Microsoft Word - siddique_proofreaded.docx                                                                                                                  Issue 2, Vol 6, 2015, 16-23 Effect of Withania somnifera leaf extract on the dietary supplementation in transgenic Drosophila model of Parkinson’s disease Yasir Hasan Siddique*1, Syed Faiz Mujtaba2, Mohammad Faisal3, Smita Jyoti1, Falaq Naz1 1Drosophila Transgenic Laboratory, Section of Genetics, Department of Zoology, Faculty of Life Sciences, Aligarh Muslim University, Aligarh, Uttar Pradesh, India. 2Photobiology Division, CSIR-Indian Institute of Toxicology Research, Lucknow, Uttar Pradesh, India. 3 Forest Entomology Division, Forest Research Institute, Dehradun, 248006, UK, India. Graphical Abstract Abstract: The role of Withania somnifera L. leaf extract was studied on the transgenic Drosophila model flies expressing normal human alpha synuclein (h-αS) in the neurons. The leaf extract was prepared in acetone and was subjected to GC-MS analysis. W. somnifera extract at final concentration of 0.25, 0.50 and 1.0 µL/mL was mixed with the diet and the flies were allowed to feed for 24 days. The effect of extract was studied on the climbing ability, lipid peroxidation and protein carbonyl content in the brains of transgenic Drosophila. The exposure of extract to PD model flies did not show any significant delay in the loss of climbing ability nor reduced the oxidative stress in the brains of transgenic Drosophila as compared to untreated PD model flies. The results suggest that W. somnifera leaf extract is not potent in reducing the PD symptoms in transgenic Drosophila model of Parkinson’s disease. Keywords: Withania somnifera; Lipid peroxidation; Protein carbonyl content; Drosophila; Climbing ability. 16 All Res. J.Biol, 2015, 6, 16-23     Introduction Parkinson’s disease (PD) is a chronic neurodegenerative disorder characterized by the progressive loss of the dopaminergic neurons of substantia nigra pars compacta in the ventral midbrain.1 One of the pathological features of PD is the presence of Lewy bodies.2 The function of alpha synuclein (αS) is not fully understood but it has been reported to bind with lipid membranes, forming an amphipathic helix.3 It has been suggested that there may be synaptic role for αS.4 Lewy bodies contain other proteins including neurofilaments and other cytoskeletal proteins, suggesting the presence of co-precipitants that might be important in aggregation.4 The aggregation of αS leads to the toxicity and oxidative stress,5 but it is still unclear whether misfolded proteins directly cause toxicity or damage cells via the formation of protein aggregates.6 The neurons being lost as the progression of PD have been reported to generate endogenous toxins (hydrogen peroxides) and free radicals that may further lead to the loss of neurons.7 In recent years, use of natural antioxidants from food and other biomaterials have been increased due to their presumed safety, nutritional and therapeutic values.8 Ayurveda has been in practice in India for more than 3500 years, and traditional healers have used this system since time immemorial for the benefit of mankind.8 The dietary habits are specific to population and vary widely hence it is necessary to study the disease- prevention potential of functional micronutrients in the regional diet.9 Withania somnifera or Ashwagandha is widely used in Ayurvedic medicine and the traditional medical system of India.10 W. somnifera (Indian Ginseng) is a subtropical under shrub that belongs to the family Solanaceae.11 Its root powder has been reported to posses free radical scavenging,12 improvement of motor neurons function,13 formation of dendrities,14 stimulating thyroid function,15 inhibition of tumor cell lines16 catecholamines and physiological abnormalities in PD model mouse,13,17 inhibits amyloid-β fibril,18 anti-diabetic,19 antigenotoxic,20 and other pharmacological properties. Most of the studies have been performed on the root extract. However, little is known about possible effects of leaf extract. Hence, an attempt has been made to study the effect of the leaf extract of W. somnifera on the PD model transgenic