Astaxanthin Scientific Review 1006 June 2010

Created by Algatechnologies
Astaxanthin and Human Health
Literature Survey
June 2010
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Algatechnologies LTD.
Kibbutz Ketura D.N Hevel Eilot 88840, Israel Tel:+972-8-6356425 Fax:+972-86356562 Website:www.algatech.com
Table of content
(I) Table of content 2
(1) Introduction 4 4
(1.1) The carotenoids 5
(2) Astaxanthin chemistry 7

(2.1) Astaxanthin – chemical structure 7
(2.2) Sources of Astaxanthin and their properties 8
(3) Bioavailability and pharmacokinetics of Astaxanthin 9

(3.1) Bioavailability 9
(3.2) Pharmacokinetics - ADME: Absorption, Distribution,
Metabolism and Elimination 9
(3.2.1) Absorption 9
(3.2.2) Distribution 10
(3.2.3) Metabolism 11
(3.2.4) Elimination 11
(4) Safety of Astaxanthin 12
(5) Anti-oxidation activity of Astaxanthin 16

(5.1) Evidences from In vitro studies 16
(5.2) Evidences from animal models (in vivo )
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(5.3) Evidences from human trials 19
(5.4) Preservation of membrane structure 20
(6) Astaxanthin and skin health 22

(6.1) Evidences from cell cultures 23
(6.2) Studies in animal models 25
(6.3) Studies in humans 26
(7) Astaxanthin and modulation of the immune system 30
(8) Astaxanthin and inflammation 32

(8.1) Studies in cell cultures 32
(8.2) Studies in rodents 34
(9) Astaxanthin and gastric ulcer 37
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(9.1) Helicobacter pylori-induced gastric ulcer 37

(9.2) Stress- and chemically-induced gastric ulcer 38
(10) Astaxanthin and the cardiovascular system 41
(10.1) Astaxanthin in prevention of atherosclorosis and dyslipidemia 41
(10.2) Astaxanthin and prevention of lipid oxidation 44
(10.3) Astaxanthin and hypertension 45
(10.4) Stroke and thrombosis damages 46
(11) Astaxanthin and muscle endurance 49

(12) Astaxanthin and anti-cancer activity 53

(12.1) Cell culture Studies 53
(13) Astaxanthin and the central nervous system 59

(13.1) Studies in cell cultures and in rodents 59
(14) Astaxanthin and eye health 63

(14.1) Studies in animal models 63
(15) Astaxanthin and Male Fertility 68
(16) Astaxanthin and the metabolic syndrome 70

(16.1) Astaxanthin and onset of Type 2 Diabetes 70
(16.2) Astaxanthin and T2D-induced nephropathy 76
(17) Summary 78
(18) Bibliography 79
3
(1) Introduction
Oxidative stress in living cells arises both as a natural result of aerobic metabolism
and as a result of environmental and pathologic conditions (Papas 1999). Reactive-
oxygen species (ROS) are regularly produced during normal metabolic processes
(Reviewed in Davies 1995, Dore 2005). These chemically unbalanced and harmful
molecules contain reduced oxygen molecules as free radicals and reactive compounds.
The strong tendency of ROS to react with neighboring molecules such as proteins, DNA,
RNA, carbohydrates, and lipids puts these molecules at risk. The results of such
“oxidative attack” may include protein and lipid peroxidation and structural changes in
DNA and RNA, which in turn may lead to damage, mutations, and even loss of function.
Environmental (air pollution, tobacco smoke, toxic chemicals or ultraviolet [UV] light),
as well as physiological (stress, inflammation, diabetes) conditions can enhance the
production of ROS. Indeed, oxidative damage has been linked to aging, atherosclerosis,
ischemia-reperfusion injury, macular degeneration of the eye, carcinogenesis, neuro-
degenerative diseases such as Alzheimer’s and Parkinson’s diseases, bacterial and viral
meningitis, and many other biological processes and diseases.
The human body has evolved a large array of endogenous antioxidant defenses against
oxidative insult. These include antioxidant enzymes that neutralize ROS prior to their
induced damage, (Matés 1999, Matés 2000, Limón-Pacheco 2009) and certain repair
enzymes that can reverse the damage produced by ROS in DNA (Wells 2009). In
addition, endogenous small molecules with antioxidant activity such as glutathione
(Fahey 1991), the hormone melatonin (Reiter RJ 1998), and uric acid (Yu 1998) are
being activated in response to oxidative stress. However, these endogenous antioxidants
do not completely protect against the sum of oxidative stresses challenging the body, and
thus there is net oxidative damage that in the long term contributes to aging and various
diseases. In addition to the body's endogenous defenses against oxidative stress, diet-
derived antioxidants including ascorbic acid (Vitamin C), alpha-tocopherol (Vitamin E),
and the carotenoids may be important in protecting against disease and age-related
phenomena (Ames 1993; Davies 1995, Halliwell 1996).
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(1.1) The Carotenoids
The group of antioxidants mostly investigated for their potential health benefits, are
the carotenoids (reviewed in Cooper 1999). Carotenoids are lipid soluble pigments
produced in some plants, algae, fungi and bacterial species that account for their red,
orange, or yellow hues. Although some crustaceans have a limited capacity to convert
closely related dietary carotenoids, other animals are unable to synthesize carotenoids,
and must acquire them from the diet.
Carotenoids are antioxidants due to their ability to quench singlet oxygen, be oxidized,
and isomerized (Edge 1997, Mortensen 1997). In addition, carotenoids absorb light due
to their special bonding structure, hence providing defense from photo-oxidative damage.
The carbon backbone of a carotenoid (Figure 1) consists of a long and uniform chain
which may contain terminal rings bearing various chemically active groups. Oxygen-free
carotenoids are called carotenes, while oxygen-containing carotenoids are called
xanthophylls. The ability of the various carotenoids to react with ROS differs greatly and
is closely related to their chemical structures (Mortensen 1997). Among the various
carotenoids, Astaxanthin is the most potent natural antioxidant. This was shown by
comparative studies in many systems as further discussed below.
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Figure 1: Structures of selected carotenoids.
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(2) Astaxanthin chemistry
(2.1) Astaxanthin – chemical structure
Astaxanthin (3,3’-dihydroxy-β-β-carotene-4,4’-dione) is a xanthophyll carotenoid,

commonly found in marine environments where it gives an orange-pink coloration to
several sea-species. As seen in Fig. 1, Astaxanthin is closely related to other well-known
carotenoids, such as β-carotene, zeaxanthin and lutein, with which it shares many of the
metabolic and physiological functions attributed to carotenoids. The presence of the
hydroxyl and keto endings on each ionone ring (Figure 1), explains some unique features
of Astaxanthin, such as its ability to be esterified, a higher anti-oxidant activity and a
more polar configuration than other carotenoids. Free Astaxanthin is particularly
sensitive to oxidation. In nature, it is found either conjugated to proteins or esterified with
one or two fatty acids, which stabilize the molecule. In the algea Haematococcus
pluvialis (H. pluvialis), the major natural source for mass production of natural
Astaxanthin, the esterified form predominates, mostly as a monoester (Lorenz 2000).
Various Astaxanthin stereoisomers are found in nature, which differ in the configuration
of the two hydroxyl groups on the molecule (Figure 1). The 3S, 3’S stereoisomer is the
main form found in H. pluvialis H. pluvialis and in wild salmon, while synthetic
Astaxanthin consists of the racemic mixture of the three enantiomers (Turujman 1997).
Synthetic Astaxanthin consist of the “un-natural” 3R-3S stereoisomer (about 50%),
which is rarely found in nature and thus, may be inactive.
Astaxanthin consists of geometric isomers as well, all-trans isomer, which is the most
abundant and the cis isomers (mainly as 9Z and 13Z) (Turujman 1997). The cis isomers,
particularly the 9Z, display higher bioavailability and potency in humans (Coral-
Hinostroza 2004). This isomer is abundant (up to 20%) in the natural Astaxanthin
complex produced by the alga H. pluvialis.
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(2.2) Sources of Astaxanthin and their properties
Astaxanthin can be commercially obtained from four major sources (reviewed in

Higuera-Ciapara 2006):
* Synthetic Astaxanthin
* Astaxanthin from Phaffia rhodozyma yeast
* Astaxanthin from crustacean by-products
* Astaxanthin from the algae Haematococcus pluvialis
The various sources differ in their yields, cost and chemical characteristics yet, of the
natural sources available, the richest source of Astaxanthin is the algae H. pluvialis ,
which can accumulate up to 50 gr (Algatechnoligies, unpublished results) of Astaxanthin
per kilogram of dry biomass (Higuera-Ciapara 2006, Ernst 2002). Moreover, Astaxanthin
produced in H. pluvialis H. pluvialis is currently the only one approved for use in humans
as a dietary supplement by U.S.-FDA, and EU-EFSA, and Japanese auhotorities. The
majority of the safety and efficacy studies were conducted with the 3S 3’S isomer of H.
pluvialis.
Astaxanthin from different sources may may be differentiated in terms of bioavailability
and metabolism; however, there are a limited number of studies comparing efficacy
based upon raw material source, and these are primarily in reference to fish coloring. One
study did demonstrate that the 9-cis isomer has much higher antioxidant potency than that
of the all-trans isomer in rat microsome and rabbit erythrocyte ghost membrane lipid
peroxidation systems, as well as in human neuroblastoma SH-SY5Y cells (Liu 2007).
.
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(3) Bioavailability and pharmacokinetics of Asthaxanthin.
(3.1) Bioavailability
Astaxanthin is not soluble in water. Its level of esterification determines Astaxanthin’s

absorption in the digestive system. When it is esterified, i.e. bound to fat or taken with
oil, Astaxanthin is readily absorbed. Other factors influencing bioavailability include
levels of oxidation, chemical structure (isomerization and stereoisomerization),
processing methods (Mendes-Pinto 2001) and raw material sources (Bowen 1999)., Free
Astaxanthin is extremely sensitive to oxidation. The esterified form of the H. pluvialis
alga is stable and displays higher bioavailability and potency. Other research indicates
that the bioavailability of Astaxanthin is significantely enhanced when esterified, and in
the presence of fats (Odberg 2003, Rufer 2008, Barbossa 1999, Okada 2009).
(3.2) Pharmacokinetics - ADME: Absorption, Distribution, Metabolism and

