The Large Jellyfish Rhizostoma luteum as Sustainable a Resource for Antioxidant Properties, Nutraceutical Value and Biomedical Applications

Jellyfish is a compartment in the marine food web that often achieves high increases of biomass and that it is starting to be explored for several human potential uses. In this paper, a recently rediscovered large jellyfish, Rhizostoma luteum, is studied for the first time to describe its organic compounds for the isolation and production of bioactive compounds in several fields of food, cosmetics, or biomedical industries. The biogeochemical composition (Carbon, Nitrogen and Sulfur content), protein and phenols content, together with their antioxidant activity, and the analysis of lipid content (identifying each of the fatty acids presented) was analyzed. The results presented here suggested this jellyfish has the highest antioxidant activity ever measured in a jellyfish, but also with high content in polyunsaturated fatty acids (PUFAs), including the essential fatty acid linoleic. The large natural biomass of Rhizostoma luteum in nature, the wide geographical spread, the fact that already its life cycle has been completed in captivity, establishes a promising positive association of this giant jellyfish species and the isolation of bioactive compounds for future use in marine biotechnology.


Introduction
One of the challenges for coming decades in nutraceutical research is to find new potential resources that will provide an easy and low-cost approach to food necessities for the coming centuries. In this sense, jellyfish is a compartment in the marine food web (the greatest biome on earth) that often achieves high increases of biomass [1]. However, only recently has this wide group of organisms been starting to be seen from a more positive point of view [2], contrasting with the deleterious impact that it has on several economic human activities such as fisheries [3], clogging of power plants [4] and tourism [5]. This dark side of jellyfish blooms has been the only vision that has prevailed in the general human perception for many decades [6]. One example of the new positive point of view of the rapid increment of biomass in jellyfish is the cannonball, Stomolophus meleagris, in the Gulf of California, where a small Mexican community obtains benefits from switching for one month from fishing fish to fishing jellyfish, processes them, and sells them to the Asiatic market [7]. Another example of the explosive increment of jellyfish is the case of Cotylorhiza tuberculata in the Mar Menor lagoon in the

Jellyfish Biomass Characterization
Biometric and average biomass data of individuals of Rhizostoma luteum are shown in Table 1. The specimens used are young medusae, with an age range from about 23-140 days (Table 1), with a proportionally increasing biomass with jellyfish age. The relation between fresh weight and diameter in Rhizostoma luteum has been described by [29], while the relationship between fresh weight (FW) and dry weight (DW) is documented for the first time for this species. The variability of the biometric measures, including the FW, the FW:diameter ratio and DW percentage values, is representative of the different growth stages of the jellyfish [30]. After lyophilization, the DW of R. luteum ranges from 3.9-31% of the FW, displaying a high variability in tissue consistency. The highest values correspond with the older jellyfish employed in this study. The first parameter that should be assessed for evaluation in its potential use as biomass is the total energy or gross energy value, which is directly related to DW. The DW of R. luteum is similar to the measured range of the FW of Cotylorhiza tuberculata (3.9-32.4%), which is higher than Aurelia sp. 1 (2.2-3%) and Rhizostoma pulmo (4.1-6.8%) [16]. These values provide important information for the potential exploitation of R. luteum jellyfish biomass. It has recently been documented using different techniques that jellyfish may practically represent the total diet of several organisms, such as leatherback sea turtles and several fish [31][32][33][34].