flies. Experimental Preparation of leaf extract: The leaves of W. somnifera were collected from the nursery of Forest Research Institute (FRI), Dehradun (Accession No: 143201).The extract was prepared according to the protocol described in an earlier published work.21 Analysis of W. somnifera extract through GC-MS: GC- MS analysis was performed using Trace GC ultra gas chromatograph connected to a Quantum XLS mass spectrometer (Thermo Scientific, FL, USA). GC was equipped with TG-5MS capillary column (30 m × 0.25 mm i.d. × 0.25 µm film thickness) consisting of a stationary phase 5% phenyl and 95% methyl polysiloxane. The injection was carried out in CT splitless mode at an injector temperature of 260°C Helium gas used as a carrier gas with a flow rate of 1.1 mL/min. The oven temperature programming was as follows: the initial oven temperature was held at 70°C for 2.0 min, and then increased to 210°C at a rate of 20°C/min and then increased to 290°C at a rate of 10°C/min held for 13.0 min. The ion source and transfer line temperature were 220°C and 290°C respectively. Identification of the compounds was performed by comparing their mass spectra with the NIST library available in the instrument. Drosophila stocks: Transgenic fly lines that expresses wild- type h-αS under UAS control in neurons ‘‘(w[*]; P{w[+mC]=UAS–Hsap/SNCA.F}’’5B and GAL4 ‘‘w[*]; P{w[+mC]=GAL4- elavL}’’3 were obtained from Bloomington Drosophila stock center (Indiana University, Bloomington, IN). When the males of UAS (upstream Activation Sequence)-Hsap/SNCA.F strains are crossed with the females of GAL4-elav. L (vice-versa) the progeny will express the human αS in the neurons.22 Drosophila culture and crosses: The flies were cultured on standard Drosophila food containing agar, corn meal, sugar and yeast at 25°C (24 ± 1).23 Crosses were set up as described in an earlier published work.23 First, the climbing assay was 17 All Res. J.Biol, 2015, 6, 16-23     performed for the PD flies and UAS-Hsap/SNCA.F (control). The other group of PD flies was exposed separately to different doses of W. somnifera extract mixed in the culture medium. W. somnifera extract was added in the medium at final concentration of 0.25, 0.50 and 1.0 µL/mL. The PD flies were also exposed to 10-3 M of L-dopamine. The UASHsap/ SNC.F acts as a control. The control flies were separately exposed to the selected doses of W. somnifera extract. Drosophila climbing assay: The climbing assay was performed as described by Pendleton et al.24 Ten flies were placed in an empty glass vial (10.5 cm × 2.5 cm). A horizontal line was drawn 8 cm above the bottom of the vial. After the flies had been acclimated for 10 min at room temperature, both controls and treated groups were assayed at random, to a total of 10 trails for each. The mean values were calculated and then averaged, and a group mean and standard error were obtained. All behavioral studies were performed at 25°C under standard lightning conditions. Lipid peroxidation assay: Lipid peroxidation assay in the brain homogenate was performed according to the procedure described by Siddique et al.25 Reagent 1 (R1) was prepared by dissolving 0.064 g of 1-methyl-2-phenylindole into 30 mL of acetonitrile to which 10 mL of methanol was added to bring the volume to 40 mL. The preparation of 37% HCl served as the reagent R2. The brain of flies was isolated under stereozoom microscope in ice cold Tris HCl (20 mM) (10 brain/group; five replicates/group). Homogenate was prepared in Tris HCl and centrifuged at 3000g for 20 min and subsequently the supernatant was collected. In a microcentrifuge tube 1300 µL of R1 was taken. A volume of 1µL of supernatant was added along with 300 µL of R2 vortexed and incubated at 45°C for 40 min. After incubation, the tubes were cooled in ice and centrifuged at 15,000g for 10 min at 4°C. All samples were read at 586 nm. Estimation of Protein Carbonyl content: The protein carbonyl content was estimated according to the protocol described by Hawkins et al.26 The brain homogenate was diluted to a protein concentration of approx. 