Neurochemistry International 65 (2014) 1–13

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Neurochemistry International

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Comparison of the neuroprotective potential of Mucuna pruriens seedextract with estrogen in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)-induced PD mice model

0197-0186/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.neuint.2013.12.001

⇑ Corresponding author. Tel.: +91 9454734930.E-mail addresses: satyndra_yadav@yahoo.co.in (S.K. Yadav), jaiprakash_biote-

ch@yahoo.co.in (J. Prakash), shikhachouhan16@gmail.com (S. Chouhan), sus-wes00@gmail.com (S. Westfall), mradul.bhu@gmail.com (M. Verma),tryambak@yahoo.com (T.D. Singh), suryasinghbhu16@gmail.com, ssingh35@bhu.a-c.in (S.P. Singh).

Satyndra Kumar Yadav a, Jay Prakash a, Shikha Chouhan a, Susan Westfall a, Mradul Verma b,Tryambak Deo Singh b, Surya Pratap Singh a,⇑a Department of Biochemistry, Faculty of Science, Banaras Hindu University, Varanasi 221005, Indiab Department of Medicinal Chemistry, Institute of Medical Science, Banaras Hindu University, Varanasi 221005, India

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

Article history:Received 23 September 2013Received in revised form 11 November 2013Accepted 2 December 2013Available online 11 December 2013

Keywords:Parkinson’s diseaseMucuna pruriensMPTPGFAPDopamineEstrogeniNOS

Parkinson’s disease (PD) is one of the most common neurodegenerative disease found in the aging pop-ulation. Currently, many studies are being conducted to find a suitable and effective cure for PD, with anemphasis on the use of herbal plants.

In Ayurveda, Mucuna pruriens (Mp), a leguminous plant, is used as an anti-inflammatory drug. In thisstudy, the neuroprotective effect of an ethanolic extract of Mp seed is evaluated in the 1-methyl-4-phe-nyl-1,2,3,6-tetrahydropyridine (MPTP) model of PD and compared to estrogen, a well reported neuropro-tective agent used for treating PD.

Twenty-four Swiss albino mice were randomly divided into four groups: Control, MPTP, MPTP + Mpand MPTP + estrogen. The behavioural recovery in both Mp and estrogen treated mice was investigatedusing the rotarod, foot printing and hanging tests. The recovery of dopamine neurons in the substantianigra (SN) region was estimated by tyrosine hydroxylase (TH), immunostaining. Additionally induciblenitric oxide synthase (iNOS) and glial fibrillary acidic protein (GFAP) immunoreactivity was evaluatedto assess the level of oxidative damage and glial activation respectively. The levels of dopamine and itsmetabolite in the nigrostriatal region were measured by HPLC.

Mp treatment restored all the deficits induced by MPTP more effectively than estrogen. Mp treatmentrecovered the number of TH-positive cells in both the SN region and the striatum while reducing theexpression of iNOS and GFAP in the SN. Treatment with Mp significantly increased the levels of dopa-mine, DOPAC and homovanillic acid compared to MPTP intoxicated mice. Notably, the effect of Mpwas greater than that elicited by estrogen.

Mp down regulates NO production, neuroinflammation and microglial activation and all of theseactions contribute to Mp’s neuroprotective activity. These results suggest that Mp can be an effectivetreatment for neurodegenerative diseases, especially PD by decreasing oxidative stress and possibly byimplementing neuronal and glial cell crosstalk.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction Parkinson’s disease (PD) is the second most common neurode-

Mucuna pruriens (Mp) is a leguminous plant belonging to thefamily Fabaceae. In Ayurveda, Mp seeds have been used as ananti-inflammatory agent (Hishika et al., 1981) and as a treatmentfor different free radical-mediated diseases including aging, rheu-matoid arthritis, diabetes and neurodegenerative diseases such asParkinson’s disease (Vaidya et al., 1978; Yadav et al., 2013).

generative disorder after Alzheimer’s disease affecting humans(Tanner and Goldman, 1996; Siderowf and Stern, 2003). It is anage-related neurodegenerative disease characterized by tremors,postural abnormalities and bradykinesia. Generally, PD is morecommon in men than in women and the risk increases with age(Baldereschi et al., 2000; Van Den Eeden et al., 2003). Chances ofPD occuring in menopausal women are drastically increased andpositively correlated with the decrease of estrogen levels (Weberand Mapstone, 2009; Alonso et al., 2010). These observations alonesuggest some interplay between the hormonal balance in womanand the development of neurodegenerative diseases.

In PD, there is progressive and selective destruction of dopami-nergic neurons in the substantia nigra (SN) brain region. Although

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the etiology of the disease remains unknown, there are many po-tential causes including genetic factors and endogenous or envi-ronmentally-derived neurotoxins. Multiple mechanisms like theubiquitin–proteasome system (UPS), oxidative stress, mitochon-drial dysfunction and inflammation are involved in the develop-ment of this disease (Seniuk et al., 1990; McNaught et al., 2004).

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) inducesdopaminergic toxicity (Mytilineou and Friedman, 1988; Songet al., 1997) and can be used as a robust model of PD in animals.After crossing the blood brain barrier, MPTP is converted into itsactive metabolite MPP+ in glial cells by the enzyme monoamineoxidase B (MAO-B). MPP+ enters dopaminergic neurons throughdopamine transporter (DAT) where it binds to complex I and im-pairs mitochondrial function. This stimulates the overproductionof free radicals which causes oxidative stress and ultimately leadsto the activation of the cell death pathways (Dauer and Przedbor-ski, 2003).

