Pharmacological Research 60 (2009) 6873

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Pharmacological Research
j our nal homepage: www. el sevi er . com/ l ocat e/ yphr s
Ginkgo biloba extract (EGb761

) inuences monoaminergic neurotransmission

via inhibition of NE uptake, but not MAO activity after chronic treatment
Christian J. Fehske, Kristina Leuner, Walter E. Mller

Department of Pharmacology, ZAFES, Biocenter, University of Frankfurt, Frankfurt, Germany

a r t i c l e i n f o
Article history:
Received 28 November 2008
Received in revised form 12 February 2009
Accepted 13 February 2009
Keywords:
Ginkgo biloba extract
EGb761

Monoaminergic neurotransmission
Norepinephrine uptake
MAO activity
a b s t r a c t
In order to explain cognition-enhancing effects of standardized Ginkgo biloba extract (EGb761

), an
increase of central monoaminergic neurotransmission has been suggested, but the underlying mech-
anisms have not yet been elucidated. Here, we conrm that the norepinephrine (NET), the serotonin
(SERT), the dopamine (DAT) uptake transporters and MAO activity are inhibited by EGb761

in vitro,
although rather high concentrations are required for inhibition of MAO-A and MAO-B activity. However,
after 14 days of daily oral treatment with 100mg/kg EGb761

only NE uptake is signicantly decreased in

NMRI mice, while 5-HT uptake and MAO activity are not affected. As synaptic dopamine clearance in the
frontal cortex is mediated by NET, not DAT, these ndings may give an explanation for the enhancement
of dopaminergic neurotransmission by EGb761

seen in animal models, presumably linked to its positive

effects on cognition and attention.
2009 Published by Elsevier Ltd.
1. Introduction
Ginkgo biloba has a long history of medical use for treating con-
ditions of cerebral dysfunction associated with brain aging and
neurodegenerative dementia [1,2]. Substantial evidence for clinical
efcacy of standardized extracts (EGb761

) exists [3,4,5,6], even if

this topic is still a matter of scientic debate [6,7,8]. Many molecular
mechanisms have been suggested to explain its ability to alleviate
or even improve cognitive impairment associated with brain aging
or dementia, such as improvement of mitochondrial dysfunction
[9,10,11], inhibition of amyloid beta-neurotoxicity [12,13], improve-
ment of membrane uidity parameters [14,15] or attenuation of
increased oxidative stress [11,16]. All these mechanisms share close
relationship to the current understanding of the neuropathology
of Alzheimers disease [16,17,18]. However, additional mechanisms
might be assumedas EGb761

alsoseems tohave behavioral effects

in man and animals not easily explainable with the mechanisms
given above [19,20,21]. For example, EGb761

has been reported

to elevate biogenic amine levels in animals [22,23], but investi-
Abbreviations: DA,
3
H-dopamine; DAT, dopamine transporters; EGb76
1
,
standardized Ginkgo biloba extract; HEPES, 4-2-hydroxyethyl-1-
piperazineethanesufonic acid; MAO, monoamine oxidase; NE,
3
H-norepinephrine;
NET, norepinephrine transporter; NMRI, Naval Medical Research Institute; SERT,
serotonin transporter; PE,
14
C-phenylethylamine.

Corresponding author at: Department of Pharmacology, Biocenter Niederursel,

University of Frankfurt, N260, 1.OG, Max-von-Laue-Str.9, 60438 Frankfurt, Germany.
Tel.: +49 69 79829373; fax: +49 69 79829374.
E-mail address: Pharmacolnat@em.uni-frankfurt.de (W.E. Mller).
gations into the underlying mechanisms have been inconclusive
so far. Earlier ndings addressing this issue found that EGb761

affects synaptosomal uptake of biogenic amines in vitro [24], but in

vivo signicance of these ndings was not demonstrated. Ramas-
samy et al. [24] also found that EGb761

at very lowconcentrations
enhances synaptosomal serotonin (5-hydroxytryptamine (5-HT))
uptake similar to the antidepressant drug tianeptine. Studies for
other uptake transporters (e.g. NET) are yet lacking. Several groups
have reported in vitro monoamine oxidase (MAO) inhibiting prop-
erties of EGb761

