Molecular Weight Of Chondroitin Sulfate Research Articles

Extraction, Physicochemical Properties, and In Vitro Antioxidant Activities of Chondroitin Sulfate from Bovine Nose Cartilage.

Beef is an important high-nutrition livestock product, and several byproducts, such as bovine cartilage, are produced during slaughter. To effectively utilize these agricultural and pastoral byproducts, combined (trypsin-papain) enzymolysis and cetylpyridine chloride purification methods were used to obtain chondroitin sulfate (CS) from the nasal cartilage of Shaanxi Yellow cattle. The effects of pH, temperature, and time on the CS yield during enzymatic hydrolysis were investigated, and the CS extraction process was optimized using response surface methodology. The best yield of CS was 21.62% under the optimum conditions of pH 6.51, temperature of 64.53°C, and enzymolysis time of 19.86 h. The molecular weight of CS from Shaanxi cattle nasal cartilage was 89.21 kDa, glucuronic acid content was 31.76 ± 0.72%, protein content was 1.12 ± 0.03%, and sulfate group content was 23.34 ± 0.08%. The nasal cartilage CS of the Yellow cattle showed strong DPPH•, •OH, and ABTS+• radical scavenging abilities and ferrous reduction ability in the experimental concentration range. This study could contribute to "turn waste into treasure" and improve the comprehensive utilization of regional characteristic biological resources.

A chondroitin sulfate purified from shark cartilage and bovine serum albumin interaction activity

Chondroitin sulfate (CS) was extracted and purified from shark cartilage, and its interaction with bovine serum albumin (BSA) were studied. The content of chondroitin sulfate in shark cartilage was 29.97 % using the 1,9-dimethyl-methylene blue method. The molecular weight of CS was determined to be 62.464 kDa by high-performance gel permeation chromatography. UV and FT-IR spectroscopy identified the characteristics of CS and its functional group information. NMR spectroscopy and disaccharide derivatization revealed that CS was predominantly composed of disulfated disaccharides, specifically ΔDi4,6S. Fluorescence quenching experiments indicated that the interaction between CS and BSA exhibited static quenching, with a binding site number of 1. The binding process was primarily mediated by van der Waals forces and hydrogen bonds. Furthermore, synchronous and 3D fluorescence spectroscopy demonstrated that CS had minimal impact on the polarity and hydrophobicity of the microenvironment surrounding Tyr and Trp residues. UV–vis absorption and circular dichroism (CD) spectroscopy demonstrated the altered structure of BSA. The molecular docking analysis revealed that CS formed hydrogen bonds and salt bridges with BSA, predominantly binding to the IIA substructure domain of BSA. Investigating the interaction between CS and BSA holds the potential for enhancing its applications in drug delivery and tissue engineering endeavors.

Quantification of orally administered chondroitin sulfate oligosaccharides in human plasma and urine.

Chondroitin sulfate (CS) has been widely administered orally to improve knee osteoarthritis (OA). CS also has various biological properties, such as anti-inflammatory, immunomodulatory, anti-oxidative, and anti-tumour activity. However, CS absorption in the digestive system and bioavailability remain controversial owing to its large molecular weight (MW). In this study, we aimed to evaluate the absorption of CS oligosaccharides (CSOS), depolymerized CS with low MW, in oral administration to humans. Four types of CS with varying MW [CS tetrasaccharide (MW. 980), CSOS-1 (MW. 1500), CSOS-2 (MW. 2800), and HMWCS (MW. 70,000)] were orally administered and quantified in plasma and urine. Exogenous CS (Exo-CS) in these samples was quantified using a high-performance liquid chromatography system equipped with a fluorescence detector. Quantitative changes of administered CS tetrasaccharide showed similar patterns in plasma and urine, therefore it was presumed that the amount of Exo-CS excreted in urine reflects its quantitative profile in blood. Considering urinary Exo-CS as a parameter of intestinal CS absorption, urinary contents of orally administered CS with varying MW were compared. Consequently, the amount of urinary Exo-CS in 24hours after administration was higher in the CSOS group than that in the HMWCS group. Additionally, in the MW distribution, urinary Exo-CS after CSOS administration showed a lower content of CSOS with a higher MW than that observed before administration. In summary, our results demonstrated for the first time that lower MW of CS is more efficiently absorbed through the digestive tract in human, and the improvement of its bioavailability is expected.