Clinical studies have shown that estrogen treatment reducesthe risk of PD (Tsang et al., 2000) and also restores the motor abil-ity in Parkinsonian mice (Benedetti et al., 2001). It has been re-ported that estrogen can protect SN dopaminergic neurons andstriatal neurotransmitter neurons in both male and female rodentsagainst MPTP and 6-OHDA induced toxicity (Quesada and Mice-vych, 2004).

Mucuna pruriens (Mp) naturally contains 4–5% L-DOPA, the pre-cursor for dopamine and the present canonical treatment for PD(levodopa). Mp also contains various alkaloids such as prurienine,prurieninine and prurienidine (Misra and Wagner, 2004). Theseeds of Mp are rich in several amino acids along with proteinsincluding globulins and albumins (Pant and Joshi, 1970), carbohy-drate, and fatty acids such as oleic acid, linoleic acid and palmiticacid (Adebowale et al., 2005). Triterpenes and sterols (b-sitosterol,ursolic acid, etc.) are also found in the root and seeds of Mp (Sid-dhuraju et al., 1996). A few studies conducted earlier have shownthat Mp extract contain NADH, Coenzyme Q10 (Manyam et al.,2004), L-DOPA (Manyam and Parikh, 2002) and also have estro-genic activity (Shahaji and Parnu, 2011). A double-blind clinicalstudy conducted previously, suggests that when L-Dopa is usedalone for longer duration it leads to the development of dyskinesiawhile the use of Mp seed powder formulation did not develop dys-kinesia (Katzenschlager et al., 2004). This suggests that Mp mightposses advantages over conventional L-Dopa preparation in longterm management of PD.

Despite the knowledge of its active components, the neuropro-tective mechanism of Mp remains elusive. Therefore, in the presentstudy, the basic molecular mechanism behind the neuroprotectiveeffect of Mp in MPTP-induced Parkinsonian mice was investigated.Further, the effectiveness of Mp’s action was compared to estrogen,a well known neuroprotective agent currently used for the treat-ment of PD. To determine the efficacy of Mp, we studied threeparameters: first, changes in motor functionality by rotarod, footprinting and hanging tests; second, oxidative stress occurring inthe nigrostriatal region by nitrite content, glutathione (GSH) andmalondialdehyde (MDA) levels and third by the immunohisto-chemical analysis of tyrosine hydroxylase (TH) expression in theSN and striatum and also, iNOS and GFAP expression in the SN. Fi-nally, the level of dopamine and its metabolites were measuredthrough HPLC.

2. Materials and methods

2.1. Animals and preparation of plant extract

Male Swiss albino mice weighing 25 ± 5 g were used in thepresent study. All experimental procedures were conducted

according to the National Guidelines on the Proper Care and Useof Animals in Laboratory Research and were approved by the Insti-tutional Ethical Committee. Animals were kept in a controlled tem-perature of 22 ± 3 �C on a 12 h:12 h light–dark cycle. Food pelletsand water were made available to these animals ad libitium (Chou-han et al., 2013). Mp seed powder was purchased from the Ayurv-eda Pharmacy, Institute of Medical Science, Banaras HinduUniversity, Varanasi, India. The ethanolic extract of Mp seed pow-der was obtained by soaking 500 g of the powdered material in1000 ml of ethanol overnight and subsequently extracting in asoxhlet apparatus using the continuous hot extraction method at60 �C for 35 h. The extract was concentrated under reduced pres-sure and stored at 4 �C. The amount of plant extract was expressedin terms of dry weight (Eze et al., 2011).

2.2. Chemicals

Acetic acid, disodium hydrogen phosphate, glutathione (GSH),reduced nicotinamide adenine dinucleotide phosphate (NADPH),potassium chloride and sodium dihydrogen phosphate were pro-cured from Sisco Research Laboratories (SRL; Mumbai, India).Streptavidin-peroxidase, normal goat serum and the DAB (3,3diaminobenzidine) system were procured from Bangalore GeneiPvt. India Ltd., Bangalore, India. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and estradiol were purchased from Sigma–Aldrich (St. Louis, MO, USA). Folin Ciocalteau reagent, hydrogenperoxide (H2O2), glutathione reductase (GR), and potassium-dichromate were purchased from Merck (Darmstadt, Germany).Primary antibodies for TH and inducible iNOS were procured fromSanta Cruz, Biotechnology (Santa Cruz, CA, USA) and the primaryantibody for GFAP was purchased from Enzo Life Science, Bioge-nuix Medsystems Pvt. Ltd. (New Delhi, India).

2.3. Acute toxicity test

To conduct the acute toxicity test, mice were divided into ninegroups with six mice in each group. The acute oral toxicity of Mpseed ethanolic extract in Swiss albino mice were carried out asper OECD (Organization for Economic Co-operation and Develop-ment) guideline 423 (OECD, 423, 2001). Briefly, ethanolic Mp seedextract was orally administered to groups of mice with increasingconcentrations: 150, 200, 250, 500, 750, 1000, 1250, 1500 and2000 mg/kg body weight. Signs of toxicity were observed in micecarefully during the first 4 h after treatment and daily thereafterfor a period of 14 days. Different parameters were observed includ-ing body weight, hair loss patterns, mortality, and signs of illness,injury, pain and allergic reactions. Food and water intake were re-corded weekly during the study period of 14 days. The observa-tions were recorded systematically and individual records weremaintained for each group (Ecobichon, 1997).

