Biopotential of Sea Cucumbers (Echinodermata) and Tunicates (Chordata) from the Western Coast of Portugal for the Prevention and Treatment of Chronic Illnesses

: In the present work, we aimed to explore the potential of two groups of marine invertebrates—sea cucumbers (Echinodermata) and ascidians (Chordata)—as sources of anti-inflammatory, anti-oxidant, and osteogenic compounds with potential to be used as pharmaceuticals and nutraceuticals for the treatment and prevention of chronic diseases. 24 extracts (ethanol, water, and ethyl acetate) from 4 species of sea cucumbers and 4 species of tunicates were produced and screened in vitro for their anti-inflammatory and anti-oxidant activities and in vivo for osteogenic activity through an assay using zebrafish larvae. Our results showed that ethanolic extracts presented anti-oxidant and anti-inflammatory activity, which revealed to be stronger in the ascidians. The osteogenic activity, which provides evidence of the bioactive potential of these organisms in preventing chronic disorders causing low bone density, was found to be strong in one species of ascidians and 3 of holothurians. This study demonstrates the high potential of extracts from these marine organisms for using as nutraceuticals in the prevention of chronic bone disorders.


Introduction
Chronic diseases are a major cause of death and morbidity globally [1]. Among these, osteoporosis and osteopenia are the most common chronic disorders related to bone [2]. Although much remains unclear about the molecular mechanism underlying the development of chronic diseases, a growing body of evidence has stressed the importance of inflammation and oxidative stress. In this context, nutraceuticals and functional foods promise to be important sources of micronutrients, antioxidants, and anti-inflammatory compounds for the prevention of chronic diseases [3]. The marine environment is considered to be a potentially inexhaustible source of bioactive compounds. Sea cucumbers (Holothuroidea) and ascidians (Ascidiacea) have gained the attention of the pharmaceutical sector due to the large amount of bioactives isolated from these groups of marine invertebrates, that have been associated with anti-inflammatory, anti-oxidant, and osteogenic activities, among others [4,5]. In the present work, we evaluated the bioactivity of extracts produced from 4 species of sea cucumbers belonging to the family Holothuriidae and 4 species of ascidians, belonging to the families Styelidae and Polyclinidae. Our aim was to explore the bioactive potential of these species for applications in the prevention and treatment of bone related chronic disorders. Anti-inflammatory activity, anti-oxidant potential, and polyphenol content were assessed for the aqueous and ethanolic extracts obtained from sea cucumbers and ascidians. Furthermore, to evaluate the potential of these species, all the extracts were screened for their pro-osteogenic bioactivity within a recently developed zebrafish-based in vivo system.

Preparation of the Extracts
A total of 24 extracts were obtained by solvent extraction from sea cucumbers and ascidians freeze-dried biomass. The biomass was ground into fine powder and solubilized in water, ethanol or ethyl acetate with a ratio 1:30 w/v. Solutions were homogenized and left for 16 h to extract soluble compounds. Extracts were centrifuged to remove insoluble material and aliquoted.

Anti-Inflammatory Activity
Anti-inflammatory activity was evaluated in the ethanolic and aqueous extracts by the cyclooxygenase-2 (COX-2) inhibitory assay, an established biomarker of inflammation processes [9].

Developmental Toxicity and Osteogenic Activity in Zebrafish Larvae
Aqueous, ethanolic and ethyl acetate extracts from sea cucumbers and ascidians were screened for their acute developmental toxicity in a 72-h exposure of zebrafish larvae. At 3 days post fertilization larvae were transferred to 6 well-plates with a density of 15 larvae per 10 mL of treatment medium/well. The larvae were treated via waterborne exposure for 72 h with different concentrations of each extract and the respective solvents as control groups (0.1% v/v ethanol for ethanol extracts, fish water for aqueous extracts; and 0.1% v/v Dimethyl sulfoxide (DMSO) for ethyl acetate extracts). Treatment medium was renewed daily (70%). All the extracts were first tested at a concentration of 100 µg/mL and toxicity was monitored during 3 days of exposure. Mortality and severe deformation were considered to be endpoints for toxicity. When the treatments were toxic, the dose was reduced to 10 or 31.6 µg/mL and the experimental exposure repeated. If no toxicity was observed, the dose increased to 200 µg/mL. The maximum non-lethal dose (MNLD) was selected to evaluate the osteogenic activity with the zebrafish operculum assay, an in vivo, phenotypic-based method that uses morphometric analysis of the opercular bone in alizarin red S-stained zebrafish larvae [10]. The area of the operculum (OpA) and the area of the head (HA) were measured and used to determine osteogenic activity.

Anti-Inflammatory Activity
The anti-inflammatory activity in the biomass of the eight species of marine organisms was measured as the percentage of inhibition of COX-2 ( Table 1). The ethanolic extracts showed higher antiinflammatory activity than the aqueous extracts, and the higher activity was reported for the ascidians. Table 1. Anti-inflammatory activity (% inhibition of COX-2) in aqueous and ethanolic extracts from 4 ascidian and four sea cucumber species. Values are presented as average ± SD. Different lowercase letters within a column correspond to statistical differences (p < 0.05) between organism species. Different uppercase letters within a row correspond to statistical differences (p < 0.05) between aqueous and ethanolic extracts. nd-not detected.