[22,25,26], but again in vivo relevance of these

ndings has been challenged by negative ndings in functional and
biochemical studies [27,22]. Accordingly, we performed a compar-
ative in vitro and in vivo study on the possible effects of EGb761

on
synaptosomal uptake of biogenic amines andonMAO-AandMAO-B
activity.
2. Materials and methods
2.1. Chemicals and radiochemicals
Agarose, sucrose and all buffer constituents, ethyl acetate,
toluene, desipramine, 5-hydroxytryptamine, pargylineHCl, and
dopamine were from SigmaAldrich at highest purity available.
FluoxetineHCl was a kind gift of STADA (Bad Vilbel, Germany),
3
H-5-HT,
14
C-5-HT (54mCi/mmol) and
14
C-phenylethylamine
(PE) (54mCi/mmol) were from GE Healthcare (Munich, Ger-
many),
3
H-norepinephrine (NE) and
3
H-dopamine (DA) from
BIOTREND

(Cologne, Germany). EGb761

, ginkgolides A, B, C
and J, bilobalide and ginkgo-avonoids were kindly provided
1043-6618/$ see front matter 2009 Published by Elsevier Ltd.
doi:10.1016/j.phrs.2009.02.012
C.J. Fehske et al. / Pharmacological Research 60 (2009) 6873 69
by Dr. Willmar Schwabe Arzneimittel GmbH (Karlsruhe, Ger-
many).
2.2. Animals and treatment procedures
Female NMRI mice (Naval Medical Research Institute; 3 months
old, body weights from 2530g) were obtained from Harlan-
Winkelmann (Borchen, Germany); female Wistar rats were from
Charles River (Sulzfeld, Germany). All animals were housed and
treated in strict accordance with the guidelines of the German Pro-
tection of Animals Act.
For acute treatment studies, the mice were randomly assigned
to groups of 10 animals per group. Animals were weighed, and
received individually dosed oral treatments according to their body
weights in 0.2% agarose as vehicle, or vehicle only. They were killed
1 or 4h after treatment for determination of acute ex vivo effects.
For subchronic treatment studies, mice were randomly assigned to
groups of 10 animals per group and received the treatments once
daily, adjusted to their body weight and were killed 24h after the
last treatment for the determination of subchronic ex vivo effects.
2.3. Synaptosomal uptake assays
The synaptosomal uptake assays were performed as published
earlier [28]. Briey, freshly obtained brain tissue from either NMRI
mice or Wistar rats was immediately homogenized with a Potter
S homogenizer (B. Braun, Melsungen, Germany) with 10 strokes
at 300 RPM in 15ml of ice-cold 0.32M sucrose solution and sub-
sequently diluted with another 10ml. The nuclear fraction was
eliminated by centrifugation (10min at 750g; 04

C) and the
supernatant again centrifuged (20min at 17400g; 04

C) to
obtain the crude synaptosomal pellet. The pellet was resuspended
in modied ice-cold Krebs-HEPES buffer (pH 7.4, NaCl 150mM, KCl
6.2mM, Na
2
HPO
4
1.2mM, MgSO
4
1.2mM, HEPES 10mM, glucose
10mM, ascorbic acid 1% and pargyline 10M) to different concen-
trations of wet weight/ml depending on the assay performed: 5-HT
50mg/ml (frontal cortex, NMRI mice), NE 35mg/ml (occipital cor-
tex, NMRI mice), DA 25mg/ml (striatum, Wistar rat). Uptake into
synaptosomes was determined in the presence of increasing con-
centrations of test substances. After preincubationat 37

C(15min),
all metabolic processes were disrupted in an ice-cold water bath
(4

C, 5min), before addition of [

3
H] labeled substrates and main
incubation at 37

C started quantiable uptake (5-HT: 4min, NE:

10min, DA: 5min). After ltration with a Brandel 24-well harvester
machine andWhatmanGF/Clters, lters were washedthree times
(NE-uptake: two times) with 2ml buffer (or ice-cold 0.9% NaCl
solution for DA-uptake). Radioactivity was determined by liquid
scintillation counting (LSC). All determinations were performed in
triplicates.
2.4. MAO activity assays
The MAO activity assay was performed as published earlier
[28]. In short, aliquots of brain homogenate were diluted 1:5 (v/v)
with assay buffer (K
2
HPO
4
50mM, DTT 10M, EDTA 2.5mM, BSA
0.5mg/ml, pH7.5), and 50l thereof were preincubated with 60l
of the test substances for 30min at RT. Subsequently, after addition
of 30l of
14
C-5-HT (65M, for MAO-A assay) or
14
C--PE (10M,
MAO-B assay) enzymatic reaction was started in a water bath at
37

C for 6min and terminated by addition of 250l 4mol/l HCl.

The
14
C-labeled deaminated products are more lipophilic and were
extracted with 1ml toluene/ethylacetate (1:1) by vigorous shaking
for 1min600l of theorganic phasewerecarefullyremovedfor LSC
quantication. Blank samples were obtained by incubating heat
inactivated brain homogenates (96

C, 10min) with radio labeled

substrates. All determinations were performed in triplicates.
2.5. MAO activity and uptake measurement in ex vivo studies
In all ex vivo studies, one or two vehicle treated control animals
were killed at the same time as the respective drug treated animals
and investigated in parallel. Data were expressed in percent of the
respective control.
2.6. Liquid scintillation counting
For determination of radioactively labeled substances, 4ml
of Lumasafe Plus

(PerkinElmer, Rodgau-Jgesheim, Germany)

were added to samples in Zinsser-Mini tubes (Zinsser Analytic,
Eschborn, Germany) and shaken for 2h on a conventional vertical
shaker. Subsequently liquid scintillation counting was performed
in a Canberra Packard Tricarb 1900 TR with 60s counting time with
optimized protocols.
2.7. Statistics
Data are presented as means SEM. To determine IC
50
values
in uptake experiments eight concentration points and GraphPad
Prism 4.03 softwares nonlinear regression analysis were used. For
statistical comparison the following levels of signicance were
used: p>0.05: n.s.; *p<0.05; **p<0.01. For statistical comparison
of different treatment groups for ex vivo effects, one-way ANOVA
with Dunnetts multiple comparison test was calculated, with the
respective control group (vehicle treated) as reference.
3. Results
First, we investigated possible effects of EGb761

on MAOactiv-
ities in vitro and ex vivo after subchronic daily treatment. We found
a preferential MAO-Aover MAO-BinhibitionbyEGb761

(about 10-
fold, see Fig. 1). However, with an IC
50
value of close to 200g/ml
inhibition of MAO-A is rather weak. This was supported by ex vivo
experiments, where no effects on MAO-A (Fig. 2A) or MAO-B activ-
ities (Fig. 2B) were found after 2 weeks of daily oral treatment with
100mg/kg EGb761

. Positive control pargyline (10mg/kg), a prefer-

ential MAO-Binhibitor, reducedMAO-Bactivity by about 50%under
these conditions.
Next, we investigated effects of EGb761

on monoaminer-
gic reuptake transporters in vitro. As shown in Fig. 3, EGb761

concentration-dependently inhibited synaptosomal uptake

of dopamine, serotonin and norepinephrine (IC
50
values:
281148g/ml, 9769g/ml and 359g/ml). To clarify
if the uptake is also affected after oral treatment with EGb761

,
we tested ex vivo effects of acute and subchronic treatment on
5-HT and NE uptake. 1 and 4h after acute treatment, 5-HT uptake
Fig. 1. EGb761

preferentially inhibits MAO-A over MAO-B activity in vitro: pargy-

line preferentially inhibits MAO-B over MAO-A (IC
50
6.71 and 592500nM, not
shown). Data are means SEM of three determinations.
70 C.J. Fehske et al. / Pharmacological Research 60 (2009) 6873
Fig. 2. After 14 days of daily oral treatment with EGb761

(100mg/kg daily) does

not affect MAO-A or MAO-B activity compared to vehicle treated control animals.
Positive control pargyline (10mg/kg for 14 days) reduced MAO-B activity by 51.3%
(p<0.05). Data are means SEM of six animals.
appeared to be higher in EGb761

