Astaxanthin (AX), a captivating red-orange pigment belonging to the carotenoid family, has garnered tremendous attention in recent years owing to its extraordinary physical properties, biochemical characteristics, and physiological effects. This remarkable compound has emerged as a promising contender in the realm of human health and well-being, prompting a surge in scientific research. In fact, the number of PubMed-indexed publications on astaxanthin has soared exponentially, skyrocketing from a mere 29 papers in 2001 to a staggering 414 papers in 2022, marking a fourteen-fold increase over the past decade. To date, more than 3500 papers on “astaxanthin” have been indexed in the PubMed database (Figure 1).
The global landscape of AX research is undergoing a notable shift, with countries worldwide, including Japan, spearheading significant advancements. Initially relegated to a modest role as a coloring agent in the aquaculture and poultry industries, AX has experienced a remarkable transformation. As the fisheries, poultry, and livestock sectors underwent structural changes, the use of AX in commercial feeds expanded exponentially. Simultaneously, novel applications in human health have propelled AX to new heights of commercial significance. Intriguingly, recent studies have begun to unveil the potential of AX in addressing health challenges stemming from societal shifts.
2. Nature and Cultural Aspects of Astaxanthin
2.1. Astaxanthin; Chemistry, History of Discovery and Structural Investigation
2.1.1. Astaxanthin; Chemical Structure and its Properties
Astaxanthin (3,3′-dihydroxy-β,β-carotene-4,4′-dione; AX) is a carotenoid with a chemical formula of C
40H
52O
4 and a molecular structure that includes two hydroxyl and two carbonyl groups. AX exhibits an orange to deep-red color due to the presence of 13 conjugated double bonds. It is important to note that in its crystalline form, astaxanthin takes on a glossy black-purple color. The molecular structure of AX is symmetrical, with two chiral carbons at the 3 and 3′ positions of both terminal β-ionone groups, giving rise to three possible optical isomers (stereoisomers): (3
S,3′
S), meso (3
R,3′
S), and (3
R,3′
R)-AX. Additionally, due to the presence of nine double bonds in the polyene moiety, there can theoretically be 512 geometric isomers. While most naturally occurring AX is in the all-trans configuration, 9-, 13-, and 15-
cis isomers have also been identified (
Figure 2).
Figure 2. Astaxanthin; structure, optical isomers and major geometric isomers.
In addition to the free form, where no hydroxyl group modifications are present, AX also occurs naturally in a form where hydroxyl groups are modified by fatty acid esters. In animals, AX may be present in a protein complex, while in bacteria, it can be found as glycosides
[4][5].
2.1.2. Astaxanthin; Discovery and History of Structural Investigation
Investigations into astaxanthin (AX) began soon after the initial discovery of carotenoids. The study of carotenoids dates back to the early nineteenth century, when carotenoids were first found and extracted from paprika (in 1817), saffron (in 1818), annatto (in 1825), carrots (in 1831), and autumn leaves (in 1837)
[5][6]. In those early years, carotenoid structures were still largely unknown, and their characterization was primarily based on their solubility and light absorption properties. AX seems to be the same as that initially called crustaceorubin by the British naturalist Henry Nottidge Moseley in 1877 and by Marian Isabel Newbigin in 1897
[7].
The early 20th century marked a major turning point in carotenoid analysis with the invention of chromatography, a revolutionary biochemical technique that became a staple in the chemistry of natural organic compounds. In 1906, Tswett successfully separated carotenes, xanthophylls, and chlorophylls from green leaves using column chromatography for the first time. Subsequently, the 1930s became known as the “golden age” of carotenoid structure elucidation. During this period, Karrer and Kuhn characterized eight carotenoids, including β-carotene, which they discovered to be a precursor of vitamin A. Their remarkable achievements earned them the Nobel Prize in Chemistry
[8]. They also elucidated the structures of lutein, zeaxanthin, and AX
[5][6].
At that time, carotenoid structural studies were conducted using elemental analysis and oxidative degradation reactions with strong oxidizers like KMnO
4. However, these techniques were not sensitive and required several grams of carotenoids in crystalline form for analysis. In the 1970s, significant improvements in analytical instrumentation, including the introduction of various spectroscopic and separation techniques such as MS,
1H-NMR,
13C-NMR, and HPLC, revolutionized the analysis of carotenoids. These advancements made it possible to analyze smaller samples more effectively. As a result, over 600 carotenoids found in nature have been structurally elucidated
[4][9].
The structure of AX was elucidated relatively early in the history of carotenoid structural determination. In 1933, Kuhn and Lederer isolated two carotenoids from the shell and eggs of the lobster species
Astacus gammarus (now known as
Homarus gammarus) and named them “astacin” (now known as astacene) and “ovoester”
[10]. In 1937, Stern and Salomon isolated a protein complex called “ovoverdin” from lobster, and in 1938, Kuhn and Sörensen further characterized “ovoverdin” and identified “ovoester” as a xanthophyll carotenoid, renaming it “astaxanthin”
[11][12]. The name “asta” is derived from “Astacus” the genus name of the lobster. Kuhn and Sörensen demonstrated that AX exhibited behavior consistent with ovoester based on its melting point (215.5–216 °C) and elemental analysis. Additionally, astacin was determined to be an oxidized artifact of AX. Based on these findings, the structure of AX was determined to be 3,3′-dihydroxy-β,β-carotene-3,3′-dione
[11]. In 1933, von Euler et al. isolated the red pigment “salmon acid” from salmon muscle
[13], and in 1973, Khare et al. showed that salmon acid was identical to AX based on MS and
1H-NMR spectral data
[14]. The search for natural sources of AX during the 1948–1950s led to its extraction from flamingo wings
[15], grasshoppers, and other insects
[16], as well as from the flower petals of the
Adonis plant
[17]. These early works on the isolation and identification of AX were published in “Nature,” one of the most prominent scientific journals, which highlights the great interest in natural pigments at that time. Subsequently, as described in
Section 2.2, AX was found to be widely distributed in microorganisms, algae, and animals. Since 1970, AX, like other carotenoids, has been characterized using various spectroscopic techniques, including MS,
1H-NMR, and
13C-NMR
[4]. Furthermore, X-ray crystallography was conducted in the 2000s
[18][19].
