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Pharmacological Research 56 (2007) 493502

Stabilization of mitochondrial function by Ginkgo biloba extract


(EGb 761)
Reham Abdel-Kader a , Susanne Hauptmann a , Uta Keil a , Isabel Scherping a ,
Kristina Leuner a , Anne Eckert b , Walter E. Muller a,
a
b

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


Neurobiology Research Laboratory, Psychiatric University Clinic, Basel, Switzerland
Accepted 10 September 2007

Abstract
A large body of data emphasizes the central role of mitochondrial dysfunction during aging and as an early event in neurodegenerative diseases.
In this study we used PC12 cells and dissociated mice brain cells, as well as isolated mitochondria to investigate the effects of EGb 761 on
mitochondrial functions. We mimicked mitochondrial abnormalities during aging by using external factors (nitrosative stress, serum deprivation
and complexes inhibitors) consequently altering mitochondrial processes, such as energy metabolism. As markers for the function of mitochondria,
ATP levels and mitochondrial membrane potential were measured.
EGb 761 alleviated mitochondrial functions in vitro at concentrations as low as 0.01 mg/ml. Treating two different age groups of mice with EGb
761 (100 mg/kg body weight for 14 days) showed beneficial effects on complexes I, IV and V of the mitochondrial respiratory chain and against
nitrosative stress. Interestingly, these effects were only observed in the aged mice group, proving higher efficacy of EGb 761 during aging.
The single components of EGb 761 showed in both cell models protection of the mitochondrial membrane potential indicating that a complementary action of the components is responsible for the versatile actions of EGb 761.
2007 Elsevier Ltd. All rights reserved.
Keywords: Ginkgo biloba extract; Mitochondria; Mitochondrial respiratory chain; Aging; Oxidative stress; Nitrosative stress

1. Introduction
The standardized Ginkgo biloba extract (EGb 761) is recommended for the treatment of geriatric memory disorders
including vascular and neurodegenerative dementia. Its use is
steadily increasing around the world. Several clinical studies
have repeatedly shown the efficacy of EGb 761 in the treatment
of mild-to-moderate dementia of different etiology, but negative
data have also been published [15].

Abbreviations: AD, Alzheimers disease; R123, Rhodamine 123; ROS,


reactive oxygen species; RNS, reactive nitrogen species; TMRE, tetramethyl-rhodamine-ethyl-ester; TTFA, thenoyl-trifluoroacetone; NaN3 , sodium
azide; NMRI, Naval Medical Research Institute; HEPES, 4-2-hydroxyethyl-1piperazineethanesufonic acid; DMEM, Dulbeccos modified Eagles medium;
SNP, sodium nitroprusside; NO, nitric oxide; PC12, pheochromocytoma cells;
COX, cytochrome oxidase.
Corresponding author at: Department of Pharmacology, 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. Muller).
1043-6618/$ see front matter 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phrs.2007.09.011

Decline in mitochondrial function is known to play a key


role in the aging process of the brain. Alteration in mitochondrial membrane potential, increase in mitochondria-based
oxidative stress and decline in respiratory chain activity are all
age-associated mitochondrial changes [611]. Moreover, mitochondrial dysfunction is highly associated with the pathogenesis
of aging-related diseases including Alzheimers disease (AD)
[1214]. A large body of data highlights the importance of mitochondrial dysfunction in cellular and animal models of AD as
well as in tissues of AD patients [1519].
Beneficial effects of EGb 761 have repeatedly been associated
with aging-related mechanisms of brain function. EGb 761 consists of two major groups of substances, the flavonoid fraction
(24%) and the terpenoid fraction (6%). The flavonoid fraction is
primarily composed of quercetin, kaempferol and isorhamnetin
glycosides and the terpenoid fraction consists of ginkgolides A,
B, C, J and bilobalide. The concentration of toxic ginkgolic acid
in the extract is restricted to 5 ppm [20]. EGb 761 exhibits versatile biochemical and pharmacological effects that were assumed
to be mainly due to its free radical scavenging properties but