Elimination
[Inasmuch as similar results are found in pre-clinical and clinical ADME studies of
Astaxanthin, this review refers only to results of clinical studies.]
(3.2.1) Absorption
Absorption of Astaxanthin starts with hydrolization of the ester moiety before the
final absorption takes place, as evident from the absence of esterified Astaxanthin in
human plasma (Odeberg 2003, Coral-Hinostroza 2004). The hydrolization of the ester
seems to be a rate-limiting step, since esterified Astaxanthin had a prolonged absorption
rate, compared to the non-esterified form (Coral-Hinostroza 2004). Following
hydrolization, Astaxanthin digestion and intestinal absorption are closely associated with
fatty acids uptake, transport and delivery, since it is a fat-soluble compound. Astaxanthin
is incorporated into chylomicrons, and distributed between very low density lipoprotein
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(VLDL), low density lipoprotein (LDL) and high density lipoprotein (HDL) and
transported to the tissues (Wang 1992).
(3.2.2) Distribution
Following intake, plasma levels reach maximal values within a few hours (Okada
2009, Odeberg 2003, Østerlie 2000, Coral-Hinostroza 2004) and both tmax and Cmax are
higher after meal or fat intake and are shorter following smoking as shown in Table 1
(Odeberg 2003, Okada 2009). Accumulation of Astaxanthin in plasma, following dose
response, shows non-linear kinetics (Rufer 2008, Coral-Hinostroza 2004). This may be
explained by prolonged absorption through the Peyer’s patch route into the lymphatic
system.
Table 1: Pharmacokinetic paramenters of Astaxanthin after administration of a single oral

dose of 48 mg Astaxanthin before and after meal to smokers and non-smokers
(mean+standard deviation). [Adapted from Okada 2009].
Astaxanthin is distributed to all tissues, and is able to pass the blood brain barrier, as
evident from studies in rodents (Aoi 2003). Studies in humans suggest that there is a
specific accumulation of Astaxanthin in VLDL chylomicrons (36-64% of total
Astaxanthin, after 6.7 h, Osterlie 2000, Coral-Hinstroza 2004), as shown in Figure 2.
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Figure 2: Astaxanthin distribution in plasma lipoproteins 6 hours after oral ingestion of a

single meal containing 100 mg Astaxanthin. [Adapted from Coral-Hinostroza (2004)]
(3.2.3) Metabolism
Kistler et al. (2002) investigated the metabolism of Astaxanthin as well as its

capacity to induce cytochrome P450 genes in primary cultures of human hepatocytes and
in two human volunteers. Four main free metabolites were found; and their reduction
products were also present as glucuronides. Based on experiments done in cultured
hepatocytes, and on the highest level of Astaxanthin reached in a clinical trial (40
mg/day, 4 weeks) (Kupcinskas 2008), Astaxanthin is not expected to affect CYP enzymes
or metabolism of drugs.
(3.2.4) Elimination
Elimination of Astaxanthin is relatively fast with elimination half life ranging

between 15.9h (Odenberg 2003) to 52h (Coral-Hinostroza 2004). In addition, studies in
rodents showed that the length of exposure to Astaxanthin is not related to plasma
concentration (Stewart 2008), and that there is a rapid elimination or catabolism of
Astaxanthin without long-term storage of astaxanthin in tissues (Petri 2007). Taken
together, these results suggest that Astaxanthin is unlikely to accumulate in the body. In
humans, a three-day follow-up of 5 volunteers who ate 1.5mg Astaxanthin in a single
meal, detected no Astaxanthin in plasma while 64% of Astaxanthin was excreted in feces
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(Elmadfa 1999). This study suggests that majority of the ingested Astaxanthin is excreted
in feces.
(4) Safety of Astaxanthin
Astaxanthin is naturally found in many animals and plants. In salmon flesh its
concentration may rang from 3 to 40 mg/kg (Turujman 1997). The main Astaxanthin
isomer identified in salmon was the 3S, 3'S stereoisomer identical to that found in H.
pluvialis . In addition to salmon, Astaxanthin is also found in crustacean species such as
krill, shrimp, crab, lobster and crawfish. Thus, humans have ingested Astaxanthin for
centuries, with amounts depending on the importance of seafood in the diet. Indeed, the
European Food Safety Authority (EFSA, 2005) estimated daily intake of Astaxanthin for
consumers with a high intake of fish and seafood, in the range of 1.6 to 4.1 mg/day.
Astaxanthin was approved by the FDA as dietary supplement in aquaculture in 1987 and
as a human dietary supplement in 2000 (Pashkew 2008). It has been marketed as a dietary
supplement for approximately 10 years without any reported adverse effects.
Furthermore, there is no evidence that consumption of Astaxanthin in foods or as a
dietary supplement has any cumulative effect that would affect its safety.
The published recommended daily intake varies among different regulatory authorities. It
is 6 mg/day in Japan, 5 mg/day in the US and 4 mg/day in Europe.
The safety of Astaxanthin either as an extract from natural sources, in its synthetic form,
or as present in algal biomass has been documented in many pre-clinical and clinical
studies. The evidences for its safety can be summarized as:
1) The long-term repeat dose exposure (1.5 and 3 months) of Astaxanthin did not result in
its bioaccumulation or any adverse effects (Ono 1999, Nishikawa 1997).
2) Many animal studies support the safety of Astaxanthin. These include specially
designed safety tests such as ADME studies, acute and subchronic (Takashi 2005)
studies (LD50 > 2000 mg/kg body weight, NOEL = 50 mg /kg/day), repeated-dose
toxicity and fertility study (Nishikawa 1997), embryotoxicity and teratology (Roche
1987), multi generation study (Roche 1987), micronucleus test (Scantox 1998) and
many efficacy-related experiments in which histological and biochemical follow-up
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was included. None of the above mentioned studies revealed any adverse effects of
Astaxanthin.
3) In vitro studies did not report of any mutagenicity (AMES test) (Takahashi 2005).
4) In multiple human clinical studies (>25) (Table 2), the safety of Astaxanthin was
confirmed at doses of up to 40 mg/day for 8 weeks or 4 mg/day for one year. Results
from these trials provide a huge margin of safety above the currently recommended
levels.
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Table 2: Findings from Astaxanthin clinical trials
14
DB-PC = double-blind placebo-controlled; wks = weeks

1
H. pluvialis biomass, 2 H. pluvialis extract, 3 Krill oil/extract, 4 H. pluvialis oil, * Multi-
vitamin formulation
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(5) Anti-oxidation activity of Astaxanthin
Astaxanthin found inside cells protects against oxidative damage by three general
mechanisms:
1) Quenching of singlet oxygen and dissipating the energy as heat.
2) Scavenging of radicals to prevent or terminate chain reactions.
3) Preservation of membrane structure thereby inhibiting membrane lipid peroxidation.
Since the antioxidative effect of Astaxanthin lies in the basis of most (if not all) of its
observed positive influence in many biological systems, numerous studies have been
conducted in an attempt to evaluate its effects and to understand their underling
mechanism(s). Astaxanthin’s protective affect was studied in many experimental systems
including in vitro solutions, membranal models (liposomes, microsomes), cell cultures,
and animal models, and in recent years, humans.
(5.1) Evidences from in vitro studies
Astaxantin’s superior potency as an anti-oxidant, compared to other carotenoides,

was demonstrated in many studies, conducted under various conditions. Representative
evidences from these in vitro studies are summarized in Tables 3 and 4. Since carotenoids
are lypophilic (oil soluble), in vivo they are expected to exert most of their antioxidant
effects in lipid environments. Such environments are found in cell membranes and
mitochondria, and are studied in vitro using the model system of liposoms (single-bilayer
phospholipid vesicles). In this respect, it is important to note that the popular method of
quantifying antioxidant capacity of a given substance (known as ORAC – Oxygen
Radical Absorbance Capacity; developed by Brunswick Labs, Norton, Massachusetts,
USA) can not be applied for Astaxanthin (nor for other carotenoids), as it is inadequate in
measuring oil soluble compounds. Comparative studies, done under lypophlic conditions,
showed that Astaxanthin has the highest singlet oxygen quenching activity (Table 3) and
free radical scavenging activity (Table 4) both in liposoms and in cell culture. Part of this
16
enhanced anti-oxidative activity can be attributed to its remarkable resistance to photo

irradiation as shown in liposoms by Oshima, et al (1993) (Figure 3).
Figure 3: Loss of antioxidant in unilamellar liposoms following photo irradiation.

[Adapted from Oshima (1993)]
Thus, as demonstrated in Tables 3 and 4, although the fold of anti-oxidative activity of

Astaxanthin compared to other carotenoids changes dramatically, depending on the
experimental conditions (e.g. ranges between 2 to up to 550 times higher than vitamin E,
Table 3), it is consistently the highest of the carotenoids.
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Table 3: Quenching of singlet oxygen potency of Astaxanthin and other carotenoids.

Experimental system Other carotenoids Astaxanthin Reference
activity rate
Chemical solution Vitamin E x80 more active Di Mascio 1991
(ORAC) Lutein x3 more active
β-caroten x2 more active
Canthaxanthin comparable
Vitamine E x100 more active Miki 1991
Zeaxanthin x10 more active
Lutein Tunaxanthin x10 more active
Canthaxanthin x10 more active
β-carotene x10 more active
Vitamine E x550 more active Shimidzu 1996
Lutein x3 more active
β-carotene comparable
Zeaxanthin comparable
Protection against Vitamine E x2 more active Oshima 1993
lipid oxidation in β-carotene x2 more active
Liposoms Canthaxanthin More stable Christopherson 1991
Protection of human β-carotene 1.5 more active Tinkler 1994
lymphoid cells against Canthaxanthin 1.5 more active
oxidation Lycopene comparable
Table 4: Scavenging of free radicals potency of Astaxanthin and other carotenoids in

vitro.
Experimental system Other carotenoids Astaxanthin Reference

activity rate
Chemical solution β-caroten x 1.5 Terao 1989
Zeaxanthin x 1.5
Zeaxanthin x2 Miki 1991
Lutein, x 3.5
Canthaxanthin x2.5
β-carotene x 4.5
Vitamine E x100
Protection against lipid Lycopene x2 Barros 2001
oxidation in Liposoms Vitamine E x2
Canthaxanthin x3-10 Rengel 2000 (+Cu)
β-carotene x2 Goto 2001
β-carotene x6 Naguib 2000
α-carotene x 2.5
Lycopene x3
Lutein x3
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Protection against lipid β-carotene comparable Nakagawa 1997 (+Fe)

oxidation in rat microsoms β-carotene x4 Palozza 1992
Vitamine E comparable
Protection against lipid Vitamine E More potent Nishigaki 1994
oxidation in rat microsoms
and organs
(5.2) Evidences from animal models (in vivo)
Astaxanthin was tested in vivo in various animal models. Early studies done in
vitamine E-deficient rats demonstrated that Astaxanthin protects their mitochondria from
damage by Fe2(+)-catalyzed lipid peroxidation, both in vivo and in vitro. This protective
effect was stronger than that of vitamin E itself (Nishigaki 1994, Kurashige 1990). That
Astaxanthin has a general protective effect against oxidative damage was demonstrated
by a study in rats that followed Nitric Oxide (NO) generation, as a marker of oxidation.
Quantification of NO end products in plasma showed a decrease in Astaxanthin-fed rats
(Hussein 2006) (Figure 4).
Figure 4: Effect of oral administration of olive oil (control) or Astaxanthin for 7 weeks on
the level of plasma oxidation markers NO2/NO3 in spontaneous hypertensive rats.
[Adapted from Hussein (2006)]
Additional evidences from animal models are given in the following references: Gross
2006, Nakajima 2008, Augusti 2008, Augusti 2009, Shen 2009, and Tripathi 2010.
(5.3) Evidences from human trials

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Oxidative damage is related to many diseases in humans, and the ability of dietary
Astaxanthin supplementation to improve or even prevent these pathologic situations is
constantly being tested in humans. Two clinical studies directly addressed the effect of
Astaxanthin on overall oxidation level: Karppi, et al. (2007) examined in a randomized
double-blind study the effect of three-month Astaxanthin supplementation (4mg daily, as
capsules) on lipid peroxidation of 20 healthy non-smoking Finnish men. It was observed
that levels of plasma 12- and 15-hydroxy fatty acids were reduced in the Astaxanthin
group only. The study indicated that intestinal absorption of Astaxanthin delivered as
capsules is adequate. The second study tested the efficacy of eight-week treatment with
Astaxanthin in 35 postmenopousal women, 21 of which suffered from high oxidative
stress (Iwabayashi 2009). Astaxanthin-treated women showed enhanced antioxidant
capacity, reduced lower limb vascular resistance, decreased blood pressure, and improved
physical and mental symptoms.
(5.4) Preservation of membrane structure
The ability of Astaxanthin to inhibit lipid peroxidation via stabilization of the

membranal structure was demonstrated in a recent comparative study that followed lipid
peroxidation in the presence of several carotenoids (McNulty 2008). Interestingely, it was
found that the nonpolar carotenoids, lycopene and β-carotene, disordered the membrane
bilayer and stimulated membrane lipid peroxidation, whereas Astaxanthin (a polar
carotenoid) preserved membrane structure and exhibited significant antioxidant activity
(>40% decrease in lipid hydroperoxide levels) (Figure 5).
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These results suggest that the antioxidant potential of carotenoids is dependent on

their distinct interactions with the membrane lipids.
Figure 5: various carotenoids (10 mol/L) were

incorporated into
dilinoleoylphosphatidylcholine membranes
and underwent lipid peroxidation at 37°C for
48 hours. [Adapted from McNulty (2008)]
Goto et al. (Goto 2001) showed that Astaxanthin was about 2-fold more effective than β-
carotene in inhibiting the production of lipid peroxides in liposoms. The analysis of the
reaction mechanisms of β-carotene and Astaxanthin suggested that Astaxanthin trapped
radicals both in the membrane and on its surface (model depicted in Figure 10), whereas
β carotene could do so only near the surface at the interior of the membrane. The
investigators suggested that the efficient antioxidant activity of Astaxanthin is due to the
unique structure of the terminal ring moiety. This structure-function relation may explain
the differences in biologic activities of the various carotenoids, with important
therapeutic implications.
21
Figure 6: Transmembrane orientation of polar carotenoids facilitates electron shuttling.

