Antioxidant Effect of Gold Nanoparticles Synthesised ...

Copyright © 2012 American Scientific Publishers All rights reserved Printed in the United States of America

Journal of Nanoneuroscience Vol. 2, 1–12, 2012

Antioxidant Effect of Gold Nanoparticles Synthesised from Curcuma Longa Restrains 1-Methyl-2-Phenyl Pyridinium Ion Induced Stress in PC12 Cells Jayshree Nellore1 ∗ , Cynthia Pauline1 , and Kanchana Amarnath2 1

Department of Biotechnology, Sathyabama University, Chennai 600119, India Department of Biochemistry, Sathyabama University, Chennai 600119, India

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Keywords: Turmeric, Gold Nanoparticles, Oxidative Stress, Parkinson’s Disease.

INTRODUCTION Parkinson’s disease is one of the most common neurodegenerative disorders, characterized by a relatively selective degeneration of dopaminergic neurons in the substantia nigra pars compacta (lsaacs, 2009). The symptoms include resting tremor, bradykinesia, postural instability, and rigidity (Tillerson et al., 2003; Oida et al., 2006). 1, methy, 4 phenyl 1,2,3,6 tetra hydro pyridine (MPTP) is a potent neurotoxin that induces Parkinson’s disease in various experimental animals including monkeys, mice, cats, dogs, rats and goldfish (Gerlach et al., 2006). MPTP per is not toxic to neurons, but it has to be oxidized to (MPP+ ) 1-methyl-2-phenyl pyridinium ion in the astrocytes by the action of monoamine oxidase for it to be active (Tillerson et al., 2003). MPP+ mediates cell death by inhibiting complex-I of electron transport chain and free radical generation leading to oxidative stress (Alcaraz-Zubeldia et al., 2001; Thomas et al., 2000) as shown by alteration in the states of antioxidant enzymes and molecules.

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Author to whom correspondence should be addressed.

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Studies have shown that biological systems have evolved with endogenous defense mechanisms to help protect against reactive oxygen species-induced cell damage. Superoxide dismutase (SOD), Catalase (CAT) and glutathione peroxidase are endogenous antioxidant enzymes which play pivotal roles in preventing cellular damage caused by reactive oxygen species (Barlow et al., 2005; Husain et al., 2005). Thus an effective and economic therapeutic molecule capable of up drifting the treatments for Parkinson’s disease, by controlling the oxidative stress, disquieting various metabolic pathways and thereby preventing the onset of complications is still the need of the era. Discovery of new molecules and manipulating those available naturally in nanosize could be appealing for their greater potential to improve health care (Dash and Shrivastava, 2009). Several pharmacological companies have won approval from the Food and Drug Administration (FDA) for the use and development of nanotechnology-based drugs in the last few years. Gold nanoparticles have found use in diagnostic and drug delivery applications (Bhumkar et al., 2007; Mohanpuria et al., 2008; Prakash et al., 2010 and Selvaraj et al., 2007; BarathManiKanth et al., 2010). Therefore, there is a growing need to develop eco-friendly nanoparticle synthesis without using

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doi:10.1166/jns.2012.1018

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The green synthesis of nanoparticles biologically inspired due to the challenges faced by chemical synthesis protocol of nanoparticles in being toxic, flammable and unstable and is expected to open new avenue to fight and prevent diseases. In this study we report the synthesis of gold nanoparticles from Curcuma longa root extract by ‘exploiting’ the reduction capabilities of varied phytochemicals present in it and was confirmed by FTIR, UV-Visible pectroscopy and TEM that interpreted the formation of gold nanoparticles by the reduction of gold salts. We extended our research to utilize the efficacy of these synthesized biocompatible gold nanoparticles (Tu-AuNPs) as an antioxidant against 1-methyl-2-phenyl pyridinium ion (MPP+ ) induced cytotoxicity and cell death in PC-12 cells. Incubation of PC-12 cells with Tu-AuNPs prevented MPP+ -induced loss in cell viability and enhanced LDH leakage in a dose-dependent manner. In addition, reduction in the level of non-protein thiol, glutathione (GSH); activities of the antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT) and glutathione S-transferase (GST) as well as the increased MDA levels have also been found to be prevented by this nanoparticles. Moreover, MPP+ exposure caused inhibition of complex I activity. In addition, MPP+ introduces apoptosis as the primary phenomena of cell death as evidenced by DAPI staining, flow cytometric analyses. Tu-AuNPs treatment, however, counteracted these changes and maintains normalcy in PC-12 cells. Our results specify that Tu-AuNPs possesses cytoprotective activity against MPP+ -induced oxidative cellular damage and prevents PC-12 cells from apoptotic death. Taken together, the above results suggest that gold nanoparticles from Curcuma longa root extract may be a candidate drug for the treatment of oxidative stressinduced neurodegenerative disease.