Figure 3 shows an Oak Ridge Thermal Ellipsoid Program (ORTEP) diagram of a single molecule obtained from a single crystal of all-trans AX.
Figure 3. Oak Ridge Thermal Ellipsoid Program (ORTEP) diagram of a single molecule and the crystal structure obtained from a crystalline sample of all-trans astaxanthin. This figure was prepared based on reference
[18].
2.1.3. The History of Astaxanthin Research in Japan
Today, AX research, including studies related to its biotechnology, is being conducted globally. However, in its early phases until the 1990s, it was predominantly carried out in Japan, where Japanese researchers made significant contributions to the field. Let us examine the history of AX research in Japan.
In Japan, AX research initially began in the field of fisheries. In the 1970s, Matsuno et al. conducted extensive research on carotenoids found in various aquatic animals, approaching the subject from the perspectives of natural product chemistry and comparative biochemistry (for detailed information, refer to other reviews
[20][21][22]). Furthermore, Hata, Katayama, et al. studied the pathway of AX production in goldfish (colored varieties of
Carassius auratus), nishikigoi (colored varieties of
Cyprinus carpio), and Japanese tiger prawn (
Marsupenaeus japonicus, formerly
Penaeus japonicus). They proposed the pathway of AX biosynthesis in these aquatic animals, considering the structures of various metabolic intermediates, using dietary carotenoids such as zeaxanthin and β-carotene
[23][24][25]. Kitahara, Hata, Hatano, Ando, et al. also contributed research on the metabolism of AX in salmon
[26][27][28][29].
In the 1980s, Matsuno, Fujita, and Miki et al. made further discoveries in AX research. They revealed the reductive metabolism of AX to tunaxanthin through their studies on the coloration of marine fish, such as sea bream and yellowtail, and the metabolism of carotenoids in their eggs
[30][31]. Additionally, Miki et al. reported that the administration of AX to aquaculture fish improved egg quality, hatching, development, and growth of fry. These findings were based on their studies of AX dynamics in fish eggs
[20].
In the 1990s, Miki et al. (1991) made a significant discovery regarding the antioxidant activity of AX. They found that AX exhibited a much stronger (more than 100 times) capability in quenching singlet oxygen than that of α-tocopherol (vitamin E) and that AX scavenged free radicals, superior to other examined carotenoids, including β-carotene and zeaxanthin, as well as α-tocopherol
[32]. Furthermore, Nishino et al. conducted research on the anti-carcinogenic effects of AX and other carotenoids
[33][34]. Prior to the 1990s, AX had primarily been studied in the field of fisheries. However, these findings sparked research on its applications in medicine and human health. Meanwhile, in 1993, the Marine Biotechnology Institute in Japan discovered an AX-producing marine bacterium, later identified as belonging to the genus
Paracoccus [35]. Subsequently, in 1995, AX biosynthesis genes, including a novel key gene for the ketolation reaction
[36], were isolated from this
Paracoccus strain, and their functions were clarified by Misawa et al.
[37], followed by the isolation of the key gene from the green alga
Haematococcus pluvialis [38]. This breakthrough led to the development of current research not only in AXs biosynthesis but also in metabolic engineering and synthetic biology for AX production. The pioneering studies conducted in the 1990s laid the foundation for AX and carotenoid research as it stands today.
Since 2000, AX has garnered significant attention in the field of preventative healthcare, particularly in relation to various lifestyle-related diseases. It has also gained recognition in the cosmetics industry for its anti-photooxidation and skin-aging effects. As a result, there have been a growing number of studies conducted worldwide on AX. In Japan, AX research and applications have been particularly prevalent in the field of ophthalmology. Considerable clinical evidence has accumulated regarding the effects of AX on eyestrain (asthenopia)
[39][40][41][42][43][44][45][46][47]. Based on these research findings, several functional food products have been launched in Japan.
2.1.4. Astaxanthin; Optical Isomers
In Section 2.1.1, it is mentioned that there are three possible optical isomers of AX. In the past, when AX was extracted from lobsters by Kuhn and Sörensen, it was considered optically inactive since it displayed minimal optical rotation
[11].
In 1975, Liaaen-Jensen et al. successfully obtained optically active AX from the green alga
Haematococcus pluvialis strain NIVA-CHL 9, now referred to as
H. lacustris strain NIVA-CHL 9. In this study,
H. lacustris will be referred to as
Haematococcus algae, unless otherwise specified
[48]. For further details, please see
Section 2Section 2.2.2.
2.2. Liaaen-Jensen et al. reduced the isolated AX from
Haematococcus algae using NaBH
4, and the resulting product exhibited a circular dichroism (CD) spectrum consistent with (3
R,3′
R)-zeaxanthin. Consequently, it was determined that AX derived from
Haematococcus algae possesses a stereoconfiguration of (3
S,3′
S)
[49][50]. Please note that the “
R” and “
S” nomenclature rules for absolute configuration were followed, where the hydroxyl group connecting the chiral carbon at the 3,3′ position to the chiral center is oriented upward (HO►), designating zeaxanthin as “
R” and AX as “
S”.
In 1976, Andrewes et al. made a significant discovery regarding AX obtained from
Phaffia yeast, specifically
Phaffia rhodozyma (currently known as
Xanthophyllomyces dendrorhous). They observed that the CD spectrum of AX from this yeast was completely opposite to that of (3
S,3′
S)-AX, indicating that the AX from
Phaffia yeast exclusively adopts the (3
R,3′
R) conformation
[51]. This finding prompted to conduct a meticulous comparison of the CD spectra of AX obtained from various marine animals. The observed differences in intensities (Δε) indicated that the AX from marine animals is a mixture of optical isomers.