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more recently it is proposed that these effects are a result of its


direct protective effects on the mitochondria.
Free radical scavenger activity of EGb 761 was demonstrated
in several in vitro and in vivo models [2124]. Decrease in cell
survival and an increase in free radical accumulation were attenuated in rat primary mixed hippocampal cell cultures by both
EGb 761 and its flavonoid fraction [25]. It was also shown that
both the flavonoid component of EGb 761 and a mixture of
flavonoids and terpenes protected cerebellar granule cells from
oxidative damage and apoptosis induced by hydroxyl radicals
[26].
Several other studies highlighted the anti-apoptotic properties
of EGb 761 and its constituents [27,28]. EGb 761 up-regulated
the anti-apoptotic protein Bcl-2 and inhibited the activation
of caspases 9 and 3 [2730]. Furthermore, ginkgolides B, J
and bilobalide inhibited staurosporine- and serum deprivationinduced apoptosis [31]. Additionally, bilobalide was able to
attenuate ROS-induced elevation of the pro-apoptotic protein
Bax [32].
In a previous study, we showed that EGb 761 protects mitochondrial function in PC12 cells by stabilizing mitochondrial
membrane potential after stimulation with hydrogen peroxide
[33]. We therefore wanted to get further information about the
mitochondria-protective effects of EGb 761 and its constituents
both in vitro and ex vivo, using different methods provoking
mitochondrial dysfunction. We investigated the influence of EGb
761 on mitochondrial function after oxidative stress, nitrosative
stress and serum deprivation. As markers for the function of
mitochondria, mitochondrial membrane potential and ATP levels were measured. Moreover, we analyzed the effects of EGb
761 after inhibition of the mitochondrial respiratory chain using
complexes inhibitors. In order to get convincing proof for a direct
protective activity of EGb 761 on the mitochondria, experiments
were not only carried out in PC12 cells and dissociated mouse
brain cells but also in isolated mitochondria obtained from EGb
761-treated mice.

2. Materials and methods


2.1. Chemicals
Rhodamine 123 (R123) and tetramethylrhodamineethylester
(TMRE) were purchased from Invitrogen (Karlsruhe, Germany).
ViaLight HT kit was purchased from Cambrex, Verviers
(Brussels, Belgium). Sodium nitroprusside (SNP), rotenone,
thenoyltrifluoroacetone (TTFA), antimycine, sodium azide
(NaN3 ), oligomycine and tributyltin were obtained from Sigma
(Munich, Germany). Bovine serum albumin was purchased from
BIO-RAD (Munich, Germany). Caspase-9 substrate II was purchased by Merck Biosciences (Darmstadt, Germany). EGb 761
and its constituents were a gift from Dr. Willmar Schwabe (Karlsruhe, Germany). EGb 761 is a standardized Ginkgo biloba
extract. This concentrated extract is standardized to contain
24% flavonol glycosides, 6% terpene trilactones (of these, 2.9%
ginkgolides A, B, C and J and 3.1% bilobalide), and <5 ppm
ginkgolic acids [34].

2.2. Cell culture


PC12 cells were cultured in Dulbeccos modified Eagles
medium (DMEM) supplemented with 10% heat-inactivated fetal
calf serum and 5% heat-inactivated horse serum, 50 units/ml
penicillin and 50 g/ml streptomycin at 37 C in a humidified
incubator containing 5% CO2 . In order to measure mitochondrial membrane potential PC12 cells were plated at a density of
2 105 cells/well in a 24-well plate, and for ATP measurements
they were plated at a density of 2 104 cells/well in a 96-well
plate.
2.3. Mice
Female NMRI (Naval Medical Research Institute) mice used
in this study were purchased from Harlan-Winkelmann GmbH
(Borchen, Germany). All animals were housed in plastic cages
with water and food ad libitum. They were maintained on a 12-h
light:12-h dark cycle. All animal procedures were conducted in
accordance to the German legal requirements and guidelines for
animal care.
2.4. Dissociated brain cells
Mice were sacrificed by decapitation and brains were quickly
dissected on ice (method modified after Stoll et al. [35]). After
removing the cerebellum, the tissue was minced in 2 ml of
medium I (NaCl 138 mM, KCl 5.4 mM, Na2 HPO4 0.17 mM,
K2 PO4 0.22 mM, glucose 5.5 mM and sucrose 58.4 mM, pH
7.35) with a scalpel and further dissociated by trituration through
a nylon mesh (pore diameter 1 mm) with a Pasteur pipette.
The resulting suspension was filtered by gravity through a
fresh nylon mesh with a pore diameter of 102 M and the
dissociated cell aggregates were washed twice with medium
II (NaCl 110 mM, KCl 5.3 mM, CaCl2 H2 O 1.8 mM, MgCl2 6
H2 O 1 mM, glucose 25 mM, sucrose 70 mM and HEPES 20 mM
[4-2-hydroxyethyl-1-piperazineethanesufonic acid], pH 7.4) by
centrifugation (400 g for 3 min at 4 C). Fifty microliters of the
suspension were used for protein determination. After centrifugation, cells were re-suspended in 6 ml DMEM and distributed
on a 48-well plate for the measurement of mitochondrial membrane potential and in a 96-well plate for ATP measurement.
The protein content was determined by the method of Lowry
et al. [36], using bovine serum albumin as standard (BIO-RAD,
Munich, Germany).
2.5. Isolated mitochondria
Mice were sacrificed by decapitation, and brain hemispheres
were rapidly dissected on ice and washed in an ice-cold buffer
(210 mM mannitol, 70 mM sucrose, 10 mM HEPES, 1 mM
EDTA, 0.45% bovine serum albumin, 0.5 mM dithiothreitol,
and complete protease inhibitor mixture tablets (Roche Diagnostics). After removing the cerebellum, the tissue samples were
homogenized in 2 ml buffer with a glass homogenizer (1015
strokes, 400 rpm), and the resulting homogenate was centrifuged
at 1400 g for 7 min at 4 C to remove nuclei and tissue par-

