tunon C Olavsdotter and L Bohlin ldquoEvaluation of anti-inflammatory activity of some Swedish medicinal plants Inhi-bition of prostaglandin biosynthesis and PAF-induced exocy-tosisrdquo Journal of Ethnopharmacology vol 48 no 2 pp 61ndash761995

[15] S Banerjee H Singh and T K Chatterjee ldquoEvaluation ofanti-diabetic and anti-hyperlipidemic potential of methano-lic extract of Juniperus Communis (L) in Streptozotocin-Nicotinamide induced diabetic ratsrdquo International Journal ofPharma and Bio Sciences vol 4 no 3 pp P10ndashP17 2013

[16] S Bais N S Gill and N Rana ldquoEffect of Juniperus communisextract on reserpine induced catalepsyrdquo Inventi Rapid Ethno-pharmacology vol 2014 no 4 pp 117ndash120 2014

[17] S Pepeljnjak I Kosalec Z Kalodera and N Blazevic ldquoAntimi-crobial activity of juniper berry essential oil (Juniperus commu-nis L Cupressaceae)rdquo Acta Pharmaceutica vol 55 no 4 pp417ndash422 2005

[18] L Chen Y Ding B Cagniard et al ldquoUnregulated cytosolicdopamine causes neurodegeneration associated with oxidativestress in micerdquo Journal of Neuroscience vol 28 no 2 pp 425ndash433 2008

[19] C K Kokate Practical Pharmacognosy Vallabh Prakashan2005

[20] K R Khandelwal Practical Pharmacognosy Techniques andExperiments 11th edition 2004

[21] G E Trease andW C Evans Pharmacognosy Bailliere TindallLondon UK 11th edition 1989

[22] OECDOCDE 423 Annex 2 ldquoProcedure to be followed foreach of the starting dosesrdquo in OECD Guideline For Testing ofChemicals Acute Oral Toxicity-Acute Toxic Class Method pp114ndash1414 2001

[23] D J EcobichnonThe Basis of Toxicity Testing CRC Press NewYork NY USA 2nd edition 1997

[24] B Costall and R J Naylor ldquoOn catalepsy and catatonia andthe predictability of the catalepsy test for neuroleptic activityrdquoPsychopharmacologia vol 34 no 3 pp 233ndash241 1974

[25] M Bishnoi K Chopra and S K Kulkarni ldquoInvolvement ofadenosinergic receptor system in an animal model of tardivedyskinesia and associated behavioural biochemical and neuro-chemical changesrdquo European Journal of Pharmacology vol 552no 1ndash3 pp 55ndash66 2006

[26] S K Kulkarni Handbook of Experimental Pharmacology Val-labh Prakashan New Delhi India 2008

[27] 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

[28] M S Moron J W Depierre and B Mannervik ldquoLevels of glu-tathione glutathione reductase and glutathione S-transferaseactivities in rat lung and liverrdquo Biochimica et Biophysica Actavol 582 no 1 pp 67ndash78 1979

[29] R Lidija S Vesna J Branka and T L Dajana ldquoEffect ofglutamate antagonists on nitric oxide production in rat brainfollowing intrahippocampal injectionrdquo Archives of BiologicalSciences vol 59 no 1 pp 29ndash36 2007

[30] O H Lowry N J Rosenbrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

[31] C W Olanow and W G Tatton ldquoEtiology and pathogenesis ofParkinsonrsquos diseaserdquoAnnual Review of Neuroscience vol 22 pp123ndash144 1999


[32] WorldHealth Organization (WHO) EssentialMedicinesWHOModel List 13th edition 2007

[33] World Health Organization Management of Substance Abusesthe Bare Facts WHO 2010

[34] A Pillai A V Terry Jr and S P Mahadik ldquoDifferential effectsof long-term treatment with typical and atypical antipsychoticson NGF and BDNF levels in rat striatum and hippocampusrdquoSchizophrenia Research vol 82 no 1 pp 95ndash106 2006

[35] J M Pierre ldquoExtrapyramidal symptoms with atypical antipsy-chotics incidence prevention and managementrdquo Drug Safetyvol 28 no 3 pp 191ndash208 2005

[36] H R Greenberg ldquoInhibition of ejaculation by chlorpro-mazinerdquo Journal of Nervous and Mental Disease vol 152 no5 pp 364ndash366 1971

[37] R M Allen ldquoDopamine hypersensitivity and tardive dyskine-siardquo The American Journal of Psychiatry vol 134 no 10 pp1154ndash1155 1977

[38] S K Kulkarni Handbook of Experimental Pharmacology Val-labh Prakashan New York NY USA 3rd edition 1999

