Effect of natural polyphenols (Pycnogenol) on oxidative stress markers in children suffering from Crohn's disease – a pilot study

Article (PDF Available)inFree Radical Research 47(8) · May 2013with 446 Reads
DOI: 10.3109/10715762.2013.807508 · Source: PubMed
Abstract
Abstract Crohn's disease (CD) is a nonspecific, chronic inflammatory disease of the gastrointestinal tract. It is supposed that in etiopathogenesis oxidative stress plays a role. However, its precise role in the active and non-active state of disease is not known yet. We conducted a pilot study focusing on the relationship between oxidative stress of Crohn´s disease in remission and the possibility to influence clinical parameters and markers of oxidative stress by polyphenolic extract, Pycnogenol(®). Compared to 15 healthy controls 15 pediatric CD patients (all were in remission according to their disease activity index - PCDAI) had reduced activity of Cu/Zn-superoxide dismutase (SOD) and increased oxidative damage to proteins. We found negative correlations between markers of inflammation (CRP, calprotectin) as well as between PCDAI and total antioxidant capacity. Activities of antioxidant enzymes, SOD and glutathione peroxidase (GPX) negatively correlated with calprotectin and PCDAI. Pycnogenol (2 mg/kg) positively influenced parameters of oxidative stress in CD patients after 10 weeks of administration.
production of reactive metabolites (oxidants) or by com-
promised antioxidant defenses, or by both. OS may be
manifested by the damage to lipids, proteins, and DNA.
Several products resulting from oxidative damage have
been established as markers of OS.
Patients with CD were previously described to have
decreased antioxidant status, elevated various markers of
OS [6,7] such as lipid hydroperoxides [8], oxidatively
damaged DNA in peripheral leukocytes, and altered activ-
ities of antioxidant enzymes [9,10]. Increased OS is
observed not only in the active phase of the disease, but
also in the remission [11,12]. Lipoperoxidation is consid-
ered a key process damaging gut mucosa, inicted by
hydroxyl radical and superoxide [13]. In the inamed
mucosa, increased concentrations of malondialdehyde
(MDA) and reactive nitrogen metabolites have been
detected. Apoptosis of inamed epithelium signicantly
correlates with expression of the enzyme xanthine oxidase
generating superoxide radical [13].
Natural dietary polyphenols belong to substances posi-
tively aecting OS and inammatory disorders. They are
able to reduce inammation by modulating signaling path-
ways in intestinal cells. They interfere with the induction
of NF-kB and MAPK signaling pathways, as well as with
the production of inammatory mediators [14].
Eect of natural polyphenols (Pycnogenol) on oxidative stress markers in children
suering from Crohn’s disease a pilot study
M. Koláček1,2, J. Muchová1, M. Dvořáková1, Z. Paduchová1, I. Žitňano1, I. Čierna3, Z. Országhová1,
D. Székyová3, N. Jajcaiová-Zedníčko4, L. Kovács3 & Z. Ďuračková1
1Institute of Medical Chemistry, Biochemistry and Clinical Biochemistry, Faculty of Medicine, Comenius University, Bratislava, Slovak
Republic, 2Institute of Medical Biochemistry, Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava, Slovak Republic,
32nd Pediatric Clinic of the Faculty of Medicine, Comenius University and Children’s University Hospital, Bratislava, Slovak Republic,
and 4Workplace of Hematology and transfusiology Department of Laboratory Medicine, Children’s University Hospital, Bratislava, Slovak
Republic
Abstract
Crohn’s disease (CD) is a nonspecic, chronic inammatory disease of the gastrointestinal tract. It is supposed that in etiopathogenesis
oxidative stress (OS) plays a role. However, its precise role in the active and non-active states of disease is not known yet. We conducted a
pilot study focusing on the relationship between OS of CD in remission and the possibility to inuence clinical parameters and markers of
OS by polyphenolic extract, Pycnogenol® (Pyc). Compared to 15 healthy controls 15 pediatric CD patients (all were in remission according
to their disease activity index – PCDAI) had reduced the activity of Cu/Zn-superoxide dismutase (SOD) and increased the oxidative damage
to proteins. We found negative correlations between markers of inammation (calprotectin, CRP) as well as between PCDAI and total anti-
oxidant capacity (TAC). Activities of antioxidant enzymes, SOD, and glutathione peroxidase (GPX) negatively correlated with calprotectin
and PCDAI. Pyc (2 mg/kg) positively inuenced the parameters of OS in CD patients after 10 weeks of administration.
Keywords: Crohn’s disease, oxidative stress, antioxidant enzymes, Pycnogenol, calprotectin, diamine oxidase, iron metabolism
Introduction
Crohn’s disease (Morbus Crohn, CD) is a nonspecic,
chronic inammatory disease of the gastrointestinal tract.
Intestine sections aected by inammation are separated
by healthy gut sections. The disease is named after an
American physician–gastroenterologist Burrill Bernard
Crohn, who characterized it in detail for the rst time in
1932. The exact cause of CD is not yet known. A number
of factors come into account: genetic, immunological, and
environmental. Symptoms of the disease include abdomi-
nal pain, diarrhea, weight loss, stunted growth of children,
anorexia, blood in stool, and fever. Disease activity in chil-
dren is expressed by the index paediatric Crohn’s disease
activity index (PCDAI). It can be in either a full or a short
form [1]. Values of the short-form PCDAI 30 corre-
spond to severe disease stage, and values 10 correspond
to a non-active state [2,3].