Specific physicochemical interactions of Astaxanthin with membranes is likely
responsible for its antioxidant properties and its biologic benefits. The transmembranous
alignment provides exposure of the polar (hydrophilic) ends of the molecule to the
internal cytoplasm and to the aqueous environment external to the cell (or the
mitochondrial matrix and the intermembrane space of mitochondria), potentially
facilitating electron transfer via the double bonds of the carbon scaffold of the compound.
The intramembranous alignment of the molecule also likely provides proximity to
vitamin C, which serves as a “sink” for accepting the radical cations generated effectively
“recharging” the capacity of the degraded carotenoid. RNS- reactive nitrogen species;
ROS- reactive oxygen species.
22
(6) Astaxanthin and skin health
Our skin is often exposed to sunlight, which contains hazardous UV light.

Excessive exposure of the skin to sunlight results in erythema, sunburn and can lead to
photo-induced inflammation, immunosuppression, aging and even carcinogenesis of skin
cells (melanoma) (Figure 7). Pre-clinical studies illustrated that dietary antioxidants, such
as vitamin E, vitamin C, or β-carotene could reduce such damage. The characteristics of
Astaxanthin, as a superior antioxidant, motivated the investigation of its potential in skin
health and cosmetics.
Figure 7: Effects of UVA, UVB and Ozone on skin.

UV rays induce the production of in situ ROS and matrix metalloproteinases (MMP).
These factors are the root of wrinkle formation as they destroy the collagen matrix in the
dermis. The skin’s repair mechanism will rebuild the damaged collagen, however, the
hindrance of skin renewal by repeated exposure to ROS and MMP leads to formation of
wrinkles. The presence of Astaxanthin attenuates the effects of ROS and MMP and
therfore allows proper regeneration of the skin.
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(6.1) Evidences from cell cultures
The ability of Astaxanthin to protect from UV-induced oxidative stress was first
documented in cell cultures (O’Connor 1998, Camera 2009, Lyons 2002, Santocono
2006). UV-induced damage usually expresses itself in a battery of cellular reactions,
including apoptosis (programed cell death), which are being followed in the presence or
absence of the tested carotenoids.
In a recent study (Camera 2009) performed in human dermal fibroblasts, it was found
that Astaxanthin exhibited a pronounced photoprotective effect and counteracted all of
the UVA-induced alterations. β-Carotene only partially prevented few of the UVA-
induced reactions, but it increased membrane damage and stimulated HO-1 expression. In
addition, uptake of Astaxanthin by fibroblasts was higher than that of β-Carotene. The
photostability of Astaxanthin in fibroblasts was much higher than that of β-Carotene.The
data indicate that Astaxanthin has a superior preventive effect towards photo-oxidative
changes in human dermal fibroblasts (Figure 8).
Figure 8: Effect of different carotenoids on irradiation-induced apoptotic cell death in

human dermal fibroblasts. While Astaxanthin (Ax) and canthaxanthin (CX) inhibited
irradiation induced cell death, β-caroten (βC) enhanced it, compared to control. Adapted
from Camera (2009).
24
Earlier comparative studies showed that Astaxanthin could be more effective than
β-carotene and lutein at preventing UV-light photo-oxidation of lipids in rat kidney
fibroblasts by a factor of up to 200 and 1000 folds, respectively (O’Connor 1998) [Table
5]. The protective effect of Astaxanthin from UV-induced DNA damage was also shown
in human skin fibroblast [Figure 9] (Lyons 2002) and in rat epithelial cells (Santocono
2006).
Table 5: Carotenoids protection from UVA radiation in rat kidney fibroblasts.

[Adapted from O’Connor (1998)]
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Figure 9: Ability of Astaxanthin to protect against UVA induced damage in human

melanocytes. [Adapted from Lyons (2002)]
Astaxanthin was also found to inhibit the over-production of melanin in melanoma

cells, an important step in their carcinogenesis (Arakane, 2002). This phenomenon is
known as hyper-pigmentation of the skin, in the form of suntan, stains or freckles. Mouse
melanoma cells cultured with Astaxanthin for 3 days had a 60% lower amount of melanin
than control cells, and the inhibitory effect was dose-dependent.
Repeated exposure of the skin to UVA irradiation elicits sagging which is mainly
attributed to its biochemical mechanism to up-regulate the expression of matrix-
metalloproteinase (MMP)-1 and skin fibroblast elastase (SFE). A recent study in cultured
human dermal fibroblast showed that Astaxanthin attenuated the UVA-induced up-
regulation of these two enzymes (Suganuma 2010) [Figure 10]
26
Figure 10: Effects of Astaxanthin (AX) on matrix-metalloproteinase (MMP)-1 activity in

UVA-exposed human fibroblasts at 48 h post-irradiation. [Adapted from Suganuma
(2010)]
(6.2) Studies in animal models
Studies in animal models were restricted to hairless UV-irradiated mice while

Astaxanthin is applied either topically (Arakane, 2002) or orally (Savoure 1995).
Astaxanthin was shown to accumulate in rat skin when given orally (Petri 2007) or
topically (Ying-Shu 2006). The first group (Arakane 2002) followed wrinkles formation,
and showed decreased irreversible wrinkling in Astaxanthin- treated mice, as compared
with control mice. Scanning electron micrographs of the ultra structure of dermal
collagen fiber bundles indicated that their structure was maintained by the application of
Astaxanthin. This paper demonstrated the potential of post-irradiation topical treatment
with Astaxanthin to reduce skin wrinkling, one of the most important cosmetic targets.
The second group (Savoure 1995), searched for UV-induced modifications in epidermal
concentrations of free polyamines, as markers for skin photo damage. UV-irradiated mice
had a higher level of the polyamine putrescine. Astaxanthin supplementation inhibited
putrescine accumulation better than retinol, and decreased the levels of two additional
27
polyamines, spermidine and spermine. These results support the role of Astaxanthin as
skin photo-protector when given orally.
(6.3) Studies in humans
Studies in humans focused on two directions: the reddening (erythema) of the skin
after exposure to UV irradiation, and prevention and smoothening of wrinkles and dry
skin for cosmetic purposes.
Erythema is a leading cause for hyper-pigmentation and was found to be reduced
by both synthetic and natural forms of Astaxanthin in healthy subjects (Yamashita, 1995)
(Figure 11). Compared to natural Astaxanthin, synthetic Astaxanthin showed lower
suppression. Furthermore, natural Astaxanthin demonstrated faster recovery times of the
erythema index, suggesting anti-inflammatory properties that together with inhibition of
melanin formation may contribute to the reduction of hyper-pigmentation. Since
Astaxanthin does not absorb UVB light, the mechanism of erythema suppression might
be related directly with oxidation of melanin that is produced in response to UV
radiation.
Figure 11: Inhibitory effect of topically applied Astaxanthin (Ax) on Erythema in human.
Melanine index was measured 1 week following UVB irradiation. Each color
corresponds to a participant in the trial. [Adapted from Yamashita (1995)]
28
Three additional studies (Yamashita 2002, Yamashita 2007, Seki 2001) were
performed in order to evaluate the effect of Astaxanthin dietary supplementation with or
without topical application on skin elasticity and moisture. The results of the studies
showed, that supplementation with Astaxanthin improves moisture and elasticity of the
skin and thereby reduces formation of fine wrinkles (Figure 12, Table 6). The mechanism
behind the results is most likely the strong antioxidant effect of Astaxanthin in
combination with the high deposition of Astaxanthin in the skin. Astaxanthin present in
the skin quenchs free radicals generated by UV-light and other inducers and thereby
protects the collagen fibrils from increased breakdown.
Figure 12: Subject response after 6 weeks of Astaxanthin supplamantation. Adapted from
Yamashita (2007).
29
Table 6: Cosmetic benefits of Astaxanthin and vitamin E on Human skin.

Adapted from Yamashita (2002)
Table 7 summarises the benefits of Astaxanthin in skin health and its mechanisms of
action.
Table 7: Astaxanthin effects on skin health – a summary.
30
(7) Astaxanthin and modulation of the immune system
In the normal human body the activities of various types of the immune system
cells, are strictly balanced. An excess of activity of one type of cells (for example, Th1
type) may result from an autoimmune disease, or from an ongoing infection. Normally,
the cell activity diminishes when the physiological need thereof is reduced. An excess
activity is thus seen when the normal reduction of cell activity is not achieved even
though the inducer has diminished.
Immune modulation aims at restoring the balance between different subsets of T
cells, so that damaging responses are suppressed. The drugs currently used to suppress
the immune system have very broad action and inhibit protective functions of the
immune system as well as pathological responses. Opportunistic infection is therefore a
common complication of immune suppressive drugs.
Upon sensitization, lymphocytes undergo proliferation by oxyradical-based
mechanisms. Through continuous resting-stimulation cycles, lymphocytes accumulate
auto-induced oxidative lesions which lead to cell dysfunction and limit their viability and
proper function of the immune response. In light of its known anti-oxidative activity, the
effect of Astaxanthin as a booster and modulator of the immunological system was
investigated in cell cultures and in rodent models.
In cultured mouse thymocytes and spleen cells, Astaxanthin stimulated cell
proliferation and immunoglobulin production of murine spleen cells, and enhanced the
release of interleukin-1α and tumor necrosis factor-α from murine peritoneal adherent
cells (Okai 1996). In similar experiments, Astaxanthin increased the production of T-
helper cell antibody and increased the number of antibody secretory cells from primed
spleen cells (Jyonouchi 1996) (Figure 13). These authors also studied the effect of
Astaxanthin in the production of immunoglobulins in vitro by human blood cells and
found that it increased the production of IgA, IgG, and IgM in response to T-dependent
stimuli (Jyonouchi 1995). Other studies done in vivo in mice have demonstrated the
immunomodulating action of Astaxanthin and other carotenoids for humoral responses to
T-dependent antigens, and suggested that the supplementation with carotenoids may be
useful to restore immune responses (Jyonouchi 1994). Moreover, comparative studies
31
done in mice showed that Astaxanthin was consistently more active than β-carotene,
lutein and canthaxanthin (Jyonouchi 1994, Bennedsen 1999, Chew 1999)(Figure 13).
Figure 13: Effect of carotenoids on number of IgM antibodies (Ab)-secreting cells

formed in cultures of Th2 and spleen cells. [Adapted from Jyonouchi (1996)]
Due to its immunomodulating action, Astaxanthin has also been suggested as a