2.4. Dose chasing experiment

The results from the acute toxicity test were used to optimizetreatment doses on a different set of mice. Safe doses were taken(25, 50, 100, 150, 200 mg/kg body-weight) of ethanolic Mp seedextract and administered to mice. Neurobehavioural parametersfor motility were conducted including the hanging, rotarod andfoot printing tests, as described previously (Yadav et al., 2013).

2.5. Treatment of the mice

After conducting acute toxicity test and dose chasing experi-ments we took fresh mice for carrying out experiments. Mice weredivided into four groups with six animals in each group. The firstgroup was treated with saline (i.p.) and used as controls. The

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Fig. 1. Dose response curve of alcoholic extract of Mp seed with respect to (a) rotarod (b) stride forepaw length test and (c) hanging test conducted in mice, at concentrationsvarying from 25 to 200 mg/kg body weight (mean ± SEM, n = 6. Significant difference ⁄⁄⁄p < 0.001 compared to control).

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second group was injected (i.p.) with MPTP (30 mg/kg bodyweight/day) twice within 16 h to induce PD. The third group wasinjected (i.p.) with MPTP (30 mg/kg body weight/day) twice within16 h and additionally treated with Mp seed extract (100 mg/kgbody weight) for 7 days prior to MPTP treatment and 7 days after(Rajasankar et al., 2007). The fourth group of mice were injected(i.p.) with MPTP (30 mg/kg body weight/day) twice within 16 hand treated with estrogen (0.1 mg/kg body weight), 7 days priorto MPTP treatment and 7 days after (Baraka et al., 2011).

2.6. Behavioural studies

After the completion of treatment, behavioural studies wereperformed to understand motor skill abnormalities in the PD mod-el. The rotarod, hanging and foot printing tests were used.

In the Rotarod experiments, test animals were trained for 3 con-secutive days before starting the experiment at a fixed speed(5 rpm). The time it took for mice to fall down was recorded, upto a maximum of 5 min. For each animal, the experiment was re-peated four times and the average time was calculated (Mannaet al., 2006). The experiment was repeated after the completionof the treatment and the time it took the mice to fall was recorded.

In hanging test, mice were placed on a horizontal grid until theygripped the grid. Following, this the grid was inverted such that themice were made to hang upside down. The hanging time was mea-sured until the mice fell itself (Mohanasundari et al., 2006; Prakashet al., 2013a). The experiment was repeated 4 times and the aver-age for all the measurements were calculated.

In the foot printing test, mice first had both their forepaw andhindpaw stained in black ink and were then allowed to walk onplain white paper. The stride length was measured during normal

walking by determining the distance between each step on thesame side of the body from the middle toe of the first step to theheel of the second step. The experiment was repeated 4 timesand the average was calculated (Yadav et al., 2013; Prakash et al.,2013a).

2.7. Decapitation and dissection of brains

After behavioural tests, one set of mice from each group wassacrificed by cervical dislocation followed by decapitation to en-sure minimal discomfort (Prakash et al., 2013a; Surendran,2007). After decapitation, the brain was removed, microdissectedon a glass plate over ice and the nigrostriatal area was collectedand stored at �80 �C until further use.

2.8. Nitrite estimation

Nitrite levels were estimated using a standard protocol (Gran-ger et al., 1996). In brief, 10% nigrostriatal tissue homogenatewas incubated with ammonium chloride and mixed with Griess re-agent. The reaction mixture was incubated at 37 �C for 30 min andthe absorbance of the supernatant was recorded at 540 nm. The ni-trite content was calculated using a standard curve for sodium ni-trite (10–100 lM) in terms of lmoles/ml.

2.9. Lipid peroxidation (LPO) and glutathione (GSH) content

LPO in the nigrostriatal tissues was determined as previouslydescribed (Ohkawa et al., 1979), with slight modifications. In brief,a 20% homogenate (0.1 ml) of nigrostriatal tissue was incubatedwith a 10% SDS solution for 5 min at room temperature. Following

Fig. 2. (a) Rotarod test of adult animals was performed in all the groups and a significant improvement in the time of stay on rotarod was found in Mp treated mice comparedto MPTP group. (b) Stride forepaw length was significantly reduced in the PD mouse compared to control. (c) Hanging test showed significant improvement in Mp treatedgroup compared to MPTP exposed group (mean ± SEM, n = 6. Significant difference ⁄⁄⁄p < 0.001 compared to control and $$p < 0.01 and $$$p < 0.001 compared to MPTP).

Fig. 3. Effect of ethanolic extract of Mp seed extract treatment on nitrite level in the nigrostriatal tissue of PQ treated mice brain (mean ± SEM, n = 6. Significant differencesare ⁄⁄p < 0.01 and ⁄⁄⁄p < 0.001 compared to control; $p < 0.05 and $$$p < 0.001 compared to MPTP).

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this, 20% acetic acid (0.6 ml) was added and the solution was incu-bated for 2–5 min. Finally, 0.8% TBA (0.6 ml) was added and thereaction mixture was kept in a 100 �C boiling water bath for 1 h.The reaction mixture was cooled, centrifuged and supernatent’sabsorbance was read at 532 nm against blank. LPO levels were ex-pressed as nmoles malondialdehyde (MDA)/mg nigrostriatal tissue.

GSH in the nigrostriatal tissue was estimated by the method ofMoron et al. (1979), using 5,5-dithiobis 2-nitrobenzoic acid(DTNB). In short, the homogenate (100 ll) was mixed with DTNB(2 ml in phosphate buffer, pH 8.0) and the volume was made upto 3.0 ml with phosphate buffer. Absorbance was recorded imme-diately at 412 nm and the GSH content was calculated in lM/mgtissue (nigrostriatal tissues) using a standard curve of GSH.