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495

Fig. 1. Protective effects of EGb 761 after NO insult in PC12 cells. (A) PC12 cells were incubated with SNP, after 30 min medium was exchanged and then EGb 761
was added for 23 h. ATP levels and mitochondrial membrane potential were measured. Data are shown as means S.E.M. (n = 818).* p < 0.05, ** p < 0.01 vs. SNP
damage, Students unpaired t-test. (B) PC12 cells were incubated with SNP for 24 h, after 30 min EGb 761 was added and ATP levels and mitochondrial membrane
potential were measured. Data are expressed as means S.E.M. (n = 618). * p < 0.05, *** p < 0.001 vs. SNP damage, Students unpaired t-test. (C) Treatment with
EGb 761 reduces the increase in caspase-9 activity after exposure to SNP. PC12 cells were incubated with EGb 761 for 22 h, followed by addition of SNP for 2 h.
Data are expressed as means S.E.M. (n = 5). + p < 0.05 vs. control untreated cells,* p < 0.05 vs. SNP damage, Students unpaired t-test.

ticles. The low-speed centrifugation step was repeated once


with the supernatant. Then, the supernatant fraction was centrifuged at 10,000 g for 5 min at 4 C to obtain a mitochondrial
pellet. The resulting pellet was re-suspended in 1 ml of icecold buffer and centrifuged again at 800 g for 3 min at 4 C
Finally, the mitochondria-enriched supernatant was centrifuged
at 10,000 g for 5 min at 4 C to obtain a mitochondrial fraction.
This fraction was re-suspended in 4 ml of ice-cold buffer. Isolated mitochondria were incubated for 1 h with SNP, rotenone,
TTFA, antimycine, NaN3 , or oligomycine before measurement
of mitochondrial membrane potential with R123 dye. The protein content was determined by the method of Lowry et al. [36]
using bovine serum albumin as standard (BIO-RAD, Munich,
Germany). The average respiratory control ratio of the isolated
mitochondria of the young mice was 10.7.

tration of 0.4 M for 15 min. The cells were washed twice


with Hanks balanced salt solution (HBSS) and the fluorescence was determined with a fluorescence reader (Perkin-Elmer,
Victor multi-label counter) at 490/535 nm. Decrease in R123
fluorescence occurs as the mitochondrial membrane potential decreases. Data are represented as percentage of baseline
(untreated) control levels of each set of experiments.
To test acute and fast changes in mitochondrial membrane
potential the fluorescence dye TMRE [38] was used in a concentration of 0.4 M for 15 min. TMRE exhibits a characteristic
increase in fluorescence at 490/590 nm after challenging mitochondria with drugs decreasing the membrane potential [39].
The mitochondrial membrane potential was recorded after addition of complexes inhibitors (rotenone, TTFA, antimycine,
oligomycine and NaN3 ). Data are represented as change in
TMRE fluorescence (delta TMRE).