Jellyfish Protein Content
The total freeze-dried tissue of 8 different batches were subjected to phosphate-buffered saline (PBS) solvent extraction treatment. The whole tissues of Rhizostoma luteum contained proteins soluble in polar solvents, specifically in aqueous solution with a mean value of 13.7 ± 3.4 mg of proteins of per g of dry weight (mean ± standard deviation, n = 8 (Batch numbers: 1, 2, 4-7, 9, 10)). The values ranged between 8.4-18.7 mg of proteins/g of DW. Compared to other species, such as Aurelia sp. 1, Cotylorhiza tuberculata and Rhizostoma pulmo, with 22, 35 and 37 mg of proteins/g of DW, respectively [16], R. luteum contained less soluble protein but in the same order of magnitude. The content of protein in jellyfish is consistently the most abundant organic fraction [38] and has been studied in other rhizostomae jellyfish such as Rhopilema asamushi [40] and from Cyanea nozakii [41] for collagen isolation and characterization, which has been suggested for multipurpose uses in cosmetics and nutraceutical sectors [42].
The biostability and biochemical properties of collagen-based tissues may be enhanced by the polyphenols through modulation of mechanisms of collagen fiber cross-linking at different levels (molecular, inter-molecular and inter-microfibrillar) [45,46]. The high phenolic content of R. luteum (this study), C. tuberculata and R. pulmo [16], all of them large rhizostomae jellyfish, may be the reason for the robust and hardened mesoglea, compared to the flexible and soft Aurelia spp.

Antioxidant Activity in Jellyfish Aqueous Soluble Extract
The total antioxidant activity of the jellyfish Rhizostoma luteum was 32,598 ± 9015 nmol of TE (Trolox Equivalent) per gram of DW in the PBS extract (means ± standard deviation, n = 8 (Batch numbers: 1, 2, 4-7, 9, 10)). The values ranged between 15,468-43,501 nmol of TE/g DW. These values of R. luteum are the highest values measured in a jellyfish so far, but in the same magnitude as other rhizostomae jellies such as R. pulmo and C. tuberculata, with antioxidant activity of 22,520 and 25,621 nmol of TE/g DW, respectively [16]. Meanwhile, Aurelia sp. 1 antioxidant activity was much lower, with measured values of 7651 nmol of TE/g DW [16].
The high values of R. pulmo and C. tuberculata compared to Aurelia sp. 1 were presumably attributed to the protein and phenols contents, although other unidentified compounds could be included [16]. Similarly, high values of antioxidant activity present in R. luteum in the present study could also be related to the protein and phenol content. The antioxidant activity referring to protein content, both in PBS extract, for R. luteum is 2379 nmol of TE/mg of proteins, showing evidence that this high antioxidant activity could be ascribed to the inherent protein properties of this jellyfish. Values of this ratio are double those reported by R. pulmo and C. tuberculata [16].

