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Mar. Drugs 2023, 21, 514. https://doi.org/10.3390/md21100514 www.mdpi.com/journal/marinedrugs
Review
Astaxanthin: Past, Present, and Future
Yasuhiro Nishida 1,*, Pernilla Christina Berg 2, Behnaz Shakersain 2, Karen Hecht 3, Akiko Takikawa 4,
Ruohan Tao 5, Yumeka Kakuta 5, Chiasa Uragami 5, Hideki Hashimoto 5, Norihiko Misawa 6 and Takashi Maoka 7,*
1 Fuji Chemical Industries, Co., Ltd., 55 Yokohoonji, Kamiich-machi, Nakaniikawa-gun,
Toyama 930-0405, Japan
2 AstaReal AB, Signum, Forumvägen 14, Level 16, 131 53 Nacka, Sweden; pernilla.berg@astareal.se (P.C.B.);
behnaz.shakersain@astareal.se (B.S.)
3 AstaReal, Inc., 3 Terri Lane, Unit 12, Burlington, NJ 08016, USA; khecht@astarealusa.com
4 First Department of Internal Medicine, Faculty of Medicine, University of Toyama, 2630 Sugitani,
Toyama 930-0194, Japan; takikawa@med.u-toyama.ac.jp
5 Graduate School of Science and Technology, Kwansei Gakuin University, 1 Gakuen-Uegahara,
Sanda 669-1330, Japan; taoruohan@kwansei.ac.jp (R.T.); yumeka-kakuta@kwansei.ac.jp (Y.K.);
chiasa.uragami@kwansei.ac.jp (C.U.); hideki-hassy@kwansei.ac.jp (H.H.)
6 Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, Suematsu,
Nonoichi-shi 921-8836, Japan; n-misawa@ishikawa-pu.ac.jp
7 Research Institute for Production Development, 15 Shimogamo-morimoto-cho, Sakyo-ku,
Kyoto 606-0805, Japan
* Correspondence: octopacy1978@gmail.com (Y.N.); maoka@mbox.kyoto-inet.or.jp (T.M.)
Abstract: Astaxanthin (AX), a lipid-soluble pigment belonging to the xanthophyll carotenoids fam-
ily, has recently garnered signicant aention due to its unique physical properties, biochemical
aributes, and physiological eects. Originally recognized primarily for its role in imparting the
characteristic red-pink color to various organisms, AX is currently experiencing a surge in interest
and research. The growing body of literature in this eld predominantly focuses on AXs distinctive
bioactivities and properties. However, the potential of algae-derived AX as a solution to various
global environmental and societal challenges that threaten life on our planet has not received exten-
sive aention. Furthermore, the historical context and the role of AX in nature, as well as its signif-
icance in diverse cultures and traditional health practices, have not been comprehensively explored
in previous works. This review article embarks on a comprehensive journey through the history
leading up to the present, oering insights into the discovery of AX, its chemical and physical at-
tributes, distribution in organisms, and biosynthesis. Additionally, it delves into the intricate realm
of health benets, biofunctional characteristics, and the current market status of AX. By encompass-
ing these multifaceted aspects, this review aims to provide readers with a more profound under-
standing and a robust foundation for future scientic endeavors directed at addressing societal
needs for sustainable nutritional and medicinal solutions. An updated summary of AXs health ben-
ets, its present market status, and potential future applications are also included for a well-
rounded perspective.
Keywords: astaxanthin; microalgae; mitochondria; SDGs; anti-aging; slow-aging; commercial
production
1. Introduction
Astaxanthin (AX), a captivating red-orange pigment belonging to the carotenoid
family, has garnered tremendous aention in recent years owing to its extraordinary
physical properties, biochemical characteristics, and physiological eects. This remarka-
ble compound has emerged as a promising contender in the realm of human health and
well-being, prompting a surge in scientic research. In fact, the number of PubMed-in-
dexed publications on astaxanthin has soared exponentially, skyrocketing from a mere 29
Citation: Nishida, Y.; Berg, P.C.;
Shakersain, B.; Hecht, K.;
Takikawa, A.; Tao, R.; Kakuta, Y.;
Uragami, C.; Hashimoto, H.;
Misawa, N.; et al. Astaxanthin: Past,
Present, and Future. Mar. Drugs
2023, 21, 514. hps://doi.org/
10.3390/md21100514
Academic Editor: João Carlos
Seram Varela
Received: 4 August 2023
Revised: 18 September 2023
Accepted: 22 September 2023
Published: 28 September 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Swierland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Aribution (CC BY) license
(hps://creativecommons.org/license
s/by/4.0/).
Mar. Drugs 2023, 21, 514 2 of 155
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).
Figure 1. Number of scientic papers on astaxanthin (AX) by the end of 2022. Number of articles in
PubMed (hps://pubmed.ncbi.nlm.nih.gov/, accessed on 30 June 2023) by year. The keyword query
“astaxanthin” was used to search the PubMed database. Note that “clinical trial” and “review” ar-
ticles were selected as article type tags on PubMed, resulting in dierences from the actual number
of clinical reports shown in Section 3.3.1.
The global landscape of AX research is undergoing a notable shift, with countries
worldwide, including Japan, spearheading signicant 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 sheries, 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 signicance. Intriguingly, recent studies have begun to unveil the potential of
AX in addressing health challenges stemming from societal shifts. Thus, AX holds the po-
tential to become one of the rare natural products capable of fullling diverse needs within
human society as we forge ahead into the future.
According to available market research and forecasts, the market for AX and its end
products is currently estimated to be valued between USD 647.1 million and USD 1633.7
million in 2021. Furthermore, revenue projections suggest that the market is expected to
reach USD 965 million to USD 3200 million by 2026 [1–3]. This indicates a projected com-
pound annual growth rate (CAGR) ranging from 8% to 16%. The rapid expansion of AX
can be beer comprehended by delving into its discovery, current applications, and how
the latest research and market trends are shaping its future.
This review article also provides a comprehensive exploration of the historical jour-
ney up to the present behind the discovery of AX, shedding light on its chemical and
physical features, its distribution in organisms and biosynthesis, and furthering its often-
overlooked signicance in human culture. Unlike previous publications, we delve into the
rich tapestry of AXs impact on various aspects of human culture, unveiling its profound
connections and implications. Furthermore, we delve into the potential contributions of
Mar. Drugs 2023, 21, 514 3 of 155
AX in addressing future social and environmental challenges, underscoring its versatility
and potential for positive transformation. By bridging the past, present, and future, this
article oers a unique perspective on the multifaceted role of AX in shaping our world.
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 C40H52O4 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): (3S,3′S), meso (3R,3′S), and (3R,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 conguration, 9-, 13-, and 15-cis isomers have also been identied (Figure 2).
Figure 2. Astaxanthin; structure, optical isomers and major geometric isomers.
In addition to the free form, where no hydroxyl group modications are present, AX
also occurs naturally in a form where hydroxyl groups are modied by fay acid esters.
In animals, AX may be present in a protein complex, while in bacteria, it can be found as
glycosides [4,5]. The detailed roles of these processes are discussed in other sections, spe-
cically in Section 2.1.6 for fay acid esters, Section 2.1.8 for carotene proteins, and Section
2.2.1 for glycosides. The physical and major spectral properties of AX are described in
Supplementary Table S1.
2.1.2. Astaxanthin; Discovery and History of Structural Investigation
Investigations into astaxanthin (AX) began soon after the initial discovery of carote-
noids. The study of carotenoids dates back to the early nineteenth century, when carote-
noids were rst found and extracted from paprika (in 1817), saron (in 1818), annao (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
Mar. Drugs 2023, 21, 514 4 of 155
called crustaceorubin by the British naturalist Henry Noidge 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, Tswe successfully separated
carotenes, xanthophylls, and chlorophylls from green leaves using column chromatog-
raphy for the rst 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 KMnO4. However, these
techniques were not sensitive and required several grams of carotenoids in crystalline
form for analysis. In the 1970s, signicant 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 ad-
vancements made it possible to analyze smaller samples more eectively. 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 struc-
tural 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 identied “ovoester” as a xantho-
phyll 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 ex-
hibited 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 ndings, 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 amingo wings [15], grasshoppers, and
other insects [16], as well as from the ower petals of the Adonis plant [17]. These early
works on the isolation and identication of AX were published in “Nature,” one of the
most prominent scientic 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 distrib-
uted 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]. Fig-
ure 3 shows an Oak Ridge Thermal Ellipsoid Program (ORTEP) diagram of a single mol-
ecule obtained from a single crystal of all-trans AX.
Mar. Drugs 2023, 21, 514 5 of 155
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 gure was pre-
pared 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 con-
ducted globally. However, in its early phases until the 1990s, it was predominantly carried
out in Japan, where Japanese researchers made signicant contributions to the eld. Let
us examine the history of AX research in Japan.
In Japan, AX research initially began in the eld of sheries. In the 1970s, Matsuno et
al. conducted extensive research on carotenoids found in various aquatic animals, ap-
proaching the subject from the perspectives of natural product chemistry and comparative
biochemistry (for detailed information, refer to other reviews [20–22]). Furthermore, Hata,
Katayama, et al. studied the pathway of AX production in goldsh (colored varieties of
Carassius auratus), nishikigoi (colored varieties of Cyprinus carpio), and Japanese tiger
prawn (Marsupenaeus japonicus, formerly Penaeus japonicus). They proposed the path-
way of AX biosynthesis in these aquatic animals, considering the structures of various
metabolic intermediates, using dietary carotenoids such as zeaxanthin and β-carotene
[23–25]. Kitahara, Hata, Hatano, Ando, et al. also contributed research on the metabolism
of AX in salmon [26–29].
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 sh, 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 sh improved egg quality, hatching, development, and growth of
fry. These ndings were based on their studies of AX dynamics in sh eggs [20].
In the 1990s, Miki et al. (1991) made a signicant discovery regarding the antioxidant
activity of AX. They found that AX exhibited a much stronger (more than 100 times) ca-
pability 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
Mar. Drugs 2023, 21, 514 6 of 155
zeaxanthin, as well as α-tocopherol [32]. Furthermore, Nishino et al. conducted research
on the anti-carcinogenic eects of AX and other carotenoids [33,34]. Prior to the 1990s, AX
had primarily been studied in the eld of sheries. However, these ndings sparked re-
search on its applications in medicine and human health. Meanwhile, in 1993, the Marine
Biotechnology Institute in Japan discovered an AX-producing marine bacterium, later
identied as belonging to the genus Paracoccus [35]. Subsequently, in 1995, AX biosynthe-
sis genes, including a novel key gene for the ketolation reaction [36], were isolated from
this Paracoccus strain, and their functions were claried by Misawa et al. [37], followed by
the isolation of the key gene from the green alga Haematococcus pluvialis [38]. This break-
through 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 signicant aention in the eld 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 eects.
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 eld of
ophthalmology. Considerable clinical evidence has accumulated regarding the eects of
AX on eyestrain (asthenopia) [39–47]. Based on these research ndings, several functional
food products have been launched in Japan. These products will be discussed in more
detail in Section 3, focusing on their industrial uses.
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 specied [48]. For further details, please see Section 2.2.2. Liaaen-Jensen et al.
reduced the isolated AX from Haematococcus algae using NaBH4, and the resulting product
exhibited a circular dichroism (CD) spectrum consistent with (3R,3′R)-zeaxanthin. Conse-
quently, it was determined that AX derived from Haematococcus algae possesses a stere-
oconguration of (3S, 3′S) [49,50]. Please note that the “R” and “S” nomenclature rules for
absolute conguration were followed, where the hydroxyl group connecting the chiral
carbon at the 3,3′ position to the chiral center is oriented upward (HO►), designating ze-
axanthin as “R” and AX as “S”. For further information regarding nomenclature rules,
please refer to Supplementary Document 1.
In 1976, Andrewes et al. made a signicant discovery regarding AX obtained from
Phaa yeast, specically Phaa rhodozyma (currently known as Xanthophyllomyces dendro-
rhous). They observed that the CD spectrum of AX from this yeast was completely oppo-
site to that of (3S,3′S)-AX, indicating that the AX from Phaa yeast exclusively adopts
the (3R,3′R) conformation [51]. This nding prompted us to conduct a meticulous com-
parison of the CD spectra of AX obtained from various marine animals. The observed dif-
ferences in intensities (Δε) indicated that the AX from marine animals is a mixture of op-
tical isomers. Supplementary Figure S2A presents the CD spectra of the three stereoiso-
mers of AX for reference.
In 1979, Vecchi and Müller successfully separated racemic AX into three optical iso-
mers 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,
Mar. Drugs 2023, 21, 514 7 of 155
shrimp, salmon, and starsh. The analysis revealed that AX in shrimp, salmon, and star-
sh comprised a mixture of the three optical isomers (3R,3′R), meso, and (3S,3′S). Fur-
thermore, Vecchi and Müller directly separated the racemic AX into the three optical iso-
mers 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) [53]. By employing this method, Vec-
chi and Müller conrmed 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
conrmed that the theoretically calculated Raman spectra accurately reproduced the ex-
perimentally 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 vibra-
tional modes. They also discussed the stability of the isomers by comparing the theoreti-
cally predicted relative energies and estimated that the ratio of the all-trans conguration
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 eective 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 physico-
chemical perspective, several properties of cis isomers, including absorption maxima, sol-
ubility in solvents, and antioxidant activity, have been shown to dier from those of the
all-trans form [4,54,58–60]. Specically, the recent reports by Honda et al. and others indi-
cate 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 Fay Acid Esters
In addition to the free form, where no hydroxyl group modications are present, AX
also occurs naturally in a form where hydroxyl groups are modied by fay 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 fay acids ranging
from C12 to C18. Esteried derivatives of AX with highly unsaturated fay 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 mo-
noesters with C16 to C18 fay acids [62–64], while in krill it occurs as diesters with highly
unsaturated fay acids such as DHA and EPA [65,66]. Therefore, when quantication of
AX is required, it is often calculated from the absorbance value based on the absorption
coecient of the free form as a tentative quantication value. For more accurate quanti-
cation, saponication should be applied, and the free AX content should be quantied by
HPLC. In other words, the value obtained by converting all of the esteried 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 pres-
ence of oxygen (Supplementary Figure S1) [67]. Therefore, to accurately quantify AX, es-
teried AX must rst undergo alkaline saponication under anoxic conditions or enzy-
matic treatment with cholesterol esterase from Pseudomonas sp. [67,68]. The enzymatic
Mar. Drugs 2023, 21, 514 8 of 155
treatment is generally more convenient as the hydrolysis reaction proceeds without arti-
facts under normal oxygen levels.