1mg/mL. About 250µL of each diluted homogenate were taken in eppendorf centrifuge tubes separately. To it 250µL of 10mM 2, 4- dinitrophenyl hydrazine (dissolved in 2.5M HCl) was added, vortexed and kept in dark for 20 min. About 125µL of 50% (w/v) trichloroacetic acid (TCA) was added, thoroughly mixed and incubated at -20°C for 15min. The tubes were then centrifuged at 4°C for 10 min at 9000 rpm. The supernatant was discarded and the pellet obtained was washed twice by ice cold ethanol: ethylacetate (1:1). Finally, the pellets were re-dissolved in 1mL of 6M guanidine hydrochloride and the absorbance was read at 370nm. Statistical analyses: The statistical analyses were done using Statistica Soft Inc. The mean values of various fly groups were statistically compared using an unpaired group of the student ‘‘t’’-test. Results and Discussion The compounds present in acetone extract of leaves of W. somnifera were identified by GC-MS analysis (Figure 1). Figure 1. GC chromatogram of acetone extract of leaves of Withania somnifera. 18 All Res. J.Biol, 2015, 6, 16-23     The GC-MS chromatogram shows the presence of 2 major compounds with highest concentrations. Their retention times (RT), molecular formula and molecular weight (MW) in the leaves of W. somnifera are presented in Table 1. Table 1. The active principles in the leaf extract of Withania somnifera with their retention time (RT), molecular formula and molecular weight. The GC-MS analysis revealed that the acetone extract is mainly composed by dibenzyl ether and arachidonic acid or phytanic acid. Figures 2 A and B show the fragmentation pattern of mass spectrum and structures of the compounds extracted from the leaf extract and confirmed through NIST library of GC-MS. Those peaks matching similarity index (SI) greater than 70% in NIST library were assigned. Some of the major peaks are either column bleeding or impurities in plant extract. Mass spectra of two compounds extracted from plant leaf with similarity index (SI) greater that 70% confirmed by NIST library. The climbing response of control flies did not change over 24 days in a time course evaluation (Figure 3). From the day 12 on, however, the response of the PD flies were significantly lower as compared to the control (p<0.05). The climbing assay was performed after 24 days of the exposure to various doses of W. somnifera. The exposure of PD flies to 0.25, 0.50 and 1.0 µL/mL of W. somnifera did not show any significant delay in the loss of climbing ability as compared to untreated PD flies (Figure 4). The mean climbing ability of control flies was 9.6 ± 0.26 (Figure 4). The PD flies and the PD flies with L-Dopa were associated with the mean climbing ability of 3.2 ± 0.06 and 6.7 ± 0.17 respectively (Figure 4). The control flies treated with 0.25, 0.50, and 1.0µl/ml of W. somnifera leaf extract were associated with mean climbing ability of 9.4 ± 0.28, 9.2 ± 0.23 and 9.5 ± 0.32, respectively (Figure 4). No. RT Name of the compound Molecular formula MW 1 7.65 Benzene, 1,1’-oxybis C14H14O 198.26 2 10.22 Ethanol, 2-(9-octadecenyloxy)- C20H40O2 312.53   Figure 2. (i and ii) Mass spectra of acetone extract with its products having the high concentration compared with NIST library.   Figure 3. Climbing ability in Parkinson disease (PD) flies and control for a period of 24 days. The values are the mean of five assays (aSignificant with respect to control p < 0.05). 19 All Res. J.Biol, 2015, 6, 16-23     Figure 4. Effects of Withania somnifera on the climbing ability. The flies were allowed to feed on the diet supplemented with Withania somnifera for 24 days and then assayed for climbing ability. The values are the mean of five assays. (asignificant with respect to control p < 0.05; bsignificant with respect to PD Flies P < 0.05). (PD – Parkinson disease model flies). The mean absorbance values for the estimation of lipid peroxidation for the control, PD flies and PD flies with L- Dopa were, 0.10 ± 0.001, 0.98 ± 0.049 and 0.32 ± 0.029 respectively, (Figure 5). The treatments of 0.25, 0.50 and 1.0 µL/mL of W. somnifera extract to PD were associated with the mean absorbance values of 0.94 ± 0.063, 0.99 ± 0.073 and 0.94 ± 0.076 and, with control, the values were 0.11 ± 0.001, 0.12 ± 0.003 and 0.10 ± 0.004, respectively (Figure 5).  