ABSTRACT
Stichopus chloronotus, a marine sea cucumber rich in n-3 PUFAs and phenolic compounds, is a deep black–green sea cucumber with yellow/ red papillae tips, which has important economic and ecological value. In this project, a process was established to simultaneous extract and prepare four different active substances (including sea cucumber polysaccharide, polypeptide, saponin and lipid) from Stichopus chloronotus. The yields and purity were also improved. Except for polysaccharides, the purity of the other three active substances was above 90%. The extraction rates were all above 60%. In addition, the functional activity of sea cucumber peptides was evaluated. The results showed that sea cucumber peptides could reduce triglyceride content in mice serum, increase glutathione content and relieve alcohol-induced liver damage. The technical research laid a technical foundation for the high value utilization of Stichopus chloronotus.
1. Introduction
Sea cucumbers (SC) belong to the phylum Echinodermata, under the class Holothuroidea, which are an ancient marine invertebrates phylum with a wide distributed all over the world (Zhang et al., Citation2017). There are about 1,500 species of SC harvested from all regions of the ocean, and new species continue to be identified (Kiew & Don, Citation2012). Over the past few decades, sea cucumber fisheries worldwide have expanded in terms of catch and value (Toral-Granda et al., Citation2008), and are often used in tonic foods and folk medicine (Senadheera et al., Citation2020), especially in Asian countries (e.g. China, Japan, Korea, Malaysia, etc.), where they are one of the most popular seafood products (Conand, Citation2014; Hossain et al., Citation2020). For example, according to a survey by the Bureau of Fisheries of the Ministry of Agriculture, the total production of sea cucumbers in China in 2020 will be about 196,500 tons, with a total economic output value of 275.42 billion yuan. Stichopus chloronotus, a dark black–green sea cucumber widely distributed in tropical Indo-Pacific waters, mainly inhabits in the Xisha Islands of Hainan Province in China, rich in n-3 PUFAs and phenolic compounds, and is one of the main edible sea cucumbers in southern China (Li et al., Citation2020). S. chloronotus contains high quality peptides, saponins, fucosylated chondroitin sulfate, fatty acids and other compounds (Mou et al., Citation2020). In addition, S. chloronotus play an essential role in the marine ecosystem, with their feeding and excretion affecting nutrient cycling and bioturbation of the sediment, making sustainable use of these marine resources essential from both an environmental and commercial perspective (Lee et al., Citation2018).
Numerous studies have shown that sea cucumbers are very low in fat and cholesterol, high in protein, and have high nutritional value. In addition, they also contain various active substances, including polysaccharides, proteins, peptides, saponins, and lipids, each with their unique effects (Janakiram et al., Citation2015a). For example, glycosaminoglycan extracted from sea cucumber have many function, such as anti-thrombotic and anti-coagulant (Suzuki et al., Citation1991), anti-tumor (Borsig et al., Citation2007), immunomodulatory (Janakiram et al., Citation2015), and anti-hyperlipidemic (Wu et al., Citation2016). Sea cucumbers contain many proteins, mainly collagen, which also have high nutritional value. Collagen can be degraded into small molecule peptides by molecular bio-enzyme technology to promote absorption, which have antihypertensive (Abedin et al., Citation2015) and immunomodulatory (He et al., Citation2016) activities. Otherwise, numerous studies have shown that the saponins extracted from sea cucumber also have many physiological activities, such as improving metabolic syndrome, promoting bone marrow hematopoiesis, anti-tumor, anti-radiation, and other activities (Zhao et al., Citation2018).
Sea cucumber active substance extracts are a high-value product in sea cucumbers (Omran & Khedr, Citation2015). Each extract has its particular efficacy and can be used to develop highly effective functional food for sea cucumber according to the suitable population, increasing the utilization several times. Currently, the extraction process of active substances from sea cucumbers is mainly enzymatic hydrolysis, and the purity of the extracts is low (Ceesay et al., Citation2019). Most extracts are a mixture of peptides, polysaccharides, lipids, and saponins. Moreover, the existing extraction process of pure components of sea cucumber is complicated and cannot realize the effective separation of active substances, which leads to high loss of raw materials, low extraction rate, difficult to mass production and cannot further develop sea cucumber food with special efficacy. In addition, most of the methods studied for extracting active substances from sea cucumbers prepare a single active substance, such as extracting peptides (Saallah et al., Citation2021) or saponins (Khattab et al., Citation2018) alone, but no study has been reported to use a complete set of methods to extract four active ingredients simultaneously.