medication for the treatment of autoimmune diseases such as multiple sclerosis,
rheumatoid arthritis and Crohn’s disease (Lignell 2001). When tested in autoimmune-
prone mice (Tomita 1993), Astaxanthin was able to delay symptoms of proteinuria and
lymphadenopathy.
Recently, a first study in humans attempted to investigate the action of dietary
Astaxanthin in modulating immune response, oxidative status and inflammation in young
healthy adult female subjects, receiving 0-8 mg/day Astaxanthin for 8 weeks in a
randomized double-blind, placebo-controlled study (Park 2010). The results showed that
Astaxanthin stimulated mitogen-induced lymphocytes proliferation, increased natural
killer cell cytotoxic activity, and increased total T and B cell subpopulations.
32
(8) Astaxanthin and inflammation
One of the ways in which an immune response becomes a pathological condition

is inflammation. Severe or chronic inflammation, such as in Crohn’s disease and ulcer
disease, involve the action of many ROS. These toxic molecules not only induce
oxidative stress, but also stimulate the expression of inflammatory genes in endothelial
cells, which in turn aggravate the inflammation. In addition, the production of ROS by
the immune cells themselves, as part of the normal immune response, along with their
high membranal content of polyunsaturated fatty acids render them especially sensitive to
oxidative damage (Chew 2009). Therefore, molecular effectors with anti-inflammatory
properties are highly valuable. Astaxanthin exhibited such properties in several studies
reviewed below.
(8.1) Studies in cell cultures
Attempts to understand the mechanism of action of Astaxanthin in inflammation

were performed in cell lines including macrophages (Lee 2003), microglia (Choi 2008),
nerve PC12 cells (Chan 2009) and human lymphocytes (Bolin 2010). Two representative
experiments are detailed below. In the first study (Lee 2003) (Figure 14), Astaxanthin
was shown to inhibit inflammation-induced nitric oxide production and inflammatory
gene expression in both stimulated cells and primary macrophages. In addition,
Astaxanthin suppressed the serum levels of NO, prostaglandin E2, tumor necrosis factor-
α, and interleukin-1 β in lipopolysaccharide-administrated mice. The importance of the
Astaxanthin-mediated suppression of the NF-kappa-B pathways stems from the seminal
role of the latter in immunity, as it activates pro-inflammatory genes encoding for NO
synthase, tumor necrosis factor-α, and several interleukins.
33
Figure 14: Effect of Astaxanthin on ROS generation in mouse macrophage cells with and
without Inflammation induction by lipopolysaccharide (LPS). [Adapted from Lee (2003)]
Similar results were found recently by Kishimoto et. al (2010)(Figure 15), showing
that Astaxanthin has inhibitory effects on macrophage activation, such as scavenger
receptors up-regulation, MMPs activation, and pro-inflammatory cytokines secretion.
Activated macrophages are located in atherosclerotic lesions, where they are responsible
for the clearance of pathogenic lipoproteins and for promotion of the inflammatory
process which is a crucial factor in cardiovascular diseases.
Figure 15: Effect of Astaxanthin on expression levels of in lipopolysacaride-stimulated

MMP-1 in activated macrophages. [Adapted from Kishimoto (2010)]
34
In the second study (Choi 2008), Astaxanthin was tested in lipopolysacaride-

stimulated microglia cells. Microglia cells are the resident macrophages and immune
surveillance cells of the central nervous system.The activation of microglia is associated
with release of NO and subsequent release of pro-inflammatory cytokines (Gonzalez-
Scarano 1999), two processes that are implicated in the development of inflammation and
neurodegenerative disorders. Specifically, the high level of NO, produced by NO
synthase (NOS), has been defined as an indicator of cytotoxicity in inflammation and
endotoxemia (Schmidt 1994). Choi, et al showed that Astaxanthin inhibited the
production of inflammatory mediators by blocking NOS activation and by the
suppression NOS protein levels (Figure 16).
Figure 16: Modulation of LPS- induced iNOS expression by astaxanthin in BV-2

microglial cells. BV-2 cells were treated with astaxanthin for 1 h, and then treated with
LPS for 6 h. Adapted from Choi (2008).
(8.2) Studies in rodents
Kurashige et. al. (1990) showed that carrageenan-induced inflammation of a rat’s

paw was significantly inhibited by intraperitoneal administration of Astaxanthin [Figure
35
17]. More recent studies investigated the efficacy of Astaxanthin in lipopolysaccharide-

(Ohgami 2003) and endotoxin- (Suzuki 2006) induced inflammation (uveitis) of rat’s eye,
or following additional infalammtion inducers (Izumi-Nagai 2008). Rats injected with
Astaxanthin showed a significant decrease in the number of infiltrating cells in the
anterior chamber. Additionally, there was a significantly lower concentration of protein,
NO, TNF-alpha and PGE2, all markers of inflammation, in the aqueous humour.
Moreover, even early stages of EIU were suppressed by injection of Astaxanthin. These
results suggest that Astaxanthin reduces ocular inflammation in eyes with EIU by
downregulating proinflammatory factors and by inhibiting the NF-kappaB-dependent
signaling pathway.
Figure 17: Effect of Astaxanthin and Vitamine E on induced inflammation of rat paw.
Adapted from Kurashige (1990).
36
Additional insight to the cellular target of Astaxanthin in inhbition of inflammation

was found in mice injected with inflammation inducers following by treatment with
Disodium Disuccinate Astaxanthin – a water soluble derivative of Astaxanthin
(Lockwood 2006). A generalized antioxidant effect was identified at the time point of
maximal neutrophilic recruitment. Moreover, Astaxanthin specifically inhibited both 5-
lipoxgenase and cyclooxygenase (COX-2) at the time point of maximal
monocyte/macrophage recruitment and activation. This interaction of Astaxanthin with 5-
lipoxygenase and the subsequent inhibition of NF-kappaB, provide the foundation for
further evaluation of its therapeutic effects on these signaling pathways which are
important in inflammation.
Currently, only one study directly checked the anti-inflammatory effect of

Astaxanthin in humans (Park 2010). Fourteen young and healthy women received 0, 2, or
8 mg Astaxanthin daily for 8 week in a randomized double-blind, placebo-controlled
study. The results showed a decrease in DNA damage biomarker after 4 week but no
affect on lipid peroxidation. Plasma C-reactive protein (an inflammation marker)
concentration was lower in subjects given 2 mg Astaxanthin, while some markers of the
immune response were enhanced (see chapter 7 above). The authors suggested that the
lack of efficacy of Astaxanthin in certain response measured may result from the fact that
in many cases greater effect of antioxidants is seen in excess amounts of oxidative stress,
in immuno-compromised individuals, while the subjects in the current study were healthy
ones.
37
(9) Astaxanthin and gastric ulcer
Gastric ulcer may be caused by several factors. Seventy to ninety percent of gastric
ulcers are estimated to be due to chronic inflammation caused by the bacteria
Helicobacter pylori (H. pylori) that colonize the antral mucosa, while the rest of the cases
are caused by drugs (e.g. NSAIDs), smoking, diet and stress. The effects of Astaxanthin
on H. pylori- and chemically- induced gastric ulcers were determined in rodents and
humans, as described below.
(9.1) Helicobacter pylori-induced gastric ulcer
In mice infected with H. pylori which are fed an Astaxanthin-rich diet, Astaxanthin
had an anti-bacterial action manifested as reduced gastric mucous, lower load and
colonization by the bacterium and shift in the immune response (Bennedsen 1999, Wang
2000) (Figure 18). The mechanism of anti-bacterial action of Astaxanthin is not known,
but it is suspected that its antioxidant properties play an important role in the protection
of the hydrophobic lining of the mucous membrane making colonization by H. pylori
much more difficult (Wadstron 2001).
38
Figure 18: Effect of Astaxanthin on H. pylori colonization of gastric mucosa and

inflammation score of gastritis in mice treated with Astaxanthin at 3.5 weeks post
inoculation. [Adapted from Bennedsen (1990)]
The single study of H. pylori-infected humans suffering from functional dyspepsia was
however, less definitive (results published in two papers by Kupcinskas 2008 and by
Andersen 2007). In this study, the ability of Astaxanthin to ameliorate gastrointestinal
discomfort (GD), along with its effect on gastric inflammatory markers and interleukins
were tested in 44 patients with functional dyspepsia. It was found that after 4 weeks of
treatment no difference between the treatment groups was observed regarding mean GD
scores of abdominal pain, indigestion and reflux syndromes. However, reduction of
reflux syndrome was achieved in the higher (40mg) dose. There were no significant
changes in the density of H. pylori or in any of the interleukins during or after treatment.
The authors suggested that the differences between positive results obtained in mice
compared to the lack of influence in humans, may be due to different dietary conditions:
the mice had a controlled diet lacking any antioxidants, while humans had a varied diet.
Hence, it is possible that when the host has access to antioxidants in his diet, the effects
of Astaxanthin is not pronounced. As these are only preliminary results, the effect of
Astaxanthin on functional dyspepsia should be investigated in additional clinical
experiments.
(9.2) Stress- and chemically-induced gastric ulcer
The effects of Astaxanthin on chemically - induced gastric ulcers were tested in

rats following treatments with various chemicals including alcohol (Kim 2005a, Kamath
2008) (Figure 19), naproxen (Kim 2005b) and acetic acid (Yang 2009). In all the studies
positive effects of Astaxanthin were demonstrated in various measurements and markers
such as decreased or complete prevention of evolution of gastric ulcerations, lower ulcer
indexes, inhibited elevation of the lipid peroxide level in gastric mucosa and increase in
the activities of radical scavenging enzymes and antioxidants following pretreatment with
Astaxanthin.
39
Figure 19: Effect of Astaxanthin on the formation of gastric mucosal lesions after oral
administration of 80% Ethanol to rats. [Adapted from Kim (2005a)]
In a comparative study (Nishiwaka 2005), β-carotene and Astaxanthin prepared

from three different sources (alga Haematococcus, the yeast Phaffia, and synthetic
Astaxanthin), were tested for their effect on stress-induced gastric ulcer in rats. It was
found that rats given Astaxanthins or β-carotene prior to stressing were appreciably
protected against the evolution of gastric ulcerations compared to control rats. Ulcer
indexes were particularly lower in the rats fed Haematococcus-extracted Astaxanthin than
those of the other groups (Figure 20).
40
Figure 20: Effect of Astaxanthin from Hematococcus (Ax(H)), synthetic Astaxanthin

(Ax(S)) or β-carotene (β-C) on ulcer development in stressed rats. HD – High dose of
Astaxanthin (40mg/rat), ND – Normal dose of Astaxanthin (8mg/rat). [Adapted from
Nishiwaka (2005)]
41
(10) Astaxanthin and the cardiovascular system
Cardiovascular diseases (CVD) include heart diseases, stroke, blood hypertension,

hardening of the arteries, and additional diseases of the circulatory system. Oxidative
stress (increased ROS and NOS) and inflammation play an important role in a number of
aspects of CVD such as endothelial dysfunction, lipid disorders, myocardial damage and
arterial fibrillation. Nutritional antioxidants, including Astaxanthin, can decrease lipid
and protein oxidation and can modulate the inflammation reaction, hence potentially
protecting against atherosclerosis and arterial stiffening (Carpenter 2003, Ellingsen
2009). Indeed, several epidemiological studies showed an association between nutritional
antioxidant intake and reduced adverse cardiovascular disease outcomes (Wilcox 2008,
Fasset 2009 and references therein).
Astaxanthin was tested regarding its effects on various cardiovascular pathologies
(reviewed in Pashkew 2008, Fasset 2009). With this regard, it should be mentioned, that
all the ischemia-reperfusion studies were done with a synthetic water-soluble derivative
of Astaxanthin – Disodium Disuccinate Astaxanthin (DDA, Cardax) that enables
intravenous and oral administration (Lockwood 2005).
(10.1) Astaxanthin in prevention of atherosclorosis and dyslipidemia
Arteriosclerosis is a process in which plaque builds up in the artery walls,

