Pharmacopsychiatry 2003; 36: 15-23
DOI: 10.1055/s-2003-40449
Original Paper
© Georg Thieme Verlag Stuttgart · New York

Effects of EGb 761® Ginkgo biloba Extract on Mitochondrial Function and Oxidative Stress

A. Eckert1 , U. Keil1 , S. Kressmann1 , K. Schindowski1 , S. Leutner1 , S. Leutz1 , W. E. Müller1
  • 1Department of Pharmacology, Biocenter, J.W. Goethe University of Frankfurt, Frankfurt am Main, Germany
Further Information

Publication History

Publication Date:
07 July 2003 (online)

As major sources of reactive oxygen species (ROS), mitochondrial structures are exposed to high concentrations of ROS and may therefore be particularly susceptible to oxidative damage. Mitochondrial damage could play a pivotal role in the cell death decision. A decrease in mitochondrial energy charge and redox state, loss of transmembrane potential (depolarization), mitochondrial respiratory chain impairment, and release of substances such as calcium and cytochrome c all contribute to apoptosis. These mitochondrial abnormalities may constitute a part of the spectrum of chronic oxidative stress in Alzheimer’s disease. Accumulation of amyloid beta (Aβ) in form of senile plaques is also thought to play a central role in the pathogenesis of Alzheimer’s disease mediated by oxidative stress. In addition, increasing evidence shows that Aβ generates free radicals in vitro, which mediate the toxicity of this peptide.

In our study, PC12 cells were used to examine the protective features of EGb 761® (definition see editorial) on mitochondria stressed with hydrogen peroxide and antimycin, an inhibitor of complex III. In addition, we investigated the efficacy of EGb 761® in Aβ-induced MTT reduction in PC12 cells. Moreover, we examined the effects of EGb 761® on ROS levels and ROS-induced apoptosis in lymphocytes from aged mice after in vivo administration.

Here, we will report that EGb 761® was able to protect mitochondria from the attack of hydrogen peroxide, antimycin and Aβ. Furthermore, EGb 761® reduced ROS levels and ROS-induced apoptosis in lymphocytes from aged mice treated orally with EGb 761® for 2 weeks.

Our data further emphasize neuroprotective properties of EGb 761®, such as protection against Aβ-toxicity, and antiapoptotic properties, which are probably due to its preventive effects on mitochondria.

