Molecularly Imprinted Technology (MIT) is a polymer technology used to prepare polymers with specific recognition and binding capabilities for target molecules. The high-molecular-weight compounds prepared using MIT that exhibit specific recognition capabilities for target molecules are known as Molecularly Imprinted Polymers (MIPs). MIPs possess the advantages of pre-determined structure-activity relationships, structural stability, and high selectivity towards target compounds. They are easy to prepare, reusable multiple times, and capable of selectively extracting target molecules and structurally similar compounds from complex samples, making them one of the new technologies for the separation of various natural products.
Based on the different ways in which functional monomers interact with templates, the current methods for preparing molecularly imprinted polymers (MIPs) can be classified into three main categories. The first is the covalent bonding method, where functional monomers and templates form complexes through covalent bonds. The main forms of bonding include boronic acid esters, Schiff bases, acetals (ketals), esters, and chelation bonds. The advantages of this method are that MIPs have uniform recognition sites and high selectivity. However, the disadvantages are that the number of template molecules that can be imprinted is limited, and due to the strong covalent bonding, it is difficult to remove the template molecules. Furthermore, during the imprinting and re-recognition processes, the binding and dissociation speeds between the template and functional monomers are slow, making it difficult to meet the requirements of rapid separation.
The second method is the non-covalent bonding method, where functional monomers and templates form complexes through non-covalent interactions, including hydrogen bonds, electrostatic attractions, hydrophobic interactions, ionic bonds, and metal coordination bonds. The advantages of this method are that the preparation process is simple, the recognition speed is fast, and template molecules are easily removed. The recognition process is closer to that of natural molecular recognition systems. However, the disadvantages are that the binding sites are not uniform, often leading to non-specific binding, which can cause peak broadening and tailing. Additionally, due to the weak non-covalent interactions, the process is strongly dependent on solvent polarity, making it difficult to prepare MIPs in water. Nevertheless, due to its broad applicability, this method is currently the most commonly used for preparing MIPs.
The third method is the sacrificial spacer method, also known as the semi-covalent method, which combines the covalent bonding and non-covalent bonding approaches. In this method, the functional monomers interact with the template through covalent bonds during polymerization, but the recognition process occurs through non-covalent interactions. The advantages are that the binding and dissociation speeds between functional monomers and template molecules are relatively fast, making it suitable for rapid recognition. However, the disadvantages are that removing template molecules can be difficult, and there may be differences in recognition sites during imprinting and re-recognition, leading to a reduction in molecular selectivity.
The composition of traditional Chinese medicine (TCM) is complex, with a wide variety of ingredients and significant differences in content. Moreover, the instability of some components makes the separation and purification of active ingredients in TCM challenging to achieve. Currently, the separation and purification of active ingredients in TCM primarily rely on techniques such as silica gel column chromatography, macroporous adsorptive resin column chromatography, gel column chromatography, high-performance countercurrent chromatography, and preparative high-performance liquid chromatography. However, these methods suffer from issues like high solvent consumption, severe environmental pollution, poor specificity, and low extraction efficiency.
Multi-template molecularly imprinted solid-phase extraction technology holds significant advantages in the separation and purification of active ingredients in TCM. It enables the simultaneous extraction of similar compounds or groups of active ingredients from TCM, thereby enhancing the stability, specificity, and efficiency of the separation and extraction process. In recent years, MIPs have been widely applied in the separation and purification of various active ingredients in TCM, including flavonoids, alkaloids, phenylpropanoids, terpenes, and others.
① Extraction of flavonoids: The enrichment and extraction of flavonoids using molecular imprinting technology can be achieved based on hydrogen bonds formed between functional monomers and phenolic hydroxyl groups in flavonoid structures, π-π interactions between aromatic rings and flavonoids, or van der Waals forces. Therefore, most molecularly imprinted polymers (MIPs) used for the separation of flavonoids belong to the non-covalent bond type. Based on the multi-template molecular imprinting solid-phase extraction technology, there have been numerous studies on flavonoids such as rutin, baicalein, baicalin, and quercetin. Most MIPs used for the separation of flavonoid active ingredients in traditional Chinese medicine belong to the non-covalent bond type, and precipitation polymerization is a commonly used polymerization method due to its simplicity. Compared to conventional methods such as ultrasonic extraction, enzymatic extraction, supercritical fluid extraction, and macroporous resin adsorption, MIPs offer a simpler extraction process and higher purity of active ingredients.
