Novel peptides from sea cucumber intestines hydrolyzed by neutral protease alleviate exercise-induced fatigue via upregulating the glutaminemediated Ca2+ /Calcineurin signaling pathway in mice.

Sea cucumber intestines are considered a valuable resource in the sea cucumber processing industry due to their balanced amino acid composition. Studies have reported that peptides rich in glutamate and branched-chain amino acids have anti-fatigue properties. However, the function of the sea cucumber intestine in reducing exercise-induced fatigue remains unclear. In this study, we enzymatically hydrolyzed low molecular weight peptides from sea cucumber intestines (SCIP) and administered SCIP orally to mice to examine its effects on exercise-induced fatigue using swimming and pole-climbing exhaustion experiments. The results revealed that supplementation with SCIP significantly prolonged the exhaustion time of swimming in mice, decreased blood lactate and urea nitrogen levels, and increased liver and muscle glycogen levels following a weight-loaded swimming test. Immunofluorescence analysis indicated a notable increase the proportion of slow-twitch muscle fiber and a significant decrease the proportion of fast-twitch muscle fiber following SCIP supplementation. Furthermore, SCIP upregulated mRNA expression levels of Ca2+ /Calcineurin upstream and downstream regulators, thereby contributing to the promotion of skeletal muscle fiber type conversion. This study presents the initial evidence establishing SCIP as a potential enhancer of skeletal muscle fatigue resistance, consequently providing a theoretical foundation for the valuable utilization of sea cucumber intestines.

Antioxidant and ACE-Inhibitory Activity of Protein Hydrolysates Produced from Atlantic Sea Cucumber (Cucumaria frondosa).

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.

Novel Peptides Derived from Sea Cucumber Intestine Promotes Osteogenesis by Upregulating Integrin-Mediated Transdifferentiation of Growth Plate Chondrocytes to Osteoblasts.

The sea cucumber intestine is a major by-product of sea cucumber processing and contains high levels of protein. In this study, we isolated and identified 28 novel osteogenic peptides from sea cucumber intestinal hydrolysis by the activity-tracking method for the first time. In vitro experimental results showed that compared with high molecular weight, the peptides from sea cucumber intestine (SCIP) with molecular weight <3 kDa more significantly promoted the proliferation and mineralized nodules of MC3T3-E1 cell and exhibited potential integrin binding capacity. In vivo experimental results showed that the SCIP supplement significantly increased the longitudinal bone length and elevated the height of the growth plate (especially the hypertrophic zone, 37.2%, p < 0.01) in adolescent mice. Further, immunofluorescence labeling results indicated that the SCIP supplement increased chondrocyte transdifferentiate to osteoblast in the growth plate close to the diaphysis. Mechanistically, transcriptome analysis revealed that the SCIP supplement induced the dedifferentiation of chondrocyte to osteoprogenitor cell via integrin-mediated histone acetylation and then redifferentiated to osteoblast via integrin-mediated Wnt/ß-catenin signaling. These results reported for the first time that sea cucumber intestine had the potential to develop into a dietary supplement for promoting osteogenic, and provide new evidence for the mechanism of dietary promotes chondrocyte to osteoblast transdifferentiation.

Characterization and Expression Analysis of Regeneration-Associated Protein (Aj-Orpin) during Intestinal Regeneration in the Sea Cucumber Apostichopus japonicus.