References

  • 1 Abe K, Saito H. Both oxidative stress-dependent and independent effects of amyloid beta protein are detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay.  Brain Res. 1999;  830 146-154
  • 2 Ahlemeyer B, Mowes A, Krieglstein J. Inhibition of serum deprivation- and staurosporine-induced neuronal apoptosis by Ginkgo biloba extract and some of its constituents.  Eur J Pharmacol. 1999;  367 423-430
  • 3 Aksenov M Y, Markesbery W R. Changes in thiol content and expression of glutathione redox system genes in the hippocampus and cerebellum in Alzheimer’s disease.  Neurosci Lett. 2001;  302 141-145
  • 4 Bastianetto S, Ramassamy C, Dore S, Christen Y, Poirier J, Quirion R. The Ginkgo biloba extract (EGb 761®) protects hippocampal neurons against cell death induced by beta-amyloid.  Eur J Neurosci. 2000;  12 1882-1890
  • 5 Bastianetto S, Zheng W H, Quirion R. The Ginkgo biloba extract (EGb 761®) protects and rescues hippocampal cells against nitric oxide-induced toxicity: involvement of its flavonoid constituents and protein kinase C.  J Neurochem. 2000;  74 2268-2277
  • 6 Beal M F. Mitochondria, free radicals, and neurodegeneration.  Curr Opin Neurobiol. 1996;  6 661-666
  • 7 Bosetti F, Brizzi F, Barogi S, Mancuso M, Siciliano G, Tendi E A, Murri L, Rapoport S I, Solaini G. Cytochrome c oxidase and mitochondrial F1F0-ATPase (ATP synthase) activities in platelets and brain from patients with Alzheimer’s disease.  Neurobiol Aging. 2002;  23 371-376
  • 8 Busciglio J, Peisman A, Wong C, Pigino G, Yuan M, Mori H, Yankner G A. Altered metabolism of the amyloid β precursor protein is associated with mitochondrial dysfunction in down's syndrome.  Neuron. 2002;  33 677-688
  • 9 Butterfield D A, Hensley K, Harris M, Mattson M, Carney J. Beta-Amyloid peptide free radical fragments initiate synaptosomal lipoperoxidation in a sequence-specific fashion: implications to Alzheimer's disease.  Biochem Biophys Res Commun. 1994;  200 710-715
  • 10 Casley C S, Canevari L, Land J M, Clark J B, Sharpe M A. Beta-amyloid inhibits integrated mitochondrial respiration and key enzyme activities.  J Neurochem. 2002;  80 91-100
  • 11 Ceruti S, Barbieri D, Veronese E, Cattabeni F, Cossarizza A, Giammarioli A M, Malorni W, Franceschi C, Abbracchio M P. Different pathways of apoptosis revealed by 2-chloro-adenosine and deoxy-D-ribose in mammalian astroglial cells.  J Neurosci Res. 1997;  47 372-383
  • 12 Chandrasekaran K, Mehrabian Z, Murray P D, Starkov A A, Spinnewyn B, Drieu K, Fiskum G. Ginkgo biloba extract (EGb 761®) and bilobalide stimulate mitochondrial gene expression and protect against ischemic and excitotoxic neuronal death.  Advances in Ginkgo biloba Extract Research. 2001;  8 139-151, ed. Y Christen, Solal editeur, Marseille
  • 13 Chandrasekaran K, Mehrabian Z, Spinnewyn B, Drieu K, Fiskum G. Neuroprotective effects of bilobalide, a component of the Ginkgo biloba extract (EGb 761®), in gerbil global brain ischemia.  Brain Res. 2001;  922 282-292
  • 14 Chen C, Wei T, Gao Z, Zhao B, Hou J, Xu H, Xin W, Packer L. Different effects of the constituents of EGb 761® on apoptosis in rat cerebellar granule cells induced by hydroxyl radicals.  Biochem Mol Biol Int. 1999;  47 397-405
  • 15 Chinopoulos C, Tretter L, Adam-Vizi V. Depolarization of in situ mitochondria due to hydrogen peroxide-induced oxidative stress in nerve terminals: inhibition of alpha-ketoglutarate dehydrogenase.  J Neurochem. 1999;  73 220-228
  • 16 Christen Y. Oxidative stress and Alzheimer disease.  Am J Clin Nutr. 2000;  71 621-629
  • 17 Dei R, Takeda A, Niwa H, Li M, Nakagomi Y, Watanabe M, Inagaki T, Washimi Y, Yasuda Y, Horie K, Miyata T, Sobue G. Lipid peroxidation and advanced glycation end products in the brain in normal aging and in Alzheimer’s disease.  Acta Neuropathol (Berl). 2002;  104 113-122
