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Unlabelled: Attention deficit hyperactivity disorder (ADHD) belongs to the neurodevelopmental disorders characterized by impulsivity, distractibility and hyperactivity. In the pathogenesis of ADHD genetic and non-genetic factors play an important role. It is assumed that one of non-genetic factors should be oxidative stress. Pycnogenol, an extract from the pine bark, consists of bioflavonoids, catechins, procyanidins and phenolic acids. Pycnogenol acts as powerful antioxidant, chelating agent; it stimulates the activities of some enzymes, like SOD, eNOS, and exhibits other biological activities. Aim: The aim of this randomized, double-blind, placebo-controlled trial was to investigate the influence of administered Pycnogenol or placebo on the level of reduced (GSH) and oxidized (GSSG) glutathione in children suffering from ADHD and on total antioxidant status (TAS). This is the first investigation of the redox glutathione state in relation to ADHD. Results: One month of Pycnogenol administration (1 mg/kg body weight/day) caused a significant decrease in GSSG and a highly significant increase in GSH levels as well as improvement of GSH/GSSG ratio in comparison to a group of patients taking a placebo. TAS in children with ADHD was decreased in comparison with reference values. Pycnogenol administration normalizes TAS of ADHD children.
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INTRODUCTION
Oxygen is fundamental for the production of energy (ATP) by
all aerobic life forms. However, oxidative processes also gen-
erate highly reactive oxygen free radicals and their reactive
metabolites in tissues. This is the reason for oxidative
stress. Oxidative stress is defined as an imbalance in antiox-
idant and pro-oxidant levels to the benefit of pro-oxidants,
which results in damage to biomolecules.
1
Oxidative stress
is important in many diseases, e.g. atherosclerosis, neuro-
degeneration or ischemia-reperfusion states.
2
It is predicted
that reactive oxygen species also play a role in biological
stress
3
and in the etiology of many psychiatric diseases.
4
©W. S. Maney & Son Ltd
Redox Report Vol. 11, No. 4, 2006
DOI 10.1179/135100006X116664
Research article
The effect of polyphenolic extract from pine bark, Pycnogenol
®
,
on the level of glutathione in children suffering from
attention deficit hyperactivity disorder (ADHD)
Monika Dvo˘ráková
1
, Monika Sivo˘nová
1
, Jana Trebatická
2
, Igor
˘
Skodá˘cek
2
,
Iveta Waczuliková
3
, Jana Muchová
1
, Zde˘nka
˘
Dura˘cková
1
1
Department of Medical Chemistry, Biochemistry and Clinical Biochemistry, Faculty of Medicine,
Comenius University, Bratislava, Slovak Republic
2
Department of Child Psychiatry, Faculty of Medicine, University Hospital, Bratislava, Slovak Republic
3
Department of Nuclear Physics and Biophysics, Division of Biomedical Physics, Faculty of Mathematics, Physics
and Informatics, Comenius University, Bratislava, Slovak Republic
Attention deficit hyperactivity disorder (ADHD) belongs to the neurodevelopmental disorders
characterized by impulsivity, distractibility and hyperactivity. In the pathogenesis of ADHD genetic
and non-genetic factors play an important role. It is assumed that one of non-genetic factors should
be oxidative stress. Pycnogenol
®
, an extract from the pine bark, consists of bioflavonoids, catechins,
procyanidins and phenolic acids . Pycnogenol
®
acts as powerful antioxidant, chelating agent; it
stimulates the activities of some enzymes, like SOD, eNOS, and exhibits other biological activities.
Aim: The aim of this randomized, double-blind, placebo-controlled trial was to investigate the
influence of administered Pycnogenol
®
or placebo on the level of reduced (GSH) and oxidized
(GSSG) glutathione in children suffering from ADHD and on total antioxidant status (TAS). This is
the first investigation of the redox glutathione state in relation to ADHD.
Results: One month of Pycnogenol
®
administration (1 mg/kg body weight/day) caused a significant
decrease in GSSG and a highly significant increase in GSH levels as well as improvement of
GSH/GSSG ratio in comparison to a group of patients taking a placebo. TAS in children with
ADHD was decreased in comparison with reference values. Pycnogenol
®
administration normalizes
TAS of ADHD children.
Keywords: Oxidative stress, ADHD, Pycnogenol, glutathione, TAS
Received 28 March 2006
Revised 1 July 2006
Accepted 12 July 2006
Correspondence to: Prof. Ing. Zde˘nka
˘
D
ura˘cková PhD, Department of
Medical Chemistry, Biochemistry and Clinical Biochemistry, Faculty
of Medicine, Comenius University, Sasinkova 2, 811 08 Bratislava,
Slovak Republic
Tel: +421 2 59357411; Fax: +421 2 59357557;
E-mail: zdenka.durackova@fmed.uniba.sk
Abbreviations: CHES, 2-(N-cyclohexamine)ethanesulphonic acid;
ADHD, attention deficit hyperactivity disorder; FDNB, fluorodinitro-
benzene; MDA, malondialdehyde; NEM, N–ethylmaleimide; GSSG,
oxidized glutathione; PCA, perchloric acid; ROS; reactive oxygen
species; GSH, reduced glutathione; SOD, superoxide dismutase; TAS,
total antioxidant status
Attention deficit hyperactivity disorder (ADHD) is
one of the most common neurodevelopmental psychi-
atric disorders in children. Children with ADHD suffer
from inattention, impulsivity and hyperactivity.