2.10. Measurement of dopamine, DOPAC and HVA

The levels of DA and its metabolites (DOPAC and HVA) wereestimated from the isolated nigrostriatal homogenate using aHPLC–electro chemical detection (ECD) system (Kim et al., 1987;Krishnamurthy et al., 2011). In brief, the brain tissue samples werehomogenized in 0.17 M perchloric acid using a Polytron homoge-nizer. Homogenates were centrifuged at 33,000�g (Biofuge Stratos,Heaureas, Germany) at 4 �C. The supernatant (20 ll) was injectedinto a HPLC pump (Model 1525, Binary Gradient Pump) having aC18 a column (Spherisorb, RP C18, 5 mm particle size, 4.6 mmi.d. � 250 mm at 30 �C) connected to an ECD (Model 2465, Waters,Milford, MA, USA) at a potential of +0.8 V with a glassy carbon

Fig. 4. Effect of ethanolic extract of Mp seed on malondialdehyde (MDA) in the PD mouse brain. The PD model mouse nigro-striatum showed elevated levels of MDAcompared to the control. The increased levels of MDA were significantly decreased after treatment with ethanolic extract of Mp seed compared to the PD mouse brain(mean ± SEM, n = 6, p < 0.05).

Fig. 5. Effect of ethanolic extract of Mp seed on glutathione (GSH) in the PD mouse brain. The PD mouse nigro-striatum had reduced levels of GSH compared to the control.The decreased levels of GSH were significantly improved after treatment with ethanolic extract of Mp seed compared to the untreated PD mouse brain (p < 0.05).

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working electrode vs. a Ag/AgCl reference electrode. The mobilephase consisted of 32 mM citric acid, 12.5 mM disodium hydrogenorthophosphate, 1.4 mM sodium octyl sulfonate, 0.05 mM EDTAand 16% (v/v) methanol (pH 4.2) at a flow rate of 1.2 ml/min. Thechromatogram was recorded and analyzed with Empower soft-ware (Version 2.0). The protein content was estimated using themethod of Lowry et al. (1951).

2.11. Cryosectioning

The cryosectioning was performed on the other set of mice fromeach group according to the method of Singh et al. (2009). In short,intracardiac perfusion was performed with normal saline followedby 4% paraformaldehyde in phosphate buffered saline (PBS) at theflow rate of 20 ml/min for 4 min. Brains were dissected coronally,post fixed in 10% paraformaldehyde and cryoprotected in a sucrosesolution (10%, 20%, 30% wt./vol) for 24 h. Thin sections (20 lm) ofthe substantia nigra and striatum region of the mouse brain wasobtained using a cryostat and used to visualize TH, iNOS and GFAPwith immunohistochemical techniques.

2.12. TH-immunoreactivity

The 20 lm sections of the SN and striatum region of mice brainwere placed on a tissue culture plate in PBS. The sections werewashed three times in PBS and submerged in blocking buffer one(methanol and H2O2 in PBS) for 15 min, followed by blocking buffertwo (2% normal goat serum in PBS) for 2 h in order to prevent non-specific labeling (Singh et al., 2009; Yadav et al., 2013). The sec-tions were incubated with a mouse monoclonal anti-TH antibody(1:1000) in PBS at 4 �C for 48 h. Following this, the sections werewashed 3 times for 15 min in PBS. The sections were subsequentlyincubated with an anti-mouse biotin conjugated secondary anti-body (1:500) for 2 h. Finally, the sections were submerged in astreptavidin peroxidase complex (1:500) for 1 h. The colour wasdeveloped with 3,3-diaminobenzidine (DAB) and the sections werepermanently mounted by dextrene pthylate xylene (DPX) treat-ment after being dehydrated in graded ethanol. The sections werevisualized under a light microscope (Olympus, Model-CX31RTSF)and the images were captured. The TH-positive cells were countedas described previously (Singh et al., 2009) with slight modifica-

Fig. 6. The effect of Mp seed extract treatment (100 mg/kg) on (a) Dopamine, (b) DOPAC, and (c) HVA levels in nigrostriatum region of all the groups (Mean ± SEM, n = 6.Significant differences are ⁄⁄p < 0.01 and ⁄⁄⁄p < 0.001 compared to control; $p < 0.05 and $$$p < 0.001 compared to MPTP).

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tions. Counting was done by unbiased means. TH-positive cellsfrom 6 slides of the SN region per animal were counted and theaverage was taken. A minimum of 3 animals per group were usedand the average per group was determined.

2.13. iNOS-immunoreactivity

Animals were anesthesized with ether and perfused. The brainwas dissected as described above. The incubation with blocking re-agents, primary antibodies, and development were performed asdescribed above for TH-immunoreactivity, however, the brain sec-tions were incubated in rabbit anti-mouse polyclonal antibodiesagainst iNOS (1:800, Santa Cruz, USA) for 48 h (Chun et al., 2002)followed by an anti-mouse biotin labelled secondary antibody(1:400 in PBS) for 2 h. The labelled sections were incubated instreptavidin (1:400 in PBS) for 2 h. and the colour was developedusing DAB substrate solution. Finally, the sections were dehydratedwith graded alcohol and mounted permanently with DPX. Inte-grated densities of iNOS were measured in the SN using the freelyavailable software Image 90 J basic (version 1.38) (Singh et al.,2011).

