Environmental Science and Pollution Research

, Volume 26, Issue 26, pp 27148–27167 | Cite as

Ginkgo biloba attenuates aluminum lactate-induced neurotoxicity in reproductive senescent female rats: behavioral, biochemical, and histopathological study

  • Sonia Verma
  • Pavitra Ranawat
  • Neha Sharma
  • Bimla NehruEmail author
Research Article


Extensive use of aluminum (Al) in industry, cooking utensils, and wrapping or freezing the food items, due to its cheapness and abundance in the environment, has become a major concern. Growing evidence supports that environmental pollutant Al promotes the aggregation of amyloid beta (Aβ) in the brain, which is the main pathological marker of Alzheimer’s disease (AD). Further, AD- and Al-induced neurotoxic effects are more common among women following reproductive senescence due to decline in estrogen. Though clinically Ginkgo biloba extract (GBE) has been exploited as a memory enhancer, its role in Al-induced neurotoxicity in reproductive senescent female rats needs to be evaluated. Animals were exposed to intraperitoneal dose (10 mg/kg b.wt) of Al and oral dose (100 mg/kg b.wt.) of GBE daily for 6 weeks. A significant decline in the Al-induced Aβ aggregates was observed in hippocampal and cortical regions of the brain with GBE supplementation, as confirmed by thioflavin (ThT) and Congo red staining. GBE administration significantly decreased the reactive oxygen species, lipid peroxidation, nitric oxide, and citrulline levels in comparison to Al-treated rats. On the contrary, a significant increase in the reduced glutathione, GSH/GSSG ratio as well as in the activities of antioxidant enzymes was observed with GBE administration. Based on the above results, GBE prevented the neuronal loss in the hippocampus and cortex, hence caused significant improvement in the learning and memory of the animals in terms of AChE activity, serotonin levels, Morris water maze, and active and passive avoidance tests. In conclusion, GBE has alleviated the behavioral, biochemical, and histopathological alterations due to Al toxicity in rats. However, molecular studies are going on to better understand the mechanism of GBE protection against the environmental toxicant Al exposure.

Graphical abstract



Aluminum Oxidative stress Reproductive senescence Aβ aggregation Memory loss Ginkgo biloba extract 





Lipid peroxidation


Reactive oxygen species








Morris Water Maze


Elevated Plus Maze


Superoxide dismutase


Glutathione peroxidase


Glutathione s-transferase


Ginkgo biloba extract



The University Research fellowship (1241/Estt-I, dated 7/2/2013) to Ms. Sonia Verma is highly appreciated.

Compliance with ethical standards

All the protocols performed were approved (PU/45/99/CPCSEA/IAEC/2018/153) by the Animal Ethical Committee (IAEC) (NIH publications; Rule No. 23-85, as revised in 1985) of Panjab University, Chandigarh, India.