The aim of this study was to investigate the process of simultaneous extraction and preparation of four different active substances (including sea cucumber polysaccharides, peptides, saponins, and lipids) from the body wall of sea cucumbers and to perform the functional evaluation of the various active substances prepared. In this study, the yield and purity of the extracted sea cucumber active substances were determined, and the effects of sea cucumber peptides and sea cucumber saponins on alcohol-induced liver damage were investigated by animal tests.
2. Materials and methods
2.1. Materials
The fresh sea cucumbers (Stichopus chloronotus) weighing 100 ± 10 g were captured in the south pacific near the coast. Then they were transported to the laboratory immediately at low temperatures. Papain and ethylenediaminetetraacetic acetic acid were purchased from Beijing Solarbio Technology Co., LTD. Cysteine hydrochloride was purchased from Dalian Meilunbio Biotechnology Co., LTD. Cysteine and lactose were purchased from Sinopharm Chemical ReagentCo., Ltd. All other reagents used in this experiment were of analytical grade.
2.2. Proximate chemical composition of the sea cucumber body wall
The Stichopus chloronotus samples were cleaned with distilled water, the body wall and visceral organs were separated, the body wall was dried and ground, and the contents of crude protein, crude fat, ash, sea cucumber polysaccharide and saponins were determined. The proximate composition of sea cucumber was carried out according to the procedures established by the Standardization Administration. The crude protein content was determined by the Kjeldahl method in GB 5009.5-2010. Fat was determined using the Soxhlet extraction method. Ash was determined by incineration at 550 °C for 24 h in a muffle furnace (GB 5009.4-2016). Carbohydrate content was measured by the phenol/sulfuric acid method. Sea cucumber polysaccharides were based on the high-performance liquid chromatography method in SC T 3049-2015. Sea cucumber saponin was referred to the Technical Specification for Inspection and Evaluation of Health Food (2003 edition).
2.3. Pretreatment of sea cucumber (SC)
The fresh sea cucumbers were slaughtered, eviscerated, and washed immediately with distilled water at 4 °C. The treated sea cucumbers were cut into small pieces (2 cm × 2 cm) with scissors and crushed with a wall breaker. Further, the crushed sea cucumbers were ground into a slurry by a colloidal mill.
2.4. Extraction of lipid from sea cucumber
A 2 L solution of ethanol at 60% volume fraction was added to 575.0 g of sea cucumber pulp (water content 89.57%), and heated to reflux for 1 h. The operation was repeated three times. Subsequently, the solutions from each repeated extraction were mixed, and the remaining solids were retained. The above-mentioned solution was concentrated to dryness, then 200 mL of water was added to dissolve it, and the insoluble material was removed by filtration. The remaining fraction was extracted for three times with petroleum ether to give two layers of solution, one with the fat removed and the other with a mixture of petroleum ether and grease. The petroleum ether containing the fat was vacuum dried to obtain sea cucumber fat.
2.5. Extraction of saponin from sea cucumber
The pH of the above degreasing mixture was adjusted to 8.0 with ammonia. 200 mL of saturated n-butanol was added to the treated solution, shaken thoroughly, and allowed to stand until the solution stratified and the upper solution was collected. This step was repeated four times. Finally, the obtained n-butanol was mixed, concentrated, and dried at 50 °C to give a pale yellow or reddish-brown crude saponin powder. The powder was dissolved in water again, adsorbed by macroporous resin (D101), and eluted with ethanol at 70% by volume to give an ethanol mixture. The mixture was concentrated to dryness to obtain sea cucumber saponins.
