含丹参的中药注射液中过敏性杂质的检测

Determination of the allergic impurities in the parenteral injection of Chinese traditional medicines containing Salvia miltiorrhiza

模拟“水煮醇沉”工艺,从丹参中制备出高度不均一的丹参残留蛋白混合物（蛋白含量约4%）,为丹参抗原;用丹参抗原免疫家兔或豚鼠均可产生特异抗体。采用超滤等方法从丹参注射液和香丹注射液中提取到大分子抗原活性杂质。利用豚鼠主动过敏反应（ASA）模型和被动皮肤过敏反应（PCA）模型证明,提取到的抗原活性杂质可以引发被丹参抗原致敏的动物的速发型过敏反应。利用丹参抗原免疫家兔得到的特异性抗体,建立了检测中药注射液中残留的丹参抗原活性杂质的酶联免疫吸附试验（ELISA）;以残留蛋白计,其线性范围为0.08~5.12μg.mL-1（r2=0.990 6）,检测限和定量限分别为0.08μg.mL-1和0.4μg.mL-1。通过对308批含丹参水溶性组分的中药注射剂的测定,在其中35批（11.4%）样品中检出丹参抗原活性杂质;说明目前丹参水溶性组分的提取工艺不能有效的保证彻底去除丹参抗原活性杂质,这可能是导致临床过敏反应的原因之一。所建立的ELISA方法可以用于指导企业的工艺改造,并可作为药品质量控制的有效方法。

The residual protein mixture （the content is 4%, approximately ）, called Salvia miltiorrhiza antigen, was extracted from the Salvia miltiorrhiza cruel materials by mimicking the alcoholdeposit extracts process. Both rabbits and guinea pigs sensitized by Salvia miltiorrhiza could produce specified antibodies. Large molecular antigenic impurities were extracted from the Danshen injection and Xiangdan injection using the centrifugal filtering method. The test results of active systemic anaphylaxis （ASA） and passive cutaneous anaphylaxis （PCA） in guinea pigs confirmed that the extracted antigenic impurities could induce the anaphylaxis reaction in the animals which were sensitized by the Salvia miltiorrhiza antigen. Using the specified antibody produced from rabbits which were sensitiyed by Salvia miltiorrhiza, ELISA test method was developed to test the residual Salvia miltiorrhiza antigenic materials contained in the parenteral Chinese traditional medicines. Calculated as residual protein, the linear range was 0.08 -5.12 μg · mL^-1 （ r2 =0. 990 6） , the detection limit and quantization limit are 0.08 μg · mL^-1 and 0.4 μg · mL^-1, respectively. 308 batches of parenteral Chinese traditional medicines containing water-soluable components of Salvia miltiorrhiza were tested, and the Salvia miltiorrhiza antigenic impurities were spotted in 35 （ 11.4% ） batches of samples. The test results show that the extracting process currently used can not remove the Salvia miltiorrhiza antigenic impurities completely, and this may be one of the reasons for anaphylactic reaction in clinics. The proposed ELISA method can be used for improving the manufacture process and for routine quality control of drug products.