System-level Study on Synergism and Antagonism of Active Ingredients in Traditional Chinese Medicine by Using Molecular Imprinting Technology

OPEN SUBJECT AREAS: PHARMACEUTICS SYSTEMS ANALYSIS Received 23 July 2014 Accepted 30 October 2014 Published 24 November 2014 System-level Study on Synergism and Antagonism of Active Ingredients in Traditional Chinese Medicine by Using Molecular Imprinting Technology Tengfei Chen1*, Jiangyong Gu2*, Xinzhuang Zhang3, Yimin Ma3, Liang Cao3, Zhenzhong Wang3, Lirong Chen2, Xiaojie Xu2 & Wei Xiao1,3 1 School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing, China, 2Beijing National Laboratory for Molecular Sciences, State Key Lab of Rare Earth Material Chemistry and Applications, College of Chemistry and Molecular Engineering, Peking University, Beijing, China, 3National Key Laboratory of Pharmaceutical New Technology for Chinese Medicine, Kanion Pharmaceutical Corporation, Lianyungang, Jiangsu Province, China. Correspondence and requests for materials should be addressed to L.R.C. (lirongchen@ pku.edu.cn); X.J.X. () or W.X. (xw_kanion@ 163.com) * These authors contributed equally to this work. In this work, synergism and antagonism among active ingredients of traditional Chinese medicine (TCM) were studied at system-level by using molecular imprinting technology. Reduning Injection (RDNI), a TCM injection, was widely used to relieve fever caused by viral infection diseases in China. Molecularly imprinted polymers (MIPs) synthesized by sol-gel method were used to separate caffeic acid (CA) and analogues from RDNI without affecting other compounds. It can realize the preparative scale separation. The inhibitory effects of separated samples of RDNI and sample combinations in prostaglandin E2 biosynthesis in lipopolysaccharide-induced RAW264.7 cells were studied. The combination index was calculated to evaluate the synergism and antagonism. We found that components which had different scaffolds can produce synergistic anti-inflammatory effect inside and outside the RDNI. Components which had similar scaffolds exhibited the antagonistic effect, and the antagonistic effects among components could be reduced to some extent in RDNI system. The results indicated MIPs with the characteristics of specific adsorption ability and large scale preparation can be an effective approach to study the interaction mechanism among active ingredients of complex system such as TCM at system-level. And this work would provide a new idea to study the interactions among active ingredients of TCM. T he mechanism of traditional Chinese medicine (TCM) was mainly caused by the interactions between complex TCM system and biological system1. TCM has been used for anti-inflammatory for thousands of years and accumulated lots of clinical experience2. TCM is a complex system which contains lots of components with diversities in chemical structures, biological activities and interactions among compounds, and the content for each component varies greatly3–5. Typically, an herbal formula which consists of several herbs would comprise hundreds of compounds and can affect the biological systems through interactions with multiple cellular targets6–11. TCM is so complicated that it’s almost impossible to explore the molecular mechanism thoroughly. It’s time-consuming and laborious to explore the efficacy of each compound. Moreover, compounds in TCM exert therapeutic effects in combination rather than as individuals8,12,13. The results of individual studies do not necessarily get the overall effect of TCM because of abundant synergistic and antagonistic effects8,14–17. To understand biology and chemistry at system level, we must identify the compounds of the systems and gain insights into emergent properties through interactions among compounds in the TCM systems. Molecularly imprinted polymer (MIP) is tailor-made adsorption material used to separate template and analogues from complex matrix18–23. The binding sites of MIP have high affinity for the template by interacting with its complementary functional groups or structural elements24–27. It can be grafted to the surface of silica beads by sol-gel process to realize semi-preparative scale, even preparative scale separation and preparation, and can retain the specific adsorption performance at the same time28–30. In recent years, several researches were carried out for caffeic acid-MIPs (CA-MIPs), and the CA-MIPs were used to determine and extract CA from complex media31–33. Therefore, a method by using MIP to selectively remove the template or a group of analogues from TCM can be a promising approach to study the emergent properties in the TCM systems. SCIENTIFIC REPORTS | 4 : 7159 | DOI: 10.1038/srep07159 1 www.nature.com/scientificreports Reduning Injection (RDNI) is a TCM injection which was prepared by Artemisia annua L., Lonicera japonica Thunb. and Gardenia jasminoides E.. It is widely used in clinical to relieve fever caused by viral infection diseases, such as upper respiratory tract infection34 and hand-foot-mouth disease35. The main constituents and contents in RDNI had been determined in previous work36. It mainly included caffeoylquinic acid compounds (CACs), coumarins, iridoids, flavonoids37. In this work, MIP was used to separate CA and CACs from RDNI. Lipopolysaccharide (LPS)-induced prostaglandin E2 (PGE2) release in RAW264.7 cells based on phenotypic assays maintained reasonable experimental efficiency and related to inflammatory diseases. So it was carried out to study the anti-inflammatory effects of separated samples and sample combinations. Combination index (CI), which was proposed by Chou and Talalay38, was used to evaluate the interaction between the components. Results Synthesis and Characterization of MIPs. In this section two kinds of polymers were synthesized by the sol-gel method. 8 polymers were prepared to optimize MIP1 (Table 1). The results of chromatographic evaluations showed that MIP1e had the highest imprinted factor (9.07) and good capacity factor (15.96) for CA (Table 2). The preparation condition of MIP2 (Table 1), which was used to separate all CACs in one step, was optimized from the composition of MIP1. The chromatographic conditions for separating CA by using MIP1 as solid phase were investigated. When the mobile phase was CH3OH-HOAc (50051, v/v), the retention time of acetone and CA were about 2 min and 19 min, respectively. But the chlorogenic acid (CGA) was not cleared out within 50 min (Fig. 1A). When CH3OHHOAc (951, v/v) was used as the mobile phase, the retention time of acetone, CA, and CGA were about 1 min, 2 min, and 11 min, respectively (Fig. 1B). The possible reason of this phenomenon was that the molecular structures of CGA, Isochlorogenic acid B (IsoB), and other CACs contain caffeic acid groups, so those compounds can partly embedded in the three-dimensional cavities of MIP. The molecular structures of CA and other 6 CACs of RDNI were shown in Fig. 2. The another reason was that the numbers of hydrogen bond donors and acceptors of CGA, IsoB and other CACs were more tha (...truncated)