CN105503843A - 一种水飞蓟素的制备方法 - Google Patents
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Abstract
本发明涉及一种从水飞蓟籽种皮中提取高纯度水飞蓟素的制备方法。该制备方法包括水飞蓟籽脱出种皮，紫外线照射，酶解，无水乙醇提取，挥去提取液中的溶剂后水洗固体得水飞蓟素粗品；无水乙醇溶解，得上样液，通过反相分配色谱，用水-乙醇水梯度洗脱，收集无水乙醇部分洗脱液，浓缩洗脱液，微波真空干燥，得产品。本发明方法工艺简单、生产效率高、操作简便，产品纯度高达93%以上，且基本无溶剂残留。
Description
技术领域
本发明涉及水飞蓟素的制备方法，更具体地，涉及一种从水飞蓟种子中提取高纯度水飞蓟素的制备方法。
背景技术
水飞蓟素(silmyarin)是一类由二氢黄酮醇与苯丙素衍生物缩合而成的黄酮木脂素类成分的混合物，主要来源是菊科植物水飞蓟(silybummarinaum(L)Gaenrt)的种子(又称水飞蓟籽)，主要成分是水飞蓟宾(silybin，silibinin)，异水飞蓟宾(isosilybin，isosilibinin)，水飞蓟亭(siliehristin)，水飞蓟宁(silidinain)四种同分异构体，呈黄色粉末或结晶状粉末。
研究发现，水飞蓟素主要具有保肝利胆的作用，可保护肝脏细胞免受毒性物质侵害，尤其是酒精及环境污染物(农药、重金属等)入侵损害肝脏，临床用于治疗肝炎、胆囊炎等；还具有强力的抗氧化、消除自由基以及抗辐射作用，能保护肝脏细胞免受自由基破坏，促进蛋白质的合成，并且在降血脂、防治动脉粥样硬化、预防脑缺血、抗血小板凝集方面均具有较好效果，被认为是潜在的防治心脑血管疾病的良好药物。因此，提出一种能有效富集水飞蓟素的制备方法是非常有必要的。
我国虽然是水飞蓟种植大国，但目前对水飞蓟素的生产制备的研究仍不够深入，由于水飞蓟素易溶于丙酮、乙酸乙酯、乙醇及甲醇、难溶于氯仿、不溶于水的特性，以及水飞蓟种子自身含有大量油性成分的原因，在制备过程中往往会用到大量有机溶剂，造成产品的溶剂残留现象，或者经碱法处理，影响了水飞蓟素的得率，要经复杂的纯化精制步骤，或者为了追求高纯度而选择了繁杂的提取精制步骤。
例如，现有技术曾提出过一种水飞蓟素的生产方法，采用蒸汽软化水飞蓟壳、乙醇提取、碱水沉淀、分离沉淀干燥即得。虽然该方法工艺简单，但由于碱性试剂会对水飞蓟素有所破坏，因而该方法制得的产品中水飞蓟素含量仅达到70％。现有技术也有提出用生物技术手段来处理水飞蓟籽的方法，将里氏木霉和康宁木霉的复合微生物发酵剂接种在水飞蓟去油种子饼粉中，保温发酵后经热风干燥，无水乙醇提取后加甲醇溶解得不溶物；或者使用纤维素酶、半纤维素酶和果胶酶复合酶进行细胞破壁、加以超声波提取，最后以大孔吸附树脂的技术进行纯化,得到了高含量的水飞蓟素。国外专利对水飞蓟原料或采用榨油方式或采用石油醚、正己烷除油，既影响水飞蓟素产品的色泽和收率又容易产生溶剂残留，要使终产品符合溶剂残留小于100ppm的标准需要非常复杂的除杂工艺。
针对现有技术中制备水飞蓟素的方法存在的上述缺陷和不足，本发明的目的即提供一种工艺简单、生产效率高、操作简便的水飞蓟素制备方法。
发明内容
本发明提供了一种从水飞蓟籽种皮中提取高纯度水飞蓟素的制备方法，包括以下步骤：
⑴取水飞蓟籽，经脱壳机脱出种皮，收集种皮，紫外线照射10-20分钟；
⑵将处理后的水飞蓟籽种皮装入酶解反应罐，加入原料重量0.5-2.0％的纤维素酶：果胶酶：碱性蛋白酶(5:4:1)复合酶，酶解30-50分钟；
⑶将酶处理后的原料装入平转浸出器，用无水乙醇提取，得提取液；
⑷挥去提取液中的溶剂，得固体，用蒸馏水水洗，得水飞蓟素粗品；
⑸用无水乙醇溶解粗品，得上样液，通过反相分配色谱，用水、25％乙醇水溶液、50％乙醇水溶液、75％乙醇水溶液和无水乙醇作为流动相依次洗脱，收集无水乙醇部分洗脱液；
⑹浓缩收集的洗脱液，微波真空干燥，得产品。
