CN107736362B - 一种海藻酸钠包埋法制备植物源杀螺剂的方法 - Google Patents
一种海藻酸钠包埋法制备植物源杀螺剂的方法 Download PDFInfo
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Abstract
本发明公开了一种海藻酸钠包埋法制备植物源杀螺剂的方法。将千金藤碱粉末用少量95％乙醇溶解，并配制15～30g/L海藻酸钠溶液；将千金藤碱加入到海藻酸钠溶液中，搅拌均匀；用注射器将混合溶液滴入5～15g/L的CaCl2溶液中，静置交联；将得到的凝胶微球清洗、干燥后冷藏备用。本发明以千金藤碱为杀螺活性成分，采用海藻酸钠包埋法制备杀螺剂，不仅具有植物源杀螺剂绿色环保的优势，而且，杀螺有效成分被包埋，在施用中遇水不易溶出，具有稳定和显著的杀螺活性。
Description
技术领域
本发明涉及一种用于杀灭钉螺的药剂制备方法，尤其涉及的是一种海藻酸钠包埋法制备植物源杀螺剂的方法。
背景技术
血吸虫病是由血吸虫成虫寄生于人体所引起的地方性疾病，主要流行于亚、非、拉美的73个国家，患病人数约2亿。血吸虫病是一种严重危害人们身体健康的疾病，它使疫区居民的日常出行以及淡水资源的使用受到各种限制。钉螺是日本血吸虫唯一的中间寄主，因此，杀灭钉螺是控制血吸虫病传播的重要环节。
目前，使用的化学杀螺剂如氯硝柳胺等，其优点是有效浓度低，毒效快，但缺点也同样明显：该杀螺剂污染环境，在水中迅速产生代谢变化，半衰期短，有效作用时间不长，对鱼类毒性很大，人接触后往往产生不良反应，而且，化学杀螺剂的长期使用可能会造成钉螺产生耐药性。因此，长期以来人们一直致力于寻找高效、低毒的杀螺药剂。
先期研究发现，千金藤碱作为一种植物提取物杀螺剂，具有效果稳定、无毒副作用、无残留、对环境友好等诸多优点，其半致死剂量LD50为6.25mg/L，是一种非常有应用前景的植物源杀螺剂(参见中国发明专利ZL201310571603.2)。但是，该杀螺剂在实际应用中容易受到限制，如采用常规的水剂或粉剂进行浸杀、喷洒方法，为使杀螺剂达到有效浓度，需要施用的药剂量大，容易对水体和其他生物造成其他可能的污染和伤害；如果采用颗粒剂进行毒饵诱杀，因为千金藤碱溶于水，以淀粉等作为常规助剂制备的颗粒剂，在实际使用中千金藤碱易于溶出，而被环境稀释，大大降低了杀螺效果。因此，合理制备剂型的选择成为制约千金藤碱在杀螺应用实践中的瓶颈因素。
发明内容
本发明的目的在于克服现有技术的不足，提供了一种海藻酸钠包埋法制备植物源杀螺剂的方法，能够实现对杀螺成分的有效包埋。
本发明是通过以下技术方案实现的，本发明包括以下步骤：
(1)将千金藤碱粉末用体积浓度95％乙醇溶解，千金藤碱粉末和乙醇的料液比为
4～8mg：1mL；
(2)配制15～30g/L海藻酸钠的水溶液；
(3)将千金藤碱加入到海藻酸钠溶液中，搅拌混合均匀；
(4)用注射器将步骤(3)的混合溶液滴入5～15g/L的CaCl2溶液中，静置交联；
(5)将得到的凝胶微球清洗、干燥后冷藏备用。
所述注射器的口径为1.6～2mm。注射器的口径能够确保制备的凝胶微球尺寸合适，配合钉螺方便食用。
所述步骤(1)的千金藤碱粉末中含有其他杀螺剂化合物，可以选用白屈菜红碱，实现灭螺增效。
本发明的杀螺活性成分是采用千金藤碱，可以仅含有千金藤碱作为唯一的杀螺活性成分，也可以包括除千金藤碱以外的其他杀螺剂化合物，如白屈菜红碱等。