Apostichopus japonicus achieves intestinal regeneration in a short period after evisceration, and multiple genes are involved in this process. The transcriptome of A. japonicus was screened for regeneration-associated protein (Aj-Orpin), a gene that is specifically upregulated during intestinal regeneration. The expression and function of Aj-Orpin were identified and investigated in this study. The 5' and 3' RACE polymerase chain reaction (PCR) was used to clone the full-length cDNA of Aj-Orpin. The open reading frame codes for a 164 amino-acid protein with an EF-hand_7 domain and overlapping signal peptides and transmembrane regions. Moreover, Aj-Orpin mRNA and protein expression during intestinal regeneration was investigated using real-time quantitative PCR and Western blot. The expression pattern of Aj-Orpin in the regenerating intestine was investigated using immunohistochemistry. The results showed that Aj-Orpin is an exocrine protein with two EF-hand-like calcium-binding domains. Expression levels were higher in the regenerating intestine than in the normal intestine, but protein expression changes lagged behind mRNA expression changes. Aj-Orpin was found to play a role in the formation of blastema and lumen. It was primarily expressed in the serosal layer and submucosa, suggesting that it might be involved in proliferation. These observations lay the foundation for understanding the role of Orpin-like in echinoderm intestinal regeneration.

Oral administration of sea cucumber (Stichopus japonicus) protein exerts wound healing effects via the PI3K/AKT/mTOR signaling pathway.

This study aimed to investigate the effect of the oral administration of sea cucumber protein (SCP) on wound healing. SCP was isolated and purified from the body wall of Stichopus japonicus. A mouse skin incision model was operated on to evaluate the wound repair effect of SCP. The histological changes in the skin at the wound sites of BALB/c mice were observed by staining with haematoxylin and eosin (H&E) and Masson's trichrome. The enzyme-linked immunosorbent assay (ELISA) was used to analyze the expression of inflammatory cytokines in BALB/c mice. The boost cell migration ability was detected by a scratch assay after HaCaT cells were cultured with digested SCP (dSCP). Western blotting and RT-PCR assays were performed to determine the mechanism of SCP promoting wound healing. As a result, the wound healing rate in the SCP high dose group was 1.3-fold, compared to that in the blank group on day 14. Also, increased epidermal thickness and 1.79-fold collagen deposition contrasted with the blank group. Additionally, SCP could up-regulate the levels of pro-inflammatory factors (IL-1ß, IL-6, TNF-α) from day 3 to 7 firstly and decreased from day 7 to 14. IL-8 expression continuously decreased while the level of anti-inflammatory factor (IL-10) increased during the healing stage. Furthermore, the cell closure area reached 67% after being treated with 50 µg mL-1 of dSCP for 48 h. Cell proliferation was associated with the dSCP-activated PI3K/AKT/mTOR pathway. Taken together, SCP can be orally used as an effective agent for wound repair.

Novel peptides from sea cucumber intestinal hydrolysates promote longitudinal bone growth in adolescent mice through accelerating cell cycle progress by regulating glutamine metabolism.

Sea cucumber intestines are recognized as a major by-product in the sea cucumber processing industry and have been shown to exhibit bioactive properties. However, whether the sea cucumber intestine is beneficial for osteogenesis remains unknown. In this study, low molecular weight peptides rich in glutamate/glutamine were obtained from sea cucumber intestines (SCIP) by enzymatic hydrolysis, and orally administered to adolescent mice to investigate the effects on longitudinal bone growth. The results showed that the SCIP supplement significantly increased the femur length and new bone formation rate by 9.6% and 56.3%, and elevated the levels of serum osteogenic markers alkaline phosphatase (ALP), Collagen I and osteocalcin (OCN). Notably, H&E staining showed that SCIP significantly increased the height of the growth plate, in which the height of the proliferation zone was elevated by 95.6%. Glutamine is a major determinant of bone growth. SCIP supplement significantly increased glutamine levels in the growth plate by 44.2% and upregulated the expression of glutamine metabolism-related enzymes glutaminase 1 (Gls1) and glutamate dehydrogenase 1 (GLUD1) in the growth plate. Furthermore, SCIP supplement upregulated growth plate acetyl coenzyme A levels to promote histone acetylation and accelerated cell cycle progression by upregulating Sox9 expression, thereby contributing to rapid chondrocyte proliferation. To the best of our knowledge, this is the first report where SCIP could enhance longitudinal bone growth via promoting growth plate chondrocyte proliferation. The present study will provide new ideas and a theoretical basis for the high-value utilization of sea cucumber intestines.

