CN110183663B - 一种芍药苷分子印迹聚合物及其制备与应用 - Google Patents
一种芍药苷分子印迹聚合物及其制备与应用 Download PDFInfo
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Abstract
本发明公开了一种芍药苷分子印迹聚合物及其制备与应用,所述聚合物按如下方法制备:取硅胶,分散在无水乙醇中,然后加入交联剂,功能单体和苯基三甲氧基硅烷,室温振荡30min后,加入芍药苷乙醇溶液,二次蒸馏水,浓HCl,室温下反应4h,过滤,滤饼先用无水乙醇洗涤,干燥,然后用体积比9:1的甲醇-乙酸洗涤至洗涤液在230nm处无紫外吸收,再用甲醇洗涤除去残留的乙酸,最后干燥至恒重,即得到芍药苷分子印迹聚合物;本发明采用溶胶-凝胶法制备具有双功能单体的芍药苷分子印迹聚合物,能很好地从芍药花提取液中富集芍药苷,富集率最高可达90%,并且能够去除芍药花提取液中的其他杂质。
Description
背景技术
芍药,是芍药科多年生宿根草本植物芍药的干燥根,苦、酸、微寒、归肝、脾经,具平肝止痛,养血调经,敛阴止汗。用于头痛眩晕,肋痛,腹痛,四肢挛痛,血虚萎黄,月经不调,自汗,盗汗的功效。其生长于山坡、山谷的灌木丛或草丛中。主要分布于安徽、黑龙江、吉林、辽宁、河北、河南、山东、山西、陕西、内蒙古等地,现在全国各地均有栽培。国内外对芍药的化学成分、药理活性及临床应用等方面研究较多,并对芍药中有效成分的提取工艺做了大量的探索研究。芍药的根含芍药试、牡丹酚、芍药花试、苯甲酸约、挥发油、月旨肪油、树脂、靴质、糖、淀粉、粘液质、蛋白质、一谷昌醇和三菇类。
现有技术中提取芍药普的方法有醇水溶液回流提取法、超临界流体萃取法,膜分离法等,虽然这些现有技术尝试了各种不同的提取和纯化方法,有些方法工艺复杂,有的需要特殊的或复杂的设备,但由于这些现有技术无法完全的破坏细胞壁的束缚,最大限度地提取出有效成分,并对有效物质进行充分合理分离,因而所得产品纯度不高,也不易规模化生产。
分子印迹技术近年来在国内外都得到不断地发展,分子印迹聚合物的设计、制备、表征及应用也逐渐趋向成熟,引起了科学界的兴趣。与其他检测相比,MIPs具有的三个主要独特特征,结构可预测性、识别特异性和应用普遍性,因此得到了广泛的关注。并已成为吸引力许多领域去探索,如净化和分离,人工抗体,药物传递和催化讲解等。由于其高物理稳定性,直接制备,有着显着的稳固性和低成本的特点。目前分子印迹技术应用到环烯醚萜苷类化合物已有比较成熟的研究,但是将分子印迹技术应用到芍药苷的却很少有文献研究。因此制备合成芍药苷分子印迹聚合物,用于能够简洁高效地识别并富集芍药苷尤为重要。
发明内容
本发明的目的在于提供一种芍药苷分子印迹聚合物及其制备方法与应用,能够将芍药苷从芍药花提取物中提取出来,专一性强,拥有广大的市场前景。
本发明采用如下技术方案:
本发明提供一种芍药苷分子印迹聚合物提取,所述聚合物按如下方法制备:取硅胶,分散在无水乙醇中,然后加入交联剂,功能单体和苯基三甲氧基硅烷(PTMOS),室温(25-30℃)振荡30min后,加入芍药苷乙醇溶液,二次蒸馏水,浓HCl(优选质量浓度36%),室温下反应4h,过滤,滤饼先用无水乙醇洗涤三次,干燥,然后用体积比9:1的甲醇-乙酸洗涤至洗涤液在230nm处无紫外吸收,再用甲醇洗涤除去残留的乙酸,最后干燥(优选在60℃条件下真空干燥)至恒重,即得到芍药苷分子印迹聚合物(MIPs);所述交联剂为正硅酸乙酯(TEOS),功能单体为3-氨丙基三乙氧基硅烷(APTES);所述PTMOS与交联剂、功能单体物质的量之比为1:1.56:0.86;所述硅胶质量用量以PTMOS物质的量计为200g/mmol,所述乙醇体积用量以PTMOS物质的量计为5-20ml/mmol,所述芍药苷乙醇溶液的浓度为0.1mol/L,所述芍药苷乙醇溶液体积用量以PTMOS物质的量计为800μl/mmol,所述浓HCl体积用量以PTMOS物质的量计为100μl/mmol,所述二次蒸馏水体积用量以PTMOS物质的量计为1ml/mmol。
进一步,所述硅胶为粒径为40-60μm(优选50μm)球形硅胶。
