CN1339440A - 一种超临界萃取虫草脱氧核苷的生产方法 - Google Patents
一种超临界萃取虫草脱氧核苷的生产方法 Download PDFInfo
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Abstract
本发明属于食用药用真菌的深加工领域,是一种超临界萃取虫草脱氧核苷的生产方法,克服了水煮、醇提、浓缩等传统工艺耗能大、时间长、烦琐的缺点,能迅速获得天然活性成份,不污染环境,而且能综合利用原料,成为食用药用真菌深加工的重要工艺,达到国际领先水平。本发明的特征是:采用乙醇作夹带剂,选择条件获得以虫草素(3’一脱氧腺苷)为主的活性成分,其纯度在50—99.9%之间,是保健食品、化妆品或药品的原料,本品有抗氧化、抗病毒、抗肿瘤,调节机体免疫力作用,也是基因工程领域的重要试剂,还可能与肾功能衰竭有关,因此本发明可以创造巨额经济效益,有广阔的开发价值。
Description
本发明属于食用药用真菌的深加工领域。
虫草属真菌大多是珍贵的食用药用真菌，近年来人工培养技术的进步使稀少的野生真菌有了经济开发的价值。虫草属中的模式种一蛹虫草可以进行大量培育，其人工成品的有效成分及药理药性，能与天然产品媲美，也具有补肺益肾，止咳化痰的作用。所以，虫草属中的许多人工培养产品有珍贵的食用药用价值。专利申请号92101051.6介绍了它们的前景和价值。其中采摘子座后的培养基，一般都被废弃，因此，以此为原料提取有效成分，变废为宝有重要的价值。
在中药提取过程时，多采取水煮、醇提、蒸煮浓缩技术，能耗大，操作烦琐，还有许多废物造成环境污染；所以，采用现代化技术提取天然活性成分有巨大的经济价值和社会价值。而超临界二氧化碳萃取技术就具备低温惰性环境，不影响有效成分活性，无毒不易燃，使用安全，不会污染环境，无溶剂残留，无重金属离子。因此，采用超临界技术提取虫草培养物中的活性成分，创造一种新工艺、新方法有实用价值。
本发明的目的是创造一种以虫草、蛹草、拟青霉、亚香棒虫草等虫草属菌株的人工培养物为原料，提取脱氧核苷类活性成分的方法，从而获得脱氧核苷单体或组份，使单体符合一类新药原药申报要求，使组份符合二类新药申报要求。
本发明的技术特征是：一种超临界萃取虫草脱氧核苷的生产方法，其特征是：以食用药用真菌虫草或蛹虫草人工培养后的发酵物为原料，采用超临界技术萃取脱氧核苷类有效成分，并以乙醇等作为夹带剂减压分离获得脱氧核苷多组份及单体，经过传统工艺进而生产出相应的胶囊、针剂等不同剂型的产品。
下面叙述本发明的技术过程：
本发明制造的虫草脱氧核苷类有效成分，其特征是：采用虫草或蛹虫草人工培养后的固体或半固体为原料，(包括超临界萃取虫草油后剩余原料)，其含水量在1-60％，最佳采用固体原料，含水量为6-10％。本发明所述原料，是指虫草及虫草属中的菌株经人工培养后的产物或采摘子座后的菌渣。本发明权利中不对菌株菌种进行限定，主要是指含有虫草素的固体或半固体的原料，如：固体发酵的培养基，半固体发酵的玉米粥培养基及液体发酵后的干燥菌粉。这些产物在含水量6-10％时，易于保存运输。例如：虫草发酵菌粉、蛹虫草菌粉、蛹草米饭培养基、拟青霉发酵菌粉及米饭培养基、亚香棒发酵菌粉等。当采用上述原料用超临界技术萃取油状物后，并不影响脱氧核苷物的提取，特别指出的是，经过萃取虫草油后的剩余原料，其含水量在1-5％左右，比通常市售的大米含水量还要低许多，不影响脱氧核苷的提取。