reducing blood flow. Total blockage of the narrowed artery results in a heart attack,
stroke and peripheral circulation problems; leading to many dysfunctions including loss
of limb functions and impaired sexual potency. Susceptibility to arteriosclerosis is
determined by a combination of genetic and environmental factors, including diet,
especially a diet high in cholesterol. High blood levels of LDL (Low Density
Lipoprotein)-cholesterol, are associated with an increased risk of arteriosclerosis and the
opposite is true for HDL (High Density Lipoprotein)-cholesterol. The combination of
high LDL and triglycerides (TG) levels and low HDL level is termed dyslipidemia and is
considered a risk factor for cardiovascular problems. LDL-cholesterol aggravates
endothelial dysfunction, mainly via reduction of blood vessels flexibility and rupture of
42
plaques accumulating in the atherosclerotic lesions, thus increasing the risk for heart
attack and stroke (reviewed in Pashkew 2008). In addition, plasma, LDL is usually not
oxidized, and when it does occur, it is believed to contribute to the development of
arteriosclerosis. Therefore, a number of pre-clinical and clinical studies tested the
efficacy of Astaxanthin in reduction of dyslipidemia and LDL oxidation.
Early studies in rodents showed that Astaxanthin (but not β-caroten) led to an
increase in blood levels of HDL (Murillo, 1992). Similarly, Astaxanthin has been
reported to decrease serum TG and increase HDL-cholesterol and adiponectin in insulin
resistant rats and in obese mice fed a high fat diet (Hussein 2007, Ikeuchi 2007)(Figures
21, 22).
Figure 21: Effect of Astaxanthin (18 weeks) on HDL (A) and triglycerides (TG) in
normal Wistar rats or hypertensive, insulin-resistant rats (SHRcp). OL-olive oil, ASX –
Astaxanthin. [Adapted from Hussein (2007)]
43
Figure 22: Effect of Astaxanthin on plasma triglyceride in mice fed high-fat diet for 60 d.
Adapted from Ikeuchi (2007).
A recent randomized double-blind trial in 61 mildely hyperlipidemic subjects

receiving doses of 0, 6, 12, 18 mg/day for 12 weeks demonstrated that Astaxanthin
consumption ameliorates triglyceride and HDL-cholesterol levels in correlation with
increased adiponectin in humans (Yoshida 2010). Adiponectin has been reported to
correlate positively with HDL-cholesterol, and inversely with triglyceride (TG) and very
low density lipoprotein (VLDL)-cholesterol (Okamoto 2006, Yoshida 2005).
Atheriosclerosis is not limited to the heart, but rather influences the entire body
blood circulation. As a result, it is a risk factor for cerebral ischemic injury as well as an
important factor in the normal function of skeletal muscles. Astaxanthin was shown to
reduce cerebral infraction and to improve locomotive activity in treated vs. control
animals (Shen 2009). In addition, Astaxanthin reduced exercise-induced oxidation
markers in treated rats muscles (Aoi 2003, Aoi 2008) and exercise-induced fatigue in
both rats (Ikeuchi 2006) and humans (Nagata 2006), indicating a better blood and oxygen
supply to the muscles.
44
(10.2) Astaxanthin and prevention of lipid oxidation
The inhibitory effects of Astaxanthin on lipid oxidation were demonstrated both in

vitro and in vivo. Mason, et al (2006) showed that Astaxanthin eliminated the lipid
peroxidation caused by the drug rofecoxib in cellular membrane models. The ability of
Astaxanthin to protect from lipid oxidation was further supported in an ex-vivo analysis
of LDL particles from humans fed Astaxanthin (Iwamoto 2000) [Figure 23]. In this
study, Astaxanthin conferred resistance to copper-induced oxidation seen as increased
LDL oxidation lag times. Astaxanthin reduced LDL oxidation in contrast to other
antioxidants lacking this effect, such as vitamine E, and lutein.
Figure 23: Effect of Astaxanthin on the lag phase of conjugated dienes formation, as a
result of oxidation, in LDL. [Adapted from Iwamoto (2000)]
An in vivo study done in rabbits (Setnikar 2005), demonstrated the inhibitory effect
of Astaxanthin not only on plasma lipid peroxidation but more important on plaque
formation on the aorta wall. However, in a recent study the latter result could not be
repeated (Augusti 2009). Augusti, et a observed that high-fat atherogenic diet given to
45
rabbits increased the serum cholesterol levels and the ratio of the intima/media area in the
aortic arch. However, these changes were not prevented by Astaxanthin. Astaxanthin
only attenuated plasma lipid peroxidation and certain antioxidant enzyme activities.
(10.3) Astaxanthin and hypertension
One of the risk factors for cardiovascular disorders is high blood pressure. Blood
pressure is highly regulated and is being determined by the relaxation level of the blood
vessels (vassodilatation), with NO being one of the major regulators. In a series of
experiments (reviewed in Yanai 2008), conducted in spontaneously hypertensive rats
(SHR), which have been widely used as a model for hypertension, Astaxanthin was
shown to exert the following anti-hypertensive effects: significant reduction in blood
pressure and delayed incidence of stroke (Figure 24), significant reduction of NO end
products and the contractile responses of the aorta to α-adrenergic receptor agonist and
angiotensin II, and finally, decreased coronary artery wall thickness compared with
control, indicating the possibility that Astaxanthin ameliorates hypertension induced
vascular remodeling.
Figure 24: Effect of chronic oral administration of Astaxanthin (Asta) on the incidence of
stroke in rats after cessation of the treatment. [Adapted from Hussein (2005)]
46
(10.4) Stroke and thrombosis damages
Failure to supply blood to the heart can lead to myocardial infarction (MI), one of
the major causes of stroke. Following MI and the accompanied ischemia, an additional
damage to tissue is caused when blood supply returns to the tissue (reperfusion). The
absence of oxygen and nutrients during the ischemic period creates a condition in which
the restoration of circulation results in inflammation and oxidative damage through the
induction of oxidative stress rather than restoration of normal function. This situation is
referred to as ischemia-reperfusion damage. The water-soluble derivative of Astaxanthin,
DDA, was assessed for its efficacy in several ischemia-reperfusion studies in rats, rabbits
and dogs (reviewed in Fasset 2009, Pashkew 2008).
In the heart muscle (myocardium) of rats it has been demonstrated that prior
treatment for 4 days with intravenous DDA (Cardax), significantly reduced the
subsequent myocardial infarct size and hence improved myocardial recovery (Gross
2004)(Figure 25). There was a correlation between this effect and the dose administered.
47
Figure 25: Mean myocardial salvage as a percentage of infarct size/area at risk. Control
set at 0% salvage. Control animals received vehicle injection alone; Cardax - treated
animals received the appropriate dose once daily I.V. for 4 days prior to experimental
infarction and IS/AAR determinations on day 5.Adapted from Gross (2004).
Lauver et. al. (2005) used prior treatment for 4 days of intravenous DDA in a rabbit
myocardial ischemia reperfusion model. Again, there was a significant reduction in
myocardial infarct size and improved myocardial salvage in the DDA-treated animals. In
addition, as an assessment of inflammatory activity, complement activation was
measured. This marker was found to be attenuated in DDA treated animals suggesting a
DDA-associated reduction in tissue inflammation. High levels of free Astaxanthin and
reduced levels of lipid peroxidation were detected in myocardial tissues of treated rats
(Gross 2006). When extended to a dog model, Gross, et al (2005), using intravenous
DDA at either 2 h or 4 days, prior to coronary artery occlusion, observed a significant
reduction in myocardial infarct size in DDA treated dogs after both treatments (Figure
26). Moreover, two out of three dogs in the 4-day treatment group had 100% cardiac
protection. These findings were recently repeated in ischemic injury study in rat liver
(Gulten 2010).
48
Figure 26: Effect of Astaxanthin (Cardax) on mean infract size (IS) as percent of area at
risk (AAR) following acute infract of dogs myocardium. 2hr, 4 days – treatment with
Cardax 2 hours or 4 days prior to infract study, respectively. Adapted from Gross (2005).
DDA has been efficient in a dog model of an additional complication of

atherosclorosis, namely, rethrombosis and platelets aggregation which are thought to be
related to ROS generation (Krötz 2004). A dose-dependent reduction in the incidence of
carotid artery rethrombosis and inhibition of ex-vivo platelets aggregation and thrombus
weight were observed.
The results of the aforementioned pre-clinical experiments raise the possibility that
Astaxanthin may have a therapeutic potential in humans.
49
(11) Astaxanthin and muscle endurance
Exercise may be accompanied by muscle damage and aches as well as fatigue,

symptoms that are attributed to several factors: 1) Increased generation of free radicals as
a result of increase in mitochondrial oxygen consumption and electron transport flux,
leading to lipid peroxidation; 2) Myoglobin and coenzymes leak out into the blood from
cells and tissues damaged by exercise, and destruction of red blood cells; 3) Consumption
and depletion of energy sources such as glycogen and 4) Production and accumulation of
metabolic products such as lactic acid. Therefore, recovery from exercise requires re-
synthesis of the leaked cell and tissue components and consumed energy sources, as well
as decomposition and removal of accumulated byproducts of metabolism. In addition,
intracellular redox imbalance may affect nutrient metabolism during action in skeletal
muscle. Due to its antioxidant activity, Astaxanthin was therefore checked for its effects
on muscle performance and exercise-induced fatigue.
Studies in mice investigated the effect of Astaxanthin on muscle lipid metabolism

in exercise (Aoi 2008), on oxidative damage induced by strenuous exercise in heart and
skeletal muscles (Aoi 2003), and on exercise fatigue (Ikeuchi 2006). In the first study,
Astaxanthin improved muscle lipid metabolism in exercise. In addition, it accelerated the
decrease of body fat accumulation with exercise training, suggesting that Astaxanthin
promoted lipid metabolism rather than glucose utilization during exercise leading to
improvement of endurance and efficient reduction of adipose tissue with training [Figure
27].
50
Figure 27: Running time to exhaustion. Exercise groups performed treadmill running at
30 m/min after 4 week of treatment. [Adapted from Aoi (2008)]
In the second study (Aoi 2003), exercise-increased biochemical markers in skeletal

muscle were reduced only in the Astaxanthin group. In addition, Astaxanthin lowered
exercise-induced activity of creatine kinase and myeloperoxidase in the skeletal muscle.
In the third study (Ikeuchi 2006) [Figure 28], the Astaxanthin-supplemented group
showed a significant increase in swimming time to exhaustion as compared to the control
group. This was reflected also by improved metabolic markers such as lower blood
lactate, higher plasma non-esterfied fatty acid and glucose and decreased fat
accumulation, compared to controls. 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.
51
Figure 28: Effect of Astaxanthin on swimming exercise in mice. The mice were given
either vehicle (•), or an Astaxanthin dose of 1.2 (□), 6 (△), or 30 (○) mg/kg body weight.
The mice were made to perform swimming exercise with weights attached to their tails
corresponding to 10% of their body weight. [Adapted from Ikeuchi (2006)]
(11.2) Studies in humans [Research in humans is summarized in Table 8 below]

