盐酸多西环素杂质C的制备及体外抗菌活性与毒性研究  

Preparation of impurity C of doxycycline hyclate and its evaluation of in vitro antimicrobial activity and toxicity

作　　者：张娅[1,2] 张锦琳 赵述强 袁耀佐 张玫 樊夏雷 王亚楼[2] 

机构地区：[1]江苏省食品药品监督检验研究院,南京210008 [2]中国药科大学药物化学教研室,南京210009

出　　处：《中国药科大学学报》2016年第4期463-468,共6页Journal of China Pharmaceutical University

摘　　要：采用高效制备液相色谱法从盐酸多西环素中分离制备高纯度杂质C,同时考察其毒性及体外抗菌活性。盐酸多西环素溶液首先经加热处理,然后采用Sapphire C18柱(21.2 mm×250 mm,5μm)分离,流动相为0.1%乙酸-乙腈(83∶17),流速为20 m L/min,收集8.4 min出峰的组分,再通过旋转蒸发、调节旋蒸后溶液p H、冷冻干燥等步骤制得目标物。采用高效液相色谱法测定目标物纯度,运用紫外光谱、红外光谱、质谱及核磁共振等手段确证其结构,比较盐酸多西环素杂质C与多西环素、美他环素、β-多西环素等相关物质对中国仓鼠肺(CHL)细胞的细胞毒性和遗传毒性、斑马鱼胚胎发育毒性以及体外抗菌活性。结果发现:制备产物经结构确证为盐酸多西环素杂质C,其纯度达到90.1%,为目前文献报道最高。在对CHL细胞的细胞毒性和遗传毒性研究实验中,盐酸多西环素杂质C、多西环素、美他环素、β-多西环素对CHL细胞均有一定的细胞毒性,畸变率均小于5%,但未见明显的遗传毒性;在斑马鱼胚胎发育毒性实验中,盐酸多西环素杂质C致畸性与致死性最强;在体外抗菌活性实验中,盐酸多西环素杂质C抗菌活性较弱,其他相关物质抗菌活性由强到弱的顺序为:美他环素、多西环素、盐酸多西环素杂质C、β-多西环素。安全性和有效性试验提示盐酸多西环素杂质C属于毒性大且无效的杂质,需要在质量标准中进行单独控制,建议修订现行《中华人民共和国药典》中盐酸多西环素及其制剂的质量标准。This study aimed to isolate and prepare highly purified impurity C from doxycycline hyclate by a pre- parative HPLC method and to inspect the toxicity and in vitro antimicrobial activity of the impurity C of doxycyc- line hyclate. The solution of doxycycline hyelate treated with heat produced a solution containing 10% of impurity C which was firstly separated by the Sapphire C18（21.2 mm ×250 mm, 5 μm） column with 0. 1% acetic acid- aeetonitrile（83 ： 17） as the mobile phase at 20 mL/min. Secondly, rotary evaporation of the eluted solution at the time of 8.4 min was performed at 50 ℃ to remove organic solvent. Then the target product was prepared after freeze drying of evaporated solution adjusting pH to 1.8 with formic acid. The target product was identified with ultraviolet absorbance （UV）, infrared （IR）, mass spectrometry （MS） and nuclear magnetic resonance （NMR）,and its purity was be determined by HPLC. Meanwhile, cytotoxicity and genotoxicity in the Chinese hamster lung ceils, toxicity on the development of zebrafish embryos and in vitro antimicrobial activity were compared among impurity C of doxycycline hyclate, doxycycline, metacycline and/3-doxycycline. Results showed that prepared product was confirmed to be the impurity C of doxycycline hyclate. Its purity was 90. 1%, which had been the highest so far. In the cellular toxic tests and genetic toxic tests of Chinese hamster lung cells, impurity C of doxy- cycline hyclate, doxycycline, metacycline and β-doxycycline were somewhat toxic to Chinese hamster lung cells. Toxicity gradually decreased from doxycycline, impurity C of doxycycline hyclate,β-doxycycline to metacy- cline from -S9mix test results; toxicity gradually decreased from doxycycline,β-doxycycline, impurity C of doxy- cycline hyclate to metacycline from ＋ Sgmix test results; the aberration rate of all the tested related substances was less than 5 %, and no obvious genotoxicity was found. According to test results of the development of zebrafish embryos, impurit

关 键 词：盐酸多西环素杂质C 制备型液相色谱 毒性 体外抗菌活性 

分 类 号：R943[医药卫生—药剂学] R96[医药卫生—药学]