Glycosaminoglycans (GAGs), including hyaluronic acid (HA), dermatan sulfate (DS) and chondroitin sulfate (CS) are essential components of the bone and cartilage tissues. CS isolated from the cartilage tissue of various animals has found application in pharmaceuticals, cosmetics and food industries. In the first part of the present work, three methods were used and compared to extract and purify glycosaminoglycans (GAGs) from the cartilage powder of a local cartilaginous marine species «Scyliorhinus canicula». One of these GAGs, chondroitin sulfate (CS), will be exploited for the development of an anti-osteoarthritis generic at the request of a collaborative pharmaceutical industry. Thus this active ingredient must meet the requirements and tests described by the European Pharmacopoeia (Ph. Eur.). These tests are treated in the second part of this work. Among the three methods that have been applied in the present work, in order to optimize the best process for GAGs preparation, enzymatic hydrolysis with papain followed by deproteinisation using trichloroacetic acid (TCA) was found the best one. The separation of the extracted GAGs using agarose gel electrophoresis, and the identification of bands by Fourier Transform Infrared (FT-IR) Spectroscopy, revealed that the cartilage GAGs of « Scyliorhinus canicula» are exclusively chondroitin sulfate (CS) and dermatane sulfate (DS), with proportions of 12.889 and 87.111% respectively, and that CS is of type C. The extraction technique with papain provides a product with GAGs content of around 90%. The TCA deproteinisation yielded the lowest level of protein (2.8%) in the extracted GAGs, less than 3%, which is the standard required by the European Pharmacopoeia (Ph. Eur.). Cetylpyridinium chloride (CPC) assay suggests that the titration technique, although is introduced by the Ph. Eur. for the determination of CS content, is not an accurate method, and that the values obtained by the optimized and validated HPLC method, described in this work, are more exact. The extracted and purified active ingredient is perfectly conform to the tests described by the Ph. Eur. The results suggest that the co-product of Scyliorhinus canicula would be a perfect source of molecules of pharmacological interest, obtained by a simple and non-agressive process.

中文翻译：

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«Scyliorhinus canicula» 副产品中的糖胺聚糖：参照欧洲药典要求的提取和纯化

糖胺聚糖 (GAG)，包括透明质酸 (HA)、硫酸皮肤素 (DS) 和硫酸软骨素 (CS)，是骨骼和软骨组织的重要成分。从各种动物的软骨组织中分离出的 CS 已在制药、化妆品和食品工业中得到应用。在本工作的第一部分，使用并比较了三种方法来从当地软骨海洋物种 «Scyliorhinus canicula» 的软骨粉中提取和纯化糖胺聚糖 (GAG)。应合作制药行业的要求，其中一种 GAG 硫酸软骨素 (CS) 将用于开发抗骨关节炎仿制药。因此，这种活性成分必须符合欧洲药典 (Ph. Eur.) 中描述的要求和测试。这些测试将在本工作的第二部分进行处理。在目前工作中已应用的三种方法中，为了优化制备 GAG 的最佳工艺，发现用木瓜蛋白酶水解然后用三氯乙酸 (TCA) 去蛋白的方法是最好的一种。使用琼脂糖凝胶电泳分离提取的 GAGs，并通过傅里叶变换红外 (FT-IR) 光谱法鉴定条带，表明《Scyliorhinus canicula》的软骨 GAGs 仅是硫酸软骨素 (CS) 和硫酸皮肤素 (DS) ，比例分别为 12.889 和 87.111%，CS 为 C 型。木瓜蛋白酶提取技术提供了 GAGs 含量约为 90% 的产品。TCA 脱蛋白在提取的 GAG 中产生最低水平的蛋白质（2.8%），低于 3%，这是欧洲药典 (Ph. Eur.) 要求的标准。氯化十六烷基吡啶 (CPC) 测定表明滴定技术虽然是由 Ph. Eur 引入的。对于 CS 含量的测定，这不是一种准确的方法，并且通过本工作中描述的优化和验证的 HPLC 方法获得的值更准确。提取和纯化的活性成分完全符合 Ph. Eur 描述的测试。结果表明，Scyliorhinus canicula 的副产品将成为具有药理学意义的分子的完美来源，通过简单且非侵入性的过程获得。对于 CS 含量的测定，这不是一种准确的方法，并且通过本工作中描述的优化和验证的 HPLC 方法获得的值更准确。提取和纯化的活性成分完全符合 Ph. Eur 描述的测试。结果表明，Scyliorhinus canicula 的副产品将成为具有药理学意义的分子的完美来源，通过简单且非侵入性的过程获得。对于 CS 含量的测定，这不是一种准确的方法，并且通过本工作中描述的优化和验证的 HPLC 方法获得的值更准确。提取和纯化的活性成分完全符合 Ph. Eur 描述的测试。结果表明，Scyliorhinus canicula 的副产品将成为具有药理学意义的分子的完美来源，通过简单且非侵入性的过程获得。