The Sea Urchin Arbacia lixula: A Novel Natural Source of Astaxanthin

Several echinoderms, including sea urchins, are valuable sources of bioactive compounds but their nutraceutical potential is largely unexplored. In fact, the gonads of some sea urchin species contain antioxidants including carotenoids and polyhydroxylated naphthoquinones (PHNQ’s), such as echinochrome A. Astaxanthin is known to have particular bioactivity for the prevention of neurodegenerative diseases. This carotenoid is produced by microalgae, while several marine invertebrates can bioaccumulate or synthetize it from metabolic precursors. We determined the carotenoid content and analyzed the bioactivity potential of non-harvested Atlantic-Mediterranean sea urchin Arbacia lixula. The comparison of methanol crude extracts obtained from eggs of farmed and wild specimens revealed a higher bioactivity in farmed individuals fed with a customized fodder. HPLC-analysis revealed a high concentration of astaxanthin (27.0 μg/mg), which was the only pigment observed. This study highlights the potential of farmed A. lixula as a new source of the active stereoisomer of astaxanthin.


Introduction
Carotenoids are naturally occurring pigments widely distributed among photosynthetic organisms, bacteria, and fungi. Some of the main biological activities and biochemical processes of carotenoids in organisms are pro-vitamin A activity, photoprotection, radical quenching, and immunological modulation [1][2][3]. Animals can obtain carotenoids directly from the diet or through biochemical conversion from dietary precursors. In particular, high carotenoid concentrations occasionally found in the reproductive organs of animals suggest that they could have a role in the reproductive process [3,4]. Carotenes (i.e., β-Carotene) and xanthophylls (i.e., β-echinenone, astaxanthin, lutein, zeaxanthin, and fucoxanthin) are known for their antioxidant activity and for their potential in the prevention and treatment of various diseases. There is increasing evidence that sea urchins, together with other echinoderms, may represent potential sources of valuable bioactive compounds [5][6][7][8][9][10][11]. However, their biotechnological potential remains largely unexplored, and research in this area is still a niche topic [12], particularly in Mediterranean species. Studies conducted on the gonads of some species of sea urchin revealed powerful antioxidant activity due to the presence

Results
The cultivation protocol has been optimized and astaxanthin-hyperproducing stocks of A. lixula can be obtained in a few months. A. lixula specimens in culture showed a significantly higher egg production and a prolonged period of reproductive ability compared with wild individuals, allowing the achievement of greater biomass from cultivated sea urchins without seasonal limitation. A substantial amount of biomass was obtained from cultivated sea urchins. A single egg collection from A. lixula in culture ranged between 4.5 and 6.9 g, with an average value (±1 SD) of 5.5 ± 0.250. The astaxanthin concentration determined in samples of cultivated A. lixula was more than 15 times higher than the concentration in wild samples (see Table 1). Table 1. Astaxanthin concentration (mean ± SD) determined in wild and farmed samples of Arbacia lixula.

Chemical Analysis
The HPLC chromatogram ( Figure 1) revealed that astaxanthin was the main pigment of the extract. The quantity of astaxanthin per dry weight was significantly greater in the eggs of farmed animals than in the eggs of wild ones (n = 5; p ≤ 0.001; Table 1).

Results
The cultivation protocol has been optimized and astaxanthin-hyperproducing stocks of A. lixula can be obtained in a few months. A. lixula specimens in culture showed a significantly higher egg production and a prolonged period of reproductive ability compared with wild individuals, allowing the achievement of greater biomass from cultivated sea urchins without seasonal limitation. A substantial amount of biomass was obtained from cultivated sea urchins. A single egg collection from A. lixula in culture ranged between 4.5 and 6.9 g, with an average value (±1 SD) of 5.5 ± 0.250. The astaxanthin concentration determined in samples of cultivated A. lixula was more than 15 times higher than the concentration in wild samples (see Table 1).

