Ginkgo biloba affords dose-dependent protection against6-hydroxydopamine-induced parkinsonism in rats: neurobehavioural,neurochemical and immunohistochemical evidences

Muzamil Ahmad,*,1 Sofiyan Saleem,*,1 Abdullah Shafique Ahmad,*,1 Seema Yousuf,*Mubeen Ahmad Ansari,* M Badruzzaman Khan,* Tauheed Ishrat,* Rajnish Kumar Chaturvedi,�Ashok Kumar Agrawal� and Fakhrul Islam*

*Neurotoxicology Laboratory, Department of Medical Elementology & Toxicology, Jamia Hamdard (Hamdard University),

Hamdard Nagar; New Delhi, India

�Developmental Toxicology Division, Industrial Toxicology Research Centre, M. G. Marg, Lucknow, Uttar Pradesh, India

Abstract

Ginkgo biloba extract (EGb), a potent antioxidant and

monoamine oxidase B (MAO-B) inhibitor, was evaluated for

its anti-parkinsonian effects in a 6-hydroxydopamine

(6-OHDA) rat model of the disease. Rats were treated with

50, 100, and 150 mg/kg EGb for 3 weeks. On day 21, 2 lL

6-OHDA (10 lg in 0.1% ascorbic acid saline) was injected

into the right striatum, while the sham-operated group

received 2 lL of vehicle. Three weeks after 6-OHDA injec-

tion, rats were tested for rotational behaviour, locomotor

activity, and muscular coordination. After 6 weeks, they were

killed to estimate the generation of thiobarbituric acid react-

ive substances (TBARS) and reduced glutathione (GSH)

content, to measure activities of glutathione-S-transferase

(GST), glutathione reductase (GR), glutathione peroxidase

(GPx), catalase, and superoxide dismutase (SOD), and to

quantify catecholamines, dopamine (DA) D2 receptor bind-

ing, and tyrosine hydroxylase-immunoreactive (TH-IR) fibre

density. The increase in drug-induced rotations and deficits

in locomotor activity and muscular coordination due to

6-OHDA injections were significantly and dose-dependently

restored by EGb. The lesion was followed by an increased

generation of TBARS and significant depletion of GSH con-

tent in substantia nigra, which was gradually restored with

EGb treatment. EGb also dose-dependently restored the

activities of glutathione-dependent enzymes, catalase, and

SOD in striatum, which had reduced significantly by lesion-

ing. A significant decrease in the level of DA and its me-

tabolites and an increase in the number of dopaminergic D2

receptors in striatum were observed after 6-OHDA injection,

both of which were significantly recovered following EGb

treatment. Finally, all of these results were exhibited by an

increase in the density of TH-IR fibers in the ipsilateral

substantia nigra of the lesioned group following treat-

ment with EGb; the lesioning had induced almost a

complete loss of TH-IR fibers. Considering our behavioural

studies, biochemical analysis, and immunohistochemical

observation, we conclude that EGb can be used as a

therapeutic approach to check the neuronal loss following

parkinsonism.

Keywords: antioxidant, dopamine, Ginkgo biloba,

6-hydroxydopamine, oxidative stress, Parkinson’s disease,

tyrosine hydroxylase.

J. Neurochem. (2005) 93, 94–104.

Received June 6, 2004; revised manuscript received November 18, 2004;accepted November 18, 2004.Address correspondence and reprint requests to Fakhrul Islam, Neu-

rotoxicology Laboratory, Department of Medical Elementology &Toxicology, Jamia Hamdard (Hamdard University), Hamdard Nagar;New Delhi 110062, India. E-mail: fislam2001@yahoo.co.in1The present address of Muzamil Ahmad, Sofiyan Saleem and AbdullahShafique Ahmad is The Johns Hopkins University, School of Medicine,ACCM Department, Baltimore MD 21205, USA. E-mail (MuzamilAhmad): mahmad7@jhmi.eduAbbreviations used: CDNB, 1-chloro-2,4-dinitrobenzene; DA, dop-

amine; DHBA, 3,4-dihydroxybenzylamine; DOPAC, 3,4-dihydroxy-

phenyl acetic acid; DTNB, 5-5¢-dithio-bis-2-nitrobenzoic acid; EDTA,ethylene-diamine tetraacetic acid; EGb, extract of Ginkgo biloba; GPx,glutathione peroxidase; GR, glutathione reductase; GSH, reducedglutathione; GSSG, oxidized glutathione; GST, glutathione-S-trans-ferase; HPLC, high performance liquid chromatography; HVA, homo-valinic acid; MAO-B, monoamine oxidase B; NADPH, nicotinamideadenine dinucleotide phosphate reduced; NBT, nitroblue tetrazolium;6-OHDA, 6-hydroxydopamine; PBS, phosphate-buffered saline; PD,Parkinson’s disease; PMS, post-mitochondrial supernatant; SOD,superoxide dismutase; TBA, thiobarbituric acid; TBARS, thiobarbituricacid reactive substances; TH, tyrosine hydroxylase; TH-IR, tyrosinehydroxylase-immunoreactive.

Journal of Neurochemistry, 2005, 93, 94–104 doi:10.1111/j.1471-4159.2005.03000.x

94 � 2005 International Society for Neurochemistry, J. Neurochem. (2005) 93, 94–104

Parkinson’s disease (PD) is one of the major neurodegener-ative disorders and often presents with symptoms of resttremor, bradykinesia, rigidity, and stooped posture. The exactcause of this disease still remains a mystery that hampers thedevelopment of proper therapeutic interventions. Despitemany approaches and efforts, to date no researchers havebeen successful in developing a cure or at least a modality tocheck the disease, and most of the therapies only providefunctional relief. Evidence suggests that immense oxidativestress, free radical formation, genetic susceptibility, andprogrammed cell death all have a role in the development ofParkinson’s. The neuropathology of the disease is based ondepigmentation and cell loss in the dopaminergic nigrostri-atal tract of the brain, with the corresponding decrease in thestriatal dopamine (DA) concentrations (reviewed by vonBohlen et al. 2004).