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Antioxidant Effect of AuNPs Synthesised from Curcuma Longa Restrains MPP+ Induced Stress in PC12 Cells toxic chemicals. Biological methods for nanoparticle synthesis using microorganisms, enzymes and plants or plant extracts have been suggested as possible eco-friendly alternatives to chemical and physical methods (Shankar et al., 2004). Using plants for nanoparticle synthesis can be advantageous over other biological processes because it eliminates the elaborate process of maintaining cell cultures and can also be suitably scaled up for large-scale nanoparticle synthesis. Turmeric (Tu) is a yellow powder, derived from the plant Curcuma longa. It has been consumed in amounts up to one g/day for thousands of years in countries such as India. Previous pharmacological studies have demonstrated its anti-tumor, antiinflammatory, anti-infectious and antioxidant activity with very low toxicity (Srinivas et al., 1992; Srinivas and Shalini, 1991). In particular curcumin protected PC12 cells against MPP (+)induced cytotoxicity and apoptosis by bcl-2-mitochondria-ROSiNOS pathway (Chen et al., 2006). Therefore we investigated the synergistic potentials of polyphenols, flavonoids, catechins, and various phytochemicals present in Turmeric for the reduction reactions of gold salts to produce gold nanoparticles which have potential applications in the diagnosis and therapy of various deadly diseases including neurodegeneration. Further the synthesized nanoparticles were characterized using UV-Vis Spectroscopy, FTIR (Fourier Transform Infra red Spectrophotometer) and TEM (Transmission electron microscope) that confirmed the formation of gold nanoparticles via reduction of gold salts, role of phytochemicals in their synthesis and revealed its various sizes, respectively. Current research in antioxidant nanomaterial has opened a new era in pharmaceutical industries. Thus in the present study we report the synthesis of highly stable nanoparticles of gold from curcuma longa (Tu-AuNPs) extract endowed with antioxidant effect against MPP+ induced oxidative stress in PC-12 cells which has not been revealed yet.

RESULTS Characterization of Tu-AuNPs

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nanoparticles using turmeric after reaction with AuCl4 solution shows that the sample is composed of a large quantity of gold nanoparticles. The gold nanoparticles formed are homogenous, predominantly spherical with an average diameter of 13 ± 5 nm, depicting a narrow size distribution. FT-IR absorption spectra can provide the information about the chemical change of the functional groups involved in bioreduction. Figure 4 shows FT-IR absorption spectra of turmeric extract before and after bioreduction. To a large extent, the band at 1101 cm−1 might be contributed by the –C–O groups of the polyols, such as flavones, terpenoids and polysaccharides in the biomass (Kang et al., 2008; Shankar et al., 2003). The disappearance of band at 1101 cm−1 after bioreduction suggested that the polyols might be partly responsible for the reduction of chloroaurate ions. FT-IR analysis of bioextract before and after the addition of gold solution also revealed the strong bands at 1021, 1443, 1634 and 3428 cm−1 . The band at 1021 cm−1 corresponds to C–N stretching vibrations of amine and at 1443 cm−1 corresponds to C–H and OH bending and 3428 cm−1 is characteristic of –NH stretching of amide (II) band. The weaker band at 1634 cm−1 corresponds to amide I, arisen due to carbonyl stretch in proteins.

In Vitro-Stability Studies Biomedical applications require stability of Tu-AuNPs over a reasonable length of time. In vitro stability tests were performed by incubating solutions of Tu-AuNPs with 10% NaCl, cysteine, histidine, Human Serum Albumin (HSA) and Bovine Serum Albumin (BSA) solutions that mimic biological environments. The stability and the identity of Tu-AuNPs were measured by recording UV absorbance after 24 h, as shown in Figure 2. The plasmon resonance band at ∼535 nm confirmed the retention of nanoparticulates in all the above mixtures. This retention indicates that the AuNPs are intact, and thereby demonstrate excellent in vitro stability in biological fluids at physiological pH. Tu-AuNPs demonstrated excellent in vitro stability under

Characterization of the synthesized gold nanoparticles was carried out before testing for their potent anti-oxidative effect in PC-12 cells. The absorption measurement of the biologically synthesized gold nanoparticles indicated that the Plasmon resonance wavelength, max is at 540 nm (Fig. 1). The morphology and size of the biologically synthesized gold nanoparticles was determined using Transmission electron microscopy (TEM). TEM pictures (Fig. 3) recorded from drop-coated films of the gold

Fig. 1. UV-VIS Spectra of gold nanoparticle synthesized using curcuma longa root extract. The inset shows two bottles with the curcuma longa root extract before and after reaction with 10−3 M HAuCl4 aqueous solutions. A color version of the inset can be seen.

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Fig. 2. UV-visible spectra of Tu-AuNPs showing in vitro stability of the nanoparticles in different medium, e.g., 10% NaCl, 0.5% cysteine, 0.2 M Histidine, 0.5% HSA, 0.5% BSA, water and in different pH after 24 h treatment.

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Antioxidant Effect of AuNPs Synthesised from Curcuma Longa Restrains MPP+ Induced Stress in PC12 Cells for their site-specific delivery as diagnostic/therapeutic agents. Hence we studied whether the turmeric derived phytochemicals, if coated on AuNPs, will show internalization within PC-12 cells. Significant internalization of Tu-AuNPs nanoparticles within PC12 cells was confirmed by the distinct 0.5 to 1.2 m spheroidal structures which were of a size comparable to the nanoparticles (Fig. 5).

The Effect Tu-AuNPs on MPP+ -Mediated Cell Injury Exposure to MPP+ caused loss in PC-12 cell viability. In order to determine whether this loss could be prevented by TuAuNPs pre-treatment, we performed MTT assay. The results in Figure 6(A) shows that pretreatment of PC-12 cells with TuAuNPs dose-dependently increased viability of MPP+ treated cells. LDH leakage is associated with cell viability and is considered as an important indicator of cellular membrane damage. Severe LDH leakage was found in the MPP+ treated PC-12 cells indicating that the loss of cell membrane integrity and cytotoxicity were caused by MPP+ on PC-12 cells (Fig. 6(B)). Tu-AuNPs pretreatment effectively and dose-dependently inhibited the membrane disruption caused by the toxin as revealed from the less LDH level outside the cells. Tu-AuNPs pre-treatment at a concentration of 100 g/ml reduced the LDH leakage to a minimum and this concentration is used in subsequent studies.

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Fig. 3. Transmission Electron Microscopy (TEM) image and size histogram of the gold nanoparticles synthesised using curcuma longa root extract at the end of the reaction.

pH 4 to 9 range, implying that these nanoparticles can be used in a wide pH range for various biomedical applications.

