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Neuropharmacology 86 (2014) 192e202

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Neuropharmacology

journal homepage: www.elsevier .com/locate/neuropharm

Neuroprotective and neurotrophic effects of Apigenin and Luteolinin MPTP induced parkinsonism in mice

Sachin P. Patil, Pankaj D. Jain, Jayant S. Sancheti, Priya J. Ghumatkar, Rufi Tambe,Sadhana Sathaye*

Department of Pharmaceutical Sciences and Technology, Institute of Chemical Technology, Nathalal Parekh Marg, Matunga, Mumbai 19, India

a r t i c l e i n f o

Article history:Received 28 January 2014Received in revised form27 May 2014Accepted 14 July 2014Available online 31 July 2014

Keywords:ApigeninLuteolinNeuroprotectiveNeurotrophicParkinson's disease

* Corresponding author. Tel.: þ91 2233612218.E-mail address: sadhanasathaye@hotmail.com (S.

http://dx.doi.org/10.1016/j.neuropharm.2014.07.0120028-3908/© 2014 Elsevier Ltd. All rights reserved.

a b s t r a c t

In the present study, we aim to investigate the neuroprotective and neurotrophic effects of naturallyoccurring polyphenols like apigenin and luteolin and also to explore the underlying mechanisms withrespect to Parkinson's disease (PD). MPTP (25 mg/kg) along with Probenecid (250 mg/kg) was admin-istrated for five consecutive days to induce parkinsonism in mice. Apigenin (5, 10 and 20 mg/kg), luteolin(10 and 20 mg/kg) and Bromocriptine (10 mg/kg) were administrated orally for 26 days including 5 daysof pretreatment. Behavioural analysis and biochemical estimation of oxidative stress biomarkers wereconducted. Tyrosine hydroxylase (TH), glial fibrillary acidic protein (GFAP) and brain derived neuro-trophic factor (BDNF) were evaluated in substantia nigra (SN) region of the brain by immunostaining.TNF-a was estimated using ELISA technique.

Our results demonstrate that apigenin and luteolin treatment improved the locomotor andmuscular activities in MPTP treated mice. TH-positive cells decreased up to 7% in MPTP treated micecompared to normal mice (P < 0.001) and were found to be protected from degeneration in apigenin(69%) and luteolin (63%) treated mice (P < 0.001). Levels of GFAP were found to be decreased in the SN ofthe brain due to apigenin and luteolin treatment as compared to MPTP mice. BDNF levels were elevatedsignificantly in apigenin and luteolin treatment groups when compared to MPTP treatment mice.

In conclusion, apigenin and luteolin protected the dopaminergic neurons probably by reducingoxidative damage, neuroinflammation and microglial activation along with enhanced neurotrophic po-tential. The above results propose both these flavonoids as promising molecules in the therapeutics ofPD.

© 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Parkinson's disease (PD) is a common, slowly progressive,neurodegenerative disease resulting from the degeneration ofdopaminergic neurons in the substantia nigra (SN), a region of thebrain that controls movement (Meissner et al., 2011). The initialsymptoms of PD include tremor at rest, muscular rigidity, brady-kinesia, postural abnormalities and instability (Jankovic, 2008).Although the symptoms can be treated using currently availabledopamine (DA) replacement strategies; the present therapy to treatPD is not effective due to prolonged progress of the disease overseveral years. Moreover, chronic treatment with DA agonist or L-DOPA results into severe motor (Dyskinesia, Motor fluctuation etc.)and non-motor adverse effects (Impulse control disorders, DA

Sathaye).

dysregulation syndrome, PD dementia, Sleep disorder) (Meissneret al., 2011). Hence the drug with benefit of having neuro-protective and/or disease-modifying effects will be a major break-through in PD treatment.

It has been widely reported that oxidative stress plays a pivotalrole in the neurodegeneration associated with PD (Jenner P. 1996;Zhang J. 1999). Autopsy of the striatum and substantia nigra parscompacta (SNpc) of PD patients revealed that oxidative stress isknown to damage lipids, proteins and DNA along with decreasedsuperoxide dismutase, catalase and glutathione levels (Jenner andOlanow, 1996; Zhang J. 1999; Serra et al., 2001). Data obtainedfrom human autopsy has also indicated the relationship betweenoxidative stress and inflammatory pathways which ultimatelyleads to neuronal death (Hartmann, 2004). The neuronal inflam-mation induces glial cell activity in SN of the brain which is a wellknown characteristic of PD pathology (Hunot S et al., 1999; Tayloret al., 2013). Another clinical research showed that nucleartranslocation of the nuclear factorekB (NF-kB) is increased in

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multiple folds in nigral dopaminergic neurons from PD subjectscompared to control subjects. It also states that NF-kB is triggeredby oxidative stress and amplifies the process of inflammation andapoptotic program (Hartmann, 2004). The drug molecules havingneuroprotective, anti-inflammatory therapeutic potential will bebeneficial in order to slow down the progression of neuro-degeneration. Additionally drugs having Neurotrophic potentialwould help to promote the neuronal cell survival which mayalleviate the disease condition (Mohapel P et al., 2005; MochizukiH 2011).