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Abd-Elhady RM, Elsheikh AM, Khalifa AE (2013) Anti-amnestic properties of Ginkgo biloba extract on impaired memory function induced by aluminum in rats. Int J Dev Neurosci 31(7):598–607Google Scholar
  2. Abu-Taweel GM, Ajarem JS, Ahmad M (2012) Neurobehavioral toxic effects of perinatal oral exposure to aluminum on the developmental motor reflexes, learning, memory and brain neurotransmitters of mice offspring. Pharmacol Biochem Behav 101(1):49–56Google Scholar
  3. Ahmeda HH, Zaazaab AM, El-Motelpb BA (2014) Zingiber officinale and Alzheimer’s disease: evidences and mechanisms. Int J Pharm Sci Rev Res 27(2)Google Scholar
  4. Amjad S, Umesalma S (2015) Protective effect of Centella asiatica against aluminium-induced neurotoxicity in cerebral cortex, striatum, hypothalamus and hippocampus of rat brain-histopathological, and biochemical approach. J Mol Biomark Diagn 6(1):1Google Scholar
  5. Barron AM, Fuller SJ, Verdile G, Martins RN (2006) Reproductive hormones modulate oxidative stress in Alzheimer’s disease. Antioxid Redox Signal 8(11–12):2047–2059Google Scholar
  6. Belviranlı M, Okudan N (2015) The effects of Ginkgo biloba extract on cognitive functions in aged female rats: the role of oxidative stress and brain-derived neurotrophic factor. Behav Brain Res 278:453–461Google Scholar
  7. Best TM, Fiebig R, Corr DT, Brickson S, Ji L (1999) Free radical activity, antioxidant enzyme, and glutathione changes with muscle stretch injury in rabbits. J Appl Physiol 87(1):74–82Google Scholar
  8. Bondy SC (2010) The neurotoxicity of environmental aluminum is still an issue. Neurotoxicology 31(5):575–581Google Scholar
  9. Boyde TRC, Rahmatullah M (1980) Optimization of conditions for the colorimetric determination of citrulline, using diacetyl monoxime. Anal Biochem 107(2):424–431Google Scholar
  10. Brann DW, Dhandapani K, Wakade C, Mahesh VB, Khan MM (2007) Neurotrophic and neuroprotective actions of estrogen: basic mechanisms and clinical implications. Steroids 72(5):381–405Google Scholar
  11. Chen C, Li B, Cheng G, Yang X, Zhao N, Shi R (2018) Amentoflavone ameliorates Aβ 1–42-induced memory deficits and oxidative stress in cellular and rat model. Neurochem Res 43(4):857–868Google Scholar
  12. Christen Y (2000) Oxidative stress and Alzheimer disease. Am J Clin Nutr 71(2):621S–629SGoogle Scholar
  13. Church WH (2005) Column chromatography analysis of brain tissue: an advanced laboratory exercise for neuroscience majors. J Undergrad Neurosci Educ 3(2):A36–A41Google Scholar
  14. Cornutiu G (2015) The epidemiological scale of Alzheimer’s disease. J Clin Med Res 7(9):657–666Google Scholar
  15. DeFeudis FV, Drieu K (2000) Ginkgo biloba extract (EGb 761) and CNS functions basic studies and clinical applications. Curr Drug Targets 1(1):25–58Google Scholar
  16. Diamond BJ, Shiflett SC, Feiwel N, Matheis RJ, Noskin O, Richards JA, Schoenberger NE (2000) Ginkgo biloba extract: mechanisms and clinical indications. Arch Phys Med Rehabil 81(5):668–678Google Scholar
  17. Ellman GL (1959) Tissue sulfhydryl groups. Arch Biochem Biophys 82:70–77Google Scholar
  18. Ellman GL, Courtney KD, Andres V Jr, Featherstone RM (1961) A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol 7(2):88–95Google Scholar
  19. Farris W, Schütz SG, Cirrito JR, Shankar GM, Sun X, George A, Leissring MA, Walsh DM, Qiu WQ, Holtzman DM, Selkoe DJ (2007) Loss of neprilysin function promotes amyloid plaque formation and causes cerebral amyloid angiopathy. Am J Pathol 171(1):241–251Google Scholar
  20. Forny-Germano L, e Silva NML, Batista AF, Brito-Moreira J, Gralle M, Boehnke SE, Coe BC, Lablans A, Marques SA, Martinez AMB and Klein WL (2014) Alzheimer’s disease-like pathology induced by amyloid-β oligomers in nonhuman primates. J Neurosci, 34(41), pp.13629–13643Google Scholar
  21. Gong QH, Wu Q, Huang XN, Sun AS, Shi JS (2005) Protective effects of Ginkgo biloba leaf extract on aluminum-induced brain dysfunction in rats. Life Sci 77(2):140–148Google Scholar
  22. Habig WH, Pabst MJ, Jakoby WB (1974) Glutathione S-transferases the first enzymatic step in mercapturic acid formation. J Biol Chem 249(22):7130–7139Google Scholar
