Neurotoxicity Research

, Volume 31, Issue 4, pp 521–531 | Cite as

Metabolic Alterations and the Protective Effect of Punicalagin Against Glutamate-Induced Oxidative Toxicity in HT22 Cells

  • Kavitha Pathakoti
  • Lavanya Goodla
  • Manjunath Manubolu
  • Tewin TencomnaoEmail author


Oxidative stress is involved in many neurological diseases, including Alzheimer’s disease. Punicalagin (PC) is a hydrolysable polyphenol derived from Punica granatum and a potent antioxidant. In this study, the neuroprotective effect of PC on glutamate-induced oxidative stress was evaluated in the mouse hippocampal cell line, HT22. PC treatment protected HT22 cells from glutamate-induced cell death in a concentration-dependent manner, potentially attenuated glutamate-induced intracellular reactive oxygen species (ROS) and restored the mitochondrial membrane depolarization. Metabolic alterations after glutamate-induced oxidative stress and the protective effect of PC were evaluated with HPLC and GC-MS profiling methods with multivariate statistical analyses. Alterations in ten metabolites were identified, including amino acids, aspartic acid, asparagine, threonine, anserine, cysteine, tryptophan, lysine, as well as fatty acids palmitic acid, stearic acid, and palmitoleic acid. Metabolic pathway analysis revealed the involvement of multiple affected pathways, such as cysteine and methionine metabolism, tryptophan metabolism, alanine, aspartate, and glutamate and fatty acid oxidation. These results clearly demonstrate that PC is a promising therapeutic agent for oxidative stress-associated diseases.


Glutamate Oxidative stress HT22 cells Punicalagin Metabolite profiling Multivariate analyses 



This study was financially supported by Chulalongkorn University (RES560530255-AS). We are also thankful to the SAIF/CRNTS Department, IIT, Bombay (India) for providing the analytical facility. The author KP is thankful to Chulalongkorn University (Rachadapisek Sompot Fund) for providing a senior postdoctoral fellowship.

Supplementary material

12640_2016_9697_Fig7_ESM.gif (78 kb)
Figure S1

HPLC chromatograms for amino acids (A) Standard (B) Control (C) Glutamate group (D) PC-GLU group (GIF 78 kb)

12640_2016_9697_MOESM1_ESM.tif (1.1 mb)
High resolution (TIFF 1152 kb)
12640_2016_9697_Fig8_ESM.gif (92 kb)
Figure S2

GC-MS chromatograms for Methanol fraction (A) Control (B) Glutamate group (C) PC-GLU group (GIF 92 kb)

12640_2016_9697_MOESM2_ESM.tif (588 kb)
High resolution (TIFF 587 kb)
12640_2016_9697_Fig9_ESM.gif (81 kb)
Figure S3

GC-MS chromatograms for chloroform fraction (A) Control (B) Glutamate group (C) PC-GLU group (GIF 80 kb)

12640_2016_9697_MOESM3_ESM.tif (546 kb)
High resolution (TIFF 546 kb)