2.14. GFAP-immunoreactivity

Sections of the SN region were pre-incubated in blocking buffer(PBS containing 2% normal goat serum) for 2 h to reduce non-spe-cific binding. Sections were then incubated overnight at RT with amonoclonal mouse anti-glial fibrillary acidic protein (GFAP) anti-body (a marker of activated astrocytes, 1:1000 dilutions). Thiswas followed by incubation with a biotinylated anti-mouse IgGantibody (1:500) for 2 h at RT and subsequently in avidin peroxi-dase (1:500 dilution) at RT for 2 h. Finally, section were stained

with DAB to visualize the immunoreactivity (DAB, Sigma). PBSwashes (3 � 15 min) were performed between each step. Sectionsstained with GFAP were dehydrated and covered with a cover slipwithout counterstaining. For quantification, images of the SN GFAPimmunostaining were taken with a 10� objective using equalacquisition conditions. Image J software was used to determinethe fraction of GFAP positive area.

2.15. Statistical analysis

For the behavioural tests, statistical analysis between groupswas done using student’s t-test. Data were expressed asmean ± standard error (SEM) for separate groups and differenceswere considered statistically significant, when p values were lessthan 0.05 (p < 0.05).

To assess the biochemical and immunohistochemical analyses,statistical analysis of the data was performed using one-way anal-ysis of variance (ANOVA) with the Student-Neuman–Keuls posthoc analyses using the Statistical Package for the Social Sciences(SPSS). Data were expressed as mean ± standard error (SEM) forand differences were considered statistically significant, when pvalues were less than 0.05 (p < 0.05).

3. Results

3.1. Acute oral toxicity assessment

The saftey of the ethanolic seed extract of Mp was determinedusing an acute oral toxicity assessment. Even at the highest dosageof 2200 mg/kg body weight, no mortality was reported. Addition-ally, there were no significant changes observed in behaviour (i.e.

Fig. 7. TH immunoreactivity of dopaminergic neurons in MPTP-induced PD phenotype in mouse substantia nigra in the presence Mp or estrogen, (n = 6 in each group). (a) THimmunoreactivity in frozen brain sections of control and treated animals. (b) Bar diagram showing number of TH positive neurons in SN region of control and treated mice.(mean ± SEM, n = 6. Significant difference ⁄p < 0.05, ⁄⁄p < 0.01 and ⁄⁄⁄p < 0.001 compared to control; $$p < 0.01 and $$$p < 0.001 compared to MPTP).

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ataxia, hyperactivity, hypoactivity) in any of the mice neither werethere any variations in general appearance. Body weight, feed in-take and water consumption were found to be normal during thecourse of study. Due to the low toxicity of the ethanolic Mp seedextract, it was assigned to class 5 (LD50 > 2200 mg/kg body weight)as recommended by the OECD.

3.2. Dose selection

The results from the oral toxicity assessment were used todetermine a safe dose of ethanolic Mp extract for conducting adose chase experiment. The efficacy of the dose was assessed14 days after treatment by observing behavioural parameters formotility, namely the rotarod (Fig. 1a), foot printing (Fig. 1b) andhanging tests (Fig. 1c). It was found that a dose of 100 mg/kg bodyweight produced significant changes in the behaviour of treatedmice, thus this dose was selected to evaluate the neuroprotectiverole of Mp.

3.3. Behavioural studies

The rotarod test demonstrates rodents’ balance and coordina-tion on a self-propelled rotating beam. The time for which MPTP-treated mice remained on the beam was significantly reduced(p < 0.001) compared to controls. Following Mp cotreatment, theMPTP-Mp mice remained on the beam significantly longer thanMPTP-alone treated mice (p < 0.001). Notably, the recovery with

Mp treatment was greater than MPTP mice cotreated with estro-gen (Fig. 2a).

The hanging experiment was also implemented to assess motil-ity in PD-modeled mice. In MPTP-treated mice, the time of grippingand hanging was significantly lower (p < 0.001) compared to con-trols. When MPTP-treated mice were cotreated with Mp, the hang-ing time was significantly increased (p < 0.001). Similar to therotarod test, the MPTP-Mp treated animals showed greaterimprovement than MPTP mice cotreated with estrogen(p < 0.001; Fig. 2b).

Finally in the foot-printing test, a significant interaction be-tween the duration of treatment and the effect of MPTP orMPTP-Mp was observed (p < 0.001). The improvements observedin walking errors for the MPTP-Mp treated mice were similar toMPTP treated mice cotreated with estrogen (Fig. 2c).

3.4. Nitrite estimation

Detectable nitrite levels were observed in the nigrostriatal re-gion of all treatment groups. The one-way ANOVA revealed thatthe variation of nitrite between all the groups was highly signifi-cant (F(3, 8) = 23.08, p < 0.001). Post-hoc analyses showed thatthe administration of MPTP significantly increased the nitrite lev-els in the Parkinsonian mouse nigrostriatal region compared tocontrol (p < 0.001). Following treatment of Mp seed extract, the ni-trite levels were significantly reduced compared to the untreatedPD mouse (p < 0.001; Fig. 3). Mp treatment induced significant de-

Fig. 8. Protective effect of Mp against MPTP toxicity in Swiss albino mice. Dopamine neurons in striatum regions were visualized with TH-immunostaining. (a) THimmunoreactivity in frozen brain sections of control and treated animals. (b) Bar diagram showing number of TH fibres in striatum region of control and treated mice. Theoptical density in the striatum were measured. Each bar represents the mean ± SEM, n = 6. Data are expressed as percentages relative to untreated controls. ⁄⁄p < 0.01compared with the control group, $$p < 0.01 and $p < 0.05 compared with the MPTP group.