Table 8: Effect of Astaxanthin on muscle endurance. Summary of clinical studies
Study Men** (no. Astaxanthin Effect checked Main outcome
(P) +training) dose+time
Lignel 20 4 mg Strengh/explosiveness No effect
2001 NT 6 months and
(P) Strengh/endurance Positive effect
tests
Sawaki 16 6 mg Lactic acid accumulation Reduced lactic acid.
2002 NT* 28 days after1,200 m run
(NA)
Fry 2004 9 8 mg Histology of muscle fibers No immediate effect.
(P) RT 21 days following weight lifting, Reduced sourness
muscle sourness after 48h +
histological changes in
fibers.
Bloomer 20 4 mg Markers of skeletal No effect
2007 RT 21 days muscle injury
(P)
Nagata 19 5 mg Respiratory-circulation Positive effect
2006 NT 14 days ability and activities of
(P/B) sympathetic nervous
system
*NA-not available, P-placebo, B-blind, NT-not trained, RT- resistance trained
52
** All studies were performed in healthy men.
As evident from Table 8, research of Astaxanthin effects on muscle endurance is

limited, with small numbers of participants in each study and overall mixed results.
Further studies will have to evaluate the influence of additional factors that most
probably affect the results such as physical fitness of the subject, his diet, the type of
exercise followed etc.
(12) Astaxanthin and anti-cancer activity
Human epidemiological studies have revealed certain protective effect of vegetable

and fruit consumption for certain types of cancer (Block 1992, Giovannucci 1999, Vainio
2006). Varieties of compounds, found in these foods, have known bioactive mechanisms
and are suspected as anticancer agents. In a landmark paper, Peto,et. al (1981) suggested
an inverse correlation between dietary intake of β carotene and cancer incidence, and
recommended further larger evaluation of this effect. However, when tested in large
human intervention trials the β-carotene hypothesis proved to be disappointing. Not only
did β-carotene supplementation offer no significant protection from lung and other
cancers, it actually increased lung cancer risk among smokers in two of the trials, maybe
due to its ability to act as a pro-oxidant under certain conditions (ATBC 1994, Hennekens
1996, Omenn 1996, Gallicchio 2008). Simillar dissapointing results were obtained for
additional carotenoids for lung cancer (Gallicchio 2008).
Because it is not a significant dietary carotenoid, epidemiological data on
Astaxanthin in disease prevention is lacking. However, it has exhibited potential
anticancer properties in vitro and in animal models.
(12.1) Cell culture Studies
Sun,et al (Sun 1998) showed that mouse tumor cells grown in the presence of
Astaxanthin showed reduced DNA synthesis rate and lower overall cell number in
compared to control cultures. Similarly, Astaxanthin inhibited murine mammary tumor
53
cell proliferation by up to 40%, in a dose-dependent fashion, when included in the culture

medium (Kim 2001), and it was effective at inhibiting the invasion of rat ascites
hepatoma cells in culture (Kozuki 2000)(Figure 29).
Figure 29: Effect of Astaxanthin on the invasion of rat ascites hepatoma (AH109A) cells
in culture.Adapted from Kozuki (2000).
The growth of human cancer cell lines has also been inhibited by Astaxanthin in
vitro. Astaxanthin-rich alga Haematococcus pluvialis extract showed significant growth-
inhibitory effects in several human colon cancer cells, accompanied by up-regulation of
apoptotic enzymes and specific enzymes phosphorylation (Palozza 2009) [Figure 30]. In
addition, a 98% inhibition of androgen-induced proliferation of human prostate cancer
cells was demonstrated in the presence of Astaxanthin (Anderson 2005).
54
Figure 30: Effects of Haematococcus pluvialis (H.P.) extract on the growth of

HCT-116 cells. [Adapted from Palozza (2009)]
55
In studies with BALB/c mice, dietary Astaxanthin inhibited the growth of

transplanted tumor cells in a dose-dependent fashion (Sun 1998). In a related study,
tumor cells growth was inhibited when dietary Astaxanthin supplementation was started
at one and three weeks prior to tumor inoculation, but not when supplementation was
begun at the same time as tumor inoculation (Jyonouchi 2000)(Figures 31,32). These
results suggest that Astaxanthin may inhibit tumor development in the early stages but
not in the later stages of progression. In other studies with mice, Astaxanthin
supplementation reduced transplanted mammary tumor growth (Chew 1999) (Figure 33)
and suppressed spontaneous liver carcinogenesis (Nishimo 1999). Dietary consumption
of egg yolks containing Astaxanthin inhibited chemically-induced tumors in mice (Lee
1997, Lee 1998).
Figure 31: Tumor weight and size after inoculation of fibrosarcoma cells in mice fed
control or Astaxanthin-supplemented diet. [Adapted from Jyonouchi (2000)]
56
Figure 32: Cytotoxic T lymphocyte activity against tumor cells after inoculation in mice
fed control or Astaxanthin-supplemented diet. [Adapted from Jyonouchi (2000)]
Figure 43: Effect of feeding with carotenoids on average mammary tumor volume on day
31 to 45 post-inoculation in mice. [Adapted from Chew (1999)]
57
A series of studies on cancer chemoprevention by natural and synthetic substances

in mice and rats revealed several carotenoids, including Astaxanthin, as effective
antitumor agents (Mori 1997). Thus, Astaxanthin was found to significantly reduce both
the incidence and proliferation of chemically-induced urinary bladder cancer in mice
(Tanaka 1994). In two related studies, the incidence and proliferation of chemically-
induced cancers of the oral cavity and colon (Prabhu 2009, Nagendraprabhu1 2009) were
significantly reduced in Astaxanthin-supplemented rats, relative to control rats [Figure
34].
Figure 34: Effect of Astaxanthin (ASX) on 1,2-dimethyl hydrazine (DMH)-induced

experimental colon carcinogenesis, expressed as no. of aberrant crypt foci in rats colon.
Pre, Post – treatment with Astaxanthin 1 week before or 1 week after induction of
carcinogenesis, respectively. [Adapted from Prabhu (2009)]
Astaxanthin has shown effectiveness against the initiation of liver carcinogenesis

in aflatoxin B1- (Gardelet 1998) and in cyclophosphamide-treated rats, in which it also
decreased oxidative stress, and DNA damage (Tripathi 2010) (Figure 35). In the later
study it was suggested that Astaxanthin may serve as a chemoprotective agent against the
toxicity of the anticancer drug cyclophosphamide. Finally, Astaxanthin also reduced
metastatic nodules and lipid peroxidation in livers of rats subjected to restraint stress
(Yang 1997, Kurihara 2002).
58
Figure 35: Effect of post-treatment with Astaxanthin (Asta) on cyclophosphamide-

induced (CP 50) carcinogenic foci in rat liver. [Adapted from Tripathi (2010)]
The possible mechanisms of action of the cancer-preventive effect of Astaxanthin are

currently not known. However, in light of it activities detailed in previous chapters,
Astaxanthin is expected to act via several pathways: as an antioxidant, as an enhancer of
immune system, as a regulator of gene expression (especially of gap junction proteins)
and in detoxification of carcinogenic compounds. Since all these pathways and their
correspondence products have been shown to be involved in cancer development and
fighting, Astaxanthin may prove to be a beneficial modulator of these reactions.
59
(13) Astaxanthin and the central nervous system
Intense metabolic activity, high levels of unsaturated fats and iron, and rich
irrigation with blood vessels make the nervous system (brain, spinal cord, and peripheral
nerves) very susceptible to oxidative damage (Halliwell 1992, Fachinetti 1998).
Oxidative stress is also implied in the pathogenesis of major neurodegenerative diseases.
These include Parkinson’s disease, Alzheimer’s disease, and amyotrophic lateral sclerosis
(ALS, “Lou Gehrig’s disease”), as well as in cases of stroke, trauma, and seizures. Hence,
a number of in vitro, in vivo and clinical studies have provided evidence that dietary
supplementation with lipid-soluble antioxidants may inhibit the onset and progression of
neurological diseases (Grant 1997, de Rijk 1997, Hajieva 2006, Zhao 2009). As
previously mentioned, Astaxanthin is able to cross the Blood Brain Barrier in mammals
(Tso 1996), and is therefore a promising candidate for research in neurological diseases.
Studies of Astaxanthin’s effect on the CNS focused on ischemic brain injury and on
degenerative pathologies, including effect on memory.
(13.1) Studies in cell cultures and in rodents
Astaxanthin has a protective effect in neuronal cells exposed to oxidative damage.

This effect was expressed in increased and dose-dependant viability of dopaminergic SH-
SY5Y cells treated with chemical oxidative agents and with Astaxanthin and was
reflected also in inhibited intracellular ROS generation and mitochondrial abnormalities
(Liu 2009). Similar results were obtained in PC12 neuronal cells treated with Astaxanthin
and oxidazing agents (Chan 2009) [Figure 36].
60
Figure 36: Effect of Astaxanthin (AX) (10, 20 µM) or Canthaxanthin (CX) (10, 20 µM)
on viability of PC12 cells, treated with H2O2 to induce cell death. [Adapted from Chan
(2009)]
When tested in rodents, Astaxanthin showed an impressive protecting effect in mice

subjected to transient cerebral ischemia, which were then followed for their learning and
memory skills in Morris water maze (Hussein 2005a). Astaxanthin-fed mice presented
better learning and memory skills. Similarly, when Astaxanthin was cerebraly-injected to
rats prior to the ischemic induction, it increased locomotor activity and reduced cerebral
infarction at 2 days after the injury, compared to vehicle (Shen 2009) [Figure 37]. To
evaluate the protective mechanisms of Astaxanthin against stroke, brain tissues were
assayed for free radical damage, apoptosis, and excitoxicity. It was shown that
Astaxanthin can reduce ischemia-related injury in brain tissue through the inhibition of
oxidative stress, reduction of glutamate release, and an anti-apoptosis effect. In addition,
Astaxanthin showed a protective effect in rats chronically fed with alcohol, in which it
antagonized an alcohol-induced neural marker (Abadie-Guedes 2008).
61
Figure 37: Effects of Astaxanthin (ATX) on cortical infarction induced by MCAo and
reperfusion. A – Volume of infraction, B – Max infraction area/slice. [Adapted from Shen
(2009)]
Finally, the impact of Astaxanthin-enriched H. pluvialis powder on auxiliary

memory improvement was assessed in normal BALB/c mice, pre-supplemented with
different powder dosages for 30 days (Zhang 2007). The results indicated that H.
pluvialis powder was associated with dose-dependent memory improvement with the low
dosage of algal powder being the best. These results suggest that Astaxanthin may have
beneficial effects in improving memory in vascular dementia, ischemic injury and in
healthy brain.
Currently, only a single clinical study was conducted, which addressed the effect of
Astaxanthin on cognitive function (Satoh 2008). In this open-label study, 10 healthy male
subjects (50–69 years of age), who complained of age-related forgetfulness, were given
12 mg Astaxanthin for 12 weeks. Compared to base level, Astaxanthin improved
response time and accuracy of several tasks [Table 9], suggesting that it might improve
higher brain function including cognition, attention, memory, information processing,
and resultant behaviors in older persons.
62
Table 9: Mean response times and accuracies (±SD) on CogHealth tasks at baseline, and
after 6 and 12 weeks of Ax treatment. [Adapted from Satoh (2008)]
Tasks (Mean ± SD)