For this purpose, natural phytoconstituents from the class offlavones such as apigenin and luteolin were selected and evaluatedfor their neuroprotective and neurotrophic activities. Flavonoidsare the largest group of polyphenols present in many plants whichare known to promote a number of physiological benefits, espe-cially in scavenging free radicals, cognitive impairment, learningand memory (Bhullar and Rupasinghe, 2013). Apigenin has beenscreened for its antiparkinson's activity in transgenic Drosophilamelanogaster and it has been reported to enhance locomotorability of these flies in the disease condition (Siddique et al., 2011).In-vitro study on the microglial cells has proved that apigenin andluteolin have inhibitory effect on inflammatory mediators, whichsuggested that they may have neuroprotective properties inneurodegenerative diseases (Rezai-Zadeh et al., 2008). Luteolinprotects dopaminergic neurons from inflammation-induced injurythrough inhibition of microglial activation (Chen et al., 2008).Another study suggested that apigenin promotes neurogenesis inadult mice (Taupin, 2009). Hence the primary objective of thepresent study was to evaluate the neuroprotective and neuro-trophic potential of the apigenin and luteolin in parkinsonisminduced mice.

In this study, MPTP (1-mehtyl-4-phenyl-1,2,3,6-tetrahydropyridine) which is known to produce biochemical,neuropathological and behavioural changes analogous to thoseobserved in idiopathic PD was used to induce parkinsonism in mice(Jenner and Olanow,1996; Przedborski and Vila, 2001). Behaviouralanalysis was done using rotarod and open field test. Dopaminergicneurodegeneration was evaluated by studying the tyrosine hy-droxylase (TH) positive cells in SNpc using immunohistochemistry.Furthermore, the oxidative stress level in the mid brain wasinvestigated by measuring the activities of superoxide dismutase(SOD), catalase (CAT), reduced glutathione (GSH) and lipid perox-idation (LPO). Inflammatory mediators like Tumour necrosis factor-a (TNF-a) and glial fibrillary acidic protein (GFAP) were examined,to ensure whether inflammation induced by MPTP was alleviatedby apigenin and luteolin. In addition the neurotrophic activity wasassessed by determining the brain derived neurotrophic factor(BDNF) levels.

2. Material and methods

Apigenin (98%) and luteolin (98%) were procured from A. K. Scientific, Inc. (USA).1-mehtyl-4-phenyl-1,2,3,6-tetrahydropyridine Hydrochloride (MPTP-HCl) waspurchased from SigmaeAldrich (USA). All other chemicals and reagents used in theexperiments were of analytical grade. Primary monoclonal mouse antibody totyrosine hydroxylase, glial fibrillary acidic protein and brain derived neurotrophicfactor were purchased from Abcam, (USA). Elisa kit for the estimation of TNF- awasprocured from eBioscience, (USA).

2.1. Animals

Adult male Swiss-albino mice (22e25 g) were procured from Haffkin institute,Parel, Mumbai. Animals were housed in the animal house of Institute of ChemicalTechnology (ICT) and had free access to food and drinking water. They were main-tained on a 12e12 h lightedark cycle, in the controlled temperature (25 ± 2 �C) andrelative humidity (50e70%). All the experimental procedures and protocols used inthe study were reviewed and approved by the Institutional Animal Ethical Com-mittee, which is registered under the Committee for the Purpose of Control andSupervision on Experiments on Animals (CPCSEA), India.

2.2. Experimental design

Animals were randomly divided into eight groups containing 10 animals in eachgroup.

Group I: Normal control, Group II: MPTP 25 mg/kg, Group III: MPTP 25 mg/kg þ Apigenin 5 mg/kg, Group IV: MPTP 25 mg/kg þ Apigenin 10 mg/kg Group V:MPTP 25 mg/kg þ Apigenin 20 mg/kg Group VI: MPTP 25 mg/kg þ Luteolin 10 mg/kg, Group VII: MPTP 25 mg/kg þ Luteolin 20 mg/kg, Group VIII: MPTP 25 mg/kg þ Bromocriptine 10 mg/kg.

Study comprises of total 26 days including five days of pre-treatment (P1eP5)followed by 21 days of post-treatment (Day 1eDay 21) (Refer Fig. 1). MPTP (25 mg/kg) along with Probenecid (250mg/kg) was administered intraperitoneally once in aday for five consecutive days (Day 1eDay 5). During MPTP administration drugtreatment in the respective groups was done 1hr prior to MPTP dosing. Behaviouralexamination included rotarod and open field test which were conducted during thepre-treatment (Day P4 and Day P5), through the course ofMPTP administration (Day4 and Day 5) and also at the end of the treatment phase (Day 20 and Day 21)respectively (Refer Fig. 1). On the last day, animals were terminally sacrificed andtheir brains were isolated for further investigation of biochemical parameters andimmunohistochemistry studies.

2.3. MPTP and drug treatment

All animals except normal control group were injected with MPTP 25 mg/kg(i.p.) along with probenecid 250 mg/kg (i.p.) to induce parkinsonian symptoms inmice (Jackson-Lewis and Przedborski, 2007; Carta et al., 2013). Probenecid wasadministered 30 min prior to MPTP administration as it decreases the clearance ofMPTP and intensifies its neurotoxicity (Carta et al., 2013). The drugs were admin-istered orally using oral gavage needle at the volume corresponding to 1 ml/100 gbody weight. Both the apigenin and luteolin were suspended in the distilled waterusing Tween 80.