SEM Analysis of Surface Characteristics of PC-12 Cells During Internalization of Tu-AuNPs Results of cellular internalization studies of Tu-AuNPs solutions are key to providing insights into their use in biomedicine. Their selective cell and nuclear targeting will provide new pathways

Fig. 4. FTIR Spectra of Turmeric extract (Blue line) and gold nanoparticles generated from turmeric (black line).


Fig. 5. SEM Images of paraffin mounted PC-12 cells. Bar represents the magnification. (A) Control cells (B) Treated with 100 g/ml of TuAuNPs for a period of 24 hrs.

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Antioxidant Effect of AuNPs Synthesised from Curcuma Longa Restrains MPP+ Induced Stress in PC12 Cells

Fig. 6. Time and dose-dependent prevention of the MPP+ -induced (A) loss of cell viability by Tu-AuNPs. Values are expressed as percent over control. (B) LDH leakage by Tu-AuNPs. Control: Time-dependent cell viability of the normal PC-12 cells incubated in DMEM up to 48 hrs. MPP+ : Time-dependent cell viability values when PC-12 cells were incubated with MPP+ (200 M) in DMEM up to 48 hrs. Tu-AuNPs1 +MPP+ , Tu-AuNPs2 + MPP+ and Tu-AuNPs3 + MPP+ : Time-dependent cell viability values when PC-12 cells were incubated with 25 g/ml, 50 g/ml, and 100 g/ml Tu-AuNPs for 30 min prior to MPP+ (200 M) administration in DMEM and the incubation continued up to 48 hrs.

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Fig. 7. Effect of Tu-AuNPs on MDA levels in PC-12 cells treated with MPP+ . Results have been given as percentage over control. Control: PC12 cells treated with DMEM, MPP+ : PC-12 cells were incubated with MPP+ (200 M) in DMEM up to 48 hrs. Tu-AuNPs1 + MPP+ , TuAuNPs2 + MPP+ and Tu-AuNPs3 + MPP+ : PC-12 cells were incubated with 25 g/ml, 50 g/ml, and 100 g/ml Tu-AuNPs for 30 min prior to MPP+ (200 M) administration in DMEM and the incubation continued up to 48 hrs. Tu-AuNPs+3 : PC-12 cells incubated with 100 g/ml Tu-AuNPs in DMEM upto 48 hrs Each value represents mean ± S.D. (Pa < 005; Pb < 005).

Effect of Tu-AuNPs on the MPP+ -Induced Lipid Peroxidation MDA level indicates the extent of lipid peroxidation. Figure 7 shows the MPP+ -induced lipid peroxidation, an indicator of membrane damage. MPP+ administration (200 M) increased the lipid peroxidation to 160% with respect to the normal cells. Tu-AuNPs treatment prior to MPP+ administration caused inhibition in the lipid peroxidation in a linear fashion. Significant membrane damage recovery has been observed at a concentration of 50 g/ml and at a concentration 100 g/ml, the damage was recovered almost to its normal leve.

Effect of Tu-AuNPs on the MPP+ -Induced oxidative Stress Figure 8 demonstrates that MPP+ treatment decreased GSH level to about 40%. Tu-AuNPs treatment prior to MPP+ administration increased the GSH level in a dose-dependent manner. GSH level was significantly increased at a concentration of 25 g/ml.

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Fig. 8. Effect of Tu-AuNPs on GSH levels in PC-12 cells treated with MPP+ . Results have been given as percentage over control. Control: PC12 cells treated with DMEM, MPP+ : PC-12 cells were incubated with MPP+ (200 M) in DMEM up to 48 hrs. Tu-AuNPs1 + MPP+ , TuAuNPs2 + MPP+ and Tu-AuNPs3 + MPP+ : PC-12 cells were incubated with 25 g/ml, 50 g/ml, and 100 g/ml Tu-AuNPs for 30 min prior to MPP+ (200 M) administration in DMEM and the incubation continued up to 48 hrs. Tu-AuNPs+3 : PC-12 cells incubated with 100 g/ml Tu-AuNPs in DMEM upto 48 hrs. Each value represents mean ± S.D. (Pa < 005; Pb < 005).


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Effect of Tu-AuNPs on the MPP+ -Induced Antioxidant Enzyme Activity Figure 9(a) shows that MPP+ treatment decreased the CAT activity by about 40%. Significant increase in CAT activity was observed at a concentration 25 g/ml and at the concentration 100 g/ml the CAT activity was reached to almost normal. Figures 9(b and c) demonstrate the effect of the Tu-AuNPs on SOD activity and GST activities,respectively. The results are almost similar to CAT activity.

Effects of Tu-AuNP’s on Complex-I Activity The activity of complex I in the mitochondrial fraction was measured spectrophotometrically as described in Materials and methods. As shown in Figure 10, 200 m MPP+ caused a significant decrease in complex I activity and pretreatment with Tu-AuNPs blocked the effect of MPP+ . These characteristics were reversed by Tu-AuNPs in a dose dependent manner.

