Critical Reviews in Food Science and Nutrition, 46:185–196 (2006)
`Copyright C(cid:1)(cid:1) Taylor and Francis Group, LLC
`ISSN: 1040-8398
`DOI: 10.1080/10408690590957188
`
`Astaxanthin: A Review of its
`Chemistry and Applications
`
`I. HIGUERA-CIAPARA, L. F ´ELIX-VALENZUELA, and F. M. GOYCOOLEA
`Centro de Investigaci´on en Alimentaci´on y Desarrollo, A.C., P.O. Box 1735. Hermosillo, Sonora. M´exico. 83000
`
`Astaxanthin is a carotenoid widely used in salmonid and crustacean aquaculture to provide the pink color characteristic
`of that species. This application has been well documented for over two decades and is currently the major market driver
`for the pigment. Additionally, astaxanthin also plays a key role as an intermediary in reproductive processes. Synthetic
`astaxanthin dominates the world market but recent interest in natural sources of the pigment has increased substantially.
`Common sources of natural astaxanthin are the green algae Haematococcus pluvialis, the red yeast, Phaffia rhodozyma,
`as well as crustacean byproducts. Astaxanthin possesses an unusual antioxidant activity which has caused a surge in the
`nutraceutical market for the encapsulated product. Also, health benefits such as cardiovascular disease prevention, immune
`system boosting, bioactivity against Helycobacter pylori, and cataract prevention, have been associated with astaxanthin
`consumption. Research on the health benefits of astaxanthin is very recent and has mostly been performed in vitro or at the
`pre-clinical level with humans. This paper reviews the current available evidence regarding astaxanthin chemistry and its
`potential beneficial effects in humans.
`
`Keywords
`
`astaxanthin, health benefits, carotenoids
`
`INTRODUCTION
`
`of natural sources of AX (algae, yeast, and crustacean byprod-
`ucts) as an alternative to the synthetic pigment which currently
`covers most of the world markets. This review paper aims to
`provide an updated overview of the most important chemical,
`biological and application aspects of this unusual carotenoid un-
`derlining its relevance to the growing industry of nutraceutical
`products.
`
`CHEMICAL STRUCTURE OF CAROTENOIDS
`
`RIMFROST EXHIBIT 1100 page 0001
`
`Astaxanthin (AX) is a pigment that belongs to the family
`of the xanthophylls, the oxygenated derivatives of carotenoids
`whose synthesis in plants derives from lycopene. AX is one
`of the main pigments included in crustacean, salmonids, and
`other farmed fish feeds. Its main role is to provide the desir-
`able reddish-orange color in these organisms as they do not
`have access to natural sources of carotenoids. The use of AX
`in the aquaculture industry is important from the standpoint
`of pigmentation and consumer appeal but also as an essential
`nutritional component for adequate growth and reproduction.
`In addition to its effect on color, one of the most important
`properties of AX is its antioxidant properties which has been
`reported to surpass those of β-carotene or even α-tocopherol
`(Miki, 1991). Due to its outstanding antioxidant activity AX
`has been attributed with extraordinary potential for protecting
`the organism against a wide range of ailments such as cardio-
`vascular problems, different types of cancer and some diseases
`of the immunological system. This has stirred great interest in
`AX and prompted numerous research studies concerning its po-
`tential benefits to humans and animals. Much work has also
`been focused on the identification, production, and utilization
`
`Address correspondence to I. Higuera-Ciapara, Centro de Investigaci´on en
`Alimentaci´on y Desarrollo. -A.C. Carretera a la Victorial Km 0.6. AP 1735
`Hermosillo, Sonora 83000 Mexico. E-mail: higuera@cascabel.ciad.mx
`
`Carotenoids comprise a family encompassing more than
`600 pigments which are synthesized de novo in higher plants,
`mosses, algae, bacteria, and fungi (Goodwin, 1980). The struc-
`ture of carotenoids is derived from lycopene (Figure 1). The
`majority are hydrocarbons of 40 carbon atoms which contain
`two terminal ring systems joined by a chain of conjugated dou-
`ble bonds or poliene system (Urich, 1994). Two groups have
`been singled out as the most important: the carotenes which
`are composed of only carbon and hydrogen; and the xantho-
`phylls which are oxygenated derivatives. In the latter, oxygen
`can be present as OH groups (as in zeaxanthin), or as oxi-groups
`(as in canthaxanthin); or in a combination of both (as in AX).
