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Hindawi Publishing Corporation Journal of Neurodegenerative Diseases Volume 2013, Article ID 972391, 8 pages http://dx.doi.org/10.1155/2013/972391 Research Article Bacopa monnieri Phytochemicals Mediated Synthesis of Platinum Nanoparticles and Its Neurorescue Effect on 1-Methyl 4-Phenyl 1,2,3,6 Tetrahydropyridine-Induced Experimental Parkinsonism in Zebrafish 1 1 2 Jayshree Nellore, Cynthia Pauline, and Kanchana Amarnath Department of Biotechnology, Sathyabama University, Jeppiaar Nagar, Rajiv Gandhi Salai Chennai-119, Chennai, Tamilnadu, India Department of Biochemistry, Sathyabama University, Chennai 600119, Tamilnadu, India Correspondence should be addressed to Jayshree Nellore; sree firstname.lastname@example.org Received 17 October 2012; Revised 2 January 2013; Accepted 17 January 2013 Academic Editor: Eng King Tan Copyright © 2013 Jayshree Nellore et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Current discovery demonstrates the rapid formation of platinum nanoparticles using leaf extract of a neurobeneficial plant, Bacopa monnieri (BmE). The nanoparticles (BmE-PtNPs) were stabilized and then coated with varied phytochemicals present within the leaf extract. es Th e nanoparticles demonstrated the same activity of Complex I, as that of oxidizing NADH to NAD using a spectrophotometric method. This suggests that BmE-PtNPs are a potential medicinal substance for oxidative stress mediated disease with suppressed mitochondrial complex I, namely, Parkinson’s disease (PD). Hence, the neuroprotective potentials of the phytochemical coated nanoparticle were explored in 1-methyl 4-phenyl 1,2,3,6 tetrahydropyridine- (MPTP-)induced experimental Parkinsonism in zebrash fi model. BmE-PtNPs pretreatment significantly reversed toxic effects of MPTP by increasing the levels of dopamine, its metabolites, GSH and activities of GPx, catalase, SOD and complex I, and reducing levels of MDA along with enhanced locomotor activity. Taken together, these findings suggest that BmE-PtNPs have protective effect in MPTP-induced neurotoxicity in this model of Parkinson’s disease via their dual functions as mitochondrial complex I and antioxidant activity. 1. Introduction via the dopamine transporter (DAT). MPP accumulates in the mitochondria and induce neuronal cell death via Parkinson’s disease (PD) is a neurodegenerative disease of several pathways, including the inhibition of complex I dopamine (DA) neurons in substantia nigra characterized activity of the respiratory chain. This contributes to both predominantly by resting tremors, bradykinesia, muscular reactive oxygen species generation and nigral cell loss. . eTh excessive production of reactive oxygen species, rigidity, and postural instability, along with several non- motor symptoms . eTh diseaseisassociatedwithaloss such as superoxide anion, hydroxyl radical and hydrogen peroxide, may either directly damage the cellular macro- of antioxidants or increase in prooxidant levels and mito- chondrial dysfunction. eTh neurotoxin 1-methyl-4-phenyl- molecule to cause cell necrosis or affect normal cellular 1,2,3,6- tetrahydropyridine (MPTP) is known to cause a signaling pathways and gene regulation to induce apoptosis . similar loss of dopaminergic neurons in the human midbrain with corresponding Parkinsonian symptoms . Several Lately, several studies demonstrated the free radical animal species have also shown sensitivity to MPTP, includ- scavenging activity, reducing the concentration of reac- tive oxygen, and nitrogen species of artificial antioxidants ing primates, mice, goldfish, and, most recently, zebrash fi . MPTP is metabolized to 1-methyl-4-phenyl pyridinium include inorganic nanoparticles possessing intrinsic antioxi- (MPP )inglial cellsinthe brain. Aeft r releasefrom dant properties and nanoparticles functionalized with natural the glia, MPP is transported into dopaminergic neurons antioxidants or antioxidant enzymes . 