The standardized Ginkgo biloba extract EGb 761® (definition see editorial) has been
shown to produce neuroprotective effects in different in vivo and in vitro models. Since EGb 761® is a complex mixture containing flavonoid glycosides, terpene
lactones (non-flavone fraction) and various other constituents, the question arises
as to which of these compounds mediates the protective activity of EGb 761®. Previous
studies have demonstrated that the non-flavone fraction was responsible for the antihypoxic
activity of EGb 761®. Thus, we examined the neuroprotective and anti-apoptotic ability
of the main constituents of the non-flavone fraction, the ginkgolides A, B, C, J and
bilobalide. In focal cerebral ischemia models, the administration of bilobalide (5
- 20 mg/kg, s. c.) 60 min before ischemia dose-dependently reduced the infarct area
in mouse brain and the infarct volume in rat brain 2 days after the onset of the injury.
30 minutes of pretreatment with ginkgolide A (50 mg/kg, s. c.) and ginkgolide B (100
mg/kg, s. c.) reduced the infarct area in the mouse model of focal ischemia. In primary
cultures of hippocampal neurons and astrocytes from neonatal rats, ginkgolide B (1
μM) and bilobalide (10 μM) protected the neurons against damage caused by glutamate
(1 mM, 1 h) as evaluated by trypan blue staining. In addition, bilobalide (0.1 μM)
was able to increase the viability of cultured neurons from chick embryo telencepalon
when exposed to cyanide (1 mM, 1h). Furthermore, we attempted to find out whether
ginkgolides A, B, and J and bilobalide were also able to inhibit neuronal apoptosis
(determined by nuclear staining with Hoechst 33 258 and TUNEL-staining). Ginkgolide
B (10 μM), ginkgolide J (100 μM) and bilobalide (1 μM) reduced the apoptotic damage
induced by serum deprivation (24h) or treatment with staurosporine (200 nM, 24h) in
cultured chick embryonic neurons. Bilobalide (100 μM) rescued cultured rat hippocampal
neurons from apoptosis caused by serum deprivation (24h), whereas ginkgolide B (100
μM) and bilobalide (100 μM) reduced apoptotic damage induced by staurosporine (300
nM, 24h). Ginkgolide A failed to affect apoptotic damage neither in serum-deprived
nor in staurosporine-treated neurons. The results suggest that some of the constituents
of the non-flavone fraction of EGb 761® possess neuroprotective and anti-apoptotic
capacity, and that bilobalide is the most potent one. In contrast, ginkgolic acids
(100 - 500 μM) induced neuronal death, which showed features of apoptosis as well
as of necrosis, but these constituents were removed from EGb 761® below an amount
of 0.0005 %. Taking together, there is experimental evidence for a neuroprotective
effect of EGb 761® that agrees with clinical studies showing the efficacy of an oral
treatment in patients with mild and moderate dementia.
References
- 1
Ahlemeyer B, Selke D, Schaper C, Klumpp S, Krieglstein J.
Ginkgolic acids induce neuronal death and activate protein phosphatase type-2C.
Eur J Pharmacol.
2001;
430
1-7
- 2
Ahlemeyer B, Möwes 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 Ahlemeyer B, Krieglstein J. Nenroprotective effects of the Ginkgo biloba extract. In:
Lawson LD, Bauer R. (Eds.)
Phytomedicine of Europe: Chemistry and Biological activity. ACS Symposium Series 691, American Chemical Society Washington DC; 1998: pp. 210-220
- 4
Backhauß C, Karkoutly C, Welsch M, Krieglstein J.
A mouse model of focal cerebral ischemia for screening neuroprotective drug effects.
J Pharmacol Meth.
1992;
27
23-27
- 5
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 β-amyloid.
Eur J Neurosci.
2000;
12
1882-1890
- 6
Bazan N G, Squinto S P, Braquet P, Panetta T, Marcheselli V L.
Platelet activating factor and polyunsaturated fatty acids in cerebral ischemia or
convulsions: intracellular PAF-binding sites and activation of a Fos/Jun/AP-1 transcriptional
signalling system.
Lipids.
1991;
26
1236-1242
- 7
Bielenberg G W, Sauer D, Nuglisch J, Beck T, Roßberg C, Mennel H D, Krieglstein J.
Effects of emopamil on postischemic blood flow and neuronal damage in rat brain.
Naunyn Schmiedeberg’s Arch Pharmacol.