上述制备方法中，所述步骤(2)的操作具体为：将处理后的水飞蓟籽种皮装入酶解反应罐，蒸汽软化10-20分钟后，将温度降至20-40℃，加入原料重量0.5-2％的纤维素酶：果胶酶：碱性蛋白酶(5:4:1)复合酶，运转搅拌器，酶解30-50分钟。
上述制备方法中，所述步骤(3)的操作具体为：将酶处理后的原料装入所述平转浸出器的物料格，装料量为所述物料格的70-80％，提取温度为20-40℃，转速为1r/60-120min，连续喷淋，提取3-5小时。
上述制备方法中，所述步骤(4)的操作具体为：真空干燥挥去溶剂，用5-8倍水水洗，得水飞蓟素粗品。
上述制备方法中，所述步骤(5)的操作具体为：用无水乙醇溶解粗品，得上样液，通过反相分配色谱，色谱柱填料为硅藻土，用水、25％乙醇水溶液、50％乙醇水溶液、75％乙醇水溶液和无水乙醇作为流动相依次洗脱，其中75％乙醇水溶液和无水乙醇中分别加入2-5％PEG400，收集无水乙醇部分洗脱液。
上述制备方法中，所述步骤(6)的操作具体为：微波真空干燥温度：20-40℃，回差温度2-4℃，真空度-0.07Mpa以上，微波功率40-80KW，干燥40-80分钟。
本发明方法制得的水飞蓟素产品纯度为93％以上，且基本无溶剂残留。
本发明还提供了上述制备方法制得的水飞蓟提取物。
本发明的制备方法具有以下优点：
1、本发明选择水飞蓟籽的种皮作为原料，避免水飞蓟籽的油脂成分对水飞蓟素的影响，使整个制备方法更为简化，利于工业大生产。
2、由于选择了酶解的方式，因而在酶解前选择了紫外线灭去植物种皮自身含有的酶成分，进而选择合适的复合酶进行种皮细胞的破壁，非常利于后续的溶剂提取，极大地提高了提取率。
3、采用平转浸出器作为提取设备，降低溶剂使用量和损耗，增大投料量，提高了生产效率，减少了生产时间。
4、本发明的精制方法在水洗的基础上通过反相分配色谱，选择硅藻土作为柱填料，选择含2-5％PEG400的无水乙醇作为目标成分的流动相，PEG400能极大地增强水飞蓟素在无水乙醇中的溶解度，通过梯度洗脱的方式除去多余杂质，得到产品纯度在93％以上的水飞蓟素产品，基本无溶剂残留。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚，下面将本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例是本发明一部分实施例，而不是全部可能性。本领域普通技术人员在没有做出创造性劳动下所得到的所有其他实施例，都落入本发明的保护范围内。
实施例1
10吨水飞蓟种子经脱壳机脱出种皮，收集种皮，得种皮4.5吨，紫外线照射15分钟后将种皮装入酶解反应罐，经100℃蒸汽软化20分钟，出料水分8％左右，将温度降至25℃，加入原料重量1.5％的纤维素酶：果胶酶：碱性蛋白酶(5:4:1)复合酶，运转搅拌器，酶解40分钟。将酶解后的原料整体移入平转浸出器的物料格，装料量为所述物料格的70-80％，提取温度为25℃，转速为1r/60-120min，用无水乙醇连续喷淋，提取3小时，得提取液；提取液浓缩至10m3后，真空干燥挥去溶剂，用8倍量水水洗，得水飞蓟素粗品。用无水乙醇溶解粗品，得上样液，通过反相分配色谱，色谱柱填料为硅藻土，用水、25％乙醇水溶液、50％乙醇水溶液、75％乙醇水溶液和无水乙醇作为流动相依次洗脱，其中75％乙醇水溶液和无水乙醇中分别加入5％PEG400，收集无水乙醇部分洗脱液。浓缩收集的洗脱液，微波真空干燥温度20-40℃，回差温度2-4℃，真空度-0.07Mpa以上，微波功率40-80KW，干燥40-80分钟，得水飞蓟素0.47吨，木脂素含量94.3％。
实施例2