In 1979, Vecchi and Müller successfully separated racemic AX into three optical isomers using high-performance liquid chromatography (HPLC) in a normal-phase system. They employed diastereomeric esters of di-(−)-camphanate for this purpose
[52]. Through this method, they were able to separate the optical isomers of AX obtained from lobster, shrimp, salmon, and starfish. The analysis revealed that AX in shrimp, salmon, and starfish comprised a mixture of the three optical isomers (3
R,3′
R), meso, and (3
S,3′
S). Furthermore, Vecchi and Müller directly separated the racemic AX into the three optical isomers using HPLC with a commercially available column called Sumichiral OA-2000. This column utilized an optically active stationary phase known as
N-3,5-dinitrobenzoyl-D-phenylglycine (refer to Supplementary Figure S2B in original paper)
[53]. By employing this method, Vecchi and Müller confirmed the existence of three stereoisomeric forms of AX in various marine animals.
2.1.5. Astaxanthin; Geometric Isomers
Most natural AX exists as a mixture of geometrical isomers, including small amounts of 9-
cis, 13-
cis, and 15-
cis forms, along with the all-trans form (
Figure 2). In 1980, Roche’s group isolated ten geometric isomers of AX by HPLC, and their UV-VIS and
1H-NMR spectral data were reported
[54]. Very recently, Yao et al. reported studies on the Raman spectra of the isomers of AX using density functional theory (DFT) calculations
[55]. They confirmed that the theoretically calculated Raman spectra accurately reproduced the experimentally recorded Raman spectra of the all-trans, 9-
cis, and 13-
cis isomers of AX. They expanded the theoretical studies to the other isomers of AX (15-
cis, 9,9′-
cis, 9,13-
cis, 9,13′-
cis, 9,15-
cis, 13,13′-
cis, and 13,15-
cis isomers) and proposed the assignment of the vibrational modes. They also discussed the stability of the isomers by comparing the theoretically predicted relative energies and estimated that the ratio of the all-
trans configuration is approximately 70%, while 9-
cis and 13-
cis isomers each account for about 10%. The other isomers make up less than 2% under thermal equilibrium conditions.
Recently, Honda et al. introduced effective methods to generate geometrical isomers of AX in a thermally dependent process
[56][57]. To date, there have been few reports on the physiological activities related to the geometric isomers of AX
[58]. From a physicochemical perspective, several properties of cis isomers, including absorption maxima, solubility in solvents, and antioxidant activity, have been shown to differ from those of the all-
trans form
[4][54][58][59][60]. Specifically, the recent reports by Honda et al. and others indicate that certain geometric isomers may have higher bioavailability in rodents and are expected to have clinical applications in the near future
[58][60][61].
2.1.6. Astaxanthin Fatty Acid Esters
In addition to the free form, where no hydroxyl group modifications are present, AX also occurs naturally in a form where hydroxyl groups are modified by fatty acid esters (details of the distributions are described in Section 2.2). AX exists in both mono- and di-ester forms, with the ester moieties commonly composed of saturated fatty acids ranging from C12 to C18. Esterified derivatives of AX with highly unsaturated fatty acids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have also been reported in marine animals. For example, AX in
Haematococcus algae occurs mainly as a series of monoesters with C16 to C18 fatty acids
[62][63][64], while in krill it occurs as diesters with highly unsaturated fatty acids such as DHA and EPA
[65][66]. Therefore, when quantification of AX is required, it is often calculated from the absorbance value based on the absorption coefficient of the free form as a tentative quantification value. For more accurate quantification, saponification should be applied, and the free AX content should be quantified by HPLC. In other words, the value obtained by converting all of the esterified AX into its free form is often used as the AX concentration. The reasons for this and the details of the analytical methods are discussed individually in Section 2.4.2. One of the most important concerns is that AX can be readily converted to “astacene” (3,3′-dihydroxy-2,3,2′,3′-tetradehydro-β,β-carotene-4,4′-dione) through oxidation in alkaline solutions in the presence of oxygen
[67]. Therefore, to accurately quantify AX, esterified AX must first undergo alkaline saponification under anoxic conditions or enzymatic treatment with cholesterol esterase from
Pseudomonas sp.
[67][68]. The enzymatic treatment is generally more convenient as the hydrolysis reaction proceeds without artifacts under normal oxygen levels.
2.1.7. Astaxanthin Aggregates
Similar to many other carotenoids, AX is believed to undergo self-aggregation in hydrated polar solvents, resulting in the formation of aggregates
[69][70]. The water concentration in the AX-solvent mixture influences the morphology of these aggregates and significantly impacts their photophysical properties. Spectroscopic analysis reports suggest that the absorption spectrum of AX aggregates is either blue-shifted (H-aggregate) or red-shifted (J-aggregate) compared to that of the monomer, reflecting the conditions during aggregation
[71][72][73].
In the J-aggregate, astaxanthin molecules are arranged from head to tail, forming a relatively relaxed aggregate. Conversely, the H-aggregate exhibits a tighter “card-pack” stacking of polyene chains, which are somewhat aligned in parallel to each other
[74]. Notably, the formation of H-aggregates is a unique characteristic of carotenoids possessing a hydroxyl group on the terminal cyclohexene ring. Introducing an O-R group in place of the hydroxyl group inhibits aggregation, thereby strongly suppressing aggregation in the ester form of AX
[75].