-treated animals compared to

control animals (20% increase 1h after, 15% increase 4h after treat-
ment). However, both failed to reach the p<0.05 signicance level
(see Fig. 4A and C). Positive controls for 5-HT and NE uptake were
the SSRI uoxetine (10mg/kg) and the tricyclic drug desipramine
(25mg/kg), both showing signicant and robust ex vivo reduction
in specic synaptosomal uptake of 5-HT (Fig. 4A andC) or NE
(Fig. 4B and D), respectively, 1 and 4h after treatment.
For subchronic treatment, animals received 100mg/kg EGb761

daily for 14 consecutive days. 24h after the last treatment specic
uptake was comparedtovehicle treatedcontrol animals (Fig. 4Eand
F). EGb761

-treatment did not affect 5-HT uptake, but NE-Uptake

was reduced signicantly by 30% (p<0.05). Again, positive controls
uoxetine and desipramine were much more active.
Fig. 3. EGb761

inhibits uptake of dopamine (IC

50
: 281148g/ml), but at
even lower concentrations of serotonin and norepinephrine (97.269 and
34.79g/ml) in vitro. Depicted are representative experiments for each neuro-
transmitter. IC
50
data are means SEM of six determinations using 10 different
EGb761

concentrations.
Since EGb761

consists of many different constituents, we

investigated if NE- and 5-HT-inhibition could be attributed to one
or more of the known pharmacologically active compounds, i.e.
ginkgolides A, B, C and J, bilobalide or the avonoid fraction. As
depicted in Fig. 5, all of the before mentioned compounds signi-
cantly inhibited 5-HT uptake at 100g/ml, but none of them was
signicantly active at 10g/ml. At 100g/ml, the avonoid frac-
tion had the strongest effect (57% reduction), followed by ginkgolid
A (48% reduction) and bilobalide (43% reduction), the other ones
only about 25% or less (Fig. 5A). NE-uptake, however, was signi-
cantly affected only by two of the compounds tested: The avonoid
fraction, at both 100 and 10g/ml, and ginkgolid A (Fig. 5B).
4. Discussion
The mechanisms behind cognition-enhancing effects of the
standardized G. biloba extract EGb761

are not fully understood

yet. A number of animal studies points towards an increase
in monoaminergic neurotransmission [26,29,30]. Earlier studies
implicated inhibition of MAO as possibly involved in these effects
[25,26,30]. However, several other studies provided evidence that
MAO inhibition by EGb761

might not be relevant in vivo. Fowler

et al. [27] showed in 10 healthy subjects receiving a commercially
available EGb761

preparation for 4 weeks. Using a PET detection

method for MAO-A and MAO-B, no signicant differences between
active treatment and placebo treated subjects were found. As the
method is specic to have a minimum sensitivity level to detect
changes of about 15%, it cannot be excluded that an even lower
level of inhibition of MAO might have occurred. Porsolt et al. [31]
reported possible effects of EGb761

in four functional tests indica-

tive of in vivo MAO inhibition and found signicant effects only in
one of the tests for the highest concentration tested (100mg/kg, 4
days, i.p.). He concluded that MAO inhibition was not the mecha-
nism primarily responsible for EGb761s antistress activity.
In the present study, we rst conrmed that EGb761

in vitro
preferentially inhibits MAO-A over MAO-B (10), although at
rather highconcentrations. However, EGb761

didnot affect ex vivo

brain MAO activities after 14 days of treatment in agreement with
the studies mentioned above.
Afewstudies have addressed possible inuences of EGb761

on
monoaminergic uptake mechanisms in different experimental set-
tings. Ramassamy et al. [32,33] reported that EGb761

prevents a
reduction of synaptosomal 5-HT or DA uptake potentially via reac-
tiveoxygenspecies, whichcanbeeasilyexplainedbythefreeradical
scavenging properties. In another investigation, they found that in
vitro and ex vivo EGb761

