Bull Vet Inst Pulawy 53, 449-453, 2009
INFLUENCE OF ETHANOL SOLUTION OF RESVERATROL
ON LEUKOCYTE COUNT AND PHAGOCYTIC ACTIVITY
IN PIGLETS
ZDENKA HOLEŠOVSKÁ, TOMÁŠ VOLNÝ1, VÁCLAV KOTRBÁČEK,
AND JAROSLAV DOUBEK
Department of Physiology and Pathophysiology, University of Veterinary and Pharmaceutical Sciences,
612 42 Brno, Czech Republic
[email protected]
1
Innovation Institute, 746 01 Opava, Czech Republic
[email protected]
Received for publication June 15, 2009
Abstract
Ten six-week-old White Large piglets were administered resveratrol (res) in a daily dose of 3 mg /kg0.75 b.w. Another ten piglets
were used as controls. Both groups were housed in individual pens, fed ad libitum a complete feed mixture. Blood samples were
collected from v. jugularis at the beginning of weeks 1 and 2 of the experiment. The total number and different types of white blood
cells (WBC) were determined. The percentage of phagocytising cells (PB values) and the phagocyte activity index (PAI) were
determined in neutrophils. The administration of res caused a significant decrease in the number of WBC (P<0.01) at the end of week
1. The decrease was recorded in the number of lymphocytes (P<0.01) and almost to half in neutrophils (P<0.01), particularly in
young cell band-forms (P<0.01). After two weeks of res administration no further reduction in leukocyte count was found. In the
controls, a significant (P<0.01) decrease in WBC, caused by the reduced number of lymphocytes (P<0.05), was observed only at the
end of the experiment and the number of neutrophils remained unchanged. The number of bands at the end of the first week was
even significantly higher (P<0.05). Res lowered PB (P<0.05) and the PAI (P<0.01) values at the end of the experiment. It can be
concluded that res caused a significant, although only temporary, suppression of leukopoiesis, with a consequent decrease in
neutrophil phagocyte activity.
Key words: lymphocytes, phagocytosis, resveratrol.
Resveratrol (trans-3, 5, 4‘-trihydroxystilbene)
was first isolated in 1940 from the underground part of
white hellebore, Veratrum. It has already been found in
over 72 species of plants (21). It belongs to
phytoalexins, i.e. secondary plant metabolites, which are
produced in response to certain fungal infections (1). In
the late 20th century, comprehensive information on
resveratrol was obtained thanks to its presence in
grapevine, Vitis vinifera (18). For research purposes,
resveratrol is usually extracted from different plants, or
it is produced synthetically (21). Resveratrol is one of
the most potent natural antioxidants, a scavenger of free
radicals (19). It belongs to tumour inhibitors (11), is
mentioned in connection with anti-inflammatory effects,
and has the ability to reduce blood fats and cholesterol
levels (9, 17). Trans-resveratrol is a very effective
phytoestrogen that modulates oestrogen metabolism (13,
15). Resveratrol experiments have so far been mainly
performed on laboratory animals, i.e. on rats, rabbits,
and mice (4, 7) or in vitro experiments (10).
In the study reported here, the effects of
resveratrol extracted from Polygonum cuspidatum on
total leukocyte, lymphocyte, and neutrophil counts in
piglets were tested. The pig as a model species was
selected because during the early postnatal period there
are very dynamic age changes in numbers, types, and
functional abilities of leukocytes. In a relatively short
time, it is possible to monitor the influence of these
extracts on given changes. Regarding the fact that
leukocytes are an important part of the developing
immune system of piglets, we expect to use the
knowledge gained in veterinary medicine. Similarity of
physiological functions of the chosen model with the
human body also allows a wider interpretation of the
results.
Material and Methods
Two litters of 26-week-old Large White breed
piglets were divided equally into a control group (C) and
an experimental group (E). In each group, there were six
males and four females (Table 1).
450
Table 1
Experiment design
Control group (n=10)
Day 0
Day 1-6
Blood sampling 1
20 ml of 15% alcohol per piglet per day
Day 7
Day 8–13
Blood sampling 2
30 ml of 15% alcohol per piglet per day
Day 14
Blood sampling 3
Piglets were fed ad libitum a complete feed mix
for piglet rearing and had free access to drinking water.