1989;
339
230-235
- 8 Chatterjee S S, Gabard B. Protective effect of an extract of Ginkgo biloba and other
hydroxyl radical scavengers against hypoxia. VIII International Congress on Pharmacology
Tokyo; 1981: pp. 483-486
- 9
Chen C, Wei T T, Gao Z H, Zhao B L, Hou J W, Xu H B, Xin W J, 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
- 10 DeFeudis F V. Ginkgo biloba extract (EGb 761®). From chemistry to the clinic. Ullstein
Medical Wiesbaden; 1998
- 11
Droy-Lefaix M T.
Effect of the antioxidant action of Ginkgo biloba extract (EGb 761®) on aging and
oxidative stress.
Age.
1997;
20
141-149
- 12
Ernst E, Pittler M H.
Ginkgo biloba for dementia: A systematic review of double-blind, placebo-controlled
trials.
Clin Drug Invest.
1999;
17
301-308
- 13
Ginsberg M D, Busto R.
Rodent models of cerebral ischemia.
Stroke.
1989;
20
1627-1642
- 14
Guidetti C, Paracchini S, Lucchini S, Cambieri M, Marzatico F.
Prevention of neuronal cell damage induced by oxidative stress in- vitro: effect of
different Ginkgo biloba extracts.
J Pharm Pharmacol.
2001;
53
387-392
- 15
Husain S R, Cillard J, Cillard P.
Hydroxyl radical scavenging activity of flavonoids.
Phytochemistry.
1987;
26
2489-2491
- 16
Janssens D, Remacle J, Drieu K, Michiels C.
Protection of mitochondrial respiration activity by bilobalide.
Biochem Pharmacol.
1999;
58
109-119
- 17
Kanowski S, Herrmann W M, Stephan K, Wierich W, Hörr 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 of multi-infarct
dementia.
Pharmacopsychiatry.
1996;
29
47-56
- 18
Karcher L, Zagermann P, Krieglstein J.
Effect of an extract of Ginkgo biloba on rat brain energy metabolism in hypoxia.
Naunyn-Schmiedeberg’s Arch Pharmacol.
1984;
327
31-35
- 19
Kleijnen J, Knipschild P.
Ginkgo biloba.
Lancet.
1992;
340
1136-1139
- 20
Krieglstein J, Beck T, Seibert A.
Influence of an extract of Ginkgo biloba on cerebral blood flow and metabolism.
Life Sci.
1986;
39
2327-2334
- 21
Krieglstein J, Ausmeier F, El-Abhar H, Lippert K, Welsch M, Rupalla K, Henrich-Noack P.
Neuroprotective effects of Ginkgo biloba constituents.
Eur J Pharm Sci.
1995;
3
39-48
- 22
Le Bars P, Katz M M, Herman 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.
J Am Med Assoc.
1997;
278
1327-1332
- 23
Le Bars P L, Velasco P M, Ferguson J M, Dessain E G, Kieser M, Hörr 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
- 24
Lugasi A, Horhavovich P, Dworschak E.
Additional information to the in vitro antioxidant activity of Ginkgo biloba.
L Phytother Res.
1999;
13
160-162
- 25
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
- 26
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
- 27
Nuglisch J, Karkoutly C, Mennel H D, Roßberg C, Krieglstein J.
Protective effect of nimodipine against ischemic neuronal damage in rat hippocampus
without changing postischemic cerebral blood flow.
J Cereb Blood Flow Metab.
1990;
10
654-659
- 28
Nunez D, Chignard M, Korth R, Le Couedic J P, Norel X, Spinnewyn B, Braquet P, Benveniste J.
Specific inhibition of paf-acether-induced platelet activition by BN 52 021 and comparison
with the paf-acether inhibitors Kadsurenone and CV 3988.
Eur J Pharmacol.
1986;
123
197-205
- 29
Oberpichler H, Beck T, Abdel-Rahman M M, Bielenberg G W, Krieglstein J.
Effects of Ginkgo biloba constituents related to protection against brain damage caused
by hypoxia.
Pharmacol Res Commun.
1988;
20
349-368
- 30
Oberpichler H, Sauer D, Roßberg C, Mennel H D, Krieglstein J.
PAF-antagonist ginkgolide B reduced postischemic neuronal damage in rat brain hippocampus.
J Cereb Blood Flow Metab.
1990;
10
133-135
- 31
Oppenheim R W.
Cell death during development of the nervous system.
Ann Rev Neurosci.
1991;
14
453-501
- 32
Oyama Y, Fuchs P A, Katayam N, Noda K.
