中药致癌性吡咯里西啶类生物碱二维靶标聚焦式分析模型的建立与评价

There are many kinds of traditional Chinese medicines (TCMs) that contained with pyrrolizidine alkaloids (PAs), and they were also widely used in China. However, a lot of PAs are with strong carcinogenicity, which bring great risk for the safe use of the TCMs that contained with PAs. While the traditional methods cannot realize the goal of simultaneously separating, analyzing and evaluating the targeted compounds in TCMs. In this project, we will establish an analysis model that can analyze and evaluate the carcinogenic PAs at the same time. Firstly, focused on the PAs in complex matrix, we will prepare the magnetic fluorescent nano-molecularly imprinted polymers (MFN-MIPs) that will be with the ability of specific recognition of PAs. The obtained MFN-MIPs will also take many advantages, such as rational designed, size controlled and easy to be separated and detected. Based on the MFN-MIPs, the first-dimensional targeted analysis unit will be established. Secondly, focused on the carcinogenic PAs and based on the principle that the carcinogenic PAs and the fluorescence system labeled by Eu3+ can be competitively combined with the fragment of DNA, we will establish the second-dimensional targeted analysis unit by using indirect fluorescent hypersensitive immune technique. Both of the two analysis units will be combined together and a binary targeted analysis model for the analysis of potential carcinogenic PAs in TCMs will be established. The established analysis model will be with the functions of analysis and evaluation of carcinogenic PAs in TCMs. This project will provide new method and idea for the analysis and evaluation of the carcinogenic compounds in TCMs.

含吡咯里西啶类生物碱（PAs）的中药种类繁多且应用广泛，但PAs中的诸多化合物具强致癌性，给含PAs中药的用药安全带来极大隐患，而传统分离分析方法难以同时实现对特定目标化合物的选择性分离分析和毒性评价。本研究拟建立一种集对中药中致癌性PAs分析和评价于一体的分析模型。首先，聚焦于中药复杂组分中的PAs，整合磁性粒子易于分离、荧光标记易于检测、分子印迹的特异选择性、计算机辅助的理性设计以及可控自由基聚合等优点，制备具有特异选择性的磁性荧光纳米分子印迹聚合物，并构建基于该聚合物的一维靶标分析单元；其次，聚焦于致癌性PAs，利用致癌性PAs和特定荧光体系二者能竞争性的与DNA结合的原理，构建竞争性荧光超敏免疫二维靶标分析单元；最后，两个分析单元共同组建成一种能同时实现对中药中致癌性PAs快速筛查和毒性评价的分析模型。该分析模型的建立可为中药致癌性成分的分析和安全性评价提供新的思路和方法。

含有吡咯里西啶类生物碱（PAs）的中药种类繁多且应用广泛，但在600多种PAs中有一半以上为毒性PAs，它们具有很强的肝毒性和致癌毒性，给含此类化合物中药的用药安全性带来极大隐患。传统的中药毒性成分分析与评价方法相对独立，且均存在工作量大、耗时长、评价效率低和成本高等问题。本研究在前期工作基础上，研究整合分子印迹特异识别、磁性颗粒易于分离和荧光易于检测等三种技术的优点，制备了四种对毒性PAs具有特异识别能力的聚合物材料（包括PAs-3D-分子印迹聚合物、PAs-分子印迹-固相微萃取介质、PAs-磁性荧光-分子印迹聚合物和强阳离子固相萃取介质等），分别采用扫描电子显微镜、透射电镜、红外光谱、热重分析、氮吸附-脱附等技术对所得介质的表观形貌、理化性质和稳定性等进行了考察；通过静态吸附实验、吸附动力学实验、吸附选择性实验等分别测定了各介质对毒性PAs的特异识别能力，优化了识别条件，并考察了分子识别的基本规律和可能的识别机制；并将所得聚合物材料用于复杂中药样品的前处理中，结合高效液相色谱法、荧光光谱法、超高效液相-质谱法和超高效液相-飞行时间质谱法等，建立了四种分析方法，所建方法简便、快速、准确，且集致癌成分分析和评价于一体，为生物碱类致癌性成分“识别-分离-分析-评价”提供了新思路；研究分别对6种典型中药材（一点红、紫草、紫菀、返魂草、千里光、款冬花）、8种中药制剂（咽炎片、止咳片、消糜栓、小儿止咳颗粒、止咳颗粒1、止咳颗粒2、急支糖浆、减肥片）和10种进口花茶（Erdbeer-Sahne、Earl Grey、Magie des Orients、Himbeer Vanille、Orange-Ingwer、Orange des Sudens、Klassisch、Mango-Kokos、Kamille和Sahne-Karamell）中的12种毒性PAs（erucifoline、europine、retrorsine、seneciphylline、heliotrine、echimidine、lasiocarpine、senecionine、intermedine、senecivernine、lycopsamine和monocrotaline等）进行了特异性分离和分析，实现了对中药中具潜在毒性PAs的快速筛查，也为其他中药毒性成分的分析和安全性评价提供了新思路。