[39] D T Dexter A E Holley W D Flitter et al ldquoIncreased levelsof lipid hydroperoxides in the Parkinsonian substantia nigra anHPLC and ESR studyrdquoMovement Disorders vol 9 no 1 pp 92ndash97 1994

[40] V Parikh M M Khan and S P Mahadik ldquoDifferential effectsof antipsychotics on expression of antioxidant enzymes andmembrane lipid peroxidation in rat brainrdquo Journal of PsychiatricResearch vol 37 no 1 pp 43ndash51 2003

[41] K D Tripathi Essentials of Medical Pharmacology JaypeeBrothers Medical Publishers 6th edition 2008

[42] K S Sandhu and A C Rana ldquoEvaluation of anti parkinsonrsquosactivity of Nigella sativa (kalonji) seeds in chlorpromazineinduced experimental animal modelrdquo International Journal ofPharmacy and Pharmaceutical Sciences vol 5 p 3 2013

[43] K J Barnham C L Masters and A I Bush ldquoNeurodegen-erative diseases and oxidative stressrdquo Nature Reviews DrugDiscovery vol 3 no 3 pp 205ndash214 2004

[44] G Nikolova ldquoOxidative stress and Parkinson diseaserdquo TrakiaJournal of Sciences vol 10 no 1 pp 171ndash172 2012

[45] I Guevara J Iwanejko A Dembinska-Kiec et al ldquoDetermina-tion of nitritenitrate in human biologicalmaterial by the simpleGriess reactionrdquo Clinica Chimica Acta vol 274 no 2 pp 177ndash188 1998

[46] V L Dawson T M Dawson D A Bartley G R Uhl and S HSnyder ldquoMechanisms of nitric oxide-mediated neurotoxicity inprimary brain culturesrdquoThe Journal of Neuroscience vol 13 no6 pp 2651ndash2661 1993

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


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Evolutionary BiologyInternational Journal of



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Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

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Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


(58) andmyrcene (83) [8] J communis can be used (tra-ditionally) for renal suppression acute and chronic cystitiscatarrh of the bladder albuminuria leucorrhoea and amen-orrhoea [9] J communis fruit can be used as being antisepticstimulant and styptic It can also be used in the treatmentof migraine infantile tuberculosis rheumatic and painfulswellings chronic Brightrsquos disease piles and nephroticdropsy of children [6] The plant was reported to have anal-gesic activity [10] antibacterial activity [11] hepatoprotectiveactivity [12] antihypercholesterolemic activity [13] antiin-flammatory activity [14] antidiabetic and antihyperlipidemicactivity [15] anticataleptic activity [16] and antimicrobial[17] activity Previous study indicates that Parkinsonrsquos diseaseoccurs due to the increased oxidative stress [18] and severalplants with antioxidant activity have been found to beeffective in the treatment of disease [8]Thus the study aimedat screening of the plant for its anti-Parkinsonrsquos activity

2 Materials and Methods

21 Animals Experiments were carried out on Wistar rats(150ndash200 g) of either sex and were obtained from the LokanThermometer (Ambala India) They were housed understandard lightdark cycle and fedwith standard pellet diet andwater ad libitum The experimental protocols were approvedby the Institutional Animal Ethics Committee and conductedaccording to the guidelines of Committee for the Purposeof Control and Supervision of Experiments on Animals(CPCSEA) New Delhi India

22 Procurement of Plant Material Standardized dry meth-anolic extract of leaves was obtained from Amsar Pvt LtdIndore (M P)The extract dose was prepared using 2 CMC(suspending agents) prior to administration

23 Drugs and Chemicals Chlorpromazine levodopa andcarbidopa were procured as gift sample from Sun pharma-ceuticals (Baddi India) All chemicals are of analytical gradeand are purchased from Spruce Enterprises Ambala Cantt(Punjab India)

24 Phytochemical Evaluation The methanolic extract of Jcommunis was screened by different chemical tests to test thepresence of the active chemical constituents such as flavon-oids alkaloids steroids terpenoids glycosides and phenoliccompounds These tests were performed to get a preliminaryidea of the chemical constituents [19ndash21]

25 Determination of LD50 LD50

of the extract was deter-mined following the Organizations for Economic Cooper-ation and Development (OECD) guidelines Acute toxicityclass method (OECD guideline no 423) and revised up-and-down method (OECD guideline no 425) were followed forthe testing of chemicals Animals were observed for 24 hoursfor toxic symptoms such as behavioral changes convulsionslocomotion and mortality If one animal showed mortalitythen the same dose was repeated again to confirm the toxicdose If mortality was not observed then the same procedurewas repeated for further higher doses [22 23]

