Squid Cartilage Research Articles

Inflammatory regulation of squid cartilage gelatin with different molecular weights for treatment of chronic wounds in diabetes

Squid, as a very important economic marine species, accounts for 5 % of the total catch of fish and cephalopods. The waste from the processing process of squid can be used for collagen extraction, which has great application value in the field of biomedical materials. Here, we obtained squid cartilage gelatin (SCG) with different molecular weights by adjusti.ng the reaction conditions and used for the treatment of chronic wounds in diabetes. SCG extracted at low temperatures and short heating times demonstrated a more intact structure, higher molecular weight, and superior gel stability. Based on cell study and transcriptome analysis, SCG with high molecular weight significantly promoted cell adhesion, because it provided more contact sites for cells, whereas small molecules of SCG could directly reduce inflammation. Animal studies have demonstrated that SCG significantly promotes diabetic wound healing as evidenced by reducing inflammation, inducing vascular regeneration, promoting tissue growth, re-epithelialization, collagen deposition and remodeling. This study elucidated the immunoregulatory mechanisms of SCG with different molecular weights, and validated its potential application in chronic wound healing in diabetes.

Preparation, typical structural characteristics and relieving effects on osteoarthritis of squid cartilage type II collagen peptides

The promoting effects of collagen and its derivatives on bone health have been uncovered. However, the structure and effects of type II collagen peptides from squid cartilage (SCIIP) on osteoarthritis still need to be clarified. In this study, SCIIP was prepared from squid throat cartilage with pretreatment by 0.2 mol/L NaOH at a liquid–solid ratio of 10:1 for 18 h and hydrolyzation using alkaline protease and flavourzyme at 50 °C for 4 h. The structure of SCIIP was characterized as a molecular weight lower than 5 kDa (accounting for 87.7 %), a high glycine level of 35.0 %, typical FTIR and CD features of collagen peptides, and a repetitive sequence of Gly–X–Y. GP(Hyp)GPD and GPAGP(Hyp)GD were separated and identified from SCIIP, and their binding energies with TLR4/MD-2 were − 8.4 and − 8.0 kcal/mol, respectively. SCIIP effectively inhibited NO production in RAW264.7 macrophages and alleviated osteoarthritis in rats through the TLR4/NF-κB pathway. Therefore, SCIIP exhibited the potential for application as an anti-osteoarthritis supplement.

PROCESSING OF SQUID (Loligo sp.) CARTILAGE WASTE AS ANTIBACTERIAL HANDWASH

Squid cartilage (Loligo sp.) is known to contain chitosan compounds that have antibacterial activity. This study aims to formulate chitosan from squid cartilage (Loligo sp.) in the form of handwash preparations with concentrations of 0.1%, 0.2%, and 0.3% which are physically and chemically stable, and see the optimum concentration in inhibiting bacterial growth. The method used is laboratory experiments through three stages of extraction, namely deproteination, demineralization, and deacetylation as well as physical quality tests and ability tests before and after cycling tests which include organoleptic tests, homogeneity tests, pH tests, viscosity tests, foam height tests and antibacterial activity tests against Staphylococcus aureus and Escherichia coli bacteria by the method of sumuran. The results obtained based on the ANOVA test on Staphylococcus aureus bacteria are 0.000 and on Escherichia coli bacteria are 0.000. Chitosan from squid cartilage (Loligo sp.) can be formulated in the form of a handwash preparation and the most effective concentration in inhibiting Staphylococcus aureus and Escherichia coli is a concentration of 0.3%.

Squid cartilage type II collagen accelerates osteoporotic fractures healing through regulating homocysteine mediated EGF and FBN1 signals