Chemical Analysis
The HPLC chromatogram ( Figure 1) revealed that astaxanthin was the main pigment of the extract. The quantity of astaxanthin per dry weight was significantly greater in the eggs of farmed animals than in the eggs of wild ones (n = 5; p ≤ 0.001; Table 1). From these results, the amount of astaxanthin production in the eggs of cultivated individuals was estimated (Table 2). Table 2. Total quantity of astaxanthin production in the eggs of cultivated individuals.

Mean Fresh Weight of Eggs Mean Dry Weight of Eggs Astaxanthin Weight per g of Dry Eggs
5.5 ± 1.5 g 0.5 ± 0.250 g 0.014 ± 0.004 g Therefore, we estimated that the total quantity of astaxanthin per dry weight of eggs was about 2.7%. From these results, the amount of astaxanthin production in the eggs of cultivated individuals was estimated (Table 2). Table 2. Total quantity of astaxanthin production in the eggs of cultivated individuals.

Mean Fresh Weight of Eggs Mean Dry Weight of Eggs Astaxanthin Weight per g of Dry Eggs
5.5 ± 1.5 g 0.5 ± 0.250 g 0.014 ± 0.004 g Therefore, we estimated that the total quantity of astaxanthin per dry weight of eggs was about 2.7%.

Radical Scavenging Activity
The aqueous ethanol extract (hereafter referred to as the extract) from A. lixula eggs exhibited marked reducing activity toward radical species when the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging ability was tested. The addition of extract concentrations of 0.1, 1, and 10 µg/mL taken from each individual resulted in a dose-dependent reduction of the purple radical DPPH into the yellow reduced form for eggs from farmed animals (7.07% ± 5.56% (mean ± SD), 10.31% ± 5.09%, and 86.40% ± 2.82%, respectively). This trend was not consistent across reared and wild individuals due to the lower activity of extracts from wild animals, which were only significantly active (47.44% ± 10.42%) at the highest concentration (Tables 3 and 4, Figure 2).

Radical Scavenging Activity
The aqueous ethanol extract (hereafter referred to as the extract) from A. lixula eggs exhibited marked reducing activity toward radical species when the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging ability was tested. The addition of extract concentrations of 0.1, 1, and 10 μg/mL taken from each individual resulted in a dose-dependent reduction of the purple radical DPPH into the yellow reduced form for eggs from farmed animals (7.07 ± 5.56% (mean ± SD), 10.31% ± 5.09%, and 86.40 ± 2.82%, respectively). This trend was not consistent across reared and wild individuals due to the lower activity of extracts from wild animals, which were only significantly active (47.44 ± 10.42%) at the highest concentration (Tables 3 and 4, Figure 2).  Table 3. RM-ANOVA (Repeated Measures-ANalysis of Variance) on transformed (x' = x + 10) percentages of DPPH (2,2-diphenyl-1-picrylhydrazyl). SS stands for Sum of Squares error estimator, df indicates the degrees of freedom, MS represents the mean square error estimator, F is the test statistics, p is the probability associated to the observed F, and G-G and H-F refer to Greenhouse-Geisser and Huynh-Feldt corrections for within-subject effects.

