Chemico-Biological Interactions 206 (2013) 239–247

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Chemico-Biological Interactions

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Mangiferin attenuates MPTP induced dopaminergic neurodegenerationand improves motor impairment, redox balance and Bcl-2/Baxexpression in experimental Parkinson’s disease mice

0009-2797/$ - see front matter � 2013 Elsevier Ireland Ltd. All rights reserved.http://dx.doi.org/10.1016/j.cbi.2013.09.016

⇑ Corresponding authors at: Department of Food Science and Nutrition, CAMS,Sultan Qaboos University, Muscat, Oman. Tel.: +968 24143604 (M.M. Essa).

E-mail addresses: drmdessa@gmail.com, drmdessa@squ.edu.om (M.M. Essa),mani_pdresearchlab@rediffmail.com (T. Manivasagam).

Mani Kavitha a, Jagatheesan Nataraj a, Musthafa Mohammed Essa b,c,⇑, Mushtaq A. Memon d,Thamilarasan Manivasagam a,⇑a Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai University, Annamalai Nagar 608 002, Tamilnadu, Indiab Department of Food Science and Nutrition, CAMS, Sultan Qaboos University, Muscat, Omanc Ageing and Dementia Research Group, Sultan Qaboos University, Muscat, Omand College of Veterinary Medicine, Washington State University, Pullman, WA, United States

a r t i c l e i n f o a b s t r a c t

Article history:Received 28 July 2013Received in revised form 4 September 2013Accepted 25 September 2013Available online 2 October 2013

Keywords:Experimental Parkinson’s diseaseMangiferinBehaviorOxidative stressAnti-apoptosis

Mangiferin, a polyphenol compound of C-glucoside, is well-known for its anti-inflammatory, antioxidant,anticancer, antidiabetic and cognitive enhancement properties. In this study, we investigated the neuro-protective effect of mangiferin against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mousemodel of Parkinson’s disease (PD), which is most popular and widely used to evaluate therapeutic impli-cations of new protective agents. Male C57BL/6 mice were orally treated with mangiferin (10, 20 and40 mg/kg body wt.) for 14 days and from 10th day onwards MPTP (30 mg/kg, i.p.) was injected for last5 days. MPTP treatment leads to enhanced oxidative stress, induction of apoptosis (upregulates theexpression of Bax, proapoptotic protein and downregulates the expression of anti-apoptotic markerBcl-2), and loss of dopominergic neurons which results in motor impairments. Results of our study con-firmed that mangiferin prevented MPTP-induced behavioral deficits, oxidative stress, apoptosis, dopami-nergic neuronal degeneration and dopamine depletion. Taken together, we conclude that mangiferinattenuates the dopaminergic neurodegeneration mainly through its potent antioxidant and antiapoptoticproperties.

� 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

PD is the second most common and progressive neurodegenera-tive disease affecting about 1% of the aged people. The diagnosis isbased on the presence of a set of cardinal motor signs that is a con-sequence of the loss of dopaminergic neurons in substantia nigra(SN) and depletion of dopamine in straitum [1]. Though the causeis not yet completely understood, oxidative stress, mitochondrialdysfunction, inflammation, proteosome dysfunction and apoptosisplay a key role in the pathophysiology of this movement disorder [2].

MPTP is a selective neurotoxin of dopaminergic neurons in theSN and has been shown to induce PD symptoms in various exper-imental animals including monkeys, mice, cats, dogs, rats, goldfishand also humans [3]. Due to its lipophilic nature, it can easily crossthe blood–brain barrier and get metabolized to 1-methyl-4-phen-ylpyridinium (MPP+) by monoamine oxidase in the glial cells,

which is selectively gathered into the mitochondria of dopaminer-gic neurons by dopamine transporter [4]. It inhibits complex I ofthe mitochondrial electron transport chain and ultimately leadsto the formation of excess reactive oxygen species (ROS) [5].Downstream effect of ROS results in disturbed mitochondrialmembrane permeability, translocation of B-cell lymphoma (Bcl-2) family proteins, increased levels of cytosolic cytochrome C(Cyt-C), activation of caspase-3 and finally lead to apoptotic lossof dopaminergic neurons [6]. There is no proper therapy for PD.

Mangifera indica and its active components have been exten-sively used in the Indian sub-continent as food additives and incosmetics and medicines. Mangiferin, a potent glucosyl xanthonecompound of M indica, has been reported to possess antidiabetic,antioxidant, antiproliferation, immunomodulatory, cardiotonicand diuretics properties [7]. The pharmacology of mangiferin hasrecently gained great attention owing to its protective functionagainst oxidative injuries as well as its ability to modulate severalkey inflammatory pathways [8] in various tissues, including thebrain. It is able to cross the blood brain barrier and has the realpotential to ameliorate the oxidative stress observed in neurode-generative disorders [9]. It is reported that the administration of

240 M. Kavitha et al. / Chemico-Biological Interactions 206 (2013) 239–247

mangiferin protected N2A cells against 1-methyl-4-phenylpyridine(MPP+)-induced cytotoxicity by restoring reduced glutathione(GSH) content and down-regulating both superoxide dismutase(SOD) and catalase (CAT) mRNA expression [10]. However its pro-tective effect against MPTP-induced toxicity in mice is not yetinvestigated.

