Polyphenol oxidase mediates (-)-epigallocatechin gallate to inhibit endogenous cathepsin activity in Apostichopus japonicus.

Apostichopus japonicus (A. japonicus) has rich nutritional value and is an important economic crop. Due to its rich endogenous enzyme system, fresh A. japonicus is prone to autolysis during market circulation and storage, resulting in economic losses. In order to alleviate this phenomenon, we investigated the effect of polyphenol oxidase (PPO) mediated (-)-epigallocatechin gallate (EGCG) on the activity and structure of endogenous cathepsin series protein (CEP) from A. japonicus. Research on cathepsin activity showed that PPO mediated EGCG could significantly reduce enzyme activity, resulting in a decrease in enzymatic reaction rate. SDS-PAGE and scanning electron microscopy results showed that PPO mediates EGCG could induce CEP aggregation to form protein aggregates. Various spectral results indicated that EGCG caused changes in the structure of CEP. Meanwhile, the conjugates formed by PPO mediated EGCG had lower thermal stability. In conclusion, PPO mediated EGCG was an effective method to inhibit the endogenous enzyme activity.

Investigation of the Presence of Fibrillin in Sea Cucumber (Apostichopus japonicus) Body Wall by Utilizing Targeted Proteomics and Visualization Strategies.

Fibrillin is an important structural protein in connective tissues. The presence of fibrillin in sea cucumber Apostichopus japonicus is still poorly understood, which limits our understanding of the role of fibrillin in the A. japonicus microstructure. The aim of this study was to clarify the presence of fibrillin in the sea cucumber A. japonicus body wall. Herein, the presence of fibrillin in sea cucumber A. japonicus was investigated by utilizing targeted proteomics and visualization strategies. The contents of three different isoforms of fibrillin with high abundance in A. japonicus were determined to be 0.96, 2.54, and 0.15 µg/g (wet base), respectively. The amino acid sequence of fibrillin (GeneBank number: PIK56741.1) that started at position 631 and ended at position 921 was selected for cloning and expressing antigen. An anti-A. japonicus fibrillin antibody with a titer greater than 1:64 000 was successfully obtained. It was observed that the distribution of fibrillin in the A. japonicus body wall was scattered and dispersed in the form of fibril bundles at the microscale. It further observed that fibrillin was present near collagen fibrils and some entangled outside the collagen fibrils at the nanoscale. Moreover, the stoichiometry of the most dominant collagen and fibrillin molecules in A. japonicus was determined to be approximately 250:1. These results contribute to an understanding of the role of fibrillin in the sea cucumber microstructure.

Fucosylated chondroitin sulfate from sea cucumber Stichopus chloronotus alleviate the intestinal barrier injury and oxidative stress damage in vitro and in vivo.

This study aimed to investigate the alleviative effects of fucosylated chondroitin sulfate from sea cucumber Stichopus chloronotus (fCSSc) on the intestinal barrier injury and oxidative stress damage in vitro and in vivo. The results showed that fCS-Sc protected the intestinal barrier and improved the antioxidant function in H2O2 damaged Caco-2 cells via up-regulating the tight junction proteins and activating Keap1-Nrf2-ARE antioxidant pathway. Furthermore, administration fCS-Sc could ameliorate the weight loss and spleen index decrease in Cyclophosphamide (Cy) treated mice, improve the expressions of ZO-1, Claudin-1, Nrf2, SOD, and NQO-1 in Cy damaged colon tissue, showing significant protective effects against intestinal barrier damage and oxidative stress in vivo. fCS-Sc intervention also alleviated the gut microbiota disorder though increasing the richness and diversity of intestinal bacteria, regulating the structural composition of gut microbiota. fCS-Sc promoted the relative abundance of beneficial microbiota and inhibited the growth of harmful bacteria. This study provided a theoretical basis for the application of fCS-Sc as a prebiotic in chemotherapy.

Physicochemical and thermal properties of pepsin- and acid-soluble collagen isolated from the body wall of sea cucumbers (Stichopus hermanni).