According to a variety of epidemiological data, the inci-
dence of ADHD in children and adolescents ranges from
3–5%. Boys are 2.5–9.0 times more likely to be diag-
nosed with ADHD than girls.
5
The molecular basis of
ADHD is not yet clear; however, it is known that in its
etiology, genetic as well as non-genetic factors play an
important role. It is believed that ADHD arises from a
complex interaction of environmental and biological
factors, with strong evidence for a genetic component.
6
Non-genetic factors include some prenatal (fetal expo-
sure to alcohol, drugs and tobacco) and birth complica-
tions, lead poisoning or head injuries.
7
Studies of ADHD neurochemistry, neuro-imaging and
genetics have supported the view that ADHD is a familial
disease involving differences in monoamine regulation
and frontal–striatal neural circuitry. Dopamine and nora-
drenaline are known to take part in the normal function of
the prefrontal cortex.
8
In the pathophysiology of ADHD,
damage to adrenaline, noradrenaline and dopamine
metabolism occurs. These changes can modify attention,
thinking and acting.
9
Damage in catecholamine’s metab-
olism is considered as one of the possible sources of free
radical formation. In vitro, catecholamines at higher con-
centrations in the presence of oxygen can be easily oxi-
dized. Oxidation of catecholamines results in
o–semiquinone formation. Under physiological condi-
tions, semiquinone reacts with ascorbic acid, glutathione
or other sulfhydryl groups to form stable thiol products.
However, when semiquinone reacts with oxygen (e.g. in
the brain), superoxide radical is formed, regenerating
o–quinones. Generated o–quinones are susceptible to fur-
ther metabolism to reactive semiquinone. In this reaction
cycle, increasing amounts of superoxide radical and sub-
sequently other reactive oxygen species are created.
10,11
In
vivo, this can lead to oxidative stress and to neurodegener-
ative processes.
9,12
During oxidative processes, H
2
O
2
is formed. Under
physiological conditions, H
2
O
2
is inactivated by catalase
or glutathione peroxidase using reduced glutathione
(GSH) as co-factor. Toxicity of H
2
O
2
is caused not only
by its oxidant power, but also because of its reactivity
with Fe
2+
and other heavy metals ions in the Fenton-type
reaction when a highly reactive hydroxyl radical (
OH)
is formed.
It is assumed that oxidative stress plays a role in the
pathophysiology of ADHD.
13
In recent years, antioxi-
dant therapy of ADHD has been discussed
14
as a supple-
ment to classical treatment by psychostimulants,
antidepressants, neuroleptics and mood stabilisators.
However, different side-effects of these psychomedica-
ments were found.
15,16
Several reports have suggested a beneficial effect of
Pycnogenol
®
on ADHD symptoms in children. First case
reports concerning positive effects following treatment
of children with ADHD with Pycnogenol
®
were col-
lected by Passwater (1998),
17
Heimann (1999),
18
and
Masao (2000).
19
An attempt to demonstrate reduction of
ADHD symptoms in adults failed in a double-blind,
placebo-controlled, comparative study with 24 adults.
20
We also found a significant improvement of ADHD
symptoms in children after 1 month of Pycnogenol
®
1
mg/kg/day administration.
14
Pycnogenol
®
(Horphag Res. Ltd, Geneva, Switzerland)
is a standardized extract from the French pine (Pinus
pinaster) bark-concentrate of polyphenols consisting of
procyanidins, catechin, taxifoline and phenolic acids.
21
It
acts as a strong scavenger of free radicals in vitro
21
and it
stimulates activities of antioxidant enzyme (Cu/Zn
superoxide dismutase).
22
We have shown
13
that Pycnogenol
®
lowers oxidative damage to DNA (expressed as 8-oxoG/10
6
G) of children suffering from ADHD. This is an impor-
tant finding as a significant increase in DNA damage
was found in ADHD children compared to a group of
healthy children.
13
Pycnogenol
®
increases vasodilatation by stimulating
the activity of endothelial NO-synthase.
23
Except for the
pathological effects of higher concentrations of nitric
oxide (neurodegeneration or neuro-inflammation), at
physiological concentrations NO plays a crucial role in
the brain (e.g. neuromodulation, neurotransmission and
synaptic plasticity).
Pycnogenol
®
influences glutathione metabolism
through elevation of glutathione peroxidase and glu-
tathione reductase activities.
24
In addition, Pycnogenol
®
can scavenge produced free radicals and so spares GSH;
this can indirectly lead to the elevation of GSH levels.
25
Glutathione is one of the most abundant intracellular
antioxidants. GSH (γ-glutamyl-cysteinyl-glycine) plays a
key role in the protection of proteins, lipids and nucleic
acids against free radical damage. There is also a direct cor-
relation between the speed of aging and the reduction of
GSH concentrations in intracellular fluids.
26,27
GSH has potent electron-donating capacity, as indi-
cated by the high negative redox potential of the
GSH/GSSG redox couple (E
0
= –0.33 V).