26 Methodology Rats were randomly allocated into fourgroups each containing 6 animals Animals of Group Iwere administered with 1 gum acacia as a vehicle andserved as control group Group II animals were administeredwith chlorpromazine 3mgkg ip (dissolved with 1 gumacacia in distilled water suspension) daily for a period of 21days and served as the negative control group Group IIIanimals received the combination of carbidopa + levodopa(1 10 ratio) (10mgkg ip) and served as standard groupGroup IVa and Group IVb animals were administered withmethanolic extract of J communis (MEJC) 100mgkg ipand 200mgkg ip respectively for a period of 21 daysChlorpromazine was given 30 minutes prior to standard andtest drug Body weight changes and behavioral assessmentswere carried out before the start of the treatment Vari-ous parameters like catalepsy (bar test) locomotor activity(actophotometer test) andmuscle activity (rotarod test) weremeasured in all animals [24ndash26] After the 21 days animalswere sacrificed and their brains were removed and weighedA 10 tissue homogenate was prepared in 01M phosphatebuffer (pH 8) for TBARS GSH nitrites and total protein

27 Biochemical Estimation

271 Lipid Peroxidation Assay (TBARS) Thiobarbituric acidreactive substances (TBARS) measurement is an index oflipid peroxidation in brain For the estimation of TBARSten percent (wv) tissue homogenate was mixed with sodiumdodecyl sulfate acetate buffer (pH 35) and aqueous solutionof thiobarbituric acid After heating at 95∘C for 60minthe red pigment produced was extracted with n-butanol-pyridine mixture and estimated by the absorbance at 532 nmAs an external standard tetramethoxypropane was usedand lipid peroxide level was expressed in terms of nmolmalondialdehyde [27]

272 Estimation of Reduced Glutathione (GSH) For the esti-mation of reduced glutathione the 1ml of tissue homogenatewas precipitated with 1ml of 10 trichloroacetic acid (TCA)To an aliquot of the supernatant 4ml of phosphate solutionand 05ml of 551015840-dithio-bis-(2-nitrobenzoic acid) (DTNB)reagent were added and absorbance was taken at 412 nm Astandard curve of reduced glutathione was prepared and theconcentration of GSH in the supernatant was determinedfrom the standard curve [28]

273 Estimation of Nitrite The production of nitric oxide(NO) in the brainmay occur due to oxidative stress and it canbe determined by estimation of nitrite level The nitrite levelwas determined spectrophotometrically with Griess reagent(01 N-1-naphthyl ethylene amine dihydrochloride 1 sul-phanilamide and 25 phosphoric acid) Brain homogenateand Griess reagent were mixed equally and this mixture wasincubated for 10min and the absorbance was measured at546 nm The standard curve of sodium nitrite was preparedand the concentration of nitrite in the supernatant wasdetermined from standard curve [29]

274 Estimation of Protein For the estimation of proteincontent of brain Lowry method was used Standard curvewas determined using bovine serum albumin [30]



3 Results








Days

Cata

leps

y (s

)


5

10

15

20

IIIIII

IVaIVb

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast




119875 lt








Days

Mea

n fa

ll-off

tim

e (s)


50

100

150

IIIIII

IVaIVb

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast



lowastlowastlowast


119875 lt 005119875 lt 001 and








0

20

40

60

80

100

IIIIII

IVaIVb

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast


lowast

Loco

mot

or ac

tivity

(cou

nts5

min

)










GroupsGSH level



mean plusmn SEM


mean plusmn SEM








4 Discussion









5 Conclusion




References
























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



3 Results








Days

Cata

leps

y (s

)


5

10

15

20

IIIIII

IVaIVb

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast




119875 lt








Days

Mea

n fa

ll-off

tim

e (s)


50

100

150

IIIIII

IVaIVb

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast



lowastlowastlowast


119875 lt 005119875 lt 001 and








0

20

40

60

80

100

IIIIII

IVaIVb

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast


lowast

Loco

mot

or ac

tivity

(cou

nts5

min

)










GroupsGSH level



mean plusmn SEM


mean plusmn SEM








4 Discussion









5 Conclusion




References
























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Days

Mea

n fa

ll-off

tim

e (s)


50

100

150

IIIIII

IVaIVb

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast



lowastlowastlowast


119875 lt 005119875 lt 001 and








0

20

40

60

80

100

IIIIII

IVaIVb

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast


lowast

Loco

mot

or ac

tivity

(cou

nts5

min

)










GroupsGSH level



mean plusmn SEM


mean plusmn SEM








4 Discussion









5 Conclusion




References
























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



GroupsGSH level



mean plusmn SEM


mean plusmn SEM








4 Discussion









5 Conclusion




References
























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


5 Conclusion




References
























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Neuroprotective Effect of Juniperus communis on Chlorpromazine