Efficient extraction and structural characterization of chondroitin sulfate by deep eutectic solvents

Chondroitin sulfate (CS) is widely distributed in animal cartilage tissues, such as thoracic cartilage, laryngeal bone, and nasal bone. The market demand for CS products is ever increasing with the global aging and aging of chronic diseases in recent years, due to the various functional characteristics, such as antioxidant, anti-inflammatory, and lipid-lowering. It is very necessary to promote the extraction of specific active substances. Natural deep eutectic solvents (NADESs) are often simply synthesized to design their structure for high extraction efficiency. The solvents can also be recycled and reused after use, in order to make them green and environmentally friendly. This study aims to improve the utilization rate of cartilage raw materials and reduce the burden of waste treatment. A natural deep eutectic solvent method was also proposed to extract the CS from the bovine laryngeal cartilage, taking betaine and choline chloride as hydrogen bond acceptors and six common natural substances as hydrogen bond donors. Twelve NADESs were prepared to combine them in pairs. Among them, NADES-3 (betaine/urea, molar ratio 1:2) had the highest extraction rate of CS, which was 16.49%. The reason was that the weak hydrogen bonds were formed in the solvent, due to the small molecular weight of urea, and then the betaine counteracted the denaturation of protein by urea, indicating the synergistic effect. The extracted CS was characterized by monosaccharide analysis, infrared spectroscopy, and molecular weight analysis, in order to confirm the extracted CS with an excellent structure. Subsequently, the effects of four single factors on CS extraction were studied, including liquid-solid ratio (10:1, 20:1, 30:1, 40:1, and 50:1 mL/g), moisture content (5%, 20%, 40%, 60%, and 80%), temperature (90, 100, 110, 120, and 130 ℃), and time (60, 90, 120, 150, and 180 min). The optimal extraction conditions were: liquid-solid ratio of 30:1 mL/g, moisture content of 40%, extraction temperature of 110 ℃, and extraction time of 120 minutes. The CS extraction rate reached 17.33%, which was 2.88 times higher than the traditional water extraction. A correlation analysis was made between the physicochemical properties of NADESs and the CS recovery rate. The extraction rate was negatively correlated with the viscosity, whereas, that was positively correlated with pH and polarity, and there was no significant correlation with the density. The density and viscosity of the solvent decreased after dilution with water, and the hydrogen bonding interaction between NADES components gradually weakened to enhance the mass transfer movement of the cartilage matrix to the solvent, thereby improving the extraction rate of CS. Excessive water disrupted the tight hydrogen bonding structure of NADESs, leading to the formation of simple aqueous solutions and the extraction efficiency of solvents. Finally, NADES-3 with a moisture content of 40% was determined as the optimal solvent for the CS extraction. Subsequently, NADES-3 was recycled to verify the reusability. The extraction rate of CS still reached 15.89% after five repetitions. The excellent extraction was maintained after multiple extractions, where some collagen residues in the solvent increased the steric hindrance between molecules, and weakened the hydrogen bonding interaction between them. The NADES extraction can effectively green-recover CS from laryngeal cartilage. This finding can provide new ideas for recyclable extractants, in order to extract bioactive compounds.