| Chondroitin sulfate (CS) is a kind of acid mucopolysaccharide with repeat disaccharide unit of [â†'4GlcUAβ1â†'3GalNAcβ1â†']. According to the variance of monosaccharide composition and location and degree of sulfo groups, CS can be divided into different classes, such as CS-A, CS-B, CS-C, CS-D and CS-E, and so on.CS is widely applied in the field of pharmaceuticals, medical biomaterials and functional foods. As a drug or a dietary supplement, CS can be used in the prevention and therapy of variant diseases, such as cardiovascular and ophthalmological diseases and osteoarthritis. In Chinese Pharmacopoeia (2010), three main sources of CS have been listed, including porcine laryngeal cartilage, nasal cartilage and trachea. The sources of CS also comprise the cartilage from marine animals, cattle, birds and edible poultry. The CS structure is of high heterogeneity. In early study people believed that CS-A and CS-C located independently. CS-A existed in the cartilage of terrestrial mammals and CS-C existed in the cartilage of marine vertebrates. The structure, contents and physiological functions of CS varied along with the species and tissue sources, resulting in different clinical effect.Alkaline enzymolysis is widely applied in the production of CS in China, which possesses the advantages of high efficiency, short production cycle and low consumption of acid, alkali and salt. But high alcohol consumption is required in the process. The CS content of the final product is generally below 90%. Ion exchange process is based on the different binding capacity between ion exchanger and different ions. Certain ions in the solution are adsorbed by the exchanger, and the components which can not be adsorbed are washed off, thus separation and purification of target substance can be achieved.In this paper we studied the preparation technology of CS:the enzymolysis was carried out by combination use of alkaline lipase and alkaline proteinase to reduce the impurities; ion exchange chromatography was used in the purification, and the refining effects of defferent resins were evaluated; the contents of CS prepared from porcine nasal cartilage, trachea, laryngeal cartilage, and cartilage of cattle, shark and chicken were determined by enzymolysis-HPLC listed in Chinese Pharmacopoeia (2010). The main research results were as follows:1. The alkaline lipase enzymolysis conditions were established:the lipase concentration was 6 U/mL, under pH 9, the enzymolysis process lasted for 30 min at 50 ℃.2. The refining effects of three kinds of resins, including D201 (Dongda), Amberlite IRA-910 (DOW) and FPA94C1 (DOW) were investigated. The results showed that Amberlite FPA94C1 (DOW) had better refining capacity.3. The technological conditions of separation and purification of CS from enzymatic hydrolysate of porcine cartilage were established:(1) Anion exchange resin FPA94C1 was used in purification of CS.(2) The static adsorption conditions were screened as follows:the initial concentration of the sample was 30 mg/mL and the pH was 6, the adsorption process lasted for 10 h at 25 ℃, using 6 mol/L NaCl as eluant.(3) The dynamic adsorption conditions were optimized as follows:the initial concentration of the sample was 30 mg/mL and the pH was 6, with the flow rate of 1 bv/h, the adsorption was achieved under 25 ℃; 6 mol/L NaCl was used as eluant, with the flow rate of 0.3 bv/h.The high-grade of CS with purity of 95% was obtained by one step alcohol precipitation.4. HPLC analysis showed that the structure of CS from the porcine nasal cartilage, trachea and laryngeal cartilage were not exactly the same. The CS-4/CS-6 disaccharide ratio of the CS standard substance specified in Chinese Pharmacopoeia (2010) is similar to the product originated from the porcine nasal cartilage, and is of large difference with the product originated from porcine trachea and laryngeal cartilage, which were similar to that from cattle source. |
PDF Full Text Request