The main objective of this work was to characterize the acid-soluble collagen (ASC) and pepsin-soluble collagen (PSC) from the body wall of the sea cucumber scientifically called, Stichopus hermanni. For the extraction of ASC and PSC, the pre-treated sea cucumber body walls were subjected to 0.5 M acetic acid and 5 g L-1 pepsin, respectively. The yield of ASC (7.30% ± 0.30%) was found to be lower than the PSC (23.66% ± 0.15%), despite both ASC and PSC having similar chemical compositions except for the quantity of protein. The collagens produced from ASC and PSC show maximum peaks on ultraviolet-visible spectroscopic profiles at wavelengths of 230 and 235 nm, respectively, with no significant difference in the maximum temperature (Tmax ) of the extracted ASC and PSC. The ASC's coloration was whiter than that of the PSC. As a result, the collagen obtained from the body wall of the sea cucumber showed promise for usage as a substitute for collagen derived from marine sources. PRACTICAL APPLICATION: The two most popular methods of collagen extraction were acid hydrolysis and enzymatic hydrolysis. To determine whether the extracted collagen is a suitable substitute for animal collagen in different industries, it is required to characterize its physicochemical qualities. This study discovered a new application for marine collagen in the food industry: The sea cucumber has collagen with a greater yield in pepsin extraction with good physicochemical qualities.

Chemical substances and their activities in sea cucumber Apostichopus japonicus: A review.

Apostichopus japonicus, also known as Stichopus japonicus, with medicinal and food homologous figures, is a globally recognized precious ingredient with extremely high nutritional value. There is no relevant review available through literature search, so this article selects the research articles through the keywords "sea cucumber" and "Apostichopus japonicus (Stichopus japonicus)" in six professional databases, such as Wiley, PubMed, ScienceDirect, ACS, Springer, and Web of Science, from 2000 to the present, summarizing the extraction, isolation, and purification methods for the four major categories (polysaccharides, proteins and peptides, saponins, and other components) of the A. japonicus chemical substances and 10 effective biological activities of A. japonicus. Included are anticoagulation, anticancer/antitumor activities, hematopoiesis, regulation of gut microbiota, and immune regulatory activities that correspond to traditional efficacy. Literature support is provided for the development of medicines and functional foods and related aspects that play a leading role in future directions.

Polyphenol extracts from Ascophyllum nodosum protected sea cucumber (Apostichopus japonicas) body wall against thermal degradation during tenderization.

To retard the protein degradation during sea cucumber processing, polyphenol extracts from Ascophyllum nodosum (PhE) was used as a potential antioxidant to maintain the structural integrity of sea cucumber body wall. Accordingly, the protection effects of PhE (0, 0.5, 1.0 and 1.5 mg PhE/g SFBW) against thermal degradation of the solid fragments of body wall (SFBW) have been investigated in order to evaluate their impact on the oxidation level and structural changes. Electronic Spin Resonance results showed that PhE could significantly inhibit the occurrence of oxidation by scavenging the free radicals. The effect of PhE on chemical analysis of soluble matters in SFBW was characterized by SDS-PAGE and HPLC. Compared with thermally treated SFBW, samples with PhE presented a decrease in protein dissolution. Thermal treatment resulted in the disintegration of collagen fibrils and fibril bundles in SFBW samples, while the density of collagen fibrils was increased, and the porosity decreased in samples with PhE. The results of FTIR and intrinsic tryptophan fluorescence confirmed that the structures of SFBW were modified by PhE. Besides, the denaturing temperature and decomposition temperature were both improved with the addition of PhE. These results suggested that PhE appeared to have a positive effect on lowering oxidation and improving thermostability and structural stability of SFBW, which could provide a theoretical basis for protecting sea cucumber body wall against degradation during thermal tenderization.

Investigating the loss of major yolk proteins during the processing of sea cucumber (Apostichopus japonicus) using an MRM-based targeted proteomics strategy.

Major yolk proteins (MYPs), one class of the main abundant proteins in sea cucumber body wall, seem to garner more attention in recent years. Herein, a method using multiple reactions monitoring mass spectrometry (MRM-MS) was deliberatively developed to perform quantification analysis of three MYPs, i.e. BAH79576.1, BAH79577.1 and PIK45784.1. Contents of MYPs in body wall of fresh and dried sea cucumbers as well as in waste liquid of boiling and steaming were determined using their corresponding signature peptides of VDEFTGIVGSLR, KLDMYPPPLAR, LDMYPPPLAR, and SGHGEVMFVDSK. The loss of MYPs in the processing of sea cucumbers was directly verified by quantitation data of MYPs in sea cucumber body wall and the waste liquids. This study not only evidenced the loss of MYPs during the processing of sea cucumbers, but also implicated the potential of recycling MYPs from the processing waste water, providing helpful suggestions in maximizing the value of sea cucumbers.

