Hyaluronic acid (HA) and chondroitin sulfate (CS) are valuable bioactive polysaccharides that have been highly used in biomedical and pharmaceutical applications. Extensive research was done to ensure their efficient extraction from marine and terrestrial by-products at a high yield and purity, using specific techniques to isolate and purify them. In general, the cartilage is the most common source for CS, while the vitreous humor is main used source of HA. The developed methods were based in general on tissue hydrolysis, removal of proteins and purification of the target biopolymers. They differ in the extraction conditions, enzymes and/or solvents used and the purification technique. This leads to specific purity, molecular weight and sulfation pattern of the isolated HA and CS. This review focuses on the analysis and comparison of different extraction and purification methods developed to isolate these valuable biopolymers from marine and terrestrial animal by-products.

The characterization of terpene-based eutectic solvent systems is performed to describe their solid–liquid phase transitions. Physical properties are measured experimentally and compared to computed correlations for deep eutectic solvents (DES) and the percentage relative error e_r for the density, surface tension, and refractive index is obtained. The thermodynamic parameters, including the degradation, glass transition and crystallization temperatures, are measured using DSC and TGA. Based on these data, the solid–liquid equilibrium phase diagrams are calculated for the ideal case and predictions are made using the semi-predictive UNIFAC and the predictive COSMO RS models, the latter with two different parametrization levels. For each system, the ideal, experimental, and predicted eutectic points are obtained. The deviation from ideality is observed experimentally and using the thermodynamic models for Thymol:Borneol and Thymol:Camphor. In contrast, a negative deviation is observed only experimentally for Menthol:Borneol and Menthol:Camphor. Moreover, the chemical interactions are analyzed using FTIR and ¹H-NMR to study the intermolecular hydrogen bonding in the systems.

Natural deep eutectic solvents (NaDES) were used to extract bioactive compounds from marine by-products: codfish bones, mussel meat, and tuna vitreous humor. NaDES were prepared using natural compounds, including lactic acid (Lac), fructose (Fru), and urea (Ur), and were characterized to define their physicochemical properties, including the viscosity, density, surface tension, and refractive index. FTIR and NMR analysis confirmed the presence of intermolecular hydrogen bonding in NaDES. The extracts obtained using these NaDES were characterized to define their composition. Results demonstrated that the extract’s composition differed highly, depending not only on the DES used, but also on the structure and composition of the raw material. Proteins and lipids were mainly present in extracts obtained from mussels, while ash content was highest in the extracts obtained from codfish bones. The biocompatibility of NaDES and the soluble fractions (SF) of the raw materials in NaDES was evaluated, and it was possible to conclude that the soluble ingredients obtained from the raw materials improved the biocompatibility of NaDES.

A green technique was developed to extract hyaluronic acid (HA) from tuna vitreous humor (TVH) for its potential application in managing dry eye disease. Deep eutectic solvents (DES) were used to extract HA and were synthesized using natural compounds (lactic acid, fructose, and urea). The DES, the soluble fraction of TVH in DES (SF), and the precipitated extracts (PE) were evaluated for their potential use in dry eye disease treatment. In vitro experiments on human corneal epithelial cell lines and the effect on dry eye-associated microorganisms were performed. The influence of the samples on the HCE viability, their intracellular reactive oxygen species (ROS) scavenging capacity, inflammatory response, and antimicrobial properties were studied. According to the results, all samples displayed an antioxidant effect, which was significantly higher for PE in comparison to SF. Most of the tested samples did not induce an inflammatory response in cells, which confirmed the safety in ophthalmic formulations. In addition, the DES and SF proved to be efficient against the studied bacterial strains, while PE did not show an antimicrobial effect. Hence, both DES and SF at defined concentrations could be used as potential compounds in dry eye disease management.

Purpose: Tear film instability, hyperosmolarity and neurosensory abnormality are the key etiological contributors to the pathophysiological process of the Dry Eye Disease (DED).¹ Hyaluronic acid (HA) and Chondroitin sulfate (CS) have shown improvements in the corneal epithelial barrier in patients with DED.² Moreover they are used in pharmaceutical applications due to their biocompatibility, viscoelasticity, lubricity and immunostimulatory effects. In this study, they are extracted from marine wastes and will be evaluated as possible DED treatment using natural, green and novel Deep Eutectic Solvents (DES) to replace time‐consuming and expensive conventional methods and maintaining the quality and purity of the isolated HA and CS. Natural compounds such as menthol and trehalose, are used in DES preparation and have shown to be effective in DED treatment.^3,4Methods: HA and CS are isolated from marine wastes using conventional methods and DES as low‐cost alternative technique. DES are prepared by heating and mixing the components at certain ratios. They are mixed with the marine sources at specific extraction conditions. The extracts are then enzymatically hydrolyzed and analyzed by HPLC and capillary electrophoresis to quantify HA and CS.Results: HA and CS are extracted from mussels, codfish bones and furtherly tuna eyeballs and shark fins using DES. Several DES were tested such as menthol:borneol, menthol:camphor, trehalose:glycerol, and significant amounts of HA and CS were extracted with high specificity. For example, 37.92 µg/g_{dry codfish bones} CS disaccharide (ΔHexAβ1‐3GalNAc4S) were extracted using menthol:borneol.Conclusion: DES are effective compounds for the extraction of valuable biopolymers HA and CS. The isolated HA and CS, as well as their therapeutic mixture with DES will be studied in vitro on conjunctival and corneal epithelial human cell lines to assess their toxicity, biocompatibility, muco‐adhesion and anti‐oxidant effects for the treatment of the DED.References: Craig, Jennifer P., et al. “TFOS DEWS II report executive summary.” The Ocular Surface 15.4 (2017): 802‐812. Limberg, Michael B., et al. “Topical application of hyaluronic acid and chondroitin sulfate in the treatment of dry eyes.” American journal of ophthalmology 103.2 (1987): 194‐197. Arita, Reiko, et al. “Effects of a warm compress containing menthol on the tear film in healthy subjects and dry eye patients.” Scientific reports 7 (2017): 45848. Fondi, Klemens, et al. “Effect of Hyaluronic Acid/Trehalose in Two Different Formulations on Signs and Symptoms in Patients with Moderate to Severe Dry Eye Disease.” Journal of ophthalmology 2018 (2018).