Phyllantus niruri Protects against Fe2+ and SNP Induced Oxidative Damage in Mitochondrial Enriched Fractions of Rats Brain

Authors: Olusola Olalekan Elekofehinti, Isaac Gbadura Adanlawo, Joao Batista Teixeira Rocha

Abstract:

The potential neuroprotective effect of Phyllantus nuriri against Fe2+ and sodium nitroprusside (SNP) induced oxidative stress in mitochondria of rats brain was evaluated. Cellular viability was assessed by MTT reduction, reactive oxygen species (ROS) generation was measured using the probe 2,7-dichlorofluoresce indiacetate (DCFH-DA). Glutathione content was measured using dithionitrobenzoic acid (DTNB). Fe2+ (10μM) and SNP (5μM) significantly decreased mitochondrial activity, assessed by MTT reduction assay, in a dose-dependent manner, this occurred in parallel with increased glutathione oxidation, ROS production and lipid peroxidation end-products (thiobarbituric acid reactive substances, TBARS). The co-incubation with methanolic extract of Phyllantus nuriri (10-200 μg/ml) reduced the disruption of mitochondrial activity, gluthathione oxidation, ROS production as well as the increase in TBARS levels caused by both Fe2+ and SNP in a dose dependent manner. HPLC analysis of the extract revealed the presence of gallic acid (20.540.01), caffeic acid (7.930.02), rutin (25.310.05), quercetin (31.280.03) and kaemferol (14.360.01). This result suggests that these phytochemicals account for the protective actions of P. niruri against Fe2+ and SNP -induced oxidative stress. Our results show that P. nuriri consist important bioactive molecules in the search for an improved therapy against the deleterious effects of Fe2+, an intrinsic producer of reactive oxygen species (ROS), that leads to neuronal oxidative stress and neurodegeneration.

Keywords: Phyllantus niruri, mitochondria, antioxidant, oxidative stress, synaptosome.

Digital Object Identifier (DOI): doi.org/10.5281/zenodo.1105629

References:

[1] B. Halliwell, and J.M.C. Gutteridge. Free radicals in biology and medicine, 4th ed. Oxford University Press, New York, 2007.
[2] A. Melo, L. Monteiro, R.M.F. Lima, D.M. deOliveira, M.D. de Carqueira and R.S. El- Balsha. Oxidative stress in neurodegenerative diseases: Oxidative medicine and Cellular Longevity. 2011. Doi:10:1155/2011/467180, 2011.
[3] R.R. Crichton, and R.J. Ward. Iron metabolism –New perspectives in view. Biochem vol. 31, pp. 11255-11264, 1992.
[4] J. Beard. Iron deficiency alters brain development and functioning. J Nutr vol. 133, pp. 1468S-1472S, 2003.
[5] R. Deregnier, C. Nelson, K. Thomas, S. Wewerka, and M. Georgieff. Neurophysiologic evaluation of auditory recognition memory in healthy newborn infants and infantsofdiabetic mothers. J Pediatr vol. 137, pp. 777 –784, 2000.
[6] C. Nelson, K. Erikson, D. Pinero, and J.L. Beard. Alterations in dopamine metabolism in iron deficient rats. J Nutr vol. 12, pp. 2282– 2288, 1997.
[7] J.P. Kamdem, O.O. Elekofehinti, H. Waseem, I.J. Kade, A.A. Boligon, M.L. Athayde, D.O. Souza, and J.B.T. Rocha. Trichiliacatigua(Catuaba) bark extract exerts Neuroprotection against oxidative stress induced by different neurotoxic agents in rat hippocampal slices. Ind. Crops Prod. vol. 50, pp. 625-632, 2013.
[8] J.K. Andersen. Iron dysregulation and Parkinson’s disease. J Alzheimer‘s Dis, vol. 6, pp. S47-52, 2004.
[9] O.O. Elekofehinti, and I.J.Kade. Aqueous extract of African Egg Plant inhibitFe2+ and SNP induced lipid peroxidation in Ratís brain- In vitro. Der Pharma. Lett. Vol. 4, pp. 1352-1359, 2012.
[10] V.V. Paithankar, K.S. Raut, R.M. Charde, and J.V. Vyas. PhyllanthusNiruri: A magic Herb. Research in Pharmacy vol. 1, pp. 1-9, 2011.
[11] A.A. Adeneye. The leaf and seed aqueous extract of Phyllanthusamarus improves insulin resistance diabetes in experimental animal studies. J Ethnopharmacol vol. 144, pp. 705–711, 2012.
[12] U. Latha, and M.G. Rajesh.Hepatoprotective effect of an Ayurvedic medicine, IndianDrugs. Vol. 36, pp. 470-473, 1999.
[13] G. Bagalkotkar, S.R. Sagineedu, and M.S. Saad. Phytochemicals from Phyllanthusniruri Linn. and their pharmacological properties, J Pharm Pharmacol. vol. 58, pp. 1559- 70, 2006.
[14] D.F. Meinerz, M.T. de Paula, B. Comparsi, M.U. Silva, A.E. Schmitz, H.C. Braga, P.S. Taube, A.L. Braga, J.B.T. Rocha, A.L. Dafre, M. Farina, J.L. Franco, and T. Posser. Protective effects of organoselenium compounds against methylmercury-induced oxidative stress in mouse brain mitochondrial-enriched fractions. Braz. J. Med. Biol. Res. vol. 44, pp. 1156-1163, 2011.
[15] A. Dreiem, C.C. Gertz, and R.F. Seegal. The effects of methylmercury on mitochondrial function and reactive oxygen species formation in rat striatal synaptosomes are age-dependent. ToxicolScivol. 87, pp. 156-162, 2005.
[16] G.L. Ellman. Tissue sulfhydryl groups. Arch BiochemBiophysvol. 82, 70-77, 1959.
[17] H. Ohkawa, N. Ohishi, and K. Yagi. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem, vol. 95, pp. 351- 35, 1979..
[18] M.M. Bradford. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem, vol. 72: pp. 248-254, 1976.
[19] MT. Lin and M. F. Beal. Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases. Nature .vol 443 2006|doi:10.1038/nature05292. Pp. 787-795.
[20] N.N. Danial, and S.J.Korsmeyer. Cell death: critical control points. Cell vol. 116, pp.205–219, 2004.
[21] R.S. Sohal, and B.H. Sohal. Hydrogen peroxide release by mitochondria increases during aging. Mech Ageing Dev, vol. 57, pp.187–202, 1991.
[22] R.S. Sohal, H.H. Ku, S. Agarwal, M.J. Forster, and H. Lal. Oxidative damage, mitochondrial oxidant generation, and antioxidant defenses during aging and in response to food restriction in the mouse. Mech Ageing Dev vol. 74, pp. 121–133, 1994.
[23] S.J. Lee, Y. Jin, H.Y. Yoon, B.O. Choi, H.C. Kim, Y.K. Oh, H.S. Kim, and W.K. Kim. Ciclopirox protects mitochondria from hydrogen peroxide toxicity. British J. Pharmacol vol. 145, pp. 469–476, 2005.
