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Research Article Effect of Astaxanthin Supplementation on Salivary IgA, Oxidative Stress, and Inflammation in Young Soccer Players

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  • University of Belgrade Faculty of Pharmacy

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The physiologic stress induced by physical activity is reflected in immune system perturbations, oxidative stress, muscle injury, and inflammation. We investigated the effect of astaxanthin (Asx) supplementation on salivary IgA (sIgA) and oxidative stress status in plasma, along with changes in biochemical parameters and total/differential white cell counts. Forty trained male soccer players were randomly assigned to Asx and placebo groups. Asx group was supplemented with 4 mg of Asx. Saliva and blood samples were collected at the baseline and after 90 days of supplementation in preexercise conditions. We observed a rise of sIgA levels at rest after 90 days of Asx supplementation, which was accompanied with a decrease in prooxidant-antioxidant balance. The plasma muscle enzymes levels were reduced significantly by Asx supplementation and by regular training. The increase in neutrophil count and hs-CRP level was found only in placebo group, indicating a significant blunting of the systemic inflammatory response in the subjects taking Asx. This study indicates that Asx supplementation improves sIgA response and attenuates muscle damage, thus preventing inflammation induced by rigorous physical training. Our findings also point that Asx could show significant physiologic modulation in individuals with mucosal immunity impairment or under conditions of increased oxidative stress and inflammation.
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Research Article
Effect of Astaxanthin Supplementation on
Salivary IgA, Oxidative Stress, and Inflammation in
Young Soccer Players
Ivana Baralic,1Marija Andjelkovic,1Brizita Djordjevic,2
Nenad Dikic,1Nenad Radivojevic,1Violeta Suzin-Zivkovic,3
Sanja Radojevic-Skodric,4and Snezana Pejic5
1Sports Medicine Association of Serbia, Lazarevacki Drum 14, 11000 Belgrade, Serbia
2Institute for Bromatology, Faculty of Pharmacy, University of Belgrade, Vojvode Stepe 450, 11000 Belgrade, Serbia
3Institute of Histology and Embryology, School of Medicine, University of Belgrade, Dr Subotica 1, 11000 Belgrade, Serbia
4Institute of Pathology, School of Medicine, University of Belgrade, Dr Subotica 1, 11000 Belgrade, Serbia
5Snezana Pejic, Department of Molecular Biology and Endocrinology, “Vinca” Institute of Nuclear Sciences,
UniversityofBelgrade,P.O.Box522,11000Belgrade,Serbia
Correspondence should be addressed to Snezana Pejic; snezana@vinca.rs
Received  March ; Accepted  June 
Academic Editor: Francesca Mancianti
Copyright ©  Ivana Baralic et al. is is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
e physiologic stress induced by physical activity is reected in immune system perturbations, oxidative stress, muscle injury, and
inammation. We investigated the eect of astaxanthin (Asx) supplementation on salivary IgA (sIgA) and oxidative stress status
in plasma, along with changes in biochemical parameters and total/dierential white cell counts. Forty trained male soccer players
were randomly assigned to Asx and placebo groups. Asx group was supplemented with  mg of Asx. Saliva and blood samples
were collected at the baseline and aer  days of supplementation in preexercise conditions. We observed a rise of sIgA levels at
rest aer  days of Asx supplementation, which was accompanied with a decrease in prooxidant-antioxidant balance. e plasma
muscle enzymes levels were reduced signicantly by Asx supplementation and by regular training. e increase in neutrophil count
and hs-CRP level was found only in placebo group, indicating a signicant blunting of the systemic inammatory response in the
subjects taking Asx. is study indicates that Asx supplementation improves sIgA response and attenuates muscle damage, thus
preventing inammation induced by rigorous physical training. Our ndings also point that Asx could show signicant physiologic
modulation in individuals with mucosal immunity impairment or under conditions of increased oxidative stress and inammation.
1. Introduction
Astaxanthin (Asx) is a red-orange carotenoid mainly pro-
duced by micro- and macroalgal species and accumulated
in many marine organisms, such as shrimps, crabs, trout,
and salmon. e polyene system gives astaxanthin its dis-
tinctive molecular structure, chemical properties, and light-
absorption characteristics []. e presence of the hydroxyl
and keto moieties on each ionone ring explains some of its
unique features such as higher antioxidant (AO) activity, a
more polar nature than other carotenoids, and ability to be
esteried []. Its both high AO potency and polar prop-
erties, make Asx an attractive nutraceutical for promising
applications in human health and nutrition. Asx has also
been attributed with extraordinary potential for protecting
the organism against a wide range of diseases [].
e mucosal immune system functions as the rst line
of defense against pathogen invasion by preventing the
attachments of infectious agents to mucosal surfaces []. An
important activity of the epithelia lining in gastrointestinal,
respiratory, and genitourinary tract is the production of
secretory IgA (sIgA), the antibody that largely dominates
Hindawi Publishing Corporation
Evidence-Based Complementary and Alternative Medicine
Volume 2015, Article ID 783761, 9 pages
http://dx.doi.org/10.1155/2015/783761
Evidence-Based Complementary and Alternative Medicine
mucosal humoral immunity []. sIgA protects mucosal
surfaces by directly cross-linking environmental microor-
ganisms or macromolecules, thus preventing their contact
with the surface of epithelial cells and hence facilitating
their elimination []. Numerous studies of saliva compo-
sition have found a decreased sIgA secretion with age [],
psychological, and occupational stresses []andalsoin
nutritional deciencies []. In addition, mounting evidence
indicates that prolonged and intensive physical exertion can
cause a decrease in sIgA concentration and secretion rate
[]. Lowered concentrations of sIgA are associated with
an increased frequency of upper respiratory tract infection
(URTI) episodes or with reduced protection against certain
infections [].