Attenuation of MPP+ -Induced Apoptosis by Tu-AuNPs

DISCUSSION The promising potential of gold nanoparticles in treating inflammatory and auto immune disease (Mukherjee et al., 2005) have J. Nanoneurosci. 2, 1–12, 2012

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DAPI is known to form fluorescent complexes with natural double-stranded DNA, showing fluorescence specificity for AT, AU and IC clusters. When DAPI binds to DNA, its fluorescence is strongly enhanced due to a highly energetic and intercalative type of interaction, but there is also evidence that DAPI binds to the minor groove, stabilized by hydrogen bonds between DAPI and acceptor groups of AT, AU and IC base pairs. MPP+ induces apoptosis in PC-12 cells. Accordingly, we investigated the ability of Tu-AuNPs to inhibit the apoptosis induced by MPP+ in PC-12 cells using DAPI staining. As shown in Figure 11, uniform PC12 with normal morphology were observed in control group (A), whereas the cells with fragmented chromatin and apoptotic bodies were noted following treatment with 200 m MPP+ (B) and these abnormalities were completely abolished by the Tu-AuNPs pre-treatment. An early event in apoptosis is the flipping of phosphatidylserine of the plasma membrane from the inside surface to the outside surface. Annexin V binds specifically to phosphatidylserine and FITC-conjugated Annexin V can be used as a fluorescent probe to label apoptotic cells. Propidium iodide (PI) is used in conjunction with Annexin V-FITC. The cell membrane integrity excludes PI in viable and apoptotic cells, whereas necrotic cells are permeable to PI. To characterize the anti-apoptotic activity of Tu-AuNPs, we further quantified apoptotic and anti-apoptotic cells using flow cytometry analysis as illustrated in Figure 12. Results suggest that after treatment with 200 m MPP+ , showed significant increases in apoptosis compared with control groups (P < 001). Apoptotic rate was 29.8 ± 2.15% (Fig. 12). When we added Tu-AuNPs (100 g/ml) for 30 min before exposing cells to 200 m MPP+ for 48 h, apoptotic rates decreased from 298 ± 215% to 15 ± 249% (P < 001).

Fig. 9. Effect of Tu-AuNPs on (A) Catalase (CAT) (B) SOD and (C) GST activity in PC-12 cells treated with MPP+ . Control: PC-12 cells treated with DMEM; MPP+ : PC-12 cells treated with MPP+ (200 M); Tu-AuNPs1 + MPP+ , Tu-AuNPs2 + MPP+ and Tu-AuNPs3 + MPP+ : PC12 cells were incubated with 25 g/ml, 50 g/ml, and 100 g/ml TuAuNPs for 30 min prior to MPP+ (200 M) administration in DMEM and the incubation continued up to 48 hrs; Tu-AuNPs+3 : PC-12 cells incubated with 100 g/ml Tu-AuNPs in DMEM upto 48 hrs. “a” indicates the significant difference between the normal control and MPP+ -treated cells, “b” indicates the significant difference between the MPP+ -treated and Tu-AuNPs pretreated groups. Each column represents mean ± S.D., n = 6; (P a < 001, P b < 001).

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Fig. 10. Effect of Tu-AuNP’s on MPP+ -induced loss in mitochondrial complex I activity. Results have been shown as % control. Control: complex-I activity of the normal PC-12 cells incubated in DMEM up to 48 hrs. MPP+ : complex-I activity when PC-12 cells were incubated with MPP+ (200 M) in DMEM up to 48 hrs. Tu-AuNPs1 + MPP+ , Tu-AuNPs2 + MPP+ and Tu-AuNPs3 + MPP+ : complex-I activity when PC-12 cells were incubated with 25 g/ml, 50 g/ml, and 100 g/ml Tu-AuNPs for 30 min prior to MPP+ (200 M) administration in DMEM and the incubation continued up to 48 hrs.

augmented greater interest to investigate the antioxidative activity of the gold nanoparticles against the MPP+ induced oxidative stress. As part of our ongoing efforts toward the design and development of biocompatible gold nanoparticles for subsequent use in medical applications, we have initiated studies on the direct intervention of phytochemicals for the production of gold nanoparticles. Our process for the production of gold nanoparticles uses the direct interaction of sodium tetrachloaurate with turmeric in aqueous media without the intervention of any external manmade chemicals. Therefore, this reaction pathway satisfies all the conditions of a 100% green chemical process (Hung et al., 2007; Jorge et al., 2003; Gardea et al., 2002). Gold nanoparticles produced by this process did not require any external chemicals for the stabilization of the nanoparticulate matrix. Phytochemicals present in turmeric are presumably responsible for the creation of a robust coating on gold nanoparticles and thus, rendering the nanoparticles stable against agglomeration. The results obtained in the synthesis and characterization of the synthesized nanoparticles is strongly supported by previously published reports on synthesis of gold nanoparticles using phytochemicals (Nune et al., 2009; Katti et al., 2009). The most important criteria for biomedical applications are the stability of gold nanoparticles over a reasonable time period (Nune et al., 2009; Katti et al., 2009). Our results from these in vitro stability studies have confirmed that the gold nanoparticles are intact and thus, demonstrate excellent in vitro stability of Tu-AuNPs in biological fluids at physiological pH. It is also remarkable that this ‘nano-compatible’ structural motif of phytochemicals in turmeric offers stability to gold nanoparticles in aqueous media for over a month. These results suggest that the green nanotechnological process reported herein provides both the production and stabilization processes under mild conditions without the intervention of any man made harsh chemicals.

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Fig. 11. Detection of the mode of cell death by DAPI staining assay. DAPI stained nuclei in (A) PC-12 cells treated with DMEM medium only; (B) PC-12 cells treated with MPP+ and (C) PC-12 cells treated with Tu-AuNPs before addition of MPP+ . Cells were observed using a microscope (original magnification X 20). The measurements were made six times.

Results of cellular internalization studies of AuNPs solutions are key to provide insights into their use in biomedicine. Their selective cell and nuclear targeting will provide new pathways for their site-specific delivery as diagnostic/therapeutic agents. A J. Nanoneurosci. 2, 1–12, 2012

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Fig. 12. Representative flow cytometric analysis of PC-12 apoptotic cells stained for Annexin V+propidium iodide (PI). (A) Distribution of PC-12 cells treated with DMEM, (B) Distribution of PC-12 cells treated with 200 M MPP+ , (C) Distribution of PC-12 cells treated with Tu-AuNPs (100 g/ml) before addition of MPP+ . In each panel, the lower right quadrant contains apoptotic cells (positive for Annexin V and negative for PI).