2.1.7. Astaxanthin Aggregates
AX is strongly lipophilic, as indicated in Supplementary Table S1. 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 inuences the morphology of these aggregates and signicantly impacts their
photophysical properties. Spectroscopic analysis reports suggest that the absorption spec-
trum of AX aggregates is either blue-shifted (H-aggregate) or red-shifted (J-aggregate)
compared to that of the monomer, reecting the conditions during aggregation [71–73].
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] (Fig-
ure 4). 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 aggre-
gation in the ester form of AX [75].
Aggregates often exhibit signicant dierences in behavior compared to non-aggre-
gated forms of biomolecules. These changes in physical properties can have a signicant
impact on their biological activity, particularly their pharmacological activity. For in-
stance, H-aggregates of carotenoids demonstrate higher photostability in aqueous solu-
tions 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]. In-
corporating 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-aggre-
gates and the preparation of complex nanosuspensions containing these two types of ag-
gregates [77]. AX aggregates, which are typically unstable, can be stabilized by incorpo-
rating 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 dierent behavior in terms of radical scavenging activity
compared to monomers in simple aqueous polar solvents [77]. This dierence may be at-
tributed 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
dierent electron transport capacities [77]. However, there are still many unresolved as-
pects regarding the physiological activity of aggregates, and further investigations are re-
quired.
Recently, researchers have isolated ve distinct forms of AX aggregates that allow for
the adjustment of intermolecular coupling between AX molecules. Time-resolved absorp-
tion 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 sin-
glet ssion, with rates of triplet generation and annihilation that can be linked to the
strength of intermolecular coupling. This nding challenges the conventional model of
singlet ssion in linear molecules [80], as it demonstrates that the triplet state of AX is
directly formed from the initial 1Bu+ (S2) photoexcited state through an ultrafast singlet
ssion 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 signicant hypsochromic shift in absorption, extending into the UV spectral region,
compared to that of the monomer. Consequently, the H-aggregate of AX eciently ab-
sorbs 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
Mar. Drugs 2023, 21, 514 9 of 155
via the ultrafast ssion process. The signicance of aggregates in natural systems is further
discussed in Section 2.1.8.
Figure 4. Predicted representative forms of astaxanthin aggregates in hydrated polar solvents. Re-
produced from Ref. [74] with permission from the Royal Society of Chemistry.
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 anity for certain proteins, such as albu-
min, 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 colora-
tion of lobster shells belonging to the species Homarus gammarus and H. americanus. Crus-
tacyanins are members of the lipocalin superfamily of proteins, as deduced from the
amino acid sequence of their subunits, which are hydrophobic ligand-binding proteins
[82–86]. The multimeric α-crustacyanin (with a maximum absorption wavelength of ap-
proximately 630 nm) and dimeric β-crustacyanin (with a maximum absorption wave-
length 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 heterodi-
mers formed by two apocrustacyanins. Apocrustacyanins comprise ve subunits: A1, C1,
and C2, each with a molecular weight of approximately 21 kDa, and A2 and A3, each with
a molecular weight of approximately 19 kDa. In lobsters (Homarus gammarus ), the major
subunits of β-crustacyanin are A2 and C1 [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 wa-
ter, as well as histidine residues, and the close interactions of the two chromophores (AX).
Mar. Drugs 2023, 21, 514 10 of 155
Since the usual maximum absorption wavelength (λmax) 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 eects during the association of β-crustacy-
anin with α-crustacyanin in lobster shells. In crustaceans, the combination of apoprotein
subunits varies among species and mutations, contributing to the variation in the colora-
tion of crustacyanins [86,87].
Another carotenoprotein that forms the exoskeleton in lobsters, similar to crustacya-
nin, 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 inter-
actions through natural H-aggregates (see Section 2.1.7.), with the chromophores ar-
ranged 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 local-
ize dierently 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-
specic genes are divided into two distinct clades within the lipocalin protein superfam-
ily. The fact that the crustacyanin gene family emerged early in the evolution of Malacos-
traca crustaceans suggests that this protein played a signicant role in the evolutionary
success of this group of arthropods [86,91]. Crustaceans, in particular, are known for their
diverse species-specic shell colors and paerns, 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 crusta-
ceans. Ovoverdins, reported as the pigment responsible for the dark green color (λmax; ca.
465–470 and 660–670 nm) of lobster ovaries and eggs [12,94–96], 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 λmax, AX may bind to two
distinct sites: one might be a weak non-specic association, and the other is a specic stoi-
chiometric association with lipoproteins. Additionally, there are at least two dierent mo-
lecular-weight proteins (ca. 700 kDa and 600 kDa) [96]. In the laer, 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” jellysh Velella velella [82,98]. There
are two types of this protein, named V600 (λmax; ca. 600 nm) and V620 (λmax; 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 subu-
nits and form a helical structure. The quaternary structures of velellacyanin and the N-
terminal peptide sequence of the subunit comprising V600 reveal similarity to apocrusta-
cyanin C [99–101]. In fact, immunocross-reactivity showed reactivity with polyclonal an-
tibodies 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 ex-
pected.
In echinoderms, two well-known carotenoproteins, as shown below, have been par-
tially characterized by X-ray structural and CD spectral analyses; however, no phyloge-
netic or functional analysis of the proteins, including detailed genetic background, has yet
been available. The common starsh Asterias rubens has “asteriarubin,” a purple-blue ca-
rotenoprotein, also present [103,104]. Asteriarubin (λmax; ca. 570 nm) is approximately 43
Mar. Drugs 2023, 21, 514 11 of 155
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 carot-
enoids 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 subu-
nits [81,103]. Reconstitution studies revealed similarities between the binding require-
ments of asteriarubin and crustacyanin; however, the tetrameric asteriarubin contains
only one carotenoid molecule, and the CD spectrum shows no exciton spliing. 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 aributed to the absence of exciton eects 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 echi-
noderms, the vivid blue skin of calcied starsh called “blue star,” Linckia laevigata, has
a blue carotenoprotein called “linckiacyanin” (λmax; 395, 612 nm), with (3S,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. How-
ever, 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-crus-
tacyanin subunit [102], it is possible that linkyacyanin is a distinct family member from
the lipocalin superfamily.
In Asia, it is easy to nd vivid pink egg clumps on the surface of rice plants and on
the walls of aqueducts for rice elds. This pigment is known as “ovorubin.” Ovorubin is
a carotenoprotein found in the perivitelline uid that surrounds the embryo of the ferti-
lized egg, which is an accessory gland of the female reproductive tract of the South Amer-
ican freshwater snail Pomacea canaliculata (Gastropoda: Ampullariidae). Ovorubin is de-
scribed as a large red AX-binding glycoprotein of approximately 330 kDa [106], and the
binding of (3S,3′S)-AX and their fay acid esters to ovorubin results in a small batho-
chromic shift (20–30 nm) to λmax 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 ad-
dition to ovorubin, there is another carotenoprotein called “alloporin” (λmax; 545 nm)
found in the soft coral Allopora californica. Alloporin is approximately 68 kDa and com-
prises four subunits with a molecular weight of approximately 17 kDa each. It has an equal
molar of (3S,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 de-
tails and functions of these chromophores, their characterization holds the key to unlock-
ing a deeper understanding of the captivating structures and extraordinary roles played
by these carotenoproteins. The journey to uncover their secrets promises to be a captivat-
ing exploration into the realms of science and discovery.
All photosynthetic plants utilize carotenoid-binding proteins as an important com-
ponent of their photosynthetic function. For example, in chloroplast thylakoid mem-
branes, 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-eciency light harvesting. The photosystem II
supercomplex (PSII) is also a pigment-protein complex that catalyzes water spliing and
oxygen-evolving reactions in photosynthesis, converting light energy into chemical en-
Mar. Drugs 2023, 21, 514 12 of 155
ergy. The PSII core complex is composed of more than 20 subunits and contains approxi-
mately 35 chlorophylls (Chl) and 12 β-carotene molecules, as well as other oxidation-re-
duction 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 MnCaO5 cluster to split water. If the rate of photo-
damage exceeds the rate of repair under excessively intense light conditions, a phenome-
non 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 eects of photoinhibition of PSII, plants have developed sys-
tems 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 ex-
cited 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 oxy-
gen-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 specic 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 as-
phalt surface in mid-summer, benets from a water-soluble AX-binding carotenoprotein
called “AstaP” [114]. AstaP (AstaP-orange1) is a secreted protein that exhibits thermally
stable 1O2 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 ex-
tensively 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 isoe-
lectric point (pI = 3.6–4.7), which is signicantly dierent from the original AstaP-orange1
(pI = 10.5) [115]. Orthologues of AstaP have also been found in diverse green algae, in-
cluding 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 specically, 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 bind-
ing to other xanthophylls and carotenes [117]. Recombinant AstaP is a ~20 kb water-solu-
ble protein that accepts carotenoids in acetone solution or embedded in biological mem-
branes. 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 inte-
grated into the photosynthetic system. The high accumulation of AX and other ketocarot-
enoids has been found to impact growth, CO2 assimilation, and photosynthetic electron
transfer in transgenic plants. Moreover, studies have demonstrated that ketocarotenoids
act specically on the thylakoid membrane and, more specically, on PSII [118,119].
Interestingly, transplastomic leuce, which has been genetically modied in its chlo-
roplast genome, has been found to predominantly accumulate AX [120]. Despite having
low levels of naturally occurring photosynthetic carotenoids [111], this leuce exhibits
normal growth similar to that of non-modied plants. Initially, the quantum yield of PSII
Mar. Drugs 2023, 21, 514 13 of 155
in this leuce is low under normal growth conditions; however, it becomes comparable to
control leaves under higher light intensities. In AX-accumulating leuce, 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
aects the assembly, photophysical properties, and function of PSII. However, the repair
mechanisms in AX-accumulating leuce 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 inuence the
physical properties of the lipid membrane itself, such as uidity and permeability to small
molecules [121]. In AX-accumulating leuce, the high accumulation of xanthophylls may
prevent the permeation of singlet oxygen produced by PSII and enable ecient quenching
within the lipid bilayer. Specically, AX and carbonyl derivatives of lutein have been ob-
served to adhere to the surface of the PSII core complex, indicating their eective quench-
ing of singlet oxygen (1O2). Essentially, the apparent photosynthetic capacity of this leuce
may be aributed to the antioxidant eect 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 carote-
noids, 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 ali-
ation with the steroidogenesis acute regulatory protein (StAR) family, featuring a distinc-
tive structural element known as the StAR-related lipid transfer domain. This domain fa-
cilitates 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, signicant advancements have been made, including the successful
heterologous expression of BmCBP in E. coli while maintaining its functions [125]. Fur-
thermore, the crystal structure of BmCBP has been determined through X-ray structural
analysis, and the binding site for xanthophylls has been identied [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 carote-
noids from crude extracts [125].
AX-protein complexes have also been observed in sh. In salmon, for instance, mus-
cle AX exhibits specic 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 excep-
tions, as mentioned in Section 2.2.4. Therefore, recent research has highlighted the in-
volvement 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 stoichio-
metric manner or not. Additionally, enzymatic degradation of carotenoids takes place in
various tissues. This degradation involves reactions catalyzed by carotenoid cleavage ox-
ygenases, resulting in the formation of apocarotenoids. A well-known example is the con-
version of β-carotene into vitamin A retinoids by the enzyme BCO1. The detailed roles of
Mar. Drugs 2023, 21, 514 14 of 155
these processes are discussed in other sections, specically in Sections 2.3.4 and 4.1 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 reac-
tive nitrogen species (RNS). Additionally, it employs indirect mechanisms by inducing a
group of antioxidant enzymes in biological systems. However, this review specically fo-
cuses on the ROS-scavenging mechanism of AX. For further information on AXs role in
the biological environment (e.g., plasma membrane and carrier proteins like HDL/LDL)
and its induction of antioxidant enzymes, such as SODs, GSTs, GPXs, and Catalase, via
the activation of the Nrf2/PGC-1α pathway, as well as its suppression of the production
of pro-inammatory cytokines by inhibiting the NFκB or JAK/STAT pathway, please refer
to the author’s other comprehensive review (see Sections 1.2, 2.1, and 2.2 of [130]).
Quenching Singlet Oxygen
Singlet molecular oxygen (1O2) is generated from the ground-state triplet molecular
oxygen (3O2: 3∑g−) through photochemical processes in biological systems [131–141]. Ap-
proximately 1O2 exists in two singlet states with dierent spins (1∑g+ and 1∆g), with 1∆g
being the lowest excited singlet state. The former has a very short lifetime, while the laer,
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
inuenced by the surrounding environment. Additionally, there is a very short-lived
dimol molecule (O2(1∆g)-O2(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 O2(1∆g) emission in the infrared
spectral region around 1270 nm.
Under normal environmental conditions, the electric dipole transition of oxygen mol-
ecules from the ground triplet state (3Σg−) to the lowest electronically excited singlet state
(1Δg) has an extremely low transition probability. This transition is forbidden due to con-
siderations of spin angular momentum, orbital angular momentum, and parity. As a re-
sult, 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
(O2•–) and hydrogen peroxide (H2O2). Furthermore, an autocatalytic reaction involving the
cyclization of peroxyl radicals can also produce singlet oxygen via a tetraoxide interme-
diate. 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 [131,138,139,141].
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 in-
stance, 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-Opi syndrome, glaucoma, cataracts, and age-related macular degenera-
tion. 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
[142].