Osteoporosis fracture (OPF) is a serious social public health concern in humans. Type II collagen from squid cartilage (SCII) has been proven to promote the healing process in normal fracture mice, however, the effect and mechanism of SCII to improve osteoporosis fracture remain unknown. In this study, type II collagen was obtained from squid laryngeal cartilage, and the effects of SCII on OPF were investigated in mice that experienced bilateral ovariectomy and open tibial fracture surgery after oral administration. The results showed that SCII supplementation significantly decreased the serum TNF-α, increased aggrecan levels on the 5 d, and elevated the serum TGF-β and Col10α levels on 13 d after fracture. Notably, H&E staining exhibited an obvious chondrocyte proliferation on 5 d, and a specific promotion of chondrocyte hypertrophic differentiation and mineralization in the SCII group on 13 d. These findings suggested that SCII promoted osteoporosis fracture healing by enhancing the proliferation and hypertrophy of chondrocyte. Mechanically, SCII supplementation significantly reduced homocysteine levels through down-regulating TMAO production and bile acid levels and activated the EGF/AKT/CylinD1 and TGFβ/Smad1/5/8 pathways, thereby recovering the delay of chondrocyte proliferation and hypertrophy in OPF mice. This study investigated the effects of SCII on osteoporosis fracture healing for the first time and provided a new theoretical basis and options for the high-value application of squid cartilage.

Green extraction and purification of chondroitin sulfate from jumbo squid cartilage by a novel procedure combined with enzyme, ultrasound and hollow fiber dialysis.

The online version contains supplementary material available at 10.1007/s13197-023-05701-7.

Effect of Squid Cartilage Chitosan Molecular Structure on the Properties of Its Monofilament as an Absorbable Surgical Suture.

Suture is an important part of surgery, and wounds closing after surgery remains a challenge for postoperative care. Currently, silk, linen fiber, and cotton are available in the market as non-absorbable suture biomaterials. So, there is an urgent need to develop a novel suture with advantageous characteristics compared to the ones available on the market. In present study, a series of ultra-high molecular weight chitosan with different DD and MV were prepared from squid cartilage by alkaline treatment and ultrasonic degradation. The corresponding chitosan monofilaments were prepared by a wet spinning process and were characterized as sutures. The effects of the DD and MV of chitosan on the properties of its monofilament were studied, including surface morphology, mechanical property, swelling ratio, ash content, in vitro enzymatic degradation, and in vitro cytotoxicity. According to the results, AS-85 was chosen to be the best suitable as an absorbable surgical suture, which was spun from squid cartilage chitosan with DD~85% and MV~1.2 × 106. The outcome of the present study might derive tremendous possibilities for the utilization of squid cartilage β-chitin for biomedical applications.

Aktivitas Antibakteri Kitosan dari Tulang Rawan Cumi-Cumi (Loligo sp.) Terhadap Bakteri Staphylococcus aureus dan Escherichia coli

Squid (Loligo sp.) is one of fisheries commodities export in Indonesia. Squid has cartilage that can be utilized in the food and non-food sectors. The inner skin of the squid cartilage contains chemical compounds are chitin and chitosan. The objectives of this research are to produce chitosan from squid cartilage and tested the antibacterial effectiveness of chitosan in bacteria Staphylococcus aureus and Escherichia coli. The research method was carried out in twophase; the manufacturing phase of chitosan from squid cartilage and the testing phase for the antibacterial activity of chitosan. The results of the first phase of this study indicate that the chitosan produced meets the quality standards of chitosan with water content 7.82% (bk), ash content 0.57% (bk), nitrogen content 3.18% (bk) and degree of deacetylation 87.43%. Further more the results obtained in the second phase showed that the higher of chitosan concentration, the greater of inhibition zone in the tested bacteria. The best antibacterial activity was found at 0.8% chitosan concentration, with an inhibition zone of 12.8 ± 0.06 mm in E. coli and 11.1 ± 0.12 mm in S. aureus. At a concentration of 0.8% showed a greater inhibitory value when compared to 70% alcohol.

Ultrasonic-Assisted Extraction and Structural Characterization of Chondroitin Sulfate Derived from Jumbo Squid Cartilage.