28
This renders
GSH an antioxidant capacity and it is a convenient co-
factor for enzymatic reactions. The reducing power of
GSH is related to its free radical scavenging, electron-
donating and sulfhydryl-donating capacity.
In addition, glutathione regulates protein activity via
GSH transferases and thioredoxin
29
and it is also the
source of amino acids for protein synthesis.
30
A deficit of
GSH would lead to degenerative processes at dopamin-
ergic terminals resulting in loss of connectivity.
9
Healthy brain cells possess high concentrations of
both enzymatic (Cu/Zn- and Mn-superoxide dismutases,
164 Dvo˘ráková, Sivonová, Trebatická et al.
GSH-peroxidase) and non-enzymatic antioxidants (glu-
tathione, vitamins C and E).
31
Under physiological con-
ditions, cells are able to cope with the toxicity of ROS
with the help of an antioxidative pool. However, free
radicals can cause peroxidation and microlesions of the
membrane in neurons. This is known to be a primary
cause of many degenerative diseases (schizophrenia,
Alzheimers disease, Parkinson’s disease).
Determination and evaluation of both GSH and GSSG
and their ratio in blood has been considered essential as an
index of several physiological and pathological situations.
A decreased level of the reduced form of glutathione con-
tributes to the disequilibration in oxidant/antioxidant bal-
ance in the organism following increased oxidative stress.
Presently, much attention is being devoted to supple-
mentation with natural polyphenols and flavonoids
showing antioxidant properties,
32,33
like Pycnogenol
®
.
23
In our pilot study,
34,35
we found increased levels of
malondialdehyde (MDA) and decreased total antioxi-
dant status (TAS) in ADHD children in comparison to a
control group. In a recent paper,
13
we found increased
levels of 8-oxodeoxyguanosine (8-oxodG) in ADHD
children. Therefore, we suggest an increased oxidative
stress in these children.
The aim of this work was to determine the antioxidant
and redox states of ADHD patients represented by
reduced and oxidized glutathione and to determine how
Pycnogenol
®
affects these markers.
PAT IENTS AND METHODS
Patients
A total of 43 out-patients (34 boys and 9 girls; aged 6–14
years) with ADHD, treated at the Child Psychiatric
Clinic of the Children’s University Hospital, were
enrolled in a randomized, double-blind and placebo-con-
trolled study. Patients were randomized to receive either
Pycnogenol
®
or placebo.
Inclusion criteria
Inclusion criteria included: early onset of ADHD (by 6–7
years), chronicity, disorders of cognitive function (inatten-
tion, distractibility), difficulty persisting with any one task,
difficulty in selective process to information, disturbance of
the executive functions (production, sequention and realiza-
tion of plans), disturbance of motivation, effort and forti-
tude, visuospacial and memory disturbance.
Exclusion criteria
Exclusion criteria included: situational hyperactivity,
pervasive developmental disorders, schizophrenia, other
psychotic disorders (mood, anxiety), personality disorder
(as unsocial behavior), personality change due to a general
medical condition, mental retardation, understimulating
environments, conduct disorder, tics, chorea and other
dyskinesias. Patients with acute inflammatory diseases,
renal and cardiovascular disorders and diabetes were
excluded from this study, too.
The Ethical Committee of the Children’s University
Hospital approved the study. Parents gave written con-
sent for participation of their children in the study.
Medication
Children were supplemented with either Pycnogenol
®
(1
mg/kg body weight/day) or placebo (with identical
shape and appearance and same number of pills/day as
in the case of Pycnogenol
®
) for 1 month. The placebo
contained lactose (58 mg) and cellulose (65 mg). Both,
Pycnogenol
®
and placebo tablets were produced by Drug
Research Institute, Modra, Slovakia.
Selection into the Pycnogenol
®
or placebo group was
carefully randomized. The ratio of these groups was
2.5:1. The sample size was estimated assuming the
power of 80% (β = 20%), the type one error (α) of 5%
and the number of controls per subject, 0.4. The recom-
mended number of patients was pre-calculated as 41 for
drug investigation and 16 subjects for placebo. We
included in the study 44 and 17 patients, respectively.
Stat Direct
®
v.2.3.7 was used for the unpaired random
allocation to intervention or control group and for the
sample size estimation.
Patients had a standard diet and were not treated with
other psychotropic drugs or with vitamins E and C dur-
ing the study period.
Patient treatment
Patients were studied at the beginning of the trial before
Pycnogenol
®
/placebo administration (0), after 1 month
of Pycnogenol
®
/placebo administration (1), and 1 month
after termination of treatment (wash-out period) (2).
Venous blood samples were taken at all investigated
periods into commercial tubes with sodium citrate as an
anticoagulant. Whole blood was used for determination
of total and oxidized glutathione. The rest of blood sam-
ples was centrifuged and plasma was aliquoted, shock
frozen and stored at –80°C until further analysis.