Carotenoids, such as lycopene, β-carotene, lutein, and AX, are highly ecient
quenchers of singlet molecular oxygen (1O2). Their reaction rate constants, typically
around 1010 M−1 s−1, approach the limit of diusion control in solvents [76,143]. The
quenching mechanism of 1O2 by carotenoids has been recently analyzed through quantum
dynamics calculations and ab initio calculations [144]. Theoretical studies suggest that the
Mar. Drugs 2023, 21, 514 15 of 155
ground-state singlet carotenoid (1Car) and 1O2 molecules can form a weakly bound com-
plex, facilitated by the donation of electron density from the carotenoid’s highest occupied
molecular orbital (HOMO) to the πg* orbitals of 1O2. The quenching of 1O2 is governed by
a Dexter-type superexchange mechanism involving charge transfer states (Car•+/O2•−).
Quantum dynamics calculations demonstrate that the quenching of 1O2 by carotenoid/O2
complexes occurs rapidly, within sub-picosecond timescales, due to strong electronic cou-
pling. This theoretical study highlights the crucial role of carotenoid cation radical species
(Car•+) in achieving ecient 1O2 quenching. Notably, AX is known for its high activity and
stability against 1O2 quenching [143].
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 1O2 quenching activity be-
comes 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 dierent
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 experi-
mental results demonstrated that the 1O2 quenching activity increased as the length of the
conjugated double bond was increased (refer to Table 1 and Supplementary Table S2)
[145,146].
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. Ad-
ditionally, AX has polar hydroxyl groups at both ends (refer to Figure 2). These molecular
frameworks might specically 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 geo-
metrical isomer have been reported. Specically, in terms of 1O2 scavenging activity, the
all-trans conguration is known to exhibit the highest activity, followed by 15-cis and 9-
cis isomers. This indicates that the rate constant for deactivating 1O2 by the cis-isomers
decreases as the cis-bond of β-carotene moves away from the center of the molecule, re-
sulting in less ecient 1O2 quenching compared to the all-trans isomers [145]. Time-re-
solved 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 sys-
tem [147,148].
In biological model membranes, such as phospholipid liposomes, several studies
have investigated the eectiveness of AX in inhibiting 1O2-induced peroxidation of phos-
pholipid 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 [149]. Nevertheless, when
evaluated based on endoproducts as outcomes, AX has been found to exert eective pro-
tective actions against phospholipid membrane peroxidation and cellular damage in-
duced by photosensitization in the presence of photosensitizers [150–154].
The reaction between AX and singlet oxygen is primarily aributed to physical
quenching, as described previously. However, a small fraction of AX does form reactive
products with 1O2. According to Nishino et al., the major products generated from this
reaction are endoperoxides, specically astaxanthin 5,6-endoperoxide or astaxanthin 5,8-
endoperoxide. These endoperoxides represent 1O2 adducts formed at the C=C bonds of
the β-end group [155].
Mar. Drugs 2023, 21, 514 16 of 155
Table 1. Singlet oxygen quenching activity of astaxanthin: comparison with common antioxidants.
1O2 Generator
EDN *
EDN *
NDPO2 *
EP *
Reference
[156]
[157]
[158]
[159]
Detection
Luminescence
Luminescence
Luminescence
Absorbance of DPBF
Solvent
CDCl3
CDCl3/
CD3OD
(2:1)
DMF/
CDCl3
(9:1)
CDCl3
CDCl3/
CD3OD (2:1)
EtOH/CHCl3
/D2O
(50:50:1)
EtOH/CHCl3/D2O
(50:50:1)
1. Carotenoids
Astaxanthin
2.2
1.8
5.4
2.2
1.8
24.0
11.7
Canthaxanthin
2.2
1.3
2.0
-
1.2
21.0
Zeaxanthin
2.0
0.73
3.4
1.9
0.12
10.0
11.2
β-Cryptoxanthin
2.0
0.27
1.7
-
-
6.0
7.0
β-Carotene
2.2
0.28
1.1
2.2
0.049
14.0
10.8
Lycopene
3.0
1.4
3.4
-
-
31.0
14.0
Capsanthin
-
-
-
-
-
-
12.1
Lutein
0.61
0.26
2.1
0.8
-
8.0
8.1
α-Carotene
0.66
0.23
0.93
-
-
19.0
10.0
Fucoxanthin
0.29
0.075
0.97
-
0.005
-
-
Tunaxanthin
-
-
-
0.15
-
-
-
2. Vitamin C
L-Ascorbic acid
-
-
0.00089
-
-
-
-
3. Vitamin E
α-Tocopherol
0.02
0.0039
0.049
-
-
0.28
0.13
β-Tocopherol
-
-
-
-
-
0.27
0.093
γ-Tocopherol
-
-
-
-
-
0.23
0.084
δ-Tocopherol
-
-
-
-
-
0.16
0.041
Trolox
-
-
0.010
-
-
-
0.042
4. Polyphenols/other phenolic antioxidants
α-Lipoic acid
0.056
0.038
0.072
-
-
0.13
0.0019
Ubiquinone-10
0.0019
0.0021
0.0068
-
-
-
0.062
BHT
-
-
0.004
-
-
-
-
Caffeic acid
-
-
0.0023
-
-
-
0.00069
Ferulic acid
-
-
-
-
-
-
0.00027
CurcuminI
-
-
0.0036
-
-
-
-
(-)-EGCG
-
-
0.0096
-
-
-
0.0051
Gallic acid
-
-
0.0023
-
-
-
-
Pyrocatechol
-
-
0.0055
-
-
-
-
Pyrogallol
-
-
0.0055
-
-
-
-
Quercetin
-
-
0.0018
-
-
-
-
Resveratrol
-
-
0.0018
-
-
-
-
Sesamin
-
-
0.0012
-
-
-
-
Capsaicin
-
-
0.0021
-
-
-
-
Probucol
-
-
0.00044
-
-
-
-
Edaravon
-
-
0.0067
-
-
-
-
* The 1O2 was generated in a dark reaction by thermodissociation from the respective endoperoxide.
EDN, 1,4-dimethylnapthalene endoperoxide; NDPO2, of 3,3′-(1,4-naphthylene) dipropionate en-
doperoxide; EP, 1-methylnaphthalene-4-propionate endoperoxide; DPBF, 1,3-diphenylisobenzofu-
ran; DMF, N,N-dimethylformamide ; (−)-EGCG, (−)-epigallocatechin gallate.
Scavenging of Free Radicals and Inhibition of Lipid Peroxidation
Carotenoids can occasionally display pro-oxidant activity by oxidizing other lipids
instead of acting as antioxidants, especially under high oxygen partial pressure or in the
absence of other antioxidants. However, in comparison to other carotenoids, AX has
shown minimal pro-oxidant activity in both simple solvent-based systems and evaluation
systems that involve phospholipid membranes [143,160]. This is in contrast to lycopene,
which is often assessed for its antioxidant properties.
Studies employing pulse radiolysis and time-resolved spectroscopy have revealed
that carotenoids engage with free radicals through dierent mechanisms [161–165]. The
preliminary products formed during the interaction between carotenoids and free radicals
indicate that electron transfer and radical addition are kinetically favored reactions. These
Mar. Drugs 2023, 21, 514 17 of 155
reactions result in the oxidation of the carotenoid to its radical cation or the generation of
carotenyl adduct radicals, such as [R-Car]• [166].
Carotenoids possess the necessary reactivity to function as antioxidants, and their
reaction rates with free radicals are comparable to those of polyunsaturated fay acids
reacting with the same oxidants [143]. When comparing the reactivity of carotenoids with
free radicals, it is observed that carotenoid radical cations can be reduced by α-, β-, and γ-
tocopherol, while lycopene and β-carotene can reduce δ-tocopherol radicals [161,167]. β-
Carotene•– transfers an electron to oxygen but not vice versa, while lycopene undergoes
reversible electron transfer with O2•– due to its more positive electronegativity resulting
from its planar geometry. Among the common carotenoids, AX radical cations are the
most easily reduced. This implies that AX radical cations can be reduced by other carote-
noids, like lycopene. Additionally, the interaction between carotenoids and other antioxi-
dants may signicantly contribute to their antioxidant activity in vivo. Skibsted and col-
leagues demonstrated that AX radical cation is eectively reduced by polyphenols such
as isoavonoids, in addition to the antioxidants mentioned earlier [168].
Interestingly, AX exhibits lower reducing abilities compared to other carotenoids.
When reacting with CCl3OO•, AX does not directly form a radical cation but instead un-
dergoes an addition of a radical, which subsequently decays to form the cation radical
[165]. Despite this, AX and canthaxanthin demonstrate greater eectiveness as antioxi-
dants compared to β-carotene or zeaxanthin in retarding hydroperoxide formation during
azo-initiated lipid peroxidation in homogeneous methyl linoleate/AMVN systems. How-
ever, the rates of AMVN-induced oxidation of AX and canthaxanthin are slower than
those of β-carotene and zeaxanthin [169].
These ndings indicate that AX and other carotenoids, when consumed in combina-
tion with multiple antioxidants rather than individually, may have a beer ability to in-
hibit lipid peroxidation through the interaction of antioxidant networks. This could pro-
vide an explanation for the suggestion that aggressive supplementation of synthetic β-
carotene at high doses may actually increase the risk of lung cancer in smokers and asbes-
tos-exposed workers [170,171]. On the other hand, simultaneous intake of green and yel-
low vegetables, which contain multiple carotenoids and antioxidant vitamins, has been
associated with a reduced risk of cancer [172–174]. Therefore, understanding the reactivity
of AX with reactive oxygen species (ROS) and free radicals, as well as the physicochemical
properties of its reaction intermediates, is crucial for a comprehensive understanding of
the true antioxidant activity of AX.
Structures of Radical Cation and Dication of Astaxanthin as Predicted Based on DFT
Calculations and Resonance Raman Spectroscopy
The results from pulse radiolysis and time-resolved spectroscopy studies demon-
strate that carotenoids possess the necessary reactivity to function as antioxidants through
kinetically favored reactions, including electron transfer and radical addition when inter-
acting with free radicals. Various tocopherols can reduce carotenoid radical cations, and
lycopene can undergo reversible electron transfer with O2•– due to its positive electroneg-
ativity. AX and canthaxanthin exhibit higher antioxidant eectiveness compared to β-car-
otene or zeaxanthin, despite slower rates of AMVN-induced oxidation.
Resonance Raman spectroscopy, along with theoretical calculations, has been uti-
lized to investigate the molecular structures of radical species of AX, revealing signicant
changes in bond orders and vibrational modes. Figure 5 displays the steady-state absorp-
tion spectra of AX in acetone with dierent amounts of FeCl3 solutions (1 mM acetone
solution) added. The addition of FeCl3 solution oxidizes AX, resulting in the formation of
a radical cation peaking around 850 nm and a dication peaking around 700 nm, depending
on the amount of FeCl3 solution added. Resonance Raman spectra of AX and its radical
species were recorded and compared with the DFT calculations in Figure 6. The DFT cal-
culations accurately replicate the ground-state (S0) Raman spectrum, while the resonance
Mar. Drugs 2023, 21, 514 18 of 155
Raman spectrum of the radical species can be seen as a combination of the calculated Ra-
man spectra of the radical cation and the dication of AX. This is due to the fact that the
resonance Raman spectrum of the ground (S0) state species was recorded using 532 nm
laser light, which resonates with the S0 → S2 absorption of AX, while the resonance Ra-
man spectrum of the radical species was recorded using 808 nm laser light, which is in
resonance with both the radical cation and dication of AX. The agreement between the
theoretically calculated Raman spectra and the experimentally observed resonance Ra-
man spectra enables a detailed discussion of the molecular structures of the radical species
of AX. As an example, Figure 7 illustrates the bond lengths of the ground (S0) state, radical
cation, and dication of AX. It is noteworthy that the bond alterations evident in the ground
(S0) state species undergo dramatic changes in the radical cation species, with all C=C
double bonds tending to elongate and all C-C single bonds tending to shrink. This sug-
gests that the bond orders of all the C=C and C-C bonds in the conjugated polyene ap-
proach 1.5. In the case of the dication species, there is a striking reversal of bond alterations
in the central part of the polyene chain, with C=C double bonds becoming C-C single
bonds and C-C single bonds becoming C=C bonds. These signicant changes in bond or-
ders are accurately reected in the vibrational modes, which were predicted based on DFT
calculations. The theoretically predicted molecular structures of the radical species of AX,
supported experimentally through resonance Raman spectroscopy, serve as a valuable
tool for exploring the functions of the radical species of AX in biological systems.
Figure 5. Steady-state absorption spectra of astaxanthin (AX) in acetone when dierent amounts of
FeCl3 solutions (1 mM acetone solution) were added. According to the addition of the FeCl3 solution,
the intensity of S0 → S2 absorption of AX around 500 nm decreases, and the new absorption bands
appear in the 600–950 nm spectral region (see inset of Figure 5). With the small amount of FeCl3
solution added, the absorption band that is associable to the radical cation of AX appears to peak
around 850 nm. With more addition of the FeCl3 solution, the radical cation of AX transforms to
dication, peaking around 700 nm. The absorption band below 400 nm is due to the absorption of
FeCl3.
Mar. Drugs 2023, 21, 514 19 of 155
Figure 6. The steady-state resonance Raman spectrum of astaxanthin (AX) in acetone recorded with
532 nm excitation laser light at room temperature (solid red line in the left panel) and the resonance
Raman spectra of radical species of AX recorded with 808 nm excitation laser light at room temper-
ature (solid red line in the right panel). The results of DFT calculations of the ground (S0) species,
radical cation, and dication of AX are also shown in each panel (solid black lines).
Figure 7. Comparison of the bond lengths of the ground (S0) state, radical cation, and dication of
astaxanthin predicted theoretically by DFT calculations.