Intensive and sustained physical activity elevates the
generation of free radicals and reactive oxygen species (ROS),
thus creating an imbalance between ROS and antioxidants
and leading to oxidative stress that not only causes lipid
peroxidation and protein oxidation but also may have a
negative impact on immune function []. Asx possesses
antioxidant, free radical scavenging, and anti-inammatory
properties that may aect human immune system and
resistance to pathogens, although most data come from in
vitro and animal studies []. ese studies showed that Asx
stimulated a delayed-type hypersensitivity (DTH) response,
the natural killer (NK) cells cytotoxic activity, and increased
concentrations of IgG and IgM and B cell population [].
No published human studies exist regarding the inuence
of Asx ingestion on exercise-induced mucosal immunity dys-
function. One human study showed that Asx could enhance
immune response and decrease a DNA oxidative damage and
inammation in healthy females []. erefore, the purpose
of the present investigation was to test the hypothesis that
Asx supplementation is eective in enhancing sIgA secretion
in young soccer players. Compared with previous research
in this eld, particular strength of this study is the fact it
was conducted during a regular competitive s eason, reecting
habitual conditions of nutrition and training program. In
addition, we determined oxidative status parameters, along
with biochemical and hematological prole, in order to
examine possible connections among mucosal immunity,
oxidative stress, inammation, and Asx supplementation in
young, healthy athletes.
2. Material and Methods
2.1. Subjects. Forty trained male soccer players, among young
selection of soccer club “Partizan,” Belgrade, were recruited
as experimental subjects. e participants met the following
exclusion criteria: smokers, subjects with recurrent respira-
tory infections, subjects taking antibiotics, subjects taking
immunosuppressive drugs, and alcohol abuse. Athletes as
well as their parents gave the written consent aer having
been explained the purpose, demands, and possible risks
associated with the study. e study was conducted according
to the guidelines laid down in Declaration of Helsinki. Exper-
imental procedures were approved by the Ethical Committee
of Sports Medicine Association of Serbia. ey were involved
in a controlled training program during a -day period over
competitive season, in which they had  to  training sessions
per week with an average weekly training of  to  hours and
took participation in national championship. ey also had
dierent aspects of trainings, including strength, resistance,
cardio, exibility, and proprioceptive training.
2.2. Research Design and Supplementation. Soccer players
were randomly assigned to Asx (𝑁=21) or placebo (P, 𝑁=
19) groups. Under double-blind procedures, subjects received
Asx ( mg per day) or placebo supplements for  days. e
Asx used in this study was a homogenized and spray dried
biomass of the green unicellular alga Haematococcus pluvialis.
Both Asx and placebo capsules were manufactured by Bioreal
(Sweden) and generously donated by Oriame, Serbia. e
dietary supplementation comprised of one capsule daily
taken orally in conjunction with a meal.
Two weeks before the rst test session, subjects reported
to the laboratory for orientation, measurement of body
composition, and cardiorespiratory tness. Body weight and
totalbodyfatweremeasuredusingtheBC-,-contact
electrodedevice(Tanita,Tokyo,Japan),whichusesbioelec-
trical impedance analysis for body composition analysis.
Height was measured to the nearest . cm with a portable sta-
diometer. Body mass index (BMI) was calculated by dividing
theweightinkilogramsbythesquareoftheheightinmeters
(kg/m2). VO2max was measured on a motor driven treadmill
(Run race, Techno gym, Italy), using an indirect calorimetry
system (Quark b, Cosmed, Italy) during an incremental
exercise test to volitional fatigue. Basic demographic and
training data were obtained through a questionnaire. Subjects
were instructed to restrain themselves from making any
drastic changes in the diet. ey agreed to avoid the use
of vitamin/mineral supplements, antioxidant supplements,
nutritional supplements, ergogenic aids, herbs, and medica-
tions known for their eect on immune function,  month
before and during the study. Subjects recorded food intake in
a -day food record before the rst exercise test session. e
food records were analyzed using CRON-O-Meter v...
soware.
Saliva and blood samples were collected at the onset of
the study and aer  days of supplementation, between :
and : a.m., aer overnight fast, before training session.
For all collection points, players were restrained from water
consumption min before sampling. e training sessions
were carried out under the same conditions,in the same place
and at the same time of the day to avoid circadian variations.
Each subject served as self-control to eliminate any biological
variability in the response to Asx supplementation.
2.3. Salivary Flow Measurement. Whole unstimulated saliva
was collected using salivettes (Sersted, Vumbrecht, Ger-
many), by placing the cotton swab under the tongue. All saliva
collections ( min, electronically timed) were made aer
players had been sitting quietly for a few minutes, leaning
forward, with their heads tilted. Immediately aer collection,
theobtainedsalivasampleswereseparatedfromthecottonby
centrifuging at  ×gforminutes.esupernatantuid
was frozen at CforlateranalysisofsIgA.Beforeandaer
Evidence-Based Complementary and Alternative Medicine
saliva collection, salivettes were weighted on an analytical
balance. e amount of saliva in grams was converted to
milliliters assuming that the specic gravity of saliva is  and
divided by  to express salivary ow in mL/min.
2.4. Measurement of sIgA Concentration and Secretion Rate.
e sIgA concentration was measured by enzyme-linked
immunosorbent assay (ELISA). Briey, the test was per-
formed on -well microtiter plates, which were coated with
goat anti-human IgA ( :  dilution, AbD Serotec, Oxford,
UK) in coating buer (. M carbonate-bicarbonate, pH .)
and kept overnight at C. On the day of analysis, plates
were incubated for  min at room temperature before being
washed with TTBS buer (Tween  Tris Buered saline,
pH.)andthenblockedwith%gelatininTTBSfor
h at room temperature. Samples were thawed and then
diluted (: ) with a sample diluent (% gelatin in TTBS).