Chondroitin sulfate (ChS) is usually used as an oral nutraceutical supplement, and has been popular in Asia, Europe, and United States for many years. In this study, a potential and sustainable source of ChS from jumbo squid (Dosidicus gigas) cartilage was explored; ultrasound-assisted extraction (UAE) was used to extract ChS from jumbo squid cartilage. The result of mass transfer coefficients based on Fick’s law showed that UAE had higher mass transfer efficacy. The response surface methodology (RSM) combined with Box–Behnken design (BBD) was employed to evaluate the effects of the extraction parameters. The optimal conditions were extraction temperature of 52 °C, extraction time of 46 min, and NaOH concentration of 4.15%. The crude extract was precipitated by 50% ethanol, which obtained a purified ChS with 23.7% yield and 82.3% purity. The purified ChS measured by energy-dispersive X-ray spectroscopy (EDX) had a carbon to sulfur molar ratio of approximately 14:1. The FTIR, 1H, and 13C NMR confirmed jumbo squid ChS were present in the form of chondroitin-4-sulfate and chondroitin-6-sulfate, with a 4S/6S ratio of 1.62. The results of this study provide an efficient process for production and purification of ChS, and are significant for the development and utilization of ChS from jumbo squid cartilage in the nutrient food or pharmaceutical industries.

A novel chondroitin sulfate E from Dosidicus gigas cartilage and its antitumor metastatic activity

Chondroitin sulfate (CS) chains containing GlcUAβ1-3GalNAc(4S,6S) (E unit) have been shown to be involved in various physiological and pathological processes. However, commercial E unit-rich CS (CS-E) is difficult to produce on a large scale due to expensive and limited squid cartilage resources. In this study, a novel CS-E (CS-nE) was isolated from the cheap and abundant cartilage of the giant squid Dosidicus gigas. The CS-nE has a surprisingly large molecular mass of 696 kDa and a relatively high E unit proportion (44.5 %). It can interact with various growth factors, including HGF, bFGF, pleiotrophin, and HB-EGF, with high affinity, and exhibits dose-dependent anti-metastatic activity. Furthermore, the E unit-rich decasaccharide selectively prepared from CS-nE has been shown to be the minimal functional domain with the strongest antitumor metastatic activity. Taken together, CS-nE will be a very promising candidate for the development of CS-E-based pharmaceutical products.

REMOVAL OF DYES IN SONGKET WASTE WATER BY USING PHOTOCATALYST Chitosan-TiO2

Photocatalyst is one of the catalysts that works when given a certain light and generally is a semiconductor that has a full valence band and an empty conduction band, such as TiO2. Synthesis of chitosan-TiO2 nanocomposite research has been carried out, nanocomposites produced from squid cartilage as chitosan-?. Nanocomposites are synthesized by combining chitosan as a supporting material and TiO2 which has a function as a high photocatalytic. Characterization of chitosan-TiO2 nanocomposites by FTIR and SEM / EDX which shows the functional groups and surface morphology of the nanocomposite. FTIR analysis shows the absorption band of O-Ti-O in the region of 678.98 cm-1 and the typical absorption of chitosan at wave numbers –OH 3425.58 cm 1, 3834.49 cm-1, and 3873.06 cm-1. From SEM / EDX analysis it can be seen that TiO2 is evenly distributed on the surface of chitosan. Then nanocomposite was applied with wrna procion in Palembang songket liquid waste. Chitosan-TiO2 nanocomposites from squid cartilage were able to degrade procion dyes in water medium with the help of UV light at the optimum wavelength of 520 nm, where the percentage of photodegradation was 54.47%. Which nanocomposite is also able to reduce the concentration of procion dyes from 5 mg / L to 1.9 mg / L with an initial acidity (pH) of 10.34 to 7.13. Key words: Photocatalyst, nanocomposites, Procion Red

Chondroitin sulfate E blocks enzymatic action of heparanase and heparanase-induced cellular responses

We examined whether chondroitin sulfates (CSs) exert inhibitory effects on heparanase (Hpse), the sole endoglycosidase that cleaves heparan sulfate (HS) and heparin, which also stimulates chemokine production. Hpse-mediated degradation of HS was suppressed in the presence of glycosaminoglycans derived from a squid cartilage and mouse bone marrow-derived mast cells, including the E unit of CS. Pretreatment of the chondroitin sulfate E (CS-E) with chondroitinase ABC abolished the inhibitory effect. Recombinant proteins that mimic pro-form and mature-form Hpse bound to the immobilized CS-E. Cellular responses as a result of Hpse-mediated binding, namely, uptake of Hpse by mast cells and Hpse-induced release of chemokine CCL2 from colon carcinoma cells, were also blocked by the CS-E. CS-E may regulate endogenous Hpse-mediated cellular functions by inhibiting enzymatic activity and binding to the cell surface.