  23. Hirata-Koizumi M, Fujii S, Ono A, Hirose A, Imai T, Ogawa K, Ema M, Nishikawa A (2011) Two-generation reproductive toxicity study of aluminium sulfate in rats. Reprod Toxicol 31(2):219–230.
  24. Humanson GL (1961) Basic procedures—animal tissue technique. Animal Tissue Techniques 1:130–132Google Scholar
  25. Kakkar V, Kaur IP (2011) Evaluating potential of curcumin loaded solid lipid nanoparticles in aluminium induced behavioural, biochemical and histopathological alterations in mice brain. Food Chem Toxicol 49(11):2906–2913Google Scholar
  26. Kawahara M (2005) Effects of aluminum on the nervous system and its possible link with neurodegenerative diseases. J Alzheimers Dis 8(2):171–182Google Scholar
  27. Keller GJ (1945) A reliable Nissl method., Bull. Int. Assoc. Med. Mus. 25: 77. (accessed March 28, 2018)
  28. Kobayashi K, Yumoto S, Nagai H, Hosoyama Y, Imamura M, Masuzawa SI, Koizumi Y, Yamashita H (1990) 26Al tracer experiment by accelerator mass spectrometry and its application to the studies for amyotrophic lateral sclerosis and Alzheimer’s disease. I. Proc Jpn Acad Ser B 66(10):189–192Google Scholar
  29. Kono Y (1978) Generation of superoxide radical during autoxidation of hydroxylamine and an assay for superoxide dismutase. Arch Biochem Biophys 186(1):189–195Google Scholar
  30. Li R, Singh M (2014) Sex differences in cognitive impairment and Alzheimer’s disease. Front Neuroendocrinol 35, 385–403.
  31. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ, Grynkiewicz G, Poenie M, Tsien RY, Folch J, Lees M, Stanley GS, Alessi DR (1951) 26. The colorimetric determination of phosphorus. J Biol Chem 193:265–275Google Scholar
  32. Luck HA (1963) Spectrophotometric method for the estimation of catalase. Methods Enzymatic Anal Academic Press, New York/Lyon, France, pp. 885–894Google Scholar
  33. Ma L, Wang S, Tai F, Yuan G, Wu R, Liu X, Wei B, Yang X (2012) Effects of bilobalide on anxiety, spatial learning, memory and levels of hippocampal glucocorticoid receptors in male Kunming mice. Phytomedicine 20(1):89–96Google Scholar
  34. McLean AC, Valenzuela N, Fai S, Bennett SA (2012) Performing vaginal lavage, crystal violet staining, and vaginal cytological evaluation for mouse estrous cycle staging identification. J Vis Exp (67) e4389.
  35. Mesbah MK, Khalifa SI, El-Gindy A, Tawfik KA (2005) HPLC determination of certain flavonoids and terpene lactones in selected Ginkgo biloba L. phytopharmaceuticals. Il Farmaco 60(6–7):583–590Google Scholar
  36. Mikhail K, Hwee SJ (2018) Alzheimer disease. In: Rakel D (ed) Integrative medicine, 4th edn. Elsevier, New York City, pp 95–107Google Scholar
  37. Miu AC, Andreescu CE, Vasiu Rand Olteanu AI (2003) A behavioral and histological study of the effects of long-term exposure of adult rats to aluminum. Int J Neurosci 113(9):1197–1211Google Scholar
  38. Morris R (1984) Developments of a water-maze procedure for studying spatial learning in the rat. J Neurosci Methods 11(1):47–60Google Scholar
  39. Nehru B, Anand P (2005) Oxidative damage following chronic aluminium exposure in adult and pup rat brains. J Trace Elem Med Biol 19(2–3):203–208Google Scholar
  40. Nehru B, Bhalla P (2006) Reversal of an aluminium induced alteration in redox status in different regions of rat brain by administration of centrophenoxine. Mol Cell Biochem 290(1–2):185–191Google Scholar
  41. Nehru B, Bhalla P, Garg A (2007) Further evidence of centrophenoxine mediated protection in aluminium exposed rats by biochemical and light microscopy analysis. Food Chem Toxicol 45(12):2499–2505Google Scholar
  42. Oh SM, Chung KH (2004) Estrogenic activities of Ginkgo biloba extracts. Life Sci 74(11):1325–1335Google Scholar
  43. Oh SM, Chung KH (2006) Antiestrogenic activities of Ginkgo biloba extracts. J Steroid Biochem Mol Biol 100(4–5):167–176Google Scholar
  44. Paglia DE, Valentine WN (1967) Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J Lab Clin Med 70(1):158–169Google Scholar
  45. Pearse AGE (1968) Histochemistry, theoretical and applied, vol 1, 3rd edn. Churchill Livingstone, London, p 660Google Scholar
  46. Raddassi K, Berthon B, Petit JF, Lemaire G (1994) Role of calcium in the activation of mouse peritoneal macrophages: induction of NO synthase by calcium ionophores and thapsigargin. Cell Immunol 153(2):443–455Google Scholar
