Journal of Natural Medicines

, Volume 65, Issue 1, pp 1–8 | Cite as

Protection of HT22 neuronal cells against glutamate toxicity mediated by the antioxidant activity of Pueraria candollei var. mirifica extracts

  • Apirada Sucontphunt
  • Wanchai De-Eknamkul
  • Ubonthip Nimmannit
  • S. Dan Dimitrijevich
  • Robert W. Gracy
Original Paper

Abstract

Neuronal degeneration is known to be due to oxidative stress acting through a pathway involving the excessive activation of glutamate receptors. We studied the neuroprotection potential of an ethyl acetate–ethanol extract of Pueraria mirifica (P. candollei var. mirifica) root (PM extract). PM extract was evaluated for its antioxidant and neuroprotective activities against glutamate toxicity in mouse hippocampal HT22 neuronal cells. The extract at concentrations of 10 and 50 μg/ml exhibited considerable antioxidant activity with significant neuroprotection, based on the microscopic observations of cell morphology and the determination of cell viability and cell number. Studies of the possible mechanisms of action indicated that the neuroprotection exerted by PM extract was related to its scavenging activity against H2O2 and related reactive oxygen species. High-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC) analyses showed that the extract contained daidzein and genistein as identified constituents, as well as additional components with antioxidant activity. While daidzein and genistein individually and in combination were observed not to be neuroprotective, we propose that the antioxidant and neuroprotective activities of PM extract are derived from the combined properties of its constituents.

Keywords

Pueraria mirifica Partially purified extract Antioxidant activity Neuroprotection Scavenging activity HT22 neuronal cells 

Notes

Acknowledgments

This work was financially supported by the Thailand Research Fund through the Royal Golden Jubilee Ph.D. Program (grant no. PHD/0104/2546) to A.S. and W.D. and the Thailand National Center for Genetic Engineering and Biotechnology (BIOTEC) (grant no. 2/2546). We would also like to thank Khao-La-Or Laboratories, Ltd., Bangkok, Thailand, for supplying the Pueraria mirifica material.

