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Effects of Astaxanthin Supplementation on Exercise-Induced Fatigue in Mice

Authors:

Abstract

The present study was designed to determine the effect of astaxanthin on endurance capacity in male mice aged 4 weeks. Mice were given orally either vehicle or astaxanthin (1.2, 6, or 30 mg/kg body weight) by stomach intubation for 5 weeks. The astaxanthin group showed a significant increase in swimming time to exhaustion as compared to the control group. Blood lactate concentration in the astaxanthin groups was significantly lower than in the control group. In the control group, plasma non-esterfied fatty acid (NEFA) and plasma glucose were decreased by swimming exercise, but in the astaxanthin group, NEFA and plasma glucose were significantly higher than in the control group. Astaxanthin treatment also significantly decreased fat accumulation. These results suggest that improvement in swimming endurance by the administration of astaxanthin is caused by an increase in utilization of fatty acids as an energy source.
Exercise-induced fatigue has been attributed to the follow-
ing factors. First, myoglobin and an energy metabolic system
coenzyme leak out into the blood from cells and tissues dam-
aged by exercise, and destruction of red blood cells occurs.
Second, exercise promotes consumption of energy sources
such as glycogen by mobilizing internal energy metabolism
to the maximum and using and depleting the energy source.
Third, through these processes, exercise causes the produc-
tion and accumulation of products of metabolism, such as
lactic acid, in the body.1—3) Therefore, recovery from exercise
fatigue requires repair of the damage that has occurred in the
body. Specifically, resynthesis of the leaked cell and tissue
components and consumed energy sources is needed, as are
decomposition and removal of accumulated byproducts of
metabolism.
During in vivo screening for endurance capacity and anti-
fatigue foods, astaxanthin was found to have a potent en-
hancing effect on endurance capacity in mice.
Astaxanthin, a red carotenoid pigment, is a biological an-
tioxidant that occurs naturally in a wide variety of living or-
ganisms. It has many highly potent pharmacological activi-
ties, such as antioxidative activity,4—7) anti-tumor and anti-
cancer effects,8) immunomodulating actions,9,10) and anti-dia-
betic9) and anti-inflammatory actions.10,11)
The chronic effects of astaxanthin on endurance capacity
have not previously been demonstrated. In the present study,
we investigated endurance capacity by administering astax-
anthin to mice and then subjecting the animals to exercise in
the form of swimming.
MATERIALS AND METHODS
Astaxanthin The astaxanthin used in this study was
AstaREAL 50F, supplied by Fuji Chemical Industry Co.,
Ltd., Toyama, Japan.
Animals Four-week-old male ddY mice (SLC, Japan)
were used. They were housed in standard cages (21.532
14 cm, 5 mice/cage) under controlled conditions of tempera-
ture (241°C), humidity (502%), and lighting (lights on
from 08:00 to 20:00). They were provided a normal diet (MR
stock, NIHON NOUSAN, Japan) and water ad libitum.
Swimming Exercise Test Protocol Experiment 1: The
mice were allowed to adapt to the laboratory housing for at
least 1 week. Forty mice were divided into four groups
(n10 per group). The mice were given either vehicle (olive
oil) or astaxanthin in doses of 1.2, 6, or 30 mg/kg body
weight by stomach intubation at 10:00 5d a week for 5
weeks. Samples were administrated in a volume of 200
m
l.
The mice were submitted to weekly swimming exercise sup-
porting constant loads (lead fish sinkers, attached to the tail)
corresponding to 10% of their body weight. The mice were
assessed to be fatigued when they failed to rise to the surface
of the water to breathe within 5 s.12) The swimming exercise
was carried out in a tank (284629 cm), filled with water
to 26 cm depth and maintained at a temperature of 30C.