2.6. Measurement of mitochondrial membrane potential


The mitochondrial membrane potential was measured using
the fluorescence dye R123 [37]. Transmembrane distribution
of the dye depends on the mitochondrial membrane potential.
The dye was added to the cell culture medium in a concen-

2.7. Determination of ATP levels with a bioluminescence


assay (ViaLightTM HT)
The kit is based upon the bioluminescent measurement of
ATP [40]. The bioluminescent method utilizes an enzyme,

Fig. 2. EGb 761 improves the reduction of mitochondrial membrane potential and ATP levels caused by SNP or H2 O2 in dissociated brain cells. (A) Dissociated
brain cells were incubated for 4 h with 2 mM SNP, EGb 761 was added 30 min after insult. Data are expressed as means S.E.M. (n = 14). ** p < 0.01, vs. SNP
damage, Students paired t-test. (B) Dissociated brain cells were incubated for 4 h with 0.2 mM SNP, EGb 761 was added 30 min after insult. Data are expressed as
means S.E.M. (n = 11). *** p < 0.001, vs. SNP damage, Students paired t-test. (C) Dissociated brain cells were incubated for 4 h with 2 mM H2 O2 , EGb 761 was
added 30 min after insult. Data are expressed as means S.E.M. (n = 9). * p < 0.05, ** p < 0.01, vs. H2 O2 damage, Students paired t-test.

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luciferase, which catalyses the formation of light from ATP


and luciferin. The emitted light is linearly related to the ATP
concentration and is measured using a luminometer. Data are
represented as percentage of baseline (untreated) control levels
of each set of experiments.
2.8. Determination of ROS levels
The mitochondrial ROS levels were determined using the
fluorescent dye dihydrorhodamine (DHR123) at a concentration
of 10 M. The dissociated mice brain cells were incubated for
15 min with the dye, followed by washing twice with HBSS
and the fluorescence was determined with a fluorescence reader
(Perkin-Elmer, Victor multi-label counter) at 485/535 nm.
Superoxide radical was measured by incubating the cells with
Dihydroethidium (DHE) for 1 h at a concentration of 40 M
and the fluorescence was determined with a fluorescence reader
(Perkin-Elmer, Victor multi-label counter) at 490/590 nm.
Fig. 3. EGb 761 protects against serum deprivation-induced damage in PC12
cells. EGb 761 was added 30 min after serum-deprivation and mitochondrial
membrane potential and ATP levels were measured after 24 h. Data are expressed
as means S.E.M. (n = 515). * p < 0.05, ** p < 0.01, *** p < 0.001 vs. serumdeprivation induced damage, Students unpaired t-test.

2.9. Measurement of caspase-9 activity


PC12 cells were incubated with EGb 761 for 22 h, followed
by addition of SNP for 2 h. Caspase-9 activity was measured

Fig. 4. EGb 761 protects the complexes of the mitochondrial respiratory chain in PC12 cells. PC12 cells were incubated with EGb 761 for 6 h followed by
treatment with complex inhibitors. (A) Rotenone 2 M. (B) TTFA 10 M. (C) Antimycin 2 M. (D) NaN3 10 mM. (E) Oligomycine 10 M. Data are expressed as
means S.E.M. (n = 411). * p < 0.05, ** p < 0.01 vs. control cells, Students unpaired t-test.

R. Abdel-Kader et al. / Pharmacological Research 56 (2007) 493502

by cleavage of the colorimetric substrate Ac-LEHD-pNA using


a photometer (Perkin-Elmer, Victor multi-label counter) at
405 nm.
2.10. Treatment of PC12 cells with EGb 761
PC12 cells were treated in three different ways. First, the protective effect of EGb 761 and its constituents on recovery after
oxidative stress was tested. Thus, PC12 cells were treated for
24 h with SNP, 0.5 mM, EGb 761 was added 30 min after the
onset of SNP exposure. Second, PC12 cells were pre-treated for
30 min with SNP, then the medium was exchanged and EGb
761 was added for 23 h. Third, PC12 cells were pre-treated
for 6 h with EGb 761, after that the mitochondrial membrane
potential was recorded and then the complexes inhibitors were
added.
2.11. Treatment of dissociated brain cells with EGb 761
Dissociated brain cells were treated in two different ways.
First dissociated brain cells were treated for 4 h with 2 mM SNP.
EGb 761 was added 30 min after the onset of SNP exposure
and then mitochondrial membrane potential was measured. For
ATP measurements 0.2 mM SNP was used. Second, dissociated
brain cells were pre-treated for 6 h with EGb 761, and then the
complexes inhibitors were added followed by the measurement
of mitochondrial membrane potential.

