鹿瓜多肽注射剂质量评价与研究
Quality evaluation and specification research of Cervus and Cucumis polypeptide injection
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摘要: 研究鹿瓜多肽注射剂的新鲜度、高相对分子质量物质、多肽含量测定、溶血与凝聚及生物学活性等指标，为鹿瓜多肽注射剂质量标准修订提供理论参考。首先，对影响终产品新鲜度的因素包括生物胺含量、黄曲霉毒素、甜瓜子原料的酸价和过氧化值等进行考察。建立丹磺酰氯柱前衍生-HPLC法测定鹿瓜多肽注射剂中8种生物胺的含量，方法学验证结果显示该法专属性、精密度、线性关系以及回收率等均良好，适用于鹿瓜多肽注射剂中生物胺类成分的检测，样品测定结果显示个别样品中检出较高浓度的尸胺；黄曲霉毒素、甜瓜子原料的酸价和过氧化值测定结果表明：部分生产原料甜瓜子存在霉变、酸败等问题，提示含动植物源性成分的多组分生化药质量标准应在新鲜度方面加强控制。其次，改进了鹿瓜多肽注射剂质量标准中高相对分子质量物质和多肽含量测定的方法。采用 Tricine-SDS-PAGE 电泳法代替凝胶色谱法测定高相对分子质量物质，提高了测定的准确性；采用试剂盒法代替福林酚法测定多肽含量，该法操作简便、专属性更强，可适用于高通量样品测定。最后，对鹿瓜多肽注射剂质量标准中缺失的溶血与凝聚、生物学活性测定项目进行了研究。研究结果显示，样品均无溶血与凝聚现象；鹿瓜多肽注射剂对单核巨噬细胞THP-1具有增殖抑制作用，且所检测样品的体外抗炎效果具有一定差异。优化后的检测方法更适用于鹿瓜多肽注射剂的质量检测，并为多组分生化药质量控制奠定了基础。Abstract: To investigate the freshness, high molecular weight substances, the determination of polypeptide, haemolysis and agglomeration, biological activity of Cervus and Cucumis polypeptide injection; to provide the direction for improving the quality of products for enterprises; furthermore, to provide reference for the revision of the quality standards of Cervus and Cucumis polypeptide injection. Firstly, we investigated the factors affecting the freshness of the injection, including biogenic amines, aflatoxins, the acid value and peroxide value of the melon seeds. The method of dansyl chloride pre-column derivatization-HPLC was used to determine the content of 8 biogenic amines in Cervus and Cucumis polypeptide injection. The method validation results showed good specificity, precision, linearity and recovery rates, which was suitable for the determination of biogenic amines in Cervus and Cucumis polypeptide injection. The results of sample determination showed that relatively higher concentrations of cadaverine were detected in the products from company B. The results of aflatoxins, acid value and peroxide value showed that the melon seeds from some companies had rancidity, mildew and other problems, indicating that the quality standards of multi-component biochemical drugs containing animal- and plant-derived components should be controlled in terms of freshness. Secondly, the methods for the determination of high molecular weight substances and polypeptides in the quality standard were improved. Tricine-SDS-PAGE electrophoresis was used instead of gel chromatography to determine the high molecular weight substances, which improved the accuracy of determination. The kits were used instead of folin-phenol for the determination of peptide content, which is easy to operate, specific and suitable for high-throughput sample determination. Finally, the haemolysis, agglomeration, and biological activity of Cervus and Cucumis polypeptide injection were studied. The results showed that no haemolysis and agglomeration were found in all samples, and the inhibitory effect of samples on THP-1 proliferation in vitro from different companies was different to some extent. In conclusion, the optimized quality standard is more suitable for the detection of Cervus and Cucumis polypeptide injection, and can lay the foundation for improving the safety of multi-component biochemical drugs.
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