6-Hydroxydopamine (6-OHDA) is a hydroxylated analogof natural neurotransmitter DA and has long been used inexperimental models to study DA function in brain and toevaluate the activity of neuroactive drugs on the centraldopaminergic system, the degeneration of which is ahallmark of PD. Direct administration of 6-OHDA into thesubstantia nigra or striatum causes degeneration of nigrostri-atal tract neurons in the brain, leading to grave behavioural,biochemical, and pathological changes typical of PD. Thesetoxic effects are attributed to the formation of variousoxidants and free radicals, lipidperoxidation, depletion ofreduced glutathione (GSH), and mitochondrial complex Ideficits (reviewed by Schober 2004).

Ginkgo biloba is an antioxidant capable of scavengingvarious reactive oxygen species, including superoxide,peroxy radical, and hydroxyl radical (reviewed by Ahlem-eyer and Krieglstein 2003), and inhibiting or reducing thefunctional and morphologic impairments observed afterlipidperoxidation (Droy-Lefaix et al. 1995; Dumont et al.1995). Ginkgo biloba has been reported to enhance theactivities of superoxide dismutase and catalase and todecrease lipidperoxidation in striatum, substantia nigra, andhippocampus, the major sites damaged in Parkinson’s andAlzheimer’s diseases (Jenner 1992; O’Brien et al. 1996).Ginkgo biloba is a potent platelet-activating factor antagon-ist. It is also likely that the flavonoid fraction, containing freeradical scavengers, is important in this respect (reviewed byAhlemeyer and Krieglstein 2003). Extract of Ginkgo biloba(EGb) exhibit protective properties against animal models ofhypoxia (Klein et al. 1997), excitotoxicity, and focal andglobal cerebral ischemia (Chandrasekaran et al. 2003, 2001).EGb is a potent inhibitor of brain monoamine oxidases(White et al. 1996; Sloley et al. 2000). Some recent studies(Smith et al. 2002; Zhou and Zhu 2000) have shown anti-apoptotic properties of EGb.

We were prompted to undertake this study because theGinkgo extracts possess diverse pharmacologic actions thatare of immense relevance to Parkinson’s disease or its animal

models. These actions include monoamine oxidase B (MAO-B) inhibition, potent antioxidation, free radical scavenging,and anti-apoptosis.

We report here that these pathologic and behaviouralchanges can be altered by treatment with EGb in a dose-dependent manner, and the biochemical benefit is restoredeffectively, thus showing that EGb can effectively be utilizedas a therapeutic tool to alleviate the Parkinson-relatedpathology. The study was an attempt toward the developmentof an anti-parkinsonian drug that can benefit both atfunctional as well as anatomical levels.

Material and methods

Chemicals

6-OHDA, apomorphine hydrochloride, GSH, oxidized glutathione

(GSSG), glutathione reductase (GR), NADPH, 1-chloro-2,4-

dinitrobenzene (CDNB), 5-5¢-dithio-bis-2-nitrobenzoic acid

(DTNB), nitroblue tetrazolium (NBT), DA, 3,4-dihydroxyphenyl

acetic acid (DOPAC), homovanilic acid (HVA), 3,4-dihydro-

xybenzylamine (DHBA), heptane sulfonic acid, bovine serum

albumin, thiobarbituric acid (TBA), ethylene-diamine tetraacetic

acid (EDTA), monoclonal tyrosine hydroxylase (TH) antibody,

anti-mouse IgG, diaminobenzidine, haloperidol were purchased

from Sigma-Aldrich Foreign Holding Chemical Company

(Bangalore, India). 3H-Spiperone was procured from NEN

(Boston, MA, USA). Other chemicals were analytical grade.

Animals and treatments

Male Wistar rats obtained from Central Animal House of Jamia

Hamdard (Hamdard University), weighing 200 ± 10 g and aged 80–

90 days at the start of the experiment, were used. Rats were housed

in groups of four animals per cage and had free access to food and

water ad libitum. Standard crude EGb was procured from the Plant

Extract Division of Saiba Industries (Mumbai, India). The extract

was applied orally by gavage in 2% gum acacia (in water), for

3 weeks prior to the lesioning.

Lesioning

After 3 weeks of EGb treatment, all the animals in experimental

and sham-operated groups were anaesthetized with 35 mg/kg

sodium pentobarbitone intraperitoneally (i.p.). Each animal was

mounted on a stereotatic stand and the skin overlying the skull was

cut to expose it, and the coordinates for the striatum (Paxinos and

Watson 1982) were measured accurately as anterio-posteror

0.5 mm, lateral 2.5 mm, dorso-ventral 4.5 mm relative to bragma

and ventral from dura with the tooth bar set at 0 mm. Thereafter,

all the animals in the experimental group were lesioned by

injecting 10 lg 6-OHDA/2 lL in 0.1% in ascorbic acid-saline into

the right striatum, whereas the sham operated group received

2.0 lL of the vehicle. The injections were made manually with the

help of a Hamilton syringe through the burr holes made for the

purpose in both the groups. The injection rate was 1.0 lL/min and

the needle was kept in place for an additional 1.0 min before being

slowly retracted. The experiments were in accordance with the

guidelines of the Animal Ethics Committee of Jamia Hamdard

(Hamdard University).

Anti-parkinsonian effects of Ginkgo biloba 95

� 2005 International Society for Neurochemistry, J. Neurochem. (2005) 93, 94–104

Experiment 1

This experiment was carried out to evaluate the pre-treatment effect

of EGb (50, 100, and 150 mg/kg body weight) for 3 weeks on the

content of thiobarbituric acid reactive substances (TBARS) and

GSH in substantia nigra; striatum was used for the assays of

enzymatic parameters. The rats were divided into eight groups, each

having 10 animals. Group 1: vehicle treated sham operated control

groups (S), received 2.0 lL of vehicle intracranially; group 2: EGb

50 mg/kg body weight treated sham-operated group (S + G1);

group 3: EGb 100 mg/kg body weight treated sham operated group

(S + G2); group 4: EGb 150 mg/kg body weight treated sham

operated group (S + G3); group 5: vehicle treated lesioned group

(L); group 6: EGb 50 mg/kg body weight treated lesioned group

(L + G1); group 7: EGb 100 mg/kg body weight treated lesioned

group (L + G2), group 8: EGb 150 mg/kg body weight treated

lesioned group (L + G3).