Figure 5. Effect of Withania somnifera on lipid peroxidation measured in the brain of transgenic Drosophila melanogaster after 24 days in various treated groups. [a) insignificant with respect to control p<0.05; b) significant with respect to PD model flies p<0.05]. No significant decrease in the mean absorbance values for lipid peroxidation was observed after the exposure to various doses of W. somnifera extract as compared to untreated PD flies (Figure 5). The mean absorbance values for the estimation of protein carbonyl content are shown in figure 6. The control flies, PD flies and PD flies + L-Dopa were associated with 0.114 ± 0.0017, 0.181 ± 0.0015 and 0.136 ± 0.0019 respectively (Figure 6). The PD flies treated with 0.25, 0.50, and 1.0 µl/ml of W. somnifera leaf extract were associated with the mean values of 0.180 ± 0.0019, 0.183 ± 0.0020 and 0.181 ± 0.002, respectively (Figure 6). The control flies treated with. 0.25, 0.50, and 1.0µl/ml of W. somnifera leaf extract were associated with the mean values of 0.112 ± 0.0011, 0.113± 0.0016 and 0.112 ± 0.001, respectively. No significant decrease in the mean absorbance value was observed as compared to PD flies, at the exposure to 0.25, 0.50 and 1.0 µL/mL of W. somnifera extract (Figure 6). Figure 6. Effect of Withania somnifera on Protein Carbonyl content measured in the brain of transgenic Drosophila melanogaster after 24 days in various treated groups.[asignificant with respect to control p<0.05; bsignificant with respect to PD model flies p<0.05]. The results of the present study reveals that the leaf extract of W. somnifera is not potent in reducing the PD symptoms in the transgenic flies expressing human α-synuclein. The accumulation of Lewy bodies together with the loss of dopaminergic neurons and the loss of climbing ability has been reported in the transgenic flies.22 20 All Res. J.Biol, 2015, 6, 16-23     In our present study the PD flies showed a progressive loss in the climbing ability as the age of the flies progresses. At 24th day the climbing ability of the flies were at the lowest, hence the duration of the treatments was selected for 24 days. The dietary supplementation of W. somnifera leaf extract did not show any significant delay in the loss of climbing ability, indicating that the extract is not potent in delaying the PD symptoms. The accumulation of the Lewy bodies leads to the toxicity and oxidative stress6. The damaging neurons as result of PD have been reported to generate hydrogen peroxide and free radicals7. Various studies on the experimental models suggest that the oxidative stress plays an important role in neurodegenerative diseases. Hence, the lipid peroxidation (LPO) and protein carbonyl content (PC) was measured in the Drosophila brains as a marker of oxidative stress. LPO represents a reliable marker of free radical generation and indicates the membrane damage4. Oxidative stress leads to the damage of lipid, protein and DNA27. The present method used for the estimation of lipid peroxidation is based on the reaction of malondialdehyde (MDA) with 1-methyl-2- phenylindole at 45°C. Two molecules of 1-methyl-2- phenylindole react with one molecule of MDA to form a stable chromophore, having a maximal absorbance at 586nm28. The results obtained for lipid peroxidation showed no reduction in the PD model flies treated with W. somnifera extract. PC content indicates the protein oxidation by the free radicals or ROS. The oxidation of protein results in the generation of carbonyl (CO) groups on protein side chains and is widely used as a marker of oxidative stress. The treatment of W. somnifera did not show any reduction in the oxidative stress markers used in the present study, hence suggesting that W. somnifera leaf extract has no effect on the oxidative stress. The results of our present study support the study carried out by Kaur et al29. The leaf extract was reported to be anti- proliferative but not anti- oxidative. The therapeutic intervention that could effectively decelerate the rate of degeneration within the substantia nigra pars compacta could add years of mobility and reduce morbidity associated with PD7. Hence the attention has been directed towards