Polymerization of chondroitin sulfate and its stimulatory effect on cartilage regeneration; a bioactive material for cartilage regeneration

Chondroitin sulfate (CS) is one of the major glycosaminoglycans (GAGs). GAGs are linear polymers comprising disaccharide residues and are found as the side chains of proteoglycans. CS has significant stimulatory effects on cell behavior and is widely used in tissue-engineered and drug delivery devices. However, it is difficult to incorporate a sufficient amount of CS into biopolymer-based scaffolds such as collagen to take full advantage of its benefit. In this study, CS has been polymerized to an 11 times higher molecular weight polymer (PCS) in an attempt to overcome this deficiency. We have previously shown that PCS was significantly more effective than CS in chondrogenesis. This study aimed to characterize the physicochemical properties of the manufactured PCS. PCS was characterized by Fourier transform infra-red (FTIR) spectroscopy together with X-ray photoelectron spectroscopy (XPS) to obtain information about its chemical structure and elemental composition. Its molecular size was measured using dynamic light scattering (DLS) and its viscoelastic properties were determined by rheology measurements. The average PCS diameter increased 5 times by polymerization and PCS has significantly enhanced viscoelastic properties compared to CS. The molecular weight of PCS was calculated from the rheological experiment to give more than an order of magnitude increase over CS molecular weight. Based on these results, we believe there is a great potential for using PCS in regenerative medicine devices.

Uronic acid carbazole assay and cetylpyridinium chloride titration depend on the chondroitin sulfate molecular weight

Chondroitin sulfate (CS) of various molecular weight (MW), up to ∼3 kDa, were produced and tested for uronic acid carbazole assay and cetylpyridinium chloride (CPC) titration showing an evident decrease in the assays depending on the CS MW. The described results for uronic acid assay by carbazole reaction and CPC titration of CS poses the problem to know the MW values before their application and to use comparable standards to obtain reliable results. Otherwise, the related quantitative data can be affected by a great error and fake certificate of analysis.

Polyamines stimulate the CHSY1 synthesis through the unfolding of the RNA G-quadruplex at the 5′-untraslated region

Glycosaminoglycans (GAGs), a group of structurally related acidic polysaccharides, are primarily found as glycan moieties of proteoglycans (PGs). Among these, chondroitin sulfate (CS) and dermatan sulfate, side chains of PGs, are widely distributed in animal kingdom and show structural variations, such as sulfation patterns and degree of epimerization, which are responsible for their physiological functions through interactions with growth factors, chemokines and adhesion molecules. However, structural changes in CS, particularly the ratio of 4-O-sulfation to 6-O-sulfation (4S/6S) and CS chain length that occur during the aging process, are not fully understood. We found that 4S/6S ratio and molecular weight of CS were decreased in polyamine-depleted cells. In addition, decreased levels of chondroitin synthase 1 (CHSY1) and chondroitin 4-O-sulfotransferase 2 proteins were also observed on polyamine depletion. Interestingly, the translation initiation of CHSY1 was suppressed by a highly structured sequence (positions -202 to -117 relative to the initiation codon) containing RNA G-quadruplex (G4) structures in 5'-untranslated region. The formation of the G4s was influenced by the neighboring sequences to the G4s and polyamine stimulation of CHSY1 synthesis disappeared when the formation of the G4s was inhibited by site-directed mutagenesis. These results suggest that the destabilization of G4 structures by polyamines stimulates CHSY1 synthesis and, at least in part, contribute to the maturation of CS chains.

Enzymatic depolymerization – An easy approach to reduce the chondroitin sulfate molecular weight

Abstract This study reports on a new approach to a well-known method to reduce the molecular weight of chondroitin sulfate (CS) using a non-specific enzyme. Here, the enzyme hyaluronidase was utilized to depolymerize raw CS (19.2 kDa) resulting in low molecular weight CS (LMWCS) as a product. A simple ultrafiltration process utilizing circular-flat membranes with different MWCO was utilized to recover the fraction of interest (5.6 kDa). LMWCS characterized by GPC/LALLS, viscometric analyses and FTIR and NMR spectroscopies. The enzymatic depolymerization kinetics was investigated and some important parameters but poorly demonstrated in the literature, such as the rate of depolymerization for bond cleavage (0.308 mol/L h); depolymerization rate constant (2.98 × 10−3 h−1); and the relationship between Mn and Mv is provided. The present work could be a basis on which to present a suitable and original method to obtain and recover LMWCS. Furthermore, it could be a useful reference work for the investigation of how to improve the biological activity of CS in biomedical and pharmaceutical devices by reducing the CS molecular weight.