Comparison of biochemical composition of commercial sea cucumbers, Apostichopus japonicus and Parastichopus californicus, under the same culture conditions.

BACKGROUND: Apostichopus japonicus and Parastichopus californicus are two of the most important and profitable commercial sea cucumbers along the North Pacific coast. This study compared the body wall production rate (BWPR), proximate composition, amino acid, fatty acid, trace element and vitamin composition, and nonspecific immune enzyme activities of A. japonicus and P. californicus cultured in an artificial pond. RESULTS: The BWPR, crude fat and ash content in the body walls of A. japonicus and P. californicus showed remarkable differences (P < 0.05). For the 18 amino acids tested, differences in the contents of 15 were significant (P < 0.05) between the two species, except for threonine, methionine and histidine, and their first limiting amino acids were both methionine+cysteine. There were seven saturated and ten unsaturated fatty acids in their body walls, and except for 18:1 and 20:1, the content differences of the other 15 fatty acids were all significant (P < 0.05). Furthermore, between the two sea cucumbers, differences in the content of seven trace elements (Cu, Fe, Mn, Zn, Cr, Ni, Se) and six vitamins (B1, B3, B5, B9, C, E) were significant (P < 0.05). The activities of superoxide dismutase (SOD), catalase (CAT), acid phosphatase (ACP) and alkaline phosphatase (AKP) also showed distinct differences (P < 0.05). CONCLUSION: There are greater differences in the biochemical compositions and contents between A. japonicus and P. californicus, each with its own unique quality advantages. A. japonicus and P. californicus have high nutritional value, which are both the superior sea cucumbers. © 2022 Society of Chemical Industry.

Characterization of a synthetic zinc-chelating peptide from sea cucumber (Stichopus japonicus) and its gastrointestinal digestion and absorption in vitro.

BACKGROUND: Zinc absorption in intestinal system could be strongly affected by the gastrointestinal digestion and absorption of zinc-chelating peptides serving as zinc carriers. In this study, a novel zinc-chelating sea cucumber synthetic peptide (SCSP) was synthesized to estimate its gastrointestinal digestion and promotive effect of zinc absorption in vitro. RESULTS: Analysis of isothermal titration calorimetry suggested that the binding of SCSP and zinc (N ≈ 1) was exothermic, with relatively weak binding affinity (K = 1.0 × 10-3  mol L-1 ). The formation of SCSP-Zn complexes brought morphological changes to the peptides confirmed by scanning electron microscopy (SEM), which also indicated 6.88% of the existence of zinc element. In addition, the SCSP-Zn complexes remained stable under simulated human gastrointestinal digestion. In an in vitro study, the SCSP-Zn complex could successfully transport through the intestinal membrane in the model of everted rat gut sacs (nearly 7.5 µM cm-2 ) as well as Caco-2 cells where the zinc transport reached 0.0014 mg mL-1 carried by SCSP. Fluorescence staining experiments revealed free zinc accumulation inside the tissues and cells treated with the SCSP-Zn complex. CONCLUSIONS: The chelation SCSP-Zn had the promotion ability of zinc absorption in vitro and ex vivo experiments, which suggested a theoretical basis for the design and production of effective zinc chelating peptides as zinc carriers to improve zinc bioavailability. © 2022 Society of Chemical Industry.

The activation effects of fucoidan from sea cucumber Stichopus chloronotus on RAW264.7 cells via TLR2/4-NF-κB pathway and its structure-activity relationship.