enhanced 5-HT uptake at very low and

potentially therapeutically relevant concentrations [24]. In accor-
dance with these ndings, in our present study we found a similar,
however smaller and not signicant trend towards an increase of
5-HT uptake 1 and 4h after acute application (Fig. 4A and C). Our in
vitro studies also conrm uptake inhibitory properties for 5-HT at
higher concentrations (Fig. 3), however, a in vitro uptake enhanc-
ing effects at low concentrations were not consistently seen in our
experiments (data not shown).
Investigations addressing noradrenergic neurotransmission
have been performed by Huguet and Tarrade [34], who found that
the age-related decrease of
2
-binding-sites in rat cerebral cortex
was prevented by G. biloba extract treatment, indicating a relative
increase of noradrenergic neurotransmission. Pointing in the same
direction, Brunello et al. [29] reported a 5-fold increase of the NE
metabolite normethanephrine (NMN) in the cerebral cortex of rats
after 14 days of treatment with G. biloba extract. In our study, we
show that Egb761

not only inhibits NE uptake in vitro, but more

importantly that this effect can also be seen ex vivo after 14 days of
daily oral treatment using a daily dose which also has been shown
C.J. Fehske et al. / Pharmacological Research 60 (2009) 6873 71
Fig. 4. Inuence of EGb761

on ex vivo uptake of 5-HT and NE. AD showacute treatment studies, E and F results after subchronic treatment. Fluoxetine reduces 5-HT uptake
by 73% 1h after treatment (A), and by 77% 4h after treatment (C). Desipramine reduces ex vivo NE uptake by 89% 1h after treatment (B), and by 92% 4h after treatment (D).
After subchronic treatment with uoxetine, 5-HT uptake is decreased by 91% (E). Desipramine treatment for 14 days results in no detectable specic NE uptake (F). Subchronic
treatment with EGb761

reduces NE uptake by 30% (F). Data are means SEM of 10 animals for vehicle control and EGb761

group, and ve animals for positive control

groups.
Fig. 5. Effects of different major compounds of EGb761

on in vitro 5-HT uptake (A)

and NE uptake (B). At 100g/ml, all of the compounds tested inhibit 5-HT uptake to
some extent, the avonoid fraction being most active. Data are means SEM of six
determinations.
to exhibit antidepressant effects under similar conditions [35,36].
Trying to attribute these effects to a particular fraction of the stan-
dardized G. biloba extract, we were able to show that the most
promising constituents seem to be the avonoid fraction.
In line with our ndings of NE uptake inhibitory properties of
EGb761

not only in vitro but also in vivo are reports on antide-

pressant activities in preclinical tests [21]. In an investigation on
age-dependant behavioral effects, EGb761

was found to reduce

immobility time in the forced swim test in 24 months old, but not
8 months old rats [35]. In another study with young rats, Porsolt
et al. [36] also found no activity of EGb761

in the forced swim

test paradigm. Yet in the same investigation, Egb761

prevented
learned helplessness, a well accepted behavioral paradigm indica-
tive of antidepressant activity [36]. Two recent reports in rats and
mice conrmed the putative antidepressant properties of EGB761

in the forced swim test and the learned helplessness paradigms

[45,46]. Supporting the notion of antidepressant-like properties of
EGb761

are observations from clinical studies, where substan-

tial improvement of impaired mood and depressive symptoms was
observed [37,38].
Treatment with the tricyclic antidepressant desipramine that
affects NE uptake preferably over 5-HT uptake, and other inhibitors
of the NE transporter have been shown to enhance cognition in
behavioral tests, supporting the idea of a link between NE uptake
72 C.J. Fehske et al. / Pharmacological Research 60 (2009) 6873
and cognition [39,40]. Moreover, in the prefrontal cortex, dopamin-
ergic synapses are different from elsewhere in the brain due to
the lack of DAT for synaptic clearance of dopamine [41,42,43].
Dopamine transporters are known to be rather promiscuous, for
example NET is known to transport DA with even higher afnities
than NE [44]. As a result, DA released in the frontal cortex also acti-
vates postsynaptic receptors of neighbored synapses until reaching
a noradrenergic nerve ending expressing NET, where it is trans-
ported across the membrane and thus cleared froma rather distant
synaptic cleft than where it was released (volume neurotransmis-
sion) [41]. Thus, it seems quite likely that EGb761

