CN113321661B - 一种高效的从河豚内脏提取和分离制备高纯度河豚毒素的方法 - Google Patents
一种高效的从河豚内脏提取和分离制备高纯度河豚毒素的方法 Download PDFInfo
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- CN113321661B CN113321661B CN202110487094.XA CN202110487094A CN113321661B CN 113321661 B CN113321661 B CN 113321661B CN 202110487094 A CN202110487094 A CN 202110487094A CN 113321661 B CN113321661 B CN 113321661B
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Abstract
本发明公开一种高效的从河豚内脏提取和分离制备高纯度河豚毒素（tetrodotoxin,简称TTX）的方法；其包括以下步骤：将干净的河豚内脏加酸性醇匀浆、加入一定量的助流动剂搅拌均匀后，按柱层析的装柱方法将材料装入层析柱中；用同一酸性醇将河豚内脏中的TTX完全洗脱出来，收集洗脱液为TTX提取液；提取液减压浓缩至水相，得到TTX提取浓缩液；TTX水溶液用离子交换树脂柱层析分离，收集含TTX高的洗脱液，用纳滤膜浓缩或减压蒸干，得到TTX粗品；TTX粗品通过溶剂沉淀和结晶，得到纯度达于98%的TTX；本发明的制备TTX的方法和所用设备简单，TTX提取和分离纯化的得率高、制备成本低，适合TTX的规模化生产；同时该方法使用的有机溶剂可全部回收重复使用，无有害溶剂和废弃物排放，是理想的安全环保的制备TTX方法。
Description
技术领域
本发明涉及一种动物体内生物活性物质的提取和分离纯化技术领域，尤其涉及河豚内脏中河豚素的制备方法。
背景技术
河豚毒素（Tetrodotoxin, TTX）最初是从河豚鱼（pufferfish, Lagocephalus sceleratus）中提取的小分子非蛋白生物碱类强神经毒素。1909年，日本学者Tawara首次从河豚鱼卵巢中提取出粗毒素并命名（Bane et al., 2014）1950年Yokoo首次从红鳍东方豚卵巢中获得纯化的TTX 结晶(Yokoo, 1950)。1964年，TTX的分子式（C11H17N3O8）和化学结构得以确定(Buchwald et al., 1964; Woodward and Gougoutas, 1964; Tsuda and Ikumaet al., 1964)。在很长一段时间里，河豚毒素被认为是河豚鱼体内特有的毒性物质，直到1964年，美国科学家Mosher等从加利福尼亚蝾螈 (Taricha torosa) 中分离得到TTX(Mosher et al., 1964)，才改变了这一说法。迄今的研究表明，TTX不仅存在于多种河豚鱼体内，还广泛存在于近140种动物中，包括脊椎动物和无脊椎动物(Lorentz et al.,1016)，如虾虎鱼(朱国萍等，2015)、蝾螈（Yotsu-Yamashita et al., 2012）、双壳类和腹足类(Laura et al., 2019)、蓝环章鱼(Whitelaw et al., 2019)、纽虫(Vlasenko et al.,2018)、线虫(毛婕，等，2020)等. 而且已经发现多种微生物可产生TTX（Chau et al.，2011；Magarlamov et al. 2017）。