Experiment 2

This experiment was carried out to evaluate the pre-treated effect of

EGb (50, 100, and 150 mg/kg body weight) for 3 weeks on

dopaminergic D2 receptor binding density and content of DA and its

metabolites, DOPAC, and HVA in the striatum. The rats were

divided into eight groups as in experiment 1, each having 10

animals.

Experiment 3

This experiment was carried out to evaluate the pre-treated effect of

EGb (50, 100, and 150 mg/kg body weight) for 3 weeks on TH

expression in the substantia nigra. The rats were divided into eight

groups as in experiment 1, each having five animals.

The behavioural parameters were performed in all the experi-

ments and only those animals that showed a threshold number of

drug-induced rotations were included in the study. All the

experiments were performed separately for all the three doses of

EGb and repeated twice.

Post-operative care

Recovery of anaesthesia took approximately 4–5 h. The rats were

kept in a well-ventilated room at 25 ± 3�C in individual cages till

they gained full consciousness and then were housed together in a

group of four animals per cage. Food and water was kept inside the

cages for the first week so that animals could easily access it without

any physical trauma due to overhead injury. Then the animals were

treated normally; food, water, and the bedding of the cages were

changed every day as usual.

Behavioural studies

Rotational behaviourOn day 23, the rats were tested for drug-induced rotational

behaviour in the Video Path Analyzer. The same procedure as used

for monitoring locomotor activity was pursued. Ipsilateral rotations

of animals were collected after giving 5.0 mg/kg D-amphetamine (in

ascorbic acid-saline) i.p. and their rotational scores were collected

over a period of 90 min. One week after amphetamine challenge, the

animals were given 0.5 mg/kg apomorphine (in ascorbic acid-

saline) subcutaneously to monitor contralateral rotations; these

scores were collected at 40-min intervals.

Locomotor activityOn day 36, all the animals were tested for locomotor activity in a

computerized animal activity monitor, Video Path Analyzer (Coul-

bourn Instruments, Allentown, PA, USA). It consists of a chamber

(50 · 50 · 35 cm), a video camera fixed over the chamber by an

adjacent rod, an activity monitor, a programmer/processor, and a

printer. Theanimalwas placed in the chamber and its locomotor activity

was monitored by the activating camera and viewed on the screen. The

activity chamber was furnished with black paper to provide contrast on

the screen. The datawas fed to the printer to print out the intervals (min),

wall hugging (s), locomotion (s), rest (s), rearing (s), stereo events

(number), rotations (clockwise), rotations (anticlockwise), and distance

travelled (cm). The activity chamber was swabbed with 10% alcohol

every time to avoid the interference due to animal odours.

Rota rod (muscular coordination)Omni Rotor (Omnitech Electronics, Inc., Columbus, OH, USA) was

used to evaluate the muscular coordination on the 40th day. It

consisted of a rotating rod, 75 mm diameter, which was divided into

four parts by compartmentalization to permit the testing of four rats

at a time. The apparatus automatically records the time in 0.1 s

when the rats fall of the rotating shaft. The speed was set at 10 r.p.m.

and cut of time was 180 s, and the drug-naıve animals were trained

on the rod, so that they could stay on it at least for the cut-off time.

Biochemical studies

Tissue preparation for antioxidant enzymes and glutathioneassaysAfter 6 weeks the animals were killed and their brains were taken

out quickly for harvesting striatum and substantia nigra by cutting

acoronal section of 1.0 mm thickness using rat brain matrix in the

light of rat brain atlas (Paxinos and Watson 1982). For enzymatic

assays, striatum was homogenized (10% w/v) in 0.01 M phosphate

buffer (pH 7.0) and centrifuged at 10 500 g for 20 min at 4�C to get

post-mitochondrial supernatant (PMS). Substantia nigra was used

for the estimation of TBARS and GSH.

Assay for thiobarbituric reactive substances, a marker oflipidperoxidationThe method of Utley et al. (1967) was modified for the estimation

of lipidperoxidation. Briefly, 0.2 mL PMS was pipetted in an

Eppendorf tube and incubated at 37 ± 1�C in a metabolic water bath

shaker for 60 min at 120 strokes up and down; another 0.2 mL was

pipetted in an Eppendorf tube and placed at 0�C incubation. After

1 h of incubation, 0.4 mL of 5% TCA and 0.4 mL of 0.67% TBA

was added in both samples (i.e. 0�C and 37�C). The reaction mixture

from the vial was transferred to the tube and centrifuged at 1125 gfor 15 min. The supernatant was transferred to another tube and

placed in a boiling water bath for 10 min. Thereafter, the test tubes

were cooled and the absorbance of the colour was read at 535 nm.

The rate of lipidperoxidation was expressed as nmol of TBARS

reactive substance formed/(h mg protein).

Assay for reduced glutathione contentReduced glutathione (GSH) was determined by the method of

Jollow et al. (1974). 0.2 mL of PMS (10% w/v) was precipitated

with 0.2 mL of sulfosalicylic acid (4%). The sample were kept at

96 M. Ahmad et al.

� 2005 International Society for Neurochemistry, J. Neurochem. (2005) 93, 94–104

4�C for at least 1 h and then subjected to centrifugation at 1200 gfor 15 min at 4�C. The assay mixture contained 0.1 mL of filtered

aliquot (10% w/v), 1.7 mL phosphate buffer (0.1 M, pH 7.4) and

0.2 mL DTNB (4 mg/1 mL of phosphate buffer, 0.1 M, pH 7.4) in a

total volume of 2.0 mL. The yellow colour developed was read

immediately at 412 nm.

Determination of glutathione-S-transferase activityGlutathione-S-transferase (GST) activity was measured by the

method of Habig et al. (1974), as described by Athar et al.(1989). The reaction mixture consisted of 0.1 M phosphate buffer

pH 6.5, 1.0 mM GSH, 1.0 mM CDNB, and 0.1 mL PMS in a final

volume of 2.0 mL. The changes in absorbance were recorded at

340 nm and the enzyme activity was calculated as nmol CDNB

conjugate formed/(min mg protein).

Determination of glutathione reductase activityGlutathione reductase (GR) activity was assayed by the method of

Carlberg and Mannervik (1975), as modified by Mohandas et al.(1984). The assay system consisted of 0.1 M phosphate buffer

pH 7.6, 0.1 mM NADPH, 0.5 mM EDTA, 1.0 mM oxidized gluta-

thione and 0.1 mL PMS in a total volume of 2.0 mL. The enzyme

activity was quantitated at room temperature by measuring the

disappearance of NADPH at 340 nm and was calculated as nmol

NADPH oxidized/(min mg protein).