2.4. Behavioural studies

2.4.1. Rotarod testMotor performance was evaluated on a rotarod equipment (Model: RR01 Plus,

Orchid Scientifics, Nashik, India), as per protocol previously described (Carter et al.,2001). In the set experiment mice were evaluated for their motor activity on arotarod at a fixed speed of 30 RPM. The time they could spend on the revolving rodwas measured. The cut off time was fixed at 60 s. All mice were pre-trained on therotarod apparatus in order to reach a stable behavioural performance. The trainingconsisted of three sessions per day for five consecutive days. The average of theretention time on the revolving rod was determined.

2.4.2. Open field testOpen-field consisted of square arena (40 cm � 40 cm) and wall (35 cm high).

The square arena was divided into 16 sub squares. The test was initiated by placingthemouse at the centre of the arena. The behaviour of themouse was then observedfor 5 min. After each test, the apparatus was thoroughly cleaned with cotton padwetted with 70% ethanol. The number of line crossings (crossing the squaresboundaries with both forepaws), rearing (standing on its hind legs), grooming(rubbing the body with paws or mouth and rubbing the head with paws) andduration of immobility were measured. During the test; video recording was carriedout and scoring was done by blind observer (Gould et al., 2009).

2.5. Perfusion and tissue processing

At the end of the experiment, animals were euthanized. Out of total animals ineach group, 4 mice were used for the immunohistochemistry and 6 for thebiochemical markers determination. The mice were perfused with 4% para-formaldehyde for immunohistochemistry analysis and with normal saline solution(37 �C) for biochemical markers estimation (Gage et al., 2012). Brains which wereisolated for biochemical estimations were rinsed in ice-cold isotonic saline andfurther processing was performed at �4 �C. Mid brain regions of the same wereisolated and these isolated brain sections were used for further investigations. Thebrain tissues were homogenized with equal volume (1 ml) of ice-cold 0.1 M phos-phate buffer saline (pH 7.4). The homogenate was then centrifuged at �4 �C(10,000 rpm; R-248Mof CPR-24 plus Instrument, Remi, India) for 15 min, and ali-quots of homogenates (0.7e0.8 ml) were used for estimations of biochemicalparameters.

2.6. Assessment of SOD, CAT and GSH

The activity of SOD in the brain homogenate (as described in 2.5) was assayed bymonitoring its ability to scavenge superoxide radicals generated by auto-oxidationof pyrogallol in the alkaline medium. SOD was determined with minor modifica-tion in the method of Nandi and Chatterjee (1988); Li (2012). Each 3 ml reactionmixture contained 2.8ml of Potassium phosphate buffer (0.1M, pH 7.4), 0.1 ml tissuehomogenate and 0.1 ml pyrogallol solution (2.6 mM in 10 mM HCl). The rate ofincrease in the absorbance at 325 nm was recorded for a period 5 min with 30sec

Fig. 1. Scheme of experimental procedure.

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interval. One unit of SOD is described as the amount of enzyme required to cause50% inhibition of pyrogallol autoxidation per 3 ml of the assay mixture.

Catalase activity was assessed by the assay method described by Sinha (1972);Aebi (1974) with some modifications. Reaction mixture consisted of 2.9 ml of10 mM H2O2 in 50 mM Potassium phosphate buffer (pH 7) followed by 0.1 ml oftissue homogenate (as described in 2.5). The rate of decrease in the absorbance at240 nmwas recorded for 3 min. The results were expressed as units of CAT activity/mg of protein.

Reduced GSHwas estimated according to Sedlak and Lindsay (1968); Smith et al.(1988) with small modifications. Each 3 ml reaction mixture consisted of 2.9 ml of5,5-Dithiobis(2-nitrobenzoic acid) prepared in Potassium phosphate buffer (0.1 M,pH 7.4) and 0.1 ml of tissue homogenate (as described in 2.5). The reaction mixturewas incubated at 37 �C for 15 min and absorbance was at 412 nm and results wereexpressed as GSH/mg protein.

2.7. Determination of LPO

The LPO content in the brain homogenate (as described in 2.5) was determinedby the spectrophotometric method as described by Okhawa et al. (1979); Reilly andAust (1999). Briefly the method describes, 0.2 ml tissue homogenate was added to0.2 ml of 8.1% SDS, 1.5 ml of 20% acetic acid solution adjusted to pH 3.5 with NaOH,and 1.5ml of 0.8% aqueous solution of TBA. The final mixture volumewas adjusted to4.0 ml with distilled water, and then heated at 95 �C for 60min in awater bath. Aftercooling, 1 ml of distilled water and 5.0 ml of the mixture of n-butanol and pyridine(15: 1, v/v) were added to the above reaction mixture and shaken vigorously. Aftercentrifugation at 4000 rpm for 10 min, the absorbance of organic layer wasmeasured at 532 nm. LPO was expressed in terms of nmol of MDA/mg of protein.

2.8. Estimation of protein concentration

Protein content in the brain homogenate (as described in 2.5) was measuredusing the dye binding method given by Bradford; where in bovine serum albumin(BSA) was used as a standard. 5 ml of tissue homogenate was added to 200 ml ofBradford reagent (SigmaeAldrich) incubated at 37� C for 15min and absorbancewasmeasured at 596 nmwith the help of microplate spectrophotometer (Epoch, Biotek,USA) (Data not shown).