Sea cucumber Stichopus chloronotus is a traditional tonic food with high nutritive value in Southern China. Fucoidan from sea cucumber Stichopus chloronotus (Fuc-Sc) is its main bio-active polysaccharide, the immune-activation effects of which have been fully investigated on RAW264.7 cells in the present study. The results indicated that Fuc-Sc could stimulate the RAW264.7 cells by promoting the production of NO, TNF-α, IL-6 and IL-10. Western blot and RT-PCR analysis revealed that TLR4 and TLR2 were involved in the recognition of Fuc-Sc and activation of downstream NF-κB signal pathway. Moreover, the chemical structure parameter molecular weight showed obvious impact on the stimulation effects of Fuc-Sc on NO production. Degraded product of Fuc-Sc with weight average molecular weight of 113.1 × 104 Da exhibited higher activities than that of intact Fuc-Sc, suggesting the existent of optimum chain length to exert its highest activities. Taken together, Fuc-Sc exerted its immunostimulating activity via TLR2/4 activation of NF-κB pathway and showed potentials to be a good immunoadjuvant.

Apostichopus japonicus Oligopeptide Induced Heterogeneity in the Gastrointestinal Tract Microbiota and Alleviated Hyperuricemia in a Microbiota-Dependent Manner.

SCOPE: This study aims to investigate the protective effect of Apostichopus japonicus oligopeptide (AJOP) on hyperuricemia, demonstrate the modulation of the gastrointestinal tract (GIT) microbiota, and clarify the underlying microbiota-dependent mechanism. METHODS AND RESULTS: Hyperuricemic mice treated with AJOP and subjected to corresponding fecal microbiota transplantation (FMT) are used to observe the beneficial effects of AJOP and microbiota. Gene transcriptions are measured using quantitative real-time PCR. The GIT (stomach, colon, cecum, and feces) microbiota is analyzed by 16S rDNA sequencing and the short-chain fatty acids are detected using GC-MS. Dietary administration of AJOP significantly alleviates hyperuricemia, regulates uric acid metabolism, inhibites the activation of the NLRP3 inflammasome and NF-κB-related signaling pathway, and restores m6A methylation levels. In addition, substantial heterogeneity is observed in GIT microbiota. Furthermore, FMT effectively alleviates hyperuricemia in mice by selectively regulating the corresponding pathways associated with AJOP treatment, indicating that the mechanism underlying the protective effects of AJOP is partly microbiota-dependent. CONCLUSION: This study demonstrates that AJOP exerts a protective effect on hyperuricemic mice by regulating uric acid metabolism, resulting in substantial heterogeneity among the GIT microbiota, thus mediating the beneficial effects in a microbiota-dependent manner.

Structural elucidation and antidiabetic activity of fucosylated chondroitin sulfate from sea cucumber Stichopus japonicas.

A fucosylated chondroitin sulfate was isolated from the body wall of sea cucumber Stichopus japonicus (FCSsj), whose structure was characterized by NMR spectroscopy and HILIC-FTMS. At the ratio of 1.00:0.26:0.65, three fucosyl residues were found: 2,4-disulfated-fucose (Fuc2,4S), 4-sulfated-fucose (Fuc4S) and 3,4-disulfated-fucose (Fuc3,4S), which were only linked to the O-3 of glucuronic acid residues (GlcA). Besides mono-fucosyl moieties, di-fucosyl branches, namely Fuc2,4Sα(1→3)Fuc4S, were also found to be attached to the O-3 of GlcA. The antidiabetic activity of FCSsj was evaluated using glucosamine induced insulin resistant (IR) Hep G2 cells in vitro. It was found that FCSsj significantly promoted the glucose uptake and glucose consumption of IR-Hep G2 cells in a dose-dependent manner, and could alleviate the cell damage. Furthermore, FCSsj could promote the glycogen synthesis in the glucosamine-induced IR-Hep G2 cells. These results provided a supplement for studying the antidiabetic activity of FCSsj.

Comparative metabolomic analysis of the body wall from four varieties of the sea cucumber Apostichopus japonicus.

The sea cucumber, Apostichopus japonicas, is an important economic species with high nutritive value. In recent years, driven by significant market demand, the sea cucumber breeding industry has developed rapidly. Body color and number of papillae are important factors that determine the value and price of sea cucumbers. In this study, metabolite profiling of four sea cucumber varieties (green, white, purple and spiny) was performed using ultra-performance liquid chromatography-quadrupole-time-of-flight-mass spectrometry (UPLC-QTOF-MS) combined with multivariate analysis. Orthogonal partial least-squares discriminant analysis (OPLS-DA) clearly discriminated the body wall metabolites of four sea cucumber varieties. Differential metabolites included fatty acids, phospholipids, and sugars. KEGG metabolic pathway analysis revealed that carbohydrate, protein, and phospholipid metabolism were highly conserved among the varieties. These results provide a comprehensive insight into differences in the metabolite profile of four A. japonicus varieties and a deeper understanding of sea cucumber varieties breeding.