Since inammation is undoubtedly associated with oxi-
dative stress (OS), it is assumed that factors involved in
the pathogenesis of CD include also OS. OS (or also redox
stress) is dened as an imbalance between oxidants and
antioxidants in favor of the oxidants, resulting in signi-
cant damage to biomolecules, organs, and the whole
organism [4,5]. OS can be caused either by excessive
Correspondence: Prof. Zdeňka Ďuračková, PhD. Institute of Medical Chemistry, Biochemistry and Clinical Biochemistry, Faculty of
Medicine, Comenius University, Sasinkova 2, 813 72 Bratislava, Slovakia. Tel: 1 421 2 59357 564, Fax: 1 421 2 59357 557.
E-mail: zdenka.durackova@fmed.uniba.sk
(Received date: 14 March 2013; Accepted date: 18 May 2013; Published online: 7 June 2013)
Free Radical Research, August 2013; 47(8): 624–634
© 2013 Informa UK, Ltd.
ISSN 1071-5762 print/ISSN 1029-2470 online
DOI: 10.3109/10715762.2013.807508
ORIGINAL ARTICLE
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Crohn’s disease, oxidative stress and Pycnogenol 625
One well-characterized source of polyphenols repre-
sents the French maritime pine (Pinus pinaster) bark
extract Pycnogenol® (Pyc). This extract is standardized to
bear 70 5% procyanidins [15]. Pyc has important anti-
oxidant and multiple biomodulating eects such as inhibi-
tion of transcription factor NF-kB [16] and cyclooxygenase
[17,18], stimulation of NO synthase, and antihypertensive
eect by inhibition of angiotensin-converting enzyme
[19,20,21], antimutagenic eects [22], alleviation of
symptoms of allergic asthma, and reduction of needs for
the use of medication [23]. Fruit juices enriched with Pyc
increased the antioxidant capacity and were shown to have
anti-inammatory activity as compared to fruit juices
without Pyc, in an in vitro study with human colon cancer
cells [24].
Our aim was to carry out a pilot study to examine the
relationship between OS and selected pathophysiological
markers in pediatric CD patients in remission and the ways
how these parameters can be inuenced by natural poly-
phenolic compounds in Pyc. We focused also on monitor-
ing new potential markers for disease progression such as
enzyme diamine oxidase (DAO) and the inammatory
marker calprotectin.
Material and methods
Patients
Fifteen pediatric patients diagnosed with CD (7 males and
8 females; age, 13—18 years) were recruited to our study.
They were registered at the 2nd Pediatric Clinic of the
Children’s University Hospital in Bratislava. Control
group consisted of 15 healthy children (6 males and 9
females; age, 12–18 years). Parents of all participants
signed an informed consent. The study was approved by
the ethics committee of the Faculty of Medicine, Come-
nius University.
Inclusion criteria: Patients with CD in remission
state of disease with a maintenance therapy of immuno-
suppresor Imuran (azathioprine [6-(1-methyl-4-nitro-
5-imidazolylthio)-purine]) were included in our project.
Exclusion criteria: Patients with abdominal abscess,
bowel obstruction, active gastrointestinal bleeding or mal-
nutrition as well as those with acute viral or bacterial
infections and other chronic inammatory disease were
excluded from the study. To the patients who were treated
with 5-aminosalicylic acid (5-ASA), administration of this
drug was stopped 2 weeks prior to the enrollment in the
project (run-in period) and during the next 12 weeks of
the project. Similarly, the use of supplementary antioxi-
dant therapy was ceased during this period. Patients
selected for the project were on a standard diet. Therapy
was adjusted according to the inclusion criteria.
After 2 weeks of run-in period, Pyc in capsules was
administered with a meal to the patients for the next
10 weeks in the morning. Capsules with Pyc powder
(2 mg/kg/day) were prepared individually according to
the body weight of patients. Patients were investigated for
another 2 weeks after discontinuation of Pyc supplementa-
tion as a wash-out period.
Biological specimen (blood, urine, and stool) were col-
lected from patients 1 day before the initiation of Pyc
administration (time 0), on the 5th week (time 5), and the
10th week (time 10) after the beginning of Pyc administra-
tion and 2 weeks after discontinuation of Pyc administra-
tion (time 12). The last intake of Pycnogenol was in the
morning before biological specimen collection.
A placebo group was not included in our project for
ethical and clinical reasons. Patients were excluded from
5-ASA treatment. Preventive anti-inammatory eect of
5-ASA was replaced by administered Pyc, known for its
anti-inammatory eects [18]. In the case of placebo
administration, patients would be without any preventive
anti-inammatory protection.
Blood samples were collected with anticoagulant hiru-
din, and blood plasma and red blood cells were obtained
by centrifugation of blood (10 min at 1 200 g). Red blood
cell hemolysate was prepared by adding 1.5 ml of ice-cold
distilled water to 0.5 ml of erythrocytes which had been
previously washed thrice in saline. Serum was obtained
from blood in the absence of anticoagulant using a stan-
dard procedure. Aliquoted biological samples were frozen
for later investigation. Blood plasma and serum were kept
at 80°C, and hemolysates, stool and urine at 20°C
until carrying out the analysis.