Discussion
The customized fodder enhanced the production and biochemical activity of astaxanthin in farmed A. lixula specimens, compared to the wild ones. The increased biochemical activity of astaxanthin extracted from farmed A. lixula resulted by testing the aqueous ethanol crude extract of eggs as a DPPH radical scavenger. In fact, we showed that astaxanthin obtained from the eggs of the cultivated A. lixula has a potent radical scavenging activity with an inhibition 33% higher than α-tocopherol, used as the benchmark, at a 10 µg/mL concentration.
Carotenoids biosynthesis is biochemically well characterized in the astaxanthin-producing Hematococcus pluvialis. In this species, astaxanthin was as the main ketocarotenoid synthesized through biosynthetic reactions starting from the precursor β-Carotene [37,38]. Considering that carotenoids found in animals can be directly accumulated from food and/or partially modified through metabolic reactions [39,40], it is very important to find a way to enhance metabolic pathways in order to optimize the production of bioactive compounds. Spirulina platensis has been proved to be a valuable source of carotenoids enhancing the pigmentation in fish [41][42][43][44] and shrimps [45,46], and its inclusion in broodstock diet is recommended to avoid carotenoid deficiency-related problems in shrimp hatcheries [47]. β-Carotene and zeaxanthin, the main carotenoids determined in S. platensis, corn, and Ulva lactuca [48], are among the reported astaxanthin precursors in animal metabolic pathways [49]. Corn and corn products are considered potential major contributors of dietary zeaxanthin and lutein [50], whereas the green macroalga U. lactuca is a good contributor of dietary β-Carotene and other carotenoids [51,52], in addition to important vitamins and minerals. Herein, the optimization of astaxanthin production was obtained through the introduction of intermediates of the biosynthetic pathway of carotenoids, using food as a carrier of target substances. The blue-green algae, S. platensis, corn, and U. lactuca were used as source of carotenoids due to their content of intermediate products of the astaxanthin biosynthetic pathway.
As previously [48] reported, astaxanthin is contained only in S. platensis at the concentration of 0.21 ± 0.02 µg/mL. The feed was administrated twice a week, and the farmed sea urchins were processed after two months of treatment. The total amount of astaxanthin found per gram of eggs was 13.5 mg/g. However, the concentration of bioaccumulated astaxanthin should have been 3.36 µg/g if all the dietary astaxanthin was kept and transferred to the eggs. It is evident that the amount of astaxanthin found in farmed A. lixula is higher than the one eventually bioaccumulated, and that it was likely de novo synthesized from the administrated precursors. Further investigations could shed light on the accumulation process and demonstrate the mechanism herein hypothesized.
Comparing the present findings with the results from the available literature [53] it is clear that the eggs of farmed A. lixula are a reliable source of astaxanthin, and a convenient alternative to some microalgae. Although the maximum amount of natural astaxanthin obtainable from microalgae up to date is 3.8 mg/g of dry matter (in the case of H. pluvialis), the production process can be very expensive due to its chemical purification from other pigments and carotenoids.

Culture System
Sea urchins were hand collected by scuba-diving from rocky shores of the Gulf of Naples, along the southern Tyrrhenian coasts of Italy. Animals were placed in a cooler and carried to the laboratory under moist conditions within 2 h.
Sea urchins used for the study were held in suspended baskets in a semi-closed recirculating system that receives very low flows of make-up seawater for water losses associated with routine tank cleaning. A centralized Life Support System (LSS) was set to maintain optimal seawater conditions at a constant temperature of 16 ± 1 • C in order to promote the accumulation of gametes and prevent unwanted spawning.

Feeding Practice
We used a mixture of the dry powdered ingredients (animal and vegetable) shaped into Ration Block Food (RBF). We obtained RBF with an average weight of 1 g and feed sea urchins twice a week for two months before the experiments. This quantity corresponded to rations tests. RBFs were always completely eaten. The diet ingredients used were natural and included mussel meal, corn, natural macroalgae (U. lactuca) and microalgae (S. platensis), fish oil, and mineral supplement.

Eggs Collection
Gametes were collected by manually pouring from the gonadal pores after animal sacrifice. This method allowed us to gather pure fresh biomass. Only eggs were selected for successive analysis. After collection, they were immediately frozen in liquid nitrogen and stored at −80 • C. The egg biomass collected from single sea urchins ranged between 4.5 and 6.9 g.

Chemical Extraction from Eggs
Extraction was performed according to Sansone et al. [54] and Cutignano et al. [55]. The extraction procedure was conducted under dark conditions, at room temperature, and under nitrogen atmosphere in order to avoid oxidation of the sample. Freeze-dried eggs (~100 mg) were extracted with 1 mL ethanol/water (3/1 v/v) mixture for 30 min. The mixture was separated by centrifugation at 4500× g, for 10 min, at 20 • C, and the supernatant was transferred to a clean tube. The pellet was resuspended in 1 mL of the ethanol/water mixture and extracted for a second time. The aqueous ethanol extract was dried in a rotary vacuum evaporator (Buchi rotavapor R-114). The dry extract was stored under nitrogen atmosphere at −20 • C prior to analysis.