Squid type II collagen as a novel biomaterial: Isolation, characterization, immunogenicity and relieving effect on degenerative osteoarthritis via inhibiting STAT1 signaling in pro-inflammatory macrophages

Collagen from marine organisms has a broad prospect in biomedical field, yet the knowledge on marine-derived type II collagen is rare. Herein, a novel type II collagen was successfully isolated from squid cartilage for the first time. After being characterized, the immunogenicity of squid type II collagen (SCII) was evaluated and compared with that of bovine type II collagen (BCII). Then investigations were further conducted for the impacts of SCII on pro-inflammatory macrophages and macrophage chemotaxis. The degenerative osteoarthritis (OA) -relieving effects of SCII were explored using OA rat model in vivo. Our results demonstrated that the isolated SCII maintained triple-superhelical structure of native collagen with high purity. Different from BCII, SCII presented no immunogenicity since it neither induced abnormal proliferation of lymphocytes in vitro nor changed the basic levels of IgM, IgG, anti-type II collagen IgG and CD4+/CD8+ lymphocytes ratio in vivo. Additionally, SCII also exerted prominent anti-inflammatory effects. SCII significantly reduced the production of pro-inflammatory cytokines by enhancing the activity of TCPTP and subsequently prompting the dephosphorylation of p-STAT1 in pro-inflammatory macrophages. Besides, it indirectly prevented hypertrophic changes of chondrocytes, and markedly impeded chemotaxis of macrophages. Moreover, inflammation condition in OA rats was significantly alleviated under treatment with SCII. These data suggested that the newly developed SCII could not only avoid the immunogenic risks of collagen derived from terrestrial animals, but more importantly, provide new choice for the control and treatment of OA.

The efficacy and safety of type E chondroitin-rich giant squid cartilage extract in knee pain symptoms: A placebo-controlled, double blind study

The efficacy and safety of type E chondroitin-rich giant squid cartilage extract in knee pain symptoms: A placebo-controlled, double blind study

Interaction of chondroitin sulfate and dermatan sulfate from various biological sources with heparin-binding growth factors and cytokines

Chondroitin sulfate (CS) and dermatan sulfate (DS) interact with various extracellular molecules such as growth factors, cytokines/chemokines, neurotrophic factors, morphogens, and viral proteins, thereby playing roles in a variety of biological processes including cell adhesion, proliferation, tissue morphogenesis, neurite outgrowth, infections, and inflammation/leukocyte trafficking. CS/DS are modified with sulfate groups at C-2 of uronic acid residues as well as C-4 and/or C-6 of N-acetyl-D-galactosamine residues, yielding enormous structural diversity, which enables the binding with numerous proteins. We have demonstrated that highly sulfated CS-E from squid cartilage, for example, interacts with heparin-binding proteins including midkine, pleiotrophin, and fibroblast growth factors expressed in brain with high affinity (Kd values in the nM range). Here, we analyzed the binding of CS and DS, which have a relatively low degree of sulfation and have been widely used as a nutraceutical and a drug for osteoarthritis etc., with a number of heparin-binding neurotrophic factors/cytokines using surface plasmon resonance (SPR) and structurally characterized the CS/DS chains. SPR showed that relatively low sulfated CS-A, DS, and CS-C also bound with significant affinity to midkine, pleiotrophin, hepatocyte growth factor, monokine-induced by interferon-γ, and stromal cell derived factor-1β, although the binding was less intense than that with highly sulfated CS-D and CS-E. These findings suggest that even low sulfated CS and/or DS chains may contain binding domains, which include fine sugar sequences with specific sulfation patterns, and that sugar sequences, conformations and electrostatic potential are more important than the simple degree of sulfation represented by disaccharide composition.