2.6. Extraction of polysaccharide and polypeptide from sea cucumber
The solid mentioned in 2.4 was rinsed with plenty of water to remove the ethanol and then dispersed in 1.5 L 0.1 mol / L sodium acetate-acetic acid buffer (pH = 6.0). 6.0 g papain, 2.2 g ethylenediamine tetraacetic acid, and 1.2 g cysteine hydrochloride were added to the solution and stirred to mix well. The resulting solution was hydrolyzed at 60 °C for 24 h, then heated in a water bath at 85°C for 15 min to inactivate the enzyme. After cooling, the supernatant was centrifuged (9000 r, 20 min), and the ethanol was added to make a volume fraction of 60% and left for 12 h. The supernatant was the crude sea cucumber peptide solution, and the precipitate was the crude sea cucumber polysaccharide. The precipitate was dissolved in water again and ultrafiltrated with an ultrafiltration membrane several times (10 KDa) (35°C, pressure 0.1 MPa). The solution was mixed, and 1.5 g activated carbon was added to the concentrate and decolorized at 80°C for 30 min, followed by rapid filtration. The insoluble material was washed with 80 °C water and repeated twice. The resulting solution was freeze-dried or spray-dried, which was sea cucumber polysaccharide. The above crude peptide solution of sea cucumber was mixed with the ultrafiltered solution and nanofiltrated using a 100 Da nanofiltration membrane (pressure 0.5 MPa-1.0 MPa). The concentrated solution was freeze-dried or spray-dried, which was the sea cucumber polypeptide.
2.7. Determination of polysaccharide, polypeptide, saponin and lipid content and extraction rate of sea cucumber extracts
2.7.1. Polysaccharide of sea cucumber
The polysaccharide of sea cucumber was determined by high-performance liquid chromatography (HPLC) using SC T 3049-2015. Sea cucumber polysaccharide powder (1.0 g) was digested with 25 mL of 0.1 M sodium acetate buffer (pH 6.0) containing papain (100 mg), EDTA (37 mg) and cysteine (22 mg) at 60 °C for 24 h. The digested mixture was centrifuged (9000×g, 10 min), and the supernatant was precipitated with potassium acetate (6.13 g). The mixture was left to stand for 12 h at 4 °C and then centrifuged (9000×g, 10 min). The precipitate was dissolved with dH2O and hydrolyzed with 1 mL of 4 M TFA at 110 °C for 8 h. The reaction solution was dried, dissolved, and neutralization with 0.3 M NaOH. The hydrolysates were heated at 70 °C for 30 min, 50 μL 2 mM lactose, 450 μL 0.5 M PMP methanol solution, and 450 μL 0.3 M NaOH. After the reaction solution was cooled, 450 μL of 0.3 M HCl was added, and the excess PMP was removed by washing with CHCl3 three times. The upper aqueous phase was filtered through a 0.45 μm membrane. These PMP derivations were quantitatively analyzed by HPLC-UV, as mentioned above.
2.7.2. Fatty acid composition
The determination of fatty acid content followed the procedure described by the International Organization for Standardization (ISO5509) (1978). Briefly, sea cucumber extracts were esterified to form fatty acid methyl esters, which were then injected into a gas chromatography system coupled to a flame ionization detector with a capillary column for quantifying fatty acid methyl esters(FAME), and each fatty acid identified and quantified by comparison with retention times and peak areas of FAME against the internal standard.
2.7.3. Amino acid analysis
The amino acid composition of sea cucumber polypeptides was determined according to GB 5009.124-2016. Sea cucumber polypeptide was added to 6 M HCl and 2% phenol and then hydrolyzed at 110 °C for 22 h under a nitrogen atmosphere. Amino acids were quantified using the automatic amino acid analyzer.
2.7.4. Dtermination of saponins in sea cucumber extracts
The determination of sea cucumber saponin content was carried out with reference to GB/T 33108-2016. The sea cucumber saponin extractive were dissolved with 2 mL TFA and hydrolyzed at 120 °C for 2 h under a nitrogen atmosphere. The reaction solution was neutralization with NaOH, and then 1 mL CHCl3 were added. After standing to stratified, CHCl3 was removed, and the process was repeated three times to obtain saponin hydrolysate. The hydrolysate was dissolved in derivatization solution (250 μL PMP and 250 μL NaOH), heated at 70 °C for 30 min, cooled, and 250 μL HCl and 1 mL CHCl3 was added. After standing to stratified, CHCl3 was removed, and this process was repeated three times. The upper aqueous phase was filtered through a 0.45 μm membrane. These PMP derivations were quantitatively analyzed by HPLC-UV, as mentioned above.
