CN1037526C - 康乐霉素c的生产方法 - Google Patents

康乐霉素c的生产方法 Download PDF

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CN1037526C
CN1037526C CN91101127A CN91101127A CN1037526C CN 1037526 C CN1037526 C CN 1037526C CN 91101127 A CN91101127 A CN 91101127A CN 91101127 A CN91101127 A CN 91101127A CN 1037526 C CN1037526 C CN 1037526C
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kanglemycin
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王南金
李群
杨贤树
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Priority to EP92103139A priority patent/EP0501399A1/en
Priority to HU9200677A priority patent/HUT63883A/hu
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Abstract

康乐霉素C是从一株奴卡氏菌的发酵液中，经过滤去菌丝，用酸调pH值至酸性，用有机酯类萃取，将萃取液浓缩后，在硅胶柱上分离用苯和甲醇混合液洗脱，收集活性部分并浓缩，在LH-20柱上纯化，用醇类有机溶剂洗脱，收集活性成分并浓缩，用醇类有机溶剂重结晶即得纯品。该化合物溶于极性有机溶剂，免疫抑制作用与环孢菌素A相似，并有抗肿瘤作用，毒性很低。本发明提取工艺简单，化学合成及结构改造较方便。

Description

康乐霉素C的生产方法
本发明涉及一种用于自身免疫溶血性贫血、肾炎、结缔组织以及脏器移植排斥反应的新免疫抑制剂康乐霉素C的生产方法。
目前开发的免疫抑制剂，其药物选择性强，专一性地调节某-免疫细胞的亚族。代表药物如环孢菌素A，能选择性抑制T辅助细胞的克隆(无性繁殖系)的增殖，在器官移植方面取得了可喜的成绩，并已广泛用于临床。然而，也发现一些副作用，如个别患者用药后出现急性和慢性肾中毒、轻度震颤、神经病变、齿龈肥厚和多毛等副作用。最近发现的FK-506属于大环内酯类抗生素(C44H19NO5)，虽然实验研究结果满意，但因生产复杂，限制了临床上广泛应用。
本发明目的在于克服现有免疫抑制剂的各种缺点，寻找更理想替代药物。康乐霉素C的化学结构新颖，有别于迄今所有的免疫抑制剂，分子量小，结构简单，便于修饰。研究表明，康乐霉素C的免疫抑制作用与环孢菌素A相似或稍强，同时有抑制肿瘤细胞的作用，有可能成为更有特点的新一代免疫抑制剂。
本发明的特点是用一株奴卡氏菌进行培养发酵，然后用有机溶剂萃取、柱层析分离和纯化，其中所说的奴卡氏菌是地中海奴卡氏菌康乐变种(Nocardia mediterranei var.kanglensis)1747-64，该菌株已于1991年2月20日由中国微生物菌种保藏管理委员会普通微生物中心保藏，保藏号为：CGMCC NO0163。所述方法其培养基是：
(1)菌种培养基(％)：KNO3 0.1、NaCl 0.5、K2HPO4 0.05、FeSO4 0.01、淀粉2、琼脂1.7、冷开水、pH7.0、28℃培养5-7天；
(2)种子培养基(％)：葡萄糖2-5、酵母粉0.1-1.5、KCl 0.1-0.5、黄豆饼粉0.1-2.5、K2HPO4 0.01-0.5、(NH4)2SO4 0.1-2、MgSO4 0.01-0.1、CaCO20.1-1、自来水100ml、自然pH。
(3)发酵培养基(％)：葡萄糖2-5，酵母粉0.5-2、黄豆饼粉0.1-1.5、蛋白胨0.05-0.5、CaCO2 0.01-1.0、自来水100ml、自然pH。
以上培养基均15磅消毒30分钟备用。将斜面挖块接种于种子培养基摇瓶中(30-70ml装量/250ml)，置180-200转/(分钟)旋转摇床，25-30℃培养24-55小时后，以5-15％接种量转入发酵培养基摇瓶(30-100ml装量/500ml)，在180-200转/(分钟)旋转摇床25-32℃培养68-96小时供提取用。将发酵液过滤后，用无机酸调pH值在1-5范围，再用乙酸丁酯萃取，萃取液浓缩后，在硅胶柱上分离，用苯和甲醇混合液(30∶1)洗脱，收集活性部分并浓缩，在LH-20柱上纯化用乙醇洗脱，收集活性部分并浓缩，用乙醇重结晶，即得纯品。康乐霉素C为黄色棒状结晶，在170℃分解变成黑色，分子量为326，旋光度为+150°(10-4)，紫外光谱中在232纳米，356纳米有最大吸收，红外光谱中在3400cm-1、1690cm-1、1650cm-1、750cm-1等处有吸收。溶于极性有机溶剂，不溶于水和非极性有机溶剂。
0.5μg/ml的康乐霉素C对小鼠脾淋巴细胞转化的抑制率达96.9％。在小鼠脾细胞培养时，康乐霉素C(40μg/ml)没有表现出毒性，在1mg/ml浓度下，对精原细胞无作用。对小鼠静脉注射(2mg/kg)没有发生死亡现象，证明毒性较低。抑制k562白血病细胞增值小于0.1μg/ml，对Hela S3细胞的抑制浓度为1μg/ml。
本发明的优点和积极效果是，目前发现的免疫抑制剂中，康乐霉素C的分子量最小，化学结构全新，提取工艺简单，化学合成及结构改造较方便，免疫抑制作用强，毒性低，因而具有应用推广价值。
图1为康乐霉素C的紫外吸收光谱。
图2为康乐霉素C的红外吸收光谱。
实施例：用一株奴卡氏菌进行培养发酵，然后用有机溶剂萃取、柱层析分离和纯化，其条件为：
(1)菌种培养基(％)：KNO3 0.1、NaCl 0.5、K2HPO4 0.05、FeSO4 0.01、淀粉2、琼脂1.7、冷开水、PH7.0、28℃培养5～7天；
(2)种子培养基(％)：葡萄糖2.5、酵母粉0.5、KCl 0.25、黄豆饼粉0.5、K2HPO4 0.02、(NH4)2SO4 0.5、MgSO4 0.02、CaCO3 0.5、自来水100ml、自然PH；
(3)发酵培养基(％)：葡萄糖3、酵母粉1、黄豆饼粉0.5、蛋白胨0.2、CaCO3 0.1、自来水100ml、自然pH。
以上培养基均15磅消毒30分种备用。将斜面挖块接种于种子培养基摇瓶中(50ml装量/250ml)，置180～200转/(分钟)旋转摇床，28℃培养48小时后，以10％接种量转入发酵培养基摇瓶(50ml装量/500ml)，在180～200转/(分钟)旋转摇床28℃培养72小时供提取用。将发酵液过滤后，用无机酸调pH值到4，再用乙酸丁酯萃取，萃取液浓缩后，在硅胶柱上分离，用苯和甲醇混合液(30∶1)洗脱，收集活性部分并浓缩，在LH-20柱上纯化用乙醇洗脱，收集活性部分并浓缩，用乙醇重结晶，即得纯品。康乐霉素C为黄色棒状结晶，在170℃分解变成黑色，分子量为326，旋光度为+150°(10-4)，紫外光谱中在232纳米，356纳米有最大吸收，红外光谱中在3400cm-1、1690cm-1、1650cm-1、750cm-1等处有吸收。溶于极性有机溶剂，不溶于水和非极性有机溶剂。