Aggregates often exhibit significant differences in behavior compared to non-aggregated forms of biomolecules. These changes in physical properties can have a significant impact on their biological activity, particularly their pharmacological activity. For instance, H-aggregates of carotenoids demonstrate higher photostability in aqueous solutions compared to monomers; however, their radical scavenging activity and ability to quench singlet molecular oxygen are much lower than those of the monomers
[75][76]. Incorporating biomacromolecules and amphiphilic compounds such as DNA, proteins (e.g., bovine serum albumin), and polysaccharides (e.g., arabinogalactan chitosan) can stabilize the formation of AX aggregates with biomacromolecules
[75][77][78]. Recent studies have reported successful incorporation of H- and J-aggregates into DNA/chitosan co-aggregates and the preparation of complex nanosuspensions containing these two types of aggregates
[77]. AX aggregates, which are typically unstable, can be stabilized by incorporating them into hydrophobic microdomains of these polymers, such as DNA/chitosan complexes, even in the absence of EtOH/water solvents. Highly aggregated molecular complexes have demonstrated different behavior in terms of radical scavenging activity compared to monomers in simple aqueous polar solvents
[77]. This difference may be attributed to the newly formed intermolecular hydrogen bonds with the biopolymer and the presence of a π-π conjugated structure in the intermolecular association. These factors may explain the variation in antioxidant activity between H- and J-aggregates due to their different electron transport capacities
[77]. However, there are still many unresolved aspects regarding the physiological activity of aggregates, and further investigations are required.
Recently, researchers have isolated five distinct forms of AX aggregates that allow for the adjustment of intermolecular coupling between AX molecules. Time-resolved absorption spectroscopic studies with sub-30 fs time-resolution have been conducted on these aggregates
[79]. Each form of AX aggregate is capable of undergoing intermolecular singlet fission, with rates of triplet generation and annihilation that can be linked to the strength of intermolecular coupling. This finding challenges the conventional model of singlet fission in linear molecules
[80], as it demonstrates that the triplet state of AX is directly formed from the initial
1B
u+ (S
2) photoexcited state through an ultrafast singlet fission process. This discovery highlights the potential use of AX aggregates, particularly the H-aggregate, as photoprotectors in biological systems. The H-aggregate of AX exhibits a significant hypsochromic shift in absorption, extending into the UV spectral region, compared to that of the monomer. Consequently, the H-aggregate of AX efficiently absorbs light in the UV—blue spectral range. Upon photoexcitation, the H-aggregate of AX can safely dissipate its energy as heat through the triplet excited state, which is formed via the ultrafast fission process. The significance of aggregates in natural systems is further discussed in Section 2.1.8.
2.1.8. Carotenoproteins: Astaxanthin-Protein Complexes
AX has hydroxyl groups at the C3 and C3′ positions and carbonyl groups at the C4 and C4′ positions. Therefore, it exhibits a high affinity for certain proteins, such as albumin, and can readily form pigment-protein complexes.
In many marine animals, AX is present in the form of protein-bound complexes. One of the most well-known examples of AX-protein complexes is seen in the blue, purple, and yellow hues of crustacean exoskeletons, which are predominantly derived from the AX-protein complex
[81]. For more information on carotenoid-protein interactions in aquatic organisms, refer to other reviews
[81].
The relationship between the structure of AX and color has been extensively studied, particularly in the case of “crustacyanins”, which contribute to the blue to purple coloration of lobster shells belonging to the species
Homarus gammarus and
H. americanus. Crustacyanins are members of the lipocalin superfamily of proteins, as deduced from the amino acid sequence of their subunits, which are hydrophobic ligand-binding proteins
[82][83][84][85][86]. The multimeric α-crustacyanin (with a maximum absorption wavelength of approximately 630 nm) and dimeric β-crustacyanin (with a maximum absorption wavelength of approximately 580–590 nm), isolated from lobster shells, exhibit blue to purple colors. The structure of α-crustacyanin has been investigated through CD spectra and X-ray crystallographic analysis of its substructure, β-crustacyanin. α-crustacyanin is a large macromolecule with a molecular weight of approximately 320 kDa, consisting of eight pairs of heterodimeric β-crustacyanin units, which are themselves composed of heterodimers formed by two apocrustacyanins. Apocrustacyanins comprise five subunits: A
1, C
1, and C
2, each with a molecular weight of approximately 21 kDa, and A
2 and A
3, each with a molecular weight of approximately 19 kDa. In lobsters (
Homarus gammarus ), the major subunits of β-crustacyanin are A
2 and C
1 [83]. Consequently, there are 16 molecules of free AX within α-crustacyanin, as each apocrustacyanin associates stoichiometrically with an equal amount of AX. X-ray crystallographic analysis of β-crustacyanin has revealed three characteristics resulting from the binding of AX: elongation of the chromophore due to the 6-s-trans planar structure, hydrogen bonding between the C4, C4′ keto group and water, as well as histidine residues, and the close interactions of the two chromophores (AX). Since the usual maximum absorption wavelength (λ
max) of the AX monomer is around 470 nm, both β- and α-crustacyanins exhibit a strong bathochromic shift in their absorption spectra as a result of the conformational change of the chromophore within the protein, resulting in a purple and blue color, respectively
[84]. Further bathochromic shifts of up to 45 nm can be observed due to aggregation effects during the association of β-crustacyanin with α-crustacyanin in lobster shells. In crustaceans, the combination of apoprotein subunits varies among species and mutations, contributing to the variation in the coloration of crustacyanins
[86][87].
The relationship between the structure of AX and color has been extensively studied, particularly in the case of “crustacyanins”, which contribute to the blue to purple coloration of lobster shells belonging to the species
Homarus gammarus and
H. americanus. Crustacyanins are members of the lipocalin superfamily of proteins, as deduced from the amino acid sequence of their subunits, which are hydrophobic ligand-binding proteins
[82][83][84][85][86]. The multimeric α-crustacyanin (with a maximum absorption wavelength of approximately 630 nm) and dimeric β-crustacyanin (with a maximum absorption wavelength of approximately 580–590 nm), isolated from lobster shells, exhibit blue to purple colors. The structure of α-crustacyanin has been investigated through CD spectra and X-ray crystallographic analysis of its substructure, β-crustacyanin. α-crustacyanin is a large macromolecule with a molecular weight of approximately 320 kDa, consisting of eight pairs of heterodimeric β-crustacyanin units, which are themselves composed of heterodimers formed by two apocrustacyanins. Apocrustacyanins comprise five subunits: A
1, C
1, and C
2, each with a molecular weight of approximately 21 kDa, and A
2 and A
3, each with a molecular weight of approximately 19 kDa. In lobsters (
H. gammarus ), the major subunits of β-crustacyanin are A
2 and C
1 [83]. Consequently, there are 16 molecules of free AX within α-crustacyanin, as each apocrustacyanin associates stoichiometrically with an equal amount of AX. X-ray crystallographic analysis of β-crustacyanin has revealed three characteristics resulting from the binding of AX: elongation of the chromophore due to the 6-s-trans planar structure, hydrogen bonding between the C4, C4′ keto group and water, as well as histidine residues, and the close interactions of the two chromophores (AX). Since the usual maximum absorption wavelength (λ
max) of the AX monomer is around 470 nm, both β- and α-crustacyanins exhibit a strong bathochromic shift in their absorption spectra as a result of the conformational change of the chromophore within the protein, resulting in a purple and blue color, respectively
[84]. Further bathochromic shifts of up to 45 nm can be observed due to aggregation effects during the association of β-crustacyanin with α-crustacyanin in lobster shells. In crustaceans, the combination of apoprotein subunits varies among species and mutations, contributing to the variation in the coloration of crustacyanins
[86][87].