Characteristics of disease activity—determination of
PCDAI index
The severity of CD was characterized by the short-form
PCDAI scores [3,25]. PCDAI values 30 correspond to
the severe stage of disease, and values 10 correspond to
the non-active state [2].
Determination of basic biochemical parameters
Concentrations of basic biochemical parameters and
parameters of iron metabolism in serum and creatinine in
urine were measured at the department of laboratory med-
icine of Children’s University Hospital in automatic ana-
lyzer COBAS Integra 800 (Roche) with a closed system
by standard methods using Roche kits. Physiological val-
ues were obtained from the department of laboratory
medicine of Children’s University Hospital.
Determination of CRP
Concentration of the very sensitive CRP was determined
in serum as a routine laboratory diagnostics in the Chil-
dren’s University Hospital by the immunochemical ana-
lyzer Vitros 5.1 Fusion Vitroc 5600 from Assist Company
(Slovakia).
Determination of calprotectin concentration in stool
A kit from NovaTec Immundiagnostica GmbH, Germany,
was used for calprotectin extraction and determination.
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626 M. Koláček et al.
Absorbance of the reaction mixture was monitored using
the microplate reader Gen5 ELISA from the Biotek
Company, USA. The concentration of calprotectin in stool
was expressed in micrograms/gram of stool.
All necessary chemicals were obtained from Lachema,
Czech Republic, Sigma-Aldrich, USA, and Lambda-Life,
Slovakia, and they were of p.a. purity.
Determination of hemoglobin
Hemoglobin (Hb) was determined using the Drabkin
method. To 5 ml of Drabkin solution (0.77 mmol/l KCN,
0.6 mmol/l K3 [Fe (CN)6], and 1 mmol/l KH2P04), 100 ml
of hemolysate was added, the solution was mixed, and
then it was allowed to stand for 15 min at room tempera-
ture. Optical density was measured spectrophotometrically
at a wavelength of 540 nm. As a reference solution Drab-
kin solution without hemolysate was used. The amount of
Hb was expressed in gram/litre.
Determination of Cu/Zn-superoxide dismutase
activity in erythrocytes
Activity of SOD was determined by the commercial kit
from Fluka, Germany, using bovine Cu/Zn-SOD as a stan-
dard (Sigma, Germany). Absorbance was monitored by
the microplate reader GEN5 Elisa from Biotek USA com-
pany. A volume of 20 ml of hemolysate was used for anal-
ysis, and 1 U of SOD activity is dened as the enzyme
activity causing 50% inhibition of WST-1 reagent reduc-
tion. SOD activity was expressed in U SOD/mg Hb.
Determination of catalase activity in erythrocytes
Catalase activity (CAT) was determined according to
Bergmeyer [26]. Determination is based on a change of
hydrogen peroxide absorbance per time at a wavelength of
240 nm. To 50 ml of 50 mmol/l PBS, pH 7.0, 250 ml H2O2
was added and diluted so that the absorbance was 0.7–1.0.
To 2 ml of this mixture 30 ml of hemolysate was added
and change of absorbance was monitored spectrophoto-
metrically (PharmaSpec UV-1700, Shimadzu, Japan) at a
wavelength of 240 nm for 1 min. CAT was calculated by
the formula:
Akat dA/DF
[] min 3

43 616 710
..
V
where dA/min is the absorbance change/1 min., DF is the
dilution factor, V is the volume of the sample, 43.6 is the
molar absorption coecient of hydrogen peroxide, and
16.7 103 is the conversion factor of U activity units to
katals. CAT activity was expressed in katals/gram of Hb.
Determination of glutathione peroxidase activity
in erythrocytes
Activity of glutathione peroxidase (GPX) was determined
by the commercial kit from Sigma-Aldrich, USA, using
an indirect method. A volume of 30 ml of hemolysate
was used for analysis. Change in absorbance was moni-
tored spectrophotometrically (UV-1700 PharmaSpec,
Shimadzu, Japan) at 340 nm. Activity of GPX was
expressed in U/g Hb.
Determination of total antioxidant capacity
of blood serum
Total antioxidant status was determined using the Trolox
Equivalent Antioxidant Capacity method (TEAC) [27].
ABTS solution (14 mmol/l ABTS, 4.9 mmol/l K2S2O8)
was diluted with deionized water until the absorbance was
0.7 0.02 at 734 nm. To 1980 ml of ABTS working solu-
tion 20 ml of serum was added and the absorbance was
recorded at the wavelength of 734 nm against deionized
water at time 0 and after 10 min. Absorbance was mea-
sured spectrophotometrically (UV-1700 PharmaSpec of
Shimadzu, Japan) at a wavelength of 734 nm. Serum anti-
oxidant capacity was determined from the analytical curve
for trolox as a standard. Total antioxidant capacity (TAC)
was expressed in mmol trolox/l.