Claims (2)

1、一种康乐霉素C的生产方法，其特征在于用一株地中海奴卡氏菌康乐变种(Nocardiamediterranei var.kanglensis)CGMCC 0163(1747-64)进行斜面培养、种子培养和发酵培养，然后从培养液中分离和纯化康乐霉素C，其中所说到的康乐霉素C为黄色棒状结晶，在170℃分解变成黑色，分子量为326，旋光度为+150°(10-4)，紫外光谱中在232纳米，356纳米有最大吸收，溶于极性有机溶剂，不溶于水和非极性有机溶剂：其中所说的斜面培养是在KNO3 0.1％，NaCl 0.5％，K2HPO4 0.05％，FeSO4 0.01％，淀粉2％，琼脂1.7％，冷开水，pH7.0中进行的；其中所说的种子培养是在葡萄糖2-5％，酵母粉0.1-1.5％，KCl 0.1-0.5％，黄豆饼粉0.1-2.5％，K2HPO4 0.01-0.5％，(NH4)2SO4 0.1-2％，MgSO4 0.01-0.1％，CaCO30.1-1％，自来水100ml，自然pH值中进行的；其中所说的发酵培养是在葡萄糖2-5％，酵母粉0.5-2％，黄豆饼粉0.1-1.5％，蛋白胨0.05-0.5％，CaCO3 0.01-1.0％，自来水100ml，自然pH中进行的。
2、按权利要求1的康乐霉素C的生产方法，其特征在于其中所说的分离和纯化是在发酵液过滤后，用无机酸调pH值1-5，再用乙酸丁酯萃取，萃取液浓缩后，在硅胶柱上分离，用苯和甲醇混合液洗脱，收集活性部分并浓缩，用乙醇重结晶。
CN91101127A 1991-03-01 1991-03-01 康乐霉素c的生产方法 Expired - Fee Related CN1037526C (zh)

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Application Number Priority Date Filing Date Title
CN91101127A CN1037526C (zh) 1991-03-01 1991-03-01 康乐霉素c的生产方法
CA002061642A CA2061642A1 (en) 1991-03-01 1992-02-21 Physiologically active kanglemycin c, process for preparing the same and use thereof
US07/839,614 US5275817A (en) 1991-03-01 1992-02-21 Physiologically active kanglemycin C
AU11198/92A AU1119892A (en) 1991-03-01 1992-02-24 Physiologically active kanglemycin c, process for preparing the same and use thereof
JP4037966A JPH0570470A (ja) 1991-03-01 1992-02-25 生理活性物質カンレマイシンｃ、その製造法及びその薬学的用途
EP92103139A EP0501399A1 (en) 1991-03-01 1992-02-25 Physiologically active kanglemycin C, process for preparing the same and use thereof
KR1019920002846A KR920018226A (ko) 1991-03-01 1992-02-25 생리활성 캉글레마이신 c, 그 제조방법 및 용도
HU9200677A HUT63883A (en) 1991-03-01 1992-02-28 Physiologically active kanglemycin c, process for producing and using same
IL101103A IL101103A0 (en) 1991-03-01 1992-02-28 Physiologically active kanglemycin c,process for preparing the same and use thereof
CS92594A CS59492A3 (en) 1991-03-01 1992-02-28 Canglemycin c, process of its preparation, its use, a pharmaceutical and astrain for said canglemycin c preparation

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CN91101127A CN1037526C (zh) 1991-03-01 1991-03-01 康乐霉素c的生产方法

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