Glutathione (GSH) is a tripeptide with a free reductive thiol functional group, responsible for the detoxification of peroxides such as hydrogen peroxide or lipid peroxides, and acting as an important anti-oxidant in cells. During the detoxification process GSH (reduced form) becomes oxidized glutathione (GSSG) which is then recycled to GSH by the enzyme glutathione reductase present in cells. The increased LPO levels in MPP+ could be due to their increased production and/or decreased destruction by antioxidants such as GSH, SOD, catalase and glutathione peroxidase and glutathione-s-transferase (Ruggiero et al., 1997; McDonagh and Hokama, 2000; Vinik et al., 2000; Kowluru and Kennedy, 2001; Chang et al., 2003). The activities of antioxidant defense enzymes in charge for scavenging free radicals and maintaining redox homeostasis such as GSH, SOD, catalase and glutathione-S-transferase are diminished during oxidative stress induced by MPP+ . An increased LPO level enhances oxidant production and impairs antioxidant defenses by multiple interacting pathways (King and Loeken, 2004). In the present study a statistically significant increase in the levels of GSH, SOD, catalase and glutathione-s-transferase in the MPP+ treated PC-12 cells with Tu-AuNPs is being proved suggesting that Tu-AuNPs prevents disruption of PC-12 cells by protecting lipids from peroxidation by ROS under oxidative stress conditions. Mitochondrial dysfunction is a consequence of oxidative damage caused by increased oxidant levels. Therefore, decreasing oxidant generation and oxidative damage should be an effective way to inhibit mitochondrial impairment. The Tu-AuNPs pretreatment also significantly protected the cells from MPP+ induced toxicity by inhibiting mitochondrial impairement. These results suggest that Tu-AuNPs may act as an effective antioxidant for preventing dopamine neuronal damage in PD (Gonzalez-Polo et al., 2004; Virmani et al., 2005). To the best of our knowledge, there is no report of any gold nanoparticles synthesized by biological means that possesses protective action against MPP+ induced apoptosis. Oxidative stress plays a foremost role in etiology of MPP+ (Yuan et al., 2007). The ability of gold nanoparticles in inhibiting the lipid from peroxidation thereby preventing the ROS generation has restored the imbalances in the antioxidants responsible for the cell dysfunction and destruction, leading to cell injury. Our result suggesting Phytochemical mediated gold nanoparticles’ potential as antioxidant is shored up with the literature

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number of studies have demonstrated that phytochemicals in tea, soya and cinnamon have the ability to penetrate the cell membrane and internalize within the cellular matrix (Mizuno et al., 2007; Sun et al., 2007). Therefore, we hypothesized that turmeric derived phytochemicals, if coated on AuNPs, will show internalization within PC-12 cells. SEM images of PC-12 cells treated with AuNPs unequivocally validated our hypothesis. Significant internalization of Tu-AuNPs nanoparticles within PC-12 cells was confirmed by the distinct 0.5 to 1.2 m spheroidal structures which were of a size comparable to the nanoparticles. The viability of PC-12 cells post-internalization suggests that the phytochemical coating renders the nanoparticles non-toxic to cells. Such a harmless internalization of Tu-AuNPs will provide new opportunities as the platform material in the fields of biodiagnostics (Nam et al., 2003), drug/DNA delivery (Paciotti et al., 2004; Prow et al., 2006), cell imaging (Bielinska et al., 2002), immunostaining (Roth, 1996), biosensing (Penn et al., 2003), and electron microscopy markers (Baschong et al., 1998). Hence, to find effective agents for Parkinson’s disease (PD) prevention and therapy, we examined the protective effects of the Tu-AuNPs in a 1-methyl-4-phenylpyridinium (MPP+ )-induced acute cellular PD model in PC-12 cells. Our results demonstrated that MPP+ -induced loss in cell viability and enhanced LDH leakage. In addition, reduction in the level of non-protein thiol, glutathione (GSH); activities of the antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT) and glutathione S-transferase (GST) as well as the increased MDA levels have also been found. We also showed that MPP+ inhibited mitochondrial function by inhibiting complex I activity. These results suggest that this in vitro cellular model successfully mimics some of the characteristics of PD. Gold nanoparticles and its derivatives are potential agents for application in the fields of biology and medicine (Salata, 2004; Jain et al., 2007; David et al., 2010). Gold nanoparticles and its derivatives are not only nanomaterials, but also they are effective antioxidants (Nie et al., 2007; Yakimovich et al., 2008) and good free radical trappers in biological systems (Shah and Vohora, 2002). Consistent with previous results on gold nanoaprticles, our results suggest that Tu-AuNPs is a powerful antioxidant with radical scavenging by interfering with the generation of LPO. This makes clear the inhibitory effect of gold nanoparticles over ROS generation during MPP+ induced oxidative stress.