Chemicals
Perchloric acid (PCA), N–ethylmaleimide (NEM),
bapthophenanthrolinedisulfonic acid (BPDS), iodoacetic
acid, 2-(N-cyclohexamine)ethanesulphonic acid (CHES),
oxidized glutathione, reduced glutathione, metacresol
Glutathione level in ADHD children 165
purple, fluorodinitrobenzene (FDNB), sodium acetate,
and acetic acid were purchased from Sigma-Aldrich
Chemical (Taufkirchen, Germany). Potassium hydroxide
(KOH) was obtained from Lachema (Brno, Czech
Republic). Ethanol and methanol were purchased from
Merck (Darmstadt, Germany).
Reagents
Solution 1 contained 12% PCA in water, 1.2 mM BPDS
in water, 40 mM NEM in water. Solution 2 contained 6%
PCA in water, 1 mM BPDS in water. Solution 3 con-
tained 3 M KOH in water, 0.3 M CHES in water. This
166 Dvo˘ráková, Sivonová, Trebatická et al.
Fig. 1. Chromatography record of HPLC determination of oxidized (A) and total (B) glutathione. HPLC conditions: 20 × 0.46 cm column Allsphere amino
(Alltech); mobile phases A and B are mixed together according to following gradient: t
0
–t
8
, 80:20 (A:B) (%); t
8
–t
30
, 1:99 (A:B) (%); t
30
–t
38
, 1:99 (A:B) (%);
t
38
–t
42
, 80:20 (A:B) (%); t
42
–t
49
, 80:20 (A:B) (%); flow rate of 1 ml/min; injection 70 µl; detection on UV detector (DeltaChrom UVD200) at 375 nm.
buffer keeps pH between 8.5–9. pH indicator 1 consisted
of 0.2 mM metacresol purple in water: a change to pur-
ple color occurs at pH 9. pH indicator 2 was 10 M
iodoacetic acid in 0.2 mM metacresol purple. The
detecting reagent was 1% FDNB in 96% ethanol. The
acetic acid solution contained 3.3 M sodium acetate,
11.025 M acetic acid in water. Mobile phase A was 80%
methanol in water. Mobile phase B was a 20% solution
of acetic acid in 80% mobile phase A. Both mobile
phases were filtered and de-aerated.
Analytical methods
Glutathione determination
Under physiological conditions, the GSH concentration
is much higher than the GSSG concentration. GSH very
easily undergoes auto-oxidation to GSSG, especially in
the presence of metals. To impede its oxidation, sub-
stances blocking thiol groups are used (N-ethyl-
maleimide, iodoacetic acid). Concentrations of total
glutathione and GSSG in whole blood were determined
separately by gradient HPLC according to a modified
method of Reed et al.
36
and detected at 375 nm.
Afterwards, the GSH concentration was determined
indirectly from values of GSSG and total glutathione:
[GSH] = [total glutathione] – (2 × [GSSG]) Eq. 1
HPLC conditions for both, total and oxidized glutathione
For the separation of total and oxidized glutathione, an
aminocolumn Allsphere amino (Alltech, Deerfield, IL,
USA) 20 × 0.46 cm was used. The size of filling mass
was 5 µm. A 20 µl aliquot of prepared sample (see
below) was directly injected onto the column. The flow
rate of the mobile phase was 1 ml/min and glutathione
was detected at 375 nm with a UV detector (DeltaChrom
UVD 200, Watrex, Czech Republic).
A multistep gradient was used with a starting ratio
80:20 (A:B) and remained isocratic for 8 min. At this
time, the composition was changed linearly to A:B
(1:99) in 30 min. This state was isocratic until 38 min
then changed to A:B (80:20) in 42 min. The separation
finished in 49 min at A:B (80:20) as shown in Figure 1.
Total glutathione (TG)
For constructing a calibration curve, standards at con-
centrations of 6.25–100 µmol/l were prepared.
Standards were then adjusted in the same way as sam-
ples – 800 µl of solution 2 was added to 200 µl of blood
sample/standard. Such modified samples were stored at
–20°C until use.
Before measurement, treated samples were cen-
trifuged at 4°C, for 6 min at 14,000 g. Then 20 µl of pH
indicator 2 was added to 200 µl of supernatant and the
pH was adjusted to 9–9.5 with solution 3. After 30 min
incubation at room temperature in the dark, 400 µl of
FDNB was added to the sample/standard. Mixed sam-
ples/standards were incubated for 24 h at 4°C. The
amount of total glutathione was measured by gradient
HPLC at 375 nm.
Total glutathione was measured as the sum of GSH
and GSSG, which accrued by spontaneous oxidation of
GSH. Total glutathione was calculated from:
[TG] = ([GSH] + {2 × [GSSG]}) × 5 (dilution) Eq. 2
where: [GSH] = concentration of reduced glutathione as
a part of total glutathione and [GSSG] = concentration of
oxidized glutathione as a part of total glutathione. The
concentration of total glutathione is expressed as µmol/l.
Oxidized glutathione (GSSG)
For constructing a calibration curve, standards at concen-
trations of 6.25–100 µmol/l were prepared. Standards
were then adjusted in the same way as samples – 800 µl of
solution 1 were added to 200 µl of blood sample/standard.
NEM binds to the –SH groups of reduced glutathione to
prevent its oxidation. Such modified samples were
stored at –20°C. Before analysis, modified samples
were centrifuged at 4°C, for 6 min at 14,000 g. Then,
20 µl of pH indicator 1 was added to 200 µl of super-
natant and the pH was adjusted to 9–9.5 with solution 3.
