CN107189081A - 一种多功能多糖水凝胶的制备方法及用途 - Google Patents
一种多功能多糖水凝胶的制备方法及用途 Download PDFInfo
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Abstract
一种多功能多糖水凝胶的制备方法及用途,将川芎红花海带的混合,粉碎后用水提醇沉的方法提取多糖;得红川海粗多糖;然后加入无水乙醇/蒸馏水溶液中,在25℃下进行搅拌2小时;(3)取NaIO4,溶于水后加入反应体系,在避光的状态下反应10小时;然后加入乙二醇,搅拌30分钟;加入无水乙醇,搅拌10分钟,抽滤,然后透析72小时,50℃下干燥12h,即得氧化的红花多糖/川穹多糖/海带多糖;然后溶于蒸馏水中配成4%的溶液,搅拌使其完全溶解;取N‑O羧甲基壳聚糖,溶于蒸馏水中配成8%的溶液,搅拌;分别将红花/川穹/海带氧化多糖和N‑O羧甲基壳聚糖两种溶液进行混合,搅拌均匀,得到多糖水凝胶。其用途为:应用于组织工程材料;应用于抗凝血活性药物辅料。
Description
技术领域
本发明属于生物材料领域,具体涉及复合水凝胶的制备技术。
背景技术
水凝胶因独特的三维网状结构,类似于生命组织材料,表面粘附蛋白质及细胞能力较弱,与血液、体液及人体组织相接触时,表现出良好的生物相容性,在细胞生长培养及医疗方面受到极大关注。目前医用合成水凝胶的高分子材料主要有丙烯酰胺、丙烯酸羟基酯,甲基丙烯酸羟基酯、聚乙烯醇、聚氧乙烯等。但常见医用水凝胶主要有自由基引发剂和交联剂进行交联的,在这类交联反应中,交联剂分解产生自由基,这些自由基引发高分子自由基链反应,从而导致高分子化合物链交联,在这里交联剂实际上起的是引发剂的作用,或者是利用交联剂分子中的官能团,与高分子化合物进行反应,通过交联剂作为桥基把聚合大分子交联起来。而应用自由基引发剂及交联剂尚有缺点,即单体残留,交联剂或引发剂残留都会引起炎症或者细胞毒性,且没有生物活性。如何改进制备材料、方法及条件克服传统医用水凝胶制备的缺点以及开发出更广泛的用途是现在研究的热点。本发明通过天然中药多糖制备具有促细胞生长和抗凝血活性多糖水凝胶的方法可以实现上述目的,并且目前尚未见到兼具有促细胞生长和抗凝血活性的复合中药多功能多糖水凝胶的报道。
发明内容
本发明的目的是提供一种朵功能多糖水凝胶的制备方法及用途。
本发明是一种多功能多糖水凝胶的制备方法,其步骤为:
(1)将川芎、红花、海带的原材料以质量比为1∶1∶1混合,粉碎后用水提醇沉的方法提取多糖;用Sevage法除多糖蛋白2次,50℃下干燥,即得红川海粗多糖;
(2)取多糖粉末,加入到干燥的反应釜中,然后加入按体积比为10ml/50ml的无水乙醇/蒸馏水混合溶液中,在25℃下进行搅拌2小时;
(3)取摩尔量相当于多糖单体摩尔量的NaIO4,溶于水后加入反应体系,在避光的状态下反应10小时;
(4)然后向反应体系中在加入与NaIO4等摩尔量的乙二醇,搅拌30分钟;加入体积相当于反应体系三倍量的无水乙醇,搅拌10分钟,抽滤,然后用截留分子量为3500的透析袋透析72小时,50℃下干燥12h,即得氧化的红花多糖/川穹多糖/海带多糖;
(5)将红花、川穹、海带氧化多糖,溶于蒸馏水中配成4%的溶液,搅拌使其完全溶解;取N-O羧甲基壳聚糖,溶于蒸馏水中配成8%的溶液,搅拌使其完全溶解;
(6)分别将红花、川穹、海带氧化多糖和N-O羧甲基壳聚糖两种溶液按体积比为1:1进行混合,200r/min搅拌均匀,得到多糖水凝胶。
以上所述的方法制得的多糖水凝胶具有促细胞生长和抗凝血作用,其用途为:应用于组织工程材料,即小直径血管组织工程血管支架,能够有效促进血管形成并抑制支架移植后的血栓形成。
以上所述的方法制得的多糖水凝胶具有促细胞生长和抗凝血作用,其用途为:应用于抗凝血活性药物辅料,能够利用3D打印制备缓释制剂。
本发明的有益之处在于:产品原料来源广泛,制备工艺简单,成本较低;红花多糖/川穹多糖/海带多糖复合水凝胶本身具有促细胞生长活性,比其它任何合成生物材料都接近活体组织,具有良好的材料生物相容性、可降解性,以及良好的材料——细胞界面及一定的空间三维结构;且具有优良的抗凝血作用,可以抑制移植血管后的凝血现象。
附图说明