Known concentrations of human secretory IgA (AbD Serotec,
Oxford,UK)andcontrolswereusedtoconstructastandard
curve. e plate was then washed before standards and
samples were added to wells (in duplicate) and incubated for
h at room temperature. e plate was washed again before
addition of diluted goat anti-human horseradish peroxidase
conjugates (AbD Serotec, Oxford, UK). Aer incubation, the
plate was washed again before addition of substrate tetram-
ethylbenzidine (AbD Serotec, Oxford, UK) and incubated for
mininthedark.ecolorimetricreactionwasstopped
via addition of  𝜇L of sulfuric acid, and the absorbance
of solution in each well was determined at  nm by using
ELx Absorbance Microplate Reader (Biotek, Winooski,
USA). Regression analysis using the relation of standard sIgA
concentrations and amount of absorbance (nm) was used to
interpolate the concentration of sIgA in the samples. sIgA is
expressed as absolute concentration and secretion rate. sIgA
secretion rate (𝜇g/min) was determined by multiplying the
absolute sIgA concentration (𝜇g/mL) with saliva ow rate
(mL/min).
2.5. Oxidative Stress Status. For determination of oxidative
status parameters, the venous blood was collected into
heparin evacuated tube (Greiner Bio-one, Kremsm¨
unster,
Austria). e blood samples were centrifuged at  g for
 min, and aliquots of plasma were stored at Cforlater
analysis.
Total antioxidant status (TAS) was determined using an
automated method developed by Erel []. A standardized
solution of Fe2+–o-dianisidine complex reacts with a stan-
dardized solution of H2O2by a Fenton-type reaction, produc-
ing hydroxyl radicals (OH). ese potent ROS oxidize the
reduced colorless o-dianisidine molecules to yellow-brown
colored dianisidyl radicals at low pH. Antioxidants in the
sample suppress the oxidation reactions and color formation.
is reaction can be monitored by spectrophotometry. e
reaction rate is calibrated with Trolox (a water-soluble ana-
logue of vitamin E, -hydroxy-,,,-tetramethylchroman-
-carboxylic acid) and was incorporated into Ilab  plus
autoanalyser (Instrumentation Laboratory, Milan, Italy). e
TAS value of the samples tested is expressed as mmolTrolox
equiv./L.
Total oxidant status (TOS) was determined according to
Erels’ method []. is assay is based on the oxidation of
ferrous ion to ferric ion in the presence of various oxidant
species in serum. e ferric ion makes a colored complex
with xylenol orange in an acidic medium. e color intensity,
which can be measured spectrophotometrically, is related
to the total amount of oxidant molecules present in the
sample.eassaywascalibratedwithH
2O2and incorporated
into Ilab  plus autoanalyser (Instrumentation Laboratory,
Milan, Italy). e results are expressed in terms of micro-
molar hydrogen peroxide equivalent per liter (𝜇molH2O2
equiv./L).
Prooxidant-antioxidant balance (PAB) was measured
according to a previously published method []. is
method is based on two dierent oxidation-reduction reac-
tions which take place simultaneously. In enzymatic reaction,
the chromogen TMB is oxidized to a colored cation by perox-
ides, and in the chemical reaction, the colored TMB cation is
reduced to a colorless compound by antioxidants. e pho-
tometric absorbance is then compared with the absorbance
given by a series of standard solutions that are made by
mixing varying proportions (–%) of  mmol/L H2O2,
as a representative of hydroperoxides, which is an indicator of
total oxidant status, with  mmol/L uric acid (in  mmol/L
NaOH), as a representative of the antioxidant capacity. is
photometric comparison was carried out using an ELISA
reader.
TMB powder ( mg) was dissolved in  mL of DMSO.
For preparation of TMB cation,  𝜇L of TMB/DMSO was
added in  mL of acetate buer (. M, pH .), and then
 𝜇L of fresh chloramine T ( mM) solution in distilled
water was added. e mixture was incubated in a dark
place for  hr at room temperature, aer which  units of
peroxidase enzyme solution were added into mL TMB
cation, dispensed in  mL, and put at C. To prepare
the TMB solution,  𝜇L of TMB/DMSO was added into
 mL of acetate buer (. M, pH .). e working solution
was prepared by mixing mL of TMB cation with  mL of
TMB solution. e mixture was incubated for  min at room
temperature, in a dark place, and it was used immediately.
e sample ( 𝜇L), standard, or blank (distilled water) were
mixed with  𝜇L of working solution, in each well of a
-well plate, which was then incubated in a dark place
at C for  min. At the end of incubation time,  𝜇L
ofNHClwasaddedintoeachwellandmeasuredin
ELISA reader at  nm, with a reference wavelength of 
or  nm (ELx Absorbance Microplate Reader, Biotek,
Winooski, USA). A standard curve was provided from the
valuesrelativetothestandardsamples.ePABvaluesofthe
unknown samples were calculated using the standard curve
and expressed in arbitrary HK units, which represent the
percentage of H2O2in standard solution, multiplied by .
2.6. Hematological and Biochemical Parameters. Hematologi-
calanalysiswasperformedinthebloodsamplescollectedinto
K-EDTA tubes (Greiner Bio-one, Kremsm¨
unster, Austria),
using an automated hematology analyzer Cell-Dyn 
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(Abbott Diagnostics, Illinois, USA). To examine biochemical
parameters, blood samples were drawn from an antecubital
vein into sample tube with serum separator gel (Greiner
Bio-one, Kremsm¨
unster, Austria) and then le to coagulate
formintoseparatetheserum,andtheobtainedsam-
ples were stored at C for later analysis. e following
items were included in the general biochemical examination:
aspartate aminotransferase (AST), alanine aminotransferase
(ALT), creatine kinase (CK), lactate dehydrogenase (LDH),
uric acid (UA), creatinine (Cre), high sensitivity C-reactive
protein (hs-CRP), total cholesterol (CHOL), HDL cholesterol
(HDL-C), and triglycerides (TG). Biochemical analysis was
performed using an ILab  Plus autoanalyser (Instrumen-
tation Laboratory, Milan, Italy) employing commercial kits
(Bioanalytica, Belgrade, Serbia). e concentration of LDL
cholesterol (LDL-C) was calculated using the formula of
Friedewald et al. [].
