Thromboembolic diseases pose a serious risk to human health worldwide. Fucosylated chondroitin sulfate (FCS) is reported to have good anticoagulant activity with a low bleeding risk. Molecular weight plays a significant role in the anticoagulant activity of FCS, and FCS smaller than octasaccharide in size has no anticoagulant activity. Therefore, identifying the best candidate for developing novel anticoagulant FCS drugs is crucial. Herein, native FCS was isolated from sea cucumber Cucumaria frondosa (FCScf) and depolymerized into a series of lower molecular weights (FCScfs). A comprehensive assessment of the in vitro anticoagulant activity and in vivo bleeding risk of FCScfs with different molecule weights demonstrated that 10 kDa FCScf (FCScf-10 K) had a greater intrinsic anticoagulant activity than low molecular weight heparin (LMWH) without any bleeding risk. Using molecular modeling combined with experimental validation, we revealed that FCScf-10 K can specifically inhibit the formation of the Xase complex by binding the negatively charged sulfate group of FCScf-10 K to the positively charged side chain of arginine residues on the specific surface of factor IXa. Thus, these data demonstrate that the intermediate molecular weight FCScf-10 K is a promising candidate for the development of novel anticoagulant drugs.

Four new mono- and trisulfated triterpene penta- and tetraosides, djakonoviosides C1 (1), D1 (2), E1 (3), and F1 (4) were isolated from the Far Eastern sea cucumber Cucumaria djakonovi (Cucumariidae, Dendrochirotida), along with six known glycosides found earlier in other Cucumaria species. The structures of unreported compounds were established on the basis of extensive analysis of 1D and 2D NMR spectra as well as by HR-ESI-MS data. The set of compounds contains six different types of carbohydrate chains including two new ones. Thus, djakonovioside C1 (1) is characterized by xylose as the second residue, that was a branchpoint in the pentasaccharide chain. Meanwhile, only quinovose and rarely glucose have been found earlier in pentasaccharide chains branched at C-2 of the second sugar unit. Djakonovioside E1 (3) is characterized by a tetrasaccharide trisulfated chain, with glucose as the second residue. So, in the series of isolated glycosides, three types of sugars in the second position were presented: the most common, quinovose-in six compounds; glucose-in three substances; and the rare xylose-in one glycoside. The set of aglycones was composed of holostane- and non-holostane-type polycyclic systems; the latter comprised normal and reduced side chains. Noticeably, isokoreoside A (9), isolated from C. djakonovi, was a single glycoside having a 9(11)-double bond, indicating two oxidosqualenecyclases are operating in the process of the biosynthesis of aglycones. Some of the glycosides from C. djakonovi, which were characterized by pentasaccharide branched chains containing one to three sulfate groups, are chemotaxonomic features of the representatives of the genus Cucumaria. The assortment of sugar parts of Cucumaria\'s glycosides was broadened with previously undescribed penta- and tetrasaccharide moieties. The metabolic network of sugar parts and aglycones is constructed based on biogenetic relationships. The cytotoxic action of compounds 1-10, isolated from C. djakonovi, against human breast cancer cell lines was investigated along with the hemolytic activity. Erythrocytes were, as usual, more sensitive to the membranolytic action of the glycosides than cancer cells. The triple-negative breast cancer MDA-MB-231 cell line was more vulnerable to the action of glycosides in comparison with the other tested cancer cells, while the MCF-7 cell line was less susceptible to cytotoxic action. Djakonovioside E1 (3) demonstrated selective action against ER-positive MCF-7 and triple-negative MDA-MB-231 cell lines, while the toxic effect in relation to normal mammary epithelial cells (MCF-10A) was absent. Cucumarioside A2-5 (6) inhibited the formation and growth of colonies of cancer cells to 44% and tumor cell migration to 85% of the control. Quantitative structure-activity relationships (QSAR) were calculated on the basis of the correlational analysis of the physicochemical properties and structural features of the glycosidic molecules and their membranolytic activity. QSAR revealed the extremely complex nature of such relationships, but these calculations correlated well with the observed SAR.

Sea cucumbers, belonging to the phylum Echinodermata, are known to possess valuable bioactive compounds that have medicinal properties. In several countries, such as Korea, China, and Japan, they are cultured in the aquaculture industries for food and medicinal purposes. Research has shown that different species of sea cucumbers each possesses unique medicinal values. As a result, we strive towards finding species with better health resilience in aquaculture system to be cultured for nutritional and medicinal purposes. In this paper, we compared the physiological and immunological parameters of three species of sea cucumbers, Cucumaria frondosa (C. frondosa), Isostychopus badionotus (I. badionotus), and Pentacta pygmaea (P. Pygmaea) from the waters of the eastern United States as they have not been studied extensively. Four different cells of sea cucumbers, phagocytic, red spherule, white spherule, and vibratile cells, that contribute to their immunity were counted. C. frondosa exhibited the highest concentrations of phagocytic cells, white spherule cells, and vibratile cells, compared to the two other species. Due to its high phagocytic cell concentration, the highest phagocytic capacity was seen in C. frondosa although it was not statistically significant. We also observed that C. frondosa had the highest total cell count and the highest concentration of coelomic protein among the three species. Lastly, C. frondosa possessed the highest lysozyme activity. Taken together, we concluded that C. frondosa is the best of the three species compared to be reared in the aquaculture systems for use in the food and biomedicine industries due to its immunological and physiological properties.