497

2.12. Treatment of animals


The treated animals received 100 mg/kg body weight EGb
761 in 0.2% agarose solution p.o. once daily for 2 weeks. The
volume given p.o. ranged between 150 and 300 l according to
body weight. The placebo group was treated with 0.2% agarose
alone. The tests were implemented 24 h after the last feeding.
2.13. Statistics
Data are given as means S.E.M. For statistical comparison
Students t-test or one-way ANOVA followed by Tukeys post
hoc test used. p 0.05 was considered statistically significant.
3. Results
We investigated the protective effect of EGb 761 on mitochondrial function after inducing both oxidative and nitrosative
stress. EGb 761 alone showed no effect on mitochondrial membrane potential or ATP levels (data not shown). EGb 761
stabilized mitochondrial membrane potential and ATP levels in
PC12 cells after exposure to the NO donor SNP. NO and its
derivative peroxynitrite cause injury to the mitochondria, inhibiting all respiratory chain complexes. This leads to a reduced ATP
formation and decreases the mitochondrial membrane potential. Removal of SNP after 30 min followed by addition of EGb
761 for 23 h improved the recovery of mitochondrial membrane
potential and ATP levels (Fig. 1A). Additionally, protective

Fig. 5. Pre-treatment with EGb 761 improved mitochondrial membrane changes caused by complex inhibitors in dissociated brain cells. Dissociated brain cells were
incubated with EGb 761 for 6 h followed by treatment with complex inhibitors. (A) Rotenone 2 M. (B) TTFA 10 M. (C) Antimycin 2 M. (D) NaN3 10 mM. (E)
Oligomycine 10 M. Data are expressed as means S.E.M. (n = 612). * p < 0.05, ** p < 0.01 vs. control cells, Students unpaired t-test.

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Fig. 6. Protective effects of EGb 761 ex vivo in dissociated brain cells and isolated mitochondria. Mice (23-month-old and 1516-month-old) were treated for 14
consecutive days with EGb 761 (100 mg/kg) p.o. daily and controls with placebo (0.2% agarose). (A) Treatment with EGb 761 shows an improvement in mitochondrial
membrane potential in dissociated brain cells of old mice only. Mitochondrial membrane potential was measured after incubating the dissociated mice brain cells
for 4 h with 2 mM SNP. Data are expressed as means S.E.M. (n = 810). ** p < 0.01 vs. placebo treated animals, Students unpaired t-test. (B) Treatment with EGb
761 shows an improvement in mitochondrial membrane potential in isolated mitochondria of old mice only. Mitochondrial membrane potential was measured after
incubating isolated mitochondria for 1 h with 2 mM SNP. Data are expressed as means S.E.M. (n = 69).* p < 0.05 vs. placebo treated controls, Students unpaired
t-test.

effects were also seen when EGb 761 was added 30 min after
SNP exposure without removal of SNP (Fig. 1B). Thus, EGb 761
was able to ameliorate mitochondrial functions after nitrosative
stress not only after removal of SNP but also during its presence.
SNP was also able to activate caspase-9 that is known to play an
important role in the mitochondria-mediated apoptotic pathway
[41]. Pre-treatment of PC12 cells with EGb 761 followed by
exposure to SNP for 2 h decreased significantly the increase in
caspase-9 activity (Fig. 1C).
To confirm these results further experiments were done using
another neuronal system. Dissociated brain cells prepared from
NMRI mice (23 months) were exposed to nitrosative stress
using the NO donor SNP and oxidative stress using H2 O2
(Fig. 2). Again EGb 761 reduced the decrease of mitochondrial
membrane potential in this cell model.
To investigate the effects of EGb 761 in the presence of
oxidative stress we measured mitochondrial function under
serum-deprived conditions. Serum deprivation is able to induce

oxidative stress, cause energy deficiency subsequently causing


mitochondrial dysfunction [4244]. EGb 761 showed protective
effects on the mitochondrial membrane potential and ATP levels
after serum deprivation (Fig. 3).
A large body of data suggests that aging causes decrease in
the activity of the mitochondrial respiratory chain. Additionally,
defects in mitochondrial respiratory chain have been observed
in brains and platelets of AD patients [15,16,45,46]. Thus, the
effects of EGb 761 on the mitochondrial respiratory chain were
examined in vitro in both PC12 cells and dissociated mice brain
cells after the addition of specific complexes (IV) inhibitors.
All respiratory chain complexes were significantly protected at
a concentration of 0.1 mg/ml in PC12 cells (Fig. 4), and at a
maximum concentration of 0.5 mg/ml in dissociated brain cells
(Fig. 5).
In order to find out if the mitochondrial protective effects
of EGb 761 differ during aging, two different age groups were
treated orally for 14 consecutive days and the protective effects