`(Figure 1).
`The poliene system gives carotenoids its distinctive molecu-
`lar structure, their chemical properties and their light-absortion
`185
`
`

`

`186
`
`I. HIGUERA-CIAPARA ET AL.
`
`Figure 1 Chemical structure of some carotenoids. Source: Urich, 1994.
`
`characteristics. Each double bond from the poliene chain may
`exist in two configurations; as geometric isomers cis or trans.
`Cis-isomers are thermodynamically less stable than the trans
`isomers. Most carotenoids found in nature are predominantly
`all trans isomers (Britton, 1995). In addition to forming ge-
`ometric isomers, and considering that each molecule has two
`(cid:2)
`chiral centers in C-3 and C-3
`, AX may present three configu-
`(cid:2)
`(cid:2)
`rational isomers: two enantiomers (3R, 3
`R and 3S, 3
`S) and a
`(cid:2)
`S) (Turujman et al., 1997) (Figure 2). From
`meso form (3R, 3
`
`(cid:2)
`
`S is the most abundant in nature
`all these isomers, the 3S, 3
`(Parajo et al., 1996). Synthetic AX consists of a racemic mix-
`ture of the two enantiomers and the meso form (Turujman et al.,
`1997). Three types of optical isomers can be found in crustacea
`(Cort´es, 1993).
`Depending on their origin, AX can be found in associa-
`tion with other compounds. It may be sterified in one or both
`hydroxyl groups with different fatty acids such as palmitic,
`oleic, estearic, or linoleic: it may also be found free, that is,
`
`RIMFROST EXHIBIT 1100 page 0002
`
`

`

`THE CHEMISTRY OF ASTAXANTHIN
`
`187
`
`Figure 2 Astaxanthin configurational isomers (a–c) and a geometric cis isomer (d). Source: Turujman et al., 1997; Osterlie et al., 1999.
`
`with the hydroxyl groups without sterification; or else, form-
`ing a chemical complex with proteins (carotenoproteins) or
`lipoproteins (carotenolipoproteins). Synthetic AX is not steri-
`fied, while found in algae is always sterified (Johnson and An,
`1991; Yuan et al., 1997). Crustacean AX on the other hand,
`is a mixture of the three forms previously described (Arango,
`1996).
`
`SOURCES OF AX
`
`Synthetic AX
`
`Synthetic AX is an identical molecule to that produced in
`living organisms and it consists of a mixture 1:2:1 of isomers
`(cid:2)
`(cid:2)
`(3S, 3S
`), (3R, 3S
`), and (3R, 3R) respectively. It is the main
`
`RIMFROST EXHIBIT 1100 page 0003
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`

`

`188
`
`I. HIGUERA-CIAPARA ET AL.
`
`carotenoid used worldwide in the aquaculture industry. Since
`1990, Roche began a large scale production of synthetic AX and
`practically fulfilled the world market for the pigment, estimated
`at 150–200 million dollars. However, the growing demand for
`natural foods and the high cost of synthetic pigments has stim-
`ulated the search for natural sources of AX with potential for
`industrialization.
`Only a few sources of microbial origin can compete econom-
`ically with synthetic AX: the green microalgae Haematococcus
`pluvialis and the red yeast Phaffia rhodozyma. Their manufac-
`turing methods have been reviewed by Johnson and An (1991),
`Nelis and De Leenheer (1991), and Parajo et al. (1996). Several
`small companies have been founded (Igene, Aquasearch, and
`Cyanotech) and are trying to compete with Roche by offering
`AX from natural sources. However, so far, these products only
`take up a very small fraction of the market due to their limited
`production (McCoy, 1999).