2. Materials and methods

2.1. Experimental animals

Male C57BL/6 mice (25–30 g) were obtained from Bangaloreand maintained in the Central Animal House, RMMC, and Annam-alai University. The animals were kept under standard conditionswith food and water ad libitum. The experimental protocols metwith the National Guidelines on the proper care and use of Animalsin Laboratory Research (Indian National Science Academy, NewDelhi, 2000) and were approved by the Animal Ethics Committeeof the Institute (Approval no: 759/2011).

2.2. Chemicals

MPTP, mangiferin, thiobarbituric acid, reduced glutathione and3,5-dithio-bis-nitrobenzoic acid (DTNB), TH and DAT primary andsecondary antibodies were purchased from Sigma Chemical Com-pany, Bangalore, India. All other reagents used were of analyticalgrade and were procured locally.

2.3. Experimental design

Animals were randomized and divided into six experimentalgroups (n = 6). Group I mice was orally administered with saline(0.5 ml) and were served as control. Group II mice received i.p.injection of MPTP (30 mg/kg body wt.) (Zhao et al., [11]) daily forfive consecutive days. Group III mice were injected orally withmangiferin (10 mg/kg body wt.) for 14 days and from the 10thday onwards, MPTP was injected as group II mice. The mice ingroups IV and V were injected orally with mangiferin, 20 and40 mg/kg body wt., respectively, for 14 days. From the tenth dayonwards MPTP was injected on the group II mice. Group VI micewere treated orally with mangiferin alone for 14 days, like groupIII mice. At the end of the experiment, 3 days after the last doseof MPTP (18th day), the behavioral tests described below were per-formed. Effective dose of mangiferin (40 mg/kg bodyweight) basedon the dopamine estimation was used for Phase II (control, MPTP,mangiferin + MPTP and mangiferin).

2.4. Estimation of dopamine and its metabolites

The levels of dopamine (DA), dihydroxyphenylacetic acid(DOPAC) and homovanillic acid (HVA) were determined by thehigh performance liquid chromatography (HPLC) with an electro-chemical detector [12]. Briefly, the straitum (ST) was sonicated inice-cold. 0.1 M perchloric acid containing 0.01% ethylene diamine-tetraacetic acid (EDTA). The supernatant collected after a spin of10,000 g for 5 min was injected (10 ll) into the HPLC system.Results were expressed in ng/mg weight of brain tissue.

2.5. Behavioral assessment

2.5.1. Open fieldA wooden square box (100 � 100 � 40 cm) was used to study

the movement and activity of mice in which the floor wascovered with a rexin cloth drawn with 25 equal squares. If theanimal crosses the central nine squares and sixteen outer squares

then it is calculated as central and peripheral movement respec-tively. Furthermore, rearing (exploratory activity) and grooming(displacement response) were manually scored, while the animalwas in the open field for 5 min [13], in normal lighting. Theequipment was cleaned with 70% alcohol and water betweentrials.

2.5.2. Swim testSwim-test was carried out by placing each animal in water tubs

(40 cm length � 25 cm width � 16 cm height) and swim score wasmeasured. Each animal was placed in the water tub to measure theswim score. The depth of water was kept at 12 cm and the temper-ature was maintained at 27 ± 2 �C. The animals were wiped dryimmediately after the experiment using a dry towel and returnedto cages kept at 27 ± 2 �C. Swim score scales were: 0, hind partsinks with head floating; 1, occasional swimming using hind limbswhile floating on one side; 2, occasional floating/swimming only;3, continuous swimming [14].

2.5.3. Hang testNeuromuscular strength was determined by the grid hang test.

Mice were lifted by their tail and slowly placed on a horizontal grid(grid 12 cm2 opening 0.5 cm2) and supported until they grabbedthe grid with both their forelegs and hind paws. The grid was theninverted so that the mice were allowed to hang upside down. Thegrid was mounted 20 cm above a hard surface, to discourage fallingor injury in case of falling. The apparatus was equipped with a3-inch wooden wall to prevent animals from moving to the upperside of the grid. Animals were allowed to stay on the grid for 30 sand 10 chances were given with intervals of 1 min and the best fallvalues were recorded. The percentage of success was recorded asmaximum time hanging/30 s � 100 [15].

2.5.4. CatalepsyBriefly, catalepsy was measured as the time the animal main-

tained an imposed position with both front limbs extended andresting on a 4 cm high wooden bar. The end point of catalepsywas considered to occur when both front paws were removed fromthe bar or if the animal moved its head in an exploratory manner. Ifthe animal maintained the imposed posture for at least 20 s it wassaid to be cataleptic and given a point. For every further 20 s thatthe animal continues to maintain the cataleptic posture an extrapoint was given. The animal is lifted by its tail and is allowed toplace its forepaws on a horizontal wooden bar. The duration takenfor the first movement of paws was measured as cataleptic time.The test was conducted accordingly [16] thrice.