2.9. Immunohistochemistry analysis

Immunohistochemistry was done on 4% paraformaldehyde-fixed, 3 mm-thickfrozen brain sections (consisting 14e15 sections) passing through the SNpc region ofbrain. Sections were fixed on poly-L-lysine coated slide and transferred throughthree changes of xylene for 30 min and then rehydrated with decreasing grades ofabsolute alcohol, 95%, 70%, 50%. Peroxidase activity was blocked by incubating with3% hydrogen peroxide in methanol for 5min. Primary monoclonal antibody to TH,GFAP and BDNF were incubated for 30 min at room temperature and then washedwith Tris buffer solution pH 7.4 for 10 min. Sections were incubated with Poly-Horseradish peroxidise (Poly-HRP) for 30 min and washed in Tris buffer solutionpH 7.4 for 10 min. Further they were incubated with substrate and examined for thecolour change to brown which appeared within 5e10 min. For immunochemicalstaining, numbers of immunopositive cells in stained sections were evaluated undera light microscope at a magnification of 400� without the examiner knowing theexperimental protocol (Fukuda et al., 1999; Matsuda et al., 2009; Sakuma et al.,2008).

2.10. Determination of TNF-a by ELISA

Brain TNF-a was estimated by using ELISA kit (eBioscience, USA) as per themethodology provided by manufacturer in the kit. Brain homogenate prepared asstated above (see 2.5) was used for the determination of TNF- a.

2.11. Statistical analysis

Data of all the results were presented as mean ± SEM. The analysis of all thestudies were done with the help of analysis of variance (ANOVA) followed byDunnett's test. For behavioural test and immunohistochemistry quantification @P < 0.05, $ P < 0.01, #P < 0.001 and for oxidative stress biomarkers *P < 0.05,**P < 0.01, ***P < 0.001 were considered to be statistically significant whencompared with MPTP group.

3. Results

3.1. Rotarod test

The results of the rotarod test are presented in Fig. 2. In pre-treatment session all animals showed relatively equal rotarod ac-tivity (50e60 s). At 4th dose of MPTP there was significant reduc-tion in rotarod performance observed inMPTP control group (~10 s)as compared to normal control (P < 0.001). In apigenin treatmentgroup, apigenin 5 mg/kg (P < 0.01) and apigenin 10, 20 mg/kg(P < 0.001) showed gradual and dose related improvement inrotarod activity. Similarly luteolin 10mg/kg (P < 0.01) and 20mg/kg(P < 0.001) showed significant increase in the motor activity. Onday 20 of the experiment slight recovery in the motor activity ofMPTP mice was observed but still significantly less than normalcontrol group animals (P < 0.001). All other treatment groupsshowed significant rotarod activity (P < 0.001). Bromocriptine(10 mg/kg), a dopaminergic agonist showed significantly improvedrotarod performance on day 4 and 20 (P < 0.001) as compared toonly MPTP treated mice.

3.2. Open field test

Fig. 3 shows the effect during pre-treatment and treatment afterMPTP administration in mice on the locomotor activity in the openfield. In the given test, lines crossed, rearing, grooming behaviouralong with immobility timewas measured. Interestingly in the pre-treatment phase apigenin and luteolin each at a dose of 20 mg/kgincreased the number of lines crossed thus enhancing the loco-motor activity P < 0.05, P < 0.01 respectively. After MPTP admin-istration all mice were found to show decreased locomotor activityby crossing less number of lines as compared to normal control.

In the treatment group on day 5 of MPTP administration api-genin 10 mg/kg (P < 0.05), apigenin 20 mg/kg (P < 0.001) andluteolin 10 mg/kg (P < 0.05), luteolin 20 mg/kg (P < 0.001) signif-icantly enhanced the locomotor activity as compared to the MPTPcontrol group (Fig. 3A). On day 21 all the treatment groupsexhibited significantly improved locomotor activity. Bor-omocriptine (10 mg/kg) treatment increased locomotion on day 5and 21 (P < 0.001).

After MPTP administration numbers of rearings were found tobe significantly decreased in MPTP control mice as compared to the

Fig. 2. Rotarod performance in different experimental groups. Data expressed as mean ± SEM. N-10; df (Between columns) ¼ 7; for Pretreatment: F ¼ 0.85; for Day 4: F ¼ 26; forDay 20: F ¼ 16. a P < 0.001 compared with normal group and @ P < 0.05, $ P < 0.01, #P < 0.001 compared with MPTP group using one-way ANOVA followed by Dunnett's test as apost-ANOVA test.

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normal control group (P < 0.001) (Fig. 3B). On day 5 apigenin20 mg/kg (P < 0.01) and luteolin 20 mg/kg (P < 0.001) showedsignificant improvement in the rearings while on day 21 all treat-ment groups showed improvement in the rearing behaviour ascompared to the MPTP control. In Bromocriptine treated animalsrearing activity on day 5 as well as on day 21 was found to besignificantly improved (P < 0.001). Similar to rearing activity thegrooming behaviour of mice treated with apigenin 20 mg/kg(P < 0.05) and luteolin 20 mg/kg (P < 0.05) was improved on day 5whereas on day 21 enhanced grooming behaviour was observed inall the treatment groups when compared to MPTP control group(Fig. 3C). Before the administration of MPTP all mice had exploredthe open arena showing lowest immobility (Fig. 3D). Apigenin20 mg/kg, luteolin 10 and 20 mg/kg were found to enhancemobility in mice during pre-treatment as compared to that of MPTPgroup mice (P < 0.01, P < 0.05, P < 0.001 respectively). MPTPadministration led to decreased mobility in all mice as compared tonormal mice. On day 5 in apigenin 10 and 20 mg/kg (P < 0.05,P < 0.001) as well as luteolin 10 and 20 mg/kg (P < 0.01, P < 0.001)treated mice indicated that immobility time was reduced signifi-cantly as compared to MPTP control group. Apigenin at 5 mg/kgdecreased the immobility although not significant as compared toMPTP group mice. All other groups showed significant increase inmobility.