TTX是已知的毒性最强的神经毒素之一，它对人类的毒性是氰化物的1200倍左右，而且没有已知的解毒剂（Lago et al.，2015）。TTX对小鼠的LD50值为10 μg/kg（腹膜内）、16μg/kg（皮下）和332 μg/kg（口服)（Kao，1966）。人体摄入TTX后，中毒症状的严重程度与剂量有关（Homaira et al.，2010）。最初症状包括舌头和嘴唇发麻（感觉异常），然后出现头痛或呕吐，并可能发展为肌肉无力和共济失调。在严重的情况下，可能由于呼吸或心力衰竭而导致死亡（Noguchi and Eebesu，2001）。TTX对人的致死剂量是1.33μg/kg（Kasteel andwesterink，2017）。TTX通过生物链传递和大量海洋与淡水养殖动物中含有TTX等问题，导致人们对环境的安全的忧虑和重视。
TTX是快速电压门控钠通道的阻断剂，能引起神经和肌肉麻醉，在医疗上可用于癌症和外科手术等各种病人的局部麻醉和无成瘾性镇痛等。TTX作为局部麻醉剂（Ogura andMori，1968），它没有明显的心血管副作用（Butterworth，2010；Stoetzer et al.，2016）或肌毒性（Padera et al.， 2006）。与临床上常用的局部麻醉剂不同的是TTX的结合位点位于膜外，它能与其它局部麻醉剂组合使用产生协同效应（Kohane et al.，1998；Berde etal.，2011；McAlvin et al.，2015）。研究人员也正在利用TTX的麻醉特性来治疗各种类型的疼痛，如严重的癌症疼痛（Hagen et al.，2008；2017），或缓解阿片类药物戒断综合征（陈素青，等，2001；Kohane et al.，2003；Shi et al.，2009）等。此外，研究发现在TTX处理的荷瘤小鼠中，肿瘤生长明显减少（El-Dayem et al.,2013）。TTX还能抑制肿瘤细胞的移动性或侵袭性，防止高转移性肿瘤和白血病的转移（Shan et al.，2014； Stock and Schwab，2015）。TTX可望在多个领域获得临床应用。
TTX的结构复杂，化学合成难度大。虽然早在2003年就完成了TTX的全人工合成（Ohyabu et al.，2003）。但人工合成的步骤太多，平均需要23-67个步骤，获得的产率含量仅有0.34%-1.82%（Chau and Cuyfikub，2011；Makarova et al.，2019）。而且在合成过程中，需要开发系列纯化方法。因此，实验室合成TTX及其类似物难以实现大规模生产（Chauand Cuyfikub，2011；Bane et al.，2014；Makarova et al.，2019）。虽然有多种微生物可以产生TTX，但产量非常低（Lago et al.，2015），其产生机制尚不清楚, 提高其产量和开发纯化方法都是待解决的难题（刘燕婷，等，2008）。因此，从动物组织中提取制备TTX，是目前TTX主要来源。
TTX为氨基全氢喹唑啉化合物（pKa 8.76），分子式为C11H17N3O8，分子量319.27，在190-200 nm有较强吸收，含有一个笼形原酸酯结构（Moczydlowski，2013），如图1所示。其分子中几乎所有碳原子都具有不对称取代，以1,2,3-胍氨基及其附近的的C-4、C-9、C-10上的羟基为活性基团。TTX晶体呈白色，无臭，没有确定的熔点，220℃以上逐渐炭化（崔建洲,等，2005），但即使在300℃也不熔化（Tsuda and Kawamura，1952）。纯TTX不溶于水和大多数有机溶剂，如甲醇、乙醇和二甲基亚砜，易溶于稀醋酸溶液，在强酸或强碱溶液中活性易受破坏（Moczydlowski，2013）。TTX在中性溶液中带正电荷，在弱酸性条件下，原酸酯、内酯形式、4-epi TTX和4，9-anhydro TTX共存（Nishikawa and Isobe，2013）。
TTX的结构与性质很独特，很难用常规的天然生物碱类物质的分离纯化方法从生物材料中提取、分离纯化和制备高纯度的TTX（Makarova et al.,2019）。最初，Tawara（1909年）用醋酸铅沉淀法从河豚卵巢的水提取物中分离出TTX，但纯度仅0.2%-4%（Moczydlowski，2013）。尽管进行了许多尝试，直到20世纪50年代初，Yokoo将粗毒素用磷钨酸、苦味酸汞、苦酮酸和苯肼等连续处理后，再经过氧化铝柱色谱和重结晶，才实现了纯晶态TTX的制备（Yokoo，1950）。随后，Tsuda和Kawamura以1000 kg河豚卵巢为原料，用马铃薯淀粉分配层析，活性炭吸附层析，滤纸层析法分离纯化TTX（Tsuda and Kawamura，1952）。