Determination of glutathione peroxidase activityGlutathione peroxidase (GPx) activity was measured according to

the procedure of Mohandas et al. (1984). The reaction mixture

consisted of 0.05 M phosphate buffer pH 7.0, 1.0 mM EDTA,

1.0 mM sodium azide, 1.4 U of 0.1 mL GR, 1.0 mM glutathione,

0.2 mM NADPH, 0.25 mM hydrogen peroxide and 0.1 mL of PMS

in a final volume of 2.0 mL. The disappearance of NADPH at

340 nm was recorded at room temperature. The enzyme activity was

calculated as nmol NADPH oxidized/(min mg protein).

Determination of superoxide dismutase activitySuperoxide dismutase (SOD) activity was measured by the method

of Beauchamp and Fridovich 1971. The reaction mixture of total

volume 1.0 mL consisted of 0.5 M phosphate buffer pH 7.4, 0.1 mL

PMS, 1.0 mM xanthine, and 57 lM NBT. It was incubated for

15 min at room temperature and reaction was initiated by the

addition of 50 mU xanthine oxidase. The rate of reaction was

measured by recording change in the absorbance at 550 nm due to

formation of formazan, a reduction product of NBT.

Determination of catalase activityCatalase activity was assayed by the method of Claiborne 1985).

Briefly, the assay mixture consisted of 0.05 M phosphate buffer

pH 7.0, 0.019 M hydrogen peroxide, and 0.05 mL PMS in a total

volume of 3.0 mL. Changes in absorbance were recorded at

240 nm. Catalase activity was calculated in terms of nmol H2O2

consumed/(min mg protein).

Markers for parkinsonism

Quantification of dopamine and its metabolitesThe method of DeVito and Wagner (1989) as described by us (Zafar

et al. 2003a,b) was used for the estimation of DA and its

metabolites, DOPAC and HVA. The striatum (20% w/v) was

sonicated in 0.4 N perchloric acid containing 100 ng/mL of the

internal standard DHBA, followed by centrifugation at 15 000 g for

10 min at 4�C and the filtrate was injected manually through a

20 lL loop over the ODS-C18 column coupled with HPLC/

Electrochemical detector (Waters, Milford, MA, USA) for separ-

ation and quantification. The mobile phase consisted of 0.1 M

potassium phosphate (pH 4.0), 10% methanol, and 1.0 mM heptane

sulfonic acid. Samples were separated on an ODS-C18 column using

a flow rate of 1.0 mL/min. The concentrations of DA and its

metabolites were calculated using a standard curve generated by

determining ratio between three known amounts of the amine or its

metabolites and a constant amount of internal standard and

represented as ng/mg of tissue.

Determination of dopaminergic D2 receptor bindingAnimals were killed by decapitation, the brain were removed and

the striatum of the right hemisphere of each brain was dissected out,

weighed, and then homogenised (5% w/v) in 40 mM Tris-HCl buffer

(pH 7.4) followed by centrifugation at 3670 g for 20 min at 4�C.Supernatant was discarded and the pellet was resuspended in the

same amount of the said buffer as the discarded supernatant and then

stored at )20�C. The binding assay was performed by the method of

our collaborating group (Agrawal et al. 1995). In brief, the

incubation mixture of 1.0 mL consisted of synaptic membrane

along with 1.0 nM 1-phenyl-4-[3H]spiperone in 40 mM Tris-HCl

(pH 7.4). A parallel incubation was carried out in the presence of

1.0 lM haloperidol to ascertain non-specific binding. The assay was

run in triplicate. Reaction mixture was incubated for 15 min at

37�C, terminated by cooling at 4�C, and filtered through glass fibre-

filters (GF/C, Whatman) through Millipore Filtration Assembly. The

filter discs were washed rapidly with 2 · 5 mL cold Tris buffer

(40 mM, pH 7.4), and transferred to scintillation vials and dried

properly. After adding 10.0 mL scintillation cocktail to vials, the

radioactivity was counted in a b-scintillation counter (WALLAC-

1410) with an efficiency of 50% for tritium. Specific binding was

calculated by subtracting non-specific binding from total binding

obtained in absence of haloperidol. Results were expressed as pmol

[3H]spiperone bound/mg protein.

Determination of proteinProtein was determined by the method of Lowry et al. (1951).

Tyrosine hydroxylase immunohistochemistryThe animals were anaesthetized under deep sodium pentobarbitone

(35.0 mg/kg) and were perfused transcardially through ascending

aorta with 100.0 mL of 0.1 M phosphate-buffered saline (PBS) at

pH 7.5 followed by 300.0 mL of 4% paraformaldehyde in 0.1 M

phosphate buffer. Brains were immediately removed and tissue

blocks including the substantia nigra were dissected out and were

further immersed in the same fixative for an additional 24 h at 4�C.Furthermore the tissues were preserved in 10%, 20%, and 30%

sucrose solution (in phosphate buffer) until they sank. The tissues

were then kept in final sucrose solution till sectioning. The fixed

tissues were embedded in OCT compound (polyvinyl glycol,

polyvinyl alcohol, and water) and frozen at )20�C. Coronal sectionsof 25 lm thicknesses were cut on a freezing cryostat (Leica) and

collected in PBS and stored at 4�C. The sections were then

Anti-parkinsonian effects of Ginkgo biloba 97

� 2005 International Society for Neurochemistry, J. Neurochem. (2005) 93, 94–104

transferred to gelatin-coated slides and immersed in wash buffer

(sodium phosphate 100 mM, sodium chloride 0.5 M, Triton X-100,

sodium azide) pH 7.4 for 20 min. Endogenous peroxidase activity

was blocked with 3% hydrogen peroxide and 10% methanol in PBS

and incubated for 30 min at room temperature. Thereafter, the slides

were washed with PBS and the sections were overlaid with 20 lL of

anti-TH antibodies (2% in PBS) and incubated for 2 h in a humid

chamber at room temperature. The slides were washed again to

remove the unbound antibodies and incubated with 20.0 lL solution

of biotinylated anti-mouse IgG (2% in PBS) for 3 h at 4�C in the

humid chamber. Then the slides were exposed to streptavidin

peroxidase and the labeled sites were visualized with a solution of

diaminobenzidine and hydrogen peroxide. Finally the sections were

dehydrated and cover slipped, viewed under a microscope, and

photomicrographs were taken.