2.7. Statistical Analysis. Statistical analyses were performed
using the PASW Statistics version . and MedCalc (version
. Soware, Belgium) soware. All data were assessed
for normality (Kolmogorov-Smirnov tests). e results are
expressed in terms of mean values and standard error (SE) for
normally distributed variables. Where distribution diered
from a normal distribution, geometric means and % con-
dence intervals were given. Subjects’ baseline characteristics
and nutritional parameters between treatment groups were
compared using independent-sample t-test. e eects of
supplementation and training were analyzed by two-way
analysis of variance (ANOVA) with repeated measures to
test for the two main eects and for the interaction between
them. When a signicant 𝑝value was obtained, Bonferroni
post hoc comparison test was employed to determine the
dierences between groups. Two-tailed 𝑝values are given
throughout.DuetothefactthatthedistributionsofsIgA
absoluteconcentrationsandsecretionrate,AST,ALT,CK,
and hs-CRP were skewed, logarithmic transformation of
these values was performed before statistical comparisons
were made.
3. Results
3.1. Subjects Characteristics. Physical characteristics for the
 soccer players randomized to Asx and P groups are
summarized in Table . No signicant dierences were found
between groups according to age, body composition, or
VO2max (𝑝 > 0.05,t-test).
According to -day food records analysis using CRON-
O-Meter v.. soware, estimated daily energy and nutrient
intake in two experimental groups were similar (𝑝 > 0.05),
Table .
3.2. Salivary Flow, sIgA Absolute Concentration, and Secretion
Rate. Salivary ow measurement was unaected by training
or supplementation (𝑝 > 0.05). e obtained values were
unchanged aer  days of training and supplementation
(Figure (a)).
ANOVA repeated measures showed a signicant inter-
action eect of supplementation and training (𝐹 = 6.221,
T : Subject characteristics at baseline.
Astaxanthin Placebo
Age (year) . ±. . ±.
Weight ( k g ) ±.  ±.
Height (cm)  ±.  ±.
Body mass index (kg/m).±. . ±.
Fat (%) . ±. . ±.
VOmax (mL/min/kg) . ±. . ±.
Values are expressed as mean ±SE.
T : Estimated daily energy and nutrient intake of soccer
players.
Asx P
Energy (kcal)  ±  ±
Protein (g)  ±.  ±.
Carbohydrates (g)  ± ±
Monosaccharides (g)  ±  ±
Fiber (g) . ±. . ±.
Fat (g)  ±±
Saturated fat (g) . ±. . ±.
Cholesterol (mg)  ±  ±
Vitamin A (IU)  ±  ±
Vitamin C (mg)  ±  ±
Vitamin E (mg) . ±. . ±.
Copper (mg) . ±. . ±.
Iron (mg) . ±. . ±.
Manganese (mg) . ±. . ±.
Selenium (𝜇g)  ±  ±
Zinc (mg) . ±. . ±.
Values are expressed as mean ±SE.
𝑝 < 0.05) on sIgA absolute concentration as well as on sIgA
secretionrate(𝐹 = 4.608,𝑝 < 0.05) (Figures (b) and (c)).
Post hoc comparison revealed a signicant increase of sIgA
absolute concentration and secretion rate in supplemented
group aer  days when compared to baseline values (𝑝<
0.05), while there were no signicant changes in P group.
3.3. Oxidative Stress Status. Oxidativestressstatusisreported
in Table . Regular soccer training had a signicant eect on
TOS, regardless of supplementation (main eect of training,
𝐹 = 108.678,𝑝 < 0.001). TOS decreased in both, the
supplementedandthePgroup,aerdaysofregular
training in comparison to baseline values. Basal TAS was
unaected by soccer training and supplementation (𝑝>
0.05). ere was a signicant decrease in PAB in Asx group
aer  days in comparison to baseline values (interaction
eect of supplementation and training: 𝐹 = 25.371,𝑝 < 0.05;
main eect of training: 𝐹 = 4.205,𝑝 < 0.001), as a result
of continuous training and supplementation. No signicant
changes were found in P group.
3.4. Hematological Parameters. Aer  days of training
and supplementation, ANOVA analysis showed a signicant
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0
0.5
1
1.5
Astaxanthin Placebo
Before supplementation
Aer supplementation
(a) Salivary ow (mL/min)
0
100
200
300
Astaxanthin Placebo
Before supplementation
Aer supplementation
(b) sIgA (𝜇g/min)
Before supplementation
Aer supplementation
0
100
200
300
400
Astaxanthin Placebo
(c) sIgA (𝜇g/min)
F : Salivary ow (a), salivary IgA concentration (b), and salivary IgA secretion rate (c) in soccer players at baseline and aer  days
of supplementation. Values are presented as mean ±SE. e dierence in relation to baseline was signicant at . ().
T : Oxidative stress status of the soccer players at baseline and aer  days of supplementation.
Astaxanthin Placebo ANOVA
Before supplementation Aer supplementation Before supplementation Aer supplementation T S T ×S
TAS (mmolTrolox
equiv./L) . ±. . ±. . ±. . ±. ns ns ns
TOS (mmol/L) . ±. . ±.∗∗∗ . ±. . ±.∗∗∗ <. ns ns
PAB (H K U ) . ±. . ±.∗∗∗ . ±. . ±. <. ns <.
