Neuroprotective Effects of Bilobalide, a Component of Ginkgo biloba Extract (EGb 761®) in Global Brain Ischemia and in Excitotoxicity-induced Neuronal Death

In this study, we compared the protective effect of bilobalide, a purified terpene lactone component of ginkgo biloba extract (EGb 761®), (definition see editorial) and EGb 761® against ischemic injury and against glutamate-induced excitotoxic neuronal death. In ischemic injury, we measured neuronal loss and the levels of mitochondrial DNA (mtDNA)-encoded cytochrome oxidase (COX) subunit III mRNA in vulnerable hippocampal regions of gerbils. At 7 days of reperfusion after 5 min of transient global ischemia, a significant increase in neuronal death and a significant decrease in COX III mRNA were observed in the hippocampal CA1 neurons. Oral administration of EGb 761® at 25, 50 and 100 mg/kg/day and bilobalide at 3 and 6 mg/kg/day for 7 days before ischemia progressively protected CA1 neurons from death and from ischemia-induced reductions in COX III mRNA. In rat cerebellar neuronal cultures, addition of bilobalide or EGb 761® protected in a dose-dependent manner against glutamate-induced excitotoxic neuronal death (effective concentration [EC₅₀] = 5 μg/ml (12 μM) for bilobalide and 100 μg/ml for EGb 761®). These results suggest that both EGb 761® and bilobalide are protective against ischemia-induced neuronal death in vivo and glutamate-induced neuronal death in vitro by synergistic mechanisms involving anti-excitotoxicity, inhibition of free radical generation, scavenging of reactive oxygen species, and regulation of mitochondrial gene expression.

Abbreviations

NMDA:N-methyl-D-aspartate

ROS:reactive oxygen species

mtDNA:mitochondrial DNA

Hippocampus - Histology - In situ hybridization - Mitochondrial mRNA - Cytochrome oxidase

