CN111635447A - 一种从牛胆汁中提取高纯度结合胆汁酸的方法 - Google Patents
一种从牛胆汁中提取高纯度结合胆汁酸的方法 Download PDFInfo
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- C07J9/005—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane containing a carboxylic function directly attached or attached by a chain containing only carbon atoms to the cyclopenta[a]hydrophenanthrene skeleton
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J41/00—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring
- C07J41/0033—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring not covered by C07J41/0005
- C07J41/0055—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring not covered by C07J41/0005 the 17-beta position being substituted by an uninterrupted chain of at least three carbon atoms which may or may not be branched, e.g. cholane or cholestane derivatives, optionally cyclised, e.g. 17-beta-phenyl or 17-beta-furyl derivatives
- C07J41/0061—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring not covered by C07J41/0005 the 17-beta position being substituted by an uninterrupted chain of at least three carbon atoms which may or may not be branched, e.g. cholane or cholestane derivatives, optionally cyclised, e.g. 17-beta-phenyl or 17-beta-furyl derivatives one of the carbon atoms being part of an amide group
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Abstract
本发明公开了一种从牛胆汁中提取高纯度结合胆汁酸的方法。以牛胆汁为原料,利用CTAB能与黏多糖类物质形成沉淀而去除黏多糖类物质,利用Zn2+和Ca2+等金属离子在碱性条件下能与大多数蛋白质反应形成沉淀而去除大部分蛋白质类物质。大部分结合胆汁酸主要以钠盐的形式存在牛胆汁中,在pH为酸性的情况下,转化为结合胆汁酸,因不溶于水而沉淀,收集得到粗胆汁酸类物质。通过层析分离的方法,得到不同种类的高纯度结合胆汁酸。通过本发明的方法获得的结合胆汁酸在产量和纯度方面符合人工合成牛黄的工业化大规模生产要求。
Description
技术领域
本发明属于制药技术领域,尤其涉及利用现代分离纯化技术方法从牛胆汁中获取高纯度结合胆汁酸的方法。
背景技术
牛黄即牛胆结石,胆汁酸是其多种有效成分中的一类,胆汁酸分为结合胆汁酸和游离胆汁酸,其中结合胆汁酸如牛磺胆酸、甘氨胆酸、牛磺去氧胆酸、甘氨去氧胆酸有抗菌、消炎等药理作用,其含量高低是区别优质或劣质牛黄的重要指标之一。
天然牛黄是自然形成的产物,质量难免存在差异,其结合胆汁酸的含量存在较大的差异,而优质的天然牛黄总结合胆汁酸含量3~7%,如优质的澳洲牛黄(结合胆汁酸含量见表1)。目前市售商品化的结合胆汁酸多为化学合成,大多数合成过程中使用的部分有机溶剂刺激性强,易对人员和环境造成不利影响,产生的一些结构相近的杂质很难去除,纯化困难,影响人工合成牛黄的功效。化学合成胆汁酸的种类不够丰富,没有甘氨去氧胆酸,无法保证人工合成牛黄的功效。现有从动物胆汁中层析分离纯化的技术,仅仅获得了牛磺胆酸和甘氨胆酸,且产量低。使用的溶剂有的毒性大,有的不易回收污染环境,不适合工业化大生产。