Determination of lipoperoxides in serum
Lipoperoxides (LPs) in serum were determined as
described by El Saadani et al. [28]. Determination is based
on the ability of peroxide to oxidize iodide (I) to iodine
(I2). Iodine in the reaction mixture then reacts with excess
iodide to form I3 with the absorption maximum at a wave-
length of 365 nm. A volume of 100 ml of serum was used
for analysis. Absorbance was measured spectrophotomet-
rically (UV-1700 PharmaSpec of Shimadzu, Japan) at
365 nm. LP concentrations were calculated by the
formula: c [nmol/ml]
ASA IDF/( )ε3
where ASA is the
absorbance of the sample, e (I3) is the molar absorption
coecient for I3 24 600 mol1·1·cm1 and DF is
the dilution factor.
Determination of free 8-isoprostanes in plasma
and urine
Free 8-isoprostanes (8-isoP) were determined using the
commercial EIA kit from Cayman Chemical Company,
USA. Fifty microliters of plasma or urine was used for
analysis. Absorbance was determined at 405 nm using
microplate reader GEN5 Elisa from Biotek, USA. The
concentration of 8-isoP was expressed in pg/ml.
Determination of diamine oxidase concentration
in serum
To determine the concentration of DAO we used the kit
from DRG Instruments, Germany. A volume of 100 ml of
serum was used for analysis. Absorbance was determined
at 450 nm by microplate reader GEN5 Elisa from Biotek,
USA. DAO concentration in the sample was expressed
in U/ml.
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Crohn’s disease, oxidative stress and Pycnogenol 627
Determination of advanced oxidation protein
products in serum
To the serum sample, the precipitating reagent (1 mol/l
MgCl2 6H2O:dextran sulfate, 1:1) was added in a ratio of
10:1 (sample:reagent). Precipitated lipids were removed
by centrifugation at 980 g for 4 min and the obtained
supernatant was used for further experiments. For the
calibration curve we used chloramine T solutions. The
microtiter plate was loaded with 200 ml of the sample 5
times diluted (40 ml of serum 1 160 ml of PBS) with 10
mmol/l PBS pH 7.4. To the wells with standard solutions,
10 ml of 1.16 mmol/l KI solution was added and the plate
was shaken for 6 min. To each well (standards and sam-
ples), 20 ml of glacial acetic acid was added and the plate
was shaken for 2 min. The absorbance of wells was mea-
sured using microplate reader GEN5 Elisa from Biotek,
USA, at a wavelength of 340 nm. The concentration of
AOPP was expressed in mmol/l from the calibration
curve.
Statistical analysis
Normal data distribution was determined by histograms
construction (StatsDirectR v.2.3.7., StatsDirect Sales,
Sale, UK). Descriptive statistics was obtained for all vari-
ables using the mean SEM for normally distributed
continuous variables, and median with interquartile range
(1st and 3rd quartiles) was calculated for data showing
departure from normality. The data without deviation
from normality were evaluated using the Student’s t-test.
For the evaluation of other data, the Mann–Whitney test
was used. To nd the dierences in PCDAI scores at
dierent times, the non-parametric Friedman test was
used and veried by the permutation tests. For parametric
linear regression, Pearson’s correlation coecient was
calculated, and for nonparametric linear regression
Spearman’s rank correlation coecient was used.
For statistical analysis, we employed the statistical pro-
gram StatsDirectR. For graphical representation of data
we used Excel 2000 (Microsoft Co.). Due to the limited
number of patients and also due to the fact that this was a
pilot study, the limit for statistical signicance was set
at p 0.1.
Results
Our project completed 14 patients, of the 15 patients
enrolled in the study. One patient was withdrawn from the
study for social reasons. The patient was a child from the
orphanage and was moved to another remote region and
hospital lost contact with him.
All remaining patients completed the study and did not
report any adverse eects during the 12 weeks of investi-
gational period.
Of the 15 CD patients, seven were without undergoing
the therapy and eight patients were taking immunosup-
pressant Imuran (azathioprine). The potential eect of
Imuran treatment on clinical status has been veried by
statistical analysis of the dierences between selected
markers of patients with CD not taking Imuran. By statis-
tical analysis, we found no dierences between CD patients
for PCDAI parameters, CRP, calprotectin, DAO, total pro-
teins, and albumin.
Main clinical and biochemical characteristics of our
patients are listed in Table I. All patients were in remission
(mean score of PCDAI was 3.93).
Table I. Basic clinical and biochemical characteristics of patients involved in our study.
