Identification of Bioactive Components of Stephania epigaea Lo and Their Potential Therapeutic Targets by UPLC-MS/MS and Network Pharmacology

Abstract

Stephania epigaea, an important traditional folk medicinal plant, elucidating its bioactive compound profiles and their molecular mechanisms of action on human health, would better understand its traditional therapies and guide their use in preclinical and clinical. This study aims to detect the critical therapeutic compounds, predict their targets, and explore potential therapeutic molecular mechanisms. This work first determined metabolites from roots, stems, and flowering twigs of S. epigaea by a widely targeted metabolomic analysis assay. Then, the drug likeness of the compounds and their pharmacokinetic profiles were screened by the ADMETlab server. The target proteins of active compounds were further analyzed by PPI combing with GO and KEGG cluster enrichment analysis. Finally, the interaction networks between essential compounds, targets, and disease-associated pathways were constructed, and the essential compounds binding to their possible target proteins were verified by molecular docking. Five key target proteins (EGFR, HSP90AA1, SRC, TNF, and CASP3) and twelve correlated metabolites, including aknadinine, cephakicine, homostephanoline, and N-methylliriodendronine associated with medical applications of S. epigaea, were identified, and the compounds and protein interactions were verified. The key active ingredients are mainly accumulated in the root, which indicates that the root is the main medicinal tissue. This study demonstrated that S. epigaea might exert the desired disease efficacy mainly through twelve components interacting via five essential target proteins. EGFR is the most critical one, which deserves further verification by biological studies.

Figure 1

Widely targeted metabolomic profiling identified the metabolites in the tissues of Stephania epigaea. (a) PCA score diagram of mass spectrometry data of each group of samples, where StR represents root, StS represents stem, and StF means flower. (b) Differential cumulative distribution of various metabolite categories in the three tissues. (c) Venn diagram of group differences. (d) Differential metabolite cluster heatmap. The cluster tree on the left side of the figure is the differential metabolite cluster tree. Different colors are the relative content. The values are obtained after standardized treatment (red represents high content, and green represents low content).

Figure 2

(a) The target-pathway-disease interaction network and (b) the target-(pathway)-target interaction network with modularity partition by Gephi with Louvain algorithm, where the nodes were targets and the edges were the shared pathways of these targets.

Figure 3

The contribution scores (CSs) of each module to various diseases.

Figure 4

The cartoon diagram of EGFR structure and interactive maps of compounds inside its active site. (a) The overall structure of EGFR (PDB ID: 1M17). (b) The ATP-binding pocket of EGFR is shown as mash surface. (c) Amino acid regions in the active site of EGFR. Molecular interactions of EGFR with (d) erlotinib, (e) 3, 4-dihydroxy-DL-phenylalanine, (f) citrulline, (g) pratensein, (h) tyrosine, (i) homostephanoline, (j) feruloyltyramine, (k) N-feruloylputrescine, (l) L-phenylalanine, (m) caffeic acid, (n) N-methylliriodendronine, and (o) aknadinine. The protein structures were shown in the rainbow-colored cartoon. The amino acid residues at the active sites were shown as colored lines with names and sequence numbers. The hydrogen bonds were shown as yellow dashed lines with distance values in angstrom. The compound structures were shown as colored sticks. Carbon atoms and carbon-carbon bonds were green colored, oxygen atoms were red colored, hydrogen atoms were grey colored, and nitrogen atoms were blue colored.

Figure 5

Integrated network for mechanisms of traditional folk medicine applications for S. epigaea. Each column represents plants, tissues, metabolites, targets, and diseases from left to right.

Figure 6

Accumulation of twelve compounds in the three tissues, where StR represents root, StS represents stem, and StF means flower.