The study aimed to investigate the effects of alcalase, papain, flavourzyme, and neutrase on the structural characteristics and bioactivity stability of Cucumaria frondosa intestines and ovum hydrolysates (CFHs). The findings revealed that flavourzyme exhibited the highest hydrolysis rate (51.88% ± 1.87%). At pH 2.0, the solubility of hydrolysate was the lowest across all treatments, while the solubility at other pH levels was over 60%. The primary structures of hydrolysates of different proteases were similar, whereas the surface hydrophobicity of hydrolysates was influenced by the types of proteases used. The hydrolysates produced by different proteases were also analyzed for their absorption peaks and antioxidant activity. The hydrolysates of flavourzyme had β-fold absorption peaks (1637 cm-1), while the neutrase and papain hydrolysates had N-H bending vibrations. The tertiary structure of CFHs was unfolded by different proteases, exposing the aromatic amino acids and red-shifting of the λ-peak of the hydrolysate. The alcalase hydrolysates showed better antioxidant activity in vitro and better surface hydrophobicity than the other hydrolysates. The flavourzyme hydrolysates displayed excellent antioxidant stability and pancreatic lipase inhibitory activity during gastrointestinal digestion, indicating their potential use as antioxidants in the food and pharmaceutical industries.

Atlantic sea cucumber is a benthic marine echinoderm found in Northwest Atlantic waters and is harvested mainly for its body wall. The body wall, along with internal organs and aquaphyrangeal bulb/flower, is a rich source of proteins, where the latter parts are often considered as processing discards. The objective of this research was to produce protein hydrolysates from sea cucumber tissues (body wall, flower, and internal organs) with bioactive properties associated with antioxidants, DNA and LDL cholesterol oxidation inhibition, and angiotensin-I-converting enzyme (ACE) inhibitory effects. The protein hydrolysates were prepared using food-grade commercial enzymes, namely Alcalase, Corolase, and Flavourzyme, individually and in combination, and found that the combination of enzymes exhibited stronger antioxidant potential than the individual enzymes, as well as their untreated counterparts. Similar trends were also observed for the DNA and LDL cholesterol oxidation inhibition and ACE-inhibitory properties of sea cucumber protein hydrolysates, mainly those that were prepared from the flower. Thus, the findings of this study revealed potential applications of sea cucumber-derived protein hydrolysates in functional foods, nutraceuticals, and dietary supplements, as well as natural therapeutics.

Seven new monosulfated triterpene glycosides, djakonoviosides A (1), A1 (2), A2 (3), and B1-B4 (4-7), along with three known glycosides found earlier in the other Cucumaria species, namely okhotoside A1-1, cucumarioside A0-1, and frondoside D, have been isolated from the far eastern sea cucumber Cucumaria djakonovi (Cucumariidae, Dendrochirotida). The structures were established on the basis of extensive analysis of 1D and 2D NMR spectra and confirmed by HR-ESI-MS data. The compounds of groups A and B differ from each other in their carbohydrate chains, namely monosulfated tetrasaccharide chains are inherent to group A and pentasaccharide chains with one sulfate group, branched by C-2 Qui2, are characteristic of group B. The aglycones of djakonoviosides A2 (3), B2 (5), and B4 (7) are characterized by a unique structural feature, a 23,16-hemiketal fragment found first in the sea cucumbers\' glycosides. The biosynthetic pathway of its formation is discussed. The set of aglycones of C. djakonovi glycosides was species specific because of the presence of new aglycones. At the same time, the finding in C. djakonovi of the known glycosides isolated earlier from the other species of Cucumaria, as well as the set of carbohydrate chains characteristic of the glycosides of all investigated representatives of the genus Cucumaria, demonstrated the significance of these glycosides as chemotaxonomic markers. The membranolytic actions of compounds 1-7 and known glycosides okhotoside A1-1, cucumarioside A0-1, and frondoside D, isolated from C. djakonovi against human cell lines, including erythrocytes and breast cancer cells (MCF-7, T-47D, and triple negative MDA-MB-231), as well as leukemia HL-60 and the embryonic kidney HEK-293 cell line, have been studied. Okhotoside A1-1 was the most active compound from the series because of the presence of a tetrasaccharide linear chain and holostane aglycone with a 7(8)-double bond and 16β-O-acetoxy group, cucumarioside A0-1, having the same aglycone, was slightly less active because of the presence of branching xylose residue at C-2 Qui2. Generally, the activity of the djakonoviosides of group A was higher than that of the djakonoviosides of group B containing the same aglycones, indicating the significance of a linear chain containing four monosaccharide residues for the demonstration of membranolytic action by the glycosides. All the compounds containing hemiketal fragments, djakonovioside A2 (3), B2 (5), and B4 (7), were almost inactive. The most aggressive triple-negative MDA-MB-231 breast cancer cell line was the most sensitive to the glycosides action when compared with the other cancer cells. Okhotoside A1-1 and cucumarioside A0-1 demonstrated promising effects against MDA-MB-231 cells, significantly inhibiting the migration, as well as the formation and growth, of colonies.