Values are expressed as mean ±SE. ANOVA: T-training, S-supplementation, and T ×S-training and supplementation interaction eect.
e dierence in relation to baseline was signicant at 𝑝 < 0.001 (∗∗∗).
increase in leukocyte count in P group (interaction eect
of supplementation and training: 𝐹 = 4.528,𝑝 < 0.05;
main eect of training: 𝐹 = 3.989,𝑝 < 0.05)butnotin
Asx group, as presented in Figure .erewasasignicant
increase in neutrophil count in P group aer  days of obser-
vational period compared to baseline values (main eect of
training: 𝐹 = 4.875,𝑝 < 0.01). ese changes were not
detected in supplemented group.
3.5. Biochemical Parameters. Biochemical parameters are
shown in Table . ANOVA revealed a signicant main eect
of training on AST (𝐹 = 57.029,𝑝 < 0.001), CK (𝐹 = 29.000,
Evidence-Based Complementary and Alternative Medicine
T : Biochemical prole of the soccer players at baseline and aer  days of supplementation.
Astaxanthin Placebo ANOVA
Before supplementation Aer supplementation Before supplementation Aer supplementation T S T ×S
AST (U/L). (.–.) . (.–.)∗∗∗ . (.–.) . (.–.)∗∗ . <. ns
ALT (U/L). (.–.) . (.–.) . (.–.) . (.–.) ns ns ns
CK (U/L) (–)  (–)∗∗  (–)  (–) <. ns ns
LDH (U/L). ±. . ±.∗∗ . ±. . ±.<. <. ns
UA (𝜇mol/L). ±. . ±. . ±. . ±. <. ns ns
Cre (𝜇mol/L). ±. . ±. . ±. . ±. <. ns ns
hs-CRP (mg/L). (.–.) . (.–.) . (.–.) . (.–.) ns ns .
CHOL (mmol/L). ±. . ±. . ±. . ±. ns ns ns
HDL-C (mmol/L). ±. . ±. . ±. . ±. ns ns ns
LDL-C (mmol/L). ±. . ±. . ±. . ±. ns ns ns
TG (mmol/L). ±. . ±. . ±. . ±. ns ns ns
Mean ±SE.; Geometric mean values (th condence interval). ANOVA: T-training, S-supplementation, and T ×S-training and supplementation interaction
eect. e dierence in relation to baseline was signicant at 𝑝 < 0.05 (), 𝑝 < 0.01 (∗∗), and 𝑝 < 0.001 (∗∗∗ ).
0
2
4
6
8
0 day 90 days 0 day
Astaxanthin Placebo
90 days
Monocytes
Lymphoc ytes
Neutrophils
6.3 ± 0.2
6.0 ± 0.3 6.0 ± 0.2
6.7 ± 0.4
Leukocyte count (109/L)
F : Total leukocyte count (denoted by numbers at the top of
the bars); neutrophil, lymphocyte, and monocyte counts in soccer
players at baseline and aer  days of supplementation.
𝑝 < 0.01), and LDH (𝐹 = 87.641,𝑝 < 0.001)plasma
levels. Post hoc comparison showed a signicant decrease in
CK activity in Asx group aer  days compared to baseline
values (𝑝 < 0.01), while a decrease in P group was not
statistically signicant. Aer  days of continuous training,
AST and LDH decreased signicantly in both Asx (𝑝 < 0.001,
𝑝 < 0.01,resp.)andPgroup(𝑝 < 0.01,𝑝 < 0.5,resp.).
In addition, a signicant main eect of supplementation was
observed on AST (𝐹 = 3.979,𝑝 < 0.05)andLDH(𝐹 = 3.995,
𝑝 < 0.05) levels, with lower activity in Asx compared to P
group.
UA and Cre levels signicantly decreased in both groups
aer  days of observational period (UA main eect of
training, 𝐹 = 5.528,𝑝 < 0.05; Cre main eect of training, 𝐹=
4.429,𝑝 < 0.05). ANOVA analysis also showed a signicant
supplementation and training interaction eect (𝐹 = 4.050,
𝑝 = 0.05)onhs-CRPlevels.Namely,aerdaysofregular
soccer training, a % increase in hs-CRP levels may be
observed in P group, but not in the Asx group. Analysis of
lipidprolerevealednoeectoftraining,supplementation,
or supplementation and training interaction.
4. Discussion
e main nding of the present study was the rise of sIgA
levels at rest aer  days of Asx supplementation which
was accompanied with a decrease in prooxidant-antioxidant
balance(PAB).Also,theincreaseinneutrophilcountand
hs-CRP level recorded in placebo group was not apparent in
supplemented group, indicating a signicant blunting of the
systemic inammatory response in the subjects taking Asx.
e observed increase in sIgA levels aer supplemen-
tation could indicate the eect of Asx on sIgA synthesis.
Previous studies reported modulatory actions of Asx on
humoral immune response such as induced production of
polyclonal antibodies G and M in murine spleen cells [],
increased IgG and IgM production in cats and dogs [,],
partially restored humoral immune response in old mice [],
andenhancedimmunoglobulinproductioninresponsetoT-
dependent stimuli in human blood cells []. Although the
mechanism of Asx action was not known, it was suggested
that it enhanced the antibody production through release of
IL-𝛼[], which is one of major regulating factors in B cell
dierentiation []. e benecial eect of Asx on mucosal
immunity, also found in our study, might be explained by its
antioxidant activity []. Immune cells are particularly sensi-
tive to oxidative stress due to a high percentage of polyun-
saturated fatty acids in their plasma membranes, and they
generally produce more oxidative products. Overproduction
of ROS can tip the oxidant/antioxidant balance, resulting in
destruction of cell membranes, proteins, and DNA []. ROS
overproduction during physical activity might also inhibit
Evidence-Based Complementary and Alternative Medicine
locomotor and bactericidal activity of neutrophils, as shown
in study of [], where NK cell cytotoxic activity reduced
the proliferation of T- and B-lymphocytes and promoted
lymphocyte apoptosis.