Image analysis of tyrosine hydroxylase immunohistochemistryThe density of TH-immunoreactive (TH-IR) fibers in the substan-

tia nigra was determined using a computerized image analysis

system (Leica Qwin 500 image analysis software) as described

earlier by our collaborating group (Chaturvedi et al. 2003). Theunbiased stereological method was implied, where a person

unknown to the experimental design carried out the image

analysis. Computerized analysis enabled the percentage area of a

selected field that was occupied by TH-IR fibers to be assessed.

This area was expressed as lm2 per total field view

(250 lm · 250 lm; 75 000 lm2). The density of TH-IR fibers

was measured in the substantia nigra in all the groups in the

ipsilateral as well as the contralateral side. Analysed values

obtained in the ipsilateral side were expressed as a percentage of

those on the intact contralateral side.

Statistics

Results are expressed as mean ± SE. ANOVA with Tukey–Kramer

post hoc analysis was used to analyze differences between the

groups. Significance was ascertained at p < 0.05.

Results

Effect of parkinsonism on behaviour activity and its

restoration by Ginkgo biloba

The ipsilateral body rotation (271.42%) induced byamphetamine and contralateral (384.84%) body rotationsinduced by apomorphine in the L groups were highlysignificant (p < 0.001) as compared to the S groups(Fig. 1a). The different doses of Ginkgo biloba has restoredthe rotation (apomorphine, 136.36%, 242.42%, 303.3%;amphetamine, 85.71%, 171.42%, 214.28%) significantlyand dose-dependently in the L + G1, L + G2, and L + G3groups, respectively, as compared to the L group.

The time spent in locomotion was significantly(p < 0.001) reduced (65.21%) and the rest time (214%)was reversed in the L group as compared to the S group.Treatment with Ginkgo biloba has significantly and dose-dependently restored the locomotion (17.39%, 34.78% and56.52%) and rest time (57.14%, 114.28%, and 185.71%) in

the groups L + G1 to L + G3 as compared to the L group,respectively (Fig. 1b).

The distance travelled (cm) was significantly (p < 0.001)decreased (71.42%) in the L group as compared to the Sgroup and there was a significant and dose-dependentrestoration (21.42%, 35.71%, and 57.14%) in distancetravelled in the L +G1, L + G2, and L + G3 groups ascompared to the L group, respectively (Fig. 1c). The numberof stereo events performed was significantly (p < 0.001)depleted (72.50%) in the L group as compared to the S groupbut it was significantly and dose-dependently recovered(15%, 37.5%, and 57.5%) by Ginkgo biloba treatment ingroups L + G1, L + G2, and L + G3 as compared to the Lgroup, respectively (Fig. 1d).

Figure 1(e) shows the significant and dose-dependentrecovery (14.28%, 33.35%, and 47.61%) on the rearingactivity of the lesioned animals due to Ginkgo bilobatreatment (L + G1, L + G2, and L + G3) as compared to theL group. The rearing activity was significantly (p < 0.001)depleted (66.66%) in the L group as compared to the Sgroup. The muscular coordination was significantly(p < 0.001) decreased (68.18%) in the L group as comparedto the S group and it was significantly and dose-dependently(18.18%, 36.63%, and 50.00%) restored in groups L + G1,L + G2, and L + G3 as compared to the L group (Fig. 1f).However, no significant effects on body rotation, locomotion,distance travel, rearing, and muscular coordination wereobserved in sham groups treated with 50, 100, and 150 mg/kg body weight of Ginkgo biloba (S + G1, S + G2, andS + G3) as compared to S.

Effect of parkinsonism on the content of thiobarbituric

acid reactive substances and reduced glutathione and

their protection by Ginkgo biloba

The content of TBARS in substantia nigra was elevated(273.74%) significantly (p < 0.001) in the L group ascompared to the S group (Fig. 2). The increased TBARSlevel was significantly and dose-dependently restored(55.53%, 129.40%, and 201.00%) in the L + G1, L + G2,and L + G3 groups as compared to the L group. Nosignificant change was observed in the S + G1 to S + G3groups as compared to the S group. On the other hand, thecontent of GSH in substantia nigra was depleted (83.58%)significantly (p < 0.001) in the L group as compared to the Sgroup and its depleted level was restored significantly anddose-dependently (18.2%, 36.60%, 59.15%) in the L + G1,L + G2, and L + G3 groups as compared to the L group(Fig. 3). No significant change was observed in the S + G1to S + G3 groups as compared to the S group.

Effect of parkinsonism on the activities of antioxidant

enzymes and their protection by Ginkgo biloba

Table 1 shows the effect of EGb on the activities of GST,GPx, and GR in striatum. The activities of all the three

98 M. Ahmad et al.

� 2005 International Society for Neurochemistry, J. Neurochem. (2005) 93, 94–104

0

4

8

12

16

20

S S+G1 S+G2 S+G3 L L+G1 L+G2 L+G3

Ro

tati

on

s to

th

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sio

n/

5 m

inu

tes

ApomorphineAmphetamine

a

a

******

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*

*

50

100

150

200

250

300

S S+G1 S+G2 S+G3 L L+G1 L+G2 L+G3

Sec

on

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5m

inu

te

LocomotionRest

***

***

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*

*

a

a

0

300

600

900

1200

1500

1800

S S+G1 S+G2 S+G3 L L+G1 L+G2 L+G3

cms/

5 m

inu

tes

*

**

***

a

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300

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500

S S+G1 S+G2 S+G3 L L+G1 L+G2 L+G3

Ste

reo

eve

nts

/ 5m

inu

tes

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**

***

a

0

50

100

150

200

250

S S+G1 S+G2 S+G3 L L+G1 L+G2 L+G3

Sec

on

ds/

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inu

tes

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*

**

a

0

55

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165

220

275

S S+G1 S+G2 S+G3 L L+G1 L+G2 L+G3

Sec

on

ds/

5 m

inu

tes

a

***

**

*

(a) (b)

(c) (d)