Carotenoids exhibit the necessary reactivity to function as antioxidants through fa-
vorable reactions with free radicals, including electron transfer and radical addition. AX
and canthaxanthin demonstrate higher antioxidant eectiveness compared to β-carotene
or zeaxanthin, despite slower rates of AMVN-induced oxidation. Resonance Raman spec-
troscopy and theoretical calculations are employed to investigate the molecular structures
of radical species of AX, uncovering signicant changes in bond orders and vibrational
modes. The agreement between the theoretically calculated Raman spectra and the exper-
imentally observed resonance Raman spectra enables a detailed discussion of the molec-
ular structures of the radical species of AX, providing valuable insights into their functions
in biological systems. Furthermore, although AX often forms fay acid esters in nature, it
has been reported that there is no essential dierence in the rst oxidation potential be-
tween AX and its n-octanoic mono- and diesters. This suggests that the AX esters have
similar scavenging rates for •OH, •CH3, and •OOH radicals compared to AX itself [175].
Mar. Drugs 2023, 21, 514 20 of 155
Biochemical Aspects of AX Properties against ROS
The intracellular localization and ROS scavenging activity of AX were presented in
an earlier review [130].
To summarize, inammation, whether acute or chronic, generates ROS and often
leads to oxidative stress in vivo. Important actions of AX include the inhibition of nuclear
translocation of NFκB, which promotes inammatory responses, and the activation of
Nrf2, a transcription factor of a group of anti-inammatory enzymes. In parallel, oxidative
stress caused by ROS can be reduced by improving mitochondrial function, which is a
major source of ROS in vivo [130,176].
In conclusion, AX could exert its typical eects in vivo by inducing multifaceted anti-
oxidant activity beyond the antioxidant activity derived from the chemical properties of
the compound itself. Their typical ecacies are shown in Section 3.
2.2. Astaxanthin; Distribution, Derivatives and Optical Structure in Nature
AX is found in a considerable number of organism species, which cover a wide taxo-
nomic variety that ranges from bacteria to several eukaryote kingdoms, i.e., fungi (yeasts),
algae, higher plants, and animals. Table 2 provides information on the approximate
amounts and isomers of AX in major species. With the exception of animals, organisms
that possess AX are capable of synthesizing it de novo through either the non-mevalonate
pathway (also known as the MEP pathway) or the mevalonate pathway. The non-meva-
lonate pathway involves the conversion of pyruvate and glyceraldehyde 3-phosphate into
1-deoxy-D-xylulose-5-phosphate (DXP) and 2-C-methyl-D-erythritol-4-phosphate (MEP)
[177]. Further details on these pathways can be found in Section 2.3.
2.2.1. Bacteria and Archaea
Among bacteria, the genus Paracoccus, belonging to the class α-Proteobacteria (Al-
phaproteobacteria) in the phylum Proteobacteria, has been found to produce (3S,3′S)-AX
[178–180]. Additionally, other bacteria such as Brevundimonas, Sphingomonas, and Alter-
erythrobacter species have also been reported to have the ability to produce AX [181–186].
In these bacteria, AX biosynthesis occurs from β-carotene through oxygenation reactions
catalyzed by two enzymes called CrtZ and CrtW. The two enzymes are β-C3-hydroxylase
[β-carotene (β-carotenoid) 3,3′-hydroxylase] and β-C4-ketolase [β-carotene (β-carotenoid)
4,4′-ketolase (oxygenase)], and facilitate multistep hydroxylation and ketolation (oxygen-
ation) reactions at the C3 and C4 positions of the β-ionone ring (β-end group), respectively
(Figure 8) [37]. The CrtZ-type β-C3-hydroxylase responsible for the hydroxylation of the
C3 and C3′ positions of the β-ionone rings generates a single stereo conguration, result-
ing in the production of (3S,3′S)-AX with a specic absolute optical conguration
[187,188].
Mar. Drugs 2023, 21, 514 21 of 155
Figure 8. Biosynthetic pathway of astaxanthin from β-carotene with bacterial enzymes. β-Carotene
(β-carotenoid) 3,3′-hydroxylase and 4,4′-ketolase are shown with blue and orange leers, respec-
tively. In this gure, the maximal levels of catalytic activities are shown concerning CrtR and CrtO.
Generally, the catalytic activity from adonixanthin to astaxanthin is weak, even with CrtW. This
pathway is based on bacterial enzymes. However, the functions of green algal BHY and BKT are the
same as those of CrtZ and CrtW, respectively.
It was interestingly found that the phylum Cyanobacteria, which are photoauto-
trophic bacteria, possess a distinct β-C3-hydroxylase, designated CrtR, which shows mod-
erate homology not to CrtZ but to CrtW [189]. Moreover, cyanobacteria were shown to
retain a distinct β-C4-ketolase, designated CrtO, that shows signicant homology to CrtI
(phytoene desaturase) [190], in addition to CrtW. Thus, their presence could theoretically
lead to the formation of AX. However, due to the substrate specicity of these enzymes,
major carotenoids are not AX but its early-stage precursors such as 3′-OH-echinenone,
echinenone, and zeaxanthin, as well as other cyanobacterium-related carotenoids such as
myxol glycosides. Thus, AX is ordinarily not present or one of trace amounts of carote-
noids in cyanobacteria [191,192], while previous reports, despite some debate regarding
analytical methods and accuracy, described the presence of AX as a constitutive carote-
noid in this phylum [193].
The reason is aributed to the extremely low or no reactivity of cyanobacterial CrtR
towards a substrate with a 4-keto-β-end group. CrtR is ordinarily likely to mediate the
synthesis of myxol 2′-fucoside by its β-C3-hydroxylase activity, while the Synechocystis sp.
PCC 6803 CrtR can convert β-carotene into zeaxanthin, as shown by functional analysis
using E. coli [189,191]. Similarly, functional analysis with E. coli demonstrated that cyano-
bacterial CrtO also exhibits very low or no reactivity towards a substrate with a 3-hy-
droxy-β-end group, since CrtO handles echinenone synthesis from β-carotene by its β-C4-
ketolase activity (Figure 8) [194]. Moreover, it was found that cyanobacterial CrtW en-
zymes generally retain much lower activity for such a substrate, compared with Paracoccus
and Brevundimonas CrtW proteins [195]. As a side note, a recent study reported that the
introduction of the crtW and crtZ genes from Brevundimonas sp. SD212 into Synechococcus
sp. PCC 7002, a type of cyanobacteria, resulted in the production and enhancement of AX
productivity [191].
Although these AX products occur mainly in their free form in these bacteria, it has
been reported that Agrobacterium aurantiacum (properly Paracoccus sp. strain N81106) pro-
duces a glycosylated AX, i.e., AX monoglucoside [196]. Additionally, Sphingomonas
Mar. Drugs 2023, 21, 514 22 of 155
astaxanthinifaciens and S. lacus PB304 also contain AX dirhamnoside and AX dideoxyglu-
coside, respectively [197–199]. The enzyme gene that forms these glycosides, crtX, has
been found in the complete gene clusters of Paracoccus sp. strain N81106 [37,200] and the
genus Sphingomonas. Such an activity of CrtX was suggested using Pantoea ananatis CrtX
with an AX-producing recombinant E. coli [201]. However, functional analysis using E.
coli has not conrmed the ability of the putative gene of Sphingomonas to mediate such
glycosylation reactions [188,198]. Furthermore, the presence of AX has also been impli-
cated in the phyla Actinomycetota and Deinococcota (Deinococcus-Thermus); however,
reliable structural analysis of AX and its biosynthetic genes in these phyla is still lacking.
Further details regarding the bacterial AX biosynthetic pathway are discussed in Section
2.3.
It is indeed plausible that AX may play a protective role in cells exposed to intense
sunlight and high levels of natural radiation. Bacteria that produce AX, such as those
found in ocean surfaces, coastal areas, and hot springs, inhabit environments where they
are subjected to these harsh conditions [181–184,196,202]. AX, with its antioxidant prop-
erties, has the potential to scavenge free radicals generated by UV radiation and protect
cells from oxidative damage. Furthermore, AXs ability to absorb and dissipate excess light
energy may also contribute to cellular photoprotection. These mechanisms suggest that
AX could serve as a natural defense mechanism against the damaging eects of sunlight
and radiation on these bacteria.
The presence of AX in halophilic archaea, which thrive in high-salinity environments
where other organisms cannot survive, has been suggested [203,204]. In particular, studies
on Halobacterium salinarum R1 have shown that depletion of the CYP174A1 gene, which
codes for a cytochrome P450 (CYP; P450), resulted in decreased production of AX. It is
noteworthy that the genome of H. salinarum does not contain genes encoding CrtZ-type
or CrtR-type β-C3-hydroxylase, CrtW-type β-C4-ketolase, or CrtO-type β-C4-ketolase,
which may be involved in AX production in other organisms [204]. This suggests that
CYP174A1 may have a role in the biosynthesis of AX in these halophilic archaea. However,
the presence and distribution of AX in archaea as a whole have yet to be fully conrmed,
and further research is needed to elucidate this aspect.
2.2.2. Eukaryotes; Fungi and Protozoa
Certain colored fungi are capable of producing carotenoids, including xanthophylls
[205]. However, the production of AX (astaxanthin) in fungi is quite limited and is cur-
rently only observed in the genus Xanthophyllomyces. The yeast Phaa rhodozyma, which is
the anamorph of Xanthophyllomyces dendrorhous, was initially isolated from exudates of
deciduous broadleaf trees at high altitudes in the northern hemisphere, where it is ex-
posed to intense UV radiation [206,207]. In recent years, similar species belonging to the
genus Xanthophyllomyces have also been discovered in the southern hemisphere, such as
in Patagonia, South Australia, and Tasmania [208–210]. The dierences in host trees and
the evolutionary distances between these Xanthophyllomyces species from the northern and
southern hemispheres suggest that they have been strongly inuenced by the ancient con-
tinental separation of the Earth, which is of great interest from an Earth science perspec-
tive [211].
Phaa yeast, including Xanthophyllomyces species, produce (3R,3′R)-AX in its free
form [51]. The biosynthesis pathway of AX in Phaa yeast is catalyzed by specic monoox-
ygenase enzymes belonging to the P450 family, such as CrtS/Asy, along with a reductase
enzyme. This pathway diers signicantly from the metabolic pathway observed in bac-
teria [212,213]. It is believed that AX plays a crucial cytoprotective role for Phaa yeast in
adapting to these harsh environments. The biosynthesis of AX in Phaa yeast is likely
induced by redox imbalances, particularly those involving the NAD(P)H/NAD(P)+ cou-
ple and the oxidative environment [214].
Mar. Drugs 2023, 21, 514 23 of 155
Overall, the production of AX in fungi, particularly in Phaa yeast, is an intriguing
phenomenon, and its biosynthesis pathway and protective role in challenging environ-
ments are subjects of scientic interest and investigation.
Several zooplankton and phytoplankton species have the ability to synthesize AX de
novo. The genera Nannochloropsis in the class Eustigmatophyceae, Aurantiochytrium in
the class Labyrinthulea, and Euglena and Trachelomonas volvocina in the class Eugleno-
phyceae, as well as certain arthropods like copepods and krill, have been reported to have
the potential for AX production [215–219] (Table 2). These ndings suggest the possibility
of industrial-scale production of AX from these organisms. With advances in biotechnol-
ogy and cultivation techniques, there is increasing interest in harnessing the AX produc-
tion capacity of these zooplankton and phytoplankton species for commercial purposes.
Industrial production of AX from these natural sources could provide a sustainable and
renewable supply of this valuable compound for various applications. However, further
research and optimization are required to fully exploit their potential and scale up the
production process eectively.
2.2.3. Eukaryotes; Algae and Higher Plants
It is worth noting that several green algae are known to accumulate extremely high
concentrations of AX in cells under high sunlight, high salinity, and starvation conditions
(Figure 9). The most well-known example is the green algae of the genus Hemacotococcus,
particularly H. lacustris (Gir.-Chantr.) Rostaf. (=H. pluvialis Flot.). Please note that this doc-
ument supports the ocial scientic name of H. lacustris (Gir.-Chantr.) Rostaf., as per the
comprehensive opinion of Ota et al. [220,221].
Descriptions of Haematococcus algae (=H. lacustris), its biology, and life cycles
emerged from the works of the German botanist Julius von Flotow in 1844 and the Amer-
ican botanist Tracy Elliot Hazen in 1899. Although early botanists described a “blood red
pigment” produced by this algae [222], it was later determined that this was AX [49,223].
Nevertheless, since the description of “H. pluvialis Flot.” can also be frequently found in
numerous publications, this species is referred to as “Haematococcus algae” in this paper
to avoid misunderstandings, unless there are exceptions in specifying the strain name.
Recent molecular genetics re-classication has revealed that H. lacustris strains are highly
genetically diverse [224], and other species previously assigned to the Haematococcus ge-
nus may be reclassied as a separate genus, possibly leaving only one species in this ge-
nus, H. lacustris [48]. The properties and industrial production of this alga are presented
in detail in Section 3.3.
In Haematococcus algae, (3S,3′S)-AX is present in the form of a fay acid ester bonded
to the hydroxyl group at the C3, C’3 position, with the main component being a monoes-
ter. Other specic species of green algae belonging to the genera Acutodesmus, Asterarcys,
Bracteacoccus, Botryococcus, Chlamydomonas, Chlorella (Chromochloris), Chlorococcum, Coelas-
trella, Monoraphidium, Neochloris, Protosiphon, Sanguina, Scenedesmus, Scotiellopsis, Tetrae-
dron, and Vischeria have also been reported to accumulate AX mainly in esters and/or free
form, as well as in protein binding forms [114,225–242]. Another example is the fay acid
esters of AX diglucoside, which have been reported from a low-temperature-tolerant al-
gae (Chlamydomonas nivalis) that grows on snowelds and glaciers in the Alps and polar
regions worldwide [229]. Interestingly, a very recent report isolated Dysmorphococcus
globosus-HI, belonging to the family Phacotaceae within the order Chlamydomonadales,
class Chlorophyceae, from the Himalayan region of northern India, and it was found that
AX accounts for about 56% of the dry intracellular weight. However, reproducibility and
further studies on this report seem to be needed [243]. Additionally, many other phyto-
plankton also produce or accumulate carotenoids, including AX [244,245]. Therefore, it is
likely that the accumulation of carotenoids in higher animals through the food chain is a
result of the presence of carotenoids in plankton [246].