Prepared samples/standards were centrifuged at 4°C,
for 15 min at 15,000 g and 50 µl of FDNB was added to
25 µl of the supernatant. Samples/standards were incu-
bated for 45 min at room temperature in the dark.
Derivatized samples/standards were dried under vac-
uum and stored at –20°C in the dark until HPLC analy-
sis. Samples/standards processed this way were stable
for several weeks. Before loading on the column, the
sample was dissolved in 200 µl of mobile phase A. The
amount of oxidized glutathione was measured by gradi-
ent HPLC at 375 nm. The concentration of GSSG was
calculated from:
[GSSG] = (P × 2 (dilution)) Eq. 3
where: P = area of the peak of the oxidized glutathione;
and [GSSG] = concentration of the oxidized glutathione.
The concentration of oxidized glutathione is expressed
as µmol/l.
Total antioxidant status
Total antioxidant status (TAS) in plasma was analysed
by standard biochemical procedures using an Hitachi
911 automatic analyser with a Randox kit (UK). The
TAS concentration is expressed in mmol/l of plasma
using of Trolox as a standard.
Glutathione level in ADHD children 167
Basic biochemical parameters
Basic biochemical parameters (bilirubin, glucose, γ-glu-
tamyl transferase, alkaline phosphatase, aspartate
aminotransferase, alanine aminotransferase, uric acid
and lipid profile) were analysed in plasma by standard
biochemical procedures using an Hitachi 911 automatic
analyser and Roche kits (Switzerland).
Statistical analysis
Descriptive statistics were obtained for all variables
using mean ± SEM for normally distributed continuous
variables, or medians and 25th and 75th interquartile
ranges (IQRs) for data showing departures from normal-
ity (according to Shapiro-Wilk’s test). Categorical vari-
ables were described using frequencies and proportions.
Due to considerable intersubject variability in the moni-
tored parameters and the unequal number of subjects in
the two groups, the baseline values differed, thus making
impossible any decision on the effect of the treatment.
Therefore, we further worked with differences of glu-
tathione concentration levels between time 0 (the begin-
ning of therapy with Pycnogenol
®
), time 1 (a month
later, at the end of the therapy), as well as between time
0 and time 2 (2 months later, including 4 weeks of wash-
out period). Standard Student’s t-test for the comparison
of raw data showed no departures from normality and
the non-parametric Mann-Whitney U-test for differences
was used as the inference test. The test for two indepen-
dent proportions was used to evaluate qualitatively the
dissimilarity of changes observed in subjects following
treatment with placebo or Pycnogenol
®
. The associations
between variables were analyzed with Pearson’s correlation
coefficients from the models of simple linear regression.
Outlying observations were censored using the typical
tests for outliers (Dixon’s Q-test and Grubbs’ test) and
smoothed by a process referred to as ‘windsorizing’. In
windsorizing, the extreme values (in our case just one
value) are not eliminated from the data set but replaced by
the value of the cut-off criterion.
37
Windsorizing is a com-
promise between the two goals of eliminating the strong
influence of extreme values on the mean while at the same
time utilizing all of the information in a data set.
For statistical analysis, we employed the statistical
program StatsDirect
®
v.2.3.7 (StatsDirect Sales, Sale,
UK). Graphical representation of data was made using
Excel 2000 (Microsoft Co.).
RESULTS
Before the trial, all values of biochemical parameters
(bilirubin, glucose, γ-glutamyl transferase, alkaline phos-
phatase, aspartate aminotransferase, alanine aminotrans-
ferase, uric acid and lipid profile) were in the physiological
range for both groups. None of these parameters changed
beyond the normal range of physiological values after 1
month of Pycnogenol
®
or placebo administration.
At the beginning of the study, the level of GSSG for
the Pycnogenol
®
group was 4.60 ± 0.09 µmol/l. One
month of Pycnogenol
®
administration caused a signifi-
cant decrease in the level of GSSG by 22.03% (3.58 ±
0.51 µmol/l; P = 0.013). In the placebo group, no signif-
icant change was observed. After the wash-out period,
the level of GSSG in the Pycnogenol
®
group increased
again (Fig. 2).
168 Dvo˘ráková, Sivonová, Trebatická et al.
Fig. 2. Oxidized glutathione level in patients suffering from ADHD after Pycnogenol
®
(filled bar) (n = 28) and placebo (empty bar) (n = 14) administration. The
level of GSSG at the beginning of the study was taken as 100%. Values represent mean in percentage ± SEM. 0, examination before the trial; 1, one month
after Pycnogenol
®
or placebo administration; 2, one month after termination of administration (wash-out period). *Denotes significance between
examination 0 and 1 (P < 0.05).
The level of GSH in the Pycnogenol
®
group at the begin-
ning of the study was 102.89 ± 19.08 µmol/l (Fig. 3). A
highly significant increase of GSH level by 26.8% was
determined in the period of the investigation 1 in com-
parison to period 0 (130.44 ± 7.94 µmol/l; P = 0.0054)
and this increase (36.4%) persisted also in the period of
the investigation 2 (140.38 ± 60.74 µmol/l; P = 0.007) in
patients taking Pycnogenol
®
. The level of GSH in
patients taking placebo was not significantly changed.