Lipid Content
Total lipid content of the jellyfish Rhizostoma luteum was around 0.94 g per 100 g of DW (standard deviation = 0.03, n = 6 (Batch numbers: 3, 5-8, 10)). This value was one order of magnitude lower than both Aurelia sp.1 and R. pulmo (4 g/100 g DW) and two orders of magnitude lower than C. tuberculata (12.3 g/100 g DW; [16]). The low values of R. luteum are probably due to the age of the specimens examined. In the case of symbiotic jellyfish such as C. tuberculata, the high lipid content could be related to the photosynthetic membranes of the zooxanthellae [30,47].
The fatty acid (FA) quantitative composition (as percentage values) of R. luteum showed that polyunsaturated fatty acids (PUFA) accounted for half of the total FA (49%), followed by saturated fatty acids (SFA), representing one-third of the total FA (about 30%), and finally monounsaturated fatty acids (MUFA), representing around 21% of the total FA (Table 2). In the case of Aurelia sp. 1, C. tuberculata and R. pulmo, the proportions were different to R. luteum, with SFA being the most abundant FA (two-thirds of the total FA), followed by PUFA (two-thirds of the total FA) and a small amount of MUFA (4-15%) [16].
Polyunsaturated fatty acids of R. luteum consisted mostly of ω-6 arachidonic (C 20:4 ), ω-3 linoleic (C 18:3 ) and the essential ω-6 linoleic (C 18:2 ) acids. Saturated fatty acids consisted mostly of stearic (C 18:0 ) and palmitic (C 16:0 ) acids. Among MUFA, oleic acid (C 18:1 ) was the prevalent FA (Table 2). This composition of fatty acids shows remarkable differences to the other three species studied previously (see Table 4 in [16]). The proportion of PUFA of both ω-6 arachidonic (C 20:4 ) and ω-3 linoleic (C 18:3 ) in R. luteum are the highest documented for jellyfish and the essential ω-6 linoleic (C 18:2 ) acid is only topped by the symbiotic jellyfish C. tuberculata [16], giving a peculiar composition of PUFA in R. luteum. In the case of MUFA oleic acid, (C 18:1 ) is in the same order of magnitude with C. tuberculata and higher than Aurelia sp.1 and R. pulmo [16]). Both ω-6 and ω-3 PUFAs were abundant in the jellyfish Rhizostoma luteum, with the ratio of ω-6 to ω-3 of 2.14. The Σ ω-6 was 33.4% while the Σ ω-3 was 15.6%. The total ω-3 PUFAs in R. luteum is similar to Aurelia sp. 1, C. tuberculata and R. pulmo [16], but the total ω-6 PUFAs in R. luteum is double the other three documented jellyfish. Therefore, the ratio ω-6 to ω-3 of the other jellyfish is lower (between 0.4 to 0.8) [16] than in R. luteum. The role of ω-3 PUFAs in a diverse biological process is well known, including development, growth, tissue, and cell homeostasis [48]. Regarding the numerous human health benefits of ω-3 PUFAs, these include antiarthritis, anti-inflammatory, antioxidant, antihypertensive, anticancer, hypo-trigleceridemic, antiaging and antidepressive effects [49]. A high ratio of ω-6 to ω-3 in the Western diet in humans appears to be related to pathogenesis of chronic disease due to its proinflammatory effects [50]. The fatty acid composition of R. luteum discovered in this study should be considered for new applications due to its nutraceutical value.

Sample Collection and Preparation
Specimens of Rhizostoma luteum jellyfish were reared in the laboratory (ICMAN-CSIC) from polyps. The origin of this living collection is from planulae gathered directly from the gonad of a free-swimming female medusa offshore of the coast of the Alboran Sea (Spain) in October 2015 (details in Kienberger et al. [29]). For the present study, a total of 303 specimens were used. After the biometric measurements (weight and diameter) were taken, and due to their small size, specimens were grouped to reach enough biomass to be analyzed and lyophilized. Samples were freeze-dried for 4 days at −55 • C using a chamber pressure of 0.110 mbar in a freeze dryer (LyoAlfa 15, Telstar Life Science Solutions, Madrid, Spain). Lyophilized samples were weighed to annotate the dry weight and stored at −20 • C until use.

Elemental Analysis
Total carbon, nitrogen, and sulfur (C, N, S) content (in wt.%, approximately 100 mg per sample) were measured using an Elementary Chemical Analyzer LECO CHNS-932.

Polar Solvent Extraction
Lyophilized samples of total jellyfish were subjected to extraction in aqueous solvent PBS. Samples were stirred with 16 volumes of PBS (2 h at 4 • C). Samples were then centrifuged at 9000× g for 30 min at 4 • C and the supernatants were essayed for protein and phenol contents and for antioxidant activity.

Protein Content
Total protein content was estimated by modified Bradford assay [51] using bovine serum albumin (BSA) as a standard.

Phenol Content
The total phenolic content was determined by using a modified Folin-Ciocalteau colorimetric method. Aliquots of extracts (100 µL) were mixed with 500 µL of Folin-Ciocalteu's phenol reagent and 500 µL of 7.5% sodium carbonate (Na 2 CO 3 ). After 2 h of incubation at room temperature in the dark, the absorbance was spectrophotometrically measured at 760 nm. Gallic acid, ranging from 2.5 to 100 µg/mL, was used as a standard. The results were expressed as gallic acid equivalents (GAE) per gram of dry extract.

