Sea cucumbers are highly used not only for the production of the bêche-de-mer, but also recent studies have been showing the high potential of these marine invertebrates on the pharmaceutical industry, as they are rich in bioactive compounds with important functions. Stichopus regalis extracts (1 mg·mL -1) were used in antioxidant, antimicrobial and antitumor assays. The lipid profile was also evaluated. No significant antioxidant activity was detected in both methanolic and dichloromethane extracts. The methanolic fraction showed the highest antimicrobial potential against Candida albicans with an IC 50 of 475.4 μg·mL -1. In the antitumor assays, the dichloromethane fraction showed a high potential for both cell lines, as revealed by the MTT method. The total fat content was 3.63% ± 0.11% and the fatty acid profile revealed the highest amount in C16:0 (9.43% ± 0.77%), C18:0 (12.43% ± 0.83%), C18:1 ω7 (5.63% ± 0.33%), EPA (12.49% ± 0.15%), DHA (7.35% ± 0.02%), ARA (19.29% ± 0.14%) and a ω3/ ω6 ratio of 1.078. These findings led us to suggest the potential use of S. regalis as a new source of bioactive compounds with pharmacological potential and its nutritional benefits for human health.
Sea cucumbers, also known as holothurians, are one of the most important seafood products in China, Japan and Singapore, where the tradition relies on eating the sea cucumbers’ body wall, known as bêche-de-mer. Further- more, the use of sea cucumbers have been raising because from a nutritional point of view, these organisms have high protein and low fat and are particularly rich in essential fatty acids that are responsible for preventing heart and degenerative diseases [
Recent studies have associated the use of sea cucumbers to the development of new therapies for the treatment of diseases, since these marine organisms have a variety of bioactive compounds with important functional activities. Moreover, various species of sea cucumbers have been used in clinical trials, since they exhibit anticoagulant, anti-inflammatory, antimicrobial, antioxidant, antitumor, tissue regeneration, among others capacities [
The development of laboratory assays in vitro has gained increasing importance in assessing the ability of sea cucumbers to different microorganisms and cell lines. Methanol, n-hexane and dichloromethane are the most used solvents in sea cucumber studies, revealing the presence of saponins and other chemical compounds [
Stichopus regalis (Cuvier, 1817), also known as Parastichopus regalis, is a holothurian species that belongs to the family Stichopodidae. It is a benthic species that can be found beyond 50 m and has a wide geographical distribution that includes the northwestern Mediterranean, eastern Atlantic from the south of the Canary Islands to the north of Ireland, and western Atlantic, Antilles and Gulf of Mexico. Stichopus regalis is the only species of Stichopodidae family to be found in the Mediterranean Sea and it is a much appreciated product in the Balearic Islands and Catalonia (NW Mediterranean), reaching 130 ?per kg and being known under the local names of “espardenya”, “llongo”, “llonguet” or “sola” [
To our knowledge, this is the first work combining the study of the biotechnological potential of this species and their important nutritional value by analyzing the lipid profile.
This study aims to provide information about the antioxidant, antimicrobial and antitumor potential of Stichopus regalis extracts and to analyze the total fat content and fatty acid profile of this economical important sea cucumber species.
A total of 15 individuals were collected by commercial fishermen from Peniche (Portugal) between 50 m and 100 m deep, in September of 2012. The samples were cleaned and stored in proper plastic recipients with sea water and transported to the Aquaculture Laboratory of the School of Tourism and Maritime Technology. Each sea cucumber was weighed and a longitudinal incision was made along the dorsal surface and the coelomic fluid and gonads were removed. Then the samples were stored at −80˚C until further use.