`
`Microalgae
`
`Numerous research reports exist concerning the study of mi-
`croalgae, particularly Haematococcus pluvialis with the aim of
`optimizing the AX production processes. The main focus of
`these efforts has been the assessment of various factors and con-
`ditions which affect algae growth and the production of AX
`(Kakizono et al., 1992; Kobayashi et al., 1992, 1993; Harker
`et al., 1995, 1996; Fabregas et al., 1998, 2000; Gong and Chen
`1998; Boussiba et al., 1999; Zhang et al., 1999; Hata et al., 2001;
`Orosa et al., 2001; and Choi et al., 2002). The recent advances
`in photobioreactor technology has been a fundamental tool to
`achieve commercial feasibility in the production of AX from
`microalgae (Olaizola, 2000) as it has allowed the development
`of culture methods with AX concentration varying from 1.5 to
`3% on a dry weight basis (Lorenz and Cysewsky, 2000). The
`production system consists of microalgae cultivation in large
`ponds under controlled conditions, followed by processing to
`break down the cell wall to increase the bioavailability of the
`carotenoid (Cyanotech, 2000) since the intact spores present low
`digestibility (Sommer et al., 1991). The biomass is finally dried
`to obtain a fine powder of reddish color. Several AX products
`currently marketed are derived from H. pluvialis microalgae and
`are being manufactured with the method previously described.
`These products may contain between 1.5 and 2.0% of AX and
`are utilized as pigments and nutrient for aquatic animals and also
`in the poultry industry for the pigmentation of broilers and egg
`yolk (Cyanotech, 2000).
`On the other hand, other algal species have been proposed
`as sources of AX but so far without much success as com-
`pared to the species previously described. Gouveia et al. (1996,
`2002) shown that Chlorella vulgaris is efficient for pigmenta-
`tion purposes with the same magnitude of synthetic pigments.
`More recently, a group of researchers has shown interest in the
`identification, extraction, and purification of carotenoids from
`the microalgae Chlorococcum sp (Li and Chen, 2001; Ma and
`
`Chen, 2001; Zhang and Lee, 2001; Yuan et al., 2002). Chloro-
`coccum seems to be a promising source of AX as well as other
`carotenoids such as canthaxanthin and adonixanthin.
`The interest shown by the aquaculture industry for natural
`sources of AX has been growing as a result of the increasing de-
`mand for fish fed with natural pigments (Guerin and Hosokawa,
`2001). In general, the microbial sources of carotenoids are com-
`parable to synthetic sources as far as pigmentation is concerned
`(Choubert and Heinrich, 1993; Gouveia et al., 1996, 2002;
`Bowen et al., 2002; Gomes et al., 2002). However, it is worth
`noting that some authors suggest that sterified AX sourced from
`algae could be twice as effective as synthetic AX for the pig-
`mentation of red seabream (Guerin and Hosokawa, 2001) in
`addition to providing a better growth rate in Penaeus monodon
`larvae (Darachai et al., 1999).
`
`Yeast
`
`For more than two decades, the red yeast Phaffia rhodozyma
`has been widely studied due to its capacity in producing AX. The
`scientific literature is very abundant in reports on this microor-
`ganism. Many of these reports have been focused on the effect of
`different nutrients or carbon sources in the culture media on the
`production of yeast biomass and AX (Kesava et al., 1998; Parajo
`et al., 1998a; Chan and Ho, 1999; Ramirez et al., 2000; An, 2001;
`Flores-Cotera and Sanchez, 2001). Other authors have been most
`interested in optimizing the conditions which favor larger AX
`yields (Parajo et al., 1998b; Vazquez and Martin, 1998; Ramirez
`et al., 2001) or in assays testing salmonid pigmentation with diets
`containing Phaffia, with a similar efficiency to that achieved us-
`ing synthetic AX (Gentles and Haard, 1991; Whyte and Sherry,
`2001). Other researchers have concentrated on the utilization of
`genetically-improved strains of the same yeast to increase AX
`yields (An et al., 1989; Adrio et al., 1993; Calo et al., 1995; Fang
`and Chiou, 1996; An, 1997). Currently the yeast is marketed in
`a fine powder form as a natural source of AX, protein, and other
`nutrients and utilized as an ingredient in salmonid feed. It is
`manufactured by natural fermentation in a carefully controlled
`environment thus effectively obtaining a product with a high
`percentage of free AX (8,000 µg/g) (Igene, 2003).