Inhibitory Effect of Chondroitin Sulfate Type E on the Binding Step of Human T-Cell Leukemia Virus Type 1

Cell surface heparan sulfate proteoglycans (HSPGs) are involved in the binding and entry of human T-cell leukemia virus type 1 (HTLV-1) into host cells, while sulfated polysaccharides such as heparin inhibit HTLV-1 infection. Chondroitin sulfate proteoglycans (CSPGs) are classified as another major type of proteoglycans. Here, we examined the effect of four types of chondroitin sulfate (CS) on HTLV-1 infection. Accordingly, a human T cell line, MOLT-4, was inoculated with cell-free HTLV-1 in the presence or absence of soluble CS, and the synthesis of reverse-transcribed HTLV-1 DNA within cells 20 h after inoculation was detected using polymerase chain reaction (PCR). Among the four types of CS (A, C, D, and E), the E type (CSE), which was derived from the squid cartilage, exhibited anti-HTLV-1 activity. Furthermore, we observed that CSE directly interacted with recombinant HTLV-1 envelope (Env) proteins and inhibited the binding of HTLV-1 virions to MOLT-4 cells, indicating that the interaction between Env and CSE plays a significant role in its anti-HTLV-1 activity. In addition, CSE inhibited syncytium formation that was induced by HTLV-1-producing cells. When CSE was mixed with the synthetic fusion inhibitor peptide corresponding to the ectodomain of the Env transmembrane subunit (TM) gp21, the HTLV-1 infection was further inhibited when compared with the inhibitory effect of each compound alone. Thus, further elucidation of the in vitro antiviral mechanism of CSE shown in this study will lead to the development of CSE-like molecules for the entry inhibition of HTLV-1.

Receptor for Advanced Glycation End Products (RAGE) Functions as Receptor for Specific Sulfated Glycosaminoglycans, and Anti-RAGE Antibody or Sulfated Glycosaminoglycans Delivered in Vivo Inhibit Pulmonary Metastasis of Tumor Cells

Altered expression of chondroitin sulfate (CS) and heparan sulfate (HS) at the surfaces of tumor cells plays a key role in malignant transformation and tumor metastasis. Previously we demonstrated that a Lewis lung carcinoma (LLC)-derived tumor cell line with high metastatic potential had a higher proportion of E-disaccharide units, GlcUA-GalNAc(4,6-O-disulfate), in CS chains than low metastatic LLC cells and that such CS chains are involved in the metastatic process. The metastasis was markedly inhibited by the pre-administration of CS-E from squid cartilage rich in E units or by preincubation with a phage display antibody specific for CS-E. However, the molecular mechanism of the inhibition remains to be investigated. In this study the receptor molecule for CS chains containing E-disaccharides expressed on LLC cells was revealed to be receptor for advanced glycation end products (RAGE), which is a member of the immunoglobulin superfamily predominantly expressed in the lung. Interestingly, RAGE bound strongly to not only E-disaccharide, but also HS-expressing LLC cells. Furthermore, the colonization of the lungs by LLC cells was effectively inhibited by the blocking of CS or HS chains at the tumor cell surface with an anti-RAGE antibody through intravenous injections in a dose-dependent manner. These results provide the clear evidence that RAGE is at least one of the critical receptors for CS and HS chains expressed at the tumor cell surface and involved in experimental lung metastasis and that CS/HS and RAGE are potential molecular targets in the treatment of pulmonary metastasis.

Specific interaction of the envelope glycoproteins E1 and E2 with liver heparan sulfate involved in the tissue tropismatic infection by hepatitis C virus

The first step in the process of infections by the hepatitis C virus (HCV) is attachment to the host cell, which is assumed to be mediated by interaction of the envelope glycoproteins E1 and E2 with cell surface glycosaminoglycans. In this study, a variety of glycosaminoglycans, heparan sulfate (HS) from various bovine tissues as well as chondroitin sulfate (CS)/dermatan sulfate from bovine liver, were used to examine the direct interaction with recombinant E1 and E2 proteins. Intriguingly, among HS preparations from various bovine tissues, only liver HS strongly bound to both E1 and E2. Since HS from liver, which is the target tissue of HCV, contains highly sulfated structures compared to HS from other tissues, the present results suggest that HS-proteoglycan on the liver cell surface appears to be one of the molecules that define the liver-specific tissue tropism of HCV infection. The interaction assay with chemically modified heparin derivatives provided evidence that the binding of the viral proteins to heparin/HS is not only mediated by simple ionic interactions, but that the 6-O-sulfation and N-sulfation are important. Heparin oligosaccharides equal to or larger than 10-mer were required to inhibit the binding. Notably, a highly sulfated CS-E preparation from squid cartilage also strongly interacted with both viral proteins and inhibited the entry of pseudotype HCV into the target cells, suggesting that the highly sulfated CS-E might be useful as an anti-HCV drug.