TAS in children with ADHD is decreased (1.02
mmol/l) in comparison to reference values of 1.1–1.7
mmol/l. After 1 month of Pycnogenol
®
administration,
TAS values increased slightly (1.05 ± 0.016 mmol/l);
however, this elevation was not statistically significant
in comparison to TAS level at the period of investigation
0. A statistically significant difference was recorded
after the wash-out period (1.09 ± 0.02 mmol/l; P =
0.002). Placebo administration had no significant effect
on TAS. TAS values ± SEM (in percentage) are shown in
Figure 4.
There was also a negative correlation between mea-
surement of TAS and concentration of oxidized glu-
tathione after Pycnogenol
®
administration (n = 26, y =
–4.457x + 9.0048, where y = GSSG concentration, x =
TAS level, r = –0.3882, P < 0.05) as shown in Figure 5.
A positive correlation between inattention score and
TAS normalizing is represented in depicted correlation (n =
33, y = –5.612x + 3.662 where y = inattention, x = TAS
Glutathione level in ADHD children 169
Fig. 3. Reduced glutathione level in patients suffering from ADHD after
Pycnogenol
®
(filled bar) (n = 28) and placebo (empty bar) (n = 15)
administration. The level of GSH at the beginning of the study was taken
as 100%. Values represent mean in percentage ± SEM. 0, examination
before the trial; 1, one month after Pycnogenol
®
or placebo administration;
2, one month after termination of administration (wash-out period).
**Significance between examination 0 and 1 after Pycnogenol
®
administration (P < 0.01);
o
significance in examination 1 between
Pycnogenol
®
and placebo (P < 0.05).
Fig. 4.TAS in patients suffering from ADHD after Pycnogenol
®
(filled
bar) (n = 42) and placebo (empty bar) (n = 17) administration. TAS at the
beginning of the study was taken as 100%. Values represent mean in
percentage ± SEM. 0, examination before the trial; 1, one month after
Pycnogenol
®
or placebo administration; 2, one month after termination of
administration (wash-out period). **Significance 0/2 after Pycnogenol
®
administration (P < 0.01).
Fig. 5. Correlation between total antioxidant status and oxidized glutathione level (n = 26, y = -4.457x + 9.0048, r = -0.3882; P < 0.05).
level, P = 0.035) as shown in Figure 6. In the placebo
group, no correlation was found.
DISCUSSION
Imbalance in the levels of pro-oxidants and antioxidants
to the benefit of pro-oxidants leads to elevated oxidative
stress. One possible mechanism to protect the cell
against free radical attack is to increase its antioxidant
pool.
Measurement of the GSH/GSSG ratio has been sug-
gested as a clinical marker in disorders in which oxidative
stress plays a role. GSH is also oxidized to GSSG in age-
ing, which is a reflection of the accumulation of oxida-
tive stress by the organism.
31
A decreased GSH/GSSG
ratio is associated with tumor progression and many
chronic diseases (gastrointestinal, cardiovascular, mus-
culoskeletal).
38
We calculated the GSH/GSSG ratio in patients with
ADHD at the beginning of the trial as 35.93. After
Pycnogenol
®
treatment, the GSH/GSSG ratio rose to
52.26 (P = 0.05). After a wash-out period, the ratio
decreased again to 42.45. In a placebo group, the ratio
GSH/GSSG was unchanged.
The concentration of GSH is also important in neu-
rodegenerative disorders. Glutathione and free radicals
have been recognized as playing a significant role in the
development and progression of many neurodegenera-
tive disorders. The brain is particularly susceptible to
free radical attack, because it generates more free radical
by-products per gram of tissue than any other organ.
Glutathione, as the brain’s important antioxidant, pro-
tects against this; for example, a significant decrease in
the level of GSH (by 27%) was observed in the cere-
brospinal fluid of drug-free schizophrenic patients.
9,12,39
The results of Castagne et al.
40
suggest that low brain
glutathione and ascorbic acid levels associated with the
dopaminergic system actively participate in the develop-
ment of some cognitive deficits affecting patients with
schizophrenia.
The results of Myhrstadt et al.
41
and Carlsen et al.
42
added regulation of GSH concentration to the list of dis-
eases prevented by the effects of polyphenols.
Whereas in ADHD disease damage to catecholamine
metabolism in the regulation of noradrenaline and
dopamine release and uptake is predicted, damage to
glutathione metabolism could share in cognitive deficit
in patients suffering from ADHD. But a positive correla-
tion between GSSG and adrenaline (n = 25, y = 1.3317x
+ 1.9984 where y = adrenaline level, x = GSSG level; P
= 0.007) and GSSG and noradrenaline (n = 25, y =
2.9185x + 8.224 where y = noradrenaline, x = GSSG
level; P = 0.003) has also been found. No correlation has
been proven 1 month after Pycnogenol
®
administration
(Dvo˘ráková et al., unpublished results).
170 Dvo˘ráková, Sivonová, Trebatická et al.
Fig. 6. Correlations between inattention score
13
in period 1 and TAS differences between periods 1 and 0 in Pycnogenol
®
group (n = 33, y = –5.612x + 3.662 where
y = inattention, x = TAS level, P = 0,035). Period 0, examination before the trial; period 1, one month after Pycnogenol
®
/placebo administration.