Myricetin and quercetin, the flavonoid constituents of Ginkgo biloba extract, greatly
reduce oxidative metabolism in both resting and Ca2+-loaded brain neurons.
Brain Res.
1994;
28
125-129
- 33
Oyama Y, Chikashia L, Ueha T, Kanemaru K, Noda K.
Ginkgo biloba extract protects brain neurons against oxidative stress induced by hydrogen
peroxide.
Brain Res.
1996;
18
349-352
- 34
Panetta T, Marcheselli V L, Braquet P, Spinnewyn B, Bazan N G.
Effect of a platelet activating factor antagonist (BN 52 021) on free fatty acids,
diacylglycerols, polyphosphoinositides and blood flow in the gerbil brain: inhibition
of ischemia-reperfusion induced cerebral injury.
Biochem Biophys Res Commun.
1987;
149
580-587
- 35
Pierre S, Jamme I, Droy-Lefaix M T, Nouvelot A, Maixent J M.
Ginkgo biloba extract (EGb 761®) protects Na,K-ATPase activity during cerebral ischemia.
NeuroReport.
1999;
10
47-51
- 36
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
733-742
- 37
Pincemail J, Thirion A, Dupuis M, Braquet P, Drieu K, Deby C.
Ginkgo biloba extract inhibits oxygen species production generated by phorbol myristate
acetate stimulated human leukocytes.
Experientia.
1987;
43
181-184
- 38
Prehn J HM, Krieglstein J.
Platelet-activating factor antagonists reduce excitotoxic damage in cultured neurons
from embryonic chick telencephalon and protect the rat hippocampus and neocortex from
ischemic injury in vivo.
J Neurosci Res.
1993;
34
179-188
- 39
Robak J, Gryglewski R J.
Flavonoids are scavengers of superoxide anions.
Biochem Pharmacol.
1998;
37
837-841
- 40
Schindowski K, Leutner S, Kressmann S, Eckert A, Müller 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
- 41
Scholtyssek H, Damerau W, Wessel R, Schminke I.
Antioxidative activity of ginkgolides against superoxide in an aprotic environment.
Chemico-Biol Interactions.
1997;
106
183-190
- 42
Shi H, Niki E.
Stoichiometric and kinetic studies on Ginkgo biloba extract and related antioxidants.
Lipids.
1998;
33
365-370
- 43
Smith M L, Bendek G, Dahlgren N, Rosen I, Wieloch T, Siesjö B K.
Models for studying long-term recovery following forebrain ischemia in the rat 2.
A 2-vessel occlusion model.
ActaNeurol Scand.
1984;
69
385-401
- 44
Tamura A, Graha D J, McCulloch J, Teasdale G M.
Focal ischemia in the rat: 1. Description of technique and early neuropathological
consequences following middle cerebral artery occlusion.
J Cereb Blood Flow Metab.
1981;
1
53-60
- 45
Thompson C B.
Apoptosis in the pathogenesis and treatment of disease.
Science.
1995;
267
1456-1462
- 46
Touvay C, Vilain B, Taylor J E, Etienne A, Braquet P.
Proof of the involvement of platelet-activating factor (PAF-acether) in pulmonary
complex immune system using a specific PAF acether receptor antagonist: BN 52 021.
Prog Lipid Res.
1986;
25
277-288
- 47
Welsch M, Nuglisch J, Krieglstein J.
Neuroprotective effect of nimodipine is not mediated by increased cerebral blood flow
after transient forebrain ischemia in rats.
Stroke.
1990;
21
96-105
- 48
Wettstein A.
Cholinesterase inhibitors and Ginkgo extracts - are they comparable in the treatment
of dementia? Comparison of published placebo-controlled efficacy studies of at least
six months’ duration.
Phytomedicine.
2000;
6
393-401
- 49
Zhou L J, Song W, Zhu X Z, Chen Z L, Yin M L, Cheng X F.
Protective effect of bilobalide on amyloid beta-peptide 25 - 35-induced PC12 cell
cytotoxicity.
Acta Pharmacol Sin.
2000;
21
75-79
Prof. Dr. Dr. Josef Krieglstein
Institut für Pharmakologie und Toxikologie
Fachbereich Pharmazie der Philipps-Universität Marburg
Ketzerbach 63
35032 Marburg
Germany
Phone: (49) 6421-2821311 (secretariat)
Fax: (49) 6421-2828918
Email: krieglst@mailer.uni-marburg.de