A Study on Development of High Functional Materials Producing Technique using By-products from Skate Processing (1) - Development of Chondroitin Sulfate Materials using Skate Cartilages

For the purpose of reuse the wasted by-products from the skate process to the health functional food or medicinal material, chondroitin sulfate was extracted from the skate cartilage with the method of hydrolysis with protease enzyme, and the contents of chondroitin sulfate and hydrolyzed protein were measured qualitatively and quantitatively. The effects of chondroitin sulfate on body weight or liver weight changes, hepatotoxicity elimination and anti-inflammatory actions were measured from in vivo test with feed-treated mice. From the hydrolytic extraction of skate cartilage with the mixture of 1% alcalase and 1% protease for 4 hours, the extraction yield of chondroitin sulfate was about 32.55%. The content and molecular weight of chondroitin sulfate was 26.63% and ., respectively and the content ratio of chondroitin sulfate to protein was measured to 1 to 2.76 with gel permeation chromatography. For the odor component, trimethylamine decreased about 30% but almost not ammonia from chondroitin sulfate with the treatment of activated carbon. From the feeding chondroitin sulfate to mice, the control effect of body and liver weights decrease was measured, anti-inflammatory action and hepatotoxicity elimination action were also measured. From these results, process operation conditions for manufacturing of chondroitin sulfate were suggested.

Effect of Molecular Sizes of Chondroitin Sulfate on Interaction with L-Selectin

Chondroitin sulfate (CS) is a glycosaminoglycan (GAG) side chain of proteoglycans (PGs) which are widely distributed in the extracellular matrix and at cell surface. CS shows a highly structural diversity in not only molecular weight (MW) but sulfonation pattern. CS has been reported to exert anti-inflammatory activity by having effects on cytokine production by helper T cells. In this study, we focused on the structures of CS chains, especially MW of CS, and investigated effect of the different MW of CS on binding affinity with L-selectin and cytokine production by murine splenocytes. Firstly, we fractionated CS by employing gel filtration chromatography and obtained several CS fractions with different MW. Then the interaction between fractionated CS and L-selectin was analyzed by surface plasmon resonance (SPR). Finally, the influence of MW of CS on cytokine production by murine splenocytes was investigated in vitro. The results showed that interferon-gamma production was significantly increased by mouse splenocytes cocultivated with CS. On the contrary, CS inhibited interleukin 5 production by murine splenocytes depending on MW of the cocultivated CS. These results strongly indicate the existence of the optimal molecular size for an anti-inflammatory effect of CS through cytokine production by murine splenocytes.

Relationship Between In Vitro Absorption of Chondroitin Sulfate (CS) and Its Molecular Weight

ObjectiveThe purpose of this study was to investigate if the molecular weight of chondroitin sulfate (CS) has an impact on its absorption in vitro, in a Caco‐2 cell model.MethodsCaco‐2 cells in 24 well plates were incubated for 2 hours with eight CS samples of differing molecular weights (7 kDa – 35 kDa). The amount of CS transported into the basolateral side of the Caco‐2 monolayer was quantitatively determined to calculate the permeability coefficients (Papp).ResultsOf the 8 samples evaluated, four samples had a Papp value of 15 × 10−6 cm sec−1 or higher indicating moderate to high absorption. Two of the four samples with higher Papp values were high molecular weight compounds (~ 35 kDa).ConclusionWithin the molecular weight range used in this experiment, the absorption of CS samples did not have a correlation with their molecular weights but, interestingly, correlation was observed between the absorption and percentage of 6‐sulfated disaccharide in the CS samples.Grant Funding Source: Pharmavite, Inc.

Isolation and Characteristic of Chondroitin Sulfate from Grass Carp Scales

Papain and alcalase were successively used to hydrolyze grass carp scales for preparation of crude polysaccharide. Chondroitin sulfate were further purified by DEAE-Sepharose Fast Flow and Sepharose 6 Fast Flow gel column chromatograph, and indentified by IR, NMR, element analysis and chemical analysis. Three polysaccharide fractions including chondroitin sulfate were fistly obtained from grass carp scales. The weight-average molecular weight of chondroitin sulfate was 46168 Da. The contents of various elements (C, H, N, S, O) in chondroitin sulfate were 28.89 %, 3.80 %, 3.36 %, 6.71 % and 47.16 %, respectively. The results of IR, NMR and chemical analysis exhibited that the sulfate group in chondroitin sulfate be O-linked in position 4 of N-acetyl-D-galactosamine.

고순도 콘드로이틴 황산의 제조

콘드로이틴황산은 상어연골로부터 알카리방법과 효소적 방법에 의해 추출하였다. 추출에 있어서 알카리의 농도, 효소인 알카라제의 농도, 온도 등에 따른 최적 추출조건을 규명하였으며 콘드로이틴 황산용 UF 막으로 막분리하여 순수한 콘드로이틴황산을 얻었다. 얻어진 콘드로이틴 황산의 분자량을 GPC로 측정하였을 때 분자량이 27만 Da이였다.