② Extraction of Alkaloids: Alkaloids are a class of nitrogen-containing organic compounds with complex and diverse structures. Imprinting template molecules can form supramolecular complexes with heavy metals, organic acids, and multi-electron groups under acidic conditions; they can also form supramolecules with tannins; cyclic peptide macromolecules can serve as host molecules to encapsulate other small molecules to form supramolecules. Due to the abundance of supramolecular forms of alkaloids, the extraction of alkaloids can be based on their properties, using acids, water, alcohols, and lipophilic organic solvents such as chloroform and benzene to disrupt the supramolecular structure of alkaloids and facilitate their extraction. Similarly, for other compounds, their physicochemical properties can be used to deduce the "imprinting template" role in the extraction process, and corresponding physicochemical methods can be selected to ultimately establish an extraction method suitable for all components of traditional Chinese medicine (TCM). This method, based on the supramolecular "imprinting template" and the entire guest system, enables precise extraction of TCM formulations.
Utilizing multi-template molecular imprinting technology, various alkaloid molecules are specifically recognized through the formation of non-covalent bonds with polymeric functional monomers. Among the numerous preparation methods for alkaloid-imprinted polymers, the choice of functional monomers is often acidic (such as methacrylic acid) and weakly basic (such as 4-VP). These functional monomers easily form stable molecularly imprinted polymers with template molecules, resulting in high reproducibility of the adsorbents. Additionally, the use of representative compounds structurally similar to the target compounds as dummy templates for preparing imprinted polymers can achieve cross-selective recognition of multiple template molecules and their structural analogues, fulfilling the purpose of separation and extraction.
③ Extraction of Phenylpropanoids: Phenylpropanoids are a naturally occurring class of compounds consisting of a benzene ring connected to three linear carbon chains (C6-C3 group), typically featuring a phenolic structure and phenolic properties. The separation and extraction of phenylpropanoids using multi-template molecular imprinting technology is mostly achieved through specific interactions such as hydrogen bonds and van der Waals forces between functional monomers and template molecular groups. Among the various polymerization methods for molecularly imprinted polymers, surface molecular imprinting is widely applied in the separation of phenylpropanoids.
In the separation and extraction of phenylpropanoids, since these compounds contain carboxylic acid groups and exhibit acidic properties, selecting basic or weakly basic functional monomers can enhance the stability of the polymers, contribute to increased adsorbent capacity, and improve overall recovery rates.
④ Extraction of Terpenoids: Terpenoids are a class of olefinic compounds whose molecular formulas are integer multiples of isoprene, naturally occurring hydrocarbons ubiquitous in plants. The separation of multiple terpenoids using multi-template molecular imprinting technology can only be achieved through van der Waals forces between the parent structure and functional monomers or through hydrogen bonding interactions with functional monomers. When utilizing molecular imprinting technology to separate terpenoids and their derivatives that do not contain functional groups, specific recognition relies primarily on the hydrophobicity of the parent structure and the van der Waals forces with functional monomers, but this recognition effect is generally weak. For terpenoids and steroidal compounds containing functional groups (such as hydroxyl and carboxyl groups), hydrogen bonding interactions with functional monomers can further enhance molecular recognition. Additionally, some terpenoids and functional monomers can form relatively stable complexes with metal ions through coordination. Therefore, molecularly imprinted polymers constructed with these complexes as template molecules exhibit higher specific recognition towards these terpenoids in the presence of metal ions.
However, despite the immense potential of molecular imprinting technology in the extraction and separation of multi-component traditional Chinese medicines, several challenges persist. For instance, the majority of interactions between MIPs and target molecules rely on hydrogen bonding, which is often limited to non-polar or weakly polar solvents. To minimize the use of organic solvents and align with the principles of green chemistry, there is a need to identify novel functional monomers suitable for use in strongly polar solvents such as water. Additionally, the current cost of implementing molecular imprinting technology is relatively high, posing a significant barrier to achieving large-scale industrial production in the near future.
References:
[1] Li Shuo, Chang Yueyue, Cheng Fangfang, Bao Beihua, Cao Yudan, Yao Weifeng, Zhang Li, Ding Anwei. Multi-template molecularly imprinted solid-phase extraction and its application in the extraction and separation of multi-components from traditional Chinese medicines. Acta Pharmaceutica Sinica, 2021, 56(03): 751-760.
[2] Chen Siyang, Li Wenjiao, Fan Qimeng, Wang Ren, Yang Yantao, Liu Wenlong, Tang Yu, Liu Runnan, Zhou Yiqun, Tang Yu, Liu Ping'an, He Fuyuan. Discussion on the supramolecular "imprinted template" action rules in the extraction process of traditional Chinese medicines. Chinese Traditional and Herbal Drugs, 2019, 50(12): 2785-2790.
[3] Hao Mengmeng, Xu Jin, Han Wei. Progress in the application of molecular imprinting technology in the extraction and purification of active ingredients from traditional Chinese medicines. Pharmaceutical and Machinery Information, 2017(23): 49-55.
About the Author
Xiaonisha, a food technology professional holding a Master's degree in Food Science, is currently employed at a prominent domestic pharmaceutical research and development company. Her primary focus lies in the development and research of nutritional foods, where she contributes her expertise and passion to create innovative products.
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