Reagents and materials
1-ethyl-3-(3-dimetylaminopropyl)-carbodiimide hydrochloride (EDC) was obtained from Shanghai Beinuo Biotechnology Co. Ltd. (Shanghai, China), N-hydroxysuccinimide (NHS) was obtained from Shanghai Dibai Biotechnology Co. Ltd. (Shanghai, China), Sodium acetate and dimethyl sulfoxide (DMSO) were obtained from Beijing Chemical Plant (Beijing, China), Ethanolamine hydrochloride was purchased from Shanghai Sanying Chemical Reagent Co. Ltd. (Shanghai, China), the protein TNF-a was provided by RD Biosciences (America), Artemisinin, Scopoletin, Arteannuin B and Artemisic acid were provided by Chengdu Ruifensi Biological Technology Co., Ltd. (Chengdu, China), Dulbecco’s phosphate buffered saline PBS buffer (PH4.7) were freshly prepared.
Plasma sample preparation
Weigh accurately 40mgEDC and 10 mg NHS, make up to 1 mL with distilled water, and dissolve. Rinse the two channels thoroughly and quickly inject the above solution. The injection time is 5 minutes and rinse with PBS buffer.
50 µg of TNF-a protein was dissolved in 100 µL of PBS, and 10 µL of the above solution was taken in three portions and diluted with sodium acetate solutions having pH values of 5.5, 6.0, and 6.5, so that the final concentration was 50 µg/ml. Reduce the flow rate to 20 µL/min and rinse the left channel for 10 minutes to determine the optimal pH of sodium acetate.
After determining the optimal pH value, 1M ethanolamine hydrochloride was injected into the two channels for 10 minutes to complete the sample fixation.
The four chemical composition Scopoletin (A), Artemisinin(B), Artemisic acid (C) and Arteannuin B (D) were divided into 12 groups according to the combination of A, B, C, D, AB, AC, AD, ABC, ABD, ACD, BCD, and ABCD, and each group was set to 6 concentrations. The control group was PBS buffer at pH 7.4.
Accurately weigh 19.2 mg of scutellaria lactone, 28.2 mg of artemisinin, 24.8 mg of artemisinin 2 and 23.4 mg of artemisinin, respectively, and dissolve them in 1 mL of DMSO (dimethyl sulfoxide). The DMSO solution in each group was gradient diluted with PBS to a final concentration of 200 µM, 66.7 µM, 22.2 µM, 7.41 µM, 2.47 µM.
Targets fishing
Known therapeutic targets for the treatment of malaria were obtained from the DrugBank database (http://www.drugbank.ca/, version, 4.3) [15]. Only the “antimalaria” was selected as the key word, and the drug-target interactions whose drugs are approved by the Food and Drug Administration, USA (FDA) for treating menstrual disorders. All target gene/ protein identifiers (IDs) were converted into the correspondinggene symbol/UniProtKB-Swiss-Prot IDto facilitate further data analyses. After removing redundant entries, 25 target genes corresponding to 15 known antimalarial drugs were retrieved.
Protein‑protein interaction (PPI) data
PPI data were imported from the following PPI databases, including Human Annotated and Predicted Protein Interaction Database (HAPPI, http://bio.informatics. iupui.edu/HAPPI/, Version 31.2) [16]. Based on the PPI network database, an interaction network of Artemisia annua candidate target groups and known antimalarial drug target groups was constructed. The distribution of target nodes in metabolic pathways and the corresponding diseases are revealed. A direct interaction network of key nodes was established, and the network was divided into modules according to the functions of the nodes. According to the malaria pathway (ko05144: Malaria) in KEGG, candidate verification molecules closely related to the malaria pathway were selected from the key nodes.
Network construction and topological analysis
Compound–target (C-T), target–pathway (T-P), and target–disease (T-D) networks about malaria were constructed using the Cytoscape 3.2 software (https://cytoscape. org/download.html), a general bioinformatics software package for data integration and visualization of biological network (Bindea et al., 2009; Smoot et al., 2011). An interaction network of Artemisia annua candidate target genes with known antimalarial drug target genes was established, consisting of 85 nodes and 298 pairs of interactions. Calculate the topological characteristic value of each node in the network, and use the median of the topological characteristic value as the card value. A total of 32 key nodes are screened. A direct interaction network of key nodes is established, and the network is processed according to the node functions. The module is divided, and the malaria pathway in Kyoto Encyclopediaof Genesand Genomes (KEGG) (ko05144: Malaria) is compared, and candidate verification molecules closely related to the malaria pathway are selected from key nodes.
Molecular docking.
The molecular structure of CDK4, NFKB1, PIK3CG, MAPK1, TNF and ITGB2 protein targets (protein species is human) is searched in the database uniprot (http://www.uniprot.org/). The structures of scopolamine and artemisinic acid are downloaded from the pubchem database (https://pubchem.ncbi.nlm.nih.gov). Chemical composition and protein structure are dehydrated and hydrotreated. Molecular docking and figures were generated Discovery Studio Visualizer software.
Probe Kd determination.
Weigh accurately 40 mg EDC and 10 mg NHS, make up to 1 mL with distilled water, and dissolve. Rinse the two channels thoroughly and quickly inject the above solution. The injection time is 5 minutes and rinse with PBS buffer. The protein TNF-a immobilized on Grafted Sensor Chips. The compound monomers and combinations are divided into 12 groups (Table 1). Each group of samples was injected at a concentration from low to high, and a control group (PBS) was set between each concentration. Regression analysis when concentration curves are separated and approaching equilibrium. The dissociation constant (Kd) and its maximum value (Bmax) were then calculated by fitting the titration curve to the single-site saturation binding equation [Y = Bmax*X/(Kd + X)] using the GraphPad Prism software (Graphpad software Incorporated, La Jolla, CA, USA).
Table 1
The compound monomers and combinations
Group | compounds |
A | Scopoletin |
B | Artemisinin |
C | Artemisic acid |
D | Arteannuin B |
AB | Scopoletin ;Artemisinin |
AC | Scopoletin; Artemisic acid |
AD | Scopoletin; Arteannuin B |
ABC | Scopoletin; Artemisinin ;Artemisic acid |
ABD | Scopoletin; Artemisinin; Arteannuin B |
ACD | Scopoletin; Artemisic acid; Arteannuin B |
BCD | Artemisinin ;Artemisic acid; Arteannuin B |
ABCD | Scopoletin; Artemisinin; Artemisic acid; Arteannuin B |