Integrated Network Pharmacology, Molecular Modeling, LC-MS Profiling, and Semisynthetic Approach for the Roots of Rubia tinctorum L. Metabolites in Cancer Treatment

Abstract

Rubia tinctorum L. is one of the most widely used plants in folk medicine, with many reported pharmacological activities. One of these valuable activities is its anticancer efficacy. The aim of this study is to explore the multilevel mechanisms of R. tinctorum metabolites in cancer treatment using network pharmacology, together with molecular docking and in vitro studies. The network pharmacology analysis enabled us to reveal the hit anticancer R. tinctorum constituents, which