Total antioxidant status (TAS) in children with ADHD
is slightly decreased when compared to physiological
values of healthy individuals. Pycnogenol
®
administra-
tion caused a slight, but insignificant, elevation of the
TAS. It was interesting to note that improvement of the
antioxidant status persisted after the wash-out period
with a significant increase compared to period 0. In spite
of the fact that Pycnogenol
®
exerts a marked antioxidant
activity in vitro in a hydrophilic as well as a lipophilic
environment,
43
in vivo (plasma) only a mild, non-signifi-
cant increase of antioxidant capacity was found after 1
month of Pycnogenol
®
administration to children with
ADHD (1 mg/kg/day). From the dose of administered
Pycnogenol
®
(1 mg/kg body weight), assuming an aver-
age molecular weight of ~300 for the individual poly-
phenols present in Pycnogenol
®
and about a 30%
absorption of polyphenols in the gastrointestinal tract,
the mean concentrations of polyphenols in blood reaches
about 10 µM. Children have higher concentrations of other
antioxidants in blood (e.g. ascorbic acid, around 50 µM;
tocopherols, 30 µM; and uric acid, 200–390 µM) than
the calculated Pycnogenol
®
contribution. From this it
follows that, under our conditions, the direct Pycnogenol
®
contribution to antioxidant capacity of plasma is not sig-
nificant when compared to the relative higher levels of
other plasma antioxidants.
We have shown a positive influence of Pycnogenol
®
on ADHD symptoms.
14
The relationship between inat-
tention and TAS normalizing has also been confirmed
(Fig. 6).
13
From this it follows that adequate intake of
antioxidants (e.g. from food) may also have a positive
influence on mental disorders like ADHD.
Similarly, a relationship between hyperactivity symptoms
and noradrenaline (n = 37, y = 0.1678x + 6.4039 where y =
hyperactivity score, x = noradrenaline level; P = 0.031) and
hyperactivity and dopamine (n = 38, y = 0.0228x + 5.5812
where y = hyperactivity score, x = dopamine level; P =
0.05) levels has been found (Dvo˘ráková et al., unpub-
lished results).
However, in addition to the direct antioxidant activity,
Pycnogenol
®
has the ability to stimulate activity of
antioxidant enzymes such as SOD,
22
through both up-
regulation of Cu/Zn-SOD protein expression
44
and
increasing its activity.
45
Stimulation of SOD expression
might persist even after discontinuation of polyphenol
administration. Even though SOD is not the main plasma
antioxidant enzyme, its elevated activity in cells may save
low-molecular weight antioxidants, leading to the increase
in their activity in plasma, which persists after termination
of Pycnogenol
®
administration. Pycnogenol
®
also indi-
rectly regenerates other antioxidant systems through
increasing glutathione reductase activity.
22
In this way,
regenerated glutathione (GSH) can also contribute to,
and increase, TAS. This is also shown in our results,
where significantly elevated levels of both TAS and
GSH persisted after the wash-out period in comparison
to period 0.
CONCLUSIONS
One month of Pycnogenol
®
administration to ADHD
children normalises total antioxidant status and
improves the redox state of the organism through a sig-
nificant decrease of GSSG levels and a highly signifi-
cant increase of GSH levels in comparison to a group
taking placebo.
ACKNOWLEDGEMENTS
This study was supported by a grant from Horphag Res.
Ltd, partly by VEGA grant No. 1/1157/04 and grant VV
MVTS 03/LF from the Ministry of Education of the
Slovak Republic, by Drug Research Institute, Modra,
Slovak Republic and by Mind & Health, Civil Association.
The authors wish to thank Assoc. Prof. P. Blazicek
(Bratislava, Slovak Republic) for the biochemical analy-
ses, Prof. Dr P. Rohdewald (University of Münster,
Germany) for his helpful comments and Mrs L.
Chandogová and L. Miková for their technical assistance.