2 Journal of Neurodegenerative Diseases Recent studies have proposed that platinum in the form research in antioxidant nanomaterial has opened a new era of nanoparticles has an activity that is similar to that of in pharmaceutical industries, we, hence forth finally, pro- oxidizing Nicotinamide adenine dinucleotide (NADH) and pose to determine the potentiality of platinum nanoparticles reducing ubiquinone (CoQ ). Platinum nanoparticles coated with phytochemicals of Bacopa monnieri leaf (BmE- also provide dual functions as mitochondrial complex I PtNPs) extract endowed with antioxidants to mitigate the to lower reactive oxygen species (ROS) generation and as oxidative damage induced by MPTP in zebrafish (Danio Superoxide dismutases (SOD)/catalase mimetics to scav- rerio). 2− enge ROS including superoxide anion (O )aswellas Thereby, in the present, we aimed to explore the neuro- hydrogen peroxide (H O )and free radicals [8, 9]. This protective role of BmE-PtNPs in MPTP-induced zebrafish PD 2 2 suggests that platinum nanoparticles can mimic a part of model. theenzymatic functionsofthe complexIandindicates their possible use in medical treatments for Parkinson’s 2. Experimental Procedure disease. 2.1. Materials. The components (natural constructs) used in With such important applications, it is imperative to the synthesis of platinum nanoparticles (PtNPs) were pro- develop platinum nanoparticles through environmentally cured from standard vendors. MPTP and Chloroplatinic acid sound and nonpolluting technologies. While a number of (H PtCl ) was purchased from Bio Corporals, Vadapalani, chemical methods  are currently available and are exten- 2 6 Chennai. Nicotinamide adenine dinucleotide (NADH), 5, sively used, the collaborations are oeft n energy intensive 5 -dithio-bis (2 nitro benzoic acid) (DTNB), nitro blue and employ toxic chemicals, thereby precluding biomed- tetrazolium, perchloric acid, potassium dihydrogen phos- ical application. With the flourishing demand on “green” phate, Acetonitrile, citric acid, 5-5 -dithiobis-p-nitrobenzoic nanotechnological processes , the el fi d of nanoparti- acid, Potassium Dihydrogen Phosphate (KH PO ), Ethylene cle synthesis has recently developed new routes. Biosyn- 2 4 diamine tetra acetic acid (EDTA), and octanesulfonic acid thetic methods employing either biological microorganisms were obtained from Southern Scientific Corporation, Chen-  or plant extracts  have emerged as simple and nai. viable alternative to chemical synthetic procedures. Using plants for nanoparticle synthesis can be advantageous over 2.1.1. Synthesis of Bacopa monnieri-Stabilized Platinum other biological processes, because they eliminate the elab- Nanoparticles (BmE-PtNPs). A general method was used to orate process of maintaining cell cultures and can also synthesize platinum nanoparticles with slight modifications be suitably scaled up for large-scale nanoparticle synthesis . The Bacopa monnieri leaves extract (BmE) was prepared . by weighing 50 g of Bacopa monnieri leaves in 250 mL beaker In this work presented here, we describe a single- along with 100 mL of distilled water and maintained at 80 C step “green synthesis” protocol for the production of well- for2hrsbeforedecanting it.Thesolutionwas filteredby defined platinum nanoparticles by utilising aqueous extract 0.45𝜇 m Milipore membrane filter and followed by 0.2 𝜇 m of Bacopa monnieri (BM) leaves. We have chosen Bacopa Millipore membrane filter. For the synthesis of platinum monnieri (BM) leaves becauseitisoeft nusedtotreat nanoparticles, 40 ml of 1 mM was reacted with 10 mL of the people with Parkinson’s disease . Studies suggest that Bacopa monnieri leaves extract in Erlenmeyer flask at room they improve circulation to the brain, as well as improving temperature. eTh formation of platinum nanoparticles was mood, cognitive function, and general neurological function conrfi med by thechangeinthe colorofthe mixturefrom . The presence of bacoside A and B in BM leaves pale yellow color to dark brown. attributes to their neurobeneficial function [ 16]. In addition The final concentration of metal nanoparticles in the to the neurobeneficial effects they exert antiamnesic [ 17], −4 solution was around6×10 M. The NPs synthesized were antioxidant , antistress , anxiolytic , memory found to be stable for more than a month when stored in enhancing , and antiulcerogenic activities . Recent closed containers, and no visible change was observed for studies suggests that Bacopa monnieri also exerts antiinflam- several days. matory  and antiarthritis activity . Hosamani and Muralidhara have reported the neuroprotective ecffi acy of Bacopa monnieri against rotenone-induced oxidative stress 2.1.2. Characterization. eTh characterization of synthesized and neurotoxicity in Drosophila melanogaster. Recent nanoparticles was carried out according to the methods studies have demonstrated that pretreatment with the BM described previously . The stability and identity of the extract protected the human neuroblastoma cell line SK-N- BmE-PtNPs were measured by recording UV absorbance. SH against H O and acrolein by modulating the activity of The absorbance peak at ∼330–380 nm conrfi med the reten- 2 2 several redox regulated proteins, that is, NF-kappaB, Sirt1, tion of nanoparticulates in all the above mixtures. The size ERK1/2, and p66Shc, so as to favor cell survival in response and shape of the nanoparticles were determined by using to oxidative stress . Based on these stipulations, we used TEM on a JEOL TEMSCAN2000EX model operating at BM extract in the present study to prove its synergistic accelerating voltage at 80 KeV. eTh sample for TEM was reduction potential in reducing platinum salts and for the prepared by putting one drop of the suspension onto standard creation of robust coating on platinum nanoparticles to carbon-coated copper grids and then drying under an IR produce stable phyto platinum nanoparticles for poten- lamp for 30 min. FTIR spectra of freeze-dried BmE-PtNPs tial applications in medicine and technology. Since recent were investigated by analyzing the sample under Brukere Journal of Neurodegenerative Diseases 3 Tensor 27 FTIR spectrometer in attenuated total reflection glutathione (GSH), superoxide dismutase (SOD), glutathione −1 mode using the spectral range of 2000–400 cm with the peroxidase (GSH-Px), catalase (CAT), and total antioxidant −1 capability by spectrophotometric methods. The Thiobarbi- resolution of 4 cm . eTh energy dispersive X-ray analysis of turic Acid Reactive Substance (TBARS) was measured to isolated nanoparticles was carried out by means of JEOL EDX analyse the MDA levels . GSH content was measured model-JSM-5610 LV. according to the method of based on thereactingwith 5, 5 -dithio-bis (2 nitro benzoic acid) (DTNB or Ellman’s 2.1.3. Oxidation of NADH by BmE-PtNPs. To investigate the reagent) to give a yellow colour compound that absorbs chemical change of BmE-PtNPs by NADH oxidation, UV- at 412 nm. eTh activity of SOD was assayed by monitoring Vis surface Plasmon resonance absorption spectra of BmE- the inhibition of the reduction of nitro blue tetrazolium by PtNPs were measured from 200 to 800 nm aeft r incubation thesampleat560nm . Catalase was analysed as the with NADH. BmE-PtNPs at 50𝜇 g/mL were incubated with rate of decrease in absorbance of H O at 240 nm/min/mg 2 2 100𝜇 M NADH in water at room temperature for 12 h. Sub- protein . Glutathione peroxidase was detected with 5-5 - sequently, the incubated mixture was centrifuged to remove dithiobis-p-nitrobenzoic acid . NADH and NAD which impede the spectrum measurement. eTh n, samples were redispersed with the equal volume of water. This washing process was repeated 10 times. 