2.5.5. Estimation of TBARSBriefly, the tissue extracts were incubated with 0.2 ml phenyl

methosulfate at 37 �C in metabolic water bath shaker. After 1 hof incubation, 0.4 ml of 5% tricarboxylic acid and 0.4 ml of 0.67%thiobarbituric acid were added. The reaction mixture was centri-fuged at 4000 rpm for 15 min, and the supernatant was boiledfor 10 min. After cooling, the samples were read at 535 nm. Therate of lipid peroxidation was expressed as nmol of thiobarbituricacid reactive substance (TBARS) formed/g tissue [17].

2.5.6. Assay of SODSOD activity was assayed using an indirect inhibition assay, in

which xanthine and xanthine oxidase serve as a superoxide gener-ator, and nitro blue tetrazolium (NBT) is used as a superoxideindicator. The assay mixture consisted of 960 ll of 50 mM sodiumcarbonate buffer (pH 10.2) containing 0.1 mM xanthine, 0.025 mMNBT, and 0.1 mM EDTA, 20 ll of xanthine oxidase and 20 ll of thebrain supernatant. Changes in absorbance were observed

M. Kavitha et al. / Chemico-Biological Interactions 206 (2013) 239–247 241

spectrophotometrically at 560 nm. The activity was expressed asunits/min/mg protein [18].

2.5.7. Determination of activity of catalaseCatalase activity was assayed by measuring the rate of decom-

position of hydrogen peroxide at 240 nm. The assay mixture con-sisted of 50 ll of 1 M Tris–HCl buffer (pH 8.0) containing 5 mMEDTA, 900 ll of 10 mM H2O2, 30 ll of MQ water and 20 ll of thebrain tissue supernatant. The rate of the decomposition of hydro-gen was observed spectrophotometrically at 240 nm. The enzymeactivity was expressed as nmol of hydrogen peroxide decom-posed/min/mg protein [19].

2.5.8. Assay of GPxThe glutathione peroxidase (GPx) assay mixture consisted of

100 ll of 1 M Tris–HCl (pH 8.0) containing 5 mM EDTA, 20 ll of0.1 M GSH, 100 ll of glutathione (GSH) reductase solution (10 U/ml), 100 ll of 2 mM NADPH, 650 ll of distilled water, 10 ll of7 mM hydroperoxide and 10 ll of the brain supernatant. Oxidationof NADPH was determined spectrophotometrically at 340 nm. Oneunit of activity was defined as the amount of GPx required to oxi-dize l lmol of NADPH per min [20].

2.5.9. Estimation of GSHThe level of GSH in the brain homogenate was measured by the

method described by Jollow et al. [21]. Brain tissue homogenatewas centrifuged at 16,000g for 15 min at 40 �C. The supernatant(0.5 ml) was added to 4 ml of ice-cold, 0.1 mM solution of 5,5-dithiobis[2-nitrobenzoicacid] in 1 M phosphate buffer (pH 8). Theoptical density was read at 412 nm in a spectrophotometer.

2.5.10. Straital monoamine oxidase-B (MAO-B) activityEstimation of MAO B activity was performed by commercially

available kit (Amplex Red Monoamine Oxidase Kit, MolecularProbes, Invitrogen, UK). Briefly, brain samples were sonicated inice cold 20 mM phosphate buffered saline (pH 7.4) and centrifugedat 13,000g for 10 min at 4 �C. Protein amount in the supernatantwas determined and a volume corresponding to 250 lg wasdiluted in assay buffer to a final volume of 500 ll. To facilitatediscrimination between MAO-A and MAO-B, samples were preincubated 30 min at room temperature with clorgyline (1 lM) asa specific MAO-A inhibitor. The fluorimetric assay started when500 ll of a reaction mixture containing Amplex Red reagent(400 lM), horseradish peroxidase (2 U/ml) and benzyl amine(2 mM) as a specific substrate for MAO-B were added. The assaywas conducted in cuvette at room temperature for 45 min. At theend of the incubation time, specific fluorescence of resurofin, theoxidation product of Amplex Red reagent, was measured using aBioteck Kontron cuvette fluorimeter (560 nm excitation, 590 nmemission). The amount of resurofin in the samples was determinedusing a resurofin standard curve, and one unit of MAO-B was de-fined as the amount of enzyme generating 1 lmol of resurofinper minute.

2.5.11. Immunohistochemical analysisFor the immunohistochemical study, ST and substantia nigra

(SN) were serially sectioned, after the slides were deparaffinizedin xylene and rehydrated in a graded series of ethanol, the slideswere boiled in citrate buffer (10 mM, pH 6.0) for 7 min followedby an additional 10 min for antigen retrieval. The latter sectionswere incubated with 0.3% hydrogen peroxide (H2O2) for 10 minat room temperature to remove the endogenous peroxidase activ-ity and then placed in blocking buffer containing 10% normal goatserum (NGS) with 0.2% Triton X-100 in 0.01 M PBS (pH 7.2) for30 min at 37 �C. In each treatment, the slides were washed at leastthree times with 0.01 M PBS each for 5 min. Sections were