Fig. 7. EGb 761 shows a pronounced effect in older mice, diminishing mitochondrial membrane potential changes evoked by different complex inhibitors. Treated
NMRI mice (23-month-old and 1516-month-old) received 100 mg/kg body weight EGb 761 p.o. once daily for 14 days. Control animals were treated with placebo
(0.2% agarose solution). Mitochondrial membrane potentials were measured after incubation of isolated mitochondria with (A) Rotenone; (B) TTFA; (C) Antimycin;
(D) NaN3 and (E) Oligomycine. Data are expressed as means S.E.M. from 7 to 8 experiments, each representing an individual animal. * p < 0.05 vs. placebo control,
Students unpaired t-test.

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499

Fig. 8. Reduction of the levels mitochondrial ROS and superoxide radical anion in EGb 761 treated mice. Treated NMRI mice (18-month-old) received 100 mg/kg body
weight EGb 761 p.o. once daily for 14 days. Control animals were treated with placebo (0.2% agarose solution). (A) Mitochondrial ROS levels (DHR fluorescence)
were lower in dissociated brain cells prepared from the EGb 761 treated group compared to the placebo treated group. Data are expressed as means S.E.M. (n = 67).
* p < 0.05 vs. placebo control, Students unpaired t-test. (B) Superoxide radical anion levels (DHE fluorescence) were lower in dissociated brain cells prepared from
the EGb 761 treated group compared to the placebo treated group. Data are expressed as means S.E.M. (n = 6). * p < 0.05 vs. placebo control, Students unpaired
t-test.

on the mitochondrial functions were tested. The dose used


(100 mg/kg body weight) has been previously shown to reduce
ROS-induced apoptosis and improve learning deficits in aged
mice [22,47]. Interestingly, EGb 761 was able to protect the
dissociated brain cells (Fig. 6A) and isolated mitochondria
(Fig. 6B) against NO induced damage in the old mice significantly, but only a slight improvement in the mitochondrial
membrane potential was observed in the young mice. The effects
of respiratory chain complexes inhibitors were also examined in
both age groups. Again, EGb 761 showed a significant protective
effect on complexes I, IV and V in the 15-month-old mice and no
effect in the 23-month-old mice, proving its higher efficiency
during aging (Fig. 7).
Further on, we compared the superoxide radical and the mitochondrial ROS levels in 18-month-old mice after treatment for
14 days with EGb 761 (100 mg/kg body weight). We observed a
significant decrease in the superoxide anion and mitochondrial
ROS levels in the EGb 761 treated group compared to the vehicle
(Fig. 8).
After noticing the protective effect of the EGb 761 extract
on the mitochondria we tested its single constituents to find out
which constituent is responsible for this effect. We stimulated
both PC12 cells and dissociated brain cells with SNP and then
tested the effectiveness of flavonoids, ginkgolides A, B, C, J or
bilobalide. The mitochondrial membrane potential was significantly improved after treatment with the single components in
Table 1
Percentage recovery of the mitochondrial membrane potential from NO insult
after treating PC12 cells with the single constituents of EGb 761
Compound

Concentration (mg/ml)
0.01

Ginkgolide A (%)
Ginkgolide B (%)
Ginkgolide C (%)
Ginkgolide J (%)
Bilobalide (%)
Flavonoids (%)

46.9
66,0
75.0
100.0
70.4
56.4

0.1

10.4**
22.9**
15.6**
16.9**
11.7**
10.8**

51.6
88.1
77.5
109.6
78.7
106.4

8.9**
17.4**
12.5**
13.9***
16.5**
35.1*

PC12 cells were incubated with SNP for 24 h, after 30 min the single constituents
were added and the percentage improvement in mitochondrial membrane potential was measured. Data are expressed as means S.E.M. (n = 6). * p < 0.05,
** p < 0.01 vs. SNP insult, Students paired t-test.