3.3. Effect on SOD, CAT and GSH

Oxidative stress evaluation included SOD, CAT and GSH analysiswhich are the major key players of natural antioxidant defencesystem (Fig. 4). MPTP administration resulted in generation ofoxidative stress because of which significant decrease in the SOD(P < 0.001), CAT (P < 0.05) activities and levels of GSH (P < 0.001)were observed inMPTP control mice in comparison to normal mice.

Apigenin 5, 10 and 20 mg/kg significantly increased the SODactivity in a dose dependant manner (P < 0.01, P < 0.001, P < 0.001respectively). Similarly luteolin 10 and 20 mg/kg both showedsignificant improvement in the SOD activity compared to MPTPtreated mice (P < 0.001). Interestingly, we found that both apigeninand luteolin improved the SOD activity as compared to normalmice.

Bromocriptine although showed protection from oxidativestress by augmenting the SOD activity as compared to only MPTPtreatment group (P< 0.001) it was less as compared to apigenin andluteolin treatment.

CAT activity was improved significantly after apigenin admin-istration at 5, 10, 20 mg/kg (P < 0.05, P < 0.001, P < 0.001) andluteolin 10 and 20 mg/kg (P < 0.01, P < 0.001) treatment. Levels ofreduced glutathione, which is a protective peptide increased aftertreatment with apigenin 5, 10, 20 mg/kg (P < 0.05, P < 0.01,P < 0.001) and luteolin 10 and 20 mg/kg (P < 0.01, P < 0.001) ascompared toMPTP group. In case of GSH, interestingly apigenin andluteolin 20 mg/kg showed major recovery in GSH levels ascompared to low doses 5 and 10 mg/kg; much higher than thecontrol group. All these antioxidant defences of the cells namelySOD along with CAT and GSH were improved after apigenin andluteolin treatment as compared to normal control mice. Apigeninand luteolin treatment remarkably improved the antioxidantdefence in comparison to bromocriptine.

3.4. Assessment of LPO

To understand the exact impairment in a cell due to oxidativestress we determined the lipid peroxidation level in the mid brainwhich was found to be elevated in the MPTP control group ascompared to normal control mice (P < 0.001) (Fig. 5). Apigenin 5,10,20 mg/kg reduced the LPO levels significantly as compared to onlyMPTP treated mice (P < 0.01, P < 0.001, P < 0.001). Treatment withluteolin 10 and 20 mg/kg also reduced the LPO level in contrast toMPTP mice (P < 0.01, P < 0.001). Bromocriptine too decreased thelevel of LPOwhen comparedwithMPTP control (P < 0.01). Apigeninand Luteolin 20 mg/kg exhibited better protection to peroxidativedamage than bromocriptine on MPTP induced damage.

3.5. Tyrosine hydroxylase

MPTP along with Probenecid administration resulted in signif-icant dopaminergic neuronal toxicity and only 7% of the Tyrosinehydroxylase-positive (TH-positive) cells were found in the SNpc ofthe MPTP group as compared to animals in the normal group(P < 0.001) (Figs. 6 and 7). In contrast, Apigenin (10 and 20 mg/kg)treated groups showed 54% and 69% TH-positive cells respectivelywith P < 0.001. A significant protection was observed with luteolin10 and 20 mg/kg treatment in which 46% and 63% of TH-positivecells were observed with P < 0.001 as compared to MPTP group.In Bromocriptine group, only 25% of the TH-positive cells survivedwhich was lower as compared to 10 and 20 mg/kg of both apigeninand luteolin.

Fig. 3. Open field performance in different experimental groups. 3A: Lines crossings; 3B: Rearing; 3C: Grooming; 3D: Immobility time. Data expressed as mean ± SEM. N-10. df(Between columns) ¼ 7. F values for Lines crossings: Pretreatment F ¼ 4.7; Day 5 F ¼ 26; Day 21 F ¼ 7.1. Rearing: Pretreatment F ¼ 3.1; Day 5 F ¼ 23; Day 21 F ¼ 12. Grooming:Pretreatment F ¼ 4.3; Day 5 F ¼ 11; Day 21 F ¼ 12. Immobility time: Pretreatment F ¼ 5.4; Day 5 F ¼ 53; Day 21 F ¼ 26. a P < 0.001 compared with normal group and @ P < 0.05, $P < 0.01, #P < 0.001 compared with MPTP group using one-way ANOVA followed by Dunnett's test as a post-ANOVA test.

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Fig. 4. Effect on SOD, CAT and GSH in mid brain. Data expressed as mean ± SEM. N-6; df (Between columns) ¼ 7; F value for SOD ¼ 37, CAT ¼ 6.6 and GSH ¼ 16. @ P < 0.05,#P < 0.001 compared with normal group and *P < 0.05, **P < 0.01, ***P < 0.001 compared with MPTP group using one-way ANOVA followed by Dunnett's test as a post-ANOVA test.