To avoid circadian variations in physical activity, swimming
exercise was performed between 11:00 and 17:00, a period
during which minimal variation of endurance capacity has
been confirmed in rats.13)
Experiment 2: The mice were given either vehicle (olive
oil), or astaxanthin in doses of 1.2, 6, or 30 mg/kg body
weight (n10 per group) by stomach intubation 5 days a
week for 3 weeks. Each of the mice had a weight attached
(5% body weight) to the tail for the duration of the swim-to-
exhaustion exercise. The mice were assessed to be fatigued
when they failed to rise to the surface of the water to breathe
within 5 s.12)
Experiment 3: The protocol was the same as above except
that the mice were made to swim for a predetermined length
of time (15 min) supporting loads corresponding to 5% of
their body weight.12) Blood samples for lactate, glucose, and
non-esterfied fatty acid (NEFA) determinations were col-
lected 7 times from the tail before the beginning and at 5-min
intervals during swimming exercise, as well as 10, 30, and
60 min after exercise. To avoid blood dilution with residual
water at the tail of the animal, the mice were quickly dried
with a towel immediately before blood collection. The mice
were immediately returned to the tank after blood sampling.
2106 Vol. 29, No. 10
© 2006 Pharmaceutical Society of JapanTo whom correspondence should be addressed. e-mail: yazawa@s.kaiyodai.ac.jp
Effects of Astaxanthin Supplementation on Exercise-Induced Fatigue in
Mice
Mayumi IKEUCHI,aTomoyuki KOYAMA,aJiro TAKAHASHI,band Kazunaga YAZAWA*,a
aLaboratory of Nutraceuticals and Functional Foods Science, Graduate School of Marine Science and Technology, Tokyo
University of Marine Science and Technology; 4–5–7 Konan, Minato-ku, Tokyo 108–8477, Japan: and bFuji Chemical
Industry Co., Ltd.; Gohkaizawa, Kamiichi-machi, Toyama 930–0397, Japan.
Received June 23, 2006; accepted August 1, 2006; published online August 9, 2006
The present study was designed to determine the effect of astaxanthin on endurance capacity in male mice
aged 4 weeks. Mice were given orally either vehicle or astaxanthin (1.2, 6, or 30 mg/kg body weight) by stomach
intubation for 5 weeks. The astaxanthin group showed a significant increase in swimming time to exhaustion as
compared to the control group. Blood lactate concentration in the astaxanthin groups was significantly lower
than in the control group. In the control group, plasma non-esterfied fatty acid (NEFA) and plasma glucose were
decreased by swimming exercise, but in the astaxanthin group, NEFA and plasma glucose were significantly
higher than in the control group. Astaxanthin treatment also significantly decreased fat accumulation. These re-
sults suggest that improvement in swimming endurance by the administration of astaxanthin is caused by an in-
crease in utilization of fatty acids as an energy source.
Key words astaxanthin; fatigue; antioxidant; exercise; endurance capacity
Biol. Pharm. Bull. 29(10) 2106—2110 (2006)
Lactic acid concentration was determined with a Kyowa
Medex commercial kit (Determiner LA, Tokyo, Japan).
NEFA was measured by the acyl-CoA synthetase and acyl-
CoA oxidase enzyme method with a commercial kit (NEFA
C-test Wako, Wako Pure Chemical Industries, Osaka Japan).
Glucose was assayed by a combination of mutase and glu-
cose oxidase with a commercial kit (Glucose CII test Wako).
The following week, these groups were further subdivided
into non-exercise and exercise groups. Exercise groups were
made to swim for 15 min supporting loads corresponding to
5% of their body weight, and immediately after swimming
were killed by dislocation of the neck. Blood samples for
creatine kinase (CK) activity were taken from the heart.
Plasma samples were refrigerated until assay, and CK activ-
ity was measured using a commercial kit (CPKII test Wako).
Liver and muscle samples from mice in both groups were re-
moved and stored at 20°C, and glycogen content was de-
termined using the method of Lo et al.14) Briefly, portions of
the muscle and liver were put into a tube containing 1.5ml of
30% KOH saturated with Na2SO4and immersed in a boiling
water bath for 30 min before glycogen was assayed using a
commercial kit (Glucose CII test Wako).
Animal studies were performed according to the regula-
tions of our laboratory in line with the 1980 guideline Notifi-
cation No. 6 of the Prime Minister’s Office of Japan.
Statistical Analysis Data are expressed as meanS.E.
Comparisons of swimming capacity between control and
treated groups were assessed using one-way analysis of vari-
ance (ANOVA) and the Tukey–Kramer Multiple Comparison
Test. The data on metabolic parameters were analyzed by the
unpaired ttest. The data on glycogen concentration were as-
sessed using two-way analysis of variance (ANOVA) fol-
lowed by Fisher PLSD post-hoc analysis. A level of p0.05
was used as the criterion for statistical significance.