Mar. Drugs 2023, 21, 514 24 of 155
Figure 9. Life cycle of Haematococcus algae. (A) Three dierent cell morphologies of a typical Haem-
atococcus algae. (B) Life cycle: When old cultures are transplanted into fresh medium, coccoid
palmelloid cells undergo cell division to form agellated cells within the mother cell wall. After
germination, agellated cells sele and form palmelloid cells. Environmental stress such as strong
light, nutrient depletion, and/or high salinity accelerates the accumulation of astaxanthin during
encystment. This gure was reproduced with some additional information, citing ref [247] under
the terms of the Creative Commons Aribution License.
In these algae, AX provides three biological advantages to the algal cells: (1) stabili-
zation of quiescent cells. Some of these algae form non-motile, extra-long-life, environ-
mentally resistant cysts called aplanospores under severe environmental stress, as de-
scribed above. These cysts are quiescent cells similar to plant seeds, with decreased chlo-
roplast volume and a high accumulation of oil droplets containing high concentrations of
AX in the cytoplasm [223,247–251]. In the case of Haematococcus algae, mature cyst cells
(also often described as aplanospores [221,252] or akinetes [224]) exhibit a deep scarlet
color (Figure 9). The biological mechanism of this unique accumulation in oil droplets is
shown in detail in Section 2.3.1, which provides an example of the biosynthesis of AX in
Haematococcus algae. AX is believed to protect the cells and their stored components, such
as lipids and DNA, from the surrounding severe environment, including intense light ex-
posure [253]. (2) In snow algae, AX surrounds and masks the chloroplasts in the algae,
thereby promoting the ability to utilize red light wavelengths, which are much more ef-
fective for CO2 uptake than green or blue light [254]. (3) Similar to the putative role in (2),
it has been reported that in Haematococcus algae, AX in green-red non-motile cyst cells,
often called palmelloids before becoming scarlet-colored mature quiescent cyst cells, is
localized in the cell center (the nuclear periphery) under normal light conditions but rap-
idly diuses to the cell periphery when exposed to strong light. In cells where AX has
completely moved to the periphery, a thin layer of AX is identied just beneath the cell
wall, and if these cells are placed in the dark, AX redistributes to the center of the cell (the
nuclear periphery) [250,255]. It has been hypothesized that this translocation may be fa-
cilitated by actin laments [255]. In all cases (1) to (3), AX likely performs a similar physi-
Mar. Drugs 2023, 21, 514 25 of 155
ological function in terms of protecting photosynthetic organs and intracellular compo-
nents from intense light. Other organisms, such as E. sanguinea, which accumulates AX
fay acid esters [217,256], and C. astaxanthina Ki-4, which produces a complex of AstaP
and AX [257], s show similar responses that accumulate AX derivatives under intense
light, suggesting that AX plays the same role in a wide variety of microalgal species.
In terrestrial higher plants, carotenoids serve as accessory pigments in photosynthe-
sis. They capture light energy as antennae pigments and also quench reactive oxygen spe-
cies (ROS) generated by photosynthetic reaction centers under intense light [112] (see also
Section 2.1.7). These primary carotenoids are typically located in the thylakoid membranes
of chloroplasts and are essential for ecient photosynthetic reactions. They are biosyn-
thesized in chloroplasts and are present within chloroplasts as well [258,259]. However,
terrestrial higher plants often accumulate carotenoids in chromoplasts or lipid globules
that are not directly involved in photosynthesis. Examples include the carotenoids found
in fruits, owers, and roots of many plants, such as lycopene in tomatoes and β-carotene
in carrot roots. These carotenoids are known as secondary carotenoids [5,259]. AX is rarely
found as a major carotenoid involved in photosynthetic function in higher plants, as men-
tioned previously. Therefore, AX is considered a secondary carotenoid rather than a pri-
mary carotenoid essential for photosynthesis. Some earlier studies reported the presence
of AX in specic higher plants [260]. However, it was later conrmed that the reddish
carotenoids found in autumn leaves were actually rhodoxanthins and their metabolites
[261,262]. Currently, the only terrestrial plants where AX has been reliably identied as a
major carotenoid are the reddish petals of certain species belonging to the genus Adonis
of the Ranunculaceae family. In these plants, (3S,3′S)-AX is present in the form of fay
acid diesters [17,263–265]. Nevertheless, there remains a possibility that higher plants con-
taining AX will be discovered in the future, as undiscovered plant species may still exist
worldwide.
2.2.4. Eukaryotes; Animals
With a few exceptions in certain arthropods, all animals lack the ability to produce
carotenoids de novo [266]. Therefore, the AX that exists is either obtained from the diet or
metabolically converted from precursor carotenoids.
The coloration of invertebrates and poikilothermic vertebrates primarily depends on
the types of chromatophores present in their integuments (i.e., skin and exoskeleton) (see
Supplementary Table S3). While there are various types of chromatophores, both carote-
noids and phenolic pteridines contribute to the red and yellow color paerns. Specically,
red chromatophores, known as erythrophores, contain ketocarotenoids like AX. Research
on animal coloration, focusing on pigments, has predominantly examined ornamental
color signals that indicate animal maturity and provide reproductive advantages. Recent
hypotheses propose that oxidative stress plays a crucial role in maintaining the honesty
of condition-dependent carotenoid-based signaling [267–269]. This is due to the potential
of carotenoids to serve as honest indicators of phenotypic quality and, consequently, as
targets for resource allocation trade-o hypotheses. In many animals, the amount of pig-
ment available in vivo is limited, and its allocation may involve a trade-o between the
animal’s mature phenotype and various essential biological functions, such as the im-
mune system and antioxidant defense [270,271].
Concurrently, pigments have been extensively linked to prey-predator relationships
[272–275]. Numerous studies have demonstrated that prey species exhibit vibrant colors
as a means to signal the presence of predator defenses, thus serving as a mechanism to
deter predators. In other words, predators advertise their defenses by being conspicuous,
which enhances predator recognition and avoidance learning through uniform signaling.
Long-wavelength color patches, such as red, orange, and yellow, are recognized as eec-
tive components of many visual warning signals, particularly when combined with black
[276]. The red color derived from ketocarotenoids, including AX, holds signicant prom-
inence, especially for mammals like us.
Mar. Drugs 2023, 21, 514 26 of 155
In Case of Invertebrates
As mentioned above, AX contributes to the colorful body color and internal organs
of invertebrates. Information on the distribution and conversion of carotenoids in aquatic
organisms is detailed in Matsuno’s comprehensive review [21]. Similarly, another review
on stereoisomers of hydroxyl groups at the C3 and C’3 positions of AX in aquatic animals
is detailed in Matsuno et al. [277]. Therefore, in this review, we only provide information
on AX in particular. Some examples of these are shown below.
In luminal animals, AX and “nor”-carotenoids, such as 2-norastaxanthin and actini-
oerythrin (Supplementary Figure S3A), are present in sea anemones [278], where the car-
bon at the 2 or 2′ position of AX is detached. AX has also been reported in jellysh and
corals [279,280]. It is taken up from crustacean-like zooplankton as a dietary source
[280,281].
In mollusks, (3S,3′S)-AX has been found in coiled mollusks such as the spindle snail
(Fusinus perplexus) and the apple snail (Pomacea canaliculata) [21]. Triton’s trumpet also
contains AX, which is produced through the oxidative metabolic conversion of β-carotene
and (3R,3′R)-zeaxanthin from starsh [281]. It is a mixture of three optical isomers that are
taken up by the starsh when they feed on it. In bivalves, (3S,3′S)-AX is present as a trace
component along with pectenolone in the ovaries of the Yesso scallop (Mizuhopecten
yessoensis) [281,282]. Additionally, some Yesso scallops have orange adductor muscles
[283]. Moreover, various colorful shells are found in the noble scallop (Mimachlamys cras-
sicostata, synonym: Chlamys nobilis), with some individuals having golden shells and scal-
lops with golden inner tissues [284]. These individuals have a high accumulation of carot-
enoids. The reasons for the dierence in coloration of these bivalves are discussed in Sec-
tion 2.3.3. Interestingly, it has been reported that these golden individuals have higher
expression of antioxidant enzymes and improved immunological responses, as well as
increased resistance to low-temperature stress compared to their brown counterparts
[285–287]. AX is present in the viscera and ovaries of cephalopods (squids and octopuses)
as a mixture of three stereoisomers [288].
In arthropods, the optical isomer composition of AX can be divided into three groups:
(1) consisting of only one isomer or more than 90% of the dominant isomer; (2) mostly
containing two isomers with a trace amount or a small proportion of another isomer; and
(3) a mixture of all three isomers, with two at similar levels and the third in larger or
smaller quantities (Table 2). While the majority of arthropods rely on dietary carotenoids,
certain crustaceans, such as shrimps, prawns, and crabs, can convert β-carotene into AX
through oxidative reactions involving echinenone, canthaxanthin, and adonirubin. With
the exception of group (1), the AX in these cases consists of a mixture of three optical iso-
mers due to the likely lack of stereospecicity in introducing hydroxyl groups at the C3
and C3′ positions [281]. In other arthropods, the conversion to AX appears to utilize β-
carotene and zeaxanthin as substrates. Acari and copepods predominantly produce
(3S,3′S)-AXs through this process [289,290]. However, it is noteworthy that Antarctic krill
exhibits signicantly higher levels of AXs with the (3R,3′R) stereo conguration [277,291].
Further details are provided in Section 2.3.3. Additionally, these AXs can exist in free, mo-
noester, and diester forms.
Furthermore, some AXs are present as protein complexes called crustacyanins [83].
Detailed information on crustacyanins was provided in Section 2.1.5. In lobsters (Homarus
gammarus), crustacyanins, including β-crustacyanin (blue) and its octamer, α-crustacyanin
(purple), and crustochrin (a yellow analog of these proteins), are found in the exoskeleton
in the form of free AX associated stoichiometrically with apoproteins. Although the exo-
skeletons of these organisms are covered by a thick cuticular layer of epidermis, the local-
ization of these characteristic carotenoproteins diers. In a study involving whitened
American lobsters (H. americanus) fed a carotenoid-decient diet, the accumulation of AX
esters initially occurs in the epidermis, which then migrates and accumulates crustacya-
nins, resulting in the formation of a thick cuticle. Finally, multiple crustacyanin molecules
are stacked like plates within the epicuticular layer to form crustochrin [292]. This study
Mar. Drugs 2023, 21, 514 27 of 155
also demonstrated that UV irradiation promotes the accumulation of these carotenopro-
teins in the exoskeleton, suggesting that these pigments may serve as either cryptic color-
ation or protection against UV radiation [292]. On the outer surface of the carapace, where
crustochrin is present, AX exists as an H1-aggregate, exhibiting a typical hypsochromic
shift and absorbing light primarily in the UV region. Therefore, crustochrin itself exhibits
a UV-shielding eect. Furthermore, the H1-aggregate of AX is considered less reactive
with other compounds since there is no intermediate state for UV-induced triplet for-
mation, and singlet ssion occurs directly from the photoexcited state of 1Bu with an ul-
trafast time scale, which may be benecial for the survival of lobsters from a physicochem-
ical perspective [79] (see Section 2.1.6). An immunohistological study conducted on rock
lobsters (Panulirus cygnus) also revealed the presence of cluster cyanine subunits not only
in the cuticle layer of the exoskeleton but also in the epithelial layer. Moreover, P. cygnus
is characterized by various paerned areas on its shell, including ne red and white spots
and horizontal white stripes along the length of the shell. Immunohistochemical staining
showed that crustacyanins were restricted to the colored areas on the carapace and the
corresponding epidermis. These results suggest that the shell color paern is formed
through the expression of crustacyanins in the epithelium and their incorporation into the
exoskeleton, rather than solely the absence of carotenoid chromophores [86].
In a study of the characterization and metabolism of carotenoproteins during embry-
onic development of the lobster H. gammarus, larvae contained carotenoproteins of three
unknown structures (blue, red, and yellow) that were thought to be metabolized and
transferred by an unknown factor (referred to as “AX”) from ovoverdin, the major carot-
enoprotein in egg yolk. Additionally, the egg yolk carotenoids were identied as free AX
and adonirubin, while during tissue formation, AX was esteried. Therefore, the diester
of AX was found to be the major carotenoid in embryos and larval tissues. This AX diester
was found to be bound to red carotene proteins. The increase in esteried AX suggests
that an early enzymatic mechanism leading to the acylation reaction occurs during em-
bryogenesis [90].
AX has also been reported in insects, including grasshoppers [16,293,294] (Table 2).
In most cases, these AX compounds are derived from their diet within the food chain.
However, there are a few exceptions among arthropods, such as spider mites (Acari; e.g.,
Tetranychus urticae) [295,296] and aphids [297–299], which have the ability to synthesize
carotenoids de novo. These organisms possess orthologous genes for enzyme proteins,
namely CrtYB and CrtI, which are involved in the carotenoid biosynthetic pathway. It is
believed that these genes originated in fungi and were acquired through lateral gene
transfer [266,296,300] (for more details, refer to Section 2.3.3).
In echinoderms, it has been reported that AX exists as a mixture of three stereoiso-
mers in starsh, sea cucumbers, and sea urchins [301–303]. From starsh, AX and its tri-
ple-bonded derivatives, namely 7,8-didehydroastaxanthin and 7,8,7′,8′-tetradehy-
droastaxanthin (refer to Supplementary Figure S3B), have been identied [280,304]. Pre-
viously, 7,8-didehydroastaxanthin and 7,8,7′,8′-tetradehydroastaxanthin were referred to
as asterinic acid, named after the scientic name of the starsh species (Asterias rubens)
[304,305].
In protochordates, the presence of (3S,3′S)-AX has been reported in sea squirts (Hal-
ocynthia rorei), along with other major carotenoids [306].