(e) (f)

Fig. 1 (a–f) The effect of extract of Ginkgo biloba (50, 100, and

150 mg/kg) for 3 weeks pre-treatment on (a) body rotations, (b)

locomotor and rest time (s), (c) distance travelled (cm), (d) number of

stereo events, (e) rearing, and (f) muscular coordination and in rats

lesioned by a single injection of 10.0 lg 6-hydroxydopamine/2.0 lL in

0.1% ascorbic acid-saline and sham received ascorbic acid-saline only

(vehicle). Each bar represents the mean ± SE of six animals and the

experiments were repeated twice. ap < 0.001 vs. S, *p < 0.05,

**p < 0.01, ***p < 0.001 vs. L.

Anti-parkinsonian effects of Ginkgo biloba 99

� 2005 International Society for Neurochemistry, J. Neurochem. (2005) 93, 94–104

enzymes were significantly decreased (p < 0.001) in the Lgroup as compared to the S group and their activities wererestored significantly and in a dose-dependent manner in theL + G1 to L + G3 groups as compared to the L group. Nosignificant change was observed in the S + G1 to S + G3groups as compared to the S group.

Effect of parkinsonism on the activities of superoxide

dismutase and catalase and their recovery by Ginkgo

biloba

The activity of SOD and catalase in striatum was significantly(p < 0.001) decreased in the L group as compared to the Sgroup and its was restored significantly and dose-dependentlyin the L + G1 to L + G3 groups as compared to the L group.No significant change was observed in the S + G1 to S + G3groups as compared to the S group (Figs 4 and 5).

Effect of parkinsonism on dopamine and its metabolites

and their protection by Ginkgo biloba

The content of DA and its metabolites, DOPAC and HVAin striatum was decreased significantly (p < 0.001) in the Lgroup as compared to the S group and EGb pre-treatmentafforded a significant and dose-dependent restoration intheir content in the L + G1 to L + G3 groups as comparedto the L group (Table 2). No significant change wasobserved in the S + G1 to S + G3 groups as compared tothe S group.

0

7

14

21

S S+G1 S+G2 S+G3 L L+G1 L+G2 L+G3

nm

ol

TBA

RS

/min

/mg

p

rote

in

*

**

***

ª

Fig. 2 The effect of extract of Ginkgo biloba (50, 100, and 150 mg/kg)

for 3 weeks pre-treatment on thiobarbituric acid reactive substances

(TBARS) content in substantia nigra in rats lesioned by a single

injection of 10.0 lg 6-hydroxydopamine/2.0 lL in 0.1% ascorbic acid-

saline and sham received ascorbic acid-saline only (vehicle). Each bar

represents the mean ± SE of six animals and the experiments were

repeated twice. ap < 0.001 vs. S, *p < 0.05, **p < 0.01, ***p < 0.001

vs. L.

0

10

20

30

40

S S+G1 S+G2 S+G3 L L+G1 L+G2 L+G3

nm

ol G

SH

/g t

issu

e

***

**

*

a

Fig. 3 Effect of extract of Ginkgo biloba (50, 100, and 150 mg/kg) for

3 weeks pre-treatment on reduced glutathione (GSH) content in sub-

stantia nigra in rats lesioned by a single injection of 10.0 lg 6-hy-

droxydopamine/2.0 lL in 0.1% ascorbic acid-saline and sham

received ascorbic acid-saline only (vehicle). Each bar represents the

mean ± SE of six animals and the experiments were repeated twice.ap < 0.001 vs. S, *p < 0.05, **p < 0.01, ***p < 0.001 vs. L.

Table 1 Effect of extract of Ginkgo biloba (EGb) on the activities of

glutathione-S-transferase (GST), glutathione reductase (GR), and

glutathione peroxidase (GPx) in striatum

Group

GST

(CDNB conjugate

formed/min/

mg protein)

GR

(nmol NADPH

oxidized/min/

mg protein)

GPx

(nmol NADPH

oxidized/min/

mg protein)

S 27.70 ± 3.03 20.00 ± 2.00 11.78 ± 1.30

S + G1 27.80 ± 2.81 20.26 ± 1.81 11.90 ± 1.01

S + G2 28.10 ± 3.20 20.38 ± 2.30 12.10 ± 1.21

S + G3 28.31 ± 3.04 20.40 ± 2.00 12.31 ± 1.24

L 7.02 ± 0.65a 5.68 ± 0.50a 3.35 ± 0.30a

(74.65%) (71.60%) (71.56%)

L + G1 11.22 ± 1.03* 9.93 ± 0.92* 4.56 ± 0.40NS

(15.16%) (21.25%) (10.27%)

L + G2 18.23 ± 1.90** 12.31 ± 1.31** 7.90 ± 0.85**

(40.46%) (33.15%) (38.62%)

L + G3 21.31 ± 2.30*** 16.15 ± 1.51*** 9.50 ± 1.00***

(51.58%) (52.35%) (52.02%)

Rats pre-treated with different doses of EGb (50, 100, and 150 mg/kg)

for 3 weeks were injected once with 10.0 lg 6-hydroxydopamine

(6-OHDA)/2.0 lL in 0.1% ascorbic acid-saline. Sham received

ascorbic acid-saline only (vehicle). 6-OHDA-lesioned group differ

significantly from sham group (ap < 0.001) and 6-OHDA-lesioned

groups + different doses of EGb-treated groups, i.e. groups L + G1 to

L + G3 differ significantly (*p < 0.05, **p < 0.01, ***p < 0.001) from

6-OHDA-lesioned group, i.e. L group. There was no significant

difference between sham and sham + drug treated groups, i.e.

S + G1 to S + G3. Each bar represents the mean ± SE of six animals

and the experiments were repeated twice.

100 M. Ahmad et al.

� 2005 International Society for Neurochemistry, J. Neurochem. (2005) 93, 94–104

Effect of parkinsonism on receptor binding and its

protection by Ginkgo biloba

The dopaminergic D2 receptor binding was significantly(p < 0.001) increased (300%) in the L group as compared tothe S group. The increased D2 receptor binding wassignificantly and dose-dependently restored (50%, 160%,

and 260%) in the L + G1, L + G2, and L + G3 groups ascompared to the L group (Fig. 6). No significant change wasobserved in the S + G1 to S + G3 groups as compared to theS group.