`
`Crustacean Byproducts
`
`Crustacean byproducts are generated during processing op-
`erations of recovering or conditioning of the edible portion
`of crabs, shrimp, and lobster. Generally, these byproducts are
`made up of mineral salts (15–35%), proteins (25–50%), chitin
`(25–35%), lipids, and pigments (Lee and Peniston, 1982). The
`carotenoid pigments contained therein have been thoroughly
`studied and quantified (Kelley and Harmon, 1972; Meyers and
`Bligh, 1981; Mandeville, 1991; Shahidi and Synowiecki, 1991;
`Olsen and Jacobsen, 1995; Gonzalez-Gallegos et al., 1997).
`The carotenoid content in shrimp and crab byproducts varies
`
`RIMFROST EXHIBIT 1100 page 0004
`
`

`

`THE CHEMISTRY OF ASTAXANTHIN
`
`189
`
`Table 1 Carotenoid contents in various sources of crustaceon biowastes
`
`Total
`astaxanthin
`(mg/100g)
`
`Astaxanthin (%)
`
`Free Monoester
`
`Diester
`
`14.77
`
`3.95
`
`19.72
`
`74.29
`
`8
`
`22.5
`
`5.6
`
`18.5
`
`69.5
`
`75.9
`
`Source
`
`Shrimp
`(P. borealis)
`Shrimp
`(P. borealis)
`Shrimp
`(P. borealis)
`Crawfish
`(P. clarkii)
`Backs snow crab
`(Ch. Opilio)
`
`amg/100g wet basis.
`
`4.97a
`
`3.09a
`
`15.3
`
`11.96
`
`Others
`carotenoids
`
`zeaxanthin
`
`—
`
`—
`
`Reference
`
`Shahidi and
`Synowiecki, 1991
`Torrisen et al., 1981
`
`Guillou et al., 1995
`
`Meyers and Bligh,
`1981
`Shahidi and
`Synowiecki, 1991
`
`40.3
`
`49.4
`
`astacene
`
`21.16
`
`5.11
`
`56.57
`
`lutein,
`zeaxanthin, astacene
`
`between 119 and 148 µg/g. AX is mainly found free or steri-
`fied with fatty acids. These byproducts may also contain small
`quantities of lutein, zeaxanthin and astacene (Shahidi and Botta,
`1994) Table 1.
`The potential utilization of shrimp, krill, crab, and langostilla
`byproducts to induce pigmentation of cultured fish has been
`tested (Coral et al., 1997). Byproducts generally contain less
`than 1000 µg/g of AX. This would imply the incorporation of
`large quantities of byproducts as feed ingredients (10–25%) in
`order to attain an efficient pigmentation process. A means of pro-
`cessing is through the transformation of this biomass into meal.
`However, the drying methods which depend on heat application
`are not suitable because of the high susceptibility of carotenoids
`to oxidative degradation under such thermal processing condi-
`tions (Olsen and Jacobsen, 1995). An additional disadvantage is
`the high ash and chitin content which significantly decrease the
`digestibility by fish and severely limit the rate of byproduct ad-
`dition to the formulations (Guillou et al., 1995; Gouveia et al.,
`1996; Lorenz, 1998b). In order to avoid this problem various
`alternative methods have been suggested so as to process crus-
`tacean byproducts. One such methods is silage, which consists
`of treating byproducts with organic or inorganic acids in order
`to protect them from bacterial decomposition and ease pigment
`recovery (Torrisen et al., 1981; Chen and Meyers, 1983; Gillou
`et al., 1995). During this treatment, calcium salts are partially
`dissolved at the low pH (4–5) due to acid addition; this results
`in AX increase in the solid fraction and a higher digestibility
`(Torrisen et al., 1981). Alternatively, the pigments have also
`been extracted with the use of vegetable or fish oils (Chen and
`Meyers, 1982a, 1982b; Meyers and Chen, 1985; Omara-Alwala
`et al., 1985; Coral et al., 1997) which can be incorporated di-
`rectly as feed ingredients. Similarly, the concurrent recovery of
`proteins and pigments in a stable complex form (carotenopro-
`tein) has also been demonstrated to be feasible and to provide
`an excellent source of pigments and aminoacids (Simpson and
`Haard, 1985; Manu-Tawiah and Haard, 1987; Simpson et al.,
`1992). The carotenoprotein complexes from crustacea provide
`a bluish-brown coloring. When these compounds are denatured
`by heat, AX is exposed and develops the typical reddish-orange
`color expected by consumers.