2.4. Estimation of Complex I Activity. ComplexIactivityin crude mitochondrial preparation from zebrafish whole brain was monitored by the decrease in absorbance at 340 nm due 2.1.4. Animals. Wild-type adult (<8monthsold)zebrasfi h to the oxidation of NADH . were obtained from specialized supplier. Animals were kept in aged tap water at 28 Cunder a14:10hlight:darkcycle. Feeding and maintenance of sh fi were done according to 2.4.1. Catecholamine Measurements. The contents of dopam- Westerfield (1995) [ 27]. Animals were acclimated for at least 2 ine and its metabolites were determined according to the weeks before the experiments. eTh procedures were approved method of Luo et al., 2009 . Briefly, whole brain tissue by the institutional animal ethics committee. was homogenised in 0.5 mL of cold perchloric acid (0.4 M). Subsequently, the sample was centrifuged at 20,000×gfor 10 min at 4 C, and the supernatant was transferred to a 2.1.5. Experimental Groups. The experimental groups used clean tube and measured for volume. One-half volume for the tests comprise the following. (a) Experimental of a solution containing 0.02 M potassium citrate, 0.3 M Parkinsonism-induced group (a single dose of MPTP potassium dihydrogen phosphate, and 0.002 M Na EDTA (225 mg/kg bwt) injected intraperitoneally (I.P)) this dosage was added and mixed thoroughly to deposit perchloric acid. was based on a previous study that has demonstrated After incubation in an ice bath for 60 min, the mix was impaired locomotor activity in adult zebrash fi [ 28]BmE- centrifuged at 15,000 g, for 20 min at 4 C. Supernatants PtNPs -control group (various concentrations such 0.3𝜇 mol, were analyzed for catecholamines, especially dopamine and 0.4𝜇 mol, and 0.5𝜇 mol/kg body weight suspended in phys- its metabolite 3.4-dihydroxyphenylacetic acid (DOPAC), by iological saline (PS) were administered once in alternative HPLC (125 mm× 3 mm I.D. column, packed with Nucleosil days for 5 days). (b) BmE-PtNPs pretreated group (BmE- 100 C 18; 3𝜇 m particle size) and electrochemical detection PtNPs + MPTP), various concentrations of BmE-PtNPs were (INTRO, ANTEC Leyden, eTh Netherlands; cell potential = administered once in alternative days for 5 days while MPTP 800mV).Themobilephase consistedof5%acetonitrile, was given on the 4th day 24 hours aeft r the injection of BmE- 10 g/L citric acid, 4 g/L KH PO , 0,1 g/L EDTA, and 0,175 g/L PtNPs. (c) Control group (PS-injected but otherwise identi- 2 4 octanesulfonic acid; pH = 3.0. cally treated sfi h served as control group): the injections were conducted using Hamilton syringes with a mean injection volume of 5𝜇 L/gbodyweight. eTh studywas carried outin 2.4.2. Locomotor Activity Assessment. The locomotor activity group of 6–8 sfi h per treatment. of zebrasfi h wasmeasuredasper theprotocolfollowedbyXia The fish were sacrificed aeft r MPTP injections to observe et al., 2010  with slight modifications. A small experimen- the eeff cts of BmE-PtNPs, 24 hours for lipid peroxidation, taltank(30cm× 10 cm× 15 cm) contained 3 L water was used the activities of antioxidant systems and complex I and 5 to assess the locomotor activity of zebrafish. A transparent days for the locomotor activity, content of dopamine and its