  • 18 Desagher S & Martinou J C. Mitochondria as the central control point of apoptosis.  Trends Cell Biol. 2000;  10 369-377
  • 19 Du G, Willet K, Mouithys-Mickalad A, Sluse-Goffart C M, Droy-Lefaix M T, Sluse F E. EGb 761® protects liver mitochondria against injury induced by in vitro anoxia/reoxygenation.  Free Radic Biol Med. 1999;  27 596-604
  • 20 Eckert A, Cotman C W, Zerfass R, Hennerici M, Muller W E. Enhanced vulnerability to apoptotic cell death in sporadic Alzheimer’s disease.  NeuroReport. 1998;  9 2443-2446
  • 21 Eckert A, Oster M, Zerfass R, Hennerici M, Müller W E. Elevated levels of fragmented DNA nucleosomes in native and activated lymphocytes indicate an enhanced sensitivity to apoptosis in sporadic Alzheimer’s disease: Specific differences to vascular dementia.  Dement Geriatr Cogn Disord. 2001;  12 98-105
  • 22 Eckert G P, Wood W G, Muller W E. Effects of aging and beta-amyloid on the properties of brain synaptic and mitochondrial membranes.  J Neural Transm. 2001;  108 1051-1064
  • 23 Forloni G, Chiesa R, Smiroldo S, Verga L, Salmona M, Tagliavini F, Angeretti N. Apoptosis mediated neurotoxicity induced by chronic application of beta amyloid fragment 25 - 35.  Neuroreport. 1993;  4 523-526
  • 24 Gibson G E. Interactions of oxidative stress with cellular calcium dynamics and glucose metabolism in Alzheimer’s disease(1,2).  Free Radic Biol Med. 2002;  32 1061-1070
  • 25 Gibson G E, Park L C, Sheu K F, Blass J P, Calingasan N Y. The alpha-ketoglutarate dehydrogenase complex in neurodegeneration.  Neurochem Int. 2000;  36 97-112
  • 26 Green D R, Reed J C. Mitochondria and apoptosis.  Science. 1998;  281 1309-1312
  • 27 Harman D. Free radical theory of aging.  Mutat Res. 1992;  275 257-266
  • 28 Hirai K, Aliev G, Nunomura A, Fujioka H, Russell R L, Atwood C S, Johnson A B, Kress Y, Vinters H V, Tabaton M, Shimohama S, Cash A D, Siedlak S L, Harris P L, Jones P K, Petersen R B, Perry G, Smith M A. Mitochondrial abnormalities in Alzheimer’s disease.  J Neurosci. 2001;  21 3017-3023
  • 29 Janssens D, Delaive E, Houbion A, Eliaers F, Remacle J, Michiels C. Effect of venotropic drugs on the respiratory activity of isolated mitochondria and in endothelial cells.  Br J Pharmacol. 2000;  130 1513-1524
  • 30 Janssens D, Delaive E, Remacle J, Michiels C. Protection by bilobalide of the ischaemia-induced alterations of the mitochondrial respiratory activity.  Fundam Clin Pharmacol. 2000;  14 193-201
  • 31 Janssens D, Michiels C, Delaive E, Eliaers F, Drieu K, Remacle J. Protection of hypoxia-induced ATP decrease in endothelial cells by Ginkgo biloba extract and bilobalide.  Biochem Pharmacol. 1995;  50 991-999
  • 32 Janssens D, Remacle J, Drieu K, Michiels C. Protection of mitochondrial respiration activity by bilobalide.  Biochem Pharmacol. 1999;  58 109-119
  • 33 Kanowski S, Herrmann W M, Stephan K, Wierich W, Horr R. Proof of efficacy of the Ginkgo biloba special extract EGb 761® in outpatients suffering from mild to moderate primary degenerative dementia of the Alzheimer type or multi-infarct dementia.  Pharmacopsychiatry. 1996;  29 47-56
  • 34 Keil U, Steiner B, Haass C, Müller W E, Eckert A. Mitochondrial dysfunction in oxidative stress-induced cell death of PC12 cells bearing APP mutations. Soc Neurosci 2002 28: abstract 193.1
  • 35 LaFerla F M, Tinkle B T, Bieberich C J, Haudenschild C C, Jay G. The Alzheimer’s A beta peptide induces neurodegeneration and apoptotic cell death in transgenic mice.  Nat Genet. 1995;  9 21-30