Table 2
Percentage recovery of the mitochondrial membrane potential from NO insult
after treating dissociated brain cells with the single constituents of EGb 761
Compound

Concentration (mg/ml)
0.01

Ginkgolide A (%)
Ginkgolide B (%)
Ginkgolide C (%)
Ginkgolide J (%)
Bilobalide (%)
Flavonoids (%)

2.6
16.3
5.3
13.8
29.5
31.0

0.05

1.6
4.8*
2.0
6.8
11.4*
8.5**

8.0
33.0
8.5
37.0
36.3
56.4

1.9**
11.2*
1.6**
12.2*
7.7**
12.7**

Dissociated brain cells were incubated with SNP for 4 h, after 30 min the single
constituents were added and the percentage improvement in mitochondrial membrane potential was measured. Data are expressed as means S.E.M. (n = 612).
* p < 0.05, ** p < 0.01 vs. SNP insult; Students paired t-test.

both PC12 cells (Table 1) and dissociated brain cells (Table 2).
A higher protection was observed in PC12 cells and the extent of
protection varied from one constituent to another. The PC12 cells
were significantly protected at concentrations of 0.01 mg/ml and
dissociated brain cells at concentrations of 0.05 mg/ml.
4. Discussion
It is postulated that mitochondrial dysfunction contributes
significantly to aging-related neurodegenerative diseases.
Increasing evidence suggests that mitochondrial dysfunction is
an early event in AD, and that impairment of mitochondrial
metabolism together with oxidative abnormalities may be an initiator for the major pathological events of AD [12,14,48]. Aging
can cause increase in ROS and RNS production, decrease in the
activity of mitochondrial respiratory chain subsequently leading
to decrease in energy production and ATP levels. In this study
we were able to show that EGb 761 can protect the mitochondria against several stress conditions playing an important role
during aging.
There is growing evidence to implicate excessive or inappropriate generation of nitric oxide (NO) in neurological and
aging-related disorders [49,50]. NO reacts with complex IV
and causes reversible inhibition of the mitochondrial respiratory chain. Complex IV may transiently increase the leakage of
superoxide anion from the electron transport chain. The super-

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oxide formed could then react with NO to generate peroxynitrite.


Mitochondrial enzymes are particularly vulnerable to attacks by
peroxynitrite that would cause irreversible injury to the mitochondria, inhibiting all complexes except complex IV. This leads
to reduced ATP formation and induction of mitochondrial permeability transition by opening of the permeability transition
pore, which decreases the mitochondrial membrane potential
[50]. EGb 761 increased ATP levels and mitochondrial membrane potential after stimulation with the NO donor SNP. In
accordance with our findings, EGb 761 was able to rescue
hippocampal cells against NO-induced neurotoxicity [25]. In
addition to stabilizing mitochondrial functions after NO insult,
EGb 761 was able to reduce the NO-induced increase in caspase9 activity. Caspase-9 is known to activate caspase-3 that finally
leads to apoptosis and cell death. In agreement with this finding
Massieu et al were able to show that caspase-3 activity decreased
after exposure to staurosporine suggesting that EGb 761 inactivates the apoptotic caspase cascade indirectly by stabilizing
mitochondrial functions [51].
Interestingly, treating young and old mice with EGb 761
showed different outcomes. EGb 761 was able to protect the
mitochondria of old mice significantly against NO induced stress
in both dissociated brain cells and isolated mitochondria with
only a slight effect in the young mice. Although EGb 761 treated
mice showed lower levels of mitochondrial ROS, the protective
effects of EGb 761 seem not only to underlie its radical scavenger
activity, but EGb 761 seems to exhibit direct effects on the mitochondria which are pronounced during aging. This hypothesis
is supported by the fact that EGb 761 improves mitochondrial
membrane potential in both PC12 cells and dissociated brain
cells after exposure to respiratory chain complex inhibitors in
vitro. Interestingly, ex vivo the aged and not the young treated
mice showed an improvement on complexes I, IV and V after
insulting with complexes inhibitors. These complexes were previously shown to play a major role in aging [52,53]. Navarro
and Boveris were able to show that the activities of complexes I
and IV were decreased by 2830% in the brains of 92-week-old
mice compared to 28-week-old mice [52]. Decline in the complexes activities was also seen in aging-related diseases such as
AD. For example a lower expression of the -subunit of ATP
synthase has been observed in AD [54], and the function of
ATP synthase -chain was altered in AD degenerating neurons
[55]. The protective effects on the respiratory chain complexes
could be explained by the ability of EGb 761 to alter mitochondrial gene expression as proposed by others. Chandrasekaran
et al. [56] showed that both bilobalide and EGb 761 protected hippocampal regions of gerbils against ischemia-induced
reductions in cytochrome oxidase (COX) subunit 3 mRNA.
Additionally, EGb 761 and bilobalide up-regulating mitochondrial gene expression of subunit 1 of NADH dehydrogenase [57].
Therefore, the effects on both complexes I and IV of the respiratory chain could be due to alteration of gene expression by
EGb 761.
Other mitochondrial alterations during aging could be the
activation of the permeability transition pore. In order to mimic
this action in vitro we incubated the cells in serum deprived
medium, which leads not only to activation of the permeability

