Professional Documents
Culture Documents
Literature Survey
June 2010
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Table of content
(1) Introduction 4 4
(1.1) The carotenoids 5
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(17) Summary 78
(18) Bibliography 79
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(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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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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* 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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(3.1) Bioavailability
[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.
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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(3.2.3) Metabolism
(3.2.4) Elimination
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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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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.
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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.
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.
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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.
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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).
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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).
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polyamines, spermidine and spermine. These results support the role of Astaxanthin as
skin photo-protector when given orally.
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)]
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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).
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Table 7 summarises the benefits of Astaxanthin in skin health and its mechanisms of
action.
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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
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done in mice showed that Astaxanthin was consistently more active than β-carotene,
lutein and canthaxanthin (Jyonouchi 1994, Bennedsen 1999, Chew 1999)(Figure 13).
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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.
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Figure 17: Effect of Astaxanthin and Vitamine E on induced inflammation of rat paw.
Adapted from Kurashige (1990).
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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.
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).
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Figure 19: Effect of Astaxanthin on the formation of gastric mucosal lesions after oral
administration of 80% Ethanol to rats. [Adapted from Kim (2005a)]
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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)]
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Figure 22: Effect of Astaxanthin on plasma triglyceride in mice fed high-fat diet for 60 d.
Adapted from Ikeuchi (2007).
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.
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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
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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.
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)]
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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.
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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).
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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).
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Figure 27: Running time to exhaustion. Exercise groups performed treadmill running at
30 m/min after 4 week of treatment. [Adapted from Aoi (2008)]
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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)]
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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
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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).
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Figure 31: Tumor weight and size after inoculation of fibrosarcoma cells in mice fed
control or Astaxanthin-supplemented diet. [Adapted from Jyonouchi (2000)]
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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)]
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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.
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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)]
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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)]
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.
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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)]
** p<0.05 vs baseline.
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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.
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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)]
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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
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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.
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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.
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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].
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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)]
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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)]
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
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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).
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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.
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.
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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.
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(17) Summary
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