erefore, under conditions of increased oxidative stress
(e.g., during disease states or physical activity), dietary
antioxidants become critical in maintaining a desirable oxi-
dant/antioxidant balance []. In our study, oxidative stress
(measured by TOS) was signicantly reduced aer  days
of regular training in both groups of soccer players, probably
due to an upregulation in the body’s enzymatic AO defense
system. However, nonenzymatic antioxidants presented in
plasma (measured by TAS) were not aected by training or
supplementation during the observational period, which is
in accordance with previous studies [,]. Antioxidants
are known to work synergistically to defend against oxidant
production. In other words, when one AO nutrient is lacking
in particular period of time, another could substitute it or it
may be regenerated by another that is in abundance []. is
may be the reason why no changes in TAS levels at rest were
observed during  days of regular training and supplemen-
tation. Mobilization of tissue antioxidants into the plasma
in order to maintain the AO status and to protect the body
against ROS may also explain, in part, why no signicant
changes in TAS levels at rest were observed in response to
training or Asx supplementation []. ese results suggest
that human organism has very potent mechanisms which
protect cells against free radicals and oxidative stress.
However, we showed that Asx supplementation resulted
in decreased prooxidant-antioxidant balance (PAB) during
regular competitive season in young soccer players. ere-
fore,webelievethatdeleteriouseectscausedbytheexcess
of ROS produced during physical activity can be additionally
attenuated by the Asx. Asx induced a better redox balance
favoring all cellular functions which depend on adequate
amounts of ROS, thereby supporting the mucosal immune
system. Our nding is in accordance with the study of Park
et al. [] who examined the possible immune-enhancing,
AO, and anti-inammatory activity of Asx in healthy women
and showed that Asx could decrease a DNA oxidative damage
and inammation and enhance immune response.
e magnitude of muscle damage, evaluated by CK and
LDH in our study, was reduced signicantly over the obser-
vational period, probably by adaptation and conditioning of
the muscle through regular training, as shown previously
by Powers and Jackson []. Asx supplementation addition-
ally attenuated exercise-induced muscle damage. Although
muscle damage represents a direct consequence of exercise
injury which mechanically disrupts muscle myobrils, tissue
damage associated to exercise-induced free radical produc-
tion and subsequent oxidative stress was also suggested by
Davies et al. [].ItcanbehypothesizedthatAsxprotectsthe
cell membranes against free radicals generated during heavy
exercise, thus preserving the functionality of muscle cells. e
unsaturated polygene chain of Asx could trap radicals in the
membrane, while the terminal rings scavenge radicals both at
thesurfaceandintheinteriorofthephospholipidmembrane
[]. Similar results were obtained in animal experiments,
reporting that Asx attenuated oxidative damage of lipids and
DNA in gastrocnemius and heart as well as the leakage of CK
into plasma [].
In this study, regular soccer training during the compet-
itiveseasonledtocertainchangesinleukocytescountonly
in placebo group. Over the time, total leukocytes count, neu-
trophil count, and hs-CRP levels had increased. e observed
elevation in hs-CRP due to the regular intensive training was
similar to the levels that have been associated with increased
risk of coronary vascular disease []. ese changes were
not detected in supplemented group, which further supports
the notion that Asx, as dietary supplement, has the ability
to suppress minor inammatory events induced by training.
Anti-inammatory action of Asx was previously detected in
human and animal studies [,,].
Evidence suggests that Asx has potential health-
promoting eects in prevention and treatment of various
diseases, such as chronic inammatory diseases, metabolic
syndrome, diabetes, cardiovascular disease, neurodegenera-
tive diseases, exercise-induced fatigue, or male infertility
[],whichlikelyinvolvesAOmechanismsanddecreased
production of inammatory mediators and cytokines. It is
already known that physiologic stress, induced by prolonged
and intensive physical activity, is reected by transient,
yet signicant immune system perturbations, along with
oxidative stress, muscle injury, and inammation.
5. Conclusions
According to the results presented in this study, Asx supple-
mentation improved sIgA response and attenuated muscle
damage, probably due to restoring redox balance, thus pre-
venting inammation induced by rigorous physical training.
Although our study focused on narrow population regarding
the age, gender, and life style, nevertheless, it showed the
modulating eects of Asx on the examined parameters.
Our ndings also indicate that Asx could show signicant
physiologic modulation in other individuals with mucosal
immunity impairment or under conditions of increased
oxidative stress and inammation. Although the currently
available data and recent ndings are very encouraging, more
extensive, well-controlled clinical trials are suggested for
other threatened categories.
Abbreviations
URTI: Upper respiratory tract infection
DTH: Delayed-type hypersensitivity
BMI: Body mass index
ELISA: Enzyme-linked immunosorbent assay
TAS: Total antioxidant status
TOS: Total oxidant status
PAB: Prooxidant-antioxidant balance
AST: Aminotransferase
ALT: Alanine aminotransferase
CK: Creatine kinase
LDH: Lactate dehydrogenase
UA: Uric aci d
Cre: Creatinine
hs-CRP: High sensitivity C-reactive protein
Evidence-Based Complementary and Alternative Medicine
CHOL: Total cholesterol
HDL-C: HDL cholesterol
TG: Triglycerides
LDL-C: LDL cholesterol
SE: Standard error
ROS: Reactive oxygen species
AMP: Adenosine monophosphate
ATP: Adenosine triphosphate.
Conflict of Interests
e authors declared that there is no conict of interests
regarding the publication of this paper.