  • 36 Le Bars P L, Katz M M, Berman N, Itil T M, Freedman A M, Schatzberg A F. A placebo-controlled, double-blind, randomized trial of an extract of Ginkgo biloba for dementia. North American EGb Study Group.  JAMA. 1997;  278 1327-1332
  • 37 Le Bars P L, Kieser M, Itil K Z. A 26-week analysis of a double-blind, placebo-controlled trial of the Ginkgo biloba extract EGb 761® in dementia.  Dement Geriatr Cogn Disord. 2000;  11 230-237
  • 38 Le Bars P L, Velasco F M, Ferguson J M, Dessain E C, Kieser M, Hoerr R. Influence of the severity of cognitive impairment on the effect of the Ginkgo biloba extract EGb 761® in Alzheimer’s disease.  Neuropsychobiology. 2002;  45 19-26
  • 39 Lin M T, Simon D K, Ahn C H, Kim L M, Beal M F. High aggregate burden of somatic mtDNA point mutations in aging and Alzheimer’s disease brain.  Hum Mol Genet. 2002;  11 133-145
  • 40 Liu Y, Schubert D. Steroid hormones block amyloid fibril-induced 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) formazan exocytosis: relationship to neurotoxicity.  J Neurochem. 1998;  71 2322-2329
  • 41 Loo D T, Copani A, Pike C J, Whittemore E R, Walencewicz A J, Cotman C W. Apoptosis is induced by beta-amyloid in cultured central nervous system neurons.  Proc Natl Acad Sci USA. 1993;  90 7951-7955
  • 42 Lyras L, Cairns N J, Jenner A, Jenner P, Halliwell B. An assessment of oxidative damage to proteins, lipids, and DNA in brain from patients with Alzheimer’s disease.  J Neurochem. 1997;  68 2061-2069
  • 43 Misonou H, Morishima-Kawashima M, Ihara Y. Oxidative stress induces intracellular accumulation of amyloid beta-protein (Abeta) in human neuroblastoma cells.  Biochemistry. 2000;  39 6951-6959
  • 44 Munch G, Thome J, Foley P, Schinzel R, Riederer P. Advanced glycation endproducts in ageing and Alzheimer’s disease.  Brain Res Brain Res Rev. 1997;  23 134-143
  • 45 Müller W E, Eckert G P, Eckert A, Scheuer K, Cairns N, Maras A, and Gattaz W F. Effects of beta-amyloid peptides on fluidity of human brain membranes at deep and superficial regions: high potencies of A-beta 1 - 42 and A-beta 1 - 43.  Amyloid. 1998;  5 10-15
  • 46 Müller W E, Koch S, Eckert A, Hartmann H, Scheuer K. Beta-Amyloid peptide decreases membrane fluidity.  Brain Res. 1995;  674 133-136
  • 47 Ni Y, Zhao B, Hou J, Xin W. Preventive effect of Ginkgo biloba extract on apoptosis in rat cerebellar neuronal cells induced by hydroxyl radicals.  Neurosci Lett. 1996;  214 115-118
  • 48 Nicholls D G, Budd S L. Mitochondria and neuronal survival.  Physiol Rev. 2000;  80 315-360
  • 49 Nunomura A, Perry G, Aliev G, Hirai K, Takeda A, Balraj E K, Jones P K, Ghanbari H, Wataya T, Shimohama S, Chiba S, Atwood C S, Petersen R B, Smith M A. Oxidative damage is the earliest event in Alzheimer disease.  J Neuropathol Exp Neurol. 2001;  60 759-767
  • 50 Orth M & Schapira A H. Mitochondria and degenerative disorders.  Am J Med Genet. 2001;  106 27-36
  • 51 Oyama Y, Chikahisa L, Ueha T, Kanemaru K, Noda K. Ginkgo biloba extract protects brain neurons against oxidative stress induced by hydrogen peroxide.  Brain Res. 1996;  712 349-352
  • 52 Pike C J, Walencewicz A J, Glabe C G, Cotman C W. Aggregation-related toxicity of synthetic beta-amyloid protein in hippocampal cultures.  Eur J Pharmacol. 1991;  207 367-368
  • 53 Reed J C. Mechanisms of apoptosis.  Am J Pathol. 2000;  157 1415-1430
  • 54 Reed J C, Jurgensmeier J M. Matsuyama S Bcl-2 family proteins and mitochondria.  Biochim Biophys Acta. 1998;  1366 127-137
  • 55 Sastre J, Millan A, Garcia de la Asuncion J, Pla R, Juan G, Pallardo  F V, O’Connor E, Martin J A, Droy-Lefaix M T, Vina J. A Ginkgo biloba extract (EGb 761®) prevents mitochondrial aging by protecting against oxidative stress.  Free Radic Biol Med. 1998;  24 298-304