[24] Q.A. Nazari, K. Mizuno, T. Kume, Y.T. Takatori, Y. Izumi, and A. Akaike. In Vivo brain oxidative stress model induced by microinjection of sodium nitroprusside in mice. J PharmacolSci, vol. 120, pp. 105 – 111, 2012.
[25] Y. Zhang, and B. Zhao. Green tea polyphenols enhance sodium nitroprusside-induced neurotoxicity in human neuroblastoma SH-SY5Y cells. J. Neurochem, vol. 86, pp. 1189–1200, 2003.
[26] J. Wang, P.S. Green, and J.W. Simpkins. Estradiol protects against ATP depletion, mitochondrial membrane potential decline and the generation of reactive oxy-gen species induced by 3-nitropropionic acid in SK-NSH human neuroblastomacells. J. Neurochem vol. 77, pp. 804–811, 2001.
[27] T.P.A. Devasagayam, J.C. Tilak, K.K. Boloor, K.S. Sane, S.S. Ghaskadbi, and R.D. Lele. “Free radicals and antioxidants in human health: current status and future prospects,” J. Ass. Physi. India. vol. 52, pp. 794–804, 2004.
[28] M. Valko, D. Leibfritz, J. Moncol, M.T. Cronin, M. Mazur, and J. Telser. Free radi-cals and antioxidants in normal physiological functions and human disease. Int J. Biochem. Cell Biol. vol. 39, pp. 44–84, 2007.
[29] O.O. Elekofehinti, J.P. Kamdem, A.A. Boligon, M.L. Athayde, S.R. Lopes, E.P. Waczuk, I.J. Kade, I.G. Adanlawo, and J.B.T. Rocha. African eggplant (Solanumanguivi Lam.) fruit with bioactive polyphenolic compounds exert in vitro antioxidant properties and inhibit cCa2+ induced mitochondrial swelling. Asian Pac. J. Trop. Biomed. vol. 3, pp. 757-766, 2013
[30] D.A. Wink, J.A. Cook, R. Pacelli, W. DeGraff, J. Gamson, J, Liebmann, et al. The effect of various nitric oxide-donor agents on hydrogen peroxide-mediated toxicity: a direct correlation between ni- tric oxide formation and protection. Arch. Biochem. Biophys. vol. 331, pp. 241– 248, 1996.
[31] S. Cardaci, G. Filomeni, G. Rotilio, and M.R. Ciriolo. Reactive oxygen species mediate p53 activation and apoptosis induced by sodium nitroprusside in SH-SY5Ycells. Molecular Pharmacol, vol. 74, pp. 1234–1245, 2008.
[32] K. Komolafe, T. M. Olaleye, O. I. Omotuyi, A.A. Boligon, M. L. Athayde, A. A. Akindahunsi, and J.B. T.Rocha. In Vitro Antioxidant Activity and Effect of ParkiabiglobosaBark Extract on Mitochondrial Redox Status. J Acupunct Meridian Stud 2013.
[33] J. Hirrlinger and R.Dringen. The cytosolic redox state of astrocytes: maintenance, regulation and functional implications for metabolite trafficking. Brain Res Rev vol. 63, pp. 1–2, 2010.
[34] K. Aoyama, K. Aoyama, M. Watabe, and T. Nakaki. Regulation of neuronal glutathione synthesis. J Pharmacol Sci. vol. 108, 227–238, 2008.
[35] R. Dringen, P.G.Pawlowski, and J. Hirrlinger. Peroxide detoxification by brain cells. J Neurosci Res vol. 79, pp. 157–165, 2005.
[36] A. Jain, J. Martensson, E. Stole, P.A. M. Auld, and A. Meister. Glutathione deficiency leads to mitochondrial damage in brain. Proc. Nati. Acad. Sci. USA vol. 88, pp. 1913-1917, 1991.
[37] L.A. Sena and N.S. Chandel. 2012. Physiological roles of mitochondrial reactive oxygen species. Molecular Cell. vol. 48, pp. 158-167, 2012.
[38] M.T. Nunez, P. Urrutia, N. Mena, P. Aguirre, V. Tapia, and J. Salazar. Iron toxicity in neurodegeneration. Biometals vol. 25, pp. 761-776, 2012.
[39] C.M. Park, J.Y. Park, K.H. Noh, J.H. Shin, and Y.S. Song. TaraxacumofficinaleWeberextract inhibit LPS-induced oxidative stress and nitric oxide production via kBmodulation in RAW 264.7 cells. J.Ethnopharmacol, vol. 133, 834–842, 2011.
[40] K.S. Kumaran, and P.S. Prince. Caffeic acid protects rat heart mitochondria against isoproterenol-induced oxidative damage. Cell stress chaperones. vol. 15, pp. 791-806, 2010.
[41] A. Im, Y. Kim, M.R. Uddin, H.W. Lee, S.W. Chae, Y.H. Kim,et al. Scutellariabaicalensis extracts and flavonoids protect rat L6 cells from antimycin A-induced mitochondrial dysfunction. Evid Based Complement Alternat Med. 2012, 517965, 2012. Epub Aug 30.