2.8. Animals and experiment
2.8.1. Animals
Seven-week-old male BALB/c mice (n = 72, weighing 20 ± 2 g) were purchased from Jinan Pengyue experimental animal breeding Co., Ltd. (Jinan, China). All mice were housed in a room at a temperature of 22 ± 2°C and relative humidity of 50 ± 10% on a 12: 12 h light–dark cycle at Ocean University Of China (Qingdao, China). After 1 week of acclimation, mice were randomly divided into the following 9 groups:(1) Con group mice were gavaged with 0.15 mL/10 g body weight of mice saline daily (n = 8); (2) Neg group mice were gavaged with 0.15 mL/10 g saline and 0.1 mL/10 g 56 degree Red Star Erguotou (n = 8); (3) Pos group mice were gavaged with 0.15 mL/10 g KingDrink (purchased from Shenzhen Neptune Health Technology Development Co., Ltd., which can promote the decomposition of alcohol and relieve liver damage caused by alcohol) and 0.1 mL/10 g 56 degree Red Star Erguotou (n = 8); (4) L-pep, M-pep, H-pep group mice were gavaged with 85, 170, 340 mg/10 g of sea cucumber peptide and 0.1 mL/10 g 56 degree Red Star Erguotou, respectively. (n = 8); (5) L-sap, M- sap, H- sap group mice were gavaged with 20, 40, 80 mg/10 g sea cucumber saponin, and 0.1 mL/10 g 56 degree Red Star Erguotou, respectively. (n = 8). The whole experimental period was 14 consecutive days.
2.8.2. Determination of malondialdehyde(MDA) in liver tissue
The mouse liver tissue was washed with saline, dried with filter paper and weighed, cut into pieces and put into a homogenizer. 0.2 M phosphate buffer was added, homogenized for 10 s (20000 r/min), stopped for 30 s, and repeated three times to prepare a 5% tissue homogenate (w/v), centrifuged for 10 min (3000 r/min.) The supernatant was assayed for malondialdehyde by assay kit (Nanjing Jiancheng Bioengineering Research Institute).
2.8.3. Determination of glutathione(GSH)
0.5 g of liver was added to 5 mL of saline and ground to the slurry. 0.5 mL slurry was mixed thoroughly with 0.5 mL of 4% sulfosalicylic acid. Centrifuge (3000 r, 10 min) and take the supernatant as the sample. The sample was a glutathione assay kit (Nanjing Jiancheng Bioengineering Research Institute).
2.8.4. Determination of triglyceride (TG)
The liver homogenates, described in 2.8.3 were taken and determined by a triglyceride assay kit (Nanjing Jiancheng Bioengineering Research Institute).
3. Results
3.1. Proximate compositions
The proximate compositions of Stichopus chloronotus body wall are shown in . The protein content (dry base) of the body wall of Stichopus chloronotus was 79.84%, which was higher than that of Apostichopus japonicus (66.60%) and similar to that of Holothuria mexicana (74.54%) (Gao et al., Citation2016). When sea cucumber is attacked, Cuvier’ sorgan and the body’s surface secrete toxins for defense. The main component of this toxin is saponins, which are relatively low in body walls (0.25%). The result is consistent with the study of Séverine Van Dyck (Van Dyck et al., Citation2010). Further, the sample contained 4.38% lipid, 12.72% ash, and 8.13% polysaccharides (dry base). The results are consistent with the conclusion that sea cucumbers are seafood containing high protein with low-fat levels (Wen et al., Citation2010).
Table 1. Proximate compositions of Stichopus chloronotus.