Mode of degradation of the chondroitin sulphate proteoglycan in rat costal cartilage

1. Chondroitin sulphate was isolated from different regions of rat costal cartilage after extensive proteolysis of the tissues. The molecular weight, determined by gel chromatography, of the polysaccharide obtained from an actively growing region (lateral zone) near the osteochondral junction was higher than that of the polysaccharide isolated from the remaining portion of the costal cartilage (medial zone). 2. In both types of cartilage the molecular weight of chondroitin sulphate, labelled with [(35)S]sulphate, remained unchanged in vivo over a period of 10 days, approximately corresponding to the half-life of the chondroitin sulphate proteoglycan. The molecular-weight distribution of chondroitin [(35)S]sulphate, labelled in vivo or in vitro, was invariably identical with that of the bulk polysaccharide from the same tissue. It is concluded that the observed regional variations in molecular-weight distribution were established at the time of polysaccharide biosynthesis. 3. In tissue culture more than half of the (35)S-labelled polysaccharide-proteins of the two tissues was released into the medium within 10 days of incubation. The released materials were of smaller molecular size than were the corresponding native proteoglycans. In contrast, the molecular-weight distribution of the chondroitin [(35)S]sulphate (single polysaccharide chains) remained constant throughout the incubation period. 4. A portion (about 20%) of the total radioactive material released from (35)S-labelled cartilage in tissue culture was identified as inorganic [(35)S]sulphate. No corresponding decrease in the degree of sulphation of the labelled polysaccharide could be detected. These findings suggest that a limited fraction of the proteoglycan molecules had been extensively desulphated. 5. It is suggested that the initial phase of degradation involves proteolytic cleavage of the proteoglycan, but the constituent polysaccharide chains remain intact. The partially degraded proteoglycan may be eliminated from the cartilage by diffusion into the circulatory system. An additional degradative process, which may occur intracellularly, includes desulphation of the polysaccharide, probably in conjunction with a more extensive breakdown of the polymer.

The molecular weight of chondroitin sulphate from human articular cartilage. Effect of age and of osteoarthritis.

The molecular weight distributions of chondroitin sulphate from normal articular cartilage of the lower femoral epiphysis from human autopsy cases of different ages and from osteoarthritic cartilage have been determined by gel chromatography on Sephadex G-200. The polysaccharide samples fell within the same molecular weight range but differed with respect to molecular weight distribution. The average molecular weight of chondroitin sulphate from normal articular cartilage was higher in newborn and young individuals than in adults or the aged. Chondroitin sulphate from osteoarthritic cartilage had a lower molecular weight than that from macroscopically normal cartilage. The observed variations in polysaccharide chain length are discussed.

Identification and concentration of the glycosaminoglycans of human articular cartilage in relation to age and osteoarthritis.

The glycosaminoglycans in articular cartilage from the femoral condyles of 119 human autopsy cases have been isolated and subsequently characterized using cetylpyridinium chloride. Using the same column method in microscale the glycosaminoglycans were estimated quantitatively in normal articular cartilage from birth to 95 years of age and in osteoarthritic cartilage. In normal cartilage chondroitinsulphate occurred as chondroitin-4-sulphate and chondroitin-6-sulphate, decreasing in ratio with age. The chondroitinsulphate had a sulphate/hexuronic acid ratio below unity, below 20 years, above unity in the higher age groups. Two types of keratansulphate and hyaluronic acid were isolated. The glycosaminoglycans isolated from osteoarthritic cartilage showed no differences from normal cartilage as to the amount of their moities of chondroitin sulphate isomers. In normal cartilage there was a significant decrease of total hexosamines from birth to 11–20 years, due to a reduction of chondroitinsulphate. In adults, an increasing ratio of the keratansulphate plus glycoprotein fraction to chondroitinsulphate was observed. Osteoarthritic cartilage showed a significant reduction of all glycosaminoglycan fractions without change of distribution. Normal cartilage taken from osteoarthritic joints was unaltered. Solubility profiles indicated decreases in molecular weight of chondroitin sulphate in normal cartilage from birth to 21–30 years, and a trend towards lower molecular weight in osteoarthritic cartilage.

A method for the determination of the molecular weight and molecular-weight distribution of chondroitin sulphate

A simple micro-method for the determination of molecular weights of chondroitin sulphate has been developed. Columns of Sephadex G-200 and Sepharose 6B were calibrated with fractions of chondroitin sulphate, the molecular weights of which had been determined by independent methods. The use of the calibrated columns for determining the molecular weights and molecular-weight distributions of unknown samples of chondroitin sulphate by gel chromatography is described. The effects of variations in the ionic strength of the eluant were studied. For the average of duplicate determinations of the molecular weight ( M w) the 95% confidence interval was M w ± 3%.