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Antioxidant Effect of AuNPs Synthesised from Curcuma Longa Restrains MPP+ Induced Stress in PC12 Cells that reports delivering the control effects of gold nanoparticles as an antioxidant (Barath Mani Kanth et al., 2010) and potential of other rare earth metals like cerium oxide to scavenge free radicals in retinal neurons (Onizawa et al., 2009). These results are also supported by the findings that suggest the noncytotoxic effect of Au (0) nanoparticles, and the ability of gold nanoparticles to reduce the production of reactive oxygen and nitrite species, which do not elicit secretion of proinflammatory cytokines TNF- and IL1-, making them suitable candidates for nanomedicine (Wang et al., 2005). The anti-oxidative effect of phytochemical mediated gold nanoparticles along with their protective effect against MPP+ induced oxidative stress may be attained through the inhibition of the stress signaling pathways or, due to the interaction of the AuNPs to the cystein-residues of the thioredoxin thereby preventing the thioredoxin-interacting protein (Txnip) from binding to it. Thioredoxin, is a highly conserved thiol reductase that act over an endogenous inhibitor, thioredoxin- interacting protein (Txnip) and is responsible for the antioxidative mechanism through the regulation of cellular redox balance (Nune et al., 2009). The potential ability of Phytochemical mediated AuNPs in this study to inhibit the oxidative stress mediated ROS generation is highly supported by existing evidences of various other nanoparticles such as Platinum nanoparticles that had an immense ability to inhibit the pulmonary inflammation led by oxidative stress as a result of cigarette smoking due to their antioxidant properties (Katti et al., 2009). The melatonin-selenium (MT-Se) nanoparticles also relapsed the ROS generated and lipid peroxidation based on which their antioxidant effect is confirmed (Wang et al., 2005). The utility of chitosan nanoparticles with an in-vitro model of acrolein-mediated cell injury using PC-12 cells effectively, and statistically, reduced damage to membrane integrity, secondary oxidative stress, and lipid peroxidation (Cho et al., 2010). The advantage of our biologically synthesized AuNPs over these nanoparticles is that biologically synthesized nanoparticles have a greater stability and do not agglomerate or aggregate.

CONCLUSIONS Nanotechnology is undergoing explosive expansions in many areas serving mankind, due to which even poorer developing countries have also decided that this new technology could represent a considered investment in future economic and social well-being that they cannot ignore. The gold nanoparticles are known for their tremendous applications in the field of therapeutics and diagnosis. In the present study we have confirmed the anti-oxidative properties of gold nanoparticles synthesized from the phytochemicals present in turmeric in PC-12 cells by balancing or inhibiting the ROS generation at oxidative stress induced by MPP+ by scavenging free radicals and thus increasing the anti-oxidant defense enzymes. The gold nanoparticles have been proven to be non-toxic in PC-12 cell lines, thereby accomplishing a sustained control over the disease progression. These potential application of gold nanoparticles preventing oxidative stress and their adverse effects, induced by MPP+ has opened up way for a new resource of cost economic alternative in the treatment of various oxidative stress-induced toxicity, such as neurodegenerative disorders, diabetics etc., Furthermore, a clear study over the mechanism and the downstream pathways through which the

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gold nanoparticles influence the control over the anti-oxidant systems and their reverse effect over oxidative stress conditions may solely contribute to its future therapeutic applications in various oxidative stress induced toxicity.

Methods Synthesis of Turmeric Initiated Biocompatible Gold Nanoparticle (Tu-AuNPs) Tu-AuNPs were prepared by (Nune et al., 2009) method with slight modifications. To a 25 ml vial was added 20 ml of doubly ionized water (DI), followed by the addition of 90 mg turmeric. The reaction mixture was stirred continuously. To the stirring mixture was added 300 l of 0.1 M NaAuCl4 solution (in DI water). The color of the mixture turned purple-red from pale yellow within 5 minutes of the addition, indicating the formation of gold nanoparticles. The reaction mixture was stirred for an additional 15 minutes. The gold nanoparticles thus formed were separated from residual turmeric immediately using a 5 micron filter and were characterized by UV-Vis absorption spectroscopy FTIR and SEM analysis.

Characterization UV-VIS Spectrophotometry Characterization of synthesized nanoparticles was carried out according to the methods described previously (Katti et al., 2009). The stability and the identity of the Tu-AuNPs were measured by recording UV absorbance after 30 min. The plasmon resonance band at ∼535 nm confirmed the retention of nanoparticulates in all the above mixtures.

Transmission Electron Microscopy (TEM) Samples for transmission electron microscopic (TEM) analysis were prepared by drop coating Tu-AuNPs solutions onto carbon coated copper TEM grids. The films on the TEM grids were allowed to stand for 2 min following which the extra solution was removed using a blotting paper and the grid is allowed to dry, prior to the measurement. TEM measurements were performed on a JEOL TEMSCAN2000EX instrument operated at an accelerating voltage at 80 keV.

Fourier Transform Infra Red Spectrophotometer (FTIR) The biocompatible colloidal gold conjugates phytochemical mediated process was analyzed using Fourier Transform Infra red Spectrophotometer. The advantage of FTIR over crystallographic techniques is its capability to provide information about the structural details of proteins in solution with greater spatial and temporal resolution. Minute quantities of gold nanoparticles were characterized through FTIR. The basic principle that governs is that the bonds and groups of bonds vibrate at characteristic frequencies. The gold nanoparticles that are exposed to infrared rays absorb infrared energy at frequencies which are characteristic to that molecule. FTIR analysis is carried out by illuminating the sample with a modulated IR beam. The gold J. Nanoneurosci. 2, 1–12, 2012

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nanoparticle transmittance and reflectance of the infrared rays at different frequencies is translated into an IR absorption plot, which is then analyzed and matched with known signatures of identified materials in the FTIR library.

In Vitro Stability Studies of Biocompatible Gold Nanoparticles In vitro stability studies of Tu-AuNPs were performed by mixing gold nanoparticles to aqueous solutions of 10% NaCl, 0.5% cysteine, 0.2 M histidine, 0.5% HSA and 0.5% BSA. The stability of the conjugates was measured by monitoring the UV absorbance over a period of 2 h, 24 h and 7 days. A negligible change in UV-vis plasmon band confirmed the retention of nanoparticulate composition in all mixtures.

Cell Cultures The cells were maintained in Dulbecco’s modified eagle’s medium (DMEM; Invitrogen) supplemented with 12.5% horse serum, 2.5% fetal bovine serum, 50 U/ml penicillin, and 5 mg/ml streptomycin—at an incubator setting of 5% CO2 and 37 C. All the experiments were carried out 24–48 h after cells were seeded. The cells were routinely harvested by trypsinization 0.25% when the cells approached sub confluent stage and were plated on 25-cm culture flasks split into 1:6.