25吨水飞蓟种子经脱壳机脱出种皮，收集种皮，得种皮9.5吨，紫外线照射20分钟后将种皮装入酶解反应罐，经110℃蒸汽软化15分钟，出料水分9％左右，将温度降至30℃，加入原料重量2.0％的纤维素酶：果胶酶：碱性蛋白酶(5:4:1)复合酶，运转搅拌器，酶解30分钟。将酶解后的原料整体移入平转浸出器的物料格，装料量为所述物料格的70-80％，提取温度为30℃，转速为1r/60-120min，用无水乙醇连续喷淋，提取5小时，得提取液；提取液浓缩至10m3后，真空干燥挥去溶剂，用6倍水水洗，得水飞蓟素粗品。用无水乙醇溶解粗品，得上样液，通过反相分配色谱，色谱柱填料为硅藻土，用水、25％乙醇水溶液、50％乙醇水溶液、75％乙醇水溶液和无水乙醇作为流动相依次洗脱，其中75％乙醇水溶液和无水乙醇中分别加入5％PEG400，收集无水乙醇部分洗脱液。浓缩收集的洗脱液，微波真空干燥温度20-40℃，回差温度2-4℃，真空度-0.07Mpa以上，微波功率40-80KW，干燥40-80分钟，得水飞蓟素1.3吨，木脂素含量93.5％。
本申请的发明人进行了大量试验，下文提供的实验结果用于表明本发明的制备方法的显著的有益效果。其中，产品中水飞蓟素的测定方法是本领域公知的方法。
实验材料：水飞蓟籽、无水乙醇、PEG400、纤维素酶、半纤维素酶、碱性蛋白酶、中性蛋白酶、果胶酶、淀粉酶、脂肪酶、硅胶、硅藻土、蒸馏水
本发明方法：按照实施例1的方法，原料取100g水飞蓟籽。
对照方法1：同本发明方法，除复合酶选择纤维素酶：半纤维素酶：果胶酶(5:3:2)。
对照方法2：同本发明方法，除复合酶选择纤维素酶：半纤维素酶：果胶酶(5:3:2)。
对照方法3：同本发明方法，除复合酶选择纤维素酶：果胶酶：中性蛋白酶(5:4:1)。
对照方法4：同本发明方法，除色谱柱填料为硅胶。
对照方法5：同本发明方法，用碱水沉淀代替反相色谱法。
实验结果见下表。
| 方法 | 木脂素含量(％) |
| 本发明实施例1的方法 | 94.3％ |
| 对照方法1 | 80.9％ |
| 对照方法2 | 81.3％ |
| 对照方法3 | 88.2％ |
| 对照方法4 | 85.6％ |
| 对照方法5 | 70.5％ |
由此可见，本发明的方法获得了极其突出的有益效果，产品纯度高，方法简单易行，利于工业化大生产。
Claims (7)
1.一种从水飞蓟籽种皮中提取高纯度水飞蓟素的制备方法，包括以下步骤：
取水飞蓟籽，经脱壳机脱出种皮，收集种皮，紫外线照射10-20分钟；
将处理后的水飞蓟籽种皮装入酶解反应罐，加入原料重量0.5-2.0%的纤维素酶：果胶酶：碱性蛋白酶（5:4:1）复合酶，酶解30-50分钟；
将酶处理后的原料装入平转浸出器，用无水乙醇提取，得提取液；
挥去提取液中的溶剂，得固体，用蒸馏水水洗，得水飞蓟素粗品；
用无水乙醇溶解粗品，得上样液，通过反相分配色谱，用水、25%乙醇水溶液、50%乙醇水溶液、75%乙醇水溶液和无水乙醇作为流动相依次洗脱，收集无水乙醇部分洗脱液；
浓缩收集的洗脱液，微波真空干燥，得产品。
2.如权利要求1所述的制备方法，所述步骤（2）的操作具体为：将处理后的水飞蓟籽种皮装入酶解反应罐，蒸汽软化10-20分钟后，将温度降至20-40℃，加入原料重量0.5-2%的纤维素酶：果胶酶：碱性蛋白酶（5:4:1）复合酶，运转搅拌器，酶解30-50分钟。
3.如权利要求1所述的制备方法，所述步骤（3）的操作具体为：将酶处理后的原料装入所述平转浸出器的物料格，装料量为所述物料格的70-80%，提取温度为20-40℃，转速为1r/60-120min，连续喷淋，提取3-5小时。
4.如权利要求1所述的制备方法，所述步骤（4）的操作具体为：真空干燥挥去溶剂，用5-8倍水水洗，得水飞蓟素粗品。
5.如权利要求1所述的制备方法，所述步骤（5）的操作具体为：用无水乙醇溶解粗品，得上样液，通过反相分配色谱，色谱柱填料为硅藻土，用水、25%乙醇水溶液、50%乙醇水溶液、75%乙醇水溶液和无水乙醇作为流动相依次洗脱，其中75%乙醇水溶液和无水乙醇中分别加入2-5%PEG400，收集无水乙醇部分洗脱液。
6.如权利要求1所述的制备方法，所述步骤（6）的操作具体为：微波真空干燥温度：20-40℃，回差温度2-4℃，真空度-0.07Mpa以上，微波功率40-80KW，干燥40-80分钟。
7.上述任一项权利要求的方法制得的水飞蓟素。
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