References

• 1 Abe K, Kawagoe J, Kogure K. Early disturbance of a mitochondrial DNA expression in gerbil hippocampus after transient forebrain ischemia.  Neurosci Lett. 1993;  153(2) 173-176
• 2 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 (6) 2268-2277
• 3 Chandrasekaran K, Liu L I, Hatanpaa K, Drieu K, Rapoport S I. Stimulation of mitochondrial gene expression by bilobalide, a component of Ginkgo biloba extract (EGb 761®). In: Packer L, Christen Y, editors Ginkgo biloba Extract (EGb 761®): Lessons from Cell Biology. Paris; Elsevier 1998: 121-128
• 4 Chandrasekaran K, Mehrabian Z, Drieu K, Fiskum G. Bilobalide and ginkgo biloba extract (EGb 761®) inhibit excitotoxic neuronal death through glycine antagonism. Soc Neurosci 26, 857.14 2000
• 5 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(2) 282-292
• 6 Chandrasekaran K, Stoll J, Brady D R, Rapoport S I. Localization of cytochrome oxidase (COX) activity and COX mRNA in the hippocampus and entorhinal cortex of the monkey brain: correlation with specific neuronal pathways.  Brain Res. 1992;  579(2) 333-336
• 7 Clark W M, Rinker L G, Lessov N S, Lowery S L, Cipolla M J. Efficacy of antioxidant therapies in transient focal ischemia in mice.  Stroke. 2001;  32(4) 1000-1004
• 8 Clemens J A. Cerebral ischemia: gene activation, neuronal injury, and the protective role of antioxidants.  Free Radic Biol Med. 2000;  28(10) 1526-1531
• 9 Clostre F. [Ginkgo biloba extract (EGb 761®). State of knowledge in the dawn of the year 2000]. Ann Pharm Fr 1999 57 Suppl 1: S8-88
• 10 Curras M C, Pallotta B S. Single-channel evidence for glycine and NMDA requirement in NMDA receptor activation.  Brain Res. 1996;  740(1 - 2) 27-40
• 11 DeFeudis F V. Ginkgo biloba extract (EGb 761®). Ullstein Medical 1998
• 12 Fiskum G, Murphy A N, Beal M F. Mitochondria in neurodegeneration: acute ischemia and chronic neurodegenerative diseases.  J Cereb Blood Flow Metab. 1999;  19(4) 351-369
• 13 Fiskum G. Mitochondrial damage during cerebral ischemia.  Ann Emerg Med. 1985;  14(8) 810-815
• 14 Flamm E S, Demopoulos H B, Seligman M L, Poser R G, Ransohoff J. Free radicals in cerebral ischemia.  Stroke. 1978;  9(5) 445-447
• 15 Glantz S. Primer of Biostatistics. New York, NY; Mc Graw-Hill 1997
• 16 Goldberg M P, Weiss J H, Pham P C, Choi D W. N-methyl-D-aspartate receptors mediate hypoxic neuronal injury in cortical culture.  J Pharmacol Exp Ther. 1987;  243(2) 784-791
• 17 Hevner R F, Wong-Riley M T. Neuronal expression of nuclear and mitochondrial genes for cytochrome oxidase (CO) subunits analyzed by in situ hybridization: comparison with CO activity and protein.  J Neurosci. 1991;  11(7) 1942-1958
• 18 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(3) 193-201
• 19 Janssens D, Remacle J, Drieu K, Michiels C. Protection of mitochondrial respiration activity by bilobalide.  Biochem Pharmacol. 1999;  58(1) 109-119
• 20 Johnson J W, Ascher P. Glycine potentiates the NMDA response in cultured mouse brain neurons.  Nature. 1987;  325(6104) 529-531
• 21 Kirino T. Delayed neuronal death in the gerbil hippocampus following ischemia.  Brain Res. 1982;  239(1) 57-69
• 22 Kleckner N W, Dingledine R. Regulation of hippocampal NMDA receptors by magnesium and glycine during development.  Brain Res Mol Brain Res. 1991;  11(2) 151-159
• 23 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 [see comments].  JAMA. 1997;  278(16) 1327-1332
• 24 Le Poncin L, Rapin J, Rapin J R. Effects of Ginkgo biloba on changes induced by quantitative cerebral microembolization in rats.  Arch Int Pharmacodyn Ther. 1980;  243(2) 236-244
• 25 Lewen A, Matz P, Chan P H. Free radical pathways in CNS injury [In Process Citation].  J Neurotrauma. 2000;  17(10) 871-890
• 26 Marcocci L, Packer L, Droy-Lefaix M T, Sekaki A, Gardes-Albert M. Antioxidant action of Ginkgo biloba extract EGb 761®.  Methods Enzymol. 1994;  234 462-475
• 27 McBain C J, Mayer M L. N-methyl-D-aspartic acid receptor structure and function.  Physiol Rev. 1994;  74(3) 723-760
• 28 Pietri S, Maurelli E, Drieu K, Culcasi M. Cardioprotective and anti-oxidant effects of the terpenoid constituents of Ginkgo biloba extract (EGb 761®).  J Mol Cell Cardiol. 1997;  29(2) 733-742
• 29 Pietri S, Seguin J R, d’Arbigny P, Drieu K, Culcasi M. Ginkgo biloba extract (EGb 761®) pretreatment limits free radical- induced oxidative stress in patients undergoing coronary bypass surgery.  Cardiovasc Drugs Ther. 1997;  11(2) 121-131
• 30 Rapin J R, Zaibi M, Drieu K. In vitro and in vivo effects of an extract of Ginkgo biloba (EGb 761®), ginkgolide B, and bilobalide on apoptosis in primary cultures of rat hippocampal neurons.  Drug Develop Res. 1998;  45(1) 23-29
• 31 Reynolds I J, Miller R J. [3H]MK801 binding to the NMDA receptor/ionophore complex is regulated by divalent cations: evidence for multiple regulatory sites.  Eur J Pharmacol. 1988;  151(1) 103-112
• 32 Reynolds I J, Miller R J. [3H]MK801 binding to the N-methyl-D-aspartate receptor reveals drug interactions with the zinc and magnesium binding sites.  J Pharmacol Exp Ther. 1988;  247(3) 1025-1031
• 33 Schousboe A, Meier E, Drejer J, Hertz L. Preparation of primary cultures of mouse (rat) cerebellar granule cells. In: Shahar A, de Vellis J, Vernadakis A, Haber B, editors A Dissection and Tiisue Culture Manual of the Nervous System. New York; Alan R Liss, Inc 1989: 203-206
• 34 Seif-El-Nasr M, El-Fattah A A. Lipid peroxide, phospholipids, glutathione levels and superoxide dismutase activity in rat brain after ischaemia: effect of ginkgo biloba extract.  Pharmacol Res. 1995;  32(5) 273-278
• 35 Spinnewyn B, Blavet N, Clostre F. [Effects of Ginkgo biloba extract on a cerebral ischemia model in gerbils] Effets de l’extrait de Ginkgo biloba sur un modele d’ischemie cerebrale chez la gerbille.  Presse Med. 1986;  15(31) 1511-1515
• 36 Spinnewyn B. Ginkgo biloba extract (EGb 761®) protects against delayed neuronal death in gerbil. In: Y.Christen, J.Costentin, M.Lacour, editors Effect of Ginkgo biloba extract (EGb 761®) on the Central Nervous System. Elsevier Paris; 1992: 113-118
• 37 Traystman R J, Kirsch J R, Koehler R C. Oxygen radical mechanisms of brain injury following ischemia and reperfusion.  J Appl Physiol. 1991;  71(4) 1185-1195
• 38 Weichel O, Hilgert M, Chatterjee S S, Lehr M, Klein J. Bilobalide, a constituent of Ginkgo biloba, inhibits NMDA-induced phospholipase A2 activation and phospholipid breakdown in rat hippocampus.  Naunyn Schmiedebergs Arch Pharmacol. 1999;  360(6) 609-615
• 39 Westman J, Drieu K, Sharma H S. Antioxidant compounds EGb 761® and BN-520 21 attenuate heat shock protein (HSP 72 kD) response, edema and cell changes following hyperthermic brain injury. An experimental study using immunohistochemistry in the rat.  Amino Acids. 2000;  19(1) 339-350
• 40 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 EGb761 and its constituents.  Toxicology. 2000;  148(2 - 3) 103-110
• 41 Zhang W R, Hayashi T, Kitagawa H, Sasaki C, Sakai K, Warita H. et al . Protective effect of ginkgo extract on rat brain with transient middle cerebral artery occlusion.  Neurol Res. 2000;  22(5) 517-521