RESULTS
Effects of Astaxanthin on Swimming Exercise In Ex-
periment 1, which involved a 10% body weight load, the
6mg/kg and 30 mg/kg astaxanthin groups showed a signifi-
cant increase in swimming time to exhaustion as compared to
the control group from the first week. In the 1.2 mg/kg astax-
anthin group, a significant increase in swimming time to ex-
haustion as compared to the control group was evident after
5 weeks (Fig. 1). In order to investigate in detail, the mice
had a weight attached 5% body weight for the duration of the
swim-to-exhaustion. With a 5% body weight load, the mice
in the astaxanthin groups again swam for significantly longer
times compared to the control group (Fig. 2).
Effects of Astaxanthin on Blood Lactate, Glucose, and
NEFA Concentration during Swimming (Experiment 3)
In the astaxanthin groups, blood lactate concentration was
significantly lower than in the control group (Fig. 3).
In the control group, plasma glucose was decreased by
15 min of swimming exercise. After the exercise ended, the
plasma glucose recovered. However, in the astaxanthin
6mg/kg, 30 mg/kg groups, plasma glucose was significantly
higher than in the control group. In the control group, plasma
NEFA concentration was decreased by 15min of swimming
exercise. In the astaxanthin 30 mg/kg group, plasma NEFA
was significantly increased by swimming exercise.
Effects of Astaxanthin on Epididymal Adipose Tissue
Weight (Experiment 3) There was no significant differ-
ence in body weight between the control group and astaxan-
thin groups for 5 weeks (control: 42.11.0 g, astaxanthin
1.2 mg/kg: 42.91.1 g, 6 mg/kg: 42.31.2 g, 30 mg/kg:
42.31.2 g). But in the 30 mg/kg astaxanthin group, epididy-
mal adipose tissue weight was significantly (p0.05) de-
creased compared to that of the control group (Fig. 4). In the
astaxanthin 1.2 mg/kg and 6 mg/kg groups, the epididymal
adipose tissue weight tended to be lower than in the control
group, but not significantly.
Effects of Astaxanthin on Liver and Muscle Glycogen
Non exercise, astaxanthin had no effect on glycogen concen-
tration in the liver and gastrocnemius muscle. Liver glycogen
contents were decreased by swimming exercise. However,
liver glycogen contents were significantly higher in the astax-
anthin 30 mg/kg groups than in the control group after swim-
ming for 15 min (Fig. 5). In the astaxanthin 6 mg/kg and
October 2006 2107
Fig. 1. Effect of Astaxanthin on Swimming Exercise in Mice (Experiment
1)
The mice were given either vehicle (), or an astaxanthin dose of 1.2 (), 6 (), or
30 () mg/kg body weight (n10 per group). The mice were made to perform swim-
ming exercise with weights attached to their tails corresponding to 10% of their body
weight. Each value represents meanS.E. p0.05, ∗∗ p0.01, ∗∗∗ p0.005 vs. con-
trol.
Fig. 2. Effect of Astaxanthin on Swimming Exercise in Mice (Experiment
2)
The mice were given either vehicle or an astaxanthin dose of 1.2, 6, or 30mg/kg
body weight for 3 weeks (n10 per group). The mice swam with weights attached to
their tails corresponding to 5% of their body weight. Each value represents meanS.E.
Significant difference from corresponding control group (p0.05, ∗∗∗ p0.005).
30 mg/kg groups gastrocnemius muscle glycogen contents
tended to decrease, but not significantly. And, gastrocnemius
muscle glycogen contents significantly higher in the astaxan-
thin 6 mg/kg and 30 mg/kg groups than in the control group
after swimming for 15 min.
Plasma CK Activity Plasma CK activity was increased
by exercise. But increased was reduced in the astaxanthin
30 mg/kg group (Fig. 6).
DISCUSSION
Many aspects of fatigue have been studied over the years,
but adequate methods for objective evaluation of fatigue have
not yet been established. In the present study, male mice ex-
ercised to fatigue, and the effect of astaxanthin supplementa-
2108 Vol. 29, No. 10
Fig. 3. Effect of Astaxanthin on Blood Lactate, Glucose, and NEFA Con-
centration during Swimming for 15 min (Experiment 3)
The mice were given either vehicle () or astaxanthin 1.2mg/kg (), 6 mg/kg (),
or 30 mg/kg () (n10 per group). The mice were made to perform swimming exer-
cise with weights attached to their tails corresponding to 5% of their body weight. Each
value represents meanS.E. p0.05, ∗∗∗ p0.005 vs. control.