Baseline 6 weeks 12 weeks
Response time (ms)
Simple Reaction 341.68 ± 94.41 303.31 ± 33.80 281.76 ± 33.56**
Choice Reaction 504.53 ± 56.84 480.63 ± 39.87 463.63 ± 26.49**
Divided Attention 494.13 ± 135.57 419.52 ± 59.32** 412.07 ± 51.97**
Working Memory 762.94 ± 141.65 732.95 ± 174.83 654.83 ± 128.42**
Delayed Recall 1008.19 ± 153.37 975.40 ± 190.75 916.77 ± 151.04**
Accuracy (%)
Working Memory 90.46 ± 7.18 95.22 ± 5.37 96.30 ± 3.94**
Delayed Recall 70.95 ± 6.42 71.19 ± 5.98 70.71 ± 8.91
** p<0.05 vs baseline.
63
(14) Astaxanthin and eye health
A primary source for oxidative stress is incoming sunlight, especially UV light,

that can lead to photo-oxidative damage to lipids and tissues. Any tissue exposed to light
is prone to undergo this photo-oxidative damage, and hence our skin and eyes are the
most vulnerable tissues and carotenoids play an essential role in their maintenance.
Furthermore, the high concentration of polyunsaturated fatty acids in the center of the
retina renders the eye a sensitive target for lipid peroxidation. Oxidative damage to the
eye and skin by UV light has been widely documented (Pappas 1999) and thus the unique
UV protection properties of Astaxanthin is believed to be very important to eye and skin
health. In order to accumulate in the retina, Astaxanthin must first penetrate the blood-
brain barrier, as was shown to occur in mammals, as well as in birds (Maher 2000, Tso
1996).
Two of the main causes of visual impairment and blindness are Age-Related
Macular Degeneration (AMD) and Age-Related Nuclear Cataracts (Guerin 2003). Both
diseases appear to be related to light-induced oxidative processes within the eye, whereas
reduced risk for AMD and nuclear cataracts is associated with a high dietary intake of
carotenoids (Lyle 1999). The protective effects of Astaxanthin against photo-oxidative
damage were demonstrated in cell cultures, in animal models and in humans (discussed in
chapter 6 above). In addition, Astaxanthin was investigated for its effect on vision and
eye function in general, as discussed below.
(14.1) Studies in animal models
Following intraperitoneal administration of Astaxanthin to rats, an Astaxanthin

content of 0.17µg/mg was measured in the rats’ retinas, indicating that Astaxanthin can
cross the Blood-Retina Barrier (Tso 1996). Upon Astaxanthin administration, the rats
were exposed to visible light for 24 hours. While control rats lost ~35% of the thickness
of the outer nuclear layer of the retina, those treated with Astaxanthin had only a 6 %
decrease. In addition, Astaxanthin was able to prevent the depletion of rhodopsin from
64
the retinas of rats treated with similar photo-damaging conditions. The effect of
Astaxanthin on endotoxin-induced uveitis (EIU, inflammatory processes of the middle
layers of the eye) was studied in rats (Ohgami 2003). Astaxanthin administrated
intravenously had a dose-dependent anti-inflammatory effect on EIU, having a possible
mechanism of suppressing the production of nitric oxide (NO). Similar results were
obtained when the inhibitory effect of Astaxanthin on EIU was demonstrated by
measuring expression of inflammatory cytokines and chemokines in the aqueous humour
(Suzuki 2005), indicating that Astaxanthin not only protects the eyes, but also blocks the
biologic pathway leading to inflammation (Figure 38).
Figure 38: Number of inflammatory cells in aqueous humour of rats with uveitis.
[Adapted from Suzuki (2005)]
The anti-inflammatory effect of Astaxanthin plays an important role in an often seen

complication of AMD called choroidal neovascularization (CNV), leading to severe
vision loss and blindness (Izumi-Nagai 2008 and ref. therein). CNV seen in AMD
develops with oxidative stress and chronic inflammation and is associated with the influx
65
of inflammatory cells including macrophages. Pharmacologic depletion of macrophages

significantly suppresse murine CNV. Indeed, when mice were pretreated with
intraperitoneal injections of Astaxanthin and then experimental CNV was induced, the
index of CNV volume was significantly suppressed by the treatment compared with
control animals (Figure 39). Astaxanthin treatment led to significant inhibition of
macrophage infiltration into CNV and inhibited in vivo and in vitro expression of
inflammation-related molecules.
Figure 39: Suppression of CNV in mice receiving Astaxanthin. Flatmounted choroids

from vehicle- and Astaxanthin (1, 10, and 100 mg/kg body weight (BW))-treated mice.
Arrowheads indicate lectin-stained CNV tissues. [Adapted from Izumi-Nagai (2008)]
Astaxanthin was demonstrated to have positive effects in additional pathologies of

the retina using animal models. These include suppressive effect of Astaxanthin on
retinal injury induced by elevated intraocular pressure in rats (Cort 2010), protection of
mice retinal cells against oxidative stress in-vitro and in-vivo (Nakajima 2008),
interaction with selenite and attenuation of Selenite-induced cataractogenesis in rats (Liao
2009) and reduction of cataract in Atlantic salmon (Waagbo 2003).
The studies done in humans focused mainly on improving vision and function, with
two studies looking directly for anti-oxidative effects. In the first research (Hashimoto
2009), the effect of Astaxanthin consumption on superoxide scavenging activity in
aqueous humor was examined in 35 subjects, classified into non-diabetic and diabetic
groups. Superoxide scavenging activity was measured before and after Astaxanthin
66
consumption (2 weeks, 6 mg/day). Results showed that superoxide scavenging activity

was increased by Astaxanthin consumption in the DM group. Although the experimental
group was small, this study points to the possible use of Astaxanthin in the restriction of
humoral oxidative stress.
In the second study, 15 subjects with nonadvanced AMD were given daily
Astaxanthin (4 mg) along with vitamin C, vitamin E, zinc, copper, lutein and zeaxanthin
for 12 months (Parisi 2008). The investigators concluded that in nonadvanced AMD,
supplementation with carotenoids and antioxidants improves the function of the central
retina.
The effect of Astaxanthin on vision and eye function was followed in several
populations including healthy young subjects (Sawaki 2002, Nakamura 2004, Iwasaki
2006), healthy subjects who complained of asthenopia (eye fatigue) (Kenji 2005),
middle-aged and old subjects complaining of presbyopia (difficulties to focus on nearby
objects with increasing age) (Kajita 2009) and visual display terminal (VDT) workers
(Nagaki 2002, Nagaki 2006). Table 10 summarizes the results of these studies.
Table 10: Effect of Astaxanthin on vision and eye function. Summary of clinical studies.
Study (P/B) Population Dose (mg)/ Main outcome in Astaxanthin group

Time (days)
Sawaki 18 healthy 6 Deep vision and critical flicker fusion
2002 28 improved. No effects on static and kinetic
(P) visual acuity.
Nakamura 49 healthy, 0-12 Improved uncorrected far visual acuity.
2004 ≥40 years 28 Accommodation time shortened. No
(NA) change in refraction, flicker fusion
frequency, or pupillary reflex.
Iwasaki 10 healthy 6 Accommodative contraction and
2006 14 relaxation times shortened. Blurred vision
(P/B) and eye dryness decreased.
Kenji 2005 40 6 Improved accommodation power and
(B) asthenopia 28 subjective symptoms of asthenopia
Kajita 2009 22 6 Improved accommodation function and
(O/NP) 45-65years 28 some subjective symptoms related to
Presbyopia presbyopia.
Nagaki 39 DVT 5 Accommodation amplitude improved. No
2002 (P/B) 28 change in critical flicker fusion.
67
Nagaki 48 DVT 6 Accommodation improved. Marked

2006 28 reduction in "heavy head" claims and
(P) other typical fatigue symptoms.
* P-placebo, B-blind, O-open, NP-no placebo, NA-not available.
This table clearly demonstrates that Astaxanthin has several positive effects on vision and
eye function, especially those related to accommodation of the lens and related symptoms
such as “eye fatigue”, difficulties to focus on nearby objects with increasing age
(presbyopia) and asthenopia symptomes (shoulder stiffness, ocular pain and headache).
Although the underling mechanism for these effects is still unknown, an interesting
hypothesis was proposed by Miyawaki, et al (2008) who showed improved blood transit
times (rheology) following Astaxanthin ingestion. The authors suggested that
Astaxanthin, via its anti-oxidative activity, may improve erythrocyte membrane
flexibility thus enabling better blood fluidity which in turn may improve eye function.
68
(15) Astaxanthin and Male Fertility
Infertility results from the coincidence of genetic disorders, environmental factors

and phatogens of the reproductive organs. Evidence has accumulated supporting the role
of ROS in the pathogenesis of sperm dysfunction among infertile men. Spermatozoa are
highly sensitive to ROS and posses little defense against oxygen damage, which induces
changes in the sperm membranal composition and harms their DNA. Due to the high
concentration of polyunsaturated fatty acids (PUFA), spermatozoa membranes are highly
vulnerable to oxidation, thus reducing their fluidity and fusogenic capacity. The
combination of Astaxanthin’s unique membranal protection and strong antioxidative
ability makes it a good candidate for intervention in male infertility treatments.
El Garem, et al (2002) evaluated the effect of Astaxanthin supplementation on

semen quality of infertile male. Twenty sub-fertile men received either 16 mg/day
Astaxanthin or placebo during three months, in addition to the conventional treatment.
Decreased ROS activity and improved sperm motility and morphology were seen in the
supplemented group, while no change was observed in the placebo group. At the end of
the treatment period, five couples out of ten successfully conceived in the Astaxanthin-
supplemented group, compared to one out of ten couples in the placebo group. The
researchers proposed that the Astaxanthin-improved quality of spermatozoa, may explain
the increased frequency of conception.
In another study, Comhaire, et al (2005) treated 30 infertile men with Astaxanthin
(16 mg/day), or placebo for three months in addition to conventional treatment. The
effects of Astaxanthin on sperm parameters, ROS level, serum hormones and pregnancy
rate were evaluated. Astaxanthin reduced ROS and Inhibin B and increased sperm linear
velocity. Pregnancy rate among the placebo cases (10.5 %) was significantly lower
compared with the Astaxanthin group (54.5 %) [Table 11].
69
Table 11: Effect of Astaxanthin on human male infertility: Semen characteristics and
pregnancy rate at baseline and after 3 months of treatment. [Adapted from Comhaire
(2005)]
Although these studies show promising findings, the results need to be confirmed in a
larger trial before recommending Astaxanthin for the complementary treatment of
infertile men.
70
(16) Astaxanthin and the metabolic syndrome
Metabolic syndrome (MS) identifies clinical symptoms and biochemical markers,

including obesity, insulin resistance, hyperglycemia, hyperlipidemia, and hypertension,
all leading to an increased risk of cardiovascular disease (CVD). These conditions cause
metabolic dysregulation, elevated fatty acids (FFA), and increased secretion of pro-
inflammatory "adipokines". Left untreated, MS causes lipotoxicity (especially to the
liver), chronic inflammation, hypertension, atherosclerosis, type 2 diabetes (T2D) and
CVD (Roberts 2009). Increasing evidence shows that MS is characterized by increased
oxidative stress (Hopps 2010). Moreover, one of the primary cellular sites for the
formation and subsequent damage of oxidative stress is the mitochondria, which are
implicated in several aspects of MS, such as insulin resistance, obesity, inflammation,
and liver abnormalities (Kim 2008).
The exact causes for MS are not fully understood, however, it is clear that genetic
predisposition in combination with high-fat diet are major risk factors (Roche 2005). In
line with the evidence regarding the pivotal role of oxidative stress in MS, Astaxanthin,
as well as other antioxidants, were tested for their effects in animal models of T2D and
obesity and in hyperlipidemic subjects. The effects of Astaxanthin on hyperlipidemia
were discussed in chapter 10, dealing with its connection to cardiovascular diseases. The
following section will describe the effects of Astaxanthin on hyperlipidemia as part of
MS and T2D.
(16.1) Astaxanthin and onset of Type 2 Diabetes
Oxidative stress induced by hyperglycemia is probably involved in tissues

resistance to insulin which may eventually deteriorate to dysfunction of pancreatic β
cells, two factors that define Type 2 Diabetes (T2D). Research is therefore conducted at
both improving tissue sensitivity to insulin and preventing the exhaustion of pancreatic
cells that leads to their final destruction. Uchiyama, et al (2002) used diabetic db/db mice,
a well-known obese model of T2D, and showed that the higher level of blood glucose
observed in db/db mice was decreased after treatment with Astaxanthin. The ability of
71
pancreatic cells to secrete insulin was preserved in the Astaxanthin-treated group. Similar
results were achieved by Hussein, et al (2007), using the hypertensive rat (SHR) model.
These rats mimic an environmentally-induced MS however, if pre-treated with
Astaxanthin, SHR rats have significantly lower levels of MS biochemical markers
including lower fasting blood glucose, higher sensitivity to insulin, improved adiponectin
level, a significant increase in HDL-cholesterol, and a significant decrease in
triglycerides, and non-esterified fatty acids (Figure 40 a,b,c). Mean blood pressure was
also reduced in hypertensive rats following treatment with Astaxanthin (Hussein 2005)
[Figure 41].
Figure 40a: Triglycerides (TG) level in Astaxanthin (ASX) or olive-oil (OL)-treated