Parameters Physiological
values Controls
Crohn’s disease patients
Before Pyc
administration
Weeks after Pyc administration
5 10 12
Age (years) 13.92 1.969
(12–18) 16.33 1.496
(13–18) 16.33 1.496 16.33 1.496 16.33 1.496
M:F 7: 8 6: 9
Duration of
disease (years) 3.53 2,774
(1–10)
Hight (m) 1.58 0.052 1.66 0.090 1.66 0.090 1.66 0.090 1.66 0.090
Weight (kg) 49.63 9.276 54.4 3.375 54.32 9,148 54.64 9.694 54.64 9.769
BMI (kg/m2) 19.66 2.311 19.76 2.019 19.69 2.150 19.74 1.967 19.73 1.959
PCDAI 10 3.93 1.730 3.92 1.847 4.08 1.977 4.85 3.211
CRP (mmol/l) 0–10 1.4 1.151 13.35 13.230*** 11.80 9.845 13.41 10.329 13.46 7.641
Stool
calprotectin (mg/g) 19.28 (0;19.50) 1 076.73***
(374.29;1 333.18)
1237.42 (442.00;
1 425.55) 1168.65 (802.82;
1 416.55) 990.31 (566.80;
1 482.15)
Diamine
oxidase (U/ml)
10 9.96 2.859 10.76 4.438 12.55 4.77012.23 3.47311.45 3.344
Total proteins (g/l) 60–85 76.50 3.459 77.86 7.260 76.72 6.219 73.33 6.85271.28 6.336
Albumin (g/l) 35–52 44.70 3.755 42.45 4.365 41.48 4.286 39.95 4.12539.25 7.847
All values are mean SD, only calprotecin is median (1st quartile; 3rd quartile). BMI – body mass index, CRP – high sensitivity C-reactive protein, F – female,
M – male, PCDAI – Paediatric Crohn’s Disease Activity Index, Pyc – pycnogenol, * – p 0.01 CD vs. Control, *** – p 0.001 CD vs. Control, p 0.1
Pyc vs. baseline (before Pyc administration), n for CD 14, n for C 15.
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628 M. Koláček et al.
As expected, patients with CD had increased CRP lev-
els and signicantly elevated the levels of calprotectin in
their stools (Table I). Pyc administration had no aect on
the levels of these inammatory markers.
We examined the level of enzyme diaminooxidase
(DAO) characterizing allergic reactions accompanying
inammation and histamine intolerance. There was no
signicant dierence in the levels of DAO between patients
with CD and controls. Pyc signicantly increased the lev-
els of DAO in patients with CD after 5 and 10 weeks of
its administration. After 2 weeks of wash-out period, the
DAO level again decreased (Table I).
Parameters related to iron in CD patients
In patients with CD we determined, as expected, decreased
concentration of iron ions in serum leading to the reduction
in transferrin saturation and to increase in free Fe binding
capacity. We found elevated soluble transferrin receptor
(sTfR) level which is understandably associated with reduced
levels of iron ions. We calculated the ratio between concen-
trations of sTfR and logarithm of ferritin concentration
(sTfR/Ferri). Patients with CD had an average index value
sTfR/Ferri 5.16 conrming a reduced iron supply (Table
II). We did not observe any dierences between total bind-
ing capacity (TBC) of Fe, transferrin and ferritin levels in
CD patients in comparison with those in controls.
After 10 weeks of administration Pyc caused reduction
in serum iron concentration, which resulted in an increase
in sTfR levels. Aected values returned to the level prior
to the Pyc treatment after stopping the supplementation
(Table II). Pyc administration reduced serum ferritin lev-
els after 5 and 10 weeks of its supplementation (from
21.51 to 17.76 and 12.84 mg/l), but this reduction was not
signicantly pronounced, probably due to large deviations
of physiological values (Table II) and the small number
of patients. Other parameters of iron metabolism were not
inuenced by Pyc.
Parameters realted to oxidative stress in CD patients
From studied markers of OS, we observed decreased SOD
activity in erythrocytes of patients with CD and increased
level of the marker of oxidative damage to proteins in
Table II. Parameters of iron metabolism in patients with CD and a control group.
Parameter
Physiological values
Controls
Crohn’s disease patients
1–14 years 15–18 years Before Pyc
administration
Weeks after Pyc administration
5 10 12
Fe (mmol/l) 4.7–19.7 M, 4.8–24.7
F, 5.9–18.3 15.69 6.32 7.29 3.65** 7.17 4.68 5.26 2.747.68 6.63
Free binding
capacity (FBC) of
Fe [mmol/l]
20–62 50.86 6.09 59.83 13.08*59.20 12.09 60.19 11.80 56.66 16.65
Transferrin
(Tran) [g/l] 1.71–3.74 2–4 3.21 0.10 2.96 0.51 3.14 0.583.07 0.52 2.98 0.64
Ferritin
(Ferri) [mg/l] 1.5–4.2 15–150 18.80 (15.95;
22.80) 12.30 (8.50;
18.35) 13.60 (11.20;
16.25) 10.10 (6.85;
16.10) 9.65 (7.75;
18.88)
Solubile transferrin
receptor
(sTfR) [mg/l]
1.5–4.2 1.2–3.2 3.56 1.67 4.70 1.67*5.10 1.67 5.16 1.584.96 1.85
Saturation of
transferrin [%] 20–50 23.51 9.12 11.31 6.49*11.02 7.53 9.06 6.52 13.66 16.26
sTfR/Ferri Index 2.78 3.67 5.16 3.67*6.31 7.40 5.38 2.38 5.11 2.57
All values are mean SD, only ferritin is median (1st quartile; 3rd quartile). Ferri – Ferritin, Pyc – pycnogenol, sTfR – Solubile transferrin receptor,
* – p 0.1 CD vs. Control, ** – p 0.01 CD vs. Control, – p 0.1 Pyc vs. baseline (before Pyc administration), n for CD 14, n for C 15.
Table III. Selected markers of oxidative stress.