  1. Berlett BS, Stadtman ER (1997) Protein oxidation in aging, disease, and oxidative stress. J Biol Chem 272:20313–20316CrossRefPubMedGoogle Scholar
  2. Bonda DJ, Wang X, Lee H-G, Smith MA, Perry G, Zhu X (2014) Neuronal failure in Alzheimer’s disease: a view through the oxidative stress looking-glass. Neurosci Bull 30:243–252CrossRefPubMedPubMedCentralGoogle Scholar
  3. Brimson JM, Brimson SJ, Brimson CA, Rakkhitawatthana V, Tencomnao T (2012) Rhinacanthus nasutus extracts prevent glutamate and amyloid-beta neurotoxicity in HT-22 mouse hippocampal cells: possible active compounds include lupeol, stigmasterol and beta-sitosterol. Int J Mol Sci 13:5074–5097CrossRefPubMedPubMedCentralGoogle Scholar
  4. Brosnan JT, Brosnan ME (2006) The sulfur-containing amino acids: an overview. J Nutr 136:1636S–1640SPubMedGoogle Scholar
  5. Campesan S, Green EW, Breda C, Satyasaikumar KV, Muchowski PJ, Schwarcz R, Kyriacou CP, Giorgini F (2011) The kynurenine pathway modulates neurodegeneration in a drosophila model of Huntington’s disease. Curr Biol 21:961–966CrossRefPubMedPubMedCentralGoogle Scholar
  6. Chen C, Pearson A, Gray JI (1992) Effects of synthetic antioxidants (BHA, BHT and PG) on the mutagenicity of IQ-like compounds. Food Chem 43:177–183CrossRefGoogle Scholar
  7. Chen PS, Li JH, Liu TY, Lin TC (2000) Folk medicine Terminalia catappa and its major tannin component, punicalagin, are effective against bleomycin-induced genotoxicity in Chinese hamster ovary cells. Cancer Lett 152:115–122CrossRefPubMedGoogle Scholar
  8. Choi DW (1988) Glutamate neurotoxicity and diseases of the nervous system. Neuron 1:623–634CrossRefPubMedGoogle Scholar
  9. Conklin SM, Runyan CA, Leonard S, Reddy RD, Muldoon MF, Yao JK (2010) Age-related changes of n-3 and n-6 polyunsaturated fatty acids in the anterior cingulate cortex of individuals with major depressive disorder prostaglandins. Leukot Essent Fatty Acids 82:111–119CrossRefGoogle Scholar
  10. Craig A, Cloarec O, Holmes E, Nicholson JK, Lindon JC (2006) Scaling and normalization effects in NMR spectroscopic metabonomic data sets. Anal Chem 78:2262–2267CrossRefPubMedGoogle Scholar
  11. Fierabracci V, Masiello P, Novelli M, Bergamini E (1991) Application of amino acid analysis by high-performance liquid chromatography with phenyl isothiocyanate derivatization to the rapid determination of free amino acids in biological samples. J Chromatogr 570:285–291CrossRefPubMedGoogle Scholar
  12. Folch J, Lees M, Stanley GHS (1957) A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem 226:497–509PubMedGoogle Scholar
  13. Gliyazova NS, Huh EY, Ibeanu GC (2013) A novel phenoxy thiophene sulphonamide molecule protects against glutamate evoked oxidative injury in a neuronal cell model. BMC Neurosci 14:93CrossRefPubMedPubMedCentralGoogle Scholar
  14. Gonzalez-Dominguez R, Garcia-Barrera T, Gomez-Ariza JL (2015a) Metabolite profiling for the identification of altered metabolic pathways in Alzheimer’s disease. J Pharm Biomed Anal 107:75–81CrossRefPubMedGoogle Scholar
  15. Gonzalez-Dominguez R, Garcia-Barrera T, Vitorica J, Gomez-Ariza JL (2015b) Deciphering metabolic abnormalities associated with Alzheimer’s disease in the APP/PS1 mouse model using integrated metabolomic approaches. Biochimie 110:119–128CrossRefPubMedGoogle Scholar
  16. Gostner JM, Becker K, Kurz K, Fuchs D (2015) Disturbed amino acid metabolism in HIV: association with neuropsychiatric symptoms. Front Psychiatry 6:97CrossRefPubMedPubMedCentralGoogle Scholar
  17. Hardy J, Selkoe DJ (2002) The amyloid hypothesis of Alzheimer’s disease: progress and problems on the road to therapeutics. Science 297:353–356CrossRefPubMedGoogle Scholar
  18. Holmquist L, Stuchbury G, Berbaum K, Muscat S, Young S, Hager K, Engel J, Munch G (2007) Lipoic acid as a novel treatment for Alzheimer’s disease and related dementias. Pharmacol Ther 113:154–164CrossRefPubMedGoogle Scholar
  19. Kang Y, Tiziani S, Park G, Kaul M, Paternostro G (2014) Cellular protection using Flt3 and PI3Kα inhibitors demonstrates multiple mechanisms of oxidative glutamate toxicity. Nat Commun 5Google Scholar
  20. Kincses ZT, Toldi J, Vecsei L (2010) Kynurenines, neurodegeneration and Alzheimer’s disease. J Cell Mol Med 14:2045–2054CrossRefPubMedPubMedCentralGoogle Scholar
  21. Krishnaiah D, Sarbatly R, Bono A (2007) Phytochemical antioxidants for health and medicine-a move towards nature. Biotechnol Mol Biol Rev 1:97–104Google Scholar