The extracts were prepared according to the adapted method by Mayachiew& Devahastin (2008) [
The total phenolic content was determined using Folin-Ciocalteu method, according to the adapted work of Yu et al. (2002) [
The DPPH free radical scavenging activity was evaluated by the described method of [
The oxygen radical absorbent capacity (ORAC) assay was performed as described by Dávalos et al. (2004) [
Antioxidant curves (fluorescence versus time) were first normalized to the curve of the blank corresponding to the same assay by multiplying original data by the factor fluorescenceblank,t = 0/fluorescencesample,t = 0. From the normalized curves, the area under the fluorescence decay curve (AUC) was calculated as:
where f0 is the initial fluorescence reading at 0 min and fi is the fluorescence reading at time i. The net AUC corresponding to a sample was calculated by subtracting the AUC corresponding to the blank. Regression equations between net AUC and antioxidant concentration were calculated for all the samples. ORAC values were expressed as Trolox equivalents (TE) by using the standard curve calculated for each assay. Final results were expressed in µmol TE/g of extract.
The antimicrobial activity of Stichopus regalis extracts was evaluated against eight microorganisms: Escheri- chia coli (ATCC 25922 and ATCC 10536), Pseudomonas aeruginosa (ATCC 27853), Bacillus subtilis (ATCC 6633) and Salmonella enteritidis (ATCC 13076) were cultured at 37˚C in Luria-broth (LB); Staphylococcus au- reus (ATCC 25923) was cultured at 37˚C in Trypticase Soy Yeast Extract medium (TSYE) and Saccharomyces cerevisiae (ATCC 9763) and Candida albicans (ATCC 10231) were cultured in Yeast Extract Peptone Dextrose (YPD) medium at 30˚C and 37˚C, respectively. All mediums were obtained from Merck (Darmstadt, Germany).
The antimicrobial assays were performed in 96 well plates, where it was added 193 µL of medium, 5 µL of microorganism inoculum and 2 µL of test samples per well and then incubated. Chloramphenicol (1 mg∙mL−1) and amphotericin B (300 µg∙mL−1) (Sigma Aldrich, Oakville, Canada) were used as positive controls and a blank for each sample was prepared. All samples were sterile filtered and the assays were performed in eight independent experiments under sterile conditions.
The ability of extracts to inhibit microbial growth was evaluated through spectrophotometric analysis at 600 nm. Results were expressed in percentage of control by the following equation:
where Abssample corresponds to the absorbance of microbial growth in the presence of the sea cucumber extracts, Absblank corresponds to the absorbance of the extracts in the respective medium and the Abscontrol corresponds to the absorbance of microbial growth without the extracts.
Fractions that showed the highest potential in reducing microbial growth were also evaluated through dose- response analysis in order to determine the IC50 values.
The cytotoxic potential (cell proliferation and viability) was tested on MCF-7 (breast carcinoma) and HepG-2 (human hepatocellular carcinoma) cell lines. Cells were cultured in RPMI 1640 medium supplemented with 10% of fetal bovine serum (FBS) and 1% antibiotic/antimycotic for HepG-2 cell line and 10% of fetal bovine serum (FBS), 1% antibiotic/antimycotic, MEM (non-essential amino acids), sodium pyruvate at 1 mM and human insulin at 10 µg∙mL−1 for MCF-7 cell line. The cell lines were maintained in culture in a CO2 incubator at 5% CO2, 95% humidity and a constant temperature of 37˚C.
The cell proliferation and cell viability studies were evaluated through the method reported by [
A dose-response assay (IC50) was performed for the fractions that showed the highest potential. The effects were revealed by the MTT and Calcein-AM methods. The results were presented as percentage of control, being calculated by the following equation:
where Fsample corresponds to the fluorescence/absorbance of the sea cucumber extracts plus cell lines and Fcontrol corresponds to the fluorescence/absorbance of DMSO (same % of the extracts) plus cell lines.
The total fat content was quantified following the modified [
where Iw is the initial weight of the vial (g), Fw is the final weight of the vial (g), and Sw is the sample weight (g).