incubated for 24 h with primary anti mouse tyrosine hydroxylase(1:1000) and anti mouse dopamine transporter (1:500) in 2%NGS, 0.2% Triton X-100 and 0.02% sodium azide in TBS. Afterwashing with 1% NGS in TBS, the sections were incubated inanti-mouse IgG-HRP-conjugated antibody (1:1000) in 1.5% NGSfor one hour. Sections were washed with PBS and exposed to diam-inobenzidine until the desired staining intensity was reached. Theintensity of TH and DAT immunoreactivity in the striatum wasquantified by optical density (OD) measurements using the MicroComputer Imaging Device (MCID) software; data were presentedas a percent of the control group values. In addition, TH and DATimmunoreactive neurons in the ST and SN were counted withthe aid of the mouse brain atlas. The number of TH and DAT immu-noreactive cells on each representative mesencephalic section wascounted for the ST and SN regions by persons who were blind tothe treatment. All raw cell counts were adjusted with a correctionformula for cell size and section thickness according to the methodof [22]. When counting was complete the slides were decoded andarranged based on treatment group. The cell counts were thenaveraged for each animal and these averages were used to calcu-late a mean ± SD for each treatment group and data were pre-sented as a percent of the control group.

2.5.12. Western blottingIn brief, striatal and nigral tissues were homogenized in an ice-

cold RIPA buffer (1% Triton 0.1% SDS, 0.5% deoxycholate, 1 mmol/lEDTA, 20 mmol/l Tris (pH 7.4), 150 mmol/l NaCl, 10 mmol/l NaF,and 0.1 mmol/l phenylmethylsulfonyl fluoride (PMSF). The homog-enate was centrifuged at 12,000 rpm/min for 15 min at 4 �C to re-move debris. Protein concentration was measured [23]. Samplescontaining 50 lg of total cellular protein were loaded and sepa-rated on 10% SDS–polyacrylamide gel electrophoresis. The gelwas then transferred onto a PVDF membrane (Millipore). Themembranes were incubated with the blocking buffer containing5% non-fat dry milk powder for 2 h to reduce non-specific bindingsites and then incubated in b-actin (rabbit polyclonal; 1:500 dilu-tion in 5% bovine serum albumin (BSA) in Tris-buffered salineand 0.05% Tween-20 (TBST), anti tyrosine hydroxylase (1:1000),anti Bax and anti Bcl 2 (1:1000) with gentle shaking overnight at4 �C. After this, membranes were incubated with their correspond-ing secondary antibodies (anti-mouse or anti-rabbit IgG conju-gated to HRP) for 2 h at room temperature. The membrane waswashed thrice with TBST for 30 min. Protein bands were visualizedby an enhanced chemiluminescence’s method using ECL-kit (Gen-Script ECL kit, USA). Bands were scanned using a scanner and quan-titated by ImageJ, a public domain Java image processing software,which of control was set to 1.

2.6. Statistical analysis

All the data were expressed as mean ± SD of the number ofexperiments (n = 6). Statistical significance was evaluated usingone-way analysis of variance (ANOVA) using SPSS version 10.0software and individual comparisons were obtained using DUN-CAN’S Multiple Range Test (DMRT).

3. Results

3.1. Dopamine metabolism

MPTP (30 mg/kg, i.p. for 5 days) produced a significant deple-tion of striatal DA, DOPAC and HVA, whereas the mice treated withmangiferin alone (40 mg/kg) showed no significant change as com-pared to controls. Pre-treatment with mangiferin significantly anddose-dependently attenuated MPTP-induced depletion of striatal

242 M. Kavitha et al. / Chemico-Biological Interactions 206 (2013) 239–247

DA and its metabolites in mice (Table 1), whereas the middle andhigh dose of mangiferin showed no significance difference as com-pared to low dose.

3.2. Behavioral analysis

Exploratory behavior in the new environment is measuredusing various tests such as the open field test and swim test. MPTPinjection led to a significant reduction in the peripheral and centralmovements along with diminished rearing and grooming activities(Fig. 1). Mangiferin pretreated and MPTP administered (group III)mice showed significant increase in the movement and activitiescompared to MPTP intoxicated (group II) mice and there was nosignificant changes were observed in mangiferin (40 mg/kg bw)alone treated mice as compared to control (P < 0.05). MPTP admin-istration significantly decreased swim score in mice when com-pared to the control mice (P < 0.05). Mangiferin pretreated andMPTP induced mice show a significant increased in swim scoreas compared to the experimental PD mice (Fig. 2).

Hang test is used to examine the neuromuscular strength of thecontrol and experimental mice. MPTP injection induced a signifi-cant increase in beam crossing duration and slipping error(P < 0.05). Mangiferin pretreated and MPTP treated mice showeda significant increase in hanging time as similar to the control mice(Fig. 3).

Catalespy test is used to observe the impairment in movementcoordination. MPTP injection decreased the cataleptic time com-pared with control mice (P < 0.05). The prophylactic administrationof mangiferin for 2 weeks markedly suppressed the MPTP-induceddiminished catalepsy reaction (P < 0.05, Fig. 4).

3.3. Effect of lipid peroxidation and enzymatic antioxidant

Intraperitoneal injection of MPTP produced a significantenhancement in the levels of TBARS and activities of SOD and cat-alase in the substania nigra of mice, whereas the animals treatedwith mangiferin alone (40 mg/kg) showed no significant changein the levels of TBARS and activities of SOD and catalase (Table 2).Treatment of animals with mangiferin significantly and dose-dependently attenuated MPTP induced oxidative stress by lower-ing the levels of TBARS and activities of SOD and catalase, whereasmangiferin at low and medium doses failed to modify the effect ofMPTP significantly as compared to high dose.