PC 12 cells with a density of 1 × 106 cells/ml were grown in Dulbecco’s modified eagle’s medium (DMEM; Invitrogen) supplemented with 12.5% horse serum, 2.5% fetal bovine serum, 50 U/ml penicillin, and 5 mg/ml streptomycin—at an incubator setting of 5% CO2 and 37 C. After trypsinization and centrifugation, cell pellets were resuspended in Hank’s balanced salt solution (HBSS) for the exposure of 100 g/ml of Tu-AuNPs and further incubated for 24 hrs at 37 C. The medium was then aspired and the cell layer was rinsed 3 times with growth medium to remove any traces of uninternalized Tu-AuNPs. About 0.5 ml of 0.1 M Trypsin-EDTA solution was added to each well to detach the cell layer from the plastic. Detached cells were dispersed in 4 ml of complete growth medium and gently pipetted out of the well. The cell suspension was transferred into a centrifuge tube and centrifuged at approximately 125× g for 5 minutes. Supernatant was discarded and cell pellet was embedded in paraffin, sectioned and examined under Scanning electron microscopy.

Determination of Cellular Viability The cell viability was determined using a modified MTT assay as described previously (Yamamoto et al., 2000). In brief, PC-12 cells were seeded in collagen coated 96 well plates at a density of 1 × 106 cells/ml. The cultures were grown for 48 h, then the medium was changed to that containing various concentrations of Tu-AuNPs (25 g/ml, 50 g/ml and 100 g/ml) or 200 m MPP+ . After incubation for up to 48 h, MTT solution (5 mg/ml in J. Nanoneurosci. 2, 1–12, 2012

Determining the Integrity of cell Membranes: The Lactate Dehydrogenase (LDH) Leakage The lactate dehydrogenase (LDH) assay is used to evaluate cellmembrane integrity because the release of this large (9–160 KD) enzyme from the cytoplasmic compartment to the supernatant of cells is indicative of membrane damage. Based on the reduction of NAD by the action of LDH to form a tetrazolium dye, the amount of LDH was measured spectrophotometrically at 492 nm. The background absorbance measured at 660 nm was subtracted from the reading at 492 nm. After cells were exposed to 200 M MPP+ in the presence of the different concentrations Tu-AuNPs (25 g/ml, 50 g/ml and 100 g/ml) for 48 h, the medium was collected, and the amount of LDH release into the medium and determination of total LDH, respectively. LDH % =

Amedium 492 nm–660 nm × 100 Atotal 492 nm–660 nm

Determination of the Parameters Related to Oxidative Stress The production of reactive oxygen species accelerates as a result of cell membrane damage which in turn drives LPO and toxic aldehyde production. Therefore a comparison of the levels of LPO following MPP+ -exposure and post-treatment with Tu-AuNP’s provides clues for examining the state of cell deterioration in response to oxidative stress. The method described earlier was used (Buege and Aust, 1978) to estimate the level of lipid peroxide following MPP+ exposure and post treatment with Tu-AuNPs leading to ROS generation. A reaction mixture was prepared using 10 l ferrous sulfate (100 mM), 10 l ascorbic acid (150 mM), 100 l Tris. Buffer (150 mM, pH 7.1), 780 l distilled water and 100 l cell lysate so that the final volume is 1.0 ml. Then the reaction mixture is incubated at 37 C for 25 min. Thiobarbituric acid (0.375%, 2 ml) was then added to the mixture and allowed to react at 100 C (in water bath) for 15 min. The reaction mixture was then centrifuged (800× g for 10 min) and the absorbance values of the supernatant obtained were measured at 532 nm against the blank. GSH content was measured according to the method of (Moron et al., 1979) based on the reacting with 5, 5’-dithio-bis (2 nitro benzoic acid) (DTNB or Ellman’s reagent) to give a yellow colour compound that absorbs at 412 nm. 0.1 ml of the cell lysate was precipitated with 5% TCA. The precipitate was removed by centrifugation. To an aliquot of the supernatant was added 2.0 ml of DTNB in 0.2 M phosphate buffer to a final volume of 3 ml. The absorbance was read at 412 nm. A standard curve was drawn using different known concentrations of GSH solution. With the help of this standard curve, GSH contents were calculated.

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Scanning Electron Microscopy Analysis of Surface Characteristics of PC-12 Cells During Internalization of Tu-AuNP’s

DMEM) was added to 96 well plates and the cells were allowed to incubate for 4 h at 37 C. After the medium had been removed, the cell and the dye crystals were solubilised by adding 200 l of dimethylsulfoxide (DMSO), and the absorbance was measured at 570 nm (540 nm as reference) with a model 550-microplate reader (Biorad). The cell viability was expressed as a percentage of the corresponding control.


Determination of Antioxidant Enzyme Activity Catalase was analysed according to the method of (Takahara et al., 1960). To 1.2 ml of phosphate buffer 0.5 ml of cell lysate was added. The enzyme reaction was started by the addition of 1.0 ml of hydrogen peroxide solution. The decrease in the absorption was measured at 240 nm in intervals for 3 min. The enzyme blank was run simultaneously with 1.0 ml of distilled water instead of Hydrogen peroxide. One unit of CAT activity is that which reduces 1 mol of hydrogen peroxide per minute. Activity of SOD was assayed by the method of (Marklund, 1984). To 0.5 ml of cell lysate, 0.25 ml of absolute ethanol 0.15 ml of chloroform were added and kept for 0.5 min at 13,000 rpm. The supernatant was used as the enzyme source. The assay mixture contained 2 ml of Tris HCL buffer, 0.5 ml pyrogallol auto oxidation after the addition of enzyme was noted at an interval of 1 min to 3 min. One unit of the enzyme is defined as the amount of enzyme which inhibits the rate of pyrogallol auto oxidation by 50%. Glutathione-S-transferase was assayed by the method of (Habig et al., 1974). The reaction mixture containing 1.0 ml of buffer, 0.1 ml of CDNB and 0.1 ml of enzyme homogenate was made up to 2.5 ml with water. The reaction mixture was pre incubated at 37 C for 5 min. 0.1 ml of GSH was added and the change in OD was measured at 340 nm for 3 min at 30 second interval.