In Case of Vertebrates
In sh, the presence of AX depends on the food chain, which means it is the result of
absorption, accumulation, or metabolism from the diet. The red color on the body surface
of many white meat sh species, apart from red meat sh, is aributed to AX and serves
as the basis for the characteristic body color of each species. The origin of these body colors
can be divided into the following paerns: (1) Salmonidae, including salmon and trout,
which contain AX in their skeletal muscles; and marine white meat shes with a red body
surface, such as red snapper; and (2) carp shes, which can metabolically convert AX from
Mar. Drugs 2023, 21, 514 28 of 155
zeaxanthin. The former accumulates AX derived from crustaceans, which are their major
food source, resulting in a mixture of three stereoisomers of AX [281]. The laer metabol-
ically converts (3R,3′R)-zeaxanthin as a precursor, leading to the presence of only (3S,3′S)-
AX [24,307]. Due to these characteristics, AX is often used as a coloring agent for snapper
and salmon, while zeaxanthin is used as a coloring agent for goldsh (colored varieties of
Carassius auratus) and Nishikigoi (colored varieties of Cyprinus carpio) [307]. Moreover,
marine sh are frequently fed zeaxanthin as a coloring agent (Supplementary Figure S5).
The metabolism and conversion to and from AX will be discussed in detail in Section 2.3.3.
In the case of salmonid sh, AX is believed to originate from minuscule arthropods
referred to as zooplankton. These zooplankton serve as prey for salmonid shes, which in
turn accumulate the pigments derived from them (refer to Table 2). Furthermore, there are
signicant dierences in carotenoid composition between land-locked and migratory
forms of salmonids due to variations in their dietary habits. Among salmonid shes, the
highest concentrations of AX have been reported in Sockeye salmon, and the ratio of AX
to total carotenoids is also high in their muscles and eggs [308] (Table 2).
Additionally, numerous sh species, particularly those belonging to the red-eshed
and salmonid categories, undergo metabolic conversion of AX into yellow xanthophylls
like tunaxanthin or salmoxanthin via a reductive pathway. These yellow pigments are
then deposited and stored in their integument (skin). Further elaboration on these pro-
cesses will be provided in the forthcoming Section 2.3.3.
In amphibians, the presence of AX has been reported in newts and frogs/toads. In
these organisms, AX exists in both free and ester forms (Table 2). It is believed that AX,
along with other carotenoids, plays a signicant role in warning coloration against pred-
ators and in the nuptial coloration that indicates individual maturity. Carotenoids respon-
sible for body coloration are thought to be present in the dermis as xanthophores and
erythrophores, as well as in liver tissue [309]. However, their specic forms in other tissues
remain unclear. Similar to red-skinned sh, AX is believed to be present in erythrospores
found in integument tissues. Since many amphibians feed on insects, it is possible that
some carotenoids derived from arthropods, mainly carotenes, accumulate intact or un-
dergo oxidative conversion to become AX. Unfortunately, many of the studies on this
topic are relatively old and were conducted before the prevalence of HPLC. With a few
exceptions, such as the Japanese newt studies, they provide limited information [309,310].
Therefore, further instrumental analysis is required to determine the precise form in
which AX exists. Recently, it was reported that certain frogs may possess enzymatic genes
that facilitate the conversion of C4-ketocarotenoids, such as AX, through oxidative metab-
olism [294] (for more details, refer to Section 2.3.3.).
In reptiles, certain lizards and snakes display vibrant ornamental colors on their skin
as a sign of maturity. These colors often include shades of yellow and red, which are at-
tributed to the accumulation of high concentrations of carotenoids and phenolic pigments,
such as pteridines. These orange to red pigments are typically found in the form of keto-
carotenoids and, in some cases, contain high levels of AX (Table 2). In reptiles, these keto-
carotenoids accumulate in the dermis within erythrophores [311–313]. Similar to reptiles,
avian species also exhibit red-orange ornamental colors derived from C4-ketocarotenoids
like AX [314]. In birds, AX has been reported in numerous species [314], most notably
contributing to the body coloration of amingos [15]. Interestingly, the ratio of AX to total
carotenoids in amingo feathers is relatively low, with canthaxanthin being the most
abundant ketocarotenoid in many species. However, all species still contain signicant
amounts of AX, particularly in the legs of American amingos [315]. While in most bird
species, AX is visually identiable in feathers, beaks, and legs, it has also been found in
the skin beneath the plumage and other body tissues (e.g., liver, blood, etc.). As with other
animals, there exists a trade-o between carotenoid ornamentation of the body surface
and other physiological functions due to the limited dietary supply of carotenoids. These
ketocarotenoids were initially believed to indicate individual maturity and be involved in
defense against oxidative stress and regulation of immunity. However, the results have
Mar. Drugs 2023, 21, 514 29 of 155
been inconsistent, and meta-analysis suggests that their presence reects individual qual-
ity rather than consistently reducing oxidative stress [269]. The metabolism of these pig-
ments is discussed in Section 2.3.3.
Considering the localization of AX in avian tissues, it is believed to coexist with col-
lagen matrices and lipids in the subcutaneous tissue, dermis, and epidermis. Unlike rep-
tiles, birds lack specic chromatophores other than melanocytes in their skin tissue, in-
cluding erythrophores (see Supplementary Table S3). In feathers, AX is presumed to be
present within the keratin matrices. Interestingly, AX is also found in the retinas of various
bird species and turtles. It has been hypothesized that AX, along with other xanthophylls,
may serve to protect retinal cells, particularly longwave-sensitive (LWS) cone cells, by act-
ing as cut-o lters against blue light, especially in situations with intense light exposure
[316–318]. Birds exhibit well-dened cone cells in the retina with oil droplets that contain
specic carotenoids, including AX (R-type). These carotenoids are believed to have a
wavelength-cuing eect on light, enhancing sensitivity in their respective wavelength
ranges [316–318]. In LWS cone cells, the dominant optical isomers of AX are (3S,3′S)-AX
[319]. In the majority of bird species, AX and other ketocarotenoids are presumed to be
present in the retina, even in those without AX pigmentation in their plumage, with a few
exceptions found in phylogenetic groups adapted to low-light environments, such as pen-
guins and owls [320–322]. Penguins and owls inhabit nocturnal or deep-diving habitats
and may have evolved colorless cone oil droplets to avoid interference with their vision,
as colored droplets could impact their visual perception [318]. The functional role and
evolutionary signicance of these cone oil droplets are discussed in greater detail in other
reviews (refer to [318,323]).
In addition to color paerns, some animals utilize polarized light paerns as a means
of communication. The biological polarizers found in nature rely on physical interactions
with light, including birefringence, dierential reection, and scaering. Interestingly,
among invertebrates, a species called the marine stomatopod crustacean, specically the
mantis shrimp (Odontodactylus scyllarus), possesses a unique biological polarizer based on
the linear dichroism of carotenoid molecules in its antennal scale. These creatures are be-
lieved to have a fundamentally dierent color recognition system from ours and to per-
ceive and experience the world of colors in a distinct manner [324]. In the mantis shrimp,
AX is deposited on the antennal scales, and the presence of AX in this dichroic compound
allows the scales to polarize light. Within these antennal scales, AX exists as an optical
mixture. By utilizing AX as a dichroic material in their antennal scales, mantis shrimps are
able to manipulate the polarization component of light rather than its color, generating
signals that vary with the direction of polarization [325]. Hence, AX plays a crucial role in
the visual function of diverse organisms, including those employing polarized light com-
munication.
In mammals, studies analyzing blood carotenoids have demonstrated that humans
are capable of absorbing AX. Additionally, mice and rats have been observed to transfer
AX to various tissues, including adipose tissue, liver, skeletal muscle, brain, and others
[250,326]. More extensive information on these ndings can be found in Section 4.1.3.
To summarize, animals discussed in Sections 2.2.3 and 2.2.4 accumulate AX on their
body surfaces, utilizing it for breeding purposes or as cryptic/warning coloration. AX is
also found in abundance in eggs. It is speculated that AX may oer protection to eggs
against light and reactive oxygen species (ROS) and is believed to be involved in repro-
ductive functions, hatching, post-hatching survival, and growth. Further details and hy-
potheses regarding these aspects can be found in Sections 3 and 5.
2.2.5. Astaxanthin Content in Various Organisms
The content of AX per 100 g in various organisms is as follows: lobster/craysh—0.1–
0.3 mg in the whole body, Sockeye salmon muscle and eggs—2.0–3.8 mg, krill—2.0–4.0
mg, Phaa yeast—20–1000 mg [327], dry biomass of Paracoccus—20–2000 mg, and mature
cyst cells of Haematococcus algae—up to 9800 mg [328,329] (Table 2).
Mar. Drugs 2023, 21, 514 30 of 155
Among the reported species, Haematococcus algae has the highest AX content by a
signicant margin. Additionally, the purity of AX as a carotenoid is also high in Haemato-
coccus algae. Therefore, the current industrial production of AX, apart from chemical syn-
thesis, primarily relies on Haematococcus algae.
As a result, most of the evidence supporting the use of AX as a dietary supplement
for humans is based on Hematococcus algae. Other sources of AX, whether biological or
synthetic, have limited applications in human consumption but are widely used in animal
feed. Additionally, some products, such as krill oil, are valued for their pharmaceutical
benets derived from other components, like omega-3 fay acids, rather than solely rely-
ing on the eects of AX. Consequently, in this article, the focus on human use of AX will
primarily revolve around Hematococcus algae.
Mar. Drugs 2023, 21, 514 31 of 155
Table 2. Astaxanthin: Summary of its presence in various organisms and its origin.
Taxon
Scientific Name
Common Name
Astaxanthin
Reference
Form †
Stereoisomer
(3R,3′R, meso, 3S,3′S)
Content (mg/100g)
Origin
Bacteria, Prtoteobacteria, Alphaproteobacteria
Paracoccus carotinifaciens
(w/. mutation)
PanaFerd-AX
Free form
3S,3′S
2180
(50.2% of total Car)
De novo
synthesis
[329,330]
Paracoccus sp. strain N81106
(NBRC 101723)
(Agrobacterium auranticum)
(w/. mutation)
N/A
Free form and glycoside
3S,3′S
~800
(63.2% of total Car)
De novo
synthesis
[331]
Brevundimonas sp. M7
(w/. mutation)
N/A
Free form **
3S,3′S **
130
De novo
Synthesis **
[186]
Sphingomonas astaxanthinifaciens
TDMA-17
N/A
Free form
3S,3′S **
96.0
(34.3% of total Car)
De novo
Synthesis **
[182]
Paracoccus haeundaensis
KCCM 10460
(Co-culture w/. Lactic Acid Bacteria)
N/A
Free form
3S,3′S **
82.1
De novo
synthesis
[332]
Paracoccus bogoriensis BOG6T (DSM16578, LMG2279)
N/A
Free form
3S,3′S
40
(10.8% of total Car)
De novo
synthesis
[183]
Brevundimonas spp.
N/A
Free form **
3S,3′S **
2.8 ~36.5
De novo
Synthesis **
[186]
Sphingomicrobium astaxanthinifaciens
CC-AMO-30B
N/A
Free form
3S,3′S **
4.0
De novo
Synthesis **
[185]
Brevundimonas sp. strain SD212
(NBRC 101024)
N/A
Free form
3S,3′S
N/A
(9.9% of total Car)
De novo
synthesis
[181]
Archaea
Halobacterium salinarium NRC-1
N/A
Free form ‡
N/D
26.5
(c.a.73% of total Car)
De novo
Synthesis *
[203]
Haloarcula hispanica ATCC 33960
N/A
Free form ‡
N/D
1.7
(c.a.1.3% of total Car)
De novo
Synthesis*
[203]
Eukaryota, Fungi
Xanthophyllomyces dendrorhous
(ATCC SD 5340)
Phaffia Yeast
Free form
3R,3′R
723.5~1247.8
(c.a. 73% of total Car)
De novo
synthesis
[327,333]
Eukaryota, Plantae
Adonis amurensis
(Reddish flower varieties)
Amur adonis,
pheasant’s eye
Fatty acid esters
3S,3′S **
~3310
(in upper red part of petal)
(c.a. 70% of total Car)
De novo
Synthesis **
[334]
Mar. Drugs 2023, 21, 514 32 of 155
Adonis annua
Autumn pheasant’s eye, blood-
drops
Fatty acid esters
3S,3′S
120 ~1000
(in dry petal)
(c.a. 75% of total Car)
De novo
Synthesis **
[17,50]
Adonis aestivalis
Summer pheasant’s eye
Fatty acid esters
3S,3′S
166
(in wet petal)
(87.4% of total Car)
De novo
synthesis
[265]
Eukaryota, Plantae, Chlorophyta
Haematococcus lacustris
Haematococcus
pluvilialis,
Haematococcus algae
Fatty acid esters
3S,3′S
~9800
(in red cyst)
(>90% of total Car)
De novo
synthesis
[64,328]
Neochloris wimmeri CCAP-213/4
N/A
Fatty acid esters
3S,3′S **
~1920
(c.a 85% of total Car)
De novo
Synthesis **
[227,228,232]
Asterarcys quadricellulare PUMCC 5.1.1
N/A
N/A
3S,3′S**
~1550
(c.a 13% of total Car)
De novo
Synthesis **
[236]
Protosiphon botryoides SAG-731/1a
N/A
Fatty acid esters
3S,3′S**
~1430
(c.a 80% of total Car)
De novo
Synthesis **
[227,228]
Scotiellopsis oocystiformis
SAG-277/1
N/A
Fatty acid esters
3S,3′S **
~1090
(c.a 70% of total Car)
De novo
Synthesis **
[227,228]
Chlorococcum sp.
N/A
Fatty acid esters
3S,3′S **
~c.a. 700
(c.a. 32% of total Car)
De novo
Synthesis **
[335–337]
Chlorella zofingiensis SAG-211/14
Chlorella
Fatty acid esters
3S,3′S **
~680
(c.a. 75% of total Car)
De novo
Synthesis **
[227,228]
Scenedesmus vacuolatus
SAG-211/15
N/A
Fatty acid esters
3S,3′S **
~270
(40–50% of total Car)
De novo
Synthesis **
[227,228]
Chlamydocapsa spp.
Strain 101-99/R2
N/A
N/A
3S,3′S **
~44.4
(20.3% of total Car)
De novo
Synthesis **
[338]
Neochloris oleoabundans UTEX#1185
N/A
Fatty acid esters
3S,3′S **
N/A
De novo
Synthesis **
[232]
Dysmorphococcus globosus-HI
N/A
Free form/
Fatty acid esters
3S,3′S **
~517,090??