S.P. Patil et al. / Neuropharmacology 86 (2014) 192e202 197

3.6. Glial fibrillary acidic protein

Fig. 8 illustrates immunostaining of GFAP particularly in theSNpc region showed brown coloured staining which representsreactive GFAP-positive astrocytes. MPTP administration signifi-cantly increased GFAP level (85%) as compared to normal animals(P < 0.001) (Fig. 9). Apigenin 10 and 20 mg/kg (P < 0.001) andluteolin 10 and 20 mg/kg (P < 0.01, P < 0.001) significantlydecreased GFAP level in comparison to MPTP group. Apigenin20 mg/kg and Luteolin 20 mg/kg showed better improvement inthe GFAP than bromocriptine.

3.7. Brain derived neurotrophic factor

In the normal control mice, BDNF level in SNpc regionwas foundto be approximately 60% while significant decrease in the BDNFimmunostaining was observed in MPTP treated group (14%,P < 0.001) (Figs. 10 and 11). Apigenin 10 and 20 mg/kg treatmentexhibited significant increase in the BDNF level when comparedwith the MPTP group (P < 0.001). Interestingly, apigenin 20 mg/kgtreatment showed significant increase in BDNF levels (77%) ascompared to normal control animals (P< 0.01). Similarly, in luteolin

10 and 20 mg/kg treatment groups 46% and 63% BDNF-positiveneurons were observed as compared to only MPTP treated ani-mals (P < 0.001).

3.8. Tumour necrosis factor-a

Neurotoxicity induced due to MPTP significantly increased theTNF-a levels in the mid brain as compared to normal animals(P < 0.001) (Fig. 12). At the dose of 5 mg/kg apigenin, no significantchange in the levels of TNF-a was observed while apigenin 10 mg/kg (P < 0.01) and 20 mg/kg (P < 0.001) significantly reduced theTNF-a levels as compared toMPTP treatedmice. Both 10 and 20mg/kg doses of luteolin significantly (P < 0.001) alleviated the levels ofTNF-a as in comparison to MPTP group. Bromocriptine could notimprove the TNF-a levels significantly.

4. Discussion

Neuroprotective and neurorescue strategies would be prom-ising as it not only slows down the progression of neuro-degeneration but also recovers the subject from the diseasecondition. Many studies state that oxidative stress followed by

Fig. 5. Effect on lipid peroxidation in mid brain. Data expressed as mean ± SEM. N-6;df (Between columns) ¼ 7; F ¼ 22. #P < 0.001 compared with normal group and*P < 0.05, **P < 0.01, ***P < 0.001 compared with MPTP group using one-way ANOVAfollowed by Dunnett's test as a post-ANOVA test.

Fig. 7. Immunostaining of tyrosine hydroxylase TH-positive neurons in SNpc. Dataexpressed as mean ± SEM. N-4; df (Between columns) ¼ 7; F ¼ 67.75. ***P < 0.001compared with normal group and @ P < 0.05, $ P < 0.01, #P < 0.001 compared withMPTP group using one-way ANOVA followed by Dunnett's test as a post-ANOVA test.

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inflammation could be the major cause behind the dopaminergicneurodegeneration in Parkinson's disease (Taylor et al., 2013). Inthe present study, we demonstrated for the first time that apigeninand its major metabolic product luteolin, most commonly foundflavones, have neuroprotective activity in MPTP mouse model ofParkinsonian disease. Results also state that apigenin and luteolinmay possess neurotrophic potential which will be beneficialparticularly in the neurodegenerative disorders. Approximately 4weeks of apigenin and luteolin treatment demonstrated multi-functional effects like protection of tyrosine hydroxylase, alleviatedoxidative stress, inflammation, enhanced neurotrophic activity andultimately improvedmotor co-ordination and locomotor behaviour

Fig. 6. TH immunohistochemistry N-4. Representative photomicrographs of TH-immunoreaMPTP þ Apigenin 10 mg/kg, E e MPTP þ Apigenin 20 mg/kg, F e MPTP þ Luteolin 10 mg/

in parkinsonism induced mice. In the given study, bromocriptine, aD2-like receptor agonist is used as a standard drug as it is clinicallyused for the treatment of PD. In addition to dopaminergic agonistactivity, bromocriptine has ability to reduce oxidative stress andmay possess neuroprotective potential (Muralikrishnan andMohanakumar, 1998). Furthermore bromocriptine has a role onPI3K-(Phosphoinositide 3-kinase) and Nrf2- (nuclear factor-E2-related factor-2) mediated upregulation of the antioxidantenzyme NQO1 (NAD(P)H quinone oxidoreductase1) (Lim et al.,2008).

ctive neurons in the SNpc. A e Normal, B e MPTP, C e MPTP þ Apigenin 5 mg/kg, D e

kg, G e MPTP þ Luteolin 20 mg/kg, H e MPTP þ Bromocriptine 10 mg/kg.

Fig. 8. GFAP immunohistochemistry N-4. Representative photomicrographs of GFAP-immunoreactive astroglial cells in the SNpc. A e Normal, B e MPTP, C e MPTP þ Apigenin5 mg/kg, D e MPTP þ Apigenin 10 mg/kg, E e MPTP þ Apigenin 20 mg/kg, F e MPTP þ Luteolin 10 mg/kg, G e MPTP þ Luteolin 20 mg/kg, H e MPTP þ Bromocriptine 10 mg/kg.