Acknowledgments
is work was nancially supported by the Ministry of Edu-
cation, Science and Technological Development, Republic of
Serbia (Grants III and III). e authors wish to
thank Dr. Ljiljana Dimitrijevic for the assistance in the data
interpretation and writing of the paper.
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... In another human study, a moderate dose of astaxanthin (8 mg per day) was administrated for a longer period (12 weeks), and a significant reduction in IL-6 and CRP was observed in healthy middle-aged non-smoking males [113]. Further to support the anti-inflammatory property of astaxanthin, a study on trained teenage soccer players showed that supplementation of a moderate dose of astaxanthin (4 mg/d) for 12 weeks resulted in a reduction of CRP [17]. ...
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The Coronavirus Disease-2019 (COVID-19) pandemic urges researching possibilities for prevention and management of the effects of the virus. Carotenoids are natural phytochemicals of anti-oxidant, anti-inflammatory and immunomodulatory properties and may exert potential in aiding in combatting the pandemic. This review presents the direct and indirect evidence of the health benefits of carotenoids and derivatives based on in vitro and in vivo studies, human clinical trials and epidemiological studies and proposes possible mechanisms of action via which carotenoids may have the capacity to protect against COVID-19 effects. The current evidence provides a rationale for considering carotenoids as natural supportive nutrients via antioxidant activities, including scavenging lipid-soluble radicals, reducing hypoxia-associated superoxide by activating antioxidant enzymes, or suppressing enzymes that produce reactive oxygen species (ROS). Carotenoids may regulate COVID-19 induced over-production of pro-inflammatory cytokines, chemokines, pro-inflammatory enzymes and adhesion molecules by nuclear factor kappa B (NF-κB), renin-angiotensin-aldosterone system (RAS) and interleukins-6- Janus kinase-signal transducer and activator of transcription (IL-6-JAK/STAT) pathways and suppress the polarization of pro-inflammatory M1 macrophage. Moreover, carotenoids may modulate the peroxisome proliferator-activated receptors γ by acting as agonists to alleviate COVID-19 symptoms. They also may potentially block the cellular receptor of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), human angiotensin-converting enzyme 2 (ACE2). These activities may reduce the severity of COVID-19 and flu-like diseases. Thus, carotenoid supplementation may aid in combatting the pandemic, as well as seasonal flu. However, further in vitro, in vivo and in particular long-term clinical trials in COVID-19 patients are needed to evaluate this hypothesis.
... In another double-blind, placebo-controlled study, a formulation containing astaxanthin, tocotrienol, and zinc significantly improved muscle strength in healthy elderly in addition to an elevation in endurance and walking distance found with exercise training alone [96]. Moreover, astaxanthin supplementation in a daily dose of 4 mg was able to prevent inflammation induced by rigorous physical training in trained male soccer players with lower neutrophil count and hs-CRP level than in a placebo group [97]. On the other hand, in a clinical trial with trained athletes, markers of inflammation (hs-CRP) and exerciseinduced skeletal muscle damage (creatine kinase) were equally unaffected by astaxanthin supplementation [98]. ...
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Carotenoids are organic, liposoluble pigments found in nature, which are responsible for the characteristic colors of ripe tomatoes, carrots, peppers, and crustaceans, among others. Palliative care provided to patients with an incurable disease is aimed at improving the patient’s quality of life through appropriate treatment of symptoms accompanying the disease. Palliative care patients with burdensome symptoms related to advanced-stage cancers are especially interested in the use of natural dietary supplements and herbal remedies to reduce symptoms’ intensity and ameliorate the quality of life. Carotenoids seem to be a group of natural compounds with particularly promising properties in relieving symptoms, mainly due to their strong antioxidant, anti-inflammatory, and neuroprotective properties. Moreover, carotenoids have been used in folk medicine to treat various diseases and alleviate the accompanying symptoms. In this narrative review, the authors decided to determine whether there is any scientific evidence supporting the rationale for carotenoid supplementation in advanced-stage cancer patients, with particular emphasis on the adjuvant treatment of cancer-related symptoms, such as neuropathic pain and cancer-related cachexia.
... Astaxanthin on Broiler e nutraceutical applications of AST previously reported include anticancer and antidiabetic applications and it has also been reported to have gastro-, hepato-, neuro-, cardio-, ocular, and skin-protective properties [63]. A study carried out by Awadh and Zangana [37] showed that AST from H. pluvialis as much as 10-40 mg/kg of feed could reduce the mortality ratio caused by the capacity of AST to enhance bird immunity by increasing the levels of Tand B lymphocytes and the production of interleukin-producing immunoglobulins and interferon [64][65][66]. AST is also effective in increasing the proportion of microorganisms, especially probiotic bacteria originating from lactic acid bacteria in the intestine and spreading to the myosin fiber network which then covers the intestinal cells, thereby inhibiting pathogenic bacteria [13]. Suppression of the growth of Gram-negative bacteria could also protect against faecal ammonia which is a gas that is harmful to poultry [67]. ...
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Recent interest in carotenoids has increased due to their antioxidant and production performance. Astaxanthin (AST) is a xanthophyll carotenoid abundantly distributed in microalgae, which is described as a highly potent antioxidant. Therefore, recent studies have tended to investigate the role of antioxidants in improving metabolic processes and physiological functioning of the body. It is now evident that AST could significantly reduce free radicals and oxidative stress and help to maintain a healthy state. Moreover, AST also could improve the performance of broiler chicken by increasing the daily feed intake, followed by improvement in the food conversion rate.