De novo
Synthesis **
[243]
Eukaryota, Chromista, Bigyra, Labyrinthulomycetes
Aurantiochytrium sp. RH-7A-7
(w/. mutation)
Labyrinthulomycetes
N/A
3S,3′S **
-470
(c.a .85% of total Car)
De novo
Synthesis*
[218]
Thraustochytrium sp. CHN-3
(FERM P-18556)
Labyrinthulomycetes
Free form **
3S,3′S **
~280
(~60% of total Car)
De novo
Synthesis *
[339]
Aurantiochytrium sp. KH-10
Labyrinthulomycetes
Fatty acid esters/
Free form
3S,3′S **
~81
(28% of total Car)
De novo
Synthesis *
[340]
Thraustochytrium sp. CHN-1
Labyrinthulomycetes
Free form
3S,3′S
50
(c.a. 50% of total Car)
De novo
Synthesis *
[341,342]
Eukaryota, Chromista, Gyrista, Eustigmatales
Mar. Drugs 2023, 21, 514 33 of 155
Nannochloropsis gaditana strain S4
(w/. mutation)
Nannochloropsis
Free form
3S,3′S?
~219
(14.4% of total Car)
De novo
Synthesis
[219]
Nannochloropsis oculata
Nannochloropsis
Free form
3S,3′S?
3.4 ng/106 cells
De novo
Synthesis
[343]
Nannochloropsis salina
Nannochloropsis
Free form
3S,3′S?
9.6 ng/106 cells
De novo
Synthesis
[343]
Eukaryota, Excavata, Euglenozoa
Euglena sanguinea
Euglena
Fatty acid
esters/free
3S,3′S
~1.9
(80% of total Car)
De novo
Synthesis
[256,344]
Trachelomonas volvocina
Euglena
Fatty acid esters/
Free form
3S,3′S *
N/A
De novo
Synthesis
[215]
Animals (Invertebrate), Coelenterata
Velella velella
By-the-wind sailor
(Jerry fish)
Free form
Mixtures of
stereoisomers
N/A
Accumulated from die-
tary Crustaceans
[98]
Aurelia aurita
(Jerry fish)
Fee form/
Fatty acid esters (minor)
N/A
12.2
(c.a.67% of total Car)
N/A
[279]
Metridium senile var. fimbriatum
Frilled anemone
(Sea anemone)
Fatty acid esters
(in ovary)
Mixtures of
stereoisomers
N/A
Oxidative metabolite of
β-carotene
[345,346]
Corynactis californica
Strawberry anemone
(Sea anemone)
Fatty acid
esters
N/A
N/A
Oxidative
metabolite of
β-carotene
[347]
Animals (Invertebrate), Mollusca, Gastropoda
Clione limacina
Sea angel
Free form
3S,3′S
0.051
(1.1% of total Car)
Oxidaive
metabolite of
zeaxanthin
[348]
Paedoclione doliiformis
Sea angel
Free form
3S,3′S
0.8
(5.5% of total Car)
Oxidaive
metabolite of
zeaxanthin
[348]
Semisulcospira libertina
Terestorial Snail
(Kawanina in Japanese)
Free form
3S,3′S
0.2
(6.5% of total Car)
Oxidaive metabolite of
zeaxanthin
[349]
Fushinus perplexu
Spindle shell
Free form
3S,3′S
0.2
(4.0% of total Car)
Oxidative
metabolite of
β-carotene
[350]
Pomacea canaliculata
Apple snail
Free form
3S,3′S
5.0 in gonad, 2.31 in egg
(~75% of total Car)
Oxidative
metabolite of
β-carotene
[351]
Animals (Invertebrate), Mollusca, Cephalopoda
Octopus vulgaris
Common octopus
Fatty acid esters/
Free form
Mixtures of stereoiso-
mers
(46:22: 32)
3.2 in liver
(c.a.80% of total Car)
Accumulated from die-
tary crustaceans
[288]
Mar. Drugs 2023, 21, 514 34 of 155
Watasenia scintillans
Firefly squid
Fatty acid esters/
Free form
Mixtures of stereoiso-
mers
(40: 6: 54)
5.0 in liver
(>90% of total Car)
Accumulated from die-
tary crustaceans
[288]
Animals (Invertebrate), Mollusca, Polyplacophora
Placiphorella japoonica
Chiton
Free form
Mixtures of stereoiso-
mers
(5:3:2)
1.25
(~34% of total Car)
Oxidative
metabolite of
β-carotene
[352]
Acanthochitona defilippii
Chiton
Free form
3S,3′S
1.55 in gonad
(~4.0% of total Car)
Oxidative
metabolite of
β-carotene
[352]
Liolophura japonica
Chiton
Free form
3S,3′S
0.8 in viscera
(~10% of total Car)
Oxidative
metabolite of
β-carotene
[352]
Animals (Invertebrate), Echinodermata
Peronella japonica
Sea urchin
Free form
Mixtures of stereoiso-
mers
(3:7:90)
~3.0 in gonad
(c.a.43% of total Car)
Oxidative
metabolite of
β-carotene
[301]
Asteria pectinifera
Starfish
Free form
Mixtures of stereoiso-
mers
(50:25:25)
~1.35
(% of total Car)
Oxidative
metabolite of
β-carotene
[305]
Asterias amurensis
Starfish
Free form
Mixtures of stereoiso-
mers
(48:25:27)
~4.64
(% of total Car)
Oxidative
metabolite of
β-carotene
[305]
Animals (Invertebrate), Arthropoda, Crustacea, Decapoda (Lobsters, rock lobsters and crawfishes)
Procambarus clarkii
Louisiana crawfish
Fatty acid ester/
Free form
Mixtures of stereoiso-
mers
7.9–19.8 in carapace
Oxidative
metabolite of
β-carotene
[353]
Pontastacus leptodactylus
(Astacus leptodactylus)
Turkish crayfish
Fatty acid esters
/Free form
Mixtures of stereoiso-
mers **
5.0 in carapace
0.13 in muscle
0.98 in intestine
(82.5% of total Car)
Oxidative
metabolite of
β-carotene **
[354]
Panulirus japonicus
Japanese Spiny Lobster
(Ise-ebi)
Free form/
Fatty acid esters
Mixtures of stereoiso-
mers
(20:20:56)
3.3 in carapace
(65% of total Car)
Oxidative
metabolite of
β-carotene
[355]
Animals (Invertebrate), Arthropoda, Crustacea, Copepoda
Tigriopus californicus
Red marine copepod
Free form
( major)
3S,3′S
(major)
~423
Oxidative
metabolite of
β-carotene
[290,356]
Animals (Invertebrate), Arthropoda, Crustacea, Eucarida
Euphausia superba
Antarctic krill
Fatty acid esters/
3R,3′R
~566 in eye
Oxidative
[291,357]
Mar. Drugs 2023, 21, 514 35 of 155
Free form
(Major, ~70%)
metabolite of
β-carotene ?
Euphausia pacifica
Pacific krill (Isada)
Fatty acid esters/
Free form
3R,3′R
(Major)
~ 252 in eye
Oxidative
metabolite of
β-carotene ?
[357,358]
Animals (Invertebrate), Arthropoda, Crustacea, Decapoda (Prawns and shrimps) ***
Pandalus borealis
Atlantic shrimp
(Northern prawn)
Fatty acid esters/
Free form
Mixtures of
stereoisomers
(25:52:23)
~28.48 in carapace
Oxidative
metabolite of
β-carotene
[62,359,360]
Penaeus japonicus
Japanese tiger prawn
(Kuruma-ebi)
Fatty acid esters/
Free form
Mixtures of stereoiso-
mers
(12:40:48)
~13 in carapace
Oxidative
metabolite of
β-carotene
[361,362]
Penaeus semisulcatus
Green tiger prawn
Fatty acid esters/
Free form
Mixtures of stereoiso-
mers
(19:44:57)
~15.6 in carapace
Oxidative
metabolite of
β-carotene
[362]
Penaeus monodon
Black tiger prawn
Fatty acid esters/
Free form
Mixtures of stereoiso-
mers
(16:43:41)
~7.3 in carapace
Oxidative
metabolite of
β-carotene
[362]
Litopenaeus vannamei
Whiteleg shrimp
Fatty acid esters/
Free form
Mixtures of stereoiso-
mers
(23:44:32)
~5.8 in carapace
Oxidative
metabolite of
β-carotene
[362]
Metapenaeus joyneri
Shiba shrimp
Fatty acid esters/
Free form
Mixtures of stereoiso-
mers
(14:46:40)
~3.3 in carapace
Oxidative
metabolite of
β-carotene
[362]
Animals (Invertebrate), Arthropoda, Crustacea, Decapoda, Brachyura (Crabs) ***
Chionoecetes japonicus
Red snow crab
(Beni-zuwai crab)
Fatty acid esters/
Free form**
Mixtures of stereoiso-
mers **
~23 in carapace
(with demineralization treatment)
Oxidative metabolite
of β-carotene?
[363]
Chionoecetes opilio
Snow crab
(Zuwai crab)
Fatty acid esters/
Free form
Mixtures of stereoiso-
mers?
~11.9 in carapace
(~91.7% of total Car)
Oxidative metabolite
of β-carotene?
[364]
Callinectes sapidus
Blue crab
Fatty acid esters/
Free form
Mixtures of stereoiso-
mers?
~9.8
(with demineralization treatment)
Oxidative metabolite
of β-carotene?
[365]
Cancer pagurus
Brown crab
Fatty acid esters/
Free form
Mixtures of stereoiso-
mers
(56:24:20)
0.37 in carapace
Oxidative metabolite
of β-carotene?
[366,367]
Animals (Invertebrate), Arthropoda, Crustacea, Decapoda (Others) ***
Paralithodes brevipes
Hanasaki crab
Fatty acid esters/
Free form
Mixtures of
stereoisomers
(26：9：6)
~2.4 in carapace
(~39.9% of total Car)
Oxidative
metabolite of
β-carotene
[368]
Paralithodes camtschaticus
Red king crab
Fatty acid esters/
Free form
Mixtures of
stereoisomers
~0.35 in carapace
(~97% of total Car)
Oxidative
metabolite of
[277,369]
Mar. Drugs 2023, 21, 514 36 of 155
(45–55：7–19：27–48)
β-carotene
Cervimunida princeps
Squat lobster
Fatty acid esters/
Free form
Mixtures of
stereoisomers
(26：9：65)
~ 0.45 in carapace
(~100% of total Car)
Oxidative
metabolite of
β-carotene
[369]
Upogebia major
Japanese mud shrimp
Fatty acid esters/
Free form
Mixtures of stereoiso-
mers
(72：21：7)
~ 0.25 in carapace
(~100% of total Car)
Oxidative
metabolite of
β-carotene
[369]
Birgus latro
Coconut crab
Fatty acid esters/
Free form
Mixtures of stereoiso-
mers
(9: 41: 50)
~ 0.3 in carapace
(~96% of total Car)
Oxidative
metabolite of
β-carotene
[370]
Asellus aquaticus
Isopoda
Free form/
Fatty acid esters?
N/A
~0.52
(~37.5% of total Car)
Oxidative
metabolite of
β-carotene ?
[371]
Pleuroncodes planipes
Red crab langostilla
Fatty acid esters/
Free form
Mixtures of stereoiso-
mers
(3–4:1:3–4)
N/A
Oxidative
metabolite of
β-carotene ?
[372]
Animals (Invertebrate), Arthropoda, Arachnida, Acari
Balaustium murorum
Red velvet mite
Free form/
Fatty acid esters
3S,3′S **
~61,530 mg/
100 g protein
(60% of total Car)
Oxidative metabolite of
zeaxanthin (De novo
Synthesis **)
[373]
Panonychus citri
Citrus red mite
Fatty acid esters
3S,3′S**
~263 mg/
100 g protein
(42.5% of total Car)
De novo
Synthesis **
[374]
Tetranychus kanzawai
Kanzawa spider mite
Fatty acid esters
3S,3′S**
Undefined
De novo
Synthesis
[375]
Tetranychus urticae
Two-spotted spider mite
Fatty acid esters
3S,3′S**
Undefined
De novo
Synthesis
[296,376]
Eylais hamata
Hydracarina
Free form/
Fatty acid esters (minor)
N/A
12.2
(c.a.67% of total Car)
N/A
[377]
Eylais extendens
Hydracarina
N/A
N/A
Undefined
(c.a.70% of total Car)
N/A
[378]
Schizonobia sycophanta
Parasite mite
Fatty acid ester
3S,3′S
Undefined
(30% of total Car)
De novo
Synthesis **
[49,289]
Animals (Invertebrate), Arthropoda, Arachnida, Araneae
Trichonephila clavata
Arachnida spider
??