Injection of MPTP alone in mice significantly reduced nigralGSH levels and GPx activities, whereas mangiferin alone treatmenthad no effect on nigral GSH and GPx in mice (Table. 2). Pre-treat-ment with high dose (40 mg/kg) of mangiferin significantly pro-tected mice against MPTP-induced GSH depletion and Gpximpaired activities, whereas low and medium doses of mangiferinshowed insignificant recovery in MPTP-induced GPx and GSHdepletion.

Table 1Dose dependent effect of mangiferin on the levels of striatal dopamine and its metabolitfollowed by DMRT.

Groups/variables Dopamine (ng/mg tissu

Control 18.65 ± 1.43MPTP 6.83 ± 0.59*

Mangiferin (10 mg/kg body weight) + MPTP 10.41 ± 0.74#

Mangiferin (20 mg/kg body weight) + MPTP 10.98 ± 0.78#

Mangiferin (40 mg/kg body weight) + MPTP 11.06 ± 0.82#

Mangiferin +(40 mg/kg body weight) 19.04 ± 1.49

* P < 0.05, compared with the control group.# P < 0.05, compared with the MPTP group.

3.4. Activity of Monoamine oxidase-B (MAO-B)

Table 3 depicts the activities of MAO-B in the control and exper-imental mice. MPTP treatment induced a marked increase in MAO-B activity as compared to control mice. Mangiferin pre-treatment(40 mg/kg bw) significantly decreased the activity of MAO-B ascompared to MPTP treated group. There is no significant changein the activity of MAO A in the animal group treated mangiferinalone.

3.5. Immunohistochemistry

Representative photomicrographs of TH and DAT immunostain-ing in the substantia nigra was shown in Fig. 5. Nigral TH andDAT-immunoreactive neurons were easily detectable in controlmice. In the substantia nigra a decrease in the number of TH andDAT immunopositive neurons was observed after MPTP treatment.In contrast, pretreatment of mangiferin (40 mg/kg bw) partiallyprevented the toxicity of MPTP by increasing the number of thenigral TH and DAT-immunopositive neurons in mice (Fig. 5a andb and Fig. 6a and b).

3.6. Western blotting

Bax and Bcl-2 protein expressions were also analyzed by immu-noblotting and quantified using b-actin as an internal protein levelcontrol. MPTP treatment markedly reduced the expressions ofnigrostriatal Bcl-2 and enhanced the Bax expression as comparedto control mice (P < 0.05). Mangiferin pre-treatment restored theexpressions of Bax and Bcl-2 in PD mice (Fig. 7a and b andFig. 8a and b).

4. Discussion

MPTP causes depletion of dopamine in mice when administeredas different acute, sub-acute and chronic paradigms. In sub-acuteparadigm, a single i.p. injection of MPTP per day (30 mg/kg) for fiveconsecutive days, leads to severe depletion of dopamine bydestroying nigral dopaminergic neurons which corroborates withour results [24]. Levels of dopamine metabolites were reducedmost strongly in the sub-acute and chronic regimens, which indi-cates that DA was released from surviving nigrostriatal terminalsand utilized (turned-over), a common compensatory mechanismobserved in PD patients and MPTP treated mice [25].

The neurotoxicity of MPTP involves in its conversion to MPP+ bymonoamine oxidase B (MAO-B), which is the main culprit thatfinally induces neuronal death. Moreover this enzyme catalyzesthe metabolism of biologically active amine compounds and par-ticipates in the oxidative deamination reaction of neurotransmit-ters such as dopamine, adrenaline, and serotonin [26]. Severalneurodegenerative diseases, such as Parkinson’s and Alzheimer’sdiseases, show high MAO-B in brain tissues [27] and selective

es in control and experimental mice. Values are given as mean ± SD (n = 6), ANOVA

e) DOPAC (ng/mg tissue) HVA (ng/mg tissue)

3.22 ± 0.23 1.49±.0.100.94 ± 0.07* 0.39 ± 0.01*

1.67 ± 0.10# 0.54 ± 0.02#

1.84 ± 0.13# 0.57 ± 0.03#

1.87 ± 0.16# 0.60 ± 0.04#

3.37 ± 0.26 1.54 ± 0.09

*#

02468

1012

No.

of

cent

ral s

quar

es

cros

sed

/5 m

inut

es

Central

Control MPTP

MPTP+Mangiferin Mangiferin

*

#

020406080

100120

No.

of

squa

res

cros

sed

5/m

inut

es

Peripheral

Control MPTP

MPTP+Mangiferin Mangiferin

* *

# #

0

5

10

15

No.

of

acti

vite

s/5

min

utes

Grooming and Rearing

Control MPTP

MPTP+Mangiferin Mangiferin

BA

C

Fig. 1. A, B and C shows the effect of Mangiferin on MPTP induced impairment of spontaneous movement and activities in open field test for 5 min. If the animal crosses thecentral nine squares and sixteen outer squares drawn on the bottom of the arena were measured by manually counting (Fig. 1A and B). Rears and grooms (Fig. 1C) weremanually counted. Values are given as mean ± SD for six mice in each group. ⁄P < 0.05, compared with the control group; #P < 0.05, compared with the MPTP group.