The fatty acid profile was performed by the adapted work of [
A 1-way ANOVA followed by a post hoc Dunnett test was used for statistical analysis [
No significant antioxidant activity was detected in the three methods under study for both organic fractions of Stichopus regalis (data not shown).
In order to evaluate the antimicrobial potential of Stichopus regalis, the methanolic and dichloromethane fractions were tested against seven microorganisms and the positive results are summarized in
In the current study, both extracts, from the body wall of Stichopus regalis were shown to exhibit in vitro significant antifungal activity against S. cerevisiae and C. albicans. The methanolic fraction showed the highest potential in growth inhibition tests against the two strains indicating the presence of bioactive compounds with antifungal properties. Numerous chemical and pharmacological studies carried out on several species of sea cucumbers indicate that these invertebrate contain triterpene glycoside (saponins) with bioactive properties as antifungal, anti-inflammatory, cytotoxic and others [
The cell viability assays showed that both in MCF-7 (
The cell proliferation tests showed once again that in both MCF-7 (
Many chemotherapeutic agents are reported to exert their anticancer effects by inducing apoptosis of cancer cells [
in sea cucumbers. In general, these organisms are known to contain a high amount of good-quality protein that has been associated with beneficial effects [
The total fat content of S. regalis was 3.63% ± 0.11%. The fatty acid profile (
Fatty acids are essential for life, due to their role as source of energy, membrane constituents, as well as meta- bolic and signaling mediators. Due to their positive impact on human health, alternative sources of polyunsaturated fatty acids (PUFA) are being pursued, with marine organisms being increasingly regarded as good alternatives [
Fatty acid | Stichopus regalis |
---|---|
∑SFAa | 24.92 ± 1.73 |
C12:0 | 0.17 ± 0.09 |
C14:0 | 2.90 ± 0.04 |
C16:0 | 9.43 ± 0.77 |
C18:0 | 12.43 ± 0.83 |
∑MUFAb | 7.65 ± 0.69 |
C18:1 ω7 | 5.63 ± 0.33 |
C18:1 ω9 | 2.02 ± 0.36 |
∑PUFAc | 42.11 ± 0.38 |
C16:3 | 1.19 ± 0.01 |
C18:3 ω3 | 1.40 ± 0.02 |
C20:2 ω6 | 0.41 ± 0.04 |
C20:4 ω6 | 19.29 ± 0.14 |
C20:5 ω3 | 12.49 ± 0.15 |
C22:6 ω3 | 7.35 ± 0.02 |
ω3/ω6 | 1.078 |
aSaturated; bMonounsaturated; cPolyunsaturated.
be consistently higher than those found in marine fish [
In summary, this study provided valuable results concerning the high biotechnological and sea food potential of Stichopus regalis. A great antifungal potential was obtained, revealing possible new sources of compounds with applications for the pharmaceutical industry. The quantification of total fat and fatty acid profile demonstrated the nutritional benefits of this sea cucumber species for human health. Although these results are preliminary, further studies are required as the bioactive compounds identification and characterization. Its potential for the pharmaceutical industry or as seasonality can affect the lipid and protein content of this species.
We would like to thank Teresa Mouga as headmaster of the School of Tourism and Maritime Technology of Peniche, for providing the authorization and support to utilize the Laboratories and other facilities, necessary to conduct this work. We would also like to thank Tiago Simões and Rita Sousa for the valuable help in the lipid profile assays and Susana Mendes for all the statistical help given to this work.
The authors declare that there’s no conflict of interests.
Author contributions to the study and manuscript preparation are as follows. Conception and design: RS. Carried out the experiments: RS, SD, SP, JS and CA. Acquisition of data: RS, SD, SP, JS and CA. Analysis and interpretation: RS, SP, CA, AP, CT and RP. Drafting the article: RS, SD, SP, JS, CA, AP, CT and RP. Statistical analysis: RS. Study supervision: CT, AP and RP. All authors read and approved the final manuscript.