, like other
inhibitors of NET also enhances dopaminergic neurotransmission
in the prefrontal cortex [42].
Our novel ndings are perfectly in line with the report by Kehr
et al. [23], who showed that a similar subchronic treatment with
EGb761

enhanced extracellular concentrations of dopamine and

norepinephrine but not of serotonin in the prefrontal cortex of the
rat. Considering the crucial role of the prefrontal dopamine for cog-
nitive function in general and working memory in specic [43] and
the cognition improving properties of other drugs that inhibit NE
uptake [47], this effect of Egb761

very likely can contribute to its

benecial properties on attention, learning, and memory function.
Taken together, our results show an additional and probably
therapeutically relevant mechanism for EGb761

. By inhibition
of NET, some cognition enhancing and mood elevating effects of
EGb761

are easily explainable.

Acknowledgement
This study was supported by Dr. Willmar Schwabe GmbH (Karl-
sruhe, Germany).
References
[1] DeFeudis FV. A brief history of EGb 761 and its therapeutic uses. Pharmacopsy-
chiatry 2003;36(Suppl. 1):S27.
[2] Zimmermann M, Colciaghi F, Cattabeni F, Di Luca M. Ginkgo biloba extract: from
molecular mechanisms to the treatment of Alzheimers disease. Cell Mol Biol
(Noisy-le-grand) 2002;4848:61323.
[3] Le Bars PL, Katz MM, BermanN, Itil TM, FreedmanAM, SchatzbergAF. Aplacebo-
controlled, double-blind, randomized trial of an extract of Ginkgo biloba for
dementia. JAMA 1997;278278:132732.
[4] Le Bars PL, Kieser M, Itil KZ. A 26-week analysis of a double-blind, placebo-
controlledtrial of theGinkgobilobaextract EGb761indementia. Dement Geriatr
Cogn Disord 2000;1111:2307.
[5] Kanowski S, Herrmann WM, Stephan K, Wierich W, Horr R. Proof of efcacy
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;2929:4756.
[6] Napryeyenko O, Borzenko I. Ginkgo biloba special extract in dementia with neu-
ropsychiatric features. A randomised, placebo-controlled, double-blind clinical
trial. Arzneimittelforschung 2007;5757:411.
[7] Schneider LS, DeKosky ST, Farlow MR, Tariot PN, Hoerr R, Kieser M. A ran-
domized, double-blind, placebo-controlled trial of two doses of Ginkgo biloba
extract in dementia of the Alzheimers type. Curr Alzheimer Res 2005;22:
54151.
[8] Birks J, Grimley EJ. Ginkgo biloba for cognitive impairment and dementia.
Cochrane Database Syst Rev 2007. CD003120.
[9] Abdel-Kader R, Hauptmann S, Keil U, Scherping I, Leuner K, Eckert A, et al. Stabi-
lizationof mitochondrial functionbyGinkgobilobaextract (EGb761). Pharmacol
Res 2007;5656:493502.