本发明生产的脱氧核苷的方法，其特征是：固体原料的粒度在20-60目之间，其虫草素的含量≥0.5mg/g，最佳在0.8mg以上/g。众所周知，虫草属中有350种之多，例如：虫草(Cordyceps Sinensis)、蛹虫草(Cordyceps militaris)、亚香棒虫草(Cordyceps Hawkesii)、蛹草拟青霉(Paecitmyces militarae)等。这些菌株经人工培养接种在米饭培养基、玉米、高梁、小麦、小米、蚕蛹、柞蚕蛹、蝙蝙蛾幼虫、蛋清等五十种以上的培养基上都能成功，虫草较难形成子实体，蛹虫草较易形成子实体。这样可把它们划为二类；一为子实体；二为发酵物(培养基，发酵菌粉)。前者可直接成为商品，上市销售；后者，如虫草发酵菌粉，蛹虫草菌粉等可作保健食品或药品，其中生产量较大的蛹草米饭培养基(菌基、菌渣)，大多没有利用。所以，上述菌株的无性发酵菌粉及人工有性生长采摘子座后的菌渣成为本发明的主要原料，以含有虫草素进行界定。
在实施技术过程中，真菌培养物要粉碎后加入萃取釜中，以20-60目为宜，太细易堵塞，太粗萃取时间长，易残留，得率低。
本发明技术特征在于采用的虫草及虫草类的人工培养物，其虫草素含量≥0.5mg/g，最佳在0.8mg/g以上，1.8mg/g以下。因为虫草素(3’-脱氧腺苷)是脱氧核苷类的主要成分，是其标志成分、活性成分。天然虫草及蛹草均低于0.05-0.08％的水平，人工培养时虫草素含量较高。目前，有报道蛹草菌粉中含量高达1.0-5.0％，或更高，所以，用虫草素限定原料有利于生产实施，有利于降低成本，符合本工艺的过程。
超临界二氧化碳萃取是指在高压条件下使二氧化碳成为液体，作为溶剂，提取中药材的成分在釜中溶出。在二氧化碳流动过程中，分离釜中的压力、温度不同使所需成分析出，结合I、II、III分离釜或分馏塔可使某一成分析出。在减压下二氧化碳挥发即可得到本发明所述脱氧核苷组份或单体，对混合组分(粗萃取物)可在釜中重新提取一次，即以50％或75％的结晶，再萃取一次；还可用通常的结晶分离法获得单一成分。本发明的方法，其特征是：采用超临界技术萃取脱氧核苷类有效成分，萃取釜压力15-50Mpa；分离釜最佳压力为5-25Mpa，但同一流程中分离釜压力始终小于萃取釜压力。一般说来，在萃取釜中采用二氧化碳流量为15-25升/h，萃取釜取温度在50-60℃左右。分离釜压力、温度根据所需条件改变。实验证明萃取温度高达100℃仍未明显破坏虫草等结构。图1所示是本发明制得的脱氧核苷组份，虫草含量在54.65％的高压液相色谱图。
图2所示是本发明制得的脱氧核苷组份，虫草素含量在75.18％时的高压液相色谱图。高峰洗出时间在6.10-6.30分，二峰在4.20-4.60分，占6.49％左右；三峰在10.48分，占4.28％；四峰在7.78分，占4.23％左右。图3是图2所示成份的紫外谱特征，217nm为4.027；在245nm有一波谷；吸收峰在259nm。其吸光度值一半时的波长在284nm左右。采用10％乙醇作夹带剂，所获成份液相色谱如图4所示，主峰只有一个，洗出时间6.11分，占75.05％，其紫外特征如图5所示，219nm为4.074，241nm波谷吸光度值为1.68；259nm峰值为2.045；峰值吸光度一半时的波长在280nm左右。图2、3与图4、5相比，虽然主要成份相近均在75％左右，由于超临界条件不同，所得副成份会有一些差异；液相分析在组成上发生变化，而紫外光谱的峰高半宽度略有推移。上述特征，构成了本技术产品的指纹特征，有别于其它方法生产的单组份或多组份。当纯度在95％左右时，主峰只有一个，其余4个吸收峰的总值在5％左右。当纯度大于98％时的标准物，其紫外特征是：波谷在225-227nm，峰值在259-260nm；峰高半值波长在278nm左右。用超临度技术获得的虫草素单体，其液相色谱只有2-3个峰，主峰＞98.6％，二峰、三峰之和为1.4％。不同液相色谱仪及洗脱条件不同时，所得主峰位置不同(洗出时间)，可用两种样品合并及分别进样扫图，以示区别。本技术单体产品的紫外特征是：波谷在227nm；峰值在259nm，半高度在274nm。因为大于98％接近于纯物质，单体纯度＞99.0％时，液相色谱可以显示一个主峰标示为100％，付成份只显示波形，而无分值；与传统产品比较二者有许多相近之处，但波形不同；必要时用红外光谱法加以区分。