*

#

0

1

2

3

4

Swim

sco

re

Swim test

Control MPTP

MPTP+Mangiferin Mangiferin

Fig. 2. The swim test performance of mice pretreated with mangiferin with orwithout MPTP. The swim test was conducted by placing each animal separately inwater tub for 3 min and swim score was measured. Swim score scales were: 0, ifhind part sinks with head floating; 1, if occasional swimming using hind limbswhile floating on one side; 2, if the occasional floating/swimming only and 3, ifcontinuous swimming. Values are given as mean ± SD for six mice in each group.⁄P < 0.05, compared with the control group; #P < 0.05, compared with the MPTPgroup.

*

#

012345

Han

ging

Tim

e(Se

cond

s) Hang test

Control MPTP

MPTP+Mangiferin Mangiferin

Fig. 3. Shows the effect of mangiferin on MPTP induced reduction in neuromus-cular strength in hang test. Animals were allowed to hang on the grid and the bestfall values were recorded. Values are given as mean ± SD for six mice in each group.⁄P < 0.05, compared with the control group; #P < 0.05, compared with the MPTPgroup.

*

#

0

2

4

6

8T

ime

(Sec

)

Catalespy

Control MPTP

MPTP+Mangiferin Mangiferin

Fig. 4. Shows the effect of mangiferin on MPTP induced rigidity or inability tocorrect an externally imposed posture by catalepsy test. Catalepsy was measured asthe time taken by the animal maintained an imposed position with both front limbsextended and resting on a 4 cm high wooden bar. If the animal maintained theimposed posture for at least 20 s and given one point, every further 20 s that theanimal continued to maintain the cataleptic posture one extra point was given, thetest was conducted for three times. Values are given as mean ± SD for six mice ineach group. ⁄P < 0.05, compared with the control group; #P < 0.05, compared withthe MPTP group.

M. Kavitha et al. / Chemico-Biological Interactions 206 (2013) 239–247 243

MAO-B inhibitors have been employed in neurodegeneration pa-tients, in which improvements have been observed[28]. Mangiferinpre-treatment significantly decreased MPTP induced enhancedactivity of MAO-B as compared to MPTP alone treated group,whereas oral treatment of mangiferin alone did not diminished

the activity of MAO-B significantly. Phytochemicals such as epigal-locatechin-3-gallate [29] and kaempferol [30] are the poor inhibi-tors of MAO-B and offer neuroprotection against MPTP inducedtoxicity by inhibiting oxidative stress and other mechanisms butnot by inhibiting MAO-B activity. Therefore, mangiferin like otherpolyphenols is likely to prevent brain MAO-B activity in PD animalsvia physiological antioxidant status improvement, rather thandirectly affecting the enzyme.

Nearly all the functional dopaminergic neurons in the SN wereTH immunopositive cells and double labeled with DAT [31]. Afterthe injections with MPTP, the protein expressions of TH and DATwere reduced owing to cell loss of dopaminergic neurons. We didnot evaluated the impact of mangiferin on MPTP induced TH andDAT activity. Nagatsu, [32] suggested that the repeated systemicadministration of MPTP diminished TH activity also. TH activityitself is markedly decreased in the SN and ST in the PD brain[33]. Borges et al. [34] reported that TH is modified by sulphydryloxidant and that this post-translational modification results in adecrease in TH catalytic function. Moreover, S-glutathionylationhas been suggested to be accelerated by ROS [35]. In fact, it was

Table 2Effect of mangiferin on MPTP induced oxidative stress in SN. Values are given as mean ± SD of six animals in each group.

Parameters TBARS nmol/g tissue GSH mg/g tissue SOD Ua/mg protein CAT Ub/mg protein GPx Uc/mg protein

Control 1.31 ± 0.11 14.69 ± 0.91 1.79 ± 0.16 1.25 ± 0.11 11.71 ± 0.80MPTP 4.10 ± 0.38* 6.73 ± 0.59* 4.05 ± 0.30* 2.38 ± 0.17* 8.26 ± 0.67*

Mangiferin (10 mg/bw) + MPTP 3.10 ± 0.23# 11.26 ± 0.77# 3.37 ± 0.23# 1.81 ± 0.14# 9.43 ± 0.69#

Mangiferin (20 mg/bw) + MPTP 3.06 ± 0.26# 11.31 ± 0.88# 3.30 ± 0.23# 1.86 ± 0.15# 9.62 ± 0.74#

Mangiferin (40 mg/bw) + MPTP 2.74 ± 0.17� 11.66 ± 0.99� 3.16 ± 0.19� 1.52 ± 0.13� 10.73 ± 0.80�

Mangiferin 1.29 ± 0.08 14.73 ± 0.99 1.84 ± 0.17 1.29 ± 0.12 11.84 ± 0.80

* P < 0.05, compared with the control group.# P < 0.05, compared with the MPTP group.