References

  1. 1.
    Cain JC (1960) Miroestrol: an oestrogen from the plant Pueraria mirifica. Nature 188:774–777PubMedCrossRefGoogle Scholar
  2. 2.
    Muangman V, Cherdshewasart W (2001) Clinical trial of the phytoestrogen-rich herb, Pueraria mirifica as a crude drug in the treatment of symptoms in menopausal women. Siriraj Hosp Gaz 53:300–309Google Scholar
  3. 3.
    Urasopon N, Hamada Y, Asaoka K, Cherdshewasart W, Malaivijitnond S (2007) Pueraria mirifica, a phytoestrogen-rich herb, prevents bone loss in orchidectomized rats. Maturitas 56:322–331PubMedCrossRefGoogle Scholar
  4. 4.
    Chansakaow S, Ishikawa T, Sekine K, Okada M, Higuchi Y, Kudo M, Chaichantipyuth C (2000) Isoflavonoids from Pueraria mirifica and their estrogenic activity. Planta Med 66:572–575PubMedCrossRefGoogle Scholar
  5. 5.
    Chansakaow S, Ishikawa T, Seki H, Sekine (née Yoshizawa) K, Okada M, Chaichantipyuth C (2000) Identification of deoxymiroestrol as the actual rejuvenating principle of “Kwao Keur”, Pueraria mirifica. The known miroestrol may be an artifact. J Nat Prod 63:173–175PubMedCrossRefGoogle Scholar
  6. 6.
    Sierens J, Hartley JA, Campbell MJ, Leathem AJC, Woodside JV (2001) Effect of phytoestrogen and antioxidant supplementation on oxidative DNA damage assessed using the comet assay. Mutat Res DNA Repair 485:169–176PubMedCrossRefGoogle Scholar
  7. 7.
    Sonee M, Sum T, Wang C, Mukherjee SK (2004) The soy isoflavone, genistein, protects human cortical neuronal cells from oxidative stress. Neurotoxicology 25:885–891PubMedCrossRefGoogle Scholar
  8. 8.
    Lof M, Weiderpass E (2006) Epidemiologic evidence suggests that dietary phytoestrogen intake is associated with reduced risk of breast, endometrial, and prostate cancers. Nutr Res 26:609–619CrossRefGoogle Scholar
  9. 9.
    Wu HJ, Chan WH (2007) Genistein protects methylglyoxal-induced oxidative DNA damage and cell injury in human mononuclear cells. Toxicol In Vitro 21:335–342PubMedCrossRefGoogle Scholar
  10. 10.
    Zeng H, Chen Q, Zhao B (2004) Genistein ameliorates beta-amyloid peptide (25–35)-induced hippocampal neuronal apoptosis. Free Radic Biol Med 36:180–188PubMedCrossRefGoogle Scholar
  11. 11.
    Simonian NA, Coyle JT (1996) Oxidative stress in neurodegenerative diseases. Annu Rev Pharmacol Toxicol 36:83–106PubMedCrossRefGoogle Scholar
  12. 12.
    Coyle JT, Puttfarcken P (1993) Oxidative stress, glutamate, and neurodegenerative disorders. Science 262:689–695PubMedCrossRefGoogle Scholar
  13. 13.
    Sokmen M, Angelova M, Krumova E, Pashova S, Ivancheva S, Sokmen A, Serkedjieva J (2005) In vitro antioxidant activity of polyphenol extracts with antiviral properties from Geranium sanguineum L. Life Sci 76:2981–2993PubMedCrossRefGoogle Scholar
  14. 14.
    Skehan P, Storeng R, Scudiero D, Monks A, McMahon J, Vistica D, Warren JT, Bokesch H, Kenney S, Boyd MR (1990) New colorimetric cytotoxicity assay for anticancer-drug screening. J Natl Cancer Inst 82:1107–1112PubMedCrossRefGoogle Scholar
  15. 15.
    Ciapetti G, Granchi D, Verri E, Savarino L, Cavedagna D, Pizzoferrato A (1996) Application of a combination of neutral red and amido black staining for rapid, reliable cytotoxicity testing of biomaterials. Biomaterials 17:1259–1264PubMedCrossRefGoogle Scholar
  16. 16.
    Ou B, Hampsch-Woodill M, Prior RL (2001) Development and validation of an improved oxygen radical absorbance capacity assay using fluorescein as the fluorescent probe. J Agric Food Chem 49:4619–4626PubMedCrossRefGoogle Scholar
  17. 17.
    Choi J, Conrad CC, Malakowsky CA, Talent JM, Yuan CS, Gracy RW (2002) Flavones from Scutellaria baicalensis Georgi attenuate apoptosis and protein oxidation in neuronal cell lines. Biochim Biophys Acta 1571:201–210PubMedGoogle Scholar
  18. 18.
    Prior RL, Wu X, Schaich K (2005) Standardized methods for the determination of antioxidant capacity and phenolics in foods and dietary supplements. J Agric Food Chem 53:4290–4302PubMedCrossRefGoogle Scholar
  19. 19.
    Li Y, Maher P, Schubert D (1998) Phosphatidylcholine-specific phospholipase C regulates glutamate-induced nerve cell death. Proc Natl Acad Sci USA 95:7748–7753PubMedCrossRefGoogle Scholar
  20. 20.
    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 2:1547–1558PubMedCrossRefGoogle Scholar
  21. 21.
    Ishige K, Schubert D, Sagara Y (2001) Flavonoids protect neuronal cells from oxidative stress by three distinct mechanisms. Free Radic Biol Med 30:433–446PubMedCrossRefGoogle Scholar
  22. 22.
    Nappi AJ, Vass E (1998) Hydroxyl radical formation resulting from the interaction of nitric oxide and hydrogen peroxide. Biochim Biophys Acta 1380:55–63PubMedGoogle Scholar
  23. 23.
    Cheton PLB, Archibald FS (1988) Manganese complexes and the generation and scavenging of hydroxyl free radicals. Free Radic Biol Med 5:325–333PubMedCrossRefGoogle Scholar
  24. 24.
    Rothe G, Valet G (1990) Flow cytometric analysis of respiratory burst activity in phagocytes with hydroethidine and 2′,7′-dichlorofluorescin. J Leukoc Biol 47:440–448PubMedGoogle Scholar

Copyright information

© The Japanese Society of Pharmacognosy and Springer 2010

Authors and Affiliations

  • Apirada Sucontphunt
    • 1
  • Wanchai De-Eknamkul
    • 2
  • Ubonthip Nimmannit
    • 3
  • S. Dan Dimitrijevich
    • 4
  • Robert W. Gracy
    • 5
  1. 1.Pharmaceutical Technology International Program, Faculty of Pharmaceutical SciencesChulalongkorn UniversityBangkokThailand
  2. 2.Department of Pharmacognosy, Faculty of Pharmaceutical SciencesChulalongkorn UniversityBangkokThailand
  3. 3.National Nanotechnology Center (NANOTEC)National Science and Technology Development Agency (NSTDA)Pathum ThaniThailand
  4. 4.Department of Integrative PhysiologyUniversity of North Texas Health Science CenterFort WorthUSA
  5. 5.The Office of Vice President for Research (VPR)University of Texas at San AntonioSan AntonioUSA

Personalised recommendations