Intertidal animals experience cycles of tidal emersion from water and are vulnerable to copper (Cu) exposure due to anthropogenic toxicant input into marine waters. Both emersion and Cu toxicity can cause damage to physiological processes like aerobic metabolism, ammonia excretion, and osmoregulation, but the interactions of the combination of these two stressors on marine invertebrates are understudied. Mixed effects of 96 h of low and high Cu exposure (20 and 200 μg/L) followed by 6 h of tidal emersion were evaluated on the intertidal sea cucumber Cucumaria miniata. The respiratory tree accumulated the highest concentrations of Cu, followed by the introvert retractor muscle, body wall, and coelomic fluid. Emersion affected accumulation of Cu, perhaps by inhibiting excretion. 200 μg/L of Cu increased lactate production in the respiratory tree, indicative of damaged aerobic metabolism. Cu diminished ammonia excretion, but emersion increased oxygen uptake and ammonia excretion upon re-immersion. The combination of the two stressors did not have any interactive effects on metabolism or ammonia excretion. Neither Cu exposure nor emersion altered ion (sodium, potassium, calcium, magnesium) content of the coelomic fluid. Overall, results of this study suggest that Cu exposure does not alter C. miniata\'s high tolerance to emersion, and some potential strategies that this species uses to overcome environmental stress are illuminated.

Sea cucumber tentacles (aquapharyngeal bulb) are a rich source of biologically active compounds, including phenolics, however they are mainly discarded as processing waste. This study evaluated free, esterified, and insoluble-bound phenolics of Atlantic sea cucumber (Cucumaria frondosa) tentacles and their antioxidant activity for the first time. Biological properties such as inhibitory activities against α-glucosidase, tyrosinase, and the formation of AGEs as well as LDL-cholesterol and DNA oxidation were investigated. The antioxidant activity of the phenolic extracts was also evaluated in a fish model system. In the UHPLC-QTOF-MS/MS analysis, 31 phenolic compounds, mainly phenolic acids and flavonoids, were identified and quantified. Among them, eight compounds were detected for the first time in any species of sea cucumber. The free phenolic fraction was the major form of phenolics, mainly protocatechuic acid, p-coumaric acid, gallic acid, ellagic acid, catechin, and quercetin, exhibiting strong antioxidant and biological activities. Fresh Atlantic salmon treated with sea cucumber phenolics delayed lipid oxidation as measured by the thiobarbituric acid reactive substances (TBARS) assay. Therefore, Atlantic sea cucumber tentacles may serve as a viable source of functional food ingredients with protective antioxidant properties.

CEL-III is a hemolytic lectin purified from the marine invertebrate Cucumaria echinata. New expression system of CEL-III was constructed, and the recombinant thioredoxin-fused CEL-III (Trx-CEL-III) showed strong hemolytic and carbohydrate-binding activity as same as authentic CEL-III. Mutation analysis of Trx-CEL-III suggested that carbohydrate binding to subdomain 1α and 2β of CEL-III might be important for the hemolytic activity.

Sea cucumber contains a wide range of bioactive compounds, including phenolics. This study investigated the free, esterified, and insoluble-bound phenolics of sea cucumber body wall as affected by high-pressure processing (HPP) pretreatment. Sea cucumber body wall was subjected to HPP (200, 400, and 600 MPa for 5, 10, and 15 min), followed by the extraction of phenolics. The contents of total phenolics and antioxidant activity were monitored. Compared to untreated samples, those treated with HPP exhibited significantly higher total phenolics, flavonoids, and antioxidant activities. Treatment of 600 MPa for 10 min offered the optimal results. The highest amount of phenolics was observed in the free phenolic fraction, followed by esterified and insoluble-bound phenolic fractions. Moreover, phenolic extracts showed inhibitory effects against cupric ion-induced low-density lipoprotein (LDL)-cholesterol oxidation, peroxyl and hydroxyl radical-induced DNA scission, α-glucosidase activity, and formation of advanced glycation end products (AGEs). Ultra-high-performance liquid chromatography equipped with a quadrupole time of fight and mass spectrometer (UHPLC-QTOF-MS/MS) identified 20 phenolic compounds, mainly phenolic acids and flavonoids, from the body wall of this species for the first time. Thus, sea cucumber may lead to the production of a multitude of value-added products.