Organ-specific Sulfation Patterns of Heparan Sulfate Generated by Extracellular Sulfatases Sulf1 and Sulf2 in Mice

Heparan sulfate endosulfatases Sulf1 and Sulf2 hydrolyze 6-O-sulfate in heparan sulfate, thereby regulating cellular signaling. Previous studies have revealed that Sulfs act predominantly on UA2S-GlcNS6S disaccharides and weakly on UA-GlcNS6S disaccharides. However, the specificity of Sulfs and their role in sulfation patterning of heparan sulfate in vivo remained unknown. Here, we performed disaccharide analysis of heparan sulfate in Sulf1 and Sulf2 knock-out mice. Significant increases in ΔUA2S-GlcNS6S were observed in the brain, small intestine, lung, spleen, testis, and skeletal muscle of adult Sulf1(-/-) mice and in the brain, liver, kidney, spleen, and testis of adult Sulf2(-/-) mice. In addition, increases in ΔUA-GlcNS6S were seen in the Sulf1(-/-) lung and small intestine. In contrast, the disaccharide compositions of chondroitin sulfate were not primarily altered, indicating specificity of Sulfs for heparan sulfate. For Sulf1, but not for Sulf2, mRNA expression levels in eight organs of wild-type mice were highly correlated with increases in ΔUA2S-GlcNS6S in the corresponding organs of knock-out mice. Moreover, overall changes in heparan sulfate compositions were greater in Sulf1(-/-) mice than in Sulf2(-/-) mice despite lower levels of Sulf1 mRNA expression, suggesting predominant roles of Sulf1 in heparan sulfate desulfation and distinct regulation of Sulf activities in vivo. Sulf1 and Sulf2 mRNAs were differentially expressed in restricted types of cells in organs, and consequently, the sulfation patterns of heparan sulfate were locally and distinctly altered in Sulf1 and Sulf2 knock-out mice. These findings indicate that Sulf1 and Sulf2 differentially contribute to the generation of organ-specific sulfation patterns of heparan sulfate.

三陸地域資源の有効利用－イカ軟骨の酵素分解によるN-acetylglucosamineの製造

三陸地域資源の有効利用－イカ軟骨の酵素分解によるN-acetylglucosamineの製造

Paradoxical effects of chondroitin sulfate-E on Japanese encephalitis viral infection

Glycosaminoglycans (GAGs) have diverse functions in the body and are involved in viral infection. The purpose of this study was to evaluate the possible roles of the E-disaccharide units GlcAβ1–3GalNAc(4,6-O-disulfate) of chondroitin sulfate (CS), a GAG involved in neuritogenesis and neuronal migration, in Japanese encephalitis virus (JEV) infection. Soluble CS-E (sCS-E) derived from squid cartilage inhibited JEV infection in African green monkey kidney-derived Vero cells and baby hamster kidney-derived BHK cells by interfering with viral attachment. In contrast, sCS-E enhanced viral infection in the mouse neuroblastoma cell line Neuro-2a, despite the fact that viral attachment to Neuro-2a cells was inhibited by sCS-E. This enhancement effect in Neuro-2a cells seemed to be related to increased viral RNA replication and was also observed in a rat infection model in which intracerebral coadministration of sCS-E with JEV in 17-day-old rats resulted in higher brain viral loads than in rats infected without sCS-E administration. These results show the paradoxical effects of sCS-E on JEV infection in different cell types and indicate that potential use of sCS-E as an antiviral agent against JEV infection should be approached with caution considering its effects in the neuron, the major target of JEV.