Fig. 4. Effect of Astaxanthin on Epididymal Adipose Tissue Weight after
Swimming Exercise for 15 min (Experiment 3)
Each value represents meanS.E. p0.05 vs. control.
Fig. 5. Effect of Astaxanthin on Liver and Muscle Glycogen after 15 min
of Swimming Exercise
The mice were given either vehicle or astaxanthin 1.2mg/kg, 6 mg/kg, or 30mg/kg
for 5 week. : no-exercise, : exercise. The mice were made to perform swimming ex-
ercise with weights attached to their tails corresponding to 5% of their body weight.
Each value represents meanS.E. p0.05, ∗∗∗ p0.005 vs. no-exercise. #p0.05 vs.
exercise control.
Fig. 6. Effect of Astaxanthin on CK Activity in Plasma after 15 min of
Swimming Exercise
The mice were given either vehicle or astaxanthin 1.2mg/kg, 6 mg/kg, or 30mg/kg
for 5 week. : non-exercise, : exercise. The mice were made to perform swimming
exercise with weights attached to their tails corresponding to 5% of their body weight.
Each value represents meanS.E. p0.05 vs. non-exercise.
tion on endurance capacity and fatigue was evaluated. Swim-
ming time was significantly prolonged by administering
astaxanthin. The present study aimed to clarify the manner
of this effect.
In the control group, plasma glucose was decreased by
swimming exercise. In the astaxanthin groups, plasma glu-
cose was significantly higher than in the control group. In ad-
dition, liver and muscle glycogen contents were significantly
higher in the astaxanthin groups than in the control group
after swimming for 15 min. These results indicate that the
supply of glucose can be used more smoothly and/or that
glucose utilization may be decreased during exercise in the
astaxanthin. The glycogen-sparing effect of astaxanthin
could provide an important survival advantage in situations
requiring extended periods of prolonged endurance exercise
because glycogen depletion is associated with physical ex-
haustion, and slower utilization of glycogen results in im-
proved endurance exercise performance. As one of the
sources of blood glucose, liver glycogen plays an important
role in controlling the availability of cellular energy. It is pos-
sible that astaxanthin may have promoted glycogenolysis re-
straint and/or gluconeogenesis. In addition, in the astaxanthin
groups, the blood lactate concentration was significantly
lower than in the control group. Lactic acid is produced as a
result of carbohydrate metabolism. These results indicate that
astaxanthin caused a decrease in glucose utilization during
exercise.
Increased fatty acid utilization during exercise reduces the
glycogen depletion rate and improves endurance exercise
performance.15) Therefore, increased fatty acid utilization is
thought to be important for endurance performance. These
increases are associated with enhanced lipolysis and sparing
of stored glycogen, resulting in a delay of complete glycogen
depletion by increasing circulating catecholamines. The en-
hanced availability of NEFA is thought to cause greater fat
metabolism in the active muscles, which in turn decreases
carbohydrate utilization and leads to increased exercise ca-
pacity.16) These concepts agree with our research results. In
the control group, plasma NEFA concentration was de-
creased by 15 min of swimming. However, in the astaxanthin
30 mg/kg group, plasma NEFA was significantly increased
by swimming. Astaxanthin activated utilization of lipid to a
greater extent than glucose as an energy source for perform-
ance. There was no significant difference in body weight be-
tween the control group and astaxanthin groups for 4 weeks.
However, in the 30mg/kg astaxanthin group, adipose tissue
weight was significantly (p0.05) decreased compared to
that of the control group. The metabolic effects of astaxan-
thin on endurance performance appear to be caused by the
increase in fatty acid utilization as an energy source, with
sparing of glycogen. The glycogen thus saved could become
an available energy source for the later stages of exercise,
thus delaying the onset of fatigue.
Improvement of cardiopulmonary function and increase of
oxygen supply to tissues as a result of an increase of hemo-
globin are commonly stated to be major factors that increase
endurance capacity. In the present study, the hemoglobin
concentration after 4 weeks of administration did not differ
from that at the start of the study (data not shown). These re-
sults indicate that astaxanthin did not influence the supply of
oxygen to tissues provided by hemoglobin.