  22. Kritis AA, Stamoula EG, Paniskaki KA, Vavilis TD (2015) Researching glutamate - induced cytotoxicity in different cell lines: a comparative/, collective analysis/study. Front Cell Neurosci 9:2015CrossRefGoogle Scholar
  23. Krug AK, Gutbier S, Zhao L, Poltl D, Kullmann C, Ivanova V, Forster S, Jagtap S, Meiser J, Leparc G, Schildknecht S, Adam M, Hiller K, Farhan H, Brunner T, Hartung T, Sachinidis A, Leist M (2014) Transcriptional and metabolic adaptation of human neurons to the mitochondrial toxicant MPP (+). Cell Death Dis 5:e1222CrossRefPubMedPubMedCentralGoogle Scholar
  24. Kulkarni AP, Mahal HS, Kapoor S, Aradhya SM (2007) In Vitro studies on the binding, antioxidant, and cytotoxic actions of punicalagin. J Agric Food Chem 55:1491–1500CrossRefPubMedGoogle Scholar
  25. Kumari S, Mehta SL, Li PA (2012) Glutamate induces mitochondrial dynamic imbalance and autophagy activation: preventive effects of selenium. PLoS One 7:e39382CrossRefPubMedPubMedCentralGoogle Scholar
  26. Kuz’mina VV, Gavrovskaia LK, Ryzhova OV (2010) Taurine. Effect on exotrophia and metabolism in mammals and fish. Zh Evol Biokhim Fiziol 46:19–27Google Scholar
  27. Lavanya G, Sivajyothi R, Manjunath M, Parthasarathy PR (2009) Fate of biomolecules during carbon tetrachloride induced oxidative stress and protective nature of Ammannia baccifera Linn.: a natural antioxidant. Int J Green Pharm 3:300–305CrossRefGoogle Scholar
  28. Lavanya G, Voravuthikunchai SP, Towatana NH (2012) Acetone extract from Rhodomyrtus tomentosa: a potent natural antioxidant. Evid Based Complement Alternat Med 2012:535479CrossRefPubMedPubMedCentralGoogle Scholar
  29. Lee DS, Ko W, Kim DC, Kim YC, Jeong GS (2014) Cudarflavone B provides neuroprotection against glutamate-induced mouse hippocampal HT22 cell damage through the Nrf2 and PI3K/Akt signaling pathways. Molecules 19:10818–10831CrossRefPubMedGoogle Scholar
  30. Lin CC, Hsu YF, Lin TC, Hsu HY (2001) Antioxidant and hepatoprotective effects of punicalagin and punicalin on acetaminophen-induced liver damage in rats. Phytother Res 15:206–212CrossRefPubMedGoogle Scholar
  31. Lin CC, Hsu YF, Lin TC (1999) Effects of punicalagin and punicalin on carrageenan-induced inflammation in rats. Am J Chin Med 27:371–376CrossRefPubMedGoogle Scholar
  32. Louzada PR, Paula Lima AC, Mendonca-Silva DL, Noel F, De Mello FG, Ferreira ST (2004) Taurine prevents the neurotoxicity of beta-amyloid and glutamate receptor agonists: activation of GABA receptors and possible implications for Alzheimer’s disease and other neurological disorders. FASEB J 18:511–518CrossRefPubMedGoogle Scholar
  33. Majid S, Khanduja KL, Gandhi RK, Kapur S, Sharma RR (1991) Influence of ellagic acid on antioxidant defense system and lipid peroxidation in mice. Biochem Pharmacol 42:1441–1445CrossRefPubMedGoogle Scholar
  34. Mally J, Szalai G, Stone TW (1997) Changes in the concentration of amino acids in serum and cerebrospinal fluid of patients with Parkinson’s disease. J Neurol Sci 151:159–162CrossRefPubMedGoogle Scholar
  35. Manjunath M, Lavanya G, Sivajyothi R, Vijaya Sarathi Reddy O (2011) Antioxidant and radical scavenging activity of Actiniopteris radiata (Sw.) link. Asian Journal of Experimental Sciences 25:73–80Google Scholar
  36. Manubolu M, Goodla L, Ravilla S, Thanasekaran J, Dutta P, Malmlof K, Obulum VR (2014) Protective effect of Actiniopteris radiata (Sw.) link. Against CCl (4) induced oxidative stress in albino rats. J Ethnopharmacol 153:744–752CrossRefPubMedGoogle Scholar
  37. Mariani E, Polidori MC, Cherubini A, Mecocci P (2005) Oxidative stress in brain aging, neurodegenerative and vascular diseases: an overview. J Chromatogr B 827:65–75CrossRefGoogle Scholar
  38. Markesbery WR (1997) Oxidative stress hypothesis in Alzheimer’s disease. Free Radic Biol Med 23:134–147CrossRefPubMedGoogle Scholar
  39. Martin V, Fabelo N, Santpere G, Puig B, Marin R, Ferrer I, Diaz M (2010) Lipid alterations in lipid rafts from Alzheimer’s disease human brain cortex. J Alzheimers Dis 19:489–502CrossRefPubMedGoogle Scholar
  40. Mattson MP (2000) Emerging neuroprotective strategies for Alzheimer’s disease: dietary restriction, telomerase activation, and stem cell therapy. Exp Gerontol 35:489–502CrossRefPubMedGoogle Scholar
  41. Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65:55–63CrossRefPubMedGoogle Scholar