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crease in nitrite content that superceeded the effect of estrogen onMPTP treated mice.

3.5. Lipid peroxidation (LPO) and glutathione (GSH) content

Following the behavioural studies, biochemical assays wereperformed to explore the effect of Mp seed extract in the improve-ment of antioxidant levels. To assess the level of lipid peroxidation,the level of MDA was examined and it was found to be significantlyvaried in response to MPTP exposure and treatment (F(3,8) = 116.75, p < 0.001). Post hoc analyses indicate that the MPTPgroup was significantly increased in the nigrostriatal region ofthe PD mouse compared to control mice (p < 0.001). In accordancewith the earlier studies, a significant reduction in the amount ofMDA was found in Mp treated PD mice (p < 0.001). Further, thereduction implemented by Mp was greater than the effect elicitedby estrogen (Fig. 4).

GSH levels also varied significantly in response to MPTP expo-sure and treatment (F(3, 20) = 19.85, p < 0.001). In particular, MPTPdecreased the GSH levels in comparison to controls in the nigro-striatal region (p < 0.001). The co-treatment of MPTP mice withMp further lead to a significant increase in GSH levels as comparedto the PD mouse (p < 0.001; Fig. 5). Again, the Mp seed extract dis-played better results compared to estrogen treatment.

3.6. Levels of dopamine and its metabolites

The effect of Mp (100 mg/kg body weight) on dopamine levelsin nigrostriatal brain regions is illustrated in Fig. 6a. Analysis byone-way ANOVA demonstrated significant differences in the dopa-mine levels of the nigrostriatal tissues between groups (F(3,20) = 249.67, p < 0.001). Post-hoc analyses showed that MPTP trea-ted mice had significantly decreased dopamine levels as comparedto controls (p < 0.001). The co-treatment of Mp implemented a sig-nificant increase in dopamine content compared to MPTP treatedanimals (p < 0.001). Again, the Mp treatment was more effectiveat rescuing dopamine levels than estrogen treatment.

The effect of repeated Mp treatments on DOPAC levels in thenigrostriatal region of MPTP treated mice is depicted in Fig. 6b.One-way ANOVA analysis revealed that there was significantchange in the DOPAC levels in response to treatment(F(3,20) = 176.26, p < 0.001). Post hoc analysis indicate that the lev-els of DOPAC in the nigrostriatal region of MPTP administeredgroup was less compared to controls (p < 0.001). However, Mptreatment at 100 mg/kg/day for 14 days significantly protectedagainst SN damage, compared to controls (p < 0.001), while estro-gen treatment invoked similar protective effects.

The effect on HVA levels in the nigrostriatal region of all theexperimental mice is depicted in Fig. 6c. One-way ANOVA analysisrevealed that, MPTP treatment yielded a significant effect on HVAlevels (F(3, 20) = 125.76). The MPTP treated group showed a signif-

Fig. 9. Representative tissue sections of the SN, immunostained with the astrocyte marker GFAP. (a) GFAP immunoreactivity in frozen brain sections of control and treatedanimals. (b) Bar diagram showing number of GFAP fibres in substantia nigra region of control and treated mice. Each column represents the mean ± SEM, n = 6. Data areexpressed as percentages relative to untreated controls. ⁄⁄p < 0.01 compared with the control group, $$p < 0.01 and $p < 0.05 compared with the MPTP group.

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icant reduction in HVA levels as compared to control (p < 0.001)whereas Mp co-treatment significantly increased the level ofHVA (p < 0.001) as compared to MPTP alone treatment. Here alsoMp showed better results than estrogen.

3.7. TH-immunoreactivity

The number of TH-positive neurons in the striatum and thenigrostriatal region was calculated by counting the number ofTH-immunoreactive cells. In both the striatum (F(3, 20) = 288.43,p < 0.001) and the substantia nigra (F(3, 8) = 71.381, p < 0.001),there was a significant effect of treatment on the number of TH-immunoreactive neurons. It was observed that MPTP treated ani-mals exhibited a significant reduction in TH-immunoreactivity aswell as the number of TH-positive neurons as compared to controls(p < 0.001). Mp treatment subsequently increased the number ofTH-positive neurons as compared to MPTP treated group(p < 0.001; Fig. 7a and b). Similarly, the fiber density of TH-positiveneurons was reduced in the striatum of the MPTP-treated group(p < 0.001) while a significant restoration of neuronal fibers wasobtained by Mp treatment in the MPTP and Mp co-treated group(p < 0.001; Fig. 8a and b).

3.8. GFAP-immunoreactivity

The number of GFAP-IR astrocytes was significantly altered inresponse to treatment (F(3, 20) = 112.43, p < 0.001). Post-hoc anal-yses indicated that the number of astrocytes was significantly de-creased as compared to controls (p < 0.001). This reduction was

restored efficiently by the treatment of Mp (p < 0.001). Addition-ally, Mp treatment more effective restored the number of GFAPneurons as compared to the estrogen treatment group (p < 0.001;Fig. 9a and b).

3.9. iNOS-immunoreactivity

Finally, the iNOS immunoreactivity was significantly altered inresponse to treatment with MPTP (F(3, 20) = 39.157, p < 0.001).After treatment with MPTP, the percent of iNOS-IR cells in the SNwas increased significantly as compared to controls (p < 0.001).Pre-treatment with Mp (100 mg/kg body weight) significantly de-creased the percentage of iNOS-IR cells, as compared to MPTP trea-ted animals (p < 0.001). Mp treatment showed similar results ascompared to PD mice cotreated with estrogen (Fig. 10a and b).