A regular fucan sulfate from Stichopus herrmanni and its peroxide depolymerization: Structure and anticoagulant activity.

Marine sulfated polysaccharides have aroused widespread concern for their various structures and bioactivities. Peroxide depolymerization is a common strategy in analysis of structures and structure-activity relationships of polysaccharides. However, confirming the depolymerization process and exact structures of the degradation products is still a considerable challenge. This study reported the structures of a fucan sulfate (FS) from sea cucumber Stichopus herrmanni and its depolymerized products (dFS) prepared by peroxide degradation. The FS was elucidated with a highly regular structure, {-3)-L-Fuc2S-(α1-}n. Structure analysis of oligosaccharides purified from dFS suggested that peroxide degradation involved in cleavage of glycosidic bonds and oxidative modification of reducing end of sugar residue, while no break in sugar ring was observed. Both FS and series of dFSs exhibited significant anticoagulant activities due to their anti-thrombin effects in presence of heparin cofactor II and their potencies were related to their molecular sizes, dFS with ∼ 20 kDa showed the strongest activity.

Immunomodulatory effects of fucosylated chondroitin sulfate from Stichopus chloronotus on RAW 264.7 cells.

Sea cucumbers were nutritional food and traditional Chinese medicine. In this study, fucosylated chondroitin sulfate from sea cucumber Stichopus chloronotus (fCS-Sc), a potential anticoagulant agent and immunological adjuvant, was investigated for its immune activation effects on RAW 264.7 macrophage for the first time. The results indicated that fCS-Sc could significantly promote the proliferation, the pinocytic activity of RAW 264.7 cells, and the production of NO, TNF-α, IL-1ß, and IL-6. The fluorescence labeling assay indicated that fCS-Sc could bind to the macrophage. Moreover, the specific pattern recognition receptor inhibition assays showed that toll-like receptor 4 (TLR4) and TLR2 were involved in the recognition of fCS-Sc. Western blot assays indicated that fCS-Sc could induce degradation of cytoplasm IκB-α, and promotion of NF-κB p65 subunit translocation to nucleus, leading to a functional improvement of macrophage through NF-κB pathway. The results suggested that fCS-Sc might served as a promising candidate of immunomodulator.

Metabolomics analysis of sea cucumber (Apostichopus japonicus) in different geographical origins using UPLC-Q-TOF/MS.

The sea cucumber (Apostichopus japonicus) has become increasingly important aquaculture with the rapid expansion of farming in China and its commercial popularity in Asia. Price varies significantly among sea cucumber species, and since visual differentiation is difficult, food fraud is likely to occur. In this study, we report an analysis of body wall metabolites of the sea cucumber (A. japonicus) from different geographical origins in Dalian, Pikou, Jinzhou and Rushan using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS). The metabolites of sea cumbers of the different geographical origins were assessed using orthogonal partial least-squares discriminant analysis (OPLS-DA) and analysed using KEGG metabolic pathway analysis. The OPLS-DA clearly discriminated the body wall metabolites of sea cucumbers from the four regions. The differential metabolites mainly included amino acids and lipids. The KEGG metabolic pathway analysis showed that lipid metabolism, amino acid metabolism and protein metabolism were closely related to geographical origin. These results can be used to discriminate the geographical origins of A. japonicus.

The effectiveness of pure Stichopus horrens extract toothpaste in reducing plaque-induced gingivitis following orthodontic bonding: A triple-blinded randomized controlled trial.