图1是本发明制备的具有促细胞生长和抗凝血作用的多功能复合多糖水凝胶(未成胶);图2是本发明制备的具有促细胞生长和抗凝血作用的多功能复合多糖水凝胶(已成胶);
图3是通过实施例1制备的红花/川穹/海带多糖水凝胶的促细胞生长活性。
具体实施方式
本发明是一种多功能多糖水凝胶的制备方法,其步骤为:
(1)将川芎、红花、海带的原材料以质量比为1∶1∶1混合,粉碎后用水提醇沉的方法提取多糖;用Sevage法除多糖蛋白2次,50℃下干燥,即得红川海粗多糖;
(2)取多糖粉末,加入到干燥的反应釜中,然后加入按体积比为10ml/50ml的无水乙醇/蒸馏水混合溶液中,在25℃下进行搅拌2小时;
(3)取摩尔量相当于多糖单体摩尔量的NaIO4,溶于水后加入反应体系,在避光的状态下反应10小时;
(4)然后向反应体系中在加入与NaIO4等摩尔量的乙二醇,搅拌30分钟;加入体积相当于反应体系三倍量的无水乙醇,搅拌10分钟,抽滤,然后用截留分子量为3500的透析袋透析72小时,50℃下干燥12h,即得氧化的红花多糖/川穹多糖/海带多糖;
(5)将红花、川穹、海带氧化多糖,溶于蒸馏水中配成4%的溶液,搅拌使其完全溶解;取N-O羧甲基壳聚糖,溶于蒸馏水中配成8%的溶液,搅拌使其完全溶解;
(6)分别将红花、川穹、海带氧化多糖和N-O羧甲基壳聚糖两种溶液按体积比为1:1进行混合,200r/min搅拌均匀,得到多糖水凝胶。
以上所述步骤(5)中氧化红川海多糖和N,O-羧甲基壳聚糖的质量比为1:2。
以上所述的方法制得的多糖水凝胶具有促细胞生长和抗凝血作用,其用途为:应用于组织工程材料,即小直径血管组织工程血管支架,能够有效促进血管形成并抑制支架移植后的血栓形成。
以上所述的方法制得的多糖水凝胶具有促细胞生长和抗凝血作用,其用途为:应用于抗凝血活性药物辅料,能够利用3D打印制备缓释制剂。
以下结合实施例对本发明作进一步详细描述。
实施例1:
(1)取川芎红花海带各250g,适当粉碎,加适量乙醇60℃下搅拌4h,进行脱脂处理,再用自来水冲洗一次,在80℃下蒸馏水浸提三次,浓缩浸提液至适量体积,用Sevage(氯仿/正丁醇=5:1)法除多糖蛋白,加三倍量无水乙醇进行醇沉,抽滤,50℃下干燥,称量,待用;
(2)取5.0g多糖粉末,加入到干燥的单口瓶中,然后加入无水乙醇/蒸馏(10ml/50ml)在25℃下进行搅拌2h,取摩尔量相当于多糖单体摩尔量(以180计算)的NaIO4,溶于10ml水后加入反应体系,在避光的状态下反应一定时间,然后向反应体系中在加入与NaIO4等摩尔量的乙二醇,搅拌30min后,加入体积相当于反应体系三倍量的无水乙醇,搅拌10min,抽滤,50℃下干燥即得氧化的多糖;
(3)向反应体系中加入体积约2倍于前述反应体系的无水乙醇,搅拌10min后抽滤,然后用截留分子量为3500的透析袋透析72h,50℃下干燥12小时,即得氧化的红川海多糖;
(4)取16g氢氧化钠固体配制成40%(25ml)氢氧化钠溶液,加5g壳聚糖粉末,混均,在-20℃下过夜保存。取20g氯乙酸溶解于100ml异丙醇,缓慢滴入上述壳聚糖溶液,约0.5h滴完。氯乙酸壳聚糖混合溶液25℃搅拌2h,然后60℃继续搅拌2h,抽滤混合液,滤饼在50℃下干燥保存,待用;
(5)分别以N,O-羧甲基壳聚糖和氧化的多糖质量比为1:1、1:2、1:3分别溶于一定量的蒸馏水中,在室温下以体积比为1:1进行混合,总体积定为4ml,并观察凝胶的形成;
(6)在37℃条件下,将粗提复方红花/川穹/海带多糖水凝胶样品CSLPH-1、CSLPH-2和CSLPH-3分别在α-MEM培养基(5 mL)中,浸泡48 h,获得各样品凝胶提取物。将WI-38细胞以1×104个细胞/孔的密度接种于96孔板中。此后,向体系中加入0.1 mL一系列水凝胶提取液。加入新鲜培养基的孔作为阳性对照。然后放置在37℃ CO2恒温培养箱中培养24 h后,采用MTT法测定细胞存活率。使用酶标仪(DNM9606,北京)在570 nm处测定并记录吸光度值。细胞存活率(%)根据下列公式计算:
细胞存活率(%)= 实验组吸光度/阳性对照吸光度×100%
如图3所示,显示了红花多糖/川穹多糖/海带多糖水凝胶有较好的促细胞生长作用。
实施例2:
(1)取5.0g多糖粉末,加入到干燥的反应釜中,然后加入无水乙醇/蒸馏(10ml/50ml)在25℃下进行搅拌2h;
(2)取摩尔量相当于多糖单体摩尔量(以180计算)的NaIO4,溶于10ml水后加入反应体系,在避光的状态下反应10h;
(3)然后向反应体系中在加入与NaIO4等摩尔量的乙二醇,搅拌30min;
加入体积相当于反应体系三倍量的无水乙醇,搅拌10min,抽滤,然后用截留分子量为3500的透析袋透析72h,50℃下干燥12h,即得氧化的红花多糖/川穹多糖/海带多糖;
(4)取4g红花、川穹、海带氧化多糖,溶于100ml蒸馏水,搅拌使其完全溶解;
(5)称取8gN-O羧甲基壳聚糖,溶于100蒸馏水中,搅拌使其完全溶解;
(6)分别将红花/川穹/海带氧化多糖和N-O羧甲基壳聚糖两种溶液按体积比为1:2进行混合,200r/min搅拌均匀(时间较短),制备成多糖凝胶;
(7)将小鼠36只,随机分为三组,以25ml/kg容量灌胃,分别给予生理盐水为模型对照组,法华令7mg/kg为阳性对照组,红花/川穹/海带多糖水凝胶1000mg/kg为实验组。1h后用直径为10mm的玻璃毛细管插入小鼠内眦球后静脉丛取血至出现血凝丝的时间,即为凝血时间。如表1显示,红花/川穹/海带多糖凝胶可显著延长小鼠凝血时间,与生理盐水对照组有显著差异。
表1 红花/川穹/海带多糖水凝胶对小鼠凝血时间(CT)的影响
。
实施例3:
(1)取5.0g多糖粉末,加入到干燥的反应釜中,然后加入无水乙醇/蒸馏(10ml/50ml)在25℃下进行搅拌2h;
(2)取摩尔量相当于多糖单体摩尔量(以180计算)的NaIO4,溶于10ml水后加入反应体系,在避光的状态下反应10h;
(3)然后向反应体系中在加入与NaIO4等摩尔量的乙二醇,搅拌30min;
加入体积相当于反应体系三倍量的无水乙醇,搅拌10min,抽滤,然后用截留分子量为3500的透析袋透析72h,50℃下干燥12h,即得氧化的红花多糖/川穹多糖/海带多糖;
(4)取4g红花、川穹、海带氧化多糖,溶于100ml蒸馏水,搅拌使其完全溶解;
(5)称取8gN-O羧甲基壳聚糖,溶于100ml蒸馏水中,搅拌使其完全溶解;
(6)分别将红花/川穹/海带氧化多糖和N-O羧甲基壳聚糖两种溶液按体积比为1:2进行混合,200r/min搅拌均匀(时间较短),将搅拌均匀的溶液以20ml/h的速度通过3D打印机打印组织工程血管支架。
Claims (4)
1.一种多功能多糖水凝胶的制备方法,其特征在于,其步骤为:
(1)将川芎、红花、海带的原材料以质量比为1∶1∶1混合,粉碎后用水提醇沉的方法提取多糖;用Sevage法除多糖蛋白2次,50℃下干燥,即得红川海粗多糖;
(2)取多糖粉末,加入到干燥的反应釜中,然后加入按体积比为10ml/50ml的无水乙醇/蒸馏水混合溶液中,在25℃下进行搅拌2小时;
(3)取摩尔量相当于多糖单体摩尔量的NaIO4,溶于水后加入反应体系,在避光的状态下反应10小时;
(4)然后向反应体系中在加入与NaIO4等摩尔量的乙二醇,搅拌30分钟;加入体积相当于反应体系三倍量的无水乙醇,搅拌10分钟,抽滤,然后用截留分子量为3500的透析袋透析72小时,50℃下干燥12h,即得氧化的红花多糖/川穹多糖/海带多糖;
(5)将红花、川穹、海带氧化多糖,溶于蒸馏水中配成4%的溶液,搅拌使其完全溶解;取N-O羧甲基壳聚糖,溶于蒸馏水中配成8%的溶液,搅拌使其完全溶解;
(6)分别将红花/川穹/海带氧化多糖和N-O羧甲基壳聚糖两种溶液按体积比为1:1进行混合,200r/min搅拌均匀,得到多糖水凝胶。
2.根据权利要求1所述的多功能多糖水凝胶的制备方法,其特征在于所述步骤(5)中氧化红川海多糖和N,O-羧甲基壳聚糖的质量比为1:2。
3.根据权利要求1中所述的多功能多糖水凝胶的制备方法,所制得的多糖水凝胶具有促细胞生长和抗凝血作用,其用途为:应用于组织工程材料,即小直径血管组织工程血管支架,能够有效促进血管形成并抑制支架移植后的血栓形成。
4.根据权利要求1中所述的多功能多糖水凝胶的制备方法,所制得的多糖水凝胶具有促细胞生长和抗凝血作用,其用途为:应用于抗凝血活性药物辅料,能够利用3D打印制备缓释制剂。
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