4. Discussion

Recent studies have suggested that Mp plays an important rolein the neuroprotection of paraquat induced mouse models of PD(Yadav et al., 2013; Prakash et al., 2013b) however, the molecularmechanism behind the neuroprotective role of Mp remains elusive.The present study investigates the neuroprotective effect of Mpand evaluates the basic mechanism of action of Mp in a PD mousemodel induced by MPTP.

MPTP is a toxin that induces not only motor deficits, but thedegeneration of nigrostriatal dopaminergic neurons rodents (Blumet al., 2001). Further, MPTP treatment creates behavioural, neuro-

Fig. 10. Effect of ethanolic extract of Mp seed on iNOS immunoreactivity, (a) Immunohistological visualization of iNOS, in substantia nigra region of mice brain. (b)Quantification of iNOS neurons in substantia nigra region of mice brain. Data are expressed as mean ± SEM, n = 6. ⁄⁄p < 0.01 compared with the control group, $$p < 0.01 and$p < 0.05 compared with the MPTP group.

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chemical and immunohistological characteristics very similar topatients with PD (RajaSankar et al., 2009).

Estrogen is known to have neuroprotective properties in similartoxin-induced mouse models of PD (Baraka et al., 2011). In thepresent study, the neuroprotective role of Mp and estrogen werecompared in terms of the damage to dopaminergic neurons in-duced by MPTP and the consequent biochemical and behaviouralparameter. This study is rationalized as Mp contains phytoestro-gens making estrogen the perfect positive control (Machadoet al., 2012), behavioural studies were conducted including therotarod, hanging and foot printing tests.

For this purpose, behavioural tests namely rotarod, hanging andnarrow beam walking were conducted that showed that impairedmotor function in MPTP treated mice was typical to the parkinson-ism phenotype. Mp treated mice showed a more significantimprovement in posture, coordinated motor skills including volun-tary movements, walking and improved forepaw stride length, ascompared to estrogen treated group. When Parkinsonian micewere treated with Mp, they showed improvement in rotarod, hang-ing time and significant enhancement in the stride length. Our re-sults suggest that the magnitude of cell loss in the SN of mice brainis significantly correlated with reduction of stride length, whichwas previously shown (Fernagut et al., 2013).

MPTP stimulates the degeneration of dopaminergic neurons inthe SN and subsequently, the loss of dopamine (Lee et al., 2012).Further, it is believed that dopamine plays a key role in body move-ment and motor control. Interestingly, various studies have shownthat different plant varieties of Mp contain different levels of L-DOPA, which is the main precursor of dopamine (Raina and Khatri,

2011). Motor functions could be mainly due to presence of L-DOPAand phytoestrogens (Shahaji and Parnu, 2011) hence supplementa-tion of these effects could indeed rescue these debilitating symp-toms of PD.

In accordance with previous studies, the treatment of PD-mod-eled mice with estrogen reared significant improvement on allmeasures of motor activity (Baraka et al., 2011). Interestingly, thepresent study shows that Mp treatment is more effective at rescu-ing the motor deficits and coordination induced by MPTP thanestrogen treatment. These results directly stipulates a potentialrole for Mp as an effective therapeutic for PD. This combined actionof all Mp’s active components could explain why it is more effec-tive than estrogen at treating PD.

In order to assess the direct effect of Mp on dopamine levels,dopamine and its metabolites (DOPAC and HVA) were quantifiedin the nigrostriatal region. The present study revealed reduced lev-els of DA, DOPAC and HVA in MPTP induced Parkinsonian mice.Similarily, these catecholamine levels also found to be decreasedin PD patients (Hinterberger, 1971; Piggott et al., 1999). The in-crease of these catecholamine levels after Mp treatment suggeststhat indeed the L-DOPA content of Mp rescues dopamine levels inthe PD mouse nigrostratum. Interestingly, Mp treatment was moreeffective than estrogen to increase the levels of catecholamine.

MPTP also elicits its neurodegenerative effects by inducing oxi-dative stress and free radical generation in the SN region (Arralet al., 2012). Further, it has been shown that oxidative stress andmitochondrial dysfunction play a major role in the etiology of PD(Napoli, 2007; Schapira, 2008). To assess the level of oxidativestress in MPTP inflicted PD mice, the level of GSH was measured.

S.K. Yadav et al. / Neurochemistry International 65 (2014) 1–13 11

In the presence of glutathione, the enzyme GSH reduces H2O2 intowater thereby eliminating one of the most damaging oxidants inthe body (Perry et al., 1982). In PD patients, the depletion of GSHis one of the earliest biochemical changes in the brain. Cells ofthe central nervous system are prone to free radical toxicity as theyhave a high amount of catecholamine oxidative metabolic activity(Fendri et al., 2006) and lack some of the antioxidant potentialpresent systemically. Together, it is predictable that oxidativedamage is key to the etiology of neurodegenerative diseases likePD.

In the present study, it was observed that the level of GSH wasreduced in the nigrostriatal region of Parkinsonian mice, confirm-ing previous findings (Rajasankar et al., 2009). This suggests thatdamage to the nigrastriatal region in the PD brain is at least par-tially due to the formation of free radicals (Hara et al., 1991; Jonesand Vale, 2000). In addition, it was observed that the treatment ofParkinsonian mice with Mp improved the expression of GSH, to agreater extent than estrogen alone.