Dry extract of resverin from Polygonum cuspidatum
roots, standardised to 50% content of trans-resveratrol
and its glycosides and 6% content of emodin, partly
soluble in water and readily soluble in ethanol, were
administered daily to the piglets through a gastric probe
according to the pattern below (Innovation Institute,
Opava).
Blood samples were collected by puncture of
the v. jugularis at the beginning of the experiment
(sampling 1), after 7-d administration of resveratrol
(sampling 2), and after 14-d administration (sampling 3).
The blood samples were tested for total white blood cell
(WBC) count using the haematological analyser
CELLTACα (Nihon Kohden, Japan), and manually for
the differential leukocyte count on blood smears stained
with May-Grünwald-Giemsa-Romanowski stain. The
following action was the phagocyte activity test (23).
After the blood samples were incubated with
microspheric hydrophile particles (2-hydroxyethyl
metacrylate particles, MSHP) and the particles absorbed
by neutrophils in the stained blood smears were counted,
and the percentage of phagocytising cells (PB values)
Experimental group (n=10)
Blood sampling 1
25 mg of resveratrol in 20 ml of 15% alcohol per
piglet per day
Blood sampling 2
37.5 mg of resveratrol in 30 ml of 15% alcohol per
piglet per day
Blood sampling 3
and the phagocyte-activity index (PAI) were determined.
The health state and the weight of the piglets were
monitored in the same intervals of the experiment.
Experiments were carried out under an
institutionally-approved procedure in accordance with
ethical principles and the Protection of Animals against
Cruelty Act and follow-up rule. Student´s t-test was used
for statistical processing of all data by the programme
Microsoft Excel (mean values and standard deviation).
Results and Discussion
Resveratrol administered to growing piglets at 3
mg/kg0.75 b.w. did not significantly influence somatic
development of the piglets. The body weight of the
resveratrol group slightly exceeded the weight of the
control group at the end of experiment but the growth of
both groups was relatively the same.
However, it influenced the dynamics of
changes in the total WBC count as well as the
representation of individual types of cells in the WBC
count (Fig. 1).
18
40
16
35
14
30
12
15
9 -1
control
experiment
20
10 .l
9
10 .l-
1
25
10
control
8
experiment
6
10
4
5
2
0
0
1
2
sampling
3
1
2
3
sampling
Fig. 1. Total WBC counts. E 1 vs 2 (P<0.01); C 1 vs 3 (P<0.01). Fig. 2. Number of neutrophils. E 1 vs 2 (P<0.01).
451
25
3
2,5
20
2
experiment
9 -1
control
10 .l
10 .l
9 -1
15
control
1,5
experiment
10
1
5
0,5
0
0
1
2
1
3
2
3
sampling
sampling
Fig. 3. Number of bands. E 1 vs 2 (P<0.01); C 1 vs 3 (P<0.01). Fig. 4. Number of lymphocytes. E 1 vs 2 (P<0.01);
E 2 vs 3 (P<0.05); C 2 vs E 2 (P<0.05).
C 1 vs 3 (P<0.05); E 1 vs 2 (P<0.01); E 1 vs 3 (P<0.05).
Table 2
Phagocyte activity
Day/Sampling
Control group
Experimental group
0/1
81.9 ± 14.89
86.9 ± 6.67
7/2
80.8 ± 8.09
77.1 ± 14.9
14/3
94.0 ± 4.9
84.4 ± 13.7
C 1 vs 3 (P<0.01); C 2 vs 3 (P<0.01); E 1 vs 2 (P<0.05); C 3 vs E 3 (P<0.05).
Table 3
Phagocyte index
Day/Sampling
Control group
0/1
10.46 ± 3.40
7/2
12.83 ± 2.53
14/3
20.68 ± 1.77
C 1 vs 3 (P<0.01); E 1 vs 3 (P<0.01); C 3 vs E 3 (P<0.01).
Compared with the group C, where the number
of WBC dropped markedly only at the end of the
experiment (P<0.01), the drop in the E occurred after
one week administration of resveratrol. That change was
mainly due to a sharp decrease (P<0.01) in the number
of neutrophils (Fig. 2) and a significant reduction
(P<0.01) in the number of lymphocytes (Fig. 4).