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Measurement of Complex I Activity Complex I activity was determined by monitoring the decrease in absorbance at 340 nm due to the oxidation of NADH (Helmerhorst et al., 2002; Schapira et al., 1990). The reaction mixture contained 250 mM sucrose, 1 mM EDTA, 50 mM Tris– HCl, pH 7.4, 2 g/ml antimycin A, 2 mM KCN, 0.15 mM coenzyme Q1, and 20–40 g mitochondrial homogenate. The total assay volume was 1 ml and the reagents were pre-warmed for 2 min at 30 C. The reaction was initiated by addition of 0.1 mM NADH and the rate of decrease in absorbance was monitored spectrophotometrically at 340 nm for 3 min. MPP+ (10 g/ml) was used to inhibit complex I activity. Absorbance was monitored for the indicated time period before and after addition of MPP+ .

DAPI Staining Assay The fluorescent dye DAPI was used to detect the nuclear fragmentation that is a characteristic of apoptotic cells. PC12 cells (5 × 103 cells/well in 12-well plates) were incubated at 37 C with 200 m MPP + for 48 h with or without pretreatment with Tu-AuNPs (50 g/ml or 100 g/ml) and then washed with PBS and fixed with 70% ethanol for 20 min. The fixed cells were washed with PBS and stained with the DNA-specific fluorochrome DAPI (1 g/ml). Following 10 min of incubation, the cells were washed with PBS, and the plates were observed under a fluorescence microscope (Olympus Optical, Japan).

Examination of MPP+ -Induced Apoptosis by Flow Cytometer PC-12 ells were incubated with 200 M MPP+ for 48 h, washed twice with PBS, adjusted to 100 L of the solution and transferred to a 1ml centrifuge tube (1 × 106 cells). Then, 5 L of

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Annexin V–FITC and 5 L of PI were added and cells were gently vortexed. Cells were then incubated for 15 min at room temperature (RT) (25 C) in the dark and 400 L of 1x binding buffer was added to each tube. Finally, cells were analyzed by flow cytometer. Cells were grown in 6-well plates for 24 h before treatment with Tu-AuNPs (100 g/ml) for 30 min before exposing them to MPP+ for 48 h. Then, 5 L of Annexin V–FITC and 5 L of PI were added and cells were incubated for 15 min at 25 C in the dark before being analyzed as described above.

Competing Interests No competing interests.

Authors’ Contributions Both the authors contribute equally to this work in co-ordinating and performing the experiments, writing the manuscript, design, interpretation and data analysis.

Acknowledgments: We would like to thank Dr. Shilpa, Assistant Professor, Sathyabama University, Chennai for guiding us to carry out the Cell line work. We also acknowledge Mr. Navukarasu, Anna university, Chennai for his immense support in analysing the samples under Scanning Electron Microscope, Mrs.Vijalakshmi, SRMC, Chennai for Flowcytometry and Mr. Baskaran, Department of Physical Chemistry, Anna University, Chennai for performing FTIR analysis for our samples. REFERENCES Alcaraz-Zubeldia M, Rojas P, Boll C, Rios C (2001) Neuroprotective effect of acute and chronic administration of copper (II) sulfate against MPP+ neurotoxicity in mice. Neurochem Res 26:59–64. Barath Mani Kanth S, Kalishwaralal K, Sriram M, Pandian SR, Youn H, Eom SH, Gurunathan S (2010) Anti-oxidant effect of gold nanoparticles restrains hyperglycemic conditions in diabetic mice. J Nanobiotechnol 8:16. Barlow BK, Lee DW, Cory-Slechta DA, Opanashuk LA (2005) Modulation of antioxidant defense systems by the environmental pesticide maneb in dopaminergic cell. J Neurotoxicity 26:63–75. Baschong W, Stierhof YD (1998) Preparation, use, and enlargement of ultrasmall gold particles in immunoelectron microscopy. Microsc Res Tech 42:66–79. Beuge JA, Aust SV (1978) Microsomal lipid peroxidation. J Methods Enzymol 52:302–310. Bhumkar DR, Joshi HM, Sastry M, Pokharkar VB (2007) Chitosan reduced gold nanoparticles as novel carriers for transmucosal delivery of insulin. J Pharm Res 24:1415–26. Bielinska A, Eichman JD, Lee I, Baker JR, Balogh L (2002) Imaging {Au-0-PAMAM} gold-dendrimer nanocomposites in cells. J Nanopart Res 4:395–403. Chang TI, Horal M, Jain S, Wang F, Patel R, Loeken MR (2003) Oxidant regulation of gene expression and neural tube development: Insights gained from diabetic pregnancy on molecular causes of neural tube defects. Dermatologia 46:538-534. Chen J, Tang XQ, Zhi JL, Cui Y, Yu HM, Tang EH, Sun SN, Feng JQ, Chen PX (2006) Curcumin protects PC12 cells against 1-methyl-4phenylpyridinium ion-induced apoptosis by bcl-2-mitochondria-ROSiNOS pathway. Apoptosis 11:943–53. Cho Y, Shi R, Borgens RB (2010) Chitosan nanoparticle-based neuronal membrane sealing and neuroprotection following acrolein-induced cell injury. J Biological Engineering 4:2.


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Received: xx Xxxx xxxx. Accepted: xx Xxxx xxxx.

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