Another carotenoprotein that forms the exoskeleton in lobsters, similar to crustacyanin, is crustochrin. Crustochrin exhibits a yellow hue with hypsochromically shifted bands, having a maximum absorption wavelength of 400–410 nm. This protein contains approximately 20 astaxanthin molecules and demonstrates typical exciton-exciton interactions through natural H-aggregates (see Section 2.1.7), with the chromophores arranged in a stack-of-cards formation
[88][89]. Interestingly, these two distinct groups of proteins (crustacyanins and cristochrins), in terms of color hue, have been found to localize differently within the lobster exoskeleton
[86][90]. This characteristic localization will be described in Section 2.2.4. Recent studies indicate that crustacyanins are restricted to Malacostraca crustaceans but are widely distributed within this group. These crustacean-specific genes are divided into two distinct clades within the lipocalin protein superfamily. The fact that the crustacyanin gene family emerged early in the evolution of Malacostraca crustaceans suggests that this protein played a significant role in the evolutionary success of this group of arthropods
[86][91]. Crustaceans, in particular, are known for their diverse species-specific shell colors and patterns, and these proteins are believed to be involved in functions such as protection through cryptic coloration, reproduction, and communication
[92][93].
Moreover, in crustaceans, AX-binding proteins are not limited to α- or β-crustacyanin alone but also exist as complexes bound to “ovoverdins” and other proteins in crustaceans. Ovoverdins, reported as the pigment responsible for the dark green color (λ
max; ca. 465–470 and 660–670 nm) of lobster ovaries and eggs
[12][94][95][96], are a complex of AX (mostly in the free form) and lipovitellin, which is a predominant glycolipoprotein found in the yolk of egg-laying organisms
[97]. Corresponding to their λ
max, AX may bind to two distinct sites: one might be a weak non-specific association, and the other is a specific stoichiometric association with lipoproteins. Additionally, there are at least two different molecular-weight proteins (ca. 700 kDa and 600 kDa)
[96]. In the latter, ovoverdin seems to form a multimer of four subunits consisting of a, b, c, and d, according to the SDS-PAGE results
[96].
Similar AX-protein complexes have also been reported to form in other organisms. For example, a blue AX-protein complex called “velellacyanin” has been isolated from the blue mantle of the blue-colored “by-the-wind-sailor” jellyfish
Velella velella [82][98]. There are two types of this protein, named V600 (λ
max; ca. 600 nm) and V620 (λ
max; ca. 620 nm), respectively, based on their maximum absorption wavelengths. The molecular weight of each is >300 kDa
[82][98]. The velellacyanins are multimeric formations of multiple subunits and form a helical structure. The quaternary structures of velellacyanin and the N-terminal peptide sequence of the subunit comprising V600 reveal similarity to apocrustacyanin C
[99][100][101]. In fact, immunocross-reactivity showed reactivity with polyclonal antibodies for not only apocrustacyanin C but also apocrustacyanin A
[102]. However, it currently remains unclear whether these velellacyanin apoproteins belong to the lipocalin superfamily, and future studies, including their origin and evolutionary position, are expected.
In echinoderms, two well-known carotenoproteins, as shown below, have been partially characterized by X-ray structural and CD spectral analyses; however, no phylogenetic or functional analysis of the proteins, including detailed genetic background, has yet been available. The common starfish
Asterias rubens has “asteriarubin”, a purple-blue carotenoprotein, also present
[103][104]. Asteriarubin (λ
max; ca. 570 nm) is approximately 43 kDa and comprises four subunits with a molecular weight of approximately 11 kDa each. The major carotenoids in this protein are AX and its acetylenic and dehydro analogues, such as 7,8-dideoxyhydroastaxanthin and 7,8,7′,8′-tetradehydroastaxanthin. These carotenoids are metabolites of AX found in echinoderms and are described in Section 2.2.4. Interestingly, the amino acid sequence shows no homology to the apocrustacyanin subunits
[81][103]. Reconstitution studies revealed similarities between the binding requirements of asteriarubin and crustacyanin; however, the tetrameric asteriarubin contains only one carotenoid molecule, and the CD spectrum shows no exciton splitting. Therefore, in this case, molecular aggregation and carotenoid-protein chromophore interactions were considered unlikely to be determinants of the bathochromic shift
[81]. This slight bathochromic shift may be attributed to the absence of exciton effects compatible with extended π conjugation due to hydrogen bonding between the terminal polar group and the protein and the co-planarity of the ring
[104]. Another type of carotenoprotein in echinoderms, the vivid blue skin of calcified starfish called “blue star”,
Linckia laevigata, has a blue carotenoprotein called “linckiacyanin” (λ
max; 395, 612 nm), with (3
S,3′
S)-AX as the major carotenoid
[105]. Although the molecular weight of linckiacyanin is quite large (>103 kDa), the main glycoprotein subunit is small, at only approximately 6 kDa. However, the minimum molecular weight of the native subunits (approximately 16 kDa) means that there are at least 200 carotenoid molecules per molecule of linkyacyanin
[105]. Since linkyacyanin showed no cross-reactivity with polyclonal antibodies for the β-crustacyanin subunit
[102], it is possible that linkyacyanin is a distinct family member from the lipocalin superfamily.