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Background: Pine bark (Pinus spp.) extract is rich in bioflavonoids, predominantly proanthocyanidins, which are antioxidants. Commercially-available extract supplements are marketed for preventing or treating various chronic conditions associated with oxidative stress. This is an update of a previously published review. Objectives: To assess the efficacy and safety of pine bark extract supplements for treating chronic disorders. Search methods: We searched three databases and three trial registries; latest search: 30 September 2019. We contacted the manufacturers of pine bark extracts to identify additional studies and hand-searched bibliographies of included studies. Selection criteria: Randomised controlled trials (RCTs) evaluating pine bark extract supplements in adults or children with any chronic disorder. Data collection and analysis: Two authors independently assessed trial eligibility, extracted data and assessed risk of bias. Where possible, we pooled data in meta-analyses. We used GRADE to evaluate the certainty of evidence. Primary outcomes were participant- and investigator-reported clinical outcomes directly related to each disorder and all-cause mortality. We also assessed adverse events and biomarkers of oxidative stress. Main results: This review included 27 RCTs (22 parallel and five cross-over designs; 1641 participants) evaluating pine bark extract supplements across 10 chronic disorders: asthma (two studies; 86 participants); attention deficit hyperactivity disorder (ADHD) (one study; 61 participants), cardiovascular disease (CVD) and risk factors (seven studies; 338 participants), chronic venous insufficiency (CVI) (two studies; 60 participants), diabetes mellitus (DM) (six studies; 339 participants), erectile dysfunction (three studies; 277 participants), female sexual dysfunction (one study; 83 participants), osteoarthritis (three studies; 293 participants), osteopenia (one study; 44 participants) and traumatic brain injury (one study; 60 participants). Two studies exclusively recruited children; the remainder recruited adults. Trials lasted between four weeks and six months. Placebo was the control in 24 studies. Overall risk of bias was low for four, high for one and unclear for 22 studies. In adults with asthma, we do not know whether pine bark extract increases change in forced expiratory volume in one second (FEV1) % predicted/forced vital capacity (FVC) (mean difference (MD) 7.70, 95% confidence interval (CI) 3.19 to 12.21; one study; 44 participants; very low-certainty evidence), increases change in FEV1 % predicted (MD 7.00, 95% CI 0.10 to 13.90; one study; 44 participants; very low-certainty evidence), improves asthma symptoms (risk ratio (RR) 1.85, 95% CI 1.32 to 2.58; one study; 60 participants; very low-certainty evidence) or increases the number of people able to stop using albuterol inhalers (RR 6.00, 95% CI 1.97 to 18.25; one study; 60 participants; very low-certainty evidence). In children with ADHD, we do not know whether pine bark extract decreases inattention and hyperactivity assessed by parent- and teacher-rating scales (narrative synthesis; one study; 57 participants; very low-certainty evidence) or increases the change in visual-motoric coordination and concentration (MD 3.37, 95% CI 2.41 to 4.33; one study; 57 participants; very low-certainty evidence). In participants with CVD, we do not know whether pine bark extract decreases diastolic blood pressure (MD -3.00 mm Hg, 95% CI -4.51 to -1.49; one study; 61 participants; very low-certainty evidence); increases HDL cholesterol (MD 0.05 mmol/L, 95% CI -0.01 to 0.11; one study; 61 participants; very low-certainty evidence) or decreases LDL cholesterol (MD -0.03 mmol/L, 95% CI -0.05 to 0.00; one study; 61 participants; very low-certainty evidence). In participants with CVI, we do not know whether pine bark extract decreases pain scores (MD -0.59, 95% CI -1.02 to -0.16; one study; 40 participants; very low-certainty evidence), increases the disappearance of pain (RR 25.0, 95% CI 1.58 to 395.48; one study; 40 participants; very low-certainty evidence) or increases physician-judged treatment efficacy (RR 4.75, 95% CI 1.97 to 11.48; 1 study; 40 participants; very low-certainty evidence). In type 2 DM, we do not know whether pine bark extract leads to a greater reduction in fasting blood glucose (MD 1.0 mmol/L, 95% CI 0.91 to 1.09; one study; 48 participants;very low-certainty evidence) or decreases HbA1c (MD -0.90 %, 95% CI -1.78 to -0.02; 1 study; 48 participants; very low-certainty evidence). In a mixed group of participants with type 1 and type 2 DM we do not know whether pine bark extract decreases HbA1c (MD -0.20 %, 95% CI -1.83 to 1.43; one study; 67 participants; very low-certainty evidence). In men with erectile dysfunction, we do not know whether pine bark extract supplements increase International Index of Erectile Function-5 scores (not pooled; two studies; 147 participants; very low-certainty evidence). In women with sexual dysfunction, we do not know whether pine bark extract increases satisfaction as measured by the Female Sexual Function Index (MD 5.10, 95% CI 3.49 to 6.71; one study; 75 participants; very low-certainty evidence) or leads to a greater reduction of pain scores (MD 4.30, 95% CI 2.69 to 5.91; one study; 75 participants; very low-certainty evidence). In adults with osteoarthritis of the knee, we do not know whether pine bark extract decreases composite Western Ontario and McMaster Universities Osteoarthritis Index scores (MD -730.00, 95% CI -1011.95 to -448.05; one study; 37 participants; very low-certainty evidence) or the use of non-steroidal anti-inflammatory medication (MD -18.30, 95% CI -25.14 to -11.46; one study; 35 participants; very low-certainty evidence). We do not know whether pine bark extract increases bone alkaline phosphatase in post-menopausal women with osteopenia (MD 1.16 ug/L, 95% CI -2.37 to 4.69; one study; 40 participants; very low-certainty evidence). In individuals with traumatic brain injury, we do not know whether pine bark extract decreases cognitive failure scores (MD -2.24, 95% CI -11.17 to 6.69; one study; 56 participants; very low-certainty evidence) or post-concussion symptoms (MD -0.76, 95% CI -5.39 to 3.87; one study; 56 participants; very low-certainty evidence). For most comparisons, studies did not report outcomes of hospital admissions or serious adverse events. Authors' conclusions: Small sample sizes, limited numbers of RCTs per condition, variation in outcome measures, and poor reporting of the included RCTs mean no definitive conclusions regarding the efficacy or safety of pine bark extract supplements are possible.
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