Species
Anti

Anti-oxidant Activity and Polyphenol Content
The anti-oxidant activity and polyphenol content are presented in Table 2. The set of three tunicate species belonging to the family Polyclinidae displayed the highest anti-oxidant activities in both extracts, with the only remarkable exceptions of the aqueous extracts of Polyclinidae ni1 and Polyclinidae ni2 with their anti-oxidant power measured by DPPH. The anti-oxidant activities determined for the holothurians were generally lower than the observed for the ascidians from the family Polyclinidae. The ethanolic extracts from the ascidians were richer in polyphenols than the aqueous extracts, while in holothurians the opposite was observed. Moreover, Polyclinidae had higher polyphenol contents compared with holothurians. Total polyphenol contents had a weak correlation with the anti-oxidant activity, even in the best case, i.e., DPPH (R 2 = 0.29). It should be noted that there are other anti-oxidant substances in tunicates [11] and sea cucumbers [12] which may be responsible for the effect reported. Comparisons with the relevant literature must consider used methodologies, measurement units, and type of extract. Further research on the seasonality and overall variation of phenolic substances and anti-oxidant activity in tunicates and holothurians is required. Table 2. Anti-oxidant activity was measured by ABTS + (µmol eq TROLOX/100 g dw), FRAP (µmol eq FeSO4/g dw), and DPPH (mg eq AA/100 g dw) and polyphenol content (mg GAE/100 g dw) in aqueous and ethanolic extracts. Values presented as average ± SD. For anti-oxidant activity, different lowercase letters within a column correspond to statistical differences (p < 0.05) between organism species. For each anti-oxidant method, different uppercase letters within a row correspond to statistical differences (p < 0.05) between aqueous and ethanolic extracts. Within the polyphenol's column, different uppercase letters correspond to statistical differences (p < 0.05) between aqueous and ethanolic extracts. nd-not detected.

Developmental Toxicity and Osteogenic Activity
Acute developmental toxicity was calculated for the ethanolic, aqueous and ethyl acetate extracts from 4 species of sea cucumbers (Holothuriidae ni1, Holothuriidae ni2, Holothuriidae ni3, Holothuriidae ni4) and 4 species of ascidians (Styelidae ni1, Polyclinidae ni1, Polyclinidae ni2, and Polyclinidae ni3). Most of the aqueous and ethanolic extracts showed no toxicity at the concentrations tested, except for the ethanol extracts of Holothuriidae ni1 that induced mortality at the higher concentration (200 µg/mL). Oppositely, all the ethyl acetate extracts induced severe mortality at the higher concentration tested (100 µg/mL), indicating the possible presence of toxic compounds in the extracts. The MNLD determined for each extract was then used to assess the osteogenic activity. Control groups did not show mortality in any of the assays performed. The area of the operculum (OpA) and the area of the head (HA) were measured, and the ratio OpA/HA was calculated for assessing osteogenic activity (Figure 1). None of the extracts significantly affected the area of the head (HA), a proxy of the size of the larva, indicating there was no significant variation on growth among treatments.The ethanol extracts from Styelidae ni1 and Polyclinidae ni1 and ni3 induced moderate to high osteogenic activity by increasing the area of the operculum, when compared with the control. The ethanol extract from Polyclinidae ni1 showed the highest osteogenic activity by inducing an increase in the size of the opercular bone of 41 ± 16.66%. The ethyl acetate extracts induced moderate pro-osteogenic effects in Holothuriidae ni2 and ni4, but Polyclinidae ni1 induced the highest osteogenic effect, with an increase of the operculum area of 28.0 ± 13.84%. Regarding sea cucumbers, treatment with the ethanol extracts from Holothuriidae ni2, Holothuriidae ni3, and Holothuriidae ni4 showed a moderate osteogenic effect. This suggests the presence of osteogenic compounds in the extracts, with higher osteogenic power in the ethanolic extracts of the ascidians Styelidae ni1, Polyclinidae ni1, and ni3 and of the Holothurians Holothuriidae ni2, ni3 and ni4. Despite many bioactive compounds being isolated from sea cucumbers and ascidians, the number of studies analyzing their potential for bone health is low and only based on in vitro studies.
Fucoidans derived from the holothurian Stichopus japonicus inhibited the differentiation of rat bone marrow macrophages into osteoclasts [13], similarly to what was observed for fucoidan extracted from brown seaweeds [14]. Furthermore, water and methanolic extracts from different species of sea cucumbers were found to be able to induce differentiation in human osteoblastic cell lines [15] and rat bone marrow mesenchymal stem cells [16]. Very few studies analyzed the osteoactive properties of compounds derived from ascidians. Calcitonin-like peptides isolated from the sea squirt Ciona intestinalis showed to be able to promote osteoblastic differentiation and mineralization in MC3T3-E1 cells [17]. Aplidin, a compound isolated from the ascidian Aplidium albicans, known for its anticancer potential, was able to reduce osteoclast differentiation when administered both alone or together with dexamethasone in both murine bone marrow primary cell culture and in an ex vivo bone organ culture model [18].
Interestingly, in the present study a significant correlation was found between the osteogenic activity and the COX-2-inhibitory activity for the extracts studied (R 2 = 0.49). However, to understand the mechanisms underlying the biological activities observed, further investigation is needed to identify the compounds responsible for the effect observed by treatment with the extracts, but also to understand the molecular pathways underlying the observed osteogenic effects.

Conclusions
In the present work, we explored the potential of two promising groups of marine invertebrates as sources of bioactive compounds to be used toward the prevention and treatment of low-bonedensity chronic diseases. The ethanolic extracts from ascidians showed the higher anti-oxidant and anti-inflammatory activities, while ethanolic extracts from two ascidians and two sea cucumbers showed high in vivo osteogenic activity by promoting bone formation in zebrafish larvae. These findings indicate that ethanolic extracts from both ascidians and sea cucumber are potential sources of compounds with health benefits, and require further research for chemical characterization of the extracts and identification of the compounds responsible for the effect reported.