3.2. Polysaccharide, polypeptide, saponins and lipid content and extraction rate of sea cucumber extracts
The content and extraction rate of polysaccharides, polypeptides, saponins and lipids extracted from the sea cucumber, Stichopus chloronotus are shown in . Among the four items studied, the purity of lipids was the highest, reaching 97.57%, and the extraction rate was 86.17%. Saponins are secondary metabolites found in animals with significant biological activities, such as sea cucumber saponins showed positive ameliorative effects on metabolic syndrome and anti-cancer activity. The content of saponins extracted by the above method was up to 92.13%, and the extraction rate was 79.15%.
Table 2. Sea cucumber polysaccharide, polypeptide, saponin and lipid content and extraction rate.
3.3. Fatty acid composition
Several studies have shown that long-chain polyunsaturated fatty acids can reduce or prevent the burden of diseases (Zhang et al., Citation2019). shows fatty acid compositions of the body wall of Stichopus chloronotus extract. A total of 21 fatty acids were identified from the body wall extracts. The fatty acid distribution we dominated by unsaturated fatty acids (81.75%), with the main fatty acid types being C20:5, C16:1,and C20:4.
Table 3. Fatty acid analysis of sea cucumber body wall extract.
In the lipids of sea cucumber extract, C20:1 accounts for 20.76% of the total MUFA. Arachidonic acid (ARA) is a ω−6 polyunsaturated fatty acid, a precursor of arachidonic acid-like substances, which is the major component of cell membrane. It can inhibit platelet aggregation, reduce thrombosis, and account for 22.55% of the total PUFA. The extract’s eicosapentaenoic acid (EPA) content accounts for 60.95% of the total PUFA. It belongs to the n-3 series of polyunsaturated fatty acids, which can regulate lipid metabolism and prevent coronary artery disease. The sum of ARA and EPA accounted for 40.62% of the total lipids.
Studies have shown that Eicosapentaenoic acid (EPA) and Docosahexaenoic acid (DHA) can promote brain cell development. In addition, an appropriate ratio of EPA and DHA is vital for brain utilization. The World Health Organization (WHO) recommends a level of 5:1–10:1 (Fats and oils in human nutrition, Citation1994). The EPA and DHA contents of sea cucumber extracts were 29.65% and 5.46%, respectively, in line with the WHO recommended level.
3.4. Amino acid composition
Sea cucumbers are critical marine resources containing high amounts of collagen in their body wall (Jin et al., Citation2019). The results showed that the essential amino acid content of Stichopus chloronotus was 19.80%, and the ratio of essential amino acids (EAA) to nonessential amino acids (NEAA) was 0.30. The determined amino acid content of glycine was 16.09%, the major component. This result is consistent with the study by wen (Wen et al., Citation2010).
There is a high level of arginine (7.282%) in Stichopus chloronotus. Studies have shown that arginine can improve brain function and delay gonad aging. It is also the primary raw material for collagen synthesis and can promote cell repair and regeneration (Mangoni et al., Citation2019). Glycine, arginine and glutamic acid can promote the activation and proliferation of NK cells and T cells and enhance immunity. The result of the amino acid analysis showed that the content of glycine, arginine and glutamic acid extracted by the above methods was 16.09%, 7.28% and 13.63%, respectively, which were high.
Hypocholesterolemic effects of low lysine/arginine protein were well documented (Bordbar et al., Citation2011). The lysine to arginine ratio of 2.21, extracted by the above method, demonstrated the immune-enhancing effect of the peptide.
3.5. Weight of mice and liver
The body weights and hepatic index of the nine groups of mice are shown in . It can be seen that there was no significant difference in the body weight of mice among the positive, model and experimental groups, and the body weight of mice in the blank group increased. There was no significant difference in hepatic index among the nine groups.
Figure 1. Body weight and hepatic index of mice.
3.6. Contents of triglyceride (TG), malonaldehyde (MDA), glutathione (GSH)
Liver damage in mice can disrupt protein synthesis and affect lipid metabolism, resulting in a considerable accumulation of TG in the serum. Therefore, TG is an important indicator reflecting liver damage in mice (Choi & Diehl, Citation2008). MDA is a metabolite of lipid peroxidation. Its content can indirectly reflect the level of active oxygen free radicals and lipid peroxidation, thus indirectly reflecting the degree of cell damage (Mousavi et al., Citation2020). GSH protects the body from oxidative damage caused by reactive oxygen species (ROS), which may be formed normally generally in metabolism (Videla & Valenzuela, Citation1982).