Mixtures of
stereoisomers
(2:1:1)
0.02
(1.9% of total Car)
Oxidative metabolite of
β-carotene
[299]
Animals (Invertebrate), Arthropoda, Insecta
Locusta migratoria
Migratory locust
Free form
Mixtures of
stereoisomers
0.25
in brown form
Oxidative metabolite of
β-carotene
[16,293]
Mar. Drugs 2023, 21, 514 37 of 155
(2:1:1)
(12.5% of total Car)
Aiolopus thalassinus tamulus
Grasshopper
Free form
Mixtures of
stereoisomers
(2:1:1)
0.09
in brown form
(3.0% of total Car)
Oxidative metabolite of
β-carotene
[293]
Schistocerca gregaria
Desert locust
Free form
Mixtures of
stereoisomers *
N/A
Oxidative metabolite of
β-carotene
[16]
Animals (Vertebrate), Fish (Salmonidae)
Oncorhynchus nerka
(Wild, anadromous form)
Sockeye salmon
Free form
(muscle/egg)/
Fatty acid esters
(skin)
Mixtures of stereoiso-
mers
(depending on AX
source)
2.6–3.8 in muscle
in egg
(% of total Car)
Accumulated from die-
tary crustaceans
[308]
Oncorhynchus nerka
(Wild, non-anadromous form)
Kokanee salmon
Free form
(muscle/egg)/
Fatty acid esters
(skin)
Mixtures of stereoiso-
mers
(depending on AX
source)
−0.8 in muscle
0.4–2.8 in skin
−1.1 in golnad
(−94% of total Car)
Accumulated from die-
tary crustaceans
[379,380]
Oncorhynchus kisutch
Coho salmon
Free form
(muscle/egg)/
Fatty acid esters
(skin)
Mixtures of stereoiso-
mers
(depending on AX
source)
1.0–2.1 in muscle
in egg
(% of total Car)
Accumulated from die-
tary crustaceans
[308]
Salvelinus alpinus
(Wild)
Arctic char
Free form
(muscle/egg)/
Fatty acid esters
(skin)
Mixtures of stereoiso-
mers
(depending on AX
source)
0.86 in muscle
in egg
(−30% of total Car)
Accumulated from die-
tary crustaceans
[308,381]
Salmo salar
(Wild)
Atlantic salmon
Free form
(muscle/egg)/
Fatty acid esters
(skin)
Mixtures of stereoiso-
mers
(depending on AX
source)
0.6–0.8 in muscle
in egg
(% of total Car)
Accumulated from die-
tary crustaceans
[308,382]
Oncorhynchus keta
Chum salmon
Free form
(muscle/egg)/
Fatty acid esters
(skin)
Mainly 3S,3′S
(in ovary)
0.1–0.5 in muscle
−0.7 in egg
0.1 in skin (male)
(4.8–90% of total Car)
Accumulated from die-
tary crustaceans
[28,29,308,383]
Oncorhynchus gorbuscha
Pink salmon
Free form
(muscle/egg)/
Fatty acid esters
(skin)
Mixtures of stereoiso-
mers
0.4–0.7 in muscle
in egg
(% of total Car)
Accumulated from die-
tary crustaceans
[308]
Oncorhynchus tshawytscha
Chinook salmon
Free form
(muscle/egg)/
Fatty acid esters
(skin)
Mixtures of stereoiso-
mers
0.54 in muscle
in egg
(% of total Car)
Accumulated from die-
tary crustaceans
[308]
Mar. Drugs 2023, 21, 514 38 of 155
Oncorhynchus masou
(Wild, anadromous form)
Masu salmon
Free form
(muscle/egg)/
Fatty acid esters
(skin)
Mixtures of stereoiso-
mers
0.3–0.8 in muscle
0.03–0.8 in skin
0.7–1.7 in egg
(1.9–80% of total Car)
Accumulated from die-
tary crustaceans
[308,380,384]
Oncorhynchus masou ishikawae
(Wild, anadromous form)
Red-spotted masu salmon
Free form
(muscle/egg)/
Fatty acid esters
(skin)
Mixtures of stereoiso-
mers
0.2 in muscle
trace in skin
N/D in egg
(1.9–68.5% of total Car)
Accumulated from die-
tary crustaceans
[380]
Oncorhynchus masou rhodurus
Biwa trout
Free form
(muscle/egg)/
Fatty acid esters
(skin)
Mixtures of
stereoisomers
0.2 in muscle
−0.1 in skin
(3.2–58.3% of total Car)
Accumulated from die-
tary crustaceans
[380]
Oncorhynchus mykiss
(Wild, pigmented phenotype)
Rainbow trout
Free form
(muscle/egg)/
Fatty acid esters
(skin)
Mixtures of
stereoisomers
trace in muscle
0.8 in skin
trace in egg
(1.9–42.3% of total Car)
Accumulated from die-
tary crustaceans
[385]
Animals (Vertebrate), Fish (Non-Salmonidae )
Sebastolobus macrochir
Broadbanded thornyhead
(Kichiji rockfish)
Fatty acid esters
(skin)
N/A
26 in skin
(>90% of total Car)
Accumulated from die-
tary crustaceans
or
Oxidative metabolite of
β-carotene/
zeaxanthin?
[386]
Plectropomus leopardus
Coral trout
(Suziara)
Fatty acid esters/
Free form
(skin)
Mixtures of stereoiso-
mers
(13:7:80)
19.5 in skin
(84.8% of total Car)
Accumulated from die-
tary crustaceans
or
Oxidative
metabolite of
β-carotene/
zeaxanthin?
[387]
Epinephelus fasciatus
Blacktip grouper
(Akahata)
Fatty acid esters
(skin)
N/A
2.27 in skin
(74% of total Car)
Accumulated from die-
tary crustaceans
or
Oxidative
metabolite of
β-carotene/
zeaxanthin?
[386]
Beryx splendens
Splendid alfonsino
(Kinmedai)
Fatty acid esters
(skin)
Mixtures of stereoiso-
mers
0.9 in skin
(c.a. 100% of total Car)
Accumulated from die-
tary crustaceans
or
Oxidative
[386]
Mar. Drugs 2023, 21, 514 39 of 155
metabolite of
β-carotene/
zeaxanthin?
Pagrus malor
Red sea bream
(Madai)
Fatty acid esters
(skin)
Mixtures of stereoiso-
mers
(38：0：62)
~2 in skin (wild)
0.25 in skin (firmed w/o. AX) /
0.98 (firmed with. AX)
(~c.a 45% of total Car)
Accumulated from die-
tary crustaceans and
supplementary pig-
ment
[277,386,388]
Carassius auratus
Goldfish
(Kingyo/Hibuna)
Free/ Fatty acid esters
(skin)
3S,3′S
0.58 (whole body)
(~47% of total Car)
Oxidative metabolite of
β-carotene/
zeaxanthin
[307]
Branchiostegus japonicus
Red tilefish
(Red amadai)
Fatty acid esters
(skin)
Mixtures of stereoiso-
mers
(24：24：52)
0.39 in skin
(35.8% of total Car)
Accumulated from die-
tary crustaceans
[389]
Animals (Vertebrate), Amphibian
Cynops pyrrhogaster
Japanese newt
Free form/
Fatty acid esters
N/A
4.55 in skin
(c.a.21% of total Car)
Oxidative
metabolite
of
β-carotene/
zeaxanthin
[309]
Salamandra salamandra
Fire salamander
Free form/
Fatty acid esters
N/A
0.23
(37.5% of total Car)
Oxidative
metabolite
of
β-carotene/
zeaxanthin
[390]
Lissotriton vulgaris
(Triturus vulgaris)
Smooth newt/common newt
Free form
N/A
0.1
(−23.5% of total Car)
Oxidative
metabolite
of
β-carotene/
zeaxanthin
[390]
Ranitomeya sirensis
Sira poison frog
Free form/
Fatty acid esters ?
N/A
N/A
(-c.a. 40% of total Car)
Oxidative
metabolite
of
β-carotene/
zeaxanthin
[294]
Bufo bufo
Common toad
Free form/
Fatty acid esters
N/A
N/D in muscle and liver
0.02 in skin
0.35 in intestine
(25.8–57.4% of total Car)
0.23 in gonads
(95.8% of total Car)
Oxidative
metabolite
of
β-carotene/
zeaxanthin
[390]
Bufotes viridis
European green toad
Free form/
N/A
N/D in muscle and liver
Oxidative
[391]
Mar. Drugs 2023, 21, 514 40 of 155
(Bufo viridis)
Fatty acid esters
0.02 in skin
0.35 in intestine
(25.8–57.4% of total Car)
0.23 in gonads
(95.8% of total Car)
metabolite
of
β-carotene/
zeaxanthin
Pelobates fuscus
European common spadefoot
toad
Free form
N/A
1.1 in liver
(19.6% of total Car)
Oxidative
metabolite
of
β-carotene/
zeaxanthin **
[390]
Melanophryniscus rubriventris
(Aposematic poison toad)
N/A
(Free form/ ester form?)
N/A
Undefined
Oxidative
metabolite of
β-carotene **
[392]
Animals (Invertebrate), Reptile
Chlamydosaurus kingii.
(the western red-frilled form)
Frillneck lizard
N/A
(Free form?)
N/A
Undefined
Oxidative
metabolite of
β-carotene
[312]
Chrysemys picta
Painted turtle
N/A
N/A
c.a 0.11 in leg skin
Oxidative
metabolite of
β-carotene
[393]
Trachemys scripta
Red-eared slider
N/A
N/A
c.a 0.06
in skin around eye spot
Oxidative
metabolite of
β-carotene
[393]
Animals (Invertebrate), Aves ***
Lagopus lagopus scoticus
Red grouse
Free form/
Fatty acid esters
N/A
317.8 in combs
N/D in plasma
(-c.a.81.6% of total Car)
Oxidative metabolite of
β-carotene
[394]
Pygoscelis papua
Gentoo penguins
Free form
N/A
2.19 in blood,
breeding adults and chicks
Accumulated from die-
tary crustaceans, fishes
[395,396]
The respective number was quoted from the reference(s), and it may vary depending on the collection location and season.†, the presence of binding forms to
carotenoproteins would not be mentioned in this table; ‡, the identication method of the compounds remains uncertain; *, the biosynthetic pathways have not
been fully characterized; **, based on the information on close species/genus; N/A; not available; N/D; not detected.*** Since astaxanthin is diversely found in the
skin, feathers, and retinas of birds, only the characteristic reports are described. ?; based on the information on close taxa. For the details of distribution in avian
species, see the other review [314].
Mar. Drugs 2023, 21, 514 41 of 155
2.3. Biosynthesis and Metabolism of Astaxanthin
2.3.1. Overview of Carotenoid Biosynthetic Pathways in Bacteria, Fungi, and Higher
Plants.
The biosynthesis of AX is entirely based on β-carotene as the common precursor.
Therefore, this document omits a detailed discussion of the metabolic pathway to β-caro-
tene. Briey, carotenoids belong to isoprenoids, which are the most diverse group of nat-
ural compounds. Isoprenoids are commonly biosynthesized from isopentenyl diphos-
phate (IPP). IPP is synthesized via the mevalonate pathway, which is present in almost all
eukaryotes (the domain Eukarya) and archaea (the domain Archaea), as well as some ac-
tinobacteria (the phylum Actinomycetota). Alternatively, the MEP (2-C-methyl-D-erythri-
tol 4-phosphate) pathway is used, which begins with pyruvate and glyceraldehyde 3-
phosphate and proceeds through 1-deoxy-D-xylulose 5-phosphate (DXP) and MEP. This
pathway is found in almost all bacteria and the chloroplasts of photosynthetic eukaryotes.
IPP is isomerized to dimethylallyl diphosphate (DMAPP) through the action of IPP iso-
merase (Idi; IDI). Subsequently, DMAPP is converted to farnesyl diphosphate (FPP) and
geranylgeranyl diphosphate (GGPP) through sequential condensation reactions with IPP.
These reactions are catalyzed by FPP synthase and GGPP synthase, respectively [397].
Figure 10 illustrates the biosynthetic pathway of carotenoids from FPP in the leaves
of higher plants, highlighting the enzyme-catalyzed reactions involved. Additionally, this
gure presents the functions of carotenoid biosynthesis enzymes derived from bacteria
and fungi [155]. According to past reports, the group of enzymes involved in the carote-
noid synthesis pathway in green algae is composed of a set of genes that share homology
with those of higher plants [398].
Figure 10. Biosynthetic pathway of carotenoids in the leaves of higher plants. Plant-type enzymes
are shown with green leers, while bacterial enzymes and fungal enzymes that can catalyze in this
pathway are wrien in pink and red, respectively.
Mar. Drugs 2023, 21, 514 42 of 155
2.3.2. Overview of Astaxanthin Biosynthetic Pathways in Bacteria, Algae and Plants
The β-C3-hydroxylation and β-C4-ketoxylation reactions involved in AX biosynthe-
sis are present in a wide taxonomic range of organisms. These reactions are mainly cata-
lyzed by membrane-integral, diiron, nonheme oxygenase superfamily enzymes, and oc-
casionally heme-dependent cytochrome P450-type monooxygenase enzymes. Detailed in-
formation regarding these enzymes is provided below.
In Proteobacteria, such as the genera Paracoccus and Brevundimonas, the genes respon-
sible for AX biosynthesis are well understood (refer to Section 2.2.1). AX is synthesized
from β-carotene through the coordinated actions of CrtZ-type β-C3-hydroxylase (β-caro-
tene 3,3′-hydroxylase; CrtZ) (EC: 1.14.15.24) and CrtW-type β-C4-ketolase (β-carotene 4,4′-
oxygenase; CrtW) (EC: 1.14.99.63; including an incorrect description on the substrate spec-
icity) with β-carotene as the initial substrate (see Figures 8 and 11) [37,399]. However,
due to variations in enzyme reactivity among dierent species, AX-producing bacteria
often accumulate signicant amounts of precursors, especially adonixanthin.
Heterologous expression of these enzyme genes is extensively conducted for astaxan-
thin production. Currently, CrtZ from Pantoea ananatis 20D3 (formerly known as Erwinia
uredovora 20D3) or Brevundimonas sp. SD212, as well as CrtW from Brevundimonas sp.
SD212, have usually been used for transgenic purposes, as they exhibit higher astaxanthin
accumulation when expressed heterologously [400,401]. Based on conserved amino acid
sequence regions and cofactors, CrtW and CrtZ display partial homology within their
conserved domains and belong to the nonheme membrane-integrated diiron nonheme
oxygenase superfamily [402,403], suggesting a possible evolutionary relationship from a
common ancestor.
Figure 11. Conversion routes from β-ring to 3-hydroxy-4-keto-β-ring in AX-biosynthesizing organ-
isms. ASY, also called CrtS, from Xanthophyllomyces dendrorhous; CrtW and CrtZ, from bacteria; BKT
and BHY, from green algae; CBFD and HBFD, from Adonis aestivalis.
On the other hand, Phaa yeast (Xanthophyllomyces dendrorhous), a basidiomycete
yeast, has a gene encoding a P450 family enzyme, astaxanthin synthase (CrtS/Asy, fungus,
EC 1.14.99.63/1.14.15.24; see also Section 2.2.2), that catalyzes the oxygenation of the C4