`
`AX IN AQUACULTURE
`
`Salmonid and crustacean coloring is perceived as a key qual-
`ity attribute by consumers. The reddish-orange color charac-
`teristic of such organisms originate in the carotenoids obtained
`from their feeds which are deposited in their skin, muscle, ex-
`oskeleton, and gonads either in their original chemical form
`or in a modified state depending on the species (Meyers and
`Chen, 1982). The predominant carotenoid in most crustacea and
`salmonids is AX (Yamada et al., 1990; Shahidi and Synowiecki,
`1991; Gentles and Haard, 1991). For instance, from the total
`carotenoids in crustacean exoskeleton, AX comprises 84–99%,
`while in the internal organs it represents 70–96% (Tanaka et al.,
`1976). In the aquatic environment, the microalgae biosynthesize
`AX which are consumed by zooplankton, insects, or crustacea,
`and later it is ingested by fish, thereby getting the natural col-
`oration (Lorenz, 1998a). Farmed fish and crustacea do not have
`access to natural sources of AX, hence the total AX intake must
`be derived from their feed.
`The use of AX and/or canthaxantin (Figure 1) as pigment-
`ing agents in aquaculture species has been well documented
`through many scientific publications for more than two decades
`(Meyers and Chen, 1982; Torrisen, 1989; Yamada et al., 1990;
`No and Storebakken, 1991; Putnam, 1991; Storebakken and No,
`1992; Smith et al., 1992; Choubert and Heinrich, 1993; Coral
`et al., 1998; Lorenz, 1998a; Gouveia et al., 2002; Bowen et al.,
`2002). Currently, the synthetic form of both pigments repre-
`sents the most important source for fish and crustacean farming
`operations. AX is available under the commercial brand name
`Carophyll PinkTM and canthaxanthin as Carophyll Red.TM Both
`of these trademarks are owned by Hoffman-LaRoche. In spite
`of the fact that canthaxanthin provides a fairly good pigmen-
`tation, AX is widely preferred over it due to the higher color
`intensity attained with similar concentrations (Storebakken and
`No, 1992). Additionally, AX is deposited in muscles more effi-
`ciently probably due to a better absorption in the digestive tract
`(Torrisen, 1989). It has also been reported that when a combina-
`tion of both carotenoids is used, a better pigmentation is obtained
`than when using either pigment separately (Torrisen, 1989; Bell
`et al., 1998). However, in a more recent study of Buttle et al.
`
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`

`190
`
`I. HIGUERA-CIAPARA ET AL.
`
`(2001) found that the absortion of these two pigments is species
`dependent. These authors found that canthaxantin is more read-
`ily deposited in the Atlantic salmon muscle (Salmo salar). Some
`researchers have geared their interest in studying the role of the
`optical and symmetry isomerism of AX on the absorption and
`distribution of these on the various tissues of salmonids. These
`studies have shown that the apparent coefficient of digestibil-
`ity of the geometric cis isomers is lower than that of all trans
`ones, therefore they are not utilized to the same extent for muscle
`pigmentation. Moreover, cis isomers tend to preferentially accu-
`mulate in the liver, while trans ones do so on muscle and plasma
`(Bjerkeng et al., 1997; Bjerkeng, 2000). Also, studies undertaken
`on rainbow trout have shown that the distribution of R/S optical
`isomers found in faeces, blood, liver, and muscle resembled that
`of the overall content of the supplied diet (Osterlie et al., 1999).