transition pore [58] but also causes energy deficiency [44]. EGb
761 was able to improve the mitochondrial functions after serum
deprivation by increasing the mitochondrial membrane potential
and the ATP levels.
Apparently, EGb 761 seems to have different mechanisms
of protecting the mitochondria and this is most probably due
to the different properties and effects of its constituents. It is
highly controversial which fraction is more active the flavonoid
or the terpenoid fraction. Hence, we decided to examine the
impact of the single constituents of EGb 761 on protection of the
mitochondrial membrane potential. Their ability to protect the
mitochondria seems to depend on the model used for induction of
mitochondrial dysfunction and on the type of cell line used. We
used both PC12 cells and dissociated brain cells and measured
mitochondrial membrane potential after the insult with SNP.
Taking into consideration the fact that the flavonoid fraction
represents 24% and the terpenoid fraction 6% of the EGb 761
extract, each component was able to protect the mitochondria
but to different extents. Ginkgolide J and the flavonoid fraction
(most abundant fraction) were the most effective and stabilized
the mitochondrial membrane potential (109 and 106% improvement, respectively) in PC12 cells, and showed relatively high
effects in dissociated brain cells. In both cell models ginkgolide
A seems to have the least impact on the mitochondria. Consistent with these findings is the fact that ginkgolide A revealed no
anti-apoptotic effect in either serum-deprived or staurosporinetreated neurons while ginkgolides B, J and bilobalide showed
anti-apoptotic properties [31].
Overall, the components of EGb 761 showed a more pronounced effect on the mitochondria in PC12 cells than in
dissociated brain cells. These discrepancies could be related to
intrinsic differences between the cell models and/or the fact that
PC12 cells were incubated for longer time periods with the single
components.
This controversy was also seen in previous studies using the
single components of EGb 761 while testing their anti-apoptotic
roles. For example, the terpene constituents were able to prevent apoptosis in cultured chick embryonic neurons as well as
in mixed cultures of neurons and astrocytes from neonatal rat
hippocampus [31], while the flavonoid constituent but not the
terpenoid fraction protected rat cerebellar granule cells against
hydroxyl-induced apoptosis [26]. Comparing two studies in
which terpenoid constituents were active against apoptosis, one
study claimed that ginkgolide B, but not bilobalide was effective and the other study revealed that bilobalide was the most
potent constituent [24]. Therefore, the methodologies employed
and cell models used seem to play a major role in the effectiveness of the single components of EGb 761. Nevertheless, each
constituent of EGb 761 seems to have protective effects on the
mitochondria in our cell models.
Concluding, several stress mechanisms were used in our
study in order to mimic mitochondrial changes during aging
which can lead to many aging-related diseases including AD
and mild cognitive impairment. EGb 761 was able to protect the
mitochondria by stabilizing the mitochondrial membrane potential and ATP levels. Our present study presented the effects of
EGb 761 in vitro in both PC12 cells and dissociated brain cells,

R. Abdel-Kader et al. / Pharmacological Research 56 (2007) 493502

additionally ex vivo treatment of young and aged mice showed


a more pronounced effect in aged mice.
Furthermore, in order to find out which of the substances in
the EGb 761 extract is responsible for its protection of the mitochondria, we tested the single components. From the results it
seems that the effects of EGb 761 are due to a complementary
action of its constituents. Further parameters should be investigated using the single components in order to elucidate the
mechanisms lying behind the mitochondrial protective activities
of EGb 761.
Our findings and the findings of others indicate substantial
neuroprotective properties of EGb 761. This agrees with some
of the clinical studies in AD where clinical benefit seems to be
associated with the progression of the disease [15].
Acknowledgment
This study was supported by Dr. W. Schwabe Arzneimittel
[Karlsruhe, Germany].
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at doi:10.1016/j.phrs.2007.09.011.
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