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. 2023 Sep 12;23(1):317.
doi: 10.1186/s12906-023-04146-x.

Combination of bioaffinity ultrafiltration-UFLC-ESI-Q/TOF-MS/MS, in silico docking and multiple complex networks to explore antitumor mechanism of topoisomerase I inhibitors from Artemisiae Scopariae Herba

Tong Chen  1 , Jingbo Hu  2 , Huan Wang  3 , Nana Tan  1 , Jianzhao Qi  4 , Xiaoling Wang  1 , Le Wang  5
Affiliations

Combination of bioaffinity ultrafiltration-UFLC-ESI-Q/TOF-MS/MS, in silico docking and multiple complex networks to explore antitumor mechanism of topoisomerase I inhibitors from Artemisiae Scopariae Herba

Tong Chen et al. BMC Complement Med Ther. .

Abstract

Background: Artemisiae Scopariae Herba (ASH) has been widely used as plant medicine in East Asia with remarkable antitumor activity. However, the underlying mechanisms have not been fully elucidated.

Methods: This study aimed to construct a multi-disciplinary approach to screen topoisomerase I (topo I) inhibitors from ASH extract, and explore the antitumor mechanisms. Bioaffinity ultrafiltration-UFLC-ESI-Q/TOF-MS/MS was used to identify chemical constitution of ASH extract as well as the topo I inhibitors, and in silico docking coupled with multiple complex networks was applied to interpret the molecular mechanisms.

Results: Crude ASH extract exhibited toxicogenetic and antiproliferative activities on A549 cells. A series of 34 ingredients were identified from the extract, and 6 compounds were screened as potential topo I inhibitors. Docking results showed that the formation of hydrogen bond and π-π stacking contributed most to their binding with topo I. Interrelationships among the 6 compounds, related targets and pathways were analyzed by multiple complex networks model. These networks displayed power-law degree distribution and small-world property. Statistical analysis indicated that isorhamnetin and quercetin were main active ingredients, and that chemical carcinogenesis-reactive oxygen species was the critical pathway. Electrophoretic results showed a therapeutic effect of ASH extract on the conversion of supercoiled DNA to relaxed forms, as well as potential synergistic effect of isorhamnetin and quercetin.

Conclusions: The results improved current understanding of Artemisiae Scopariae Herba on the treatment of tumor. Moreover, the combination of multi-disciplinary methods provided a new strategy for the study of bioactive constituents in medicinal plants.

Keywords: Artemisiae Scopariae Herba; In silico docking; LC-MS; Multiple complex networks; Topoisomerase I inhibitors.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
ASH extract promotes viability deficiency and apoptosis of A549 cells. a, Cytotoxicity of ASH extract to A549 cells by SRB assay. Cells were treated with ASH extract for 48 h, and that quercetin was used as positive control. b, Nuclear morphological changes by Hoechst 33,342 staining. The phase contrast (top) and fluorescence (bottom) images were collected using fluorescence microscopy
Fig. 2
Fig. 2
Potential topo I inhibitors identified from ASH extract. a, ESI-MS/MS spectrum and fragmentation pathways of eupatilin. b, Chemical structures of 6 potential topo I inhibitors from ASH extract
Fig. 3
Fig. 3
Binding profiles of topo I inhibitors from ASH extract. PDB ID of topo I is 1T8I. a, Interactions of hydroxygenkwanin and topo I. b, Interactions of chlorogenic acid and topo I. Light pink line indicates π-alkyl ineraction. Dark pink line indicates π-π stacked. Orange line indicates π-anion interaction. Light green line indicates carbon hydrogen bond. Green line indicates van der Waals force. Dark green line indicates conventional hydrogen bond
Fig. 4
Fig. 4
Ingredient-pathway interaction (IPI) network. Purple nodes indicate topo I inhibitors from ASH extract. Green nodes indicate the pathways related to topo I inhibitors from ASH extract. Gray lines indicate their connections
Fig. 5
Fig. 5
Degree distribution diagram of IPI network. The symbol k indicates degree, and that P(k) indicates degree distribution
Fig. 6
Fig. 6
Ingredient-ingredient interaction (TTI) network. Purple nodes indicate topo I inhibitors from ASH extract. Gray lines indicate their internal connections
Fig. 7
Fig. 7
Bipartite graph of the ingredient-pathway bimodal (IPB) network
Fig. 8
Fig. 8
Degrees sorted by descending orders for nodes of IPB network
Fig. 9
Fig. 9
Pathway-pathway interaction (PPI) network. Green nodes indicate the pathways related to topo I inhibitors from ASH extract. Gray lines indicate their internal connections
Fig. 10
Fig. 10
Features of the PPI network. a, Average clustering coefficient for nodes in the PPI network. The x-axis indicates degree k, and that y-axis indicates the clustering coefficient C(k). b, Centrality analysis of nodes in the PPI network, including degree centrality (Cd), betweenness centrality (Cb) and closeness centrality (Cc)
Fig. 11
Fig. 11
Topo I inhibitory binding assay by agarose gel electrophoresis. Lane “D” represents pure pBR322 DNA (0.175 µg). “T” is the mixture of topo I (0.5 U) and pBR322 DNA (0.175 µg). The same amounts of topo I and DNA were added to all the other lanes. Lanes “A” represents the addition of ASH extract (2.0 µg), and that “C” indicates camptothecin (1.4 µg, positive control). Lanes “M”, “Q”, “I”, represent the addition of the mixture of 1.0 µg quercetin and 1.4 µg isorhamnetin, quercetin (1.0 µg), isorhamnetin (1.4 µg), respectively
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