本发明还提供一种芍药苷印迹聚合物在富集芍药苷中的应用,所述的应用方法为:将芍药苷分子印迹聚合物加入芍药花提取液中,在60℃、40Hz下超声吸附2h,取样在4000rmp下离心20min,取上清液检测在300nm处的吸光度,根据芍药苷的标准曲线,获得上清液中芍药苷的含量,进而获得富集率。
芍药花提取物制备方法:选择干燥的芍药花,捣碎,研磨成粉末(无颗粒感即可);取芍药花粉末,用纯净水浸没,25℃、30Hz下超声提取30min;重复提取3次,过滤,合并滤液,得到芍药花的水提取液;在芍药花的水提取液中加入二氯甲烷萃取,获得上层水相和下层有机相,下层有机相重复萃取3次;收集起三次萃取的水相,下层有机相再加入乙酸乙酯萃取,获得下层水相和上层有机相,上层有机相同样重复萃取3次,收集3次萃取后的下层水相;最后将二氯甲烷萃取的水相与乙酸乙酯萃取的水相合并,用旋转蒸发仪温度设置为60℃进行旋干,得到芍药花的提取物;取芍药花提取物用甲醇溶解,即芍药花提取液;所述纯净水体积用量以芍药花粉末重量计为5ml/g。
与现有技术相比,本发明的有益效果主要体现在:
(1)本发明采用溶胶-凝胶法制备具有双功能单体的芍药苷分子印迹聚合物,能很好地从芍药花提取液中富集芍药苷,富集率最高可达90%,并且能够去除芍药花提取液中的其他杂质。
(2)本发明制备芍药苷分子印迹聚合物最大限度地提取出有效成分,并对有效物质进行充分合理分离,所得产品纯度高,可达90%,易规模化生产。
附图说明
图1是洗脱后的溶液以及芍药苷标准溶液的液相图;系列1:洗脱后溶液液相图,系列2:栀子苷标准溶液液相图。
图2是芍药苷的标准曲线。
具体实施方式
下面结合具体实施方式对本发明的技术方案作进一步说明。
本发明所述室温是指25-30℃。本发明实施例所用芍药苷按照《中国药典》2010版采集制备。
实施例1
1、芍药苷分子印迹聚合物的制备
(1)采用溶胶-凝胶法制备芍药苷分子印迹聚合物,具体过程如下:称取200g粒径大约为50μm球形硅胶,分散在10ml无水乙醇中,然后加入交联剂TEOS3ml(1.56mmol),功能单体APTES(0.86mmol)和PTMOS(1.0mmol)各200μL,室温振荡30min后,加入0.1mol/L的芍药苷(0.08mmol)乙醇溶液800μL,二次蒸馏水1ml,100μL浓HCl(浓度36%),室温下反应4h,过滤,得到滤饼5.6g。滤饼先用无水乙醇洗涤三次,60℃干燥,然后用甲醇-乙酸(9:1,v/v)洗脱滤饼去除聚合物中的模板分子,直至洗涤液在230nm处无紫外吸收,用甲醇洗涤印迹聚合物三次以除去残留的乙酸,最后在60℃条件下真空干燥至恒重,即得到芍药苷分子印迹聚合物(MIPs)3.8g。
2、芍药苷分子印迹聚合物的应用
将20mg芍药苷分子印迹聚合物加入10ml芍药苷乙醇标准液(100mg/l)中,在60℃、40Hz下超声吸附2h,取样在4000rmp下离心20min,取上清液检测在300nm处的吸光度,根据芍药苷的标准曲线,获得上清液中芍药苷的含量10mg/l,进而获得富集率90%。
本发明所述芍药苷标准曲线的绘制方法为:将芍药苷用乙醇溶解配制成不同浓度梯度标准溶液(0、5、10、15、20、mg/L),在300nm处测吸光值,以芍药苷浓度为横坐标,以吸光值为纵坐标,绘制芍药苷标准曲线,结果见图2。
将洗脱后的溶液以及芍药苷标准溶液进行液相分析,结果见图1。证明芍药苷的分子印迹聚合物可以很好地将芍药苷从西红花中提取出来,并且专一性很强。色谱柱:C18柱(150*4.6mm,5μm;日本岛津);流动相:甲醇-0.1%磷酸溶液(34∶66,v/v);流速1.0ml/min;进样体积100μL;检测波长230nm。
实施例2
1、芍药苷分子印迹聚合物的制备
(1)采用溶胶-凝胶法制备芍药苷分子印迹聚合物,具体过程如下:称取200g粒径大约为50μm球形硅胶,分散在10ml无水乙醇中,然后加入交联剂TEOS6ml(3.12mmol),功能单体APTESl(1.72mmol)和PTMOS(2.0mmol)各400μL,室温振荡30min后,加入0.1mol/L的芍药苷(0.16mmol)乙醇溶液1.6mL,二次蒸馏水1ml,200μL浓HCl,室温下反应4h,得到产物3.3g。产物先用无水乙醇洗涤三次,干燥。然后用甲醇-乙酸(9:1v/v)洗脱聚合物中的模板分子,直至洗涤液在230nm处无紫外吸收,用甲醇洗涤印迹聚合物三次以除去残留的乙酸,最后在60℃条件下真空干燥至恒重,即得到对芍药苷分子印迹聚合物(MIPs)2.3g。
