咖啡酸分子烙印聚合物的制备及其在金银花抗炎作用评价中的应用研究

Preparation of Caffeic Acid Molecular Imprinted Polymer and Its Application in Evaluation of Anti-inflammatory Effect of Lonicerae Japonicae Flos

目的:制备咖啡酸分子烙印聚合物(MIP)并特异性地去除金银花提取物中的咖啡酸,考察咖啡酸去除前后金银花提取物抑制前列腺素E2(PGE2)释放的变化,从整体上评价其抗炎活性。方法:采用溶胶-凝胶法,以粒径为62~105μm的二氧化硅微珠为载体、咖啡酸为模板分子、(3-氨丙基)三乙氧基硅烷为功能单体、四乙氧基硅烷为交联剂、四氢呋喃为溶剂,合成了咖啡酸MIP。以此MIP作为液相色谱固定相,以甲醇-乙酸(500∶1和9∶1)为流动相,特异性去除了金银花提取物中的咖啡酸。通过脂多糖刺激巨噬细胞RAW264.7释放PGE2实验评价去除咖啡酸前后金银花提取物的抗炎作用。结果:溶胶-凝胶法制备的咖啡酸MIP能够从复杂体系中特异性地分离和富集微量的咖啡酸,对咖啡酸的容量因子和烙印效率分别为15.8和9.7,最终从金银花提取物2.6 g中分离了咖啡酸146μg,纯度为92%,回收率为89%。金银花提取物和去除咖啡酸的提取物在质量浓度为100、200、400μg·mL–1时对脂多糖诱导的RAW264.7细胞释放PGE2的抑制率分别为5.8%、35.6%、62.5%和5.4%、13.3%、57.5%,去除微量咖啡酸以后的提取物在3个质量浓度时的抑制率较金银花提取物均有所降低。结论:咖啡酸是金银花提取物抗炎活性的一个重要成分,咖啡酸分子烙印聚合物可以实现金银花提取物中微量咖啡酸的特异性分离,分子烙印技术能够在保证中药完整性的前提下评价中药的药理活性。

Objective:To prepare a molecularly imprinted polymer(MIP),specifically remove caffeic acid(CA)from Lonicerae Japonicae Flos extract,investigate the inhibitory effect on the release of prostaglandin E2(PGE2)of Lonicerae Japonicae Flos extract before and after CA removal,and evaluate its anti-inflammatory activity on the whole.Methods:CA MIP had been prepared through the sol-gel coating method by using CA,silica beads(62-105μm),tetrahydrofuran,3-aminopropyltriethoxysilane,and tetraethoxysilane as a template,supporting matrix,solvent,functional monomer,and cross-linker,respectively.The specific separation of CA from Lonicerae Japonicae Flos extract was achieved by using this MIP as the liquid chromatographic stationary phase with methanol-acetic acid(500:1 and 9:1)as the mobile phase.The release of PGE2 from RAW264.7 cells stimulated by lipopolysaccharide(LPS)was investigated to evaluate the anti-inflammatory effects of the extract before and after CA was removed.Results:The CA MIP prepared by the sol-gel method could specifically separate and enrich small amounts of CA from complex systems.The capacity factor and imprinted factor for CA were 15.8 and 9.7,respectively.Finally,146μg CA was separated from the extract of Lonicerae Japonicae Flos of 2.6 g,and the purity and recovery of CA were 92%and 89%.The inhibition rate of PGE2 release of Lonicera Japonica Flos extracts and the extracts without CA in 100,200,and 400μg·mL–1 were 5.8%,35.6%,and 62.5%,as well as 5.4%,13.3%,and 57.5%,respectively.After the removal of trace CA from the extract of Lonicerae Japonicae Flos,the inhibition rate of PGE2 production was reduced.Conclusion:CA is an important component of the anti-inflammatory activity of Lonicerae Japonicae Flos extract.The MIP can achieve the specific separation of trace CA in Lonicerae Japonicae Flos extract.The molecular imprinting technology can evaluate the pharmacological activity of traditional Chinese medicine on the premise of ensuring the integrity of traditional Chinese medicine.