HPLC Analysis
Pigment analysis was conducted by High Performance Liquid Chromatography (HPLC) on an aliquot of the aqueous ethanol extract (10 mg), according to the method described by Brunet et al. [56]. Briefly, the extract was injected in a reversed-phase column (C8 Kinetex column; 50 mm × 4.6 mm; 2.6 µm particle size, Phenomenex ® , Wilmington, NC, USA) of the Hewlett Packard series 1100 HPLC (Hewlett Packard, Wilmington, NC, USA). Pigments were detected by diode-array spectroscopy (spectrum data collected in the range of 350-750 nm) using a Hewlett Packard photodiode array detector, model DAD series 1100, and absorbance chromatogram was reported at 440 nm. Identification and quantification of pigments were carried out using pigment standards from the D.H.I. Water & Environment (Horsholm, Denmark) [57].

DPPH-Assay
The 2,2-di(4-tert-octylphenyl)-1-picrylhydrazyl (DPPH) was used for the radical scavenging assay (Sigma Aldrich Co., Steinheim, Germany, cat. 257621). Various concentrations (0.1, 1, and 10 µg/mL) of eggs extract were mixed with a final concentration of DPPH of 0.1 mM in methanol, allowed to react for 30 min in the dark, and absorbance was measured at 517 nm using a microplate reader. Results were presented as a percentage of DPPH reduction with respect to the methanol negative control and α-tocopherol used as a positive control.

Experimental Design and Analysis
The study demonstrated two experimental hypotheses derived from the stated aims. The first one (H 11 ) predicted antioxidant activity by eggs extract with respect to the statistical control made of methanol. H 11 was tested at three different doses or concentrations of the eggs extract. The second one (H 12 ) predicted higher antioxidant activity by the eggs extract from reared sea urchins with respect to those from wild ones. Both experimental hypotheses were tackled through a Repeated Measures (RM) ANOVA, while the relative intensity of the antioxidant effect was tested across three different doses by an array of a priori comparisons coded as contrast following Neter et al. [58,59]. All p values were taken as measures of the relative strength of the evidence against the null hypothesis, H 0 [59,60]. Individual sea urchins acted as subjects onto which repeated measures (i.e., one extract for each dose level) were taken. Hence, dose of eggs extract (D) was the within-subject or blocking factor, with three levels (low, corresponding to 0.1 µg/mL; medium, 1 µg/mL; and high, 10 µg/mL). Wild versus reared (W vs. R) individuals was the between-subjects factor, with two levels (wild and reared). There were five sea urchin individuals (subjects) within each W vs. R level, for a total of 10 individuals in the experiment. There was one single observation per cell in this design. The response variable was the percentage of antioxidant activity with respect to α-Tocopherol, transformed as x' = x + 10 to account for negative values in the DPPH test output. The assumption of homogeneity of variances was ensured via Levene's test, while that of sphericity was tested through Maucley's W test.

Conclusions
Our results demonstrated the double advantage of improving both the available biomass and the concentration of natural astaxanthin in A. lixula eggs. The astaxhantin concentration found in the eggs of farmed A. lixula was more than 15 times higher than that found in eggs from wild samples. In addition, our approach allows us to base future exploitation of A. lixula on cultured stocks without placing any burden on the wild populations. The determination of the pathway leading to the biosynthesis of astaxanthin in this echinoderm exceeds the aims of the present work. However, complete understanding may allow for the further optimization of natural astaxanthin production. Future investigations will allow the transfer of this innovative feeding practice on other sea urchin species to induce the biosynthesis of other bioactive products for nutraceutical and cosmeceutical applications.