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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].
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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.
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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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.
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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
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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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.
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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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,
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