本发明方法，其特征是：乙醇作夹带剂，乙醇浓度在5-99％，直至采用市售的无水乙醇作夹带剂。最佳浓度是10-30％的乙醇。乙醇、丙烷和水都可以作为夹带剂，丙烷有很好发展前景，而水作夹带剂，所费时间较长。由于是溶剂分配原则分离的，所以，提取的得率尚待提高。一般来说，初步实验得率为10-20％，改善实验条件(时间、流量、温度、压力和分馏塔载体)可以提高产品得率，从而达到30-50％以上，或更高。其原理在本专利权利范围内组合即成。如采用延长分离时间、加大压差、连续多次萃取或采用分馏塔等。
使用二氧化碳萃取，结构相似成分会在分离釜中析出，可以是多组份的混合物，但主要成分虫草素采用本方案经一次分离可达50％以上，即能符合二类药品生产要求，经常规处理就能进行新药实验研究，为产品正式上市奠定了工业化生产的基础。
在本发明实施过程中获得的脱氧核苷组份，仍溶在水中，乙醇混合液中或丙烷液体中，尚需采用活性碳吸附、除杂，有机溶剂去杂、重结晶或离子树脂处理去杂、冷冻干燥或真空干燥得到粉状原料。从而成为含虫草素为50-99.9％的粉状原料，再加以辅剂，生产成片剂或胶囊，每粒含3’-脱氧腺苷5-200mg，最佳在25-100mg之间。
以超临界技术生产的脱氧核苷粉状物，加工制成冻干针剂，每支虫草素含量12.5mg-200mg，最佳在25-100mg/支。
上述组份具有抗肿瘤，抗病毒和免疫调节作用，也是基因工程领域的重要试剂，还可能肾功能衰竭有关，成为肾衰治疗辅助剂，因此本发明有广阔应用前景。
用本发明技术生产虫草类脱氧核苷，具有以下优点：①综合使用了人工培养的虫草类原料；②提高工业价值；③超临界技术无污染，易实行；④不损害活性天然成分的结构；⑤有显著的应用价值。
下面举例说明本技术过程：
例一，称取蛹虫草米饭培养基0.75公斤，粉碎过20-60目筛，虫草素含量1.4mg/g，装入萃取釜中50℃，35Mpa，20L/h，平衡15分钟后流动萃取。分离釜压力6Mpa，30℃，在2小时后采样得水状物约40ml(下层)，上层为油状物。分三次加入10％乙醇各200ml作夹带剂，每次萃取1-2小时，萃取液540ml左右，合并加活性炭除杂，真空浓缩，得淡黄色颗料状物235mg，虫草素含量75.05％。得率约16.79％。成品符合二类新药申报要求。如欲提高得率可以重复使用夹带剂提取。
上述采用2L萃取釜获得，可重复加样萃取或采用中试96升设备，能获得更多结晶，采用常规方法(范碧亭主编：中药药剂学，上海科技出版社1997年一版，348页，401页)，生产含量在25mg/片.粒的普通片剂或胶囊。
实例二，称取蛹虫草固体发酵菌粉0.75公斤，粉碎过筛(虫草素含量＞1.0mg/g)，装入萃取釜中60℃，30Mpa，15L/h，平衡后流动萃取，首先获得虫草油与水的混合物，分液漏斗去油，再用夹带剂70％乙醇萃取，每次加入200ml或用连续加入夹带剂的方法提取。分离釜II条件为35℃，5Mpa，20L/h，得淡黄色液体600ml，按常规方法除杂、浓缩、结晶，得淡黄色颗粒状物245mg，用有机试剂除杂，得白色结晶160mg其纯度为98.6％。
实例三
采用本法制得的虫草素单体500mg，溶于50ml重蒸馏水中，经玻璃滤器G6过滤，装入无菌青霉素小瓶内，每瓶2.5ml，在无菌室内的冻干机上经24小时处理，成白色海绵状物体，加盖密封，即成虫草素冻干针剂25mg/支。
参考文献：
陈顺志等：虫草素的红外与紫外光谱特征，中国生化药物杂志，1996年第期第37页。
Claims (8)
1、一种超临界萃取虫草氧核苷的生产方法，其特征是：以食用药用真菌虫草或蛹虫草人工培养后的发酵物为原料，采用超临界技术萃取脱氧核苷类有效成分，并以乙醇等作为夹带剂减压分离获得脱氧核苷多组份及单体，经过传统工艺进而生产出相应的胶囊，针剂等不同剂型的产品。
2、根据权利要求1所述的方法，其特征是：采用虫草或蛹虫草人工培养后的固体或半固为原料，(包括超临界萃取虫草油后的剩余原料)，其含水量在1-60％，最佳采用固体原料，含水量为6-10％。