The serum levels of TG, MDA and GSH in mice are shown in . It can be seen that serum TG levels were significantly decreased in L-ep M-pep and H-ep groups compared with the Neg group. Serum TG levels were reduced to different degrees in the Pos L-sap, M-sap and H-sap groups, but the differences were insignificant (P > 0.05). Compared with the Neg group, serum MAD levels were reduced in the Pos and other groups, and serum MAD levels in the H-sap group were significantly different from those in the Neg group (P < 0.05). In contrast, serum MAD levels in the remaining groups were insignificantly different from those in the Neg group. In terms of GSH content, the Pos, L-pep, M-pep, H-pep and M-sap groups showed a significant increase in GSH content compared to the Neg group.
Figure 2. Contents of TG, MDA and GSH in serum of mice.
From the above experimental results, it can be seen that sea cucumber peptide can reduce the serum triglyceride content and increase the content of reduced glutathione in mice, alleviating the alcohol-induced liver damage .
Table 4. Amino acid composition.
4. Discussion
In conclusion, we prepared sea cucumber polypeptides, lipids, polysaccharides and saponins from Stichopus chloronotus. The result showed that the extracted sea cucumber polypeptides had good quality and high nutritional components of sea cucumber lipids. Through animal experiments we can conclude that: sea cucumber peptides can reduce serum triglyceride content, increase glutathione content, and reduce alcoholic liver damage in mice.
Acknowledgments
Our research was supported by the Taishan Scholar Foundation of Shandong Province (No.tsqn202103033). We would like to thank Dejun Yu and Hu Hou for their assistance with the animal studies and the technical support provided.
Disclosure statement
No potential conflict of interest was reported by the author(s).
Correction Statement
This article has been republished with minor changes. These changes do not impact the academic content of the article.
References
- Abedin, M. Z., Karim, A. A., Gan, C. Y., Ghazali, F. C., Barzideh, Z., Zzaman, W., & Zaidul, I. S. M. (2015). Identification of angiotensin I converting enzyme inhibitory and radical scavenging bioactive peptides from sea cucumber (Stichopus vastus) collagen hydrolysates through optimization. International Food Research Journal, 22, 1074–1082. https://doi.org/10.1089/jmf.2019.0066
- Bordbar, S., Anwar, F., & Saari, N. (2011). High-value components and bioactives from sea cucumbers for functional foods–a review. Marine Drugs, 9(10), 1761–1805. https://doi.org/10.3390/md9101761
- Borsig, L., Wang, L., Cavalcante, M. C., Cardilo-Reis, L., Ferreira, P. L., Mourąo, P. A., Esko, J. D., & Pavąo, M. S. (2007). Selectin blocking activity of a fucosylated chondroitin sulfate glycosaminoglycan from sea cucumber. Effect on tumor metastasis and neutrophil recruitment. Journal of Biological Chemistry, 282(20), 14984–14991. https://doi.org/10.1074/jbc.M610560200
- Ceesay, A., Nor Shamsudin, M., Aliyu-Paiko, M., Ismail, I. S., Nazarudin, M. F., & Mohamed Alipiah, N. (2019). Extraction and Characterization of Organ Components of the Malaysian Sea Cucumber Holothuria leucospilota Yielded Bioactives Exhibiting Diverse Properties. BioMed Research International, 2019, 2640684. https://doi.org/10.1155/2019/2640684
- Choi, S. S., & Diehl, A. M. (2008). Hepatic triglyceride synthesis and nonalcoholic fatty liver disease. Current Opinion in Lipidology, 19(3), 295–300. https://doi.org/10.1097/MOL.0b013e3282ff5e55
- Fats and oils in human nutrition. (1994). WHO and FAO joint consultation: fats and oils in human nutrition. Nutr Rev., 53(7), 202–5. https://doi.org/10.1111/j.1753-4887.1995.tb01552.x
- Gao, Y., Li, Z., Qi, Y., Guo, Z., Lin, Y., Li, W., Hu, Y., & Zhao, Q. (2016). Proximate composition and nutritional quality of deep sea growth sea cucumbers (Stichopus japonicus) from different origins. Journal of the Science of Food and Agriculture, 96(7), 2378–2383. https://doi.org/10.1002/jsfa.7353
- He, L. X., Zhang, Z. F., Sun, B., Chen, Q. H., Liu, R., Ren, J. W., Wang, J. B., & Li, Y. (2016). Sea cucumber (Codonopsis pilosula) oligopeptides: immunomodulatory effects based on stimulating Th cells, cytokine secretion and antibody production. Food & function, 7(2), 1208–1216. https://doi.org/10.1039/C5FO01480H
- Hossain, A., Dave, D., & Shahidi, F. (2020). Northern Sea Cucumber (Cucumaria frondosa): A Potential Candidate for Functional Food, Nutraceutical, and Pharmaceutical Sector. Marine Drugs, 18(5), 1–27. https://doi.org/10.3390/md18050274.