Parameters Controls
Crohn’s disease patients
Before Pyc
administration
Weeks of Pyc administration
5 10 12
SOD (U/mg Hb) 63.00 6.012 37.19 9.453*** 46.66 17.59845.98 10.06842.21 16.331
GPX (U/mg Hb) 42.8 15.332 37.8 20.372 51.7 33.11851.6 38.64839.3 33.388
LP (nmol/l) 42.76 10.094 42.53 15.409 30.75 7.69030.72 8.32427.49 7.001
8-isoP in urine (ng/mmol Crea) 246.95 88.359 219.18 101.103 199.21 54.270 156.53 60.617174.39 92.379
AOPP (mmol/ml) 34.63 8.821 48.39 21.327*33.57 10.42938.70 10.445 36.50 7.097
All values are mean SD. 8-isoP – free 8-isoprostanes, AOPP advanced oxidation protein products, Crea – creatinine, GPX – glutathione peroxidase,
LP lipoperoxides, Pyc – pycnogenol, SOD – superoxide dismutase, * – p 0.1 CD vs. Control, *** – p 0.001 CD vs. Control, – p 0.1 Pyc vs.
baseline (before Pyc administration), ∆∆∆p 0.001 Pyc vs. baseline (before Pyc administration), n for CD 14, n for C 15.
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Crohn’s disease, oxidative stress and Pycnogenol 629
serum, advanced oxidation protein products (AOPPs).
Other parameters of OS (CAT, GPX, TAC, LP, and 8-isoP
in plasma and in urine) were not dierent in CD patients
in comparison with the controls.
Pyc administered to patients with CD signicantly
increased SOD and GPX activities in erythrocytes after 5
and 10 weeks of supplementation. After 2 weeks of wash-
out period, activities of mentioned enzymes showed the
tendency to return back to original activities. Pyc caused
reduction in AOPP concentration after 5 weeks of its
administration and LP levels in serum after 5 and 10
weeks of supplementation. This reduction persisted also
after wash-out period. Pyc reduced levels of 8-isoP in
urine after 10 weeks of its administration (Table III). Lev-
els of 8-isoP in plasma were not signicantly aected,
although the trend of their reduction was observed and
reached signicance only after the wash-out period (from
42.77 to 32.30 pg/ml, p 0.1). The CAT activity and TAC
of serum were not aected by the administered Pyc.
Correlations between the parameters
We have found a number of signicant correlations
between the studied variables. CRP is positively corre-
lated with PCDAI (r 0.586, p 0.029) (Figure 1A). The
trend of a positive correlation between calprotectin and
PCDAI was not signicant (r 0.392, p 0.1105). Despite
expectations, the correlation between calprotectin and
CRP was not signicant (r 0.075, p 0.789). However,
CRP positively correlated with ferritin (r 0.457,
p 0.086) (Figure 1B).
TAC of serum negatively correlated with disease
activity characterized by PCDAI score (r 0.829,
p 0.0002) and with the inammatory markers CRP
(r 0.491, p 0.0138) and calprotectin (r 0.577,
p 0.025) (Figure 2).
From markers of OS, we found negative correlations
between SOD activity and calprotectin level (r 0.369,
p 0.10) and also between PCDAI and GPX activity
(r 0.457, p 0.10) (Figure 3). Free plasma 8-isoP
negatively correlated with parameters of iron metabolism
(the total as well as the free iron-binding capacity, sTfR,
and transferrin) and positively correlated with ferritin
(Table IV).
Unexpectedly, we found a positive correlation between
DAO activity and CRP (r 0.8898, p 0.01 and with
PCDAI, (r 0.5466, p 0.0855) (Figure 4).
Discussion
CD is one of the diseases in which OS plays a key role in
pathogenesis. OS is characterized by conditions in which
the level of antioxidant capacity is depressed and oxidative
damage to biomolecules is increased, or both. It is assumed
that OS and its consequences are rather causally related to
Figure 1. Correlations between inammatory markers and PCDAI. CRP – high sensitivity C-reactive protein, Ferri ferritin, PCDAI
Paediatric Crohn’s Disease Acitvity Index, r Spearman’s rank correlation coecient.
Figure 2. Correlations between antioxidant ability and disease activity and inammatory markers respectively. Calp – calprotectin, CRP
high sensitivity C-reactive protein, PCDAI – Paediatric Crohn’s Disease Acitvity Index, TAC – total antioxidant capacity, r Pearson’s
correlation coecient.
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630 M. Koláček et al.
inammatory processes of the disease than representing a
consequence of inammatory processes [29].
Although patients enrolled in the study were in situa-
tion of disease remission according to PCDAI score, sig-
nicantly elevated levels of CRP and fecal calprotectin
suggested persisting inammatory process.
Except for the activity of SOD and AOPP levels, we
have not found any signicant dierences in parameters
of OS between patients and the control group. This is con-
sistent with the observation that patients in the active
phase have elevated LP levels and reduced TAC, while for
the those in the remission phase of the disease these
parameters are comparable with those of healthy control
groups [8,30]. In conrmation with the literature [7],
where increased AOPP levels were found in both, in active
phase and in remission, we observed increased AOPP lev-
els in the patients in remission phase.
In case of activities of antioxidant enzymes in eryth-
rocytes, data in the literature are controversially discussed.