In Asia, it is easy to find vivid pink egg clumps on the surface of rice plants and on the walls of aqueducts for rice fields. This pigment is known as “ovorubin”. Ovorubin is a carotenoprotein found in the perivitelline fluid that surrounds the embryo of the fertilized egg, which is an accessory gland of the female reproductive tract of the South American freshwater snail
Pomacea canaliculata (Gastropoda: Ampullariidae). Ovorubin is described as a large red AX-binding glycoprotein of approximately 330 kDa
[106], and the binding of (3
S,3′
S)-AX and their fatty acid esters to ovorubin results in a small bathochromic shift (20–30 nm) to λ
max 510 nm
[81]. The protein is also an oligomer composed of three subunits of approximately 28, 32, and 35 kDa and is a very high-density glycosylated lipo-carotenoprotein (VHDL) with phospholipids, sterols, and carotenoids as ligands. It is highly glycosylated. This protein provides the egg with resistance against sun radiation and oxidation of lipids
[107] and is thought to play an important physiological role in the storage, transport, and protection of carotenoids during snail embryogenesis
[108]. In addition to ovorubin, there is another carotenoprotein called “alloporin” (λ
max; 545 nm) found in the soft coral
Allopora californica. Alloporin is approximately 68 kDa and comprises four subunits with a molecular weight of approximately 17 kDa each. It has an equal molar of (3
S,3′
S)-AX bound to it
[109][110]. This seems to be similar to asteriarubin.
The authors eagerly anticipate a future where the mysteries behind the physical-chemical properties and mechanisms of the chromophores found within these remarkable AX-containing carotenoproteins are unraveled. Aiming to shed light on the intricate details and functions of these chromophores, their characterization holds the key to unlocking a deeper understanding of the captivating structures and extraordinary roles played by these carotenoproteins. The journey to uncover their secrets promises to be a captivating exploration into the realms of science and discovery.
All photosynthetic plants utilize carotenoid-binding proteins as an important component of their photosynthetic function. For example, in chloroplast thylakoid membranes, pigment molecules such as carotenoids and chlorophyll function within the light-harvesting protein complex (LHC), an antenna pigment-protein complex bound to the photosystem, to achieve extremely high-efficiency light harvesting. The photosystem II supercomplex (PSII) is also a pigment-protein complex that catalyzes water splitting and oxygen-evolving reactions in photosynthesis, converting light energy into chemical energy. The PSII core complex is composed of more than 20 subunits and contains approximately 35 chlorophylls (Chl) and 12 β-carotene molecules, as well as other oxidation-reduction cofactors required for electron transfer
[111][112]. PSII is especially sensitive to light-induced damage (photodamage) among the components of photosynthesis because it is an extremely oxidative reagent with a high enough potential for the light-excited P680 Chl molecule, which is utilized in the MnCaO
5 cluster to split water. If the rate of photodamage exceeds the rate of repair under excessively intense light conditions, a phenomenon called photoinhibition of PSII occurs. Photoinhibition of PSII is caused by reactive oxygen species (ROS) that are generated during the excitation energy transfer and electron transport processes. In particular, the repair system of photodamaged PSII is sensitive to various ROS. To reduce the effects of photoinhibition of PSII, plants have developed systems to quench or suppress the production of ROS. Non-photochemical quenching (NPQ) is one of these defense mechanisms in plants. One of the most important mechanisms is the involvement of carotenoids, which directly quench singlet oxygen generated by excited chlorophyll, or xanthophylls, which enhance heat dissipation of excess light energy through the xanthophyll cycle. Thus, it is clear that carotenoids play a pivotal role in oxygen-evolving photosynthesis. These carotenoids are present in membrane proteins in the thylakoid membrane or directly in the thylakoid membrane
[111][112], while some coexist with water-soluble proteins. One of those groups is the orange carotenoid proteins, which are widely distributed in cyanobacteria
[113]. However, there is no direct evidence that AX in the specific protein complex is involved in naïve plants’ photosynthetic function; however, certain green plants have AX-binding proteins as a photoprotector. For instance, the microalgae
Coelastrella astaxanthina Ki-4 (
Scenedesmaceae sp. Ki-4), isolated from the asphalt surface in mid-summer, benefits from a water-soluble AX-binding carotenoprotein called “AstaP”
[114]. AstaP (AstaP-orange1) is a secreted protein that exhibits thermally stable
1O
2 quenching activity
[114], which is induced through exposure to strong light. The deduced N-terminal amino acid sequence of AstaP reveals that it represents a new class of carotenoid-binding proteins homologous to the fasciclin family proteins, with extensively N-glycosylated regions
[114]. A related green alga,
Scenedesmus sp. Oki-4N, has three comparable AstaP orthologs. However, AstaP-pinks has no glycosyl residues, while AstaP-orange2 has a glycosylphosphatidylinositol (GPI) anchor motif and a higher isoelectric point (pI = 3.6–4.7), which is significantly different from the original AstaP-orange1 (pI = 10.5)
[115]. Orthologues of AstaP have also been found in diverse green algae, including
Chlamydomonas reinhardtii and
Chlorella variabilis, which are also induced by light irradiation
[116]. These results are unique examples of how the use of water-soluble AX in photosynthetic organisms is a novel strategy to protect cells from severe photooxidative stress. The native form of AstaP (AstaP-orange1) from
C. astaxanthina Ki-4 binds to AX quite specifically, whereas the expression of a correctly folded recombinant protein in
E. coli and the evaluation of its binding to the protein revealed that it is also capable of binding to other xanthophylls and carotenes
[117]. Recombinant AstaP is a ~20 kb water-soluble protein that accepts carotenoids in acetone solution or embedded in biological membranes. It then has the property of forming carotenoid-protein complexes with apparently equal stoichiometry
[117].