- Janakiram, N. B., Mohammed, A., Bryant, T., Lightfoot, S., Collin, P. D., Steele, V. E., & Rao, C. V. (2015a). Improved innate immune responses by Frondanol A5, a sea cucumber extract, prevent intestinal tumorigenesis. Cancer prevention research (Philadelphia. Pa), 8(4), 327–337. https://doi.org/10.1158/1940-6207.CAPR-14-0380
- Janakiram, N. B., Mohammed, A., & Rao, C. V. (2015b). Sea Cucumbers Metabolites as Potent Anti-Cancer Agents. Marine Drugs, 13(5), 2909–2923. https://doi.org/10.3390/md13052909
- Jin, H. X., Xu, H. P., Li, Y., Zhang, Q. W., & Xie, H. (2019). Preparation and Evaluation of Peptides with Potential Antioxidant Activity by Microwave Assisted Enzymatic Hydrolysis of Collagen from Sea Cucumber Acaudina Molpadioides Obtained from Zhejiang Province in China. Marine Drugs, 17(3). https://doi.org/10.3390/md17030169.
- Khattab, R. A., Elbandy, M., Lawrence, A., Paget, T., Rae-Rho, J., Binnaser, Y. S., & Ali, I. (2018). Extraction, Identification and Biological Activities of Saponins in Sea Cucumber Pearsonothuria graeffei. Combinatorial Chemistry & High Throughput Screening, 21(3), 222–231. https://doi.org/10.2174/1386207321666180212165448
- Kiew, P. L., & Don, M. M. (2012). Jewel of the seabed: sea cucumbers as nutritional and drug candidates. International Journal of Food Sciences and Nutrition, 63(5), 616–636. https://doi.org/10.3109/09637486.2011.641944
- Lee, S., Ford, A. K., Mangubhai, S., Wild, C., & Ferse, S. C. (2018). Effects of sandfish (Holothuria scabra) removal on shallow-water sediments in Fiji. PeerJ, 6, e4773. https://doi.org/10.7717/peerj.4773
- Li, Q., Jiang, S., Shi, W., Qi, X., Song, W., Mou, J., & Yang, J. (2020). Structure characterization, antioxidant and immunoregulatory properties of a novel fucoidan from the sea cucumber Stichopus chloronotus. Carbohydrate Polymers, 231, 115767. https://doi.org/10.1016/j.carbpol.2019.115767
- Mangoni, A. A., Rodionov, R. N., McEvoy, M., Zinellu, A., Carru, C., & Sotgia, S. (2019). New horizons in arginine metabolism, ageing and chronic disease states. Age and Ageing, 48(6), 776–782. https://doi.org/10.1093/ageing/afz083
- Mou, J., Li, Q., Shi, W., Qi, X., Song, W., & Yang, J. (2020). Chain conformation, physicochemical properties of fucosylated chondroitin sulfate from sea cucumber Stichopus chloronotus and its in vitro fermentation by human gut microbiota. Carbohydrate Polymers, 228, 115359. https://doi.org/10.1016/j.carbpol.2019.115359
- Mousavi, S. R., Jafari, M., Rezaei, S., Agha-Alinejad, H., & Sobhani, V. (2020). Evaluation of the effects of different intensities of forced running wheel exercise on oxidative stress biomarkers in muscle, liver and serum of untrained rats. Lab Animal, 49(4), 119–125. https://doi.org/10.1038/s41684-020-0503-7
- Omran, N. E., & Khedr, A. M. (2015). Structure elucidation, protein profile and the antitumor effect of the biological active substance extracted from sea cucumber Holothuria polii. Toxicology and Industrial Health, 31(1), 1–8. https://doi.org/10.1177/0748233712466135