[10] Eckert A, Keil U, Scherping I, Hauptmann S, Muller WE. Stabilization of
mitochondrial membrane potential and improvement of neuronal energy
metabolismby Ginkgo biloba extract EGb 761. Ann NYAcad Sci 2005;10561056:
47485.
[11] Sastre J, Millan A, Garcia dlA, Pla R, Juan G, Pallardo, et al. AGinkgo biloba extract
(EGb 761) prevents mitochondrial aging by protecting against oxidative stress.
Free Radic Biol Med 1998;2424:298304.
[12] Luo Y, SmithJV, ParamasivamV, Burdick A, Curry KJ, BufordJP, et al. Inhibitionof
amyloid-beta aggregation and caspase-3 activation by the Ginkgo biloba extract
EGb761. Proc Natl Acad Sci USA 2002;9999:12197202.
[13] Bastianetto S, Ramassamy C, Dore S, Christen Y, Poirier J, Quirion R. The
Ginkgo bilobaextract (EGb761) protects hippocampal neurons against cell death
induced by beta-amyloid. Eur J Neurosci 2000;1212:188290.
[14] Stoll S, Scheuer K, Pohl O, Muller WE. Ginkgo biloba extract (EGb 761)
independently improves changes in passive avoidance learning and brain
membrane uidity in the aging mouse. Pharmacopsychiatry 1996;2929:
1449.
[15] Ramassamy C, Girbe F, Christen Y, Costentin J. Ginkgo biloba extract EGb 761
or trolox C prevent the ascorbic acid/Fe2+ induced decrease in synaptosomal
membrane uidity. Free Radic Res Commun 1993;1919:34150.
[16] Smith JV, Luo Y. Elevation of oxidative free radicals in Alzheimers disease
models can be attenuated by Ginkgo biloba extract EGb 761. J Alzheimers Dis
2003;55:287300.
[17] Hauptmann S, Keil U, Scherping I, Bonert A, Eckert A, Muller WE. Mitochon-
drial dysfunction in sporadic and genetic Alzheimers disease. Exp Gerontol
2006;4141:66873.
[18] Eckert A, Keil U, Marques CA, Bonert A, Frey C, Schussel K, et al. Mitochondrial
dysfunction, apoptotic cell death, and Alzheimers disease. BiochemPharmacol
2003;6666:162734.
[19] Maclennan KM, Darlington CL, Smith PF. The CNS effects of Ginkgo biloba
extracts and ginkgolide B. Prog Neurobiol 2002;6767:23557.
[20] DeFeudis FV, Drieu K. Ginkgo biloba extract (EGb 761) and CNS functions: basic
studies and clinical applications. Curr Drug Targets 2000;11:2558.
[21] Muller WE, Chatterjee SS. Cognitive and other behavioral effects of EGb 761 in
animal models. Pharmacopsychiatry 2003;36(Suppl. 1):S2431.
[22] Sloley BD, Urichuk LJ, Morley P, Durkin J, Shan JJ, Pang PK, et al. Identication
of kaempferol as a monoamine oxidase inhibitor and potential neuropro-
tectant in extracts of Ginkgo biloba leaves. J Pharm Pharmacol 2000;5252:
4519.
[23] Kehr J, Noldner M, YoudimMB. Effects of chronic administrationof Ginkgobiloba
extract (EGb761