In the active phase of CD, Krzystek-Korpacka et al. [11]
observed reduction in catalase and GPX activities; how-
ever, expression of superoxide dismutase (SOD) was
increased. Activities of CAT and GPX were inversely pro-
portional to the disease activity. Anemia causes reduction
in CAT activity. Phylactos et al. [10] observed reduction
in activities of SOD and GPX. In contrast, other authors
[8,31] observed an increase in the activity of GPX in
patients with CD. The activity of antioxidant enzymes
cannot be interpreted without assessing markers indica-
tive for oxidative damage to the organism. We observed
reduction in intracellular antioxidant protection due to
decreased activity of SOD. Negative correlations between
SOD and Calp as well as between GPX and PCDAI scores
conrm a causal involvement of OS (represented by
reduced activity of antioxidant enzymes) in the pathophys-
iology even in the quiescent stage of the disease with low
PCDAI score.
Negative correlations between TAC, disease activity
(PCDAI), and markers of inammation (CRP, calpro-
tectin) as well as a positive correlation between free
8-isoP levels and CRP levels conrm that there is a
relationship between inammation in CD patients and
OS in accordance with conclusions by Rezaie et al.
[29], not only in the acute phase but also in the remis-
sion phase of the disease. Other authors observed also
positive correlations between disease activity and
lipoperoxidation [8].
Calprotectin is a protein produced by neutrophils and
is considered a progressive marker of inammatory dis-
ease. Elevated concentrations of calprotectin in stool were
detected in patients with colorectal cancer, inammatory
bowel disease, and gastrointestinal bacterial infection.
Signicantly increased fecal calprotectin concentrations
were observed in patients with CD (children and adults)
compared with those of healthy individuals [32]. This
marker could be used to monitor success of a treatment [33],
and could replace the invasive examination—endoscopy
[34,35].
There is no published clinical data available yet on the
eects of Pyc on CD. In an experimental model of colitis
in rats induced by 2,4,6-nitrobenzene sulfonic acid, Pyc
administration showed a dose-dependent signicant
reduction of the extent and severity of the damage to
intestinal mucosa, corresponding to a reduction of
myeloperoxidase activity (MPO) by 73% at the dose of
0.5 mg/kg and up to 93% at 10 mg/kg [36]. Several
in vitro and in vivo animal and human studies have been
carried out to examine the eect of dierent plant poly-
phenols on IBD, using pure polyphenolic standards
as well as standardized natural plant extracts. A number
of polyphenol species have been suggested to show
Table IV. Correlations of parameters of iron metabolism with CRP
and 8-isoP in plasma.
Correlation between
parameters r p
Ferri vs. CRP 0.608 0.043
TBC Fe vs. 8-isoP 0.564 0.0015
FBC Fe vs. 8-isoP 0.576 0.003
sTfR vs. 8-isoP 0.5437 0.0183
Tran vs. 8-isoP 0.643 0.0001
Ferri vs. 8-isoP 0.353 0.0249
8-isoP – free 8-isoprostanes, CRP – high sensitivity C-reactive protein, FBC
Fe free bindig capacity of iron, Ferri – ferritin, TBC Fe total binding
capacity of iron, Tran – transferrin, sTfR – solubile transferrin receptor,
r Pearson’s correlation coecient.
Figure 3. Correlations between calprotectin, disease activity and activities of antioxidant enzymes. Calp – calprotectin, GPX – glutathione
peroxidase activity, Hb – haemoglobin, SPCDAI – Paediatric Crohn’s Disease Activity Index, SOD – Cu/Zn-superoxide dismutase activity,
r Pearson’s correlation coecient.
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Crohn’s disease, oxidative stress and Pycnogenol 631
anti-inammatory eects in bowel disorders [15]. For
example, ellagic acid diminished the severity and exten-
sion of the intestinal injuries in rats with CD induced by
intracolonal administration of 2,4,6-trinitrobenzene sul-
fonic acid [37]. Pyc itself showed the ability to reduce
markers of OS in several conditions, not limited to inam-
matory pathologies [17,38–46]. In the present study, after
5 weeks of administration, Pyc decreased the levels of
AOPP to the level comparable with healthy controls.
Also markers of oxidative damage to lipids were found
to be improved. The level of 8-isoP was decreased in both
plasma and urine. Whether this results from Pyc inhibit-
ing eicosanoid-generating enzymes [18] or from its anti-
oxidant properties remains speculative at this stage. Also
the concentration of LPs was decreased after Pyc admin-
istration, which is consistent with our previous results
obtained in our model system of liposomes [47]. TAC
was not inuenced by the administration of Pyc after 5
and 10 weeks of administration which is in accordance
with our previous results found in children with ADHD
[39]. However, in adult men suering from erectile dys-
function, improvement of antioxidant capacity deter-
mined using FRAP method was observed after 3 months
of Pyc administration [38]. In our study, Pyc increased
SOD activity already after 5 weeks of treatment. How-
ever, our results do not allow us to judge whether this
increase in activity is associated with increased expres-
sion or post-translational modication. In previous
experiments, we found that Pyc increases the expression
of SOD in the brain of diabetic rats [48]. Pyc also sig-
nicantly elevated the levels of glutathione redox enzyme
activities in diabetic rats [49].