  47. Ramassamy C (2006) Emerging role of polyphenolic compounds in the treatment of neurodegenerative diseases: a review of their intracellular targets. Eur J Pharmacol 545(1):51–64Google Scholar
  48. Selvamani A, Sohrabji F (2010) The neurotoxic effects of estrogen on ischemic stroke in older female rats is associated with age-dependent loss of insulin-like growth factor-1. J Neurosci 30(20):6852–6861Google Scholar
  49. Sharma N, Nehru B (2015) Characterization of the lipopolysaccharide induced model of Parkinson’s disease: role of oxidative stress and neuroinflammation. Neurochem Int 87:92–105Google Scholar
  50. Sharma DR, Sunkaria A, Bal A, Bhutia YD, Vijayaraghavan R, Flora SJS, Gill KD (2009) Neurobehavioral impairments, generation of oxidative stress and release of pro-apoptotic factors after chronic exposure to sulphur mustard in mouse brain. Toxicol Appl Pharmacol 240(2):208–218Google Scholar
  51. Sharma S, Verma S, Kapoor M, Saini A, Nehru B (2016) Alzheimer’s disease like pathology induced six weeks after aggregated amyloid-beta injection in rats: increased oxidative stress and impaired long-term memory with anxiety-like behavior. Neurol Res 38(9):838–850Google Scholar
  52. Shepherd JK, Grewal SS, Fletcher A, Bill DJ, Dourish CT (1994) Behavioural and pharmacological characterisation of the elevated “zero-maze” as an animal model of anxiety. Psychopharmacology 116(1):56–64Google Scholar
  53. Smith RW (1996) Kinetic aspects of aqueous aluminum chemistry: environmental implications. Coord Chem Rev 149:81–93Google Scholar
  54. Smith JV, Luo Y (2004) Studies on molecular mechanisms of Ginkgo biloba extract. Appl Microbiol Biotechnol 64(4):465–472Google Scholar
  55. Sood PK, Verma S, Nahar U, Nehru B (2015) Neuroprotective role of Lazaroids against aluminium chloride poisoning. Neurochem Res 40(8):1699–1708Google Scholar
  56. Tang MX, Jacobs D, Stern Y, Marder K, Schofield P, Gurland B, Andrews H, Mayeux R (1996) Effect of oestrogen during menopause on risk and age at onset of Alzheimer’s disease. Lancet 348(9025):429–432Google Scholar
  57. Tarozzi A, Bartolini M, Piazzi L, Valgimigli L, Amorati R, Bolondi C, Djemil A, Mancini F, Andrisano V, Rampa A (2014) From the dual function lead AP2238 to AP2469, a multi-target-directed ligand for the treatment of Alzheimer’s disease. Pharmacol Res Perspect 2(2):e00023Google Scholar
  58. Thenmozhi AJ, Raja TRW, Janakiraman U, Manivasagam T (2015) Neuroprotective effect of hesperidin on aluminium chloride induced Alzheimer’s disease in Wistar rats. Neurochem Res 40(4):767–776Google Scholar
  59. Thirunavukkarasu SV, Venkataraman S, Raja S, Upadhyay L (2012) Neuroprotective effect of Manasamitra vatakam against aluminium induced cognitive impairment and oxidative damage in the cortex and hippocampus of rat brain. Drug Chem Toxicol 35(1):104–115Google Scholar
  60. Walesiuk A, Trofimiuk E, Braszko JJ (2005) Gingko biloba extract diminishes stress-induced memory deficits in rats. Pharmacol Rep 57(2):176–187Google Scholar
  61. Walton JR (2006) Aluminum in hippocampal neurons from humans with Alzheimer’s disease. Neurotoxicology 27(3):385–394Google Scholar
  62. Walton JR (2012) Cognitive deterioration and associated pathology induced by chronic low-level aluminum ingestion in a translational rat model provides an explanation of Alzheimer’s disease, tests for susceptibility and avenues for treatment. Int J Alzheimers Dis 2012;2012Google Scholar
  63. Wills ED (1966) Mechanisms of lipid peroxide formation in animal tissues. Biochem J 99(3):667–676Google Scholar
  64. Yokel RA, Florence RL (2006) Aluminum bioavailability from the approved food additive leavening agent acidic sodium aluminum phosphate, incorporated into a baked good, is lower than from water. Toxicology 227(1–2):86–93Google Scholar
  65. Zagni E, Simoni L, Colombo D (2016) Sex and gender differences in central nervous system-related disorders. Neurosci J.
  66. Zahler WL, Cleland WW (1968) A specific and sensitive assay for disulfides. J Biol Chem 243(4):716–719Google Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Biophysics, South CampusPanjab UniversityChandigarhIndia

Personalised recommendations