`In spite of the fact that AX is widely used with the sole purpose
`of attaining a given pigmentation, it has many other important
`functions in fish related mainly to reproduction: acceleration of
`sexual maturity, increasing fertilization and egg survival, and
`a better embryo development (Putnam, 1991). It has also been
`demonstrated that AX improves liver function, it increases the
`defense potential against oxidative stress (Nakano et al., 1995)
`and has a significant influence on biodefense mechanisms (Amar
`et al., 2001). Similarly, several other physiological and nutri-
`tional studies have been performed in crustaceans, mainly on
`shrimp, which have suggested that AX increases tolerance to
`stress, improves the immune response, acts as an intracellular
`protectant, and has a substantial effect on larvae growth and
`survival (Gabaudan, 1996; Darachai et al., 1999). Chien et al.,
`(2003) proposed that AX is a “semi-essential” nutrient for tiger
`shrimp (Penaeus monodon) because the presence of this com-
`pound can be critical to the animal when it is physiologically
`stressed due to environmental changes.
`According to the above information, the use of AX in the
`aquaculture industry is important not only from the standpoint
`of pigmentation to increase consumer acceptance but also as
`a necessary nutrient for adequate growth and reproduction of
`commercially valuable species.
`
`AX AS AN ANTIOXIDANT
`
`Normal aerobic metabolism in organisms generates oxidative
`molecules, that is, free radicals (molecules with unpaired elec-
`trons) such as hydroxyls and peroxides, as well as reactive oxy-
`gen species (singlets) which are needed to sustain life processes.
`However, excess quantities of such compounds are dangerous
`due to their very high reactivity because they may react with var-
`ious cellular components such as proteins, lipids, carbohydrates,
`and DNA (Di Mascio et al., 1991). This situation may cause ox-
`idative damage through a chain reaction with devastating effects
`causing protein and lipid oxidation and DNA damage in vivo.
`This constant free radical attack against an organism is known
`as oxidative stress (Maher, 2000). Such damage has been associ-
`ated with different diseases such as macular degeneration due to
`
`the aging process, retinopathy, carcinogenesis, arteriosclerosis,
`and Alzheimer disease, among other ailments (Maher, 2000). In
`order to control and reduce oxidation, the human body generates
`its own enzymatic antioxidants such as super oxide dismutase,
`catalase, and peroxidase, as well as other molecules with antiox-
`idant activity. However, in many cases, these compounds are not
`enough to provide suitable protection against oxidative stress.
`Many studies have shown that oxidation can also be inhibited
`by consuming proper quantities of antioxidants like vitamin E
`(Burton et al., 1982).
`An antioxidant is a molecule which has the ability to remove
`free radicals from a system either by reacting with them to pro-
`duce other innocuous compounds or disrupting the oxidation
`reactions (Britton, 1995). Water soluble dietary antioxidants in-
`clude vitamin C, and lipophilic antioxidants include vitamin E
`(α-tocopherol) and carotenoids such as β-carotene and AX. β-
`carotene has been thoroughly studied, but lately AX has drawn
`more and more attention due to its multiple functions and its
`great antioxidant potential.
`The potential effects of carotenoids on human health have
`been associated with their antioxidant properties. Persons who
`ingest a higher concentration of carotenoids have a lower risk of
`chronic diseases such as cardiovascular diseases, cataract de-
`velopment, macular degeneration, and some types of cancer
`(Ziegler, 1991; Mayne, 1996). Numerous studies have shown the
`antioxidant activity of antioxidants by quenching active oxygen
`species and free radicals in vitro and in vivo through well known
`mechanism (Burton and Ingold, 1984; Terao, 1989; Lee and
`Min, 1990; Di Mascio et al., 1991; Miki, 1991; Tsuchiya et al.,
`1992; Palozza and Krinsky, 1992; Kobayashi and Sakamoto,
`1999; Rengel et al., 2000). However, antioxidants can also act as
`prooxidants, that is, substances that can induce oxidative stress.
`Recent reviews on the subject have summarized the available
`data and experimental evidence on the antioxidant/prooxidant
`activity of carotenoids in different lipid systems (Palozza, 1998;
`Haila, 1999; Young and Lowe, 2001).
`Even when current knowledge of the mechanism by virtue
`of which carotenoids act as prooxidants is still controversial, a
`general mechanism has been described in which at high oxygen
`partial pressure, a carotenoid radical could react with oxygen
`to generate a carotenoid-peroxyl radical. This is an autoxida-
`tion process and such radical could act as a pro-oxidant by
`promoting oxidation of unsaturated lipids (Haila, 1999). Ma-
`jor factors involved in carotenoids prooxidant activity include
`oxygen partial pressure, carotenoid concentration, as well as
`the interaction with other antioxidant species, as reviewed by
`Palozza (1998). Thus, it has been demonstrated that the choice
`of experimental conditions in in vitro studies can greatly affect
`the antioxidant/prooxidant activity of these compounds (Haila,
`1999).