Effects of chronic prometryn exposure on antioxidative status, intestinal morphology, and microbiota in sea cucumber (Apostichopus japonicus).

Prometryn is an occasional triazine herbicide used in aquaculture to kill algae. However, deposition of prometryn at the bottom of the pond poses a potential threat to aquatic animals, especially benthos, such as the sea cucumber. This study investigated the toxic effects of prometryn oral exposure on antioxidants, and the intestinal histomorphology and microbiome of sea cucumbers. Results showed that the accumulation of prometryn in the intestine, respiratory tree, and body wall decreased sequentially under the same level. Severe pathological damages were observed in the intestines of sea cucumbers fed with 0.080 and 1.595 g/kg prometryn (measured concentration). Moreover, hydrogen peroxide (H2O2) and malondialdehyde (MDA) concentrations were significantly increased in prometryn treatment groups compared to the control group (P < 0.05), while the catalase (CAT) activity was significantly decreased (P < 0.05) in the coelomic fluid of treatment groups. At the phylum level, the abundance of Proteobacteria was significantly higher in the 0.080 g/kg treatment group than in the control group. In addition, prometryn exposure reduced the diversity of intestinal microflora in sea cucumbers. In conclusion, these results suggest that prometryn has potential toxicity to sea cucumber. Therefore, the harm of prometryn deposited in the sediment to aquatic animals must be a concern in aquaculture.

Dietary Supplementation with Exogenous Sea-Cucumber-Derived Ceramides and Glucosylceramides Alleviates Insulin Resistance in High-Fructose-Diet-Fed Rats by Upregulating the IRS/PI3K/Akt Signaling Pathway.

Endogenous ceramide is considered to be associated with the progress of insulin resistance. However, the effects of dietary exogenous glucosylceramides and ceramides on insulin resistance are unclear. A model of fructose-induced male Sprague Dawley rats was used to compare the effects of sea-cucumber-derived glucosylceramides and ceramides on insulin resistance. Both glucosylceramides and ceramides significantly improved glucose tolerance, reduced the concentrations of serum glucose and glycosylated hemoglobin, and alleviated the accompanied hypertension. Ceramides significantly enhanced glycogen levels in skeletal muscle, whereas glucosylceramides significantly increased the hepatic glycogen levels. Moreover, glucosylceramides alleviated insulin resistance by inhibiting gluconeogenesis, promoting glycogen synthesis and insulin signal transduction in the liver; meanwhile, ceramides were mainly due to the promotion of glycogen synthesis and insulin signal transduction in skeletal muscle. Additionally, glucosylceramides and ceramides effectively attenuated inflammation in adipose tissue. These results indicate that glucosylceramides and ceramides have potential value in the prevention and alleviation of insulin resistance.

Characterization of a Coproduct from the Sea Cucumber Cucumaria frondosa and Its Effects on Visceral Adipocyte Size in Male Wistar Rats.

Sea cucumbers have been shown to have potential health benefits and are a rich source of several bioactive compounds, particularly triterpenoid saponins. However, most studies concentrate on the body wall, and little is known about the health effects of the coproducts. The objectives of this study were to determine the nutritional composition of a coproduct from the sea cucumber Cucumaria frondosa and the effects of the dietary consumption of this coproduct on cardiometabolic health in rats. Chemical, biochemical, and nutritional analyses were performed to characterize this coproduct. Forty (40) male Wistar rats were then equally divided into four groups and fed a purified control diet or a diet enriched with 0.5%, 1.5%, or 2.5% (by protein) of coproduct. After 28 days of feeding, the rats were sacrificed. Body and tissue weight, body composition, epididymal adipocyte diameter, plasma and hepatic lipids, glycemia, and insulinemia were measured at the end of the 28-day experiment. Analysis of the coproduct revealed high levels of protein, omega-3 fatty acids, minerals, and saponins. The 1.5% group had significantly smaller epididymal adipocytes vs. the control. We conclude that dietary administration of this sea cucumber coproduct at 1.5% doses decreases visceral adiposity, potentially decreasing the risk of cardiometabolic dysfunction. The coproduct's saponin content may contribute to the observed effects, but the impact of other components cannot be ruled out.