normal Wistar or SHRcp rats, ex vivo. [Adapted from Hussein (2007)]
72
Figure 40b: High-density lipoprotein (HDL)-cholesterol level in Astaxanthin (ASX) or

olive-oil (OL)-treated normal Wistar or SHRcp rats, ex vivo. [Adapted from Hussein
(2007)]
Figure 40c: Non esterified fatty acids (NEFA) level in Astaxanthin (ASX) or olive-oil
(OL)-treated normal Wistar or SHRcp rats, ex vivo. [Adapted from Hussein (2007)]
73
Figure 41: Effects of Oral Administration of Astaxanthin (ASX) on mean blood pressure
in SHR Each data point represents the mean S.E.M. of 8 rats per group. [Adapted from
Hussein (2005)]
Additionally, Astaxanthin showed significant effects on the white adipose tissue by

decreasing the size of the fat cells. These results suggest that Astaxanthin ameliorates
insulin resistance by mechanisms involving the increase of glucose uptake, and by
modulating the level of circulating lipid metabolites and adiponectin. Complamentry to
these results are the findings obtained in the obese rat model (Ikeuchi 2007). Obese rats
treated with Astaxanthin presented an inhibition of the increase in body weight and
weight of adipose tissue resulting from high-fat diet feeding. In addition, Astaxanthin
reduced liver weight, liver TG, plasma TG and total cholesterol. Similar results were
obtained by Kim, et al (2009) in mice fed high-fat diet. The authors suggested that
Astaxanthin may be of value in reducing obesity and MS in humans. A support to this
notion comes from a recent study in humans (Yoshida 2010), showing that administration
of Astaxanthin to mildly hypertensive subjects resulted in increase in HDL-cholesterol
and adiponectin.
Mitochondria are a major source for ROS production and one of the first to be
damaged by them. Oxidative stress leading to mitochondrial dysfunction is a critical
factor in MS and the efficacy of Astaxanthin on mitochondrial function was hence
74
investigated in Hela cell line using Astaxanthin’s concentration close to what can be
achieved using supplements and/or diet (Wolf 2010). It was found that Astaxanthin
decreased physiologically occurring oxidative stress and protected cultured cells against
strong oxidative stress induced with a respiratory inhibitor. Moreover, Astaxanthin
improved the maintenance of a high mitochondrial membrane potential and stimulated
respiration. Astaxanthin at nanomolar concentrations was effective in maintaining
mitochondria in a reduced state. Additionally, Astaxanthin improved the ability of
mitochondria to remain in a reduced state under oxidative challenge. Supportive evidence
to the protective effect of Astaxanthin on mitochondria was obtained in two additional
studies in cell cultures: Liu, et al (2009) showed that in dopaminergic SH-SY5Y cells
Astaxanthin accumulated in mitochondria (Figure 42) and inhibited cellular oxidative
toxicity via mitochondria-targeted protective mechanism (Figure 43).
Figure 42: Astaxanthin accumulation in SH-SY5Y cells. Astaxanthin content in different

fractions was determined by HPLC analysis and expressed as % of the added total
concentration. ND – Not detected. [Adapted from Liu (2009)]
75
Figure 43: Protective effect of Astaxanthin on DHA-OOH or 6-OHDA- induced damage

to mitochondria of SH-SY5Y cells. Mitochondrial damage was followed by determining
the amount of mitochondrial cytochrom C release to the cytosol. [Adapted from Liu
(2009)]
In a more diabetes-related system, Manabe et. al. (2008) showed that Astaxanthin
protects kidney mesangial cells from hyperglycemia-induced oxidative signaling through
accumulation in the mitochondria and reduction of the production of ROS-modified
proteins in them (Figure 44). Taken together, these results suggest that Astaxanthin may
improve MS via its beneficial effects on mitochondria function.
Figure 44: Astaxanthin (ASX) inhibites glucose-induced production of oxidative

Stress-modified proteins in mitochondria of normal human mesangial cells (NHMCs).
[Adapted from Manabe (2008)]
76
(16.2) Astaxanthin and T2D-induced nephropathy
As mentioned above, ROS may be involved not only in the onset of T2D, but also
in many of its complications. One of these common complications is kidney dysfunction
called diabetic nephropathy, caused by chronic damage to the capillaries in the kidney.
Astaxanthin was investigated for its effect on characteristic histological, biochemical and
genetic features of nephropathy. In a recent study (Kim YJ 2009), the protective action of
Astaxanthin against high-glucose-induced oxidative stress was examined in proximal
tubular epithelial cells (PTECs). The efficacy of Astaxanthin was assessed by measuring
several key markers and activities, including lipid peroxidation, total reactive species
(RS), superoxide (*O(2)), nitric oxide (NO*), and peroxynitrite (ONOO(-)). Results
showed that Astaxanthin effectively suppressed all the above mentioned oxidative
damage markers (Kim 2009). Similar protective effect of Astaxanthin against progression
into diabetic nephropathy was found by Nakano et. al (2008) in ODS rats. The effect of
Astaxanthin could be further enhanced in combination with vitamine E.
Finally, it was shown by Naito, et al (2004) that Astaxanthin prevented the
progression of diabetic nephropathy induced by oxidative stress in diabetic db/db mice.
Astaxanthin-treated mice showed a lower level of blood glucose and a significantly
smaller mesangial area compared to non-treated db/db group. The increases in urinary
albumin (Figure 45) and DNA oxidation marker at 12 weeks of treatment were
significantly inhibited by supplementation with Astaxanthin. The results suggested that
the antioxidative activity of Astaxanthin reduced oxidative stress on the kidneys and
prevented renal cell damage.
77
Figure 45: Effect of Astaxanthin on urinary albumin (representing a diabetic nephropathy

marker). Db/m – normal rats, db/ab – diabetic rats. [Adapted from Naito (2004)]
In an effort to reveal the mechanism underlying these effects, the profile of gene
expression patterns in glomerular cells of Astaxanthin-treated diabetic mice were
followed (Naito 2006). The analysis of the most affected genes (3.1%) showed that the
mitochondrial oxidative phosphorylation pathway was the most significantly affected
pathway. The affected genes were associated with proteins located in the mitochondrial
inner membrane, and the expression levels of these genes were decreased in mice treated
with Astaxanthin as compared to the levels in the controls. In addition, the expression of
many genes associated with oxidative stress, collagen synthesis, and transforming growth
factor-β signaling was enhanced in the diabetic mice, and this enhancement was slightly
inhibited in the Astaxanthin-treated mice. This genetic approach stresses the likelihood
that Astaxanthin affects T2D outcomes not only by counteracting ROS but also by
modulating the expression of related genes.
78
(17) Summary
As seen in this review, Astaxanthin supplementation is a practical strategy in

protecting the body from oxidative damage, which has impact in a number of health
conditions. Natural Astaxanthin ingested in the presence of fat or oilhas greater
bioavailability and is distributed systemically. Thanks to its exceptionally high
antioxidative potency, Astaxanthin can exert its effects in relatively low concentrations,
thus making it suitable to serve as dietary supplement and in functional food/beverages.
Astaxanthin is completely safe, as evident from numerous clinical, pre-clinical and acute
toxicity studies. Promising findings suggest that Astaxanthin may improve the function of
many physiologic systems both as a preventive and as a therapeutic treatment. These
beneficial effects are believed to result from a concerted action of Astaxanthin via a
number of pathways rather than acting merely as an antioxidant. Thus, accumulating
evidence from various systems suggests that Astaxanthin acts via scavenging reactive
oxide species (ROS), inhibiting lipid peroxidation in membranes and stabilizing the
structure and fluidity of the latters and modulating the expression of genes such as
structural (connexins) genes, mitochondrial and inflammatory ones. Current and future
research in these fields will undoubtedly shed more light on Astaxanthin potential in
human health.
79
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Astaxanthin and Human Health

(I) Table of content 2

(2) Astaxanthin chemistry 7

(3) Bioavailability and pharmacokinetics of Astaxanthin 9

(4) Safety of Astaxanthin 12

(5) Anti-oxidation activity of Astaxanthin 16

(6) Astaxanthin and skin health 22

(7) Astaxanthin and modulation of the immune system 30

(8) Astaxanthin and inflammation 32

(9) Astaxanthin and gastric ulcer 37

(9.1) Helicobacter pylori-induced gastric ulcer 37

(11) Astaxanthin and muscle endurance 49

(12) Astaxanthin and anti-cancer activity 53

(13) Astaxanthin and the central nervous system 59

(14) Astaxanthin and eye health 63

(15) Astaxanthin and Male Fertility 68

(16) Astaxanthin and the metabolic syndrome 70

(1.1) The Carotenoids

Figure 1: Structures of selected carotenoids.

(2) Astaxanthin chemistry

(2.1) Astaxanthin – chemical structure

Astaxanthin (3,3’-dihydroxy-β-β-carotene-4,4’-dione) is a xanthophyll carotenoid,

(2.2) Sources of Astaxanthin and their properties

Astaxanthin can be commercially obtained from four major sources (reviewed in

(3) Bioavailability and pharmacokinetics of Asthaxanthin.

Astaxanthin is not soluble in water. Its level of esterification determines Astaxanthin’s

(3.2) Pharmacokinetics - ADME: Absorption, Distribution, Metabolism and

Table 1: Pharmacokinetic paramenters of Astaxanthin after administration of a single oral

Figure 2: Astaxanthin distribution in plasma lipoproteins 6 hours after oral ingestion of a

Kistler et al. (2002) investigated the metabolism of Astaxanthin as well as its

Elimination of Astaxanthin is relatively fast with elimination half life ranging

Table 2: Findings from Astaxanthin clinical trials

DB-PC = double-blind placebo-controlled; wks = weeks

(5) Anti-oxidation activity of Astaxanthin

(5.1) Evidences from in vitro studies

Astaxantin’s superior potency as an anti-oxidant, compared to other carotenoides,

enhanced anti-oxidative activity can be attributed to its remarkable resistance to photo

Figure 3: Loss of antioxidant in unilamellar liposoms following photo irradiation.

Thus, as demonstrated in Tables 3 and 4, although the fold of anti-oxidative activity of

Table 3: Quenching of singlet oxygen potency of Astaxanthin and other carotenoids.

Table 4: Scavenging of free radicals potency of Astaxanthin and other carotenoids in

Experimental system Other carotenoids Astaxanthin Reference

Protection against lipid β-carotene comparable Nakagawa 1997 (+Fe)

(5.2) Evidences from animal models (in vivo)

(5.3) Evidences from human trials

(5.4) Preservation of membrane structure

The ability of Astaxanthin to inhibit lipid peroxidation via stabilization of the

These results suggest that the antioxidant potential of carotenoids is dependent on

Figure 5: various carotenoids (10 mol/L) were

Figure 6: Transmembrane orientation of polar carotenoids facilitates electron shuttling.

(6) Astaxanthin and skin health

Our skin is often exposed to sunlight, which contains hazardous UV light.

Figure 7: Effects of UVA, UVB and Ozone on skin.

(6.1) Evidences from cell cultures

Figure 8: Effect of different carotenoids on irradiation-induced apoptotic cell death in

Table 5: Carotenoids protection from UVA radiation in rat kidney fibroblasts.

Figure 9: Ability of Astaxanthin to protect against UVA induced damage in human

Astaxanthin was also found to inhibit the over-production of melanin in melanoma

Figure 10: Effects of Astaxanthin (AX) on matrix-metalloproteinase (MMP)-1 activity in

(6.2) Studies in animal models

Studies in animal models were restricted to hairless UV-irradiated mice while

(6.3) Studies in humans

Table 6: Cosmetic benefits of Astaxanthin and vitamin E on Human skin.