Effective Components-Targets-Mechanism-Chinese Prescription Strategy (ETMC), A New Strategy for Chinese Prescription Development: A Case Study in Danlou Tablet

10 Background: Chinese prescription is a combination medicine used by Chinese medical workers to 11 treat various diseases and has saved countless human lives. By studying the effective substances, 12 therapeutic targets, and mechanism of Chinese prescription, Western researchers can better 13 understand the great value of Chinese prescription. 14 Methods: In this study, a new strategy was proposed for the development of Chinese prescription: 15 the effective components (E)-targets (T)-mechanism (M)-Chinese prescription (C) strategy, namely 16 the ETMC strategy. 17 Results: The proposed strategy used the chemical compositions of the Chinese prescription as the 18 source of ligands to predict the corresponding targets for discovering the mechanism of Chinese 19 prescription, which was helpful to further screen out effective substances from the chemical 20 compositions of Chinese prescription. Here an example on development of Danlou tablet was 21 performed to introduce the application of ETMC. 22 Conlusions: A novel strategy for Chinese prescription development, ETMC, along with its 23 application was provided in the current study, which contributed to the further development, wider 24 application and internationalization of Chinese prescription.


Conlusions:
A novel strategy for Chinese prescription development, ETMC, along with its 23 application was provided in the current study, which contributed to the further development, wider 24 application and internationalization of Chinese prescription. 25 26 Background 27 Chinese prescription (CP), also known as Chinese formula (CF), is a special kind of medicine that 28 has been clinically used in China for thousands of years, contributed to the prosperity and civilization 29 of the China and the surrounding countries (Luan et al., 2020). Its advantages of definite curative 30 effects and acceptable adverse effects have gradually attracted the attention of Western medical 31 workers on CP (Xiong et al., 2017). But the concern of Western medical workers on CP has not yet 32 turned into full recognition, due to the lack of clear effective components, specific targets and exact 33 therapeutic mechanism for CP and even Chinese medicine (Chan et al., 2010;Hao et al., 2017). With 34 the rise of network pharmacology, this disadvantaged situation of CP has been partially improved 35 (Luo et al., 2020;Tao et al., 2013). However, the range of effective components, targets, and 36 mechanisms of the CP predicted by the classic network pharmacology strategy is too large to be 37 further narrowed, which makes it difficult to choose a suitable research direction from it, which 38 hinders the basic research and modernization of CP. However, the effective ingredients, targets, and 39 mechanisms determined by this strategy of studying the effective ingredients and mechanisms of 40 traditional Chinese medicine compound prescriptions through network pharmacology is too large to 41 be further narrowed, which makes it difficult for medical workers to choose a suitable research 42 direction from them, thereby Inhibit basic research and modernization of CP. Therefore, it is 43 imperative to propose a new CP development strategy that can help focus research directions. 44 Here, we propose the effective components (E)-targets (T)-mechanism (M)-Chinese prescription (

Molecular Docking 78
The three-dimensional geometric coordinates of the X-ray crystal structures of selected targets, 79 ACHE (PDB ID:

Prediction and Selection of Targets Corresponding to Chemical Compositions of DTL 103
Next, target fishing was performed to predict corresponding targets of all chemical compositions of 104 DLT. The results showed that a total of 529 targets were predicted, including the same target 105 predicted by different chemical components. Chinese medicine has the advantage of "multiple targets 106 effect". And the "multiple targets effect" here not only refers to multiple targets corresponding to 107 multiple chemical components, but also refers to a single target corresponding to multiple chemical 108 components. According to the "single-target superposition" theory of Chinese medicine, different 109 chemical compositions of traditional Chinese medicine can be combined with a single target one after 110 another in superposition effects of concentration and time differences, which is one of the key 111 reasons why traditional Chinese medicine can play a highly effective and long-lasting therapeutic 112 effect (Cai et al., 2015). Therefore, intersection analysis was performed on targets predicted by 113 different chemical compositions of DLT, and those targets that had at least five intersections were 114 selected as key targets for subsequent research (Figure 2). 115

Protein-protein Interaction Network (PPI) and Gene Ontology (GO) Analysis 116
In order to clarify the connection between key targets selected above and biological functions, PPI 117 network complex was constructed with selected key targets and their corresponding chemical 118 compositions of DLT (Figure 3). Gene ontology analysis (GO) is a commonly used for defining 119 genes or protein product to identify unique biological properties of high-throughput transcriptome or 120 genome data (Ashburner et al., 2000). Therefore, GO analysis was performed after construction of 121 PPI network. Results showed that about half of all key targets were particularly enriched in 122 regulation of lipid metabolic process and the oxidation-reduction process, which were the two main 123 biological processes that DLT could potentially regulate (Figure 3). 124

Effective Components Screening from Chemical Compositions of DLT 125
Furthermore, effective components of DLT were screened out from chemical compositions of DLT 126 via molecular docking. Results showed that certain targets were regulated by corresponding chemical 127 components of DLT, whose regulatory effects were better than the original ligands of the targets 128 ( Figure 4 and Supplementary Table 1). So far, the number of potential targets of DLT has been 129 significantly limited, and effective components of certain targets were determined among several 130 chemical compositions of DLT. 131 Chinese medicine has gradually attracted widespread attention from Chinese and Western medical 143 researchers due to its excellent efficacy and minimal side effects. Revealing the effective components 144

The Relationship between Key Targets and Effective Components of DLT against
of Chinese medicine not only helps the further development of Chinese medicine, but also facilitates 145 the modernization and internationalization of Chinese medicine. In this study, a new strategy for 146 Chinese prescription development, ETMC, was proposed to screen out the effective components with 147 potential therapeutic effects from the complex chemical compositions of traditional Chinese medicine, 148 which contributed to clear directions for Chinese medicine researches. 149 CP is a special administration mode of Chinese medicine based on the theory of traditional Chinese 150 medicine, which is a combination of different Chinese medicines to exert better, longer-lasting and 151 safer effects than using a single Chinese medicine alone. However, the unclear pharmacodynamic 152 mechanism and targets of CP limits the expansion of its indications, and this obstacle can be 153 eliminated through the proposed ETMC strategy, a combined application of computer simulation 154 technology, network pharmacology and bioinformatics. ETMC strategy is beneficial for 155 determination of the pharmacodynamic targets corresponding to effective components in CP. And 156 biological processes that involve these targets are useful to determine the mechanism CP. 157 Multiple studies reported that the association of chemical compositions of CP with potential targets 158 can be predicted through classic network pharmacology research process. However, it is difficult to 159 determine fewer but more valuable effective components and key targets from a large number of 160 chemical components and potential targets in the prediction results of network pharmacology. In the 161 example of applying the ETMC strategy in this study, a few effective components of DLT and their 162 targets closely related to cardiovascular diseases were identified. Most of effective components of 163 DLT are able to regulate multiple targets, which was consistent with "single-target superposition" 164 theory of Chinese medicine. And targets corresponding to effective components of DLT have also 165 been reported to be closely related to cardiovascular disease, the indication for DLT.