� P < 0.05, compared with the mangiferin (low and middle dose) + MPTP treated group.a Enzyme concentration required for 50% inhibition of nitrobluetetrazolium reduction in 1 min.b Nanomoles of hydrogen peroxide consumed per minute.c Nanomoles of NADH oxidized per minute.

Table 3shows the effect of mangiferin on MPTP induced enhanced activity of straitalmonamine oxidase B (MAO-B). Values are given as mean ± SD (n = 6).

Groups/variable Control MPTP MPTP + mangiferin (10 mg/kg bw) Mangiferin (10 mg/kg bw)

Monoamine oxidase-B (mU/mg protein) 3.19 ± 0.24 5.34 ± 0.41* 4.25 ± 0.30# 3.02 ± 0.23

* P < 0.05, compared with the control group.# P < 0.05, compared with the MPTP group. ANOVA followed by DMRT.

AControl MPTP Mangiferin + MPTP Mangiferin

B

*

#

020406080

100120

No

of .

TH

IR

neu

rons

TH Immunoreactivity

Control MPTP

MPTP+Mangiferin Mangiferin

Fig. 5. a, b shows the quantification of TH-immunoreactivity (TH -IR) neurons only at the surface of immunolabeled SN tissue were counted manually using light microscopy(40� magnification, images analyzed by Micro Computer Imaging Device) by an individual blinded to the treatment group. The mean value for TH-IR determined for eachgroup, and was expressed as a percentage of that matched control mice. Values are presented as mean ± SD of three mice per group. ⁄P < 0.05, compared with the controlgroup. #P < 0.05, compared with the MPTP group.

244 M. Kavitha et al. / Chemico-Biological Interactions 206 (2013) 239–247

reported that antioxidants exert a protective effect on TH immuno-reactivity [36]. Levites et al., [29] reported that administration ofpolyphenols rich green tea extract (GTE) and EGCG, a majorpolyphenol of GTE offers prevention against the MPTP-induceddepletion of TH protein and TH activity. The numerous anatomo-physiopathological similarities between the nigral degenerationin MPTP animal models of PD and the human disease is suggestivefor the involvement of a specific unknown neurotoxin in PD, whichis uptaken by the DAT into the dopaminergic neuron. DAT expres-sion has been studied in living humans and most of these studiesshowed differences in the DAT levels [37]. Bezard et al., [38] re-ported that individual differences in developing Parkinson’s dis-ease in human may be related to differences of uptake throughthe DAT of a yet unidentified neurotoxin. Immunohistochemistrystudies of our present study revealed the neuroprotective ability

of mangiferin, since it protects neuronal damage induced by MPTPdue to its antioxidant, anti-inflammatory and cell signaling modu-lating properties [8].

To explore the antioxidant action of mangiferin, through inhib-iting oxidative stress, we determined the activity of the antioxidantenzymes, SOD, catalase, GSH-Px and the levels of lipid peroxidationproduct and GSH. It was reported that the MPTP metabolism andbiological interaction results in the formation of superoxide anions[39]. Most predominant free radical formed in the brain is superox-ide anions, which induces peroxidation of lipids [40]. TBARS are theimportant markers of lipid peroxidation process and their levelsare reported to have enhanced after MPTP administration [41].

Excess superoxide anion formed due to MPTP administration iseffectively scavenged by increased SOD activity and this inductionis mainly due to oxidative insult of neurotoxin on mitochondrial

AControl MPTP Mangiferin + MPTP Mangiferin

B

*

#

020406080

100120

No

of .

DA

T I

R n

euro

ns DAT Immunoreactivity

Control MPTP

MPTP+Mangiferin Mangiferin

Fig. 6. a, b shows the quantification of DAT-immunoreactivity (DAT -IR) neurons only at the surface of immunolabeled SN tissue were counted manually using lightmicroscopy (40� magnification, images analyzed by Micro Computer Imaging Device) by an individual blinded to the treatment group. The mean value for DAT-IRdetermined for each group, and was expressed as a percentage of that matched control mice. Values are presented as mean ± SD of three mice per group. ⁄P < 0.05, comparedwith the control group. #P < 0.05, compared with the MPTP group.

β

Bax

-

Bcl-

actin

2

Rel

ativ

e in

tens

ity

0.

1.

2.

3.

(Fol

d of

con

trol

)

1

00.5

11.5

22.5

33.5

2

Con

MP

ontro

PTP+

A

*

Bax

Imm

rol

P+Ma

3

B

#

Mangi

unobl

giferi

4

oblott

erin

ottinging in

MPT

Man

in ST

PTP

angife

*

Bcl

ST

iferin

#

-2

Fig. 7. a, b shows significantly enhanced the expression of Bax and diminished theexpression of Bcl-2, while mangiferin treated significantly diminished the expres-sion of Bax and elevated the expression of Bcl-2, in MPTP treated ST. Immunoblotdata quantified by using b-actin as an internal control and the values are expressedas arbitrary units and given as mean ± SD of four experiments in each group⁄P < 0.05 compared to control, #P < 0.05 compared to MPTP alone treated group.