It is well documented that a bout of aerobic physical exer-
cise markedly increases O2uptake and consumption due to
the increased skeletal muscle energy requirement. This in-
creased O2consumption further augments the generation of
reactive oxygen species (ROSs) when the scavenging capac-
ity of both nonenzymatic and enzymatic defense mechanisms
is overwhelmed. This is especially the case during an acute
bout of exhaustive exercise. ROSs have been reported to
cause modifications in cellular biochemical components such
as protein, lipid, and DNA.17—19) Furthermore, ROSs, like
lactate anion and protons, have been suggested to be impli-
cated in oxidative skeletal muscle fatigue. It is reported that
ROS alter such transport systems as potassium transport and
thus contribute to the onset of fatigue.20) Polyunsaturated
fatty acids are another ROS target, and their peroxidation
may lead to fluidity and permeability alterations. Moreover,
lactate anion, independently from proton and thus pH modifi-
cations, may decrease muscle force production by inhibiting
Ca2release from the sarcoplasmic reticulum and/or by
changing ionic strength.21) Astaxanthin has been reported to
be more effective than other antioxidants, such as vitamin E
and
b
-carotene, in preventing lipid peroxidation in solutions
and in various biologic membrane systems.4—6) Astaxanthin
can attenuate exercise-induced damage in mouse skeletal
muscle and heart, including an associated neutrophil infiltra-
tion that induces further damage.22) In the present study,
plasma CK concentration after exercise was increased. But
the increase in plasma CK activity was inhibited by treatment
with astaxanthin. It is possible to say that astaxanthin does to
free radicals, but stabilizes membranes, delays muscle fa-
tigue, and thereby enhances endurance.
In conclusion, our data suggest that astaxanthin may have
beneficial effects on endurance capacity. The administration
of astaxanthin causes an increase in utilization of fatty acids
as an energy source, which spares glycogen.
However, comprehensive chemical and pharmacological
research is required to determine the mechanism by which
astaxanthin affects endurance capacity.
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2110 Vol. 29, No. 10
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... This mechanism can enhance the utilization of fatty acids and stimulate fat-burning during sports training. The results showed that astaxanthin could improve endurance capacity and reduce fatigue in swimming, and this situation may be triggered by increasing the use of fatty acids as an energy source (Ikeuchi et al. 2006). The 7-week-old male mice were treated orally with 5 mg, 15 mg, and 30 mg of astaxanthin per kilogram of body weight and 45 min of moderate-intensity swimming training each day for 4 weeks. ...
... This mechanism can enhance the utilization of fatty acids and stimulate fat-burning during sports training. The results showed that astaxanthin could improve endurance capacity and reduce fatigue in swimming, and this situation may be triggered by increasing the use of fatty acids as an energy source (Ikeuchi et al. 2006). The 7-week-old male mice were treated orally with 5 mg, 15 mg, and 30 mg of astaxanthin per kilogram of body weight and 45 min of moderate-intensity swimming training each day for 4 weeks. ...
... The majority of natural astaxanthin today is derived from Streptococcus sanguinis, which is more stable than synthetic astaxanthin. Due to its potent effects on oxidation resistance, fatigue resistance, inflammation resistance, and immune regulation, natural astaxanthin is well-liked in the functional food sector [9][10][11][12]. Astaxanthin exerted a function to reduce inflammation and improve gut microbiota in hepatopathy mice. ...
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The present study was undertaken to determine the effects of endurance training on glycogen kinetics during exercise. A new model describing glycogen kinetics was applied to quantitate the rates of synthesis and degradation of glycogen. Trained and untrained rats were infused with a 25% glucose solution with 6-3H-glucose and U-14C-lactate at 1.5 and 0.5 microCi x min(-1) (where 1 Ci=3.7 x 10(10) Bq), respectively, during rest (30 min) and exercise (60 min). Blood samples were taken at 10-min intervals starting just prior to isotopic infusion, until the cessation of exercise. Tissues harvested after the cessation of exercise were muscle (soleus, deep, and superficial vastus lateralis, gastrocnemius), liver, and heart. Tissue glycogen was quantitated and analyzed for incorporation of 3H and 14C via liquid scintillation counting. There were no net decreases in muscle glycogen concentration from trained rats, whereas muscle glycogen concentration decreased to as much as 64% (P < 0.05) in soleus in muscles from untrained rats after exercise. Liver glycogen decreased in both trained (30%) and untrained (40%) rats. Glycogen specific activity increased in all tissues after exercise indicating isotope incorporation and, thus, glycogen synthesis during exercise. There were no differences in muscle glycogen synthesis rates between trained and untrained rats after exercise. However, training decreased muscle glycogen degradation rates in total muscle (i.e., the sum of the degradation rates of all of the muscles sampled) tenfold (P < 0.05). We have applied a model to describe glycogen kinetics in relation to glucose and lactate metabolism during exercise in trained and untrained rats. Training significantly decreases muscle glycogen degradation rates during exercise.