`Information is not available on antioxidant/prooxidant mech-
`anisms of carotenoids with structures different from β-carotene.
`As far as astaxanthin is concerned, only information accounting
`for its antioxidant activity is available. It has been reported that
`it has a antioxidant activity, as high as 10 times more than other
`
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`

`THE CHEMISTRY OF ASTAXANTHIN
`
`191
`
`carotenoids such as zeaxanthin, lutein, canthaxantin, and β-
`carotene; and 100 times more that α-tocopherol. Thus, AX has
`been dubbed a “super vitamin E” (Miki, 1991). This property has
`caused great interest and a growing number of publications have
`appeared on the subject. Naguib (2000) measured the antioxi-
`dant activity of various carotenoids using a novel fluorometric
`assay procedure. These authors found that AX has a higher
`antioxidant activity than lutein, licopene, α and β-carotene, and
`α-tocopherol. In order to explain such high activity they propose
`that, depending on the solvent type, astaxanthin exists in an
`equilibrium, with the enol form of the ketone, thus the resulting
`dihydroxy conjugated polyene system possesses a hydrogen
`atom capable of breaking the free radical reaction in a similar
`way to that of α-tocopherol. Goto et al. (2001) reported that AX
`is twice more effective than β-carotene to inhibit the production
`of peroxides induced by ADP and Fe2+
`in liposomes. Similarly,
`other studies have shown the superior antioxidant activity of
`AX in relation to other carotenoids (Terao, 1989; Lee and Min,
`1990; Miki, 1991). The natural functions of carotenoids are
`determined by their physicochemical properties which depend
`on their molecular structure. Carotenoids react rapidly with free
`radicals and their reactivity depends on the length of the poliene
`system and the terminal rings (Lee and Min, 1990; Britton, 1995;
`Miller et al., 1996; Goto et al. 2001). Other authors have reported
`different findings. For instance, Mortensen et al. (1997) have
`proposed that the mechanism and rate of free radical scavenging
`is dependent on the nature of the free radicals rather than on the
`structure of the carotenoids. Thus, caution must be exercised
`when studying and comparing the antioxidant activity since
`results will be dependent on the experimental conditions set
`forth.
`
`BENEFITS OF AX AS A HUMAN DIETARY
`SUPPLEMENT
`
`Manufacturers of natural AX have long tried to penetrate the
`aquaculture market niche with very little or no success at all. In
`recent years, their attention has shifted towards another growing
`industry: the nutraceuticals market (McCoy, 1999). Currently
`there is a wide variety of AX products sold in health food stores
`in the form of nutritional supplements. Most of these products
`are manufactured from algae or yeast extracts. Due to their high
`antioxidant properties these supplements have been attributed
`with potential properties against many diseases. Thus, research
`on the actual benefits of AX as a dietary supplement is very
`recent and basically has thus far has been limited to in vitro
`assays or pre-clinical trials.
`
`Anticancer Activity
`
`Activity of carotenoids against cancer has been the focus of
`much attention due to the association between low levels of
`these compounds in the body and cancer prevalence. Several
`
`research groups have studied the effect of AX supplementa-
`tion on various cancer types showing that oral administration
`of AX inhibits carcinogenesis in mice urinary bladder (Tanaka
`et al., 1994), in the oral cavity (Tanaka et al. 1995a) and rat colon
`(Tanaka et al., 1995b). This effect has been partially attributed to
`suppression of cell proliferation. Furthermore, Jyonouchi et al.,
`(2000) found that when mice were inoculated with fibrosarcoma
`cells, the dietary administration of AX suppresses tumor growth
`and stimulates the immune response against the antigen which
`expresses the tumor. AX activity against breast cancer has also
`been studied in female mice. Chew et al. (1999) fed mice with
`a diet containing 0, 0.1% and 0.4% AX, β-carotene or can-
`thaxanthin during three weeks before inoculating the mammary
`fat pad with tumor cells. Tumor growth inhibition by AX was
`shown to be dependent on the dose and more effective than the
`other two carotenoids tested. It has also been suggested that
`AX attenuates the liver metastasis induced by stress in mice
`thus promoting the immune response though the inhibition of
`lipid peroxidation (Kurihara et al., 2002). Kang et al. (2001)
`also reported that AX protects the rat liver from damage in-
`duced by CCl4 through the inhibition of lipid peroxidation and
`the stimulation of the cell antioxidant system. Additionally, the
`effects of AX and other carotenoids on proliferation of human
`breast cancerous cells have also been studied. This study showed
`that β-carotene and lycopene are more effective than AX in in-
`hibiting the proliferation of MCF-7 cell line in vitro (Li et al.,
`2002).