Antioxidant Activity
The total antioxidant activity was determined spectrophotometrically by the ABTS free radical decolorization assay developed by Re et al. [52], with some modifications.
In brief, a solution of the radical cation ABTS + was prepared by mixing a solution of ABST (2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (7 mM) and a solution of potassium persulfate (2.45 mM) in H 2 O. The mixture was kept in the dark overnight before use. Then, the ABTS + solution was diluted with EtOH to an absorbance of 0.70 ± 0.02 at 734 nm. Trolox was used as standard. The samples were prepared by adding 100 µL of extracts to 2 mL of ABTS + solution and the control by adding 100 µL of PBS solution to 2 mL of ABTS + solution. The measure at the absorbance at 734 nm was registered after 6 min of reaction. The percentage of inhibition of the absorbance was calculated by the following equation: where A 0 is the absorbance of the control and A 1 is the absorbance of the tested samples. Results were expressed as nmol of Trolox equivalents (TE) per gram of sample.

Total Lipid Extraction
Total lipids were extracted using the modified Bligh and Dyer method [53]. Lyophilized powder (100 mg) was mixed with a total of 10 mL solvent added in the sequence of chloroform, methanol, water, to achieve a final chloroform/methanol/water ratio of 1:2:0.8 (by volume). Samples were shaken for 15 s after addition of each solvent, and incubated overnight at 4 • C. After centrifugation at 6500× g for 10 min, the supernatant was transferred into a separating funnel, and phase separation of the biomass-solvent mixtures was achieved by adding chloroform and water to obtain a final chloroform/methanol/water ratio of 2:2:1.8 (by volume). After settling, the bottom phase was collected and evaporated under vacuum.

Fatty Acid Profiles Determination
Fatty acid methyl esters (FAMEs) were obtained using boron trifluoride (BF 3 ) according to [54] with some modifications. Total lipid extract was saponified at 90 • C for 20 min with 0.5 M KOH in methanol (3 mL). Forty-nine micrograms of the internal standard (methyl tricosanoate) were added before saponification. The fatty acids were methylated by adding 14% BF 3 in MeOH (2 mL) and heating at 90 • C for 10 min. After cooling, the mixture was extracted with hexane (1 mL × 2). After separation, the hexane layer was collected, taken to dryness under vacuum, dissolved in 1.0 mL of CH 2 Cl 2 and analyzed by gas chromatography-mass spectrometry (GC-MS).

GC-MS Analysis
The analyses were performed on a GC-MS system consisting of high-resolution SYNAPT G2 (Waters, Milford, MA, USA) instrument equipped with a quadrupole-time-of-flight (QTOF) analyzer using atmospheric pressure ionization (API) in positive ionization mode. Compounds were separated on a DB-5 capillary column Agilent J&W DB-5ms column (250 µm × 30 m, 0.25 µm film) (Agilent, Santa Clara, CA, USA). The GC parameters were as follows: the column temperature was maintained at 90 • C for 1 min, then raised to 200 • C (20 • C/min) and to 300 • C (5 • C/min), and held at 300 • C for 2 min. Fatty acids were identified by comparison of retention time, molecular formula obtained by their high-resolution molecular ion [M + H] + , and mass spectral data with FAME standards (Supelco-37) and a NIST library.

Conclusions
The potential use of the jellyfish Rhizostoma luteum as biomass, evaluating their nutraceutical value and antioxidant properties, has never been evaluated until the present study. The results presented here suggest this jellyfish to be an excellent candidate for the potentially sustainable production of nutraceutical, cosmeceutical and biomedical natural products due to the highest antioxidant activity ever measured in a jellyfish, but also for their high content in PUFAs, including the essential fatty acid linoleic. Because the present work has been performed with young medusae, further work should be carried out with adult specimens. The large natural biomass of Rhizostoma luteum in nature, the wide geographical spread, and the fact that already its life cycle has been completed in captivity establishes a promising positive potential of this giant jellyfish species and the isolation of bioactive compounds for future use in marine biotechnology.