S.P. Patil et al. / Neuropharmacology 86 (2014) 192e202 199

Considering limitations of the conventional MPTPmousemodel,in the present study we followed an alternative mouse model, inwhich co-administration of MPTP with probenecid resulted inapoptosis and the major loss of striatal dopaminergic neurons forseveral months (Jackson-Lewis and Przedborski, 2007; Carta et al.,2013). Probenecid reduces the excretion of MPTP which ultimatelyleads to severe neurotoxicity. After administration of MPTP, in thebrain it gets converted from MPDPþ to MPPþ which is an activetoxic metabolite of MPTP by the enzyme monoamine oxidase B(MAO-B) (Przedborski and Vila, 2001). MPPþ has a high affinity for

Fig. 9. Level of GFAP in SNpc. Data expressed as mean ± SEM. N-4; df (Betweencolumns) ¼ 7; F ¼ 47. ***P < 0.001 compared with normal group and @ P < 0.05, $P < 0.01, #P < 0.001 compared with MPTP group using one-way ANOVA followed byDunnett's test as a post-ANOVA test.

Dopamine transporter (DAT) through which it gains access todopaminergic neurons and due its deleterious effects leads todegeneration of neurons (Przedborski and Vila, 2001). The selectiveMAO-B inhibition assay of apigenin and luteolin was performed atthe concentration ranging from 1 to 100 mM. It was observed thatboth apigenin and luteolin did not demonstrate any inhibitoryMAO-B activity evincing no interference in the development ofneurotoxic effects of MPTP.

Oxidative stress mechanism gets more prominent in the processof ageing, thus is the most important risk factor for developing PD(Cui et al., 2012). Mitochondrial dysfunction along with damagedproteins and lipids due to oxidative stress plays a key role in PDpathological process (Perier and Vila, 2012). MPTP treatmentinduced rigorous free radical generation and oxidative stress in theSN region which led to degeneration of dopaminergic neurons(Przedborski and Vila, 2001). In the present study, determination ofactivities of SOD, CAT and levels of GSH and LPO in the mid brain, asmarker of oxidative stress, has confirmed the same.

SOD and CAT are the major enzymes which scavenge the reac-tive oxygen radicals (Weydert and Cullen, 2009). Thus, concomitantrise in both SOD and CAT due to apigenin and luteolin treatmentindicates an excellent protection from oxidative stress as producedafter MPTP administration. GSH levels are depleted during oxida-tive stress making the cells vulnerable to oxidative damage(Johannsen et al., 1991). MPTP treatment reduced the activity ofGSH which was found to be enhanced by apigenin and luteolintreatment. Apigenin and luteolin showed significant scavenging ofreactive oxygen species which may be due to their polyphenolicnature leading to overall reduction in oxidative stress. Bromocrip-tine also lowered the oxidative stress which supports findings ofprevious studies (Muralikrishnan and Mohanakumar, 1998; Limet al., 2008).

Free radicals generated due to MPTP, reacts with free oxygenmolecule to form peroxy radicals which damages the lipid mem-branes, thus resulting in lipid peroxidation (Halliwell and Chirico,1993). MDA is a diagnostic marker for lipid peroxidation and usedhere as an oxidative stress biomarker of PD (Reilly and Aust, 1999).In the present study, the levels of MDA and hence lipid peroxidationin the nigrostriatal region of MPTP treated mice were significantlyelevated. The treatment of parkinsonian mice with apigenin and

Fig. 10. BDNF immunohistochemistry N-4. Representative photomicrographs of BDNF imunoreactivity in the SNpc region. A e Normal, B e MPTP, C e MPTP þ Apigenin 5 mg/kg, De MPTP þ Apigenin 10 mg/kg, E e MPTP þ Apigenin 20 mg/kg, F e MPTP þ Luteolin 10 mg/kg, G e MPTP þ Luteolin 20 mg/kg, H e MPTP þ Bromocriptine 10 mg/kg.

S.P. Patil et al. / Neuropharmacology 86 (2014) 192e202200

luteolin, reduced MDA levels and protected from lipid peroxidationand further cellular degeneration. Reduced levels of LPO can becorrelated to promising free radical scavenging activity of apigeninand luteolin.

Toxicity of apigenin and luteolin treatment was assessed withrespect to liver. Serum levels of SGOT and SGPT were analysed.However no significant alterations were found (Data not shown).

In our study we also found that MPTP treatment increased thelevels of GFAP immunoreactivity which is in accordance with theprevious findings (Morale et al., 2006). GFAP is an intermediatefilament protein that is known to be localized to astrocytes. The

Fig. 11. Level of BDNF in SNpc N-4; df (Between columns) ¼ 7; F ¼ 40. Data expressedas mean ± SEM. **P < 0.01, ***P < 0.001 compared with normal group and @ P < 0.05, $P < 0.01, #P < 0.001 compared with MPTP group using one-way ANOVA followed byDunnett's test as a post-ANOVA test.

upregulation of GFAP following injury and astrogliosis has been alongstanding pathological observation in PD (Maragakis andRothstein, 2006). Treatment with apigenin and luteolin demon-strated lower levels of astrocyte activation, with less immuno-staining for GFAP. TNF-a, a pro-inflammatory mediator plays amajor role in initiating and regulating the cytokine cascade duringan inflammatory response (Gearing et al., 1994; Whitton, 2007).Several genetic and pharmacological studies suggest that inhibitionof TNF-a leads to improvement of the disease state (Tweedie et al.,2007). In our study levels of TNF-a were also found to be elevated

Fig. 12. Effect on TNF-a in mid brain. Data expressed as mean ± SEM. N-6; df (Betweencolumns) ¼ 7; F ¼ 12. ***P < 0.001 compared with normal group and @ P < 0.05, $P < 0.01, #P < 0.001 compared with MPTP group using one-way ANOVA followed byDunnett's test as a post-ANOVA test.