Recently, recombinant plants have been developed that possess ketocarotenoids. These ketocarotenoids are typically lacking or completely absent from the photosynthetic system found in higher plants. However, it appears that they have been successfully integrated into the photosynthetic system. The high accumulation of AX and other ketocarotenoids has been found to impact growth, CO
2 assimilation, and photosynthetic electron transfer in transgenic plants. Moreover, studies have demonstrated that ketocarotenoids act specifically on the thylakoid membrane and, more specifically, on PSII
[118][119].
Interestingly, transplastomic lettuce, which has been genetically modified in its chloroplast genome, has been found to predominantly accumulate AX
[120]. Despite having low levels of naturally occurring photosynthetic carotenoids
[111], this lettuce exhibits normal growth similar to that of non-modified plants. Initially, the quantum yield of PSII in this lettuce is low under normal growth conditions; however, it becomes comparable to control leaves under higher light intensities. In AX-accumulating lettuce, in addition to β-carotene, echinenone and canthaxanthin are bound to the PSII monomer, while the normal binding of photosynthetic carotenoids is absent. This lack of normal carotenoid binding affects the assembly, photophysical properties, and function of PSII. However, the repair mechanisms in AX-accumulating lettuce enable the maintenance of PSII function despite photodamage. The high antioxidant capacity of AX, its esters, and other ketocarotenoids accumulated in thylakoid membranes is believed to provide protection against reactive oxygen species (ROS) generated during oxygenic photosynthesis
[111].
When carotenoids and xanthophylls are highly accumulated, they can influence the physical properties of the lipid membrane itself, such as fluidity and permeability to small molecules
[121]. In AX-accumulating lettuce, the high accumulation of xanthophylls may prevent the permeation of singlet oxygen produced by PSII and enable efficient quenching within the lipid bilayer. Specifically, AX and carbonyl derivatives of lutein have been observed to adhere to the surface of the PSII core complex, indicating their effective quenching of singlet oxygen (
1O
2). Essentially, the apparent photosynthetic capacity of this lettuce may be attributed to the antioxidant effect of AX and its derivatives, compensating for the absence of essential naturally occurring carotenoids
[111].
The Silkworm,
Bombyx mori larvae, possesses a carotenoid-binding protein (BmCBP) in their silk glands. This protein has an apparent molecular weight of 33 kDa and binds carotenoids in a 1:1 molar ratio. Lutein constitutes ninety percent of the bound carotenoids, although it also binds to other carotenoids
[122]. In its cytoplasmic form, BmCBP acts as a non-internalized lipophorin receptor, binding to lutein and functioning as a “transport carrier”
[123]. The deduced amino acid sequence of BmCBP indicates its affiliation with the steroidogenesis acute regulatory protein (StAR) family, featuring a distinctive structural element known as the StAR-related lipid transfer domain. This domain facilitates lipid translocation and recognition
[122]. BmCBP demonstrates the ability to bind various carotenoids, including dietary AX, and transport them to the silkworm cocoon
[124]. In recent years, significant advancements have been made, including the successful heterologous expression of BmCBP in
E. coli while maintaining its functions
[125]. Furthermore, the crystal structure of BmCBP has been determined through X-ray structural analysis, and the binding site for xanthophylls has been identified
[126].
In recent years, carotenoid-binding proteins, including AstaP from the carotenoid binding protein (CBP) group, have emerged as promising antioxidant nanocarriers with potential applications in biomedicine
[127]. The binding ability of AstaP to carotenoids also indicates its potential industrial utility in the recovery and concentration of carotenoids from crude extracts
[125].
AX-protein complexes have also been observed in fish. In salmon, for instance, muscle AX exhibits specific binding to the surface of actomyosin, a protein present in skeletal muscles. Interestingly, the binding between this protein and AX does not appear to be stereoselective, meaning it does not show preference based on the spatial arrangement of AX molecules
[128][129].
In general, animals are unable to synthesize carotenoids de novo, with a few exceptions, as mentioned in Section 2.2.4. Therefore, recent research has highlighted the involvement of transport proteins in the absorption of carotenoids from the intestinal tract and their transfer to various tissues. These transport proteins likely share functions with other carotenoids and lipids, such as cholesterol. However, the precise binding modes between these transport proteins and AX remain unclear, although it is presumed that the binding is relatively loose. It is still uncertain whether the binding occurs in a stoichiometric manner or not. Additionally, enzymatic degradation of carotenoids takes place in various tissues. This degradation involves reactions catalyzed by carotenoid cleavage oxygenases, resulting in the formation of apocarotenoids. A well-known example is the conversion of β-carotene into vitamin A retinoids by the enzyme BCO1. The detailed roles of these processes are discussed in other sections, specifically in Section 2.3.4 and Section 4.1 of original paper for mammals.
2.1.9. Astaxanthin as a Powerful Antioxidant
AX is believed to exert its antioxidant activity through both direct quenching and scavenging of reactive chemical species, such as reactive oxygen species (ROS) and reactive nitrogen species (RNS). Additionally, it employs indirect mechanisms by inducing a group of antioxidant enzymes in biological systems. However, this research specifically focuses on the ROS-scavenging mechanism of AX.
Quenching Singlet Oxygen
Singlet molecular oxygen (
1O
2) is generated from the ground-state triplet molecular oxygen (
3O
2:
3∑
g−) through photochemical processes in biological systems
[130][131][132][133][134][135][136][137][138][139][140]. Approximately
1O
2 exists in two singlet states with different spins (
1∑
g+ and
1∆
g), with
1∆
g being the lowest excited singlet state. The former has a very short lifetime, while the latter, although short-lived, has a longer lifetime than the former. Therefore, the term “singlet oxygen” generally refers to the
1∆
g state. The lifetime of singlet oxygen is also strongly influenced by the surrounding environment. Additionally, there is a very short-lived dimol molecule (O
2(
1∆
g)-O
2(
1∆
g)) that forms as a result of the reaction between two singlet oxygen molecules. This dimol molecule can be detected through luminescence in the red spectral region, which corresponds to twice the energy of O
2(
1∆
g) emission in the infrared spectral region around 1270 nm.