`
`Prevention of Cardiovascular Diseases
`
`The risk of developing arteriosclerosis in humans correlates
`positively with the cholesterol content bound to Low Density
`Lipoprotein (LDL) or “bad cholesterol” (Golstein and Brown,
`1977). Many studies have documented that high levels of LDL
`are related to prevalence of cardiovascular diseases such as
`angina pectoris, myocardial infarction, and brain thrombosis
`(Maher, 2000). Inhibition of oxidation of LDL has been pos-
`tulated as a likely mechanism through which antioxidants could
`prevent the development of arteriosclerosis. Several studies have
`looked at carotenoids, mainly β-carotene and canthaxanthin, as
`inhibitors of LDL oxidation (Carpenter et al., 1997). However
`such studies have produced conflicting results as some authors
`have suggested otherwise (Gaziano et al. 1995). With respect
`to AX, there has been very little research focused toward their
`ability to prevent coronary disease. Iwamoto et al. (2000) per-
`formed in vivo and ex vivo studies and their results suggest that
`AX inhibits the oxidation of LDL which presumably contributes
`to arteriosclerosis prevention. Miki et al. (1998) proposed the
`manufacture of a drink containing AX whose antioxidant action
`on LDL would be useful for the prevention of arteriosclerosis,
`ischemic heart disease or ischemic encephalopathy. While it is
`feasible that oxidation of LDL may be decreased by antioxidant
`consumption, more research is needed to establish the true effect
`on coronary heart disease (Jialal and Fuller, 1995).
`
`RIMFROST EXHIBIT 1100 page 0007
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`192
`
`I. HIGUERA-CIAPARA ET AL.
`
`AX Effect Against Helicobacter Pylori Infections
`
`H. pylori is considered an important factor inducing acute
`gastritis, peptic ulcers, and stomach cancer in humans. The an-
`tibacterial action of AX has been shown in mice infected with
`this bacterium. When mice are fed with an AX rich diet, the
`gastric mucous inflammation is reduced as well as the load and
`colonization by the bacterium (Bennedsen et al., 1999; Wang
`et al., 2000). Thus, the development of products for therapeu-
`tic and prophylactic treatment of the mucous membrane of the
`gastrointestinal system caused by H. pylori has been proposed
`(Wadstron and Alejung, 2001). The mechanism of AX action to
`produce this effect is not known but it is suspected that its antiox-
`idant properties play an important role in the protection of the
`hydrophobic lining of the mucous membrane making coloniza-
`tion by H. pylori much more difficult (Wadstron and Alejung,
`2001). The use of AX could represent a new and attractive strat-
`egy for the treatment of H. pylori infections.
`
`AX as a Booster and Modulator of the Immunological System
`
`The group led by Jyonouchi et al. has performed the large
`majority of investigations regarding the potential activity of AX
`as a booster and modulator of the immunological system. AX
`increases the production of T-helper cell antibody and increases
`the number of antibody secretory cells from primed spleen cells
`(Jyonouchi et al., 1996). These authors also studied the effect
`of AX in the production of immunoglobulins in vitro by human
`blood cells and found that it increases the production of IgA, IgG,
`and IgM in response to T-dependent stimuli (Jyonouchi et al.,
`1995). Other studies performed in vivo using mice have shown
`the immunomodulating action of AX and other carotenoids for
`humoral responses to T-dependent antigens, and suggested that
`the supplementation with carotenoids may