plastic film was placed in front of the tank and divided the metabolites in zebrafish brain. tank into four segments. Fish were placed individually in the tank and their behavior was video recorded for 5 min 2.2. Preparation of Brain Homogenates. The whole brain aer ft a 10 min habituation period. Spontaneous swimming tissue homogenates were prepared in 0.1 M phosphate buffer activity was measured by recording the distance traveled, mean speed, and number of times the subject moved from and centrifuged at 3000× gfor30 min.eTh supernatantswere used in the experiments. Protein concentration was estimated one section into another during a 5 min observation. by Lowry’s method . 3. Results and Discussion 2.3. MDA, GSH, SOD, GSH-Px, Catalase, and Total Antiox- idant Capability Assay. The brain supernatants were then Our new process for the production of Bacopa monnieri subjected to the measurement of malondialdehyde (MDA), phytochemicals coated platinum nanoparticles (BmE-PtNPs) 4 Journal of Neurodegenerative Diseases (A) (B) 200 300 400 500 600 700 800 Wavelength (nm) (a) (b) Base (157) Pt Si Al 30 1015 Na Cl Mg 0123456789 2000 1800 1600 1400 1200 1000 800 600 400 (keV) (c) (d) Figure 1: Characterization of Bacopa monnieri phytochemicals coated platinum nanoparticles (BmE-PtNPs). (a) UV-Vis Spectra of BmE- 2− PtNPs.Theinset showstwo bottleswiththe Bacopa monnieri leaf extract before (A) and aeft r (B) reaction with 1 mM PtCl ions for 3 hrs at 95 C. A color version of the inset can be seen. (b) TEM images of BmE-PtNPs. (c) EDAX of BmE-PtNPs. (d) FTIR spectra of BmE-PtNPs. uses a direct interaction of Chloroplatinic acid with Bacopa energy dispersive X-ray (EDX) spectrometers illustrated the monnieri leaf extract in aqueous media without the interven- purity of the platinum, with the spectra showing a strong tion of any external man-made chemicals and, hence, 100% Pt signal (Figure 1(c)). Prominent bands were observed in biogenic.Thecolloidal solution of BmE-PtNPsshowedavery theFTIRspectra (Figure 1(d)) at 616, 887, 1015, 1049, 1270, −1 intense brown color which indicates the reduction of plat- 1389, and 1705 cm , these peaks are assigned to alcohols C–N inum ions (Figure 1(a) (inset)). The formation of BmE-PtNPs stretching vibration of aliphatic amines, phenolic groups, C– was further confirmed by tracing the reaction with UV- N stretching vibration of aromatic amines, germinal methyls, Visible spectroscopy. eTh absorption spectrum of the brown C=C groups or aromatic rings, and carbonyl groups, respec- platinum collides prepared by biogenic process showed a sur- tively.Signicfi antpeaks were notobservedinthe amideI −1 −1 face plasmon absorption band with a maximum of∼340 nm (1640 cm ) or amide II (1540 cm )regions that arechar- (Figure 1(a)). TEM (Figure 1(b)) analysis exposes mostly acteristic of proteins/enzymes accountable for the reduction spherical shaped platinum nanoparticles of approximate size of metal ions to nanoparticles by biological processes. eTh se of 5–20 nm. Under careful observation, it was evident that resultsindicatethatphytochemicalsofBMleafextract like the edges of the particles were lighter than the centers, flavanoids that have functional groups of amines, alcohols, suggesting that some bioorganic compounds such as proteins ketones, aldehydes, and carboxylic acid are robustly coated in Bacopa monnieri leaf extract capped the platinum NPs over the platinum nanoparticles synthesized. Because of the contributing to excellent robustness against agglomeration phytochemical coating and the redox chemistry of BmE- . The result obtained in the synthesis and characterization PtNPs, it is possible that they are biologically active as of nanoparticles is strongly supported by previously pub- antioxidants . lished report on the synthesis of platinum nanoparticles using To determine if BmE-PtNPs can oxidize NADH, 100𝜇 M phytochemicals . The compositional analysis through NADH was incubated with 50𝜇 g/mL BmEPt nps for 3 h and Full scale counts: 1600 Abs Journal of Neurodegenerative Diseases 5 2.25 2 140 $$ ‡‡ 1.75 $$ ∗ †† # $‡ 1.5 ∗∗ 100 †† 1.25 0.75 0.5 0.25 200 260 320 380 440 500 560 620 680 Wavelength (nm) 3 hrs 6 hrs Figure 2: Change in absorption spectra at 3 h and 6 h, respectively. 