- Purcell, S. W. (2014). Value, market preferences and trade of Beche-de-mer from Pacific Island sea cucumbers. PLoS One, 9(4), e95075. https://doi.org/10.1371/journal.pone.0095075
- Saallah, S., Roslan, J., Julius, F. S., Saallah, S., Mohamad Razali, U. H., Pindi, W., Sulaiman, M. R., Pa’ee, K. F., & Mustapa Kamal, S. M. (2021). Comparative Study of The Yield and Physicochemical Properties of Collagen from Sea Cucumber (Holothuria scabra), Obtained through Dialysis and the Ultrafiltration Membrane. Molecules, 26(9). https://doi.org/10.3390/molecules26092564
- Senadheera, T. R. L., Dave, D., & Shahidi, F. (2020). Sea Cucumber Derived Type I Collagen: A Comprehensive Review. Marine Drugs, 18(9), 1–44. https://doi.org/10.3390/md18090471
- Suzuki, N., Kitazato, K., Takamatsu, J., & Saito, H. (1991). Antithrombotic and anticoagulant activity of depolymerized fragment of the glycosaminoglycan extracted from Stichopus japonicus Selenka. Thrombosis and haemostasis, 65(4), 369–373. https://doi.org/10.1055/s-0038-1648154
- Toral-Granda, V., Lovatelli, A., & Vasconcellos, M. (2008). Sea cucumbers. A global review of fisheries and trade. Australian Institute of Marine Science.
- Van Dyck, S., Gerbaux, P., & Flammang, P. (2010). Qualitative and quantitative saponin contents in five sea cucumbers from the Indian ocean. Marine Drugs, 8(1), 173–189. https://doi.org/10.3390/md8010173
- Videla, L. A., & Valenzuela, A. (1982). Alcohol ingestion, liver glutathione and lipoperoxidation: metabolic interrelations and pathological implications. Life Sciences, 31(22), 2395–2407. https://doi.org/10.1016/0024-3205(82)90743-3
- Wen, J., Hu, C., & Fan, S. (2010). Chemical composition and nutritional quality of sea cucumbers. Journal of the Science of Food and Agriculture, 90(14), 2469–2474. https://doi.org/10.1002/jsfa.4108
- Wu, N., Zhang, Y., Ye, X., Hu, Y., Ding, T., & Chen, S. (2016). Sulfation pattern of fucose branches affects the anti-hyperlipidemic activities of fucosylated chondroitin sulfate. Carbohydrate polymers, 147, 1–7. https://doi.org/10.1016/j.carbpol.2016.03.013
- Zhang, T. T., Xu, J., Wang, Y. M., & Xue, C. H. (2019). Health benefits of dietary marine DHA/EPA-enriched glycerophospholipids. Progress in Lipid Research, 75, 100997. https://doi.org/10.1016/j.plipres.2019.100997
- Zhang, X., Sun, L., Yuan, J., Sun, Y., Gao, Y., Zhang, L., Li, S., Dai, H., Hamel, J. F., Liu, C., & Yu, Y. (2017). The sea cucumber genome provides insights into morphological evolution and visceral regeneration. PLOS Biology, 15(10), e2003790. https://doi.org/10.1371/journal.pbio.2003790
- Zhao, Y. C., Xue, C. H., Zhang, T. T., & Wang, Y. M. (2018). Saponins from Sea Cucumber and Their Biological Activities. Journal of Agricultural and Food Chemistry, 66(28), 7222–7237. https://doi.org/10.1021/acs.jafc.8b01770