INTRODUCTION: Gingivitis is one of the commonest problems faced by patients with fixed appliances (FA) as there is close relation between the appliances to gingival sulcus. Stichopus horrens (SH) is a sea cucumber from the Indo-Pacific that has medical healing properties which have been traditionally used. OBJECTIVE: To assess the effects of toothpaste containing aqueous SH extract on plaque-induced gingivitis following orthodontic bond-up and to identify the optimal concentration of SH. METHODS: A single-centred; triple-blinded randomized controlled trial conducted in 40 patients with FA. Participants were randomly assigned to one of the four groups with toothpaste which has concentration of SH extract of 0%, 3%, 6% or 9%. The statistician, the participants and the researchers involved in data collection were kept blinded from the allocation. Gingival Index (GI) and Bleeding on Probing (BOP) for each group were taken at day 0,7,14 and 30. RESULTS: 9% of SH-containing toothpaste (SHCT) showed most substantial result as there were significance difference of GI (P = 0.020) from Day 7 to 14 and from Day 0 to 14 (P = 0.020). There was also significance difference of BOP from Day 0 to 14 (P = 0.022) and from Day 0 to 30 (P = 0.027). Significant difference was seen in 3% of SHCT group with the decrease of GI (P = 0.004) from Day 1 to 14. There were no significant difference noted for 0% and 6% SHCT. CONCLUSION: The 9% SHCT is the most effective concentration to reduce both the gingival inflammation (up to day 14) and bleeding on probing (up to day 30).

Collagen fibrils of sea cucumber (Apostichopus japonicus) are heterotypic.

Sea cucumbers attracted increasing interest due to its nutritional functions. Collagen is the most important structural biomacromolecule in sea cucumber body wall, and is highly related to the textual properties and food quality of sea cucumber. In this study, the type of constituent collagens of sea cucumber collagen fibrils was investigated, employing a commercially important species Apostichopus japonicus as the material. Proteomics and bioinformatics analysis revealed that collagen fibrils of A. japonicas are heterotypic. Two clade A and one clade B fibrillar collagens and two FACIT collagens were identified from the fibrils. Besides, the heterogeneity was also revealed in the pepsin-solubilized collagen (PSC) of A. japonicus by using the proteomics strategy. It implied that the previous conclusions on the type of sea cucumber collagen deduced from SDS-PAGE analysis should be rechecked. The results provided novel insight into the composition of sea cucumber collagen fibrils.

Release of antidiabetic peptides from Stichopus japonicas by simulated gastrointestinal digestion.

Sea cucumber (Stichopus japonicus) is a high-protein food with the potential to release certain peptides through enzymolysis. This work is to explore the characteristics of peptides released from Stichopus japonicus protein in the process of digestion. Hydrolysates were obtained by gastrointestinal digestion and fractioned to <3, 3-10, 10-30 and >30 kDa fractions. Fifty-eight peptides from <3 kDa fraction were characterized using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Hydrolysates could improve glucose uptake of 3 T3-L1 cells and high insulin-induced insulin-resistant Hep G2 cells. Molecular docking showed that the released peptides had similar binding mode with anagliptin, a dipeptidyl peptidase IV (DPP-IV) inhibitor. The <3 kDa fraction in gastro and intestinal digestion showed the greatest DPP-IV inhibitory potency (IC50 0.51 and 0.52 mg/mL, respectively). The results indicated that sea cucumber could be used as a functional food to release antidiabetic peptides through gastrointestinal digestion.

Hot water pretreatment-induced significant metabolite changes in the sea cucumber Apostichopus japonicus.

Hot water pretreatment of sea cucumbers potentially changes nutritional benefits. This study aimed to quantify hot water pretreatment-induced changes in metabolite profiles of sea cucumber body walls. ICP-OES, GC-MS, and LC-MS analyses of untreated- (UT-BW), hot water-treated body walls (HW-BW) of Apostichopus japonicus, and the hot water extract (HW-E) determined significant losses of minerals (25-50% w/w), protein (~11% w/w), carbohydrate (33% w/w), saponins (~41% w/w), and spermidine (100%), a potential antipsychotic from hot water-treated samples. Multivariate comparisons of HW-BW with UT-BW and HW-BW with HW-E showed increases in amino acids and fatty acids, suggesting hot water-induced degradation or transformation or easier extraction of protein, lipid or other components. Presence of 80 to 88.5% of compounds in the HW-E and lower DHA, EPA and glycerophospholipids levels in HW-BW suggested extraction of these metabolites. These data indicate that novel processing technologies are required to preserve the full nutritional benefits of sea cucumbers.