𝜇 𝜇 BmEPt nps were incubated with NADH in water at room temper- ature. The concentrations of platinum in BmE-PtNPs and NADH MDA GSH-Px were 50𝜇 g/mL and 100𝜇 M, respectively. CAT GSH SOD (a) 6 h, respectively. The absorbance decreased and increased with time at 340 and 260 nm, respectively (Figure 2). This ∗∗∗ observation indicated that BmE-PtNPs oxidized. NADH to NAD . This is because the bands at 340 and 80 260 nm are from the n-𝜋 transition of dihydronicotinamide ∗ ∗ part and𝜋 -𝜋 transition of the adenine ring, respectively. This result demonstrates that BmE-PtNPs have an activity similar to mitochondrial NADH : Ubiquinone oxidoreduc- tase, which is concurrence with the earlier published results of pectin protected platinum nanoparticles . This suggests that BmE-PtNPs are a potential medicinal substance for oxidativestressmediateddisease with suppressed mitochon- drial complex I, namely, Parkinson’s disease (PD). Oxidativestresswas generatedinzebrasfi hbyexposureto 𝜇 𝜇 MPTP, which is an intracellular free radical-generating com- pound resulting in corresponding Parkinsonian symptoms (b) . The intraperitoneal administration of a single dose of MPTP (225 mg/kg bwt) resulted in a profound increase in the Figure 3: (a) Eeff cts of Bacopa monnieri phytochemicals coated levels of MDA, diminished activities of antioxidant defense platinum nanoparticles (BmE-PtNPs) on the content/activity of MDA, GSH, SOD, GSH-Px, and CAT in the MPTP zebrash fi brain. mechanism in charge for scavenging free radicals and main- ∗#†$‡ Data were shown as mean ± SEM.𝑛=6 –8, 𝑃 < 0.05 , taining redox homeostasis such as SOD, CAT, GPx, GSH, ∗∗##††$$‡‡ ∗∗∗###†††$$$‡‡‡ 𝑃 < 0.01 , 𝑃 < 0.001 versus MPTP group. and complex I were observed in experimental Parkinsonism- (b) Eeff cts of Bacopa monnieri phytochemicals coated platinum induced group (MPTP) (Figures 3(a) and 3(b)). The BmE- nanoparticles (BmE-PtNPs) on the activity of complex I in the PtNPs concentrations tested were 0.3, 0.4, and 0.5𝜇 mol, MPTP zebrash fi brain. Data were shown as mean ± SEM.𝑛=6 –8, respectively.TheMDA levels were signicfi antly decreasedby ∗ ∗∗ ∗∗∗ 𝑃<0.05 , 𝑃<0.01 , 𝑃<0.001 versus MPTP group. 0.4𝜇 mol of BmE-PtNPs (Figure 3(a)). This makes clear the inhibitory effect of BmE-PtNPs over ROS generation during MPTP-induced oxidative stress. Glutathione (GSH) is a tripeptide with a free reductive thiol functional group, responsible for the detoxification of such as GSH, SOD, catalase, and glutathione peroxidase are peroxides such as hydrogen peroxide or lipid peroxides and diminished during oxidative stress induced by MPTP. In acting as an important antioxidant in cells. During the detox- the present study, a statistically significant increase in the ification process, GSH (reduced form) becomes oxidized levels of GSH, SOD, catalase, and glutathione peroxidase in glutathione (GSSG) which is then recycled to GSH by the the MPTP treated zebrasfi h with 0.4 𝜇 mol of BmE-PtNPs is enzyme glutathione reductase present in cells. eTh increased being proved. This study demonstrates that BmE-PtNPs act as MDAlevelsinMPTPcould be duetotheir increased reductive catalyst, by the ability to scavenge ROS, superoxide production and/or decreased destruction