表1优质澳洲牛黄结合胆汁酸含量统计表
发明内容
为了解决现有技术中存在的上述问题,本发明的目的是提供一种从牛胆汁中提取高纯度的结合胆汁酸且适合大批量工业化生产的方法。采用现代分离纯化技术从牛胆汁中获取四种高纯度结合胆汁酸,解决了现有化学合成法和层析分离纯化法的各种问题,所用溶剂安全性好、易回收、价格低廉,工艺步骤简单,产量高,产品纯度高,种类齐全,适合大批量工业化生产,能有效保证人工合成牛黄的功效。
为了实现上述目的,本发明采用如下技术方案:
第一方面,提供一种从牛胆汁中提取高纯度结合胆汁酸的方法,所述结合胆汁酸为牛磺胆酸、甘氨胆酸、牛磺去氧胆酸、甘氨去氧胆酸,以牛胆汁为原料进行以下处理:
①黏多糖的去除:每升牛胆汁中加入80~120ml 0.02%的CTAB溶液,搅拌4~6min,静置20~40min,过滤去除沉淀,得滤液;
②蛋白质的去除:用氢氧化钠将步骤①的滤液pH调至碱性,向每升滤液中加入15~25ml 0.02%的硫酸锌或氯化锌溶液,15~25ml 0.01%的氯化钙或硝酸钙溶液形成金属盐类复合物,过滤去除沉淀,得滤液;
③结合胆汁酸混合物的获取:用浓盐酸将步骤②中滤液pH调至酸性,搅拌2~5min,静置20~40min,离心收集沉淀物;
④结合胆汁酸的分离:将步骤③收集的沉淀物与硅胶以(5~6):(3~4)的比例直接拌样,干燥上样;硅胶层析柱内径17~25cm,装柱硅胶量4~6kg,高度30~40cm;以乙酸乙酯/甲醇/甲酸为洗脱液进行洗脱,洗脱速度5~8ml/s。
优选的,上述步骤②中,调节步骤①中滤液pH的碱性范围为:10~10.5,所述步骤③中,调节步骤②滤液pH的酸性范围为:1~2。
优选的,上述步骤④中,将步骤③收集的沉淀物与100-200目硅胶以5:4的比例直接拌样,干燥上样;硅胶层析柱内径22cm,装柱硅胶量5kg,装柱100-200目硅胶高度35cm。
优选的,上述步骤④中,拌样硅胶和装柱硅胶目数均为100~200目,单次上样量500~600g,拌样硅胶量300~400g,拌样硅胶高度4~6cm,具体洗脱方式:先后经过乙酸乙酯/甲醇/甲酸体积比=(50~90):(0.7~1.3):(0.7~1.3)洗脱45~65L得甘氨去氧胆酸,(70~130):(1.4~2.6):(0.7~1.3)洗脱30~50L得甘氨胆酸,随后(55~105):(14~26):(0.7~1.3)洗脱14~26L得牛磺去氧胆酸,最后经过(52~98):(18~32):(0.7~1.3)洗脱28~52L得牛磺胆酸。
进一步的,上述步骤④中,单次上样量500g,拌样硅胶量400g,拌样硅胶高度5cm,具体洗脱方式:先后经过乙酸乙酯/甲醇/甲酸体积比=70/1/1洗脱50L得甘氨去氧胆酸,100/2/1洗脱40L得甘氨胆酸,随后80/20/1洗脱20L得牛磺去氧胆酸,最后经过75/25/1洗脱40L得牛磺胆酸。
和现有技术相比,本发明具有以下有益效果:
牛胆汁除水分外,含有胆汁酸、胆色素、黏多糖类、蛋白质类以及无机盐等物质。本发明利用CTAB能与黏多糖类物质形成沉淀而去除黏多糖类物质,利用Zn2+和Ca2+等金属离子在碱性条件下能与大多数蛋白质反应形成沉淀而去除大部分蛋白质类物质。大部分结合胆汁酸主要以钠盐的形式存在牛胆汁中,在pH为酸性的情况下,转化为结合胆汁酸,因不溶于水而沉淀,收集得到粗胆汁酸类物质。通过层析分离的方法,得到不同种类的结合胆汁酸。按以上步骤1L牛胆汁可提取牛磺胆酸14.2~16.3g,甘氨胆酸10.2~13.3g,牛磺去氧胆酸3.5~4.9g,甘氨去氧胆酸2.8~3.9g,平均提取率依次为:78.9%、74.7%、72.1%、69.5%,纯度范围依次为:99.1~99.3%、99.2~99.6%、98.0~98.3%、98.1~98.7%,符合人工合成牛黄的工业化大规模生产要求,并可以保证其疗效。
具体实施方式
通过以下详细说明可以进一步理解本发明的特点和优点。所提供的实施例仅是对本发明方法的说明,而不以任何方式限制本发明揭示的其余内容。
利用液相梯度洗脱方法并结合蒸发光检测器检测制备获得的结合胆汁酸纯度。具体检测方法如下:采用XAqua C18色谱柱(250mm×4.6mm,5μm),以乙腈为流动相A,0.2%三氟乙酸溶液为流动相B,进行梯度洗脱,1~30min:A(%)25→52,B(%)75→48;30~40min:A(%)52,B(%)48;40~44min:A(%)52→98,B(%)48→2;44~57min:A(%)98,B(%)2,流动相流速1.0ml/min,用蒸发光散射检测器检测,雾化温度35℃,漂移管温度80℃,气体流速1.8L/min,供试品和对照品均采用甲醇溶解并超声处理。
【实施例1】