目前，制备TTX的工艺一般包括，以河豚鱼内脏组织为原料，用乙酸水溶液为提取溶剂，再用超声波或/和加热辅助提取。提取物经阳离子交换柱层析-活性炭柱纯化，酸溶碱沉使TTX析出，最后用制备型高效液相色谱（HPLC）纯化和重结晶。主要有关TTX的提取及纯化方法的报道总结如表2。
表2 文献报道的TTX提取及纯化工艺 。
然而，这些提取和分离制备方法存在以下几个方面的问题：
1）提取效率低。TTX很难从组织中提取出来和提取干净，用传统的浸泡提取、或超声波辅助的提取方法提取效率低、提取溶剂用量大。
2）提取溶剂多数用酸性水，导致提取液中含大量的蛋白质等水溶性物质，使后续的TTX分离纯化过程需要除去蛋白质，难以分离纯化。同时分离纯化的步骤多，回收率很低。
3）大部分报道限于实验室研究，实验方法和结果没有经过检验。难以重复和用于TTX的生产。
因此，开发简单高效合的能用于大量制备的TTX提取分离和纯化方法与工艺十分重要。
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发明内容
本发明的目的是，提供一种提取效率高、设备投资低、操作简便的从河豚内脏提取和分离制备高纯度河豚毒素的方法。
为达到以上技术目的，本发明采用的技术方案如下：
一种从河豚内脏中提取和制备高纯度河豚毒素的方法，其包括以下步骤：
（1）将新鲜或解冻的河豚内脏组织切成小块，加入一定量体积的酸性醇在匀浆机中匀浆；
（2）在上述（1）获得的匀浆液中加入一定量的助流动剂或助滤剂，搅拌混合均匀后、按柱层析的装柱方法将提取物装入层析柱中；静置一定时间后，用上述同样的酸性醇按柱层析洗脱的方法将材料中的杂质和TTX先后洗脱出来。用HPLC测定洗脱液中的TTX含量，至洗脱液中无TTX为止。收集含TTX的洗脱液为TTX提取液。
（3）将上述（2）的TTX提取液在真空浓缩仪中减压浓缩至水相，回收乙醇用于重复使用。水相用有机溶剂萃取除去脂溶性物质，得到的水相为TTX提取浓缩液。
（4）上述（3）中得到的TTX提取浓缩液用预处理好的离子交换树脂柱层析分离。样品上样后，先后用中性铵盐和水洗脱杂质，再用酸性醇洗脱TTX。收集含TTX的洗脱液，用纳滤浓缩仪浓缩至小体积，再用真空浓缩仪浓缩干。得到TTX粗品。
（5）上述（4）中得到的TTX粗品，用水和有机溶剂多次反复酸溶碱沉或结晶，得到高纯度的TTX。TTX经HPLC分析，纯度为98%以上。
具体地，步骤（1）中，所述河豚内脏为河豚卵巢或肝脏，将卵巢或肝脏切成小块后，加入2.0倍体积的95%酸性醇，在匀浆机中将河豚内脏组织打成匀浆。
优选地，步骤（2）中，所述的在匀浆中加入的助流动剂或助滤剂为硅藻土。在河豚内脏组织匀浆中加入内脏重量0.5-1.0倍重量的硅藻土，搅拌混合均匀后，将混合物缓慢流入层析柱中。静置1小时待材料中的TTX全部溶解后，用95%酸性醇洗脱提取其中的TTX，至洗脱液中无TTX为止。
优选地，步骤（3）中，将TTX提取液用真空浓缩仪减压浓缩至水相，回收乙醇用于重复使用。水相用有机溶剂萃取除去脂溶性物质，得到的水相为TTX水溶液。
优选地，步骤（4）中，所述的TTX水溶液用预先处理好或再生好的D-152弱酸性阳离子交换树脂柱层析分离。上样后，依次用中性铵盐、纯水、低浓度醋酸（0.1-1.0%）溶液和较高浓度的醋酸溶液洗脱杂质，然后用3-5%醋酸溶液洗脱TTX。
优选地，步骤（5）中，所述的TTX的洗脱液用截留分子量为100-300纳滤膜浓缩仪浓缩至小体积，再用真空浓缩仪进一步将浓缩液蒸干。获得的浓缩物用碱性醇和碱性水洗脱杂质，用酸水溶解除去不溶物，将溶液调至碱性（pH8-9），置于4oC冰箱内结晶，得到高纯度的TTX。
与现有技术相比较，本发明具有如下优势：
1）本发明的TTX提取和分离制备方法，其中的提取步骤采用高效节能的柱层析提取新方法，提取过程一步完成，提取效率高。用7倍体积的提取溶剂可将原料中的TTX全部提取干净。