一种海藻酸钠包埋法制备植物源杀螺剂的方法制备得到的杀螺剂。
所述杀螺剂为凝胶微球，所述杀螺剂的直径为1.6～2mm。便于钉螺食用。
本发明利用海藻酸钠包埋制备杀螺剂，包埋法是近年来迅速发展起来的一种细胞或酶的新兴固定化技术，具有操作简单，对细胞或酶的活性影响较小，效率高等特点。海藻酸钠是一类天然高分子多糖类物质，对微生物无毒害作用。
本发明相比现有技术具有以下优点：本发明以千金藤碱为杀螺活性成分，采用海藻酸钠包埋法制备杀螺剂，不仅具有植物源杀螺剂绿色环保的优势，而且，杀螺有效成分被包埋，在施用中遇水不易溶出，具有稳定和显著的杀螺活性。
附图说明
图1是海藻酸钠包埋制备的千金藤碱杀螺剂不同粒径凝胶微球；
图2是海藻酸钠包埋制备的千金藤碱杀螺剂成品图；
图3是HPLC法测定千金藤碱的标准曲线。
具体实施方式
下面对本发明的实施例作详细说明，本实施例在以本发明技术方案为前提下进行实施，给出了详细的实施方式和具体的操作过程，但本发明的保护范围不限于下述的实施例。
本实施例检测以海藻酸钠包埋法制备的杀螺剂在水中浸泡后随时间溶出的情况，以推测杀螺活性成分在凝胶微球中被包埋效果的情况。
首先制备杀螺剂：
(1)称取100mg的千金藤碱药剂，用20mL的95％乙醇溶解，制备浓度分别为0.005g/L、0.01g/L、0.02g/L、0.025g/L的溶液，定容于1mL容量瓶中备用；
(2)配制20g/L的海藻酸钠溶液100mL，在摇床上振摇30分钟混匀；
(3)将千金藤碱按不同配比加入到海藻酸钠溶液中，搅拌均匀；
(4)用2.5mL注射器将步骤(3)的混合溶液滴入10g/L的CaCl2溶液中，静置交联；
(5)将得到的凝胶微球用超纯水清洗小球，如图1和图2所示，自然干燥后放入自封袋中，4℃冰箱内保存备用；
(6)取上述不同浓度的对照品溶液，采用海藻酸钠和CaCl2包埋法，制备并挑选直径约为2mm的凝胶微球100粒，放入10mL的纯水中，分别在0h、2h、3h、6h、12h、24h、48h取浸泡液100μL，定容于1mL容量瓶中备用；
(7)采用HPLC法测定千金藤碱含量，仪器设备包括高效液相色谱仪(Waters 600)，C18色谱柱型号为Kromasil 100-5(4.6mm×250mm，5μm)，UV2489检测器，25μL的SGE微量进样针(Australia)。色谱条件：流动相为1％乙酸(A)-乙腈(B)，梯度洗脱，用0.22μm微孔滤膜过滤供试样品，紫外检测波长为282nm，流速为1mL.min-1，柱温25℃。
(8)分别取步骤(1)中的0.025g/L、0.02g/L、0.01g/L、0.005g/L对照品溶液，进样分析，三次重复。以峰面积的平均值为Y，以进样量为X(μg)，进行线性回归处理，线性方程为Y＝5E+06X-11786，R2＝0.9938，结果见图3。
(9)精确吸取步骤(1)中的0.005g/L、0.01g/L、0.02g/L、0.025g/L对照品溶液20μL，采用HPLC法分析，连续进样6次，测定千金藤碱的峰面积；计算RSD值分别为1.83％、1.24％、0.97％、0.88％、0.99％、1.61％，表明仪器精密度良好。
(10)取0.01g/L千金藤碱对照品溶液制备的凝胶微球，分别收集3h、6h、12h、24h浸泡液，采用HPLC法分析，测定千金藤碱的峰面积，计算RSD值分别为1.03％、0.91％、0.83％、1.07％，表明该分析方法在24h内稳定性良好。