3、根据权利要求1所述的方法，其特征是：固体原料粒度为20-60目，其虫草素的含量≥0.5mg/g；最佳0.8mg/g以上，18mg/g以下。
4、根据权利要求1所述的方法，其特征是：采用超临界技术萃取脱氧苷类有效成分，萃取釜压力15-50Mpa；分离釜，最佳压力为5-25Mpa，但同一流程中分离釜压力始终小于萃取釜压力。
5、根据权利要求1所述的方法，其特征是：乙醇作夹带剂，乙醇浓度在5-99％，最佳乙醇浓度为10％以上直到采用市装的无水醇作夹带剂。
6、根据权利要求1所述的方法，其特征是：本发明分离获得成份相近的几种脱氧核苷，如：3’-脱氧腺苷、3’-氨基-3’-脱氧腺苷，成为多组份物，但主要成份虫草素(3-脱氧腺苷)的比例在30-99.9％之间，最佳50-99.9％之间。
7、根据权利要求1所述的方法，其特征是：采用超临界技术制备的脱氧核苷类粉状原料，加工成片剂或胶囊，每粒含3’-脱氧腺苷5-200mg，最佳在25-100mg之间。
8、根据权利要求1所述的方法，其特征是：以超临界技术制备的脱氧核苷类粉状原料，加工成冻干针剂，每支含量12.5-200mg，最佳在25-100mg/支。
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005030231A1 (fr) * | 2003-09-28 | 2005-04-07 | Shaosheng Sun | Extraction de l'aweto, fabrication et utilisation |
| CN101124988B (zh) * | 2007-09-25 | 2010-10-06 | 江苏瑞迪生科技有限公司 | 从蛹虫草中提取精制虫草素和虫草多糖的方法 |
| CN101406491B (zh) * | 2008-11-26 | 2011-04-06 | 吉林大学 | 一种水包油型虫草乳剂及其制备工艺 |
| CN102349934A (zh) * | 2011-10-24 | 2012-02-15 | 周伯扬 | 一种虫草菌丝体有效成分的提取工艺 |
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| CN1086732A (zh) * | 1993-07-03 | 1994-05-18 | 李春燕 | 北冬虫夏草提取液及其制备方法 |
| US5582828A (en) * | 1995-03-15 | 1996-12-10 | Ching-Yuang Lin | Active fractions of Cordyceps sinensis and method of isolation thereof |
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2001
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005030231A1 (fr) * | 2003-09-28 | 2005-04-07 | Shaosheng Sun | Extraction de l'aweto, fabrication et utilisation |
| CN101124988B (zh) * | 2007-09-25 | 2010-10-06 | 江苏瑞迪生科技有限公司 | 从蛹虫草中提取精制虫草素和虫草多糖的方法 |
| CN101406491B (zh) * | 2008-11-26 | 2011-04-06 | 吉林大学 | 一种水包油型虫草乳剂及其制备工艺 |
| CN102349934A (zh) * | 2011-10-24 | 2012-02-15 | 周伯扬 | 一种虫草菌丝体有效成分的提取工艺 |
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