2、芍药苷分子印迹聚合物的应用
将20mg芍药苷分子印迹聚合物加入10ml芍药苷乙醇标准液(100mg/l)中,在60℃、40Hz下超声吸附2h,取样在4000rmp下离心20min,取上清液检测在300nm处的吸光度,根据芍药苷的标准曲线,获得上清液中芍药苷的含量22.8mg/l,进而获得富集率77.2%。
实施例3
1、芍药苷分子印迹聚合物的制备
(1)采用溶胶-凝胶法制备芍药苷分子印迹聚合物,具体过程如下:称取200g粒径大约为50μm球形硅胶,分散在10ml无水乙醇中,然后加入交联剂TEOS1.5ml(0.78mmol),功能单体APTESl(0.43mmol)和PTMOS(0.5mmol)各100μL,室温振荡30min后,加入0.1mol/L的芍药苷(0.04mmol)乙醇溶液400μL,二次蒸馏水1ml,50μL浓HCl,室温下反应4h,得到产物4.4g。产物先用无水乙醇洗涤三次,干燥。然后用甲醇-乙酸(9:1v/v)洗脱聚合物中的模板分子,直至洗涤液在230nm处无紫外吸收。用甲醇洗涤印迹聚合物三次以除去残留的乙酸,最后在60℃条件下真空干燥至恒重,即得到对芍药苷分子印迹聚合物(MIPs)2.8g。
2、芍药苷分子印迹聚合物的应用
将20mg芍药苷分子印迹聚合物加入10ml芍药苷乙醇标准液(100mg/l)中,在60℃、40Hz下超声吸附2h,取样在4000rmp下离心20min,取上清液检测在300nm处的吸光度,根据芍药苷的标准曲线,获得上清液中芍药苷的含量30.5mg/l,进而获得富集率69.5%。
Claims (6)
1.一种芍药苷分子印迹聚合物,其特征在于所述聚合物按如下方法制备:取硅胶,分散在无水乙醇中,然后加入交联剂,功能单体和苯基三甲氧基硅烷,室温振荡30min后,加入芍药苷乙醇溶液,二次蒸馏水,浓HCl,室温下反应4h,过滤,滤饼先用无水乙醇洗涤,干燥,然后用体积比9:1的甲醇-乙酸洗涤至洗涤液在230nm处无紫外吸收,再用甲醇洗涤除去残留的乙酸,最后60℃真空干燥至恒重,即得到芍药苷分子印迹聚合物;所述交联剂为正硅酸乙酯,所述功能单体为3-氨丙基三乙氧基硅烷;所述硅胶为粒径为40-60μm的球形硅胶。
2.如权利要求1所述芍药苷分子印迹聚合物,其特征在于所述苯基三甲氧基硅烷与交联剂、功能单体物质的量之比为1:1.56:0.86,所述硅胶质量用量以苯基三甲氧基硅烷物质的量计为200g/mmol。
3.如权利要求1所述芍药苷分子印迹聚合物,其特征在于所述乙醇体积用量以苯基三甲氧基硅烷物质的量计为5-20ml/mmol,所述芍药苷乙醇溶液的浓度为0.1mol/L,所述芍药苷乙醇溶液体积用量以苯基三甲氧基硅烷物质的量计为800μl/mmol,所述浓HCl体积用量以苯基三甲氧基硅烷物质的量计为100μl/mmol,所述二次蒸馏水体积用量以苯基三甲氧基硅烷物质的量计为1ml/mmol。
4.一种权利要求1所述 芍药苷分子 印迹聚合物在富集芍药苷中的应用。
5.如权利要求4所述的应用,其特征在于所述的应用方法为:将芍药苷分子印迹聚合物加入芍药花提取液中,在60℃、40Hz下超声吸附2h,取样在4000rmp下离心20min,取上清液即为富集芍药苷的溶液。
6.如权利要求5所述的应用,其特征在于所述芍药花提取物制备方法:选择干燥的芍药花,捣碎,研磨成粉末;取芍药花粉末,用纯净水浸没,25℃、30Hz下超声提取30min;重复提取3次,过滤,合并滤液,得到芍药花的水提取液;在芍药花的水提取液中加入二氯甲烷萃取,获得上层水相和下层有机相,下层有机相重复萃取3次,收集起三次萃取的水溶液,下层有机相再加入乙酸乙酯萃取,获得下层水相和上层有机相,上层有机相同样重复萃取3次,收集3次萃取的下层水相;最后将二氯甲烷萃取的水相和乙酸乙酯萃取的水相合并,用旋转蒸发仪温度设置为60℃进行旋干,得到芍药花的提取物;取芍药花提取物用甲醇溶解,即芍药花提取液;所述纯净水体积用量以芍药花粉末重量计为5ml/g。
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