Effect of parkinsonism on tyrosine hydroxylase

immunohistochemistry and protection by Ginkgo biloba

The immunohistochemical analysis of ipsilateral substantianigra has shown a marked depletion in the staining of TH inthe L group as compared to the S group and a pronouncedrestoration was observed in the number of ipsilateralsubstantia nigra neurons in the EGb treated lesioned groups(Fig. 7). We observed a significant and dose-dependentincrease in the percentage of the TH-IR neurons in theL + G1, L + G2, and L + G3 groups as compared to the Lgroup (Fig. 8). The number of TH-IR neurons in theipsilateral side was analyzed as a percentage of the TH-IRneurons in the intact contralateral side.

Discussion

The present study demonstrates the beneficial effects ofGinkgo biloba in Parkinsonian rats. Our findings confirm that

0

4

8

12

S S+G1 S+G2 S+G3 L L+G1 L+G2 L+G3

nm

ol f

orm

azan

form

ed/

min

/mg

pro

tein

**

**

***

ª

Fig. 4 Superoxide dismutase activity in striatum was protected by

pre-treatment with different doses of extract of Ginkgo biloba (50,

100, and 150 mg/kg) for 3 weeks. Rats were lesioned by a single

injection of 10.0 lg 6-hydroxydopamine/2.0 lL in 0.1% ascorbic

acid-saline and sham received ascorbic acid-saline only (vehicle).

Enzyme activity measurement and animals treatments are described

in Materials and Methods. Each bar represents the mean ± SE of

six animals and the experiments were repeated twice. ap < 0.001

vs. S, **p < 0.01 vs. L.

0

3

6

9

S S+G1 S+G2 S+G3 L L+G1 L+G2 L+G3

nm

ol H

2O

2 c

on

su

me

d/

min

/mg

pro

tein

*

**

***

ª

Fig. 5 Catalase activity in striatum was protected by pre-treatment

with different doses of extract of Ginkgo biloba (50, 100, and 150 mg/

kg) for 3 weeks. Rats were lesioned by a single injection of 10.0 lg

6-hydroxydopamine/2.0 lL in 0.1% ascorbic acid-saline and sham

received ascorbic acid-saline only (vehicle). Enzyme activity meas-

urement and animals treatments are described in Materials and

Methods. Each bar represents the mean ± SE of six animals and the

experiments were repeated twice. ap < 0.001 vs. S, *p < 0.05,

**p < 0.01, ***p < 0.001 vs. L.

Table 2 Effect of Ginkgo biloba on the content of dopamine (DA),

3,4-dihydroxyphenyl acetic acid (DOPAC), and homovanilic acid

(HVA) in striatum

Group DA

(ng/mg protein)

DOPAC

(ng/mg protein)

HVA

(ng/mg protein)

S 6.12 ± 0.63 0.94 ± 0.85 0.80 ± 0.81

S + G1 6.19 ± 0.59 1.00 ± 0.10 0.87 ± 0.90

S + G2 6.25 ± 0.60 1.00 ± 0.08 0.87 ± 0.07

S + G3 6.35 ± 0.65 1.02 ± 0.09 0.89 ± 0.10

L 1.60 ± 0.13a 0.24 ± 0.02a 0.18 ± 0.02a

(73.85%) (74.76%) (77.50%)

L + G1 2.30 ± 0.20* 0.37 ± 0.03* 0.33 ± 0.03**

(11.43%) (13.82%) (18.75%)

L + G2 3.30 ± 0.29* 0.53 ± 0.05** 0.46 ± 0.48**

(27.77%) (30.85%) (35.00%)

L + G3 4.50 ± 0.04*** 0.72 ± 0.07*** 0.63 ± 0.05**

(47.38%) (51.06) (56.25%)

Animals were pre-treated with different doses of extract of Ginkgo

biloba (50, 100, and 150 mg/kg) for 3 weeks were injected once with

10.0 lg 6-hydroxydopamine (6-OHDA)/2.0 lL in 0.1% ascorbic acid-

saline. Sham received ascorbic acid-saline only (vehicle). Dopamine

and their metabolites were analysed by HPLC (Waters). 6-OHDA-le-

sioned group differ significantly from sham group (ap < 0.001) and

6-OHDA-lesioned groups + different doses of EGb-treated groups, i.e.

groups L + G1 to L + G3 differ significantly (*p < 0.05, **p < 0.01,

***p < 0.001) with 6-OHDA-lesioned group, i.e. L group. There was no

significant difference between sham and sham + drug treated groups,

i.e. S + G1 to S + G3. Each bar represents the mean ± SE of six

animals and the experiments were repeated twice.

Anti-parkinsonian effects of Ginkgo biloba 101

� 2005 International Society for Neurochemistry, J. Neurochem. (2005) 93, 94–104

the increase in amphetamine-induced ipsilateral and apomor-phine-induced contralateral rotation in 6-OHDA-lesionedrats is a reliable marker for the nigrostriatal DA depletion

(Fig. 1a). Previous reports have shown that rotation due toamphetamine and apomorphine is only possible when thelesion is complete or nearly complete, whereas the mildlylesioned rats do not rotate significantly (Przedborski et al.1995). We report here an appreciable decrease in drug-induced rotation and a significant restoration of striatal DAand its metabolites following treatment with EGb. Ginkgobiloba is a potent inhibitor of MAO (Sloley et al. 2000),which would prevent the degradation of DA and increase itsavailability. The behavioural defects following the lesionmay, in turn, be restored by the pool of DA now madeavailable by this pathway as observed in our results. Ourfindings are further supported by an earlier report in whichdeprenyl, a MAO-B inhibitor, delayed the disability in aParkinson’s patient (Knoll 1983; Parkinson’s study group1989; Myllyla et al. 1992). MAO-B inhibitors are alsoreported to decrease DA re-uptake (Riederer and Jellinger1983). A partial recovery of striatal DA content withpre-treatment with EGb in an MPTP model of parkinsonismhas been previously reported (Wu and Zhu 1999).