(11)取0.01g/L千金藤碱对照品溶液制备的凝胶微球，浸泡处理12h，重复6次，取浸泡液采用HPLC法分析，计算RSD值分别为1.53％、1.10％、0.61％、1.10％、0.99％、0.87％，表明该分析方法的重复性良好。
(12)取步骤(6)中不同时间点的浸泡溶液各20μL，HPLC法进样分析，根据步骤(8)中的标准曲线，计算浸泡液中的千金藤碱含量、凝胶微球中包埋的千金藤碱含量，以及凝胶微球中包埋的千金藤碱占浸泡前原制备小球中千金藤碱总量的百分率，结果见表1。结果表明，海藻酸钠包埋制备的凝胶微球浸泡48h后，千金藤碱保留量比例均高于90％以上，杀螺活性成分在浸泡后不易溶出。
表1不同浸泡时间杀螺剂凝胶小球中千金藤碱含量分析
本实施例为以千金藤碱为植物源制备得灭螺剂的药效实验。
(1)试验条件：温度26±1℃，相对湿度65±5％。
(2)供试钉螺：安徽省现场采集的湖北钉螺，筛选40只阴性成螺作为供试钉螺。
(3)将20只供试钉螺放入烧杯，取水溶液浸没钉螺，投入本实施例制备的杀螺剂凝胶微球50粒左右；用塑料窗纱盖于烧杯口以防钉螺爬出，同时设阴性对照，一天后，倒去溶液，恢复饲养48小时，用敲击法鉴定供试钉螺的死亡数。
(4)另取20只，同上述试验浸杀48小时，恢复培养48小时，计算死亡数。
(5)计算钉螺杀灭死亡率，试验结果如表2所示。
表2杀螺剂杀灭钉螺试验的结果统计表
(6)结果评价：按农药检(生测)函[2005]77号《农药登记卫生用杀螺剂灭螺药效试验方法和评价》(试行)中4.灭螺药效评价指标进行评价：室内浸杀试验，浓度为50g/L和100g/L的本实施例制备的千金藤碱凝胶微球杀螺剂作用1天，湖北钉螺的死亡率均为100％。50g/L和100g/L的海藻酸钠包埋制备的千金藤碱凝胶微球杀螺剂对湖北钉螺有显著的杀灭作用。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。
Claims (3)
1.一种使用植物源杀螺剂杀灭钉螺的方法，其特征在于，包括以下步骤：
(1)将千金藤碱粉末用体积浓度95％的乙醇溶解，千金藤碱粉末和乙醇的料液比为4～8mg：1mL；
(2)配制15～30g/L的海藻酸钠溶液，混匀备用；
(3)将千金藤碱加入到海藻酸钠溶液中，搅拌均匀；
(4)用注射器将步骤(3)的混合溶液滴入5～15g/L的CaCl2溶液中，静置交联，所述注射器的口径为1.6～2mm；
(5)将得到的凝胶微球清洗、干燥后冷藏备用；
(6)将供试钉螺放入烧杯，取水溶液浸没钉螺，投入所述步骤(5)制备的杀螺剂凝胶微球；用塑料窗纱盖于烧杯口以防钉螺爬出，同时设阴性对照，一天后，倒去溶液，恢复饲养48小时，用敲击法鉴定供试钉螺的死亡数；
(7)所述步骤(6)的室内浸杀试验，浓度为50g/L和100g/L的千金藤碱凝胶微球杀螺剂作用1天，钉螺的死亡率均为100％。
2.根据权利要求1所述的一种使用植物源杀螺剂杀灭钉螺的方法，其特征在于，所述步骤(1)的千金藤碱粉末中含有其他杀螺剂化合物。
3.根据权利要求1所述的一种使用植物源杀螺剂杀灭钉螺的方法，其特征在于，所述杀螺剂为凝胶微球，所述杀螺剂的直径为1.6～2mm。
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