by antioxidants anion radicals (O ), and hydrogen peroxide (H O ). This 2 2 2 such as GSH, SOD, catalase, and glutathione peroxidase . data is in good accordance with the previous studies where The activities of antioxidant defense enzymes in charge for platinum nanoparticles are known to act as a SOD/catalase scavenging free radicals and maintaining redox homeostasis mimetics to extend the lifespan of C. elegans, extended Abs Control (%) Control (%) Control Control MPTP MPTP 0.3 mol BmE-PtNPs 0.3 mol BmE-PtNPs MPTP + MPTP 0.4 mol BmE-PtNPs 0.4 mol BmE-PtNPs + MPTP MPTP 0.5 mol BmE-PtNPs 0.5 mol BmE-PtNPs + MPTP + MPTP 6 Journal of Neurodegenerative Diseases ∗∗ ‡‡‡ ∗∗∗ ††† †† Control MPTP 0.4 𝜇 mol BmE-PtNPs + MPTP Control 0.4 𝜇 mol BmE-PtNPs MPTP + MPTP DA DOPAC (a) HVA Figure 4: Effects of Bacopa monnieri phytochemicals coated plat- 0.09 inum nanoparticles (BmE-PtNPs) on the contents of dopamine, DOPAC, and HVA in the MPTP zebrafish brain. Data were shown 0.08 ∗†‡ ∗∗††‡‡ ∗∗∗†††‡‡‡ as mean + SEM.𝑛=6 –8, 𝑃<0.05 , 𝑃<0.01 , 𝑃< 0.07 0.001 versus MPTP group. 0.06 0.05 0.04 the lifespan of the roundworm Caenorhabditis elegans[39– 41], inhibited pulmonary inflammation in mice exposed 0.03 to cigarette smoke , inhibited cell growth of human 0.02 tongue carcinoma cells , and furthermore ameliorated neurological function and brain damage aer ft ischemic stroke 0.01 . The mitochondrial respiratory chain, especially at com- Control 0.4 𝜇 mol BmE-PtNPs MPTP plexes I, is thought of as a primary site of ROS generation. + MPTP In some oxidative stress diseases such as Parkinson’s disease, (b) excessive ROS generation is responsible for pathogenesis due to the suppression of complex I. In the current study a significant inhibition of complex I activity was observed in the experimental Parkinsonism-induced group (Figure 3(b)), which was attenuated by the pretreatment of various concen- trations of BmE-PtNPs (Figure 3(b)). However, 0.4𝜇 mol of BmE-PtNPs demonstrated a noteworthy effect on restoring thecomplex Iactivityaswellasthe levels of GSH, SOD, catalase,and glutathioneperoxidaseinthe MPTP treated zebrafish. This result demonstrates that BmE-PtNPs serve dual functions as mitochondrial complex I to lower ROS gen- eration and as SOD/catalase mimetics to scavenge generated excessive ROS. Postmortem studies provided evidence for the decrease in the content of dopamine (DA) and its metabolites dihydrox- yphenylacetic acid (DOPAC), and homovanillic acid (HVA) in the brains of Parkinson’s disease. Our results showed that Control MPTP 0.4 𝜇 mol BmE-PtNPs + MPTP theDA, DOPAC, andHVA contents in MPTP zebrasfi h were markedly lower than those of control sfi h, and BmE-PtNPs (c) increasedDA, DOPAC, andHVA levels (Figure 4). In Parkinson’s disease, the most debilitating symptom Figure 5: Effects of Bacopa monnieri phytochemicals coated plat- of the disease is the loss of motor control. Figure 5 shows inum nanoparticles (BmE-PtNPs) on zebrafish brain locomotor the results for the locomotion activity. MPTP administration activity. Data were shown as mean± SEM.𝑛 =6–8;one-wayANOVA results in a signicfi ant reduction in the total movement test was performed. (ng/g) Mean speed (m/s) Number of line crossings Distance (m) Journal of Neurodegenerative Diseases 7 distance,meanvelocity, andmeandistanceper movement  R. Endo, T. Saito, A. Asada, H. Kawahara, T. Ohshima, and S. I. Hisanaga, “Commitment of 1-methyl-4-phenylpyrinidinium in zebrafish compared to the control animals. This n fi ding ion-induced neuronal cell deathby proteasome-mediated points the correlate loss of dopamine due to MPTP neuro- degradation of p35 cyclin-dependent kinase 5 activator,” toxicity. 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