It is well known that in piglets the number of
neutrophils decreases in the first weeks of their life in
favour of lymphocytes (28). However, we do not believe
that the reduction in the number of neutrophils in the
group E can be explained by this ontogenetic
development only. In this case the same sharp drop
would also appear in the group C. Because in the group
E we found a significant decrease in both the total
number (Fig. 3) and percentage of representation of
young-bands forms of neutrophils, the role of resveratrol
should be assumed. Fig. 3 also documents the different
development of the band number of both groups. While
in the group C this parameter increased in connection
with age, in the group E this development trend was
Experimental group
10.39 ± 2.62
13.92 ± 3.88
16.57 ± 3.95
interrupted after 1 week of resveratrol application and
the number of bands reached a significantly lower level
in comparison with controls (P<0.05).
There are many in vitro experiments, which
demonstrated the inhibitory effect of resveratrol on
blood cell proliferation. For instance, 10-4 M
concentration of resveratrol (23 mg.l-1) in the incubation
medium inhibited human peripheral blood mononuclear
cell proliferation (2). At the lower concentration (10-5
M)–2.3 mg.l-1, the inhibitory effect was not observed.
The dose-dependent inhibitory effect of resveratrol on
the growth cycle of acute myeloid leukaemia cells was
also described by Estrov (8).
In this respect, King et al. (13) postulated that
future research should focus on transfer of in vitro
findings into in vivo models. In experiments on
laboratory animals, synthetic resveratrol was added to
diets at various concentrations to test its effects,
including its possible toxicity. Juan et al. (12)
administered 20 mg of resveratrol per kg body weight
daily to rats for a month. They found a slight increase in
452
liver enzymes in the experimental animals but no
symptoms of its toxicity. Toxicity symptoms were
observed at a concentration of 3,000 mg/kg of body
weight (6), which demonstrates that resveratrol is well
metabolised in the organism. The daily dose of 37.5 mg
per piglet in our experiment was far from these toxic
doses. However, it was high in comparison with the
beneficial intake of resveratrol recommended for human
organism. This beneficial effect related to the wine
consumption – the so-called French paradox - was
observed during long-term administration of about 1-2
mg of resveratrol a day (16). By comparison, one litre of
red wine contains 5–10 mg of total stilbene
transformable to resveratrol.
Previous experiments on rats showed that 4
mg/kg0.75 of resveratrol administered to rats for only a
week significantly decreased the number and functional
activity of thrombocytes (7). In our study, comparable
doses of resveratrol at 3 mg/kg0.75 to piglets after one
week’s administration caused suppression of the
majority of examined values. In the second week of the
experiment, we found no significant change, although
resveratrol administration per unit of metabolic body
weight remained unchanged.
Apart from the temporary suppression of
leukopoiesis, resveratrol also significantly affected
phagocyte activity. This activity in the group E showed
no increase during the experiment compared with the
group C (Table 2). It reached the minimum level after
one-week’s administration of resveratrol. At the end of
week 2 of the experiment, the resveratrol-supplemented
piglets had a significantly lower percentage of PB and
also PAI (P<0.05 and P<0.01, respectively). We believe
that these findings coincided with the lower number of
more active phagocytic bands (Tables 2 and 3).
In order to confirm this assumption, other data
about immunology should be collected. However, our
results are in agreement with the former work by
Cavallaro et al. (3). In their study, resveratrol showed a
significant dose-dependent inhibitory effect on some
activities of polymorphonuclear leukocytes. Recently,
Kohnen et al. (14) also reported the inhibitory effect of
resveratrol on equine neutrophil myeloperoxidase and
Tou and Urbizo (22) demonstrated the inhibitory
influence of structurally similar diethylstilbestrol and
resveratrol in the presence of ethanol on the
degranulation of neutrophils, hence its antiinflammatory effect. Phagocyte activity is linked with
activation of reactive oxygen species and nitric oxide by
phagocytes. Ciz et al. (5) documented that resveratrol in
wine, like other polyphenols, decreased the release of
this product. The scavenging activity of this compound
is important in the protection of cells and tissues against
oxidative damage but may be also related to attenuation
of phagocyte responses.
It is clear from the results that one week
administration of resveratrol at a daily dose of 3
mg/kg0.75 body weight has no significant effect on the
growth of piglets but causes temporarily suppression of
their leukopoiesis. This resulted in drop of the total
WBC count and mainly in the occurrence of young
forms of neutrophils. We suppose that like in vitro
experiments, resveratrol administered per os in
relatively high doses can inhibit the growth cycle of
leukocytes, including their activity. On the one hand, the
effects on leukocyte count were not noticeable after the
second week of administration of resveratrol but the
phagocyte activity of neutrophils at the end of the
experiment was still significantly lower.
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