) on levels of dopamine, noradrenaline and serotonin in the

prefrontal cortex of the awake rat. Planta Med 2006;7272:9611089.
[24] Ramassamy C, Christen Y, Clostre F, Costentin J. The Ginkgo bilobaextract,
EGb761, increases synaptosomal uptake of 5-hydroxytryptamine: in-vitro and
ex-vivo studies. J Pharm Pharmacol 1992;4444:9435.
[25] White HL, Scates PW, Cooper BR. Extracts of Ginkgo biloba leaves inhibit
monoamine oxidase. Life Sci 1996;5858:131521.
[26] Wu WR, Zhu XZ. Involvement of monoamine oxidase inhibitioninneuroprotec-
tive and neurorestorative effects of Ginkgo bilobaextract against MPTP-induced
nigrostriatal dopaminergic toxicity in C57 mice. Life Sci 1999;6565:
15764.
[27] Fowler JS, Wang GJ, Volkow ND, Logan J, Franceschi D, Franceschi M, et al. Evi-
dence that Ginkgo bilobaextract does not inhibit MAO A and B in living human
brain. Life Sci 2000;6666:L1416.
[28] Muller WE, Rolli M, Schafer C, Hafner U. Effects of hypericum extract (LI
160) in biochemical models of antidepressant activity. Pharmacopsychiatry
1997;30(Suppl. 2):1027.
[29] BrunelloN, Racagni G, Clostre F, DrieuK, Braquet P. Effects of anextract of Ginkgo
biloba on noradrenergic systems of rat cerebral cortex. Pharmacol Res Commun
1985;1717:106372.
[30] Pardon MC, Joubert C, Perez-Diaz F, Christen Y, Launay JM, Cohen-Salmon C.
In vivo regulation of cerebral monoamine oxidase activity in senescent con-
trols and chronically stressed mice by long-term treatment with Ginkgo biloba
extract (EGb 761). Mech Ageing Dev 2000;113113:15768.
[31] Porsolt RD, Roux S, Drieu K. Evaluation of a Ginkgo biloba extract (EGb 761)
in functional tests for monoamine oxidase inhibition. Arzneimittelforschung
2000;5050:2325.
[32] Ramassamy C, Clostre F, Christen Y, Costentin J. Prevention by a Ginkgo biloba
extract (GBE 761) of the dopaminergic neurotoxicity of MPTP. J PharmPharma-
col 1990;4242:7859.
[33] Ramassamy C, Naudin B, Christen Y, Clostre F, Costentin J. Prevention by
Ginkgo biloba extract (EGb 761) and trolox C of the decrease in synaptoso-
mal dopamine or serotonin uptake following incubation. Biochem Pharmacol
1992;4444:2395401.
[34] Huguet F, Tarrade T. Alpha 2-adrenoceptor changes during cerebral ageing. The
effect of Ginkgo biloba extract. J Pharm Pharmacol 1992;4444:247.
[35] Angoli A, Rapain JR, Scapagnini V, Weitbrecht WV, editors. Effects of Ginkgo
biloba extract on organic cerebral impairment. London Paris: J Libbey; 1985.
pp. 3542.
[36] Porsolt RD, Martin P, Lenegre A, Fromage S, Drieu K. Effects of an extract of
Ginkgo biloba (EGB 761) on learned helplessness and other models of stress
in rodents. Pharmacol Biochem Behav 1990;3636:96371.
[37] Hoerr R. Behavioural and psychological symptoms of dementia (BPSD): effects
of EGb 761. Pharmacopsychiatry 2003;36(Suppl. 1):S5661.
[38] Scripnikov A, Khomenko A, Napryeyenko O. Effects of Ginkgo biloba extract EGb
761 on neuropsychiatric symptoms of dementia: ndings from a randomised
controlled trial. Wien Med Wochenschr 2007;157:295300.
[39] Lapiz MD, Bondi CO, Morilak DA. Chronic treatment withdesipramine improves
cognitive performance of rats in an attentional set-shifting test. Neuropsy-
chopharmacology 2007;3232:100010.
[40] Arnsten AF, Li BM. Neurobiology of executive functions: catecholamine
inuences on prefrontal cortical functions. Biol Psychiatry 2005;5757:
137784.
[41] Stahl SM. Neurotransmission of cognition. Part 1. Dopamine is a hitchhiker in
frontal cortex: norepinephrine transporters regulate dopamine. J Clin Psychia-
try 2003;6464:45.
[42] Bymaster FP, Katner JS, Nelson DL, Hemrick-Luecke SK, Threlkeld PG, Heiligen-
stein JH, et al. Atomoxetine increases extracellular levels of norepinephrine
and dopamine in prefrontal cortex of rat: a potential mechanism for ef-
C.J. Fehske et al. / Pharmacological Research 60 (2009) 6873 73
cacy in attention decit/hyperactivity disorder. Neuropsychopharmacology
2002;2727:699711.
[43] Seamans JK, Yang CR. The principal features and mechanisms of dopamine
modulation in the prefrontal cortex. Prog Neurobiol 2004;7474:158.
[44] Bonisch H, Bruss M. The norepinephrine transporter in physiology and disease.
In: Handbook of experimental pharmacology; 2006. pp. 485524.
[45] Sakakibara H, Ishida K, Grundmann O, Nakajima J, Seo S, Butterweck V, et al.
Antidepressant effect of extracts fromGinkgobilobaleaves inbehavioral models.
Biochem Pharm Bull 2006;29:176770.
[46] Kalkunte SS, Gingh AP, Chaves FC, Gianfagna TJ, Pundir VS, Jaiswal
AK, et al. Antidepressant and antistress activity of GCMS character-
ized lipophilic extracts of Ginkgo biloba leaves. Phytother Res 2007;21:
10615.
[47] Stahl SM. Neurotransmissionof cognition. Part 2. SelectiveNRIs aresmart drugs:
exploiting regionally selective actions on both dopamine and norepinephrine
to enhance cognition. J Clin Psychiatry 2003;64(2), 110.1.