Under normal environmental conditions, the electric dipole transition of oxygen molecules from the ground triplet state (
3Σ
g−) to the lowest electronically excited singlet state (
1Δ
g) has an extremely low transition probability. This transition is forbidden due to considerations of spin angular momentum, orbital angular momentum, and parity. As a result, singlet oxygen is typically generated through interaction with photosensitizers such as porphyrins and chlorophylls. Additionally, it is believed that singlet oxygen can be generated in the absence of light through the Haber-Weiss reaction involving superoxide (O
2•–) and hydrogen peroxide (H
2O
2). Furthermore, an autocatalytic reaction involving the cyclization of peroxyl radicals can also produce singlet oxygen via a tetraoxide intermediate. Therefore, the Russell mechanisms facilitate the generation of singlet oxygen from lipid peroxyl radicals. Enzymatic reactions, such as those catalyzed by myeloperoxidase in monocytes, can also lead to the production of singlet oxygen
[130][137][138][140].
The production of singlet oxygen is indeed harmful to biological tissues. This is due to its ability to readily oxidize and modify lipids, proteins, and nucleic acids, which are vital for biological functions, thereby causing their loss of function through Diels-Alder reactions. Consequently, singlet oxygen has been implicated in several diseases. For instance, it has been strongly suggested that singlet oxygen is involved in light-exposed skin and ocular tissues, contributing to conditions such as skin aging, skin cancer, Porphyria, Smith-Lemli-Opitz syndrome, glaucoma, cataracts, and age-related macular degeneration. Moreover, even in the absence of light exposure, singlet oxygen’s involvement is strongly suspected in the onset or exacerbation of diabetes mellitus and bronchial asthma
[141].
Carotenoids, such as lycopene, β-carotene, lutein, and AX, are highly efficient quenchers of singlet molecular oxygen (
1O
2). Their reaction rate constants, typically around 10
10 M
−1 s
−1, approach the limit of diffusion control in solvents
[76][142]. The quenching mechanism of
1O
2 by carotenoids has been recently analyzed through quantum dynamics calculations and ab initio calculations
[143]. Theoretical studies suggest that the ground-state singlet carotenoid (
1Car) and
1O
2 molecules can form a weakly bound complex, facilitated by the donation of electron density from the carotenoid’s highest occupied molecular orbital (HOMO) to the π
g* orbitals of
1O
2. The quenching of
1O
2 is governed by a Dexter-type superexchange mechanism involving charge transfer states (Car
•+/O
2•−). Quantum dynamics calculations demonstrate that the quenching of
1O
2 by carotenoid/O
2 complexes occurs rapidly, within sub-picosecond timescales, due to strong electronic coupling. This theoretical study highlights the crucial role of carotenoid cation radical species (Car
•+) in achieving efficient
1O
2 quenching. Notably, AX is known for its high activity and stability against
1O
2 quenching
[142].
Experimental evidence supports the theoretical understanding that as the polyene chain length of carotenoids increases, i.e., the conjugated π-electron system becomes more extended, the HOMO level of carotenoids decreases, and their
1O
2 quenching activity becomes stronger. For instance, Conn and Edge et al. conducted an evaluation of the singlet oxygen scavenging activity of various β-carotene and lycopene analogs with different lengths of conjugated π-electron systems. They extended the conjugated double bonds from 7,7′-dihydro-β-carotene (
n = 7) to dodecapreno-β-carotene (
n = 19), and the experimental results demonstrated that the
1O
2 quenching activity increased as the length of the conjugated double bond was increased (refer to
Table 1)
[144][145].
AX (
n = 13) possesses two expanded π-electron systems due to the presence of C4, C4′ diketo groups connected to the
n = 11 conjugated polyene of C40 carotenoids. Additionally, AX has polar hydroxyl groups at both ends (refer to
Figure 2). These molecular frameworks might specifically contribute to the expression of AXs superior antioxidant activity while maintaining its molecular stability.
Comparisons of the activity of cis-isomers, such as β-carotene, with the all-
trans geometrical isomer have been reported. Specifically, in terms of
1O
2 scavenging activity, the all-
trans configuration is known to exhibit the highest activity, followed by 15-
cis and 9-
cis isomers. This indicates that the rate constant for deactivating
1O
2 by the
cis-isomers decreases as the
cis-bond of β-carotene moves away from the center of the molecule, resulting in less efficient
1O
2 quenching compared to the all-
trans isomers
[144]. Time-resolved resonance Raman studies have shown that all β-carotene isomers share a common triplet state, characterized by a twist in the central carbon-carbon double bond compared to the ground state. This structural variation may have an impact on the conjugated system
[146][147].
In biological model membranes, such as phospholipid liposomes, several studies have investigated the effectiveness of AX in inhibiting
1O
2-induced peroxidation of phospholipid membranes. However, the observed activity of AX in these studies sometimes falls below expectations, such as exhibiting a lower reaction rate constant compared to β-carotene, which can vary depending on the detection system
[148]. Nevertheless, when evaluated based on endoproducts as outcomes, AX has been found to exert effective protective actions against phospholipid membrane peroxidation and cellular damage induced by photosensitization in the presence of photosensitizers
[149][150][151][152][153].
The reaction between AX and singlet oxygen is primarily attributed to physical quenching, as described previously. However, a small fraction of AX does form reactive products with
1O
2. According to Nishino et al., the major products generated from this reaction are endoperoxides, specifically astaxanthin 5,6-endoperoxide or astaxanthin 5,8-endoperoxide. These endoperoxides represent
1O
2 adducts formed at the C=C bonds of the β-end group
[154].
Table 1. Singlet oxygen quenching activity of astaxanthin: comparison with common antioxidants [k(109M−1s−1)].