S.P. Patil et al. / Neuropharmacology 86 (2014) 192e202 201

due to MPTP treatment. Both apigenin and luteolin decreased thelevels of TNF-a supporting the previous results which suggest thatapigenin and luteolin down regulates the inflammatory cascade(Rezai-Zadeh et al., 2008; Funakoshi-Tago et al., 2011). Importantly,apigenin and luteolin treatment showed significant reduction inthe levels of TNF-a as compared to bromocriptine.

TH is the rate-limiting enzyme responsible for conversion of L-DOPA to dopamine (Daubner et al., 2011). The measurement of TH-immunoreactivity is thus a measure of the functionality of dopa-minergic neurons and fibres present in the SN (Fukuda et al., 1999).In the present study, MPTP treatment imposed a significantreduction in TH-immunoreactivity. It is believed that the mainmechanism of dopaminergic neuronal loss in these animals isoxidative stress generated in response to MPTP exposure(Przedborski and Vila, 2001). Apigenin and luteolin (10 and 20 mg/kg) significantly protected the dopaminergic neurons and fibres ascompared to only MPTP treated mice. This protection can becorrelated to antioxidant and anti-inflammatory activities of api-genin and luteolin. Our results also suggested that bromocriptineprotected the dopaminergic neurons in the SN but to a lesser extentas compared to apigenin and luteolin treatment. This activity ofbromocriptine could be because of its role on oxidative stress.

It is well known that neuronal development and survival re-quires neurotrophic support (Binder and Scharfman, 2004). BDNFand GDNF (glial cell derived neurotrophic factors) are the mostpromising neurotrophic factors in the neurorescue or restorativetreatment of neurodegenerative diseases (Weissmiller and Wu,2012). Nowadays increased attention is paid towards strategiesaimed at inducing the expression of such endogenous neurotrophicfactors or enhancing their signalling as alternative therapeuticoptions for PD. In our study we have observed that treatment withapigenin and luteolin increased BDNF levels in parkinsonian micebrain. Interestingly at the highest dose of apigenin and luteolin20 mg/kg BDNF level was found to be increased in contrast tonormal level while bromocriptine showed no increment in BDNFlevel. Our findings suggest that apigenin and luteolin both possessneurotrophic potential and may have helped in dopaminergic cellsurvival.

Locomotor dysfunction is a kind of clinical symptom of PD(Jankovic, 2008). In the behavioural analysis animals treated withapigenin and luteolin exhibited better grip strength and muscularco-ordination in the rotarod test whereas in the open field test,increased locomotor activity was observed in comparison withMPTP group. Apigenin and luteolin treatment may have protectedthe dopaminergic cells from degeneration in the SN of mice brainwhich resulted in enhanced motor co-ordination, locomotion andoverall behavioural activity. Bromocriptine, being a D2-like recep-tor agonist significantly improved the behavioural activities ascompared to apigenin and luteolin. Interestingly, during pretreat-ment phase, apigenin and luteolin at high doses showed increasedlocomotor activity in the open field test and it could be correlated totheir antianxiety potential (Viola et al., 1995; Salgueiro et al., 1997).

Overall bromocriptine, being a dopaminergic receptor agonisthas majorly shown good behavioural activity/symptomatic relief incomparison to apigenin and luteolin in the early phase of disease.Conversely in case of biochemical markers apigenin and luteolinshowed better reversal as compared to bromocriptine which couldbe correlated to their additive effect of neuroprotective (antioxi-dant, antineuroinflammatoy) and neurotrophic potential.

In view of this, combination of drugs which serve symptomaticrelief as well as delays the progression of the disease could be arational approach to treat Parkinson's like neurodegenerativedisorders.

The many studies of flavonoids in foods have been conducted todate provide a good indication of brain distribution and their

therapeutic efficacy (Youdim et al., 2003, 2004; Datla et al., 2001;Manach et al., 2004). Flavonoids reach the plasma concentrationof maximum 1e5 mmol/lit indicating that they are biologicallyactive at very low concentration (Manach et al., 2004). Thus dietrich in flavonoids can contribute to the numerous beneficial effectson human health. However detail pharmacokinetic and bio-distribution profile alongwith the safety of these flavonoids need tobe investigated in further studies.

In summary, apigenin and luteolin have neuroprotective roleagainst the MPTP-induced parkinsonian mouse model. This neu-roprotective effect could be attributed to their strong antioxidantpotential and inhibitory role on different key events involved inneuroinflammation. Apigenin and luteolin increased BDNF levels inthe SNpc which demonstrated their neurotrophic potential. Thefindings of the study endorse apigenin and luteolin as the pro-spective molecules in treating PD.

Acknowledgement

This work was supported by Department of Science and Tech-nology (DST), New Delhi, India. Authors would like to thank Mr.Rahul Chaudhari, Mr. Gauresh Somani, Ms. Divya Kanchan, Mr.Devang Sarvaiya, Mr. Madhav Seervi and Mr. Sagar Bachhav fortheir assistance in the experiment.

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