Also antioxidant enzymes (SOD, GPX, glutathione
reductase, and glutathione S-transferase) in liver of dia-
betic rats were ameliorated after Pyc supplementation
[50]. In our study, the activity of CAT was not aected by
Pyc. However, in some animal experiments with CCl4-
induced hepatotoxicity, except SOD, GPX and CAT activ-
ities were increased [51].
Transferrin is traditionally considered an antioxidant
due to its ability to bind iron, thus preventing catalysis of
free radical reactions (Fenton reaction and Haber–Weiss
reaction). Patients with CD are known to have reduced
concentrations of the Fe. Therefore, it is not clear whether
the potential antioxidant capacity of transferrin is respon-
sible for the negative correlation of transferrin with 8-isoP
or some other reasons are involved. Ferritin is one of the
acute-phase proteins, increasing its levels during inam-
mation. This could explain the positive correlation with
8-isoP. Positive correlation of ferritin with 8-isoP as well
as its positive correlation with CRP point rather to a
prooxidant action of ferritin [52,53].
The inuence of Pyc on status of some trace elements
was investigated by our research group in relation to atten-
tion-decit hyperactivity disorder in children [46]. It was
found that Pyc decreased the level of iron in serum of
ADHD children. Reduction of iron level was also observed
in our present study in CD children after 10 weeks of Pyc
administration. Although procyanidins in Pyc are sug-
gested to chelate certain metals, however, whether this is
true for its metabolites prevailing in humans remains
unknown. On the other hand, the positive eect of Pyc on
metabolism of iron in CD patients can be supported by the
trend of decreasing ferritin level with potential prooxidant
properties and of increasing level of transferrin with
potential antioxidant action (Table II).
DAO characterizes histamine intolerance. Histamine
intolerance is highly probable in patients with DAO
activity 3 U/ml, less probable in patients with DAO
activity 10 U/ml, and improbable in patients with
DAO activity 10 U/ml [54]. DAO is an enzyme found
primarily in the intestinal mucosa. Circulating DAO
activity correlates with intestinal mucosa activity and the
status of the intestinal mucosa and with activity index
[55,56]. Activity of this enzyme could be considered a
marker of disease activity since it inversely correlates
with the wall permeability of the small intestine. The
increase in intestinal epithelial permeability precedes
relapse of CD. We measured the enzyme levels not its
activity. However, according to the manufacturer data,
there is a correlation between its level and its activity
(r 0.837); therefore, we can compare our results with
results of the study measuring directly DAO activity. Sig-
nicantly reduced DAO activity in serum was observed
in patients with CD compared to controls [57]. However,
we have not detected a signicant dierence. This can be
Figure 4. Correlations between inammation, PCDAI and the level of diamine oxidase. CRP – high sensitivity C-reactive protein, DAO –
diamine oxidase, PCDAI – Paediatric Crohn’s Disease Acitvity Index, r Pearson’s correlation coecient.
Free Radic Res Downloaded from informahealthcare.com by IBI Circulation - Ashley Publications Ltd on 09/16/13
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632 M. Koláček et al.
explained by dierences in the size of experimental
groups as well as by the fact that our patients were in
remission. Surprisingly, the observed positive correlation
of DAO activity with CRP and PCDAI scores (not with
calprotectin) assumes the induction of increased DAO
activity by existing inammation in CD patients. How-
ever, these assumptions need to be conrmed in further
studies with a larger number of patients enrolled.
We have found increased level of DAO after 5 and 10
weeks of Pyc administration. The inuence of Pyc on
DAO activity has not been published yet. However, anti-
histaminic activity of Pyc was investigated in dierent
models. Pyc dose-dependently reduced histamine release
from rat peritoneal mast cells triggered by anti-dinitrop-
henyl IgE [58] or by compound 48/80 and calcium iono-
phore A-23187 [59].
Conclusion
Our results conrm important role of OS, especially
imbalance in antioxidant enzymes activities, in chronic
inammation of CD patients. Administered Pycnogenol
had no impact on markers of inammation (CRP, cal-
protectin) and disease activity (PCDAI score), but posi-
tively modied markers of iron metabolism, with
increased transferrin and reduced ferritin levels. Pycnog-
enol reduces OS (it increased activities of antioxidant
enzymes such as SOD and GPX, reduces the level of
markers of oxidative damage to lipids (lipoperoxides,
8-isoP) and proteins (AOPP)), and increases the level of
DAO. Pycnogenol may represent a useful and tolerable
supplement for reducing secondary eects of inamma-
tion in CD patients.
Acknowledgement
Pycnogenol was kindly provided by Horphag Res. Ltd.,
Geneva, Switzerland. The authors thank to Dr. Frank
Schonlau, PhD. from Horphag. Res. for his kind contribu-
tion to manuscript preparation, to Mrs. L. Chandogová
and D. Opálená for their technical cooperation, to Mrs. L.
Míková for her help with references, and to the nurses
from 2nd Pediatric Clinic of the Children’s University
Hospital and Faculty of Medicine CU for their help during
collection of biological material.
Declaration of interest
The authors report no declarations of interest. The authors
alone are responsible for the content and writing of the
paper.
The study was conducted with the nancial support
from the Ministry of Health grant 2007/16-UK-01, grants
VEGA 1/4310/07 and 1/0224/08 and Mind and Health,
civil association.
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