... Astaxanthin (AX) is a safe, lipid-soluble, bioavailable natural carotenoid compound present in several microorganisms and various aquatic and nonaquatic species including crustaceans, fish or the flamingo. A major source of AX is Haematococcus pluvialis, a green microalga with high AX content [1][2][3][4]. AX is a potent nutraceutical widely used as a nutritional supplement with antioxidant and anticancer actions. AX was shown to prevent diabetes, cardiovascular diseases, neurodegenerative disorders, and stimulate immunization [5,6]. ...
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Astaxanthin is a lipid-soluble carotenoid influencing lipid metabolism, body weight, and insulin sensitivity. We provide a systematic analysis of acute and chronic effects of astaxanthin on different organs. Changes by chronic astaxanthin feeding were analyzed on general metabolism, expression of regulatory proteins in the skeletal muscle, as well as changes of excitation and synaptic activity in the hypothalamic arcuate nucleus of mice. Acute responses were also tested on canine cardiac muscle and different neuronal populations of the hypothalamic arcuate nucleus in mice. Dietary astaxanthin significantly increased food intake. It also increased protein levels affecting glucose metabolism and fatty acid biosynthesis in skeletal muscle. Inhibitory inputs innervating neurons of the arcuate nucleus regulating metabolism and food intake were strengthened by both acute and chronic astaxanthin treatment. Astaxanthin moderately shortened cardiac action potentials, depressed their plateau potential, and reduced the maximal rate of depolarization. Based on its complex actions on metabolism and food intake, our data support the previous findings that astaxanthin is suitable for supplementing the diet of patients with disturbances in energy homeostasis.
... Park et al. have observed that AST diminished the expression of COX-2, iNOS, and ICAM-1 proteins in the streptozotocin (STZ)-induced diabetic rats, secondary to diminished inflammation [28]. Another study has demonstrated that AST suppressed inflammation secondary by excessive physical activity [29] and UV light, diminishing the expression of iNOS and COX-2 proteins and mRNAs [30]. ...
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Immunoglobulin (Ig), found in serum and in fluids on the body's mucosal surfaces, is an important soluble mediator of host defense against bacterial and viral infections. Serum Ig levels do not change appreciably with acute exercise or after moderate exercise training. However, recent reports show low serum Ig levels in athletes, compared with clinical norms, during prolonged periods (i.e., months) of intensive exercise training. Production of serum antibodies to specific antigens appears to be enhanced following moderate exercise training; the effects of intensive training on specific antibodies are not yet known. In contrast to serum Ig, the concentration of salivary IgA, a marker of secretory Ig, decreases acutely after intensive exercise and over prolonged periods of intensive training in elite athletes. The mechanisms responsible for the decreased serum Ig and secretory IgA levels in elite athletes are not currently known but may involve neurohormonal factors related to physical and psychological stress resulting from intensive daily exercise. It has been suggested that exercise-induced decreases in secretory IgA may be one mechanism related to the high incidence of upper respiratory tract infection among athletes who train intensively on a daily basis over prolonged periods of time.
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The immune system plays an essential role in maintaining the body’s overall health and resistance to diseases. It comprises two branches, known as the innate or antigen-nonspecific branch, and the adaptive or antigen-specific branch. A truly effective immune defence is based on a balance of the different arms of the whole immune system. The human immune response system is very complex and carotenoids have been reported to have effects on many different aspects. To understand the significance of this it is necessary to have a working knowledge of the immune system. An outline of the main features and principles is given below, but the non-specialist reader is recommended to consult a modern biology or biochemistry textbook, or an introductory book on immunology.
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Astaxanthin is a potent antioxidant carotenoid and may play a role in modulating immune response in cats. Blood was taken from female domestic shorthair cats (8-9 mo old; 3.2 ± 0.04 kg body weight) fed 0, 1, 5 or 10mg astaxanthin daily for 12 wk to assess peripheral blood mononuclear cell (PBMC) proliferation response, leukocyte subpopulations, natural killer (NK) cell cytotoxic activity, and plasma IgG and IgM concentration. Cutaneous delayed-type hypersensitivity (DTH) response against concanavalin A and an attenuated polyvalent vaccine was assessed on wk 8 (prior to vaccination) and 12 (post-vaccination). There was a dose-related increase in plasma astaxanthin concentrations, with maximum concentrations observed on wk 12. Dietary astaxanthin enhanced DTH response to both the specific (vaccine) and nonspecific (concanavalin A) antigens. In addition, cats fed astaxanthin had heightened PBMC proliferation and NK cell cytotoxic activity. The population of CD3(+) total T and CD4(+) T helper cells were also higher in astaxanthin-fed cats; however, no treatment difference was found with the CD8(+) T cytotoxic and MHC II(+) activated lymphocyte cell populations. Dietary astaxanthin increased concentrations of plasma IgG and IgM. Therefore, dietary astaxanthin heightened cell-mediated and humoral immune responses in cats.
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No information is available on the possible role of astaxanthin on immune response in domestic canine. Female Beagle dogs (9-10 mo old; 8.2 ± 0.2 kg body weight) were fed 0, 10, 20 or 40 mg astaxanthin daily and blood sampled on wk 0, 6, 12, and 16 for assessing the following: lymphoproliferation, leukocyte subpopulations, natural killer (NK) cell cytotoxicity, and concentrations of blood astaxanthin, IgG, IgM and acute phase proteins. Delayed-type hypersensitivity (DTH) response was assessed on wk 0, 12 and 16. Plasma astaxanthin increased dose-dependently and reached maximum concentrations on wk 6. Dietary astaxanthin enhanced DTH response to vaccine, concanavalin A-induced lymphocyte proliferation (with the 20mg dose at wk 12) and NK cell cytotoxic activity. In addition, dietary astaxanthin increased concentrations of IgG and IgM, and B cell population. Plasma concentrations of C reactive protein were lower in astaxanthin-fed dogs. Therefore, dietary astaxanthin heightened cell-mediated and humoral immune response and reduced DNA damage and inflammation in dogs.