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The anticancer activities of beta-carotene, astaxanthin and canthaxanthin against the growth of mammary tumors were studied in female eight-wk-old BALB/c mice. The mice were fed a synthetic diet containing 0, 0.1 or 0.4% beta-carotene, astaxanthin or canthaxanthin. After 3 weeks, all mice were inoculated with 1 x 10(6) WAZ-2T tumor cells into the mammary fat pad. All animals were killed on 45 d after inoculation with the tumor cells. No carotenoids were detectable in the plasma or tumor tissues of unsupplemented mice. Concentrations of plasma astaxanthin (20 to 28 mumol/L) were greater (P < 0.05) than that of beta-carotene (0.1 to 0.2 mumol/L) and canthaxanthin (3 to 6 mmol/L). However, in tumor tissues, the concentration of canthaxanthin (4.9 to 6.0 nmol/g) was higher than that of beta-carotene (0.2 to 0.5 nmol/g) and astaxanthin (1.2 to 2.7 nmol/g). In general, all three carotenoids decreased mammary tumor volume. Mammary tumor growth inhibition by astaxanthin was dose-dependent and was higher than that of canthaxanthin and beta-carotene. Mice fed 0.4% beta-carotene or canthaxanthin did not show further increases in tumor growth inhibition compared to those fed 0.1% of each carotenoid. Lipid peroxidation activity in tumors was lower (P < 0.05) in mice fed 0.4% astaxanthin, but not in those fed beta-carotene and canthaxanthin. Therefore, beta-carotene, canthaxanthin and especially astaxanthin inhibit the growth of mammary tumors in mice; their anti-tumor activity is also influenced by the supplemental dose.
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The antioxidant activities of astaxanthin and related carotenoids have been measured by employing a newly developed fluorometric assay. This assay is based on 4,4-difluoro-3,5-bis(4-phenyl-1, 3-butadienyl)-4-bora-3a,4a-diaza-s-indacene (BODIPY 665/676) as an indicator; 2,2'-azobis-2,4-dimethylvaleronitrile (AMVN) as a peroxyl radical generator; and 6-hydroxy-2,5,7, 8-tetramethylchroman-2-carboxylic acid (Trolox) as a calibrator in an organic and liposomal media. By employing this assay, three categories of carotenoids were examined: namely, the hydrocarbon carotenoids lycopene, alpha-carotene, and beta-carotene; the hydroxy carotenoid lutein; and the alpha-hydroxy-ketocarotenoid astaxanthin. The relative peroxyl radical scavenging activities of Trolox, astaxanthin, alpha-tocopherol, lycopene, beta-carotene, lutein, and alpha-carotene in octane/butyronitrile (9:1, v/v) were determined to be 1.0, 1.0, 1.3, 0.5, 0.4, 0.3, and 0.2, respectively. In dioleoylphosphatidyl choline (DOPC) liposomal suspension in Tri-HCl buffer (pH 7.4 at 40 degrees C), the relative reactivities of astaxanthin, beta-carotene, alpha-tocopherol, and lutein were found to be 1.00, 0.9, 0.6, and 0.6, respectively. When BODIPY 665/676 was replaced by 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a, 4a-diaza-s-indacene-3-undecanoic acid (BODIPY 581/591 C(11)) as an indicator, astaxanthin showed the highest antioxidant activity toward peroxyl radicals. The relative reactivities of Trolox, astaxanthin, alpha-tocopherol, alpha-carotene, lutein, beta-carotene, and lycopene were determined to be 1.0, 1.3, 0.9, 0.5, 0.4, 0.2, and 0.4, respectively.