  42. Murphy TH, Miyamoto M, Sastre A, Schnaar RL, Coyle JT (1989) Glutamate toxicity in a neuronal cell line involves inhibition of cystine transport leading to oxidative stress. Neuron 2:1547–1558CrossRefPubMedGoogle Scholar
  43. Paglia G, Stocchero M, Cacciatore S, Lai S, Angel P, Alam MT, Keller M, Ralser M, Astarita G (2016) Unbiased metabolomic investigation of Alzheimer’s disease brain points to dysregulation of mitochondrial aspartate metabolism. J Proteome Res 15:608–618CrossRefPubMedGoogle Scholar
  44. Qin XY, Cao C, Cawley NX, Liu TT, Yuan J, Loh YP (2016 Apr.) Cheng Y (2016) decreased peripheral brain-derived neurotrophic factor levels in Alzheimer’s disease: a meta-analysis study (N = 7277). Mol Psychiatry 26Google Scholar
  45. Redjems-Bennani N, Jeandel C, Lefebvre E, Blain H, Vidailhet M, Gueant JL (1998) Abnormal substrate levels that depend upon mitochondrial function in cerebrospinal fluid from Alzheimer patients. Gerontology 44:300–304CrossRefPubMedGoogle Scholar
  46. Reilmann R, Rolf LH, Lange HW (1995) Decreased plasma alanine and isoleucine in Huntington’s disease. Acta Neurol Scand 91:222–224CrossRefPubMedGoogle Scholar
  47. Ripps H, Shen W (2012) Review: taurine: a "very essential" amino acid. Mol Vis 18:2673–2686PubMedPubMedCentralGoogle Scholar
  48. Rosemberg DB, da Rocha RF, Rico EP, Zanotto-Filho A, Dias RD, Bogo MR, Bonan CD, Moreira JC, Klamt F, Souza DO (2010) Taurine prevents enhancement of acetylcholinesterase activity induced by acute ethanol exposure and decreases the level of markers of oxidative stress in zebrafish brain. Neuroscience 171:683–692CrossRefPubMedGoogle Scholar
  49. Seeram NP, Henning SM, Zhang Y, Suchard M, Li Z, Heber D (2006) Pomegranate juice ellagitannin metabolites are present in human plasma and some persist in urine for up to 48 hours. J Nutr 136:2481–2485PubMedGoogle Scholar
  50. Shadrina MI, Slominsky PA, Limborska SA (2010) Molecular mechanisms of pathogenesis of Parkinson’s disease. Int Rev Cell Mol Biol 281:229–266CrossRefPubMedGoogle Scholar
  51. van der Goot AT, Zhu W, Vazquez-Manrique RP, Seinstra RI, Dettmer K, Michels H, Farina F, Krijnen J, Melki R, Buijsman RC, Ruiz Silva M, Thijssen KL, Kema IP, Neri C, Oefner PJ, Nollen EA (2012) Delaying aging and the aging-associated decline in protein homeostasis by inhibition of tryptophan degradation. Proc Natl Acad Sci U S A 109:14912–14917CrossRefPubMedPubMedCentralGoogle Scholar
  52. Vauzour D, Vafeiadou K, Rodriguez-Mateos A, Rendeiro C, Spencer JPE (2008) The neuroprotective potential of flavonoids: a multiplicity of effects. Genes Nutr 3:115–126CrossRefPubMedPubMedCentralGoogle Scholar
  53. Wang X, Wang W, Li L, Perry G, Lee HG, Zhu X (2014) Oxidative stress and mitochondrial dysfunction in Alzheimer’s disease. Biochim Biophys Acta 1842:1240–1247CrossRefPubMedGoogle Scholar
  54. Weon JB, Yang HJ, Lee B, Yun B-R, Ahn JH, Lee HY, Ma CJ (2011) Neuroprotective activity of the methanolic extract of Lonicera japonica in glutamate-injured primary rat cortical cells. Pharmacogn Mag 7:284–288CrossRefPubMedPubMedCentralGoogle Scholar
  55. Xia J, Psychogios N, Young N, Wishart DS (2009) MetaboAnalyst: a web server for metabolomic data analysis and interpretation. Nucleic Acids Res 37:W652–W660CrossRefPubMedPubMedCentralGoogle Scholar
  56. Xia J, Wishart DS (2010) MetPA: a web-based metabolomics tool for pathway analysis and visualization. Bioinformatics 26:2342–2344CrossRefPubMedGoogle Scholar
  57. Yi L, Shi S, Wang Y, Huang W, Xia ZA, Xing Z, Peng W, Wang Z (2016) Serum metabolic profiling reveals altered metabolic pathways in patients with post-traumatic cognitive impairments. Sci Rep 6:21320CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Kavitha Pathakoti
    • 1
    • 2
  • Lavanya Goodla
    • 3
  • Manjunath Manubolu
    • 4
  • Tewin Tencomnao
    • 1
    Email author
  1. 1.Department of Clinical Chemistry, Faculty of Allied Health SciencesChulalongkorn UniversityBangkokThailand
  2. 2.Department of BiologyJackson State UniversityJacksonUSA
  3. 3.South China Institute of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and HealthChinese Academy of SciencesGuangzhouChina
  4. 4.Division of Environmental Health Sciences, College of Public HealthThe Ohio State UniversityColumbusUSA

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