Environmentally Friendly Valorization of Solieria filiformis (Gigartinales, Rhodophyta) from IMTA Using a Biorefinery Concept.

Marine macroalgae (seaweed) are an excellent source of novel bioactive metabolites. The biorefinery concept applied to seaweed facilitates the extraction of many chemical constituents from the same biomass ensuring that the resource is used fully, generating few residues through a succession of extraction steps. In the present study, the biomass of the carragenophyte Solieria filiformis (Rhodophyta, Gigartinales) cultured in an integrated multi-trophic aquaculture (IMTA) system was evaluated to obtain valuable products by a biorefinery approach. Enzymatic-assisted extraction (EAE) and microwave-assisted extraction (MAE) were the eco-friendly technologies used to ensure an environmentally friendly valorization of the biomass. Three valuable products were successfully recovered: a water-soluble extract rich in proteins and sulfated polysaccharides suitable as a food supplement; a lipid fraction rich in polyunsaturated fatty acids (PUFAs) with potential to be used in the nutraceutical industry; and a pure ι-carrageenan with a powerful antiviral activity against Herpes simplex virus (EC50 = 6.3 µg mL-1) comparable to the commercial antiviral acyclovir (EC50 = 3.2⁻5.4 µg mL-1).

The first description of complete invertebrate arginine metabolism pathways implies dose-dependent pathogen regulation in Apostichopus japonicus.

In this study, three typical members representative of different arginine metabolic pathways were firstly identified from Apostichopus japonicus, including nitric oxide synthase (NOS), arginase, and agmatinase. Spatial expression analysis revealed that the AjNOS transcript presented negative expression patterns relative to those of Ajarginase or Ajagmatinase in most detected tissues. Furthermore, Vibrio splendidus-challenged coelomocytes and intestine, and LPS-exposed primary coelomocytes could significantly induce AjNOS expression, followed by obviously inhibited Arginase and AjAgmatinase transcripts at the most detected time points. Silencing the three members with two specific siRNAs in vivo and in vitro collectively indicated that AjNOS not only compete with Ajarginase but also with Ajagmatinase in arginine metabolism. Interestingly, Ajarginase and Ajagmatinase displayed cooperative expression profiles in arginine utilization. More importantly, live pathogens of V. splendidus and Vibrio parahaemolyticus co-incubated with primary cells also induced NO production and suppressed arginase activity in a time-dependent at an appropriate multiplicity of infection (MOI) of 10, without non-pathogen Escherichia coli. When increasing the pathogen dose (MOI = 100), arginase activity was significantly elevated, and NO production was depressed, with a larger magnitude in V. splendidus co-incubation. The present study expands our understanding of the connection between arginine's metabolic and immune responses in non-model invertebrates.

The improvement of the endogenous antioxidant property of stone fish (Actinopyga lecanora) tissue using enzymatic proteolysis.

The stone fish (Actinopyga lecanora) ethanolic and methanolic tissue extracts were investigated for total phenolic contents (TPCs) as well as antioxidant activity using 2,2-diphenyl-1-picrylhydrazyl (DPPH(•)) radical scavenging activity and ferric reducing antioxidant power (FRAP) assays. Both extracts showed low amount of phenolics (20.33 to 17.03 mg of gallic acid equivalents/100 g dried sample) and moderate antioxidant activity (39% to 34% DPPH(•) radical scavenging activity and 23.95 to 22.30 mmol/100 mL FeSO4 FRAP value). Enzymatic proteolysis was carried out in order to improve the antioxidant activity using six commercially available proteases under their optimum conditions. The results revealed that the highest increase in antioxidant activity up to 85% was obtained for papain-generated proteolysate, followed by alcalase (77%), trypsin (75%), pepsin (68%), bromelain (68%), and flavourzyme (50%) as measured by DPPH(•) radical scavenging activity, whilst for the FRAP value, the highest increase in the antioxidant activity up to 39.2 mmol/100 mL FeSO4 was obtained for alcalase-generated proteolysate, followed by papain (29.5 mmol/100 mL FeSO4), trypsin (23.2 mmol/100 mL FeSO4), flavourzyme (24.7 mmol/100 mL FeSO4), bromelain (22.9 mmol/100 mL FeSO4), and pepsin (20.8 mmol/100 mL FeSO4). It is obvious that proteolysis of stone fish tissue by proteolytic enzymes can considerably enhance its antioxidant activity.