2）本发明的TTX提取和分离制备方法，提取溶剂采用高浓度的酸性醇进行柱层析提取，提取液中水溶性杂质少，不含蛋白质等大分子水溶性物质，大大简化了后续的TTX的分离纯化。同时，提取溶剂可回收，重复使用。
3）本发明的TTX提取和分离制备方法，过程简单，步骤少，分离纯化的回收率高，TTX纯品的总体得率高。同时TTX的制备成本低。
4）本发明的TTX提取和分离制备方法，所用设备为普通的层析柱和浓缩等常规设备，设备投资很低。
5）本发明的TTX提取和分离制备方法，提取和分离纯化过程全部在室温下进行；有机溶剂用量少，且可以回收重复使用，节能环保。
附图说明
为了易于说明，本发明由下述的具体实施及附图作以详细描述。
图1为本发明的TTX提取分离和制备的工艺流程图。
图2 为制备的TTX样品的HPLC分析。
具体实施方式
本发明的“高效的从河豚内脏提取和分离制备高纯度河豚毒素的方法”工艺流程如下：
1）将新鲜的河豚卵巢15千克用清水漂洗和除杂后，用绞肉机搅碎，加入河豚卵巢重量的2-3倍酸性（1%醋酸）无水乙醇和0.6倍的硅藻土搅拌均匀，均匀装入ø200 x 1500 mm的层析柱中，静置1小时后，用酸性乙醇洗脱，直至层析洗脱液中无TTX为止。收集层析洗脱液即为TTX提取液。得含TTX 1236.0 mg的提取液，TTX提取率96%。
（2）将TTX提取液减压浓缩至水相，回收的乙醇可重复使用。浓缩液用1/2-1/10体积的醋酸乙酯萃取除去低极性杂质得TTX提取浓缩液。得含TTX 1168mg的提取浓缩液，TTX回收率90.5%。
（3）将15千克河豚卵巢提取浓缩液用D-152弱酸性阳离子交换树脂柱层析（ø100 x1000 mm）分离，上样后先后用0.2%磷酸铵溶液、纯水和0.5%醋酸水各洗脱3BV，洗去杂质。然后用2%醋酸水溶液洗脱至无TTX，TTX的回收率为93.6%。收集TTX集中段，用纳滤浓缩至小体积，减压浓缩干，得TTX粗品，TTX的回收率为90.4%。TTX含量低的洗脱相再用D-152弱酸性阳离子交换树脂柱层析浓缩回收TTX。
（4）TTX粗品用碱性乙醇和碱性水洗脱3次，沉淀用反复用酸溶碱沉3次，得纯化的TTX，TTX的回收率为80.7%。
（5）纯化的TTX用HPLC分析和测定含量，得TTX 815.6 mg。HPLC分析，TTX纯度≥98%（如图2）。TTX的总回收率为63.3%。
Claims (1)
1.一种高效的从河豚鱼内脏提取和分离制备河豚毒素的方法，其特征在于包括以下步骤：
（1）将新鲜的河豚卵巢15千克用清水漂洗和除杂后，用绞肉机搅碎，加入河豚卵巢重量的2-3倍酸性（1%醋酸）无水乙醇和0.6倍的硅藻土搅拌均匀，均匀装入ø200 x 1500 mm 的层析柱中，静置1小时后，用酸性乙醇洗脱，直至层析洗脱液中无TTX为止，收集层析洗脱液即为TTX提取液，得含TTX 1236.0 mg的提取液，TTX提取率96%；
（2）将TTX提取液减压浓缩至水相，回收的乙醇可重复使用，浓缩液用1/2-1/10体积的醋酸乙酯萃取除去低极性杂质得TTX提取浓缩液，得含TTX 1168mg的提取浓缩液，TTX回收率90.5%；
（3）将15千克河豚卵巢提取浓缩液用D-152弱酸性阳离子交换树脂柱层析（ø100 x1000 mm）分离，上样后先后用0.2%磷酸铵溶液、纯水和0.5%醋酸水各洗脱3BV，洗去杂质，然后用2%醋酸水溶液洗脱至无TTX，TTX的回收率为93.6%，收集TTX集中段，用纳滤浓缩至小体积，减压浓缩干，得TTX粗品，TTX的回收率为90.4%，TTX含量低的洗脱相再用D-152弱酸性阳离子交换树脂柱层析浓缩回收TTX；
（4）TTX粗品用碱性乙醇和碱性水洗脱3次，沉淀酸溶碱沉3次，得纯化的TTX，TTX的回收率为80.7%；
（5）纯化的TTX用HPLC分析和测定含量，得TTX 815.6 mg，HPLC分析，TTX纯度≥98%，TTX的总回收率为63.3%。
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