以10L牛胆汁为原料,加入0.9L 0.02%CTAB溶液,搅拌4min,静置25min,过滤去除沉淀,得滤液10.86L,用氢氧化钠将滤液pH调至10,加180ml 0.02%氯化锌溶液和180ml0.01%氯化钙溶液形成金属盐类复合物,过滤得滤液,用浓盐酸调pH至1,搅拌2min、静置20min,离心收集沉淀物510g,取510g沉淀物与340g硅胶(100目)直接拌样,干燥,得拌样硅胶,等分两次上样,每次拌样高度5.8cm,硅胶层析柱内径18cm,装柱硅胶(100目)量4.5kg,高度37cm,以乙酸乙酯/甲醇/甲酸为洗脱液进行洗脱,洗脱速度5~8ml/s,上柱层析。经过乙酸乙酯:甲醇:甲酸=72:1:1洗脱48L得甘氨去氧胆酸,90:1.8:1洗脱35L得甘氨胆酸,随后72:18:1洗脱18L得牛磺去氧胆酸,最后经过66:25:1洗脱30L得牛磺胆酸。统计结果如表2。
表2实施例1中制备结合胆汁酸结果
注:提取率=B/A*100%。
【实施例2】
以20L牛胆汁为原料,加入2.2L 0.02%CTAB溶液,搅拌6min,静置35min,过滤去除沉淀,得滤液22.12L,用氢氧化钠将滤液pH调至10.5,加入460ml 0.02%氯化锌溶液和460ml 0.01%氯化钙溶液形成金属盐类复合物,过滤得滤液,用浓盐酸调pH至2,搅拌5min、静置40min,离心收集沉淀物1030g,取1030g沉淀物与720g硅胶(200目)直接拌样,干燥,得拌样硅胶,等分两次上样,每次拌样高度4.2cm,硅胶层析柱内径24cm,装柱硅胶(200目)量5.5kg,高度34cm,以乙酸乙酯/甲醇/甲酸为洗脱液进行洗脱,洗脱速度5~8ml/s,上柱层析。每次经过乙酸乙酯:甲醇:甲酸=65:1:1洗脱55L得甘氨去氧胆酸,110:2:1洗脱45L得甘氨胆酸,随后80:20:1洗脱24L得牛磺去氧胆酸,最后经过72:25:1洗脱48L得牛磺胆酸。统计结果如表3。
表3实施例2中制备结合胆汁酸结果
【实施例3】
以10L牛胆汁为原料,加入1.1L 0.02%CTAB溶液,搅拌6min,静置40min,过滤去除沉淀,得滤液11.95L,用氢氧化钠将滤液pH调至10.5,加入250ml 0.02%硫酸锌溶液和250ml 0.01%氯化钙溶液形成金属盐类复合物,过滤得滤液,用浓盐酸调pH至2,搅拌5min、静置40min,离心收集沉淀物520g,取520g沉淀物与400g硅胶(200目)直接拌样,干燥,得拌样硅胶,高度5.3cm,硅胶层析柱内径22cm,装柱硅胶量(200目)5.3kg,高度37cm,以乙酸乙酯/甲醇/甲酸为洗脱液进行洗脱,洗脱速度5~8ml/s,上柱层析。经过乙酸乙酯/甲醇/甲酸=72:1.2:1洗脱53L得甘氨去氧胆酸,105:2:1洗脱44L得甘氨胆酸,随后82:22:1洗脱22L得牛磺去氧胆酸,最后经过78:25:1洗脱42L得牛磺胆酸。统计结果如表4。
表4实施例3中制备结合胆汁酸结果
将250L牛胆汁置于发酵罐内,加7.5kg牛磺酸,蒸汽灭菌后,加2.5L含有大肠杆菌的种子液,控制发酵温度36~38℃,罐内压力0.45~0.5bar,pH值6~8,搅拌机转速50r/min,发酵80h,得发酵液250L,加300L饱和氢氧化钙溶液,搅拌20min、煮沸15min、冷却,过滤得沉淀物(干重)12.7kg,向沉淀物中加10kg胆红素,1.5kg胆酸,1kg去氧胆酸,0.15kg硫酸锌和0.15kg硫酸镁置于分散机中,并加100L纯化水,搅拌1.5h,真空冷冻干燥并粉碎,得中间体25.1kg。取2kg中间体,加2L上述发酵牛胆汁,0.8g亚硫酸氢钠,2L纯化水以及54g牛磺胆酸,50g甘氨胆酸,14g牛磺去氧胆酸,14g甘氨去氧胆酸,4g羟丙甲基纤维素(型号SH-K15M),搅拌15min,静置30min,用盐酸调pH至6.2,偏轴定向旋转培育得到大小均一的类球形物,干燥得成品。经检测,除质量指标符合2015年中国药典标准一部第70页中牛黄检测标准外,还利用液相梯度洗脱方法并结合蒸发光检测器检测,牛磺胆酸、甘氨胆酸、牛磺去氧胆酸、甘氨去氧胆酸含量分别为:1.92%、1.62%、0.53%,0.45%,结合胆汁酸总含量为4.52%,与优质天然牛黄基本一致。
Claims (5)
1.一种从牛胆汁中提取高纯度结合胆汁酸的方法,所述结合胆汁酸为牛磺胆酸、甘氨胆酸、牛磺去氧胆酸、甘氨去氧胆酸,其特征在于,以牛胆汁为原料进行以下处理:
②蛋白质的去除:用氢氧化钠将步骤的滤液pH调至碱性,向每升滤液中加入15~25ml 0.02%的硫酸锌或氯化锌溶液,15~25ml 0.01%的氯化钙或硝酸钙溶液形成金属盐类复合物,过滤去除沉淀,得滤液;
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