During stressful conditions, free radicals are generated and re-act with a free oxygen molecule on the membrane lipid to form aperoxy radical, thus resulting in lipid peroxidation. MDA is a diag-nostic marker for lipid peroxidation and used here as a biomarkerof PD. In the present study, the levels of MDA and hence lipid per-oxidation in the nigrastriatal region of MPTP treated mice were sig-nificantly elevated, supporting previous studies in rodents (Guptaet al., 2010) and monkeys. The treatment of PD mice with Mp, re-duced MDA levels, suggesting that Mp prevents lipid peroxidation.Further, the effect of Mp on MDA levels was greater than estrogenalone.

TH is the rate-limiting enzyme responsible for converting L-DOPA into dopamine. The measurement of TH-immunoreactivityis thus a measure of the functionality of dopaminergic neuronsand fibers present in the SN and striatum, respectively. In the pres-ent study, MPTP treatment imposed a significant reduction in TH-immunoreactivity, a loss that was dependent on the length ofMPTP exposure. These results are supported by earlier reportsshowing selective dopaminergic neuronal loss following exposureto several PD-inducing neurotoxins (Lee et al., 2012). It is believedthat the main mechanism of dopaminergic neuronal loss in theseanimals was oxidative stress generated in response to MPTP expo-sure (Blum et al., 2001). Mp treatment produced a significantimprovement in the number of dopaminergic neurons and fibersas compared to the untreated PD mice, which further support theantioxidant properties of Mp.(Tripathi and Upadhyay, 2001) Ourresults also suggest that estrogen protects the dopaminergic neu-rons in the SN to a similar extent as Mp treatment. Notably the re-sults obtained here are quiet contrary to those obtained by Kastureet al. (2009), which showed no significant effect of Mp treatmenton MPTP mice model. We believe that these discrepancies in theresults are due to the dose regime and Mp extract preparation bythe previous researchers.

Recently, many studies have considered the involvement of NOin the neurotoxic action of MPTP. It was found that MPTP increasesthe level of NO production in the brain, which leads to enhanceddopaminergic neuronal damage (Jackson-Lewis and Smeyne,2005). NO is regarded as a very reactive free radical (Halliwelland Gutteridge, 1999), despite reacting with only a small rangeof compounds (Butler et al., 1995). NO is much less reactive thanOH (Butler et al., 1995), but NO has been suggested to react withsuperoxide anions to produce a very reactive molecule peroxynit-rile (ONOO-) (Beckman et al., 1990; Ischiropoulos et al., 1992),which can subsequently produce OH radicals (Van der Vliet et al.,1994). Thus, it may be suggested that the OH radical generationin the present study results from ONOO- formation in the brain fol-lowing the administration of MPTP. Treatment with Mp seed ex-tract inhibits NO radical production resulting in lower levels of

ONOO radicals in the brain and lower production of reactive OH.Finally, the treatment with Mp seed extract was more efficientthan estrogen in lowering NO levels.

Activated astrocytes express a high level of iNOS, which ismainly responsible for NO production mediating dopaminergicneuronal injury. GFAP is a marker of activated astrocytes, thusheightened expression of GFAP can be associated with an enhancedproduction of NO. Indeed, MPTP treatment increased the level ofGFAP immunoreactivity in the present study which is in accor-dance with previous study (Morale et al., 2006). In addition, MPTPmice treated with estrogen demonstrated lower levels of astrocyteactivation, inhibition of MPTP-induced iNOS-derived nitrites aswell as reduced cell death and DA toxicity (Morale et al., 2006).Interestingly, estrogen modulates neuronal signals in astrocytesand glial cells leading to neuronal protective effects. Importantly,Mp treatment showed much better neuroprotective capabilitiesthan estrogen. From the present study, it is suggested that Mp seedextract potentially implements its neuroprotective effect on glialcells and stimulates communication with neurons to protect themfrom oxidative damage, ultimately enhancing longevity.

5. Conclusion

The ethanolic extract of Mp seed used in this study has a neuro-protective role against the MPTP-induced Parkinsonian mousemodel. The ethanolic extract of Mp demonstrated a strong antiox-idant potential against free radicals generated in the MPTP model.In addition, Mp treatment improved motor behaviour of Parkinso-nian mice. Fundamentally, Mp induced the level of catecholaminesand stimulated antioxidant potential in the nigrostriatal region.Mp treatment improved the expression of TH in the SN and striatalregions and recovered normal expression levels of iNOS and GFAPin MPTP treated animals. This demonstrates Mp’s propensity to aidin the recovery from neuronal injury and oxidative stress. Alto-gether, this study demonstrates that this herbal product from In-dia’s natural medical system (Ayurveda) could be used for thedevelopment of therapeutics against PD and other debilitatingneurodegenerative diseases by both reversing the symptoms andcorrecting the underlying cause.

Conflicts of interest

Authors declare that there is no conflict of interest.

Acknowledgments

Authors are thankful to the Head, Department of Biochemistryand Zoology, BHU for providing the basic departmental and cryo-stat facility, respectively. We sincerely thank Council of Scientificand Industrial Research (No. 37 (1518)/11/EMR-II), New Delhi, In-dia for providing financial support for this work and also for pro-viding fellowship to Satyndra Kumar Yadav. Authorsacknowledge Indian Council of Medical Research, New Delhi forproviding fellowship to Jay Prakash and Department of Scienceand Technology, New Delhi, for providing fellowship to ShikhaChouhan.

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