Actinopyga lecanora hydrolysates as natural antibacterial agents.

Actinopyga lecanora, a type of sea cucumber commonly known as stone fish with relatively high protein content, was explored as raw material for bioactive peptides production. Six proteolytic enzymes, namely alcalase, papain, pepsin, trypsin, bromelain and flavourzyme were used to hydrolyze A. lecanora at different times and their respective degrees of hydrolysis (DH) were calculated. Subsequently, antibacterial activity of the A. lecanora hydrolysates, against some common pathogenic Gram positive bacteria (Bacillus subtilis and Staphylococcus aureus) and Gram negative bacteria (Escherichia coli, Pseudomonas aeruginosa, and Pseudomonas sp.) were evaluated. Papain hydrolysis showed the highest DH value (89.44%), followed by alcalase hydrolysis (83.35%). Bromelain hydrolysate after one and seven hours of hydrolysis exhibited the highest antibacterial activities against Pseudomonas sp., P. aeruginosa and E. coli at 51.85%, 30.07% and 30.45%, respectively compared to the other hydrolysates. Protein hydrolysate generated by papain after 8 h hydrolysis showed maximum antibacterial activity against S. aureus at 20.19%. The potent hydrolysates were further fractionated using RP-HPLC and antibacterial activity of the collected fractions from each hydrolysate were evaluated, wherein among them only three fractions from the bromelain hydrolysates exhibited inhibitory activities against Pseudomonas sp., P. aeruginosa and E. coli at 24%, 25.5% and 27.1%, respectively and one fraction of papain hydrolysate showed antibacterial activity of 33.1% against S. aureus. The evaluation of the relationship between DH and antibacterial activities of papain and bromelain hydrolysates revealed a meaningful correlation of four and six order functions.

High-value components and bioactives from sea cucumbers for functional foods--a review.

Sea cucumbers, belonging to the class Holothuroidea, are marine invertebrates, habitually found in the benthic areas and deep seas across the world. They have high commercial value coupled with increasing global production and trade. Sea cucumbers, informally named as bêche-de-mer, or gamat, have long been used for food and folk medicine in the communities of Asia and Middle East. Nutritionally, sea cucumbers have an impressive profile of valuable nutrients such as Vitamin A, Vitamin B1 (thiamine), Vitamin B2 (riboflavin), Vitamin B3 (niacin), and minerals, especially calcium, magnesium, iron and zinc. A number of unique biological and pharmacological activities including anti-angiogenic, anticancer, anticoagulant, anti-hypertension, anti-inflammatory, antimicrobial, antioxidant, antithrombotic, antitumor and wound healing have been ascribed to various species of sea cucumbers. Therapeutic properties and medicinal benefits of sea cucumbers can be linked to the presence of a wide array of bioactives especially triterpene glycosides (saponins), chondroitin sulfates, glycosaminoglycan (GAGs), sulfated polysaccharides, sterols (glycosides and sulfates), phenolics, cerberosides, lectins, peptides, glycoprotein, glycosphingolipids and essential fatty acids. This review is mainly designed to cover the high-value components and bioactives as well as the multiple biological and therapeutic properties of sea cucumbers with regard to exploring their potential uses for functional foods and nutraceuticals.