  • 56 Schindowski K, Leutner S, Kressmann S, Eckert A, Muller W E. Age-related increase of oxidative stress-induced apoptosis in mice prevention by Ginkgo biloba extract (EGb 761®).  J Neural Transm. 2001;  108 969-978
  • 57 Schindowski K, Leutner S, Müller W E, Eckert A. Age-related changes of apoptotic cell death in human lymphocytes.  Neurobiol Aging. 2000;  21 661-670
  • 58 Schüssel K, Leutner S, Cairns N J, Eckert A, Müller W E. Increased activities of antioxidant enzymes in brains from Alzheimer’s disease patients.  Pharmacopsychiatry. 2001;  34 200
  • 59 Schwartz-Bloom R D, Ahrens R, Galeffi F, Lingle K, Zhang L. Do benzodiazepines protect neurons by acting at the GABAA receptor or directly at the mitochondria?. ACNP 41th meeting, Puerto Rico 2002, abstract 291
  • 60 Schwartz-Bloom R D, Sah R. γ-Aminobutyric acidA neurotransmission and cerebral ischemia.  J Neurochem. 2001;  77 353-371
  • 61 Selkoe D J. Alzheimer's disease is a synaptic failure.  Science. 2002;  298 789-795
  • 62 Semlitsch H V, Anderer P, Saletu B, Binder G A, Decker K A. Cognitive psychophysiology in nootropic drug research: effects of Ginkgo biloba on event-related potentials (P300) in age-associated memory impairment.  Pharmacopsychiatry. 1995;  28 134-142
  • 63 Song W, Guan H J, Zhu Z X, Chen Z L, Yin M L, Cheng X F. Protective effects of bilobalide against nitric oxide-induced neurotoxicity in PC12 cells.  Acta Pharmacol Sin. 2000;  21 415-420
  • 64 Stoll S, Scheuer K, Pohl O, Müller W E. Ginkgo biloba extract (EGb 761®) independently improves changes in passive avoidance learning and brain membrane fluidity in the aging mouse.  Pharmacopsychiatry. 1996;  29 144-149
  • 65 Susin S A, Zamzami N, Kroemer G. Mitochondria as regulators of apoptosis: doubt no more.  Biochim Biophys Acta. 1998;  1366 151-165
  • 66 Tatton W G, Chalmers-Redman R M. Mitochondria in neurodegenerative apoptosis: an opportunity for therapy?.  Ann Neurol. 1998;  44 (Suppl.1) 134-141
  • 67 Tendi E A, Bosetti F, Dasgupta S F, Stella A M G, Drieu K, Rapoport S I. Ginkgo biloba Extracts EGb 761® and bilobalide increase NADH dehydrogenase mRNA level and mitochondrial respiratory control ratio in PC12 cells.  Neurochem Int. 2002;  27 319-323
  • 68 Wadia J S, Chalmers-Redman R ME, Ju W JH, Carlile G W, Phillips J L, Fraser A D, Tatton W G. Mitochondrial membrane potential and nuclear changes in apoptosis caused by serum and nerve growth factor withdrawal: time course and modification by (-)-deprenyl.  J Neurosci. 1998;  18 932-947
  • 69 Wei T, Ni Y, Hou J, Chen C, Zhao B, Xin W. Hydrogen peroxide-induced oxidative damage and apoptosis in cerebellar granule cells: protection by Ginkgo biloba extract.  Pharmacol Res. 2000;  41 427-433
  • 70 Xin W, Wei T, Chen C, Ni Y, Zhao B, Hou J. Mechanisms of apoptosis in rat cerebellar granule cells induced by hydroxyl radicals and the effects of EGb 761® and its constituents.  Toxicology. 2000;  148 103-110
  • 71 Yao Z, Drieu K, Papadopoulos V. The EGb 761® Ginkgo biloba extract rescues the PC12 neuronal cells from beta-amyloid-induced cell death by inhibiting the formation of beta- amyloid-derived diffusible neurotoxic ligands.  Brain Res. 2001;  889 181-190
  • 72 Zhou L J, Song W, Zhu X Z, Chen Z L, Yin M L, Cheng X F. Protective effects of bilobalide on amyloid beta-peptide 25 - 35-induced PC12 cell cytotoxicity.  Acta Pharmacol Sin. 2000;  21 75-79
  • 73 Zhou L J, Zhu X Z. Reactive oxygen species-induced apoptosis in PC12 cells and protective effect of bilobalide.  J Pharmacol Exp Ther. 2000;  293 982-988

PD Dr. Anne Eckert

Dept. of Pharmacology

Biocenter

University of Frankfurt

Marie-Curie-Str. 9

D-60439 Frankfurt

Germany

Phone: (+49) 69 79829377

Fax: (+49) 69 79829374

Email: A.Eckert@em.uni-frankfurt.de