β-

Rel

ativ

e in

tens

ity

Bax

Bcl-2

actin

(Fol

d of

con

trol

)

x

2

tin

00.5

11.5

22.5

3

C

MPT

ontrol

1

*Imm

P+Man

#

Bax

mun

A 2

unob

giferin

oblott

rin

Blotting

3

ing in

MPTP

Mangif

*

in SN

TP

#

Bcl-

SN

erin

4

#

2

Fig. 8. a, b shows significantly enhanced the expression of Bax and diminished theexpression of Bcl-2, while mangiferin treated significantly diminished the expres-sion of Bax and elevated the expression of Bcl-2, in MPTP treated SN. Immunoblotdata quantified by using b-actin as an internal control and the values are expressedas arbitrary units and given as mean ± SD of four experiments in each group⁄P < 0.05 compared to control, #P < 0.05 compared to MPTP alone treated group.

M. Kavitha et al. / Chemico-Biological Interactions 206 (2013) 239–247 245

electron transport chain. SOD formed hydrogen peroxide, a lesstoxic free radical by utilizing superoxide anion, which is scavengedby catalase and GPx. So the enhanced activity of SOD is more effec-tive, if it is followed by the enhanced activities of catalase and GPx.Our results show similar pattern in SOD and catalase activitiesafter MPTP administration. In the present study, MPTP-treated ani-mals shows marked depletion of GSH and GPx in the SN, this is insupport of previous findings [42,43]. The decrease in GSH appears

to be an early component of the disease that initiates a cascade ofevents leading to oxidative stress and may render the nigrostriataldopaminergic system sensitive to toxins [44]. Pretreatment ofmangiferin produces a notable improvement in the levels of GSHand GPx, due to its potent antioxidant function.

In the present study, MPTP treatment significantly caused alter-ations in motor function observed by the results of various behav-ioral tests (open field, hang test, swim test, catalepsy). There was ahigh degree of correlation between dopaminergic neuron degener-ation and motor impairment in MPTP-induced Parkinson model

246 M. Kavitha et al. / Chemico-Biological Interactions 206 (2013) 239–247

[14]. Nigrostriatal pathway is a neural pathway that connects theSN with the striatum via dopaminergic neurons. The primary func-tion of SN appears to be the control of motor function, and it formsa part of ‘motor circuit’. Disruption of such a system may result invarious symptoms such as tremor, muscular rigidity, akinesia, andbradykinesia seen in Parkinson’s disease [45]. The neurotransmit-ter, DA plays a key role in the body movement and motor control.Degeneration of DA neurons concomitantly decreases the releaseof DA neurotransmitter that results in profound deficits in the mo-tor functions. Reduced levels of catecholamines and excess oxida-tive stress are the contributing factors of neurodegeneration in PDand this leads to the loss of motor function seen in the patientswith PD [46]. Treatment with mangiferin improves the behavioralstatus of MPTP intoxicated mice due to its neuroprotective effectby enhancing depleted dopamine and minimizing the other delete-rious effect of neurotoxins. Clinical and experimental research hasshown that increased physical activity in both patient and animalmodels of PD result in improved motor function and increased stri-atal DA concentrations [47]. Behavioral patterns were establishedas a sensitive index of nigrostriatal pathway function, normaliza-tion of these behaviors by mangiferin treatment is therefore anindicator of therapeutic efficacy of mangiferin.

In normal individuals, approximately 2400 nigral cells dieannually, but, in a brain of Parkinson’s patient approximately240,000 nerve cells in SN die per year [48]. It is reported that sys-temic injection of MPTP in mice produced death of dopaminergicneurons in the substantia nigra [49] by inducing apoptosis. Bcl-2family members play an important role in MPP+ and MPTP inducedapoptotic cell death [50]. Neurotoxicity results in a reduction ofanti-apoptotic protein Bcl-2, which correlates with an increase inpro-apoptotic protein Bax [51]. In our study, MPTP induction sig-nificantly increased the expression of Bax and reduced the Bcl-2,which were consistent with previous studies [52]. Increased levelsof ROS induce Bax to permeabilize the external mitochondrialmembrane [53], leading to the release of cytochrome C and theactivation of caspases, The pathway of extracellular signal-regu-lated protein kinase (Erk) which is essential for apoptotic neuronaldeath, can be stimulated and modulated by increases in the levelsof intracellular calcium, protein kinase A, diacylglycerol and cAMP[54]. The antiapoptotic Bcl-2 protein over expression and proapop-totic Bax ablation by mangiferin have been shown to prevent theneurotoxicity and apoptosis induced by MPTP. Also in line withour results, several reports have demonstrated that mangiferintreatment in neuronal cells and in vivo has protective activity viaregulation of Bcl-2 family proteins, including Bcl-2 and Bax. Fol-lowing excitotoxic stimulus, mangiferin impede the upregulationof Bax in the cytosol, thus intercepting apoptotic death whichdepends on caspases and/or AIF since a Bax-induced pro-oxidantstate is critical for cytochrome C release during programmed neu-ronal death. [55].

In conclusion, our results clearly demonstrate that mangiferinconfer protection against MPTP toxicity and its protective mecha-nism could be partly associated with its anti-oxidative and anti-apoptotic effect. In future, mangiferin may be a useful therapeuticagent for the treatment of idiopathic PD.

Conflict of interest statement

There are no competing interests.

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