the identification of the inhibitors of αS aggregations or free radicals scavengers30. In this regard the leaf extract of W. somnifera did not show any protective effect against the PD symptoms in the transgenic model flies expressing h- αS. In mouse model of Parkinson’s disease the leaf extract has shown to increase the levels of superoxide dismutase, catalase and malondialdehyde, reduced levels of glutathione and glutathione peroxidase in the mid brain and corpus striatum thus suggesting it as an antioxidative and protective against PD symptoms13. There are various neurotoxins that have provided valuable information about the pathophysiology of PD but paraquat and rotenone failed to produce any obvious signs of dopaminergic damage31. The process of damaging the dopaminergic neurons by the various neurotoxins and α-Synuclein aggregation is entirely different31. However, the roots of W. somnifera contain steroid lactones (Withanolides), phytosterols and alkaloids (ashwagandhine, ashwaghandhinine, withasomine, visamine, somniferine, somniferinine)8, 32, 33. Our GC-MS analysis reveals the absence of these compounds in the leaf extract of W. somnifera. This may be the possible reason of not showing any protective effects against the PD symptoms in the flies. The above compounds have been shown to have neuroprotective and neuro regenerative potential32. Our earlier study with Eucalyptus citriodora leaf extract showed a protective effect in the PD model flies expressing h- αS34. The natural antioxidants such as apigenin,35 curcumin,28 nordihydroguaiaretic acid,23 ascorbic acid,34 and grape extract,36 are potent in delaying the loss of climbing ability and reduction in the oxidative stress in the brains of PD model flies. The dietary supplementation of the flavonoids showed improvement in cognitive function possibly by protecting vulnerable neurons, enhancing existing neuronal function or by stimulating neuronal regeneration.30 The complex brain of Drosophila is capable of learning and memory. Almost all the major classes of ion channels, receptors and neurotransmitters similar to humans composed of specialized cell types are found in Drosophila brain22. There are reports for the presence of novel and known withanolides from W. somnifera leaf extracts16, 37. Various pharmacological studies have attributed linkage of therapeutic actions to one or the other type of withanolides. A significant qualitative as well as quantitative difference 21 All Res. J.Biol, 2015, 6, 16-23     between the leaf and root tissue, particularly with respect to secondary metabolites, has been reported in the GC-MS analysis37. However, in our GC-MS analysis, no peaks corresponding to the withanolides were observed according to the NIST library. C12H10O is arachidic acid, a saturated fatty acid38 and C20H40O2 is phytanic acid, a branched fatty acid, and its metabolites have been reported to bind or activate transcription factors39. The antioxidant and free radical scavenging potential have been reported due to the presence of the withanolides/withanone. But there are reports of having no antioxidant potential in the leaf extract of W. somnifera29. No one scientific approach is likely to single handedly solve all the mysteries of neurodegenerative disease and hence multidisciplinary research approaches utilizing many model systems will be required for elucidating the neuropathogenesis with the disease40. The complex brain of Drosophila consists of all major classes of ion channels, receptors, and neurotransmitters found in humans40. The neural complexity and proteomic analysis have revealed that more than 70% of the disease related loci in humans have a clear ortholog in Drosophila41. Hence, the Drosophila is the most ideal organism for studying the neurodegenerative diseases. The variation in the compounds in the GC-MS gram may be due to the type of solvent used in the preparation of extract or part from where the leaves were collected. Age of the leaves may also have some effects. In our present study with leaf extract of W. somnifera, the extract was not found to be potent in reducing the PD symptoms, in the transgenic flies expressing h- αS. Acknowledgments We are thankful to the Chairman of the Department of Zoology for providing the laboratory facilities. The flies for the experiments were purchased from the Bloomington Drosophila Stock Centre, Department of Biology, Indiana University, Bloomington, IN, USA. References     1. Kartika, B., Muralidharan, P., and Rahman, H. (2010). Herbal treatment of Parkinsonism: A Review. Int. J. Pharma. Sci. Rev. Res., 5, 185-191. 