The locomotor deficits observed in 6-OHDA-lesioned ratsin our studies were restored following exposure to EGbprobably due to partial restoration of striatal DA levels(Table 2). These findings are further strengthened by thenormalization of denervation-related super sensitivity of DAD2 receptors in the striatum by the EGb. It is well documentedthat the denervation-related up regulation of these receptors isa compensatory mechanism for DA deficit (Hu et al. 1990;Hudson et al. 1993; Schwarting and Huston 1996). Theseeffects are not just a functional benefit, but rather an outcomeof the increasing number of surviving dopaminergic neurons,clearly shown in our results in which we observed a dose-

0

65

130

195

260

S S+G1 S+G2 S+G3 L L+G1 L+G2 L+G3

pm

ol

3H

-sp

ipero

ne

bo

un

d/g

pro

tein

**

*

***

a

Fig. 6 Effect of extract of Ginkgo biloba (50, 100, and 150 mg/kg) for

3 weeks pre-treatment on dopaminergic D2 receptor binding in stria-

tum in rats lesioned by a single injection of 10.0 lg 6-hydroxydop-

amine/2.0 lL in 0.1% ascorbic acid-saline and sham received ascorbic

acid-saline only (vehicle). Each bar represents the mean ± SE of six

animals and the experiments were repeated twice. ap < 0.001 vs. S,

*p < 0.05, **p < 0.01, ***p < 0.001 vs. L.

Fig. 7 (a–e) Effect of 3 weeks of pre-treatment of extract of Ginkgo

biloba (EGb) on tyrosine hydroxylase in ipsilateral substantia nigra in

rats lesioned by a single injection of 10.0 lg 6-hydroxydopamine/

2.0 lL in 0.1% ascorbic acid-saline and sham received ascorbic acid-

saline only (vehicle). The expression of tyrosine hydroxylase was

almost negligible in lesioned group (b) as compared to sham group (a),

whereas the lesioned group pre-treated with 50, 100, and 150 mg/kg

body weight of EGb (c–e) has shown a dose-dependent staining of

tyrosine hydroxylase. However, the sham group has shown no dis-

cernible change in tyrosine hydroxylase staining. Scale bar is 150 lm

and magnification is 4 ·.

0

30

60

90

120

S L L+G1 L+G2 L+G3

% o

f T

H-I

R n

euro

ns

in

sub

stan

tia

nig

ra ***

**

*

a

Fig. 8 Effect of extract of Ginkgo biloba on the density of tyrosine

hydroxylase-immunoreactive (TH-IR) fibers in ipsilateral substantia

nigra in 6-hydroxydopamine-lesioned animals (ratio lesioned/intact

side). Density of TH-IR fibers was calculated, averaging six sections

per rat and three rats per group. ap < 0.001 vs. S, *p < 0.05,

**p < 0.01, ***p < 0.001 vs. L.

102 M. Ahmad et al.

� 2005 International Society for Neurochemistry, J. Neurochem. (2005) 93, 94–104

dependent increase in the number of TH-IR positive fibers inipsilateral substantia nigra due to EGb treatment.

We previously reported an inverse relationship betweenlipidperoxidation and GSH activities and its related enzymes,along with the activities of catalase and superoxide dismutasein parkinsonism (Zafar et al. 2003a,b). This has been furthersupported by other reports (Kumar et al. 1995; Perumal et al.1992; Cohen 1984). A reduction in GSH may impair H2O2

clearance and promote OH formation, thus increasing thefree radical load, which triggers oxidative stress andconsequently disrupts homeostasis. As all antioxidantdefences are interrelated (Sun 1990), disruption of the microenvironment by a single factor, oxidative stress in this case,can shift the entire balance and lead to a catastrophe. In viewof our findings, it is reasonable to infer that the depletion inGSH triggers lipidperoxidation, leading to the degenerationof nigrostriatal neurons, which, in turn, would deplete DAand, subsequently, its metabolites. Conversely, GSH isconverted to GSSG, which is reconverted to GSH by GR,thus maintaining the pool of GSH, which, in conjunctionwith the reductant nicotinamide adenine dinucleotide phos-phate reduced (NADPH), can reduce lipid peroxidase, freeradicals, and H2O2. The increase in the content of GSH anddecrease in the extent of lipidperoxidation with the treatmentof EGb, in our study, is in concordance with earlier reports(Perumal et al. 1992; Roghani and Behzadi 2001; Zafar et al.2003a,b), where antioxidants had been used for the similarmodels of PD.

Our findings are in agreement with previous reports thatoxidant radicals inactivate GR and GPx (Huang and Philbert1996). GPx plays a predominant role in removing excess freeradicals and hydroperoxides and is a major defence systemagainst oxidative stress in brain (Imam and Ali 2000).Meanwhile, GST catalyzes the detoxification of oxidizedmetabolites of catecholamine (O-quinone) and may serve asan antioxidant system preventing degenerative cellularprocesses (Baez et al. 1997). The enzymes that remove bothsuperoxide and H2O2 protect the cells against intermediatesof oxygen generated during normal aerobic metabolism, butwhen the production of O2 and H2O2 crosses the normalthreshold, the system is compromised. SOD converts super-oxide into H2O2 (Fridovich 1975; Freeman and Crapo 1982);and catalase, which is found at very low levels in the brain,removes H2O2 in the form of H2O. Our laboratory and othershave reported the role of antioxidants in providing protectionagainst 6-OHDA-induced deleterious effects (Zafar et al.2003a,b; Perumal et al. 1992; Roghani and Behzadi 2001).

In conclusion, we report here that the 6-OHDA-inducedlesions produce the characteristic alterations in the beha-vioural pattern of our experimental model, which is furthercorroborated by the biochemical alterations in the variousclassical parameters used in characterizing Parkinson andParkinson-related disorders. Furthermore, pre-treating ratswith EGb before lesioning leads to a restoration of compro-

mised behavioural activity and cellular integrity by reversingthe effect and re-storing near normal levels of TH expression,of DA levels, and of the various enzymatic and nonenzymaticmarkers of lipidperoxidation. Therefore, Ginkgo bilobaappears to act via antioxidant, free radical scavenging,MAO-B-inhibiting, and DA-enhancing mechanisms thatrescue the compromised cells within the dopaminergiclesions. Further studies to understand the sequence of eventswould be worth investigating.

Acknowledgements

The technical assistance of Messers Iqbal, Anil, and Dharamvir is

greatly acknowledged. D-Amphetamine was kindly gifted by

Professor S. B. Vohora, the then Head of our department.

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