2. Samii, A., Nutt, J. G., and Ransom, B. R. (2004). Parkinson’s disease. Lancet, 363, 1783-1793. 3. Clayton, D. F., and George, J. M. (1998). The synucleins: A family of proteins involved in synaptic function, plasticity, neurodegeneration and disease. Trends in neurosci., 21, 249- 254. 4. Cookson, M. P. (2005). The biochemistry of Parkinson’s disease. Ann. Rev. Biochem., 74, 29-52. 5. Conway, K. A., Harper, J. D., and Lansbury, P. T. (1998). Accelerated in vitro fibril formation by a mutant α-synuclein linked to early-onset Parkinson disease. Nat. Med., 4, 1318- 1320. 6. Dauer, K., and Przedborski S. (2003). Parkinson disease: mechanisms and models. Neuron, 39, 889-909. 7. Fahn, S. (1989). The endogenous toxin hypothesis of the etiology of Parkinson’s disease and apilot trial of high dosage antioxidants in an attempt to slow the progression of the illness. Annals of the NY Acad. Sci., 570, 186-196. 8. Jayaraj, R. L., Ravindar, D. J., Manigandan, K., Padarthi, P. K., and Namasivayam E. (2012). An overview of Parkinson’s disease and oxidative stress: Herbal Scenario. Neuropatho. Dis., 1, 95-122. 9. Devasagayam, T. P. A., Tilak, J. C., Boloor, K. K., Sane, K., Ghaskadbi, S., and Lele, R. (2004). Free radicals and antioxidants in human health: Current status and future prospects. J. Ass. Phy. India., 52,794-804. 10. Mishra, L. C., Singh, B. B., and Dagenais, S. (2000). Scientific basis for the therapeutic use of Withania somnifera (Ashwagandha): A Review. Alt. Med. Rev., 5, 334-336. 11. Sankar, S. R., Manivagsagam, T., Krishnamurti, A., and Ramanathan M. (2007). The neuroprotective effect of Withania somnifera root extract in MPTP- Intoxicated mice: an analysis of behavioural and biochemical variables. Cell Mol. Bio. Lett., 12, 473-481. 12. Panda, S., and Kar, A. (1997). Evidence for free radical scavenging activity of ashwagandha root powder in mice. Indian J. Physio. Pharmaco., 41, 424-426. 13. RajaSankar, S., Manivasagam, T., Sankar, V., Prakash, S., Muthusamy, R., Krishnamurti, A., and Surendran, S. (2009). Withania somnifera root extract improves catecholamines and physiological abnormalities seen in a Parkinson’s disease model mouse. J. Ethnopharmacol., 125, 369-373. 14. Chihiro, T., Tomoharu, K., and Komatsu, K. (2000). Dendrite extension by methanol extract of Ashwagandha (roots of Withania somnifera) in SK-NSH cells. Neuroreport., 11, 1981-1985. 15. Panda, S., and Kar A. (1999). Withania somnifera and Bauhinia purpurea in the regulation of circulating thyroid 22 All Res. J.Biol, 2015, 6, 16-23     hormone concentrations in female mice. J. Ethnopharmacol., 67, 233-239. 16. Jayaprakasan, B., Zhang, Y., Seeram, N. P., and Nair, M. G. (2003). Growth inhibition of human tumor cell lines by winthanolides from Withania somnifera leaves. Lif. Sci., 74, 125-132. 17. Ahmad, M., Saleem, S., Ahmad, A. S., Ansari, M. A., Yousuf, S., Hoda, M. N., and Islam, F. (2005). Neuroprotective effects of Withania somnifera on 6- hydroxydopamine induced Parkinsonism in rats. Human Exp. Toxicol., 24, 137-147. 18. Kumar, S., Harris, R. J., Seal, C. J., and Okello, E. J. (2012). An Aqueous Extract of Withania somnifera Root Inhibits Amyloid β Fibril Formation in vitro. Phytothera. Res., 26, 113-117. 19. Udayakumar, R., Kasthurirengan, S., Mariashibu, T. S., Rajesh, M., Anbazhagan, V. R., Kim, S. C., Ganapathi, A., and Choi, C. W. (2009). Hypoglycaemic and hypolipdaemic effects of Withania somnifera root and leaf extracts on alloxan-induced diabetic rats. Intern. J. Mol. Sci., 10, 2367- 2382. 20. Rani, G., Kaur, K., Wadhwa, R., Kaul, S. C., and Nagpal, A. C. (2005). Evaluation of antigenotoxicity of leaf extract of Ashwagandha. Food Chem. Toxicol., 43, 95-98. 21. Siddique, Y. H., Ara, G., Beg, T., Faisal, M., Ahmad, M., and Afzal, M. (2008). Antigenotoxic role of Centella asiatica L. extract against cyproterone acetate induced genotoxic damage in cultured human lymphocytes. Toxicol. in vitro., 22, 10-17. 22. Feany, M. B., and Bender, W. W. (2000). A Drosophila model of Parkinson’s disease. Nature., 404, 394-398. 23. Siddique, Y. H., Ara, G., Jyoti, S., and Afzal, M. (2012). The dietary supplementation of nordihydroguaiaretic acid (NDGA) delayed the loss of climbing ability in Drosophila model of Parkinson’s disease. J. Diet. Suppl., 9, 1-8. 24. Pendleton, R. G., Parvez, F., Sayed, M., and Hillman, R. (2002). Effects of pharmacological agents upon a transgenic model of Parkinson’s disease in Drosophila melanogaster. Pharmacol. Exp. Therap., 300, 91-96. 25. Siddique, Y. H., Ara, G., and Afzal, M. (2012). Estimation of lipid peroxidation induced by hydrogen peroxide in cultured human lymphocytes. Dose Resp., 10, 1- 10. 26. Hawkins, C. L., Morgan, P. E., Davies, M. J. (2009)Quantification of protein modification by oxidants. Free Rad. Bio. Med., 46, 965-988. 27. Ryter, S. W., Kim, H. P., Hoetzel, A., Park, J. W., Nakahira, K., Wang, X., and Choi, A. M. K. (2007). Mechanisms of cell death in oxidative stress. Antioxi. redox signal., 9, 49-89. 28. Siddique, Y. H., Ara, G., Jyoti, S., and Afzal, M. (2012). Protective effect of curcumin in transgenic Drosophila melanogaster model of Parkinson’s disease. Alter. Med. Stud., 2, e3. 29. Kaur, K., Rani, G., Widodo, N., Nagpal, A., Taira, K., Kaul, S. C., and Wadhwa, R. (2004). Evaluation of the anti- proliferative and anti-oxidative activities of leaf extract from in vivo and in vitro raised Ashwagandha. Food Chem. Toxicol., 42, 2015-2020. 30. Amer, D. A. M., Irvine, G.B., and El-Agnaf, O. M. A. (2006). Inhibitors of α-synuclein oligomerization and toxicity: a future therapeutic strategy for Parkinson’s disease and related disorders. Exp. Brain Res., 173, 223-233. 31. Rojo, A.I., Cavada, C., de Sagarra, M. R., and Cuadrado, A. (2007). Chronic inhalation of rotenone or paraquat does not induce Parkinson's disease symptoms in mice or rats. Exp. Neurol., 208, 120-126. 32. Tohda, C., Kuboyama, T., and Komatsu K. (2005). Search for natural products related to regeneration of the neuronal network. Neurosignals., 14, 34-45. 33. Tohda, C., Kuboyama, T., and Komatsu, K. (2000). Dendrite extension by methanol extract of Ashwagandha (roots of Withania somnifera) in SK-N-SH cells. Neuroreport., 11, 1981-1985. 34. Siddique, Y. H., Mujtaba, S. F., Jyoti, S., Naz, F. (2013). GC–MS analysis of Eucalyptus citriodora leaf extract and its role on the dietary supplementation in transgenic Drosophila model of Parkinson’s disease. Food Chem. Toxicol., 55, 29- 35. 35. Siddique, Y. H., Jyoti, S., Naz, F., and Afzal, M. (2011). Protective effect of apigenin in transgenic Drosophila melanogaster model of Parkinson’s disease. Pharmacologyonline., 3, 790-795. 36. Long, J., Gao, H., Sun, L., Liu, J., and Zhao-Wilson, Xi. (2009). Grape extract protects mitochondria from oxidative damage and improves locomotor dysfunction and extends lifespan in a Drosophila Parkinson's disease model. Rejuven. Res., 12, 321-331. 37. Chatterjee, S., Srivastava, S., Khalid, A., Singh, N., Sangwan, R. S., Sidhu, O. P., Roy, R., Khetrapal, C. L., and Tuli, R. (2010). Comprehensive metabolic fingerprinting of Withania somnifera leaf and root extracts. Phytochemistry., 71, 1085-1094. 38. Beare-Rogers, J. L., Dieffenbacher, A., and Holm, J. V. (2001). Lexicon of lipid nutrition (IUPAC Technical Report). Pure App. Chem., 73, 685-744. 39. Gloerich, J., van Vlies, N., Jansen, G. A., Denis, S., Ruiter, J. P. N., van Werkhoven, M. A., Duran, M., Vaz, F. M., Wanders, R. J. A., and Ferdinandusse, S. (2005). A phytol-enriched diet induces changes in fatty acid metabolism in mice both via PPARα-dependent and - independent pathways. J. Lipid Res., 46, 716-726. 40. Palladino, M. J., and Celotto, A. M. (2005). Drosophila: A “model” model system to study neurodegeneration. Mol. Interv., 5, 292-303. 41. Reiter, L. T., Potocki, L., Chien, S., Gribskov, M., and Bier, E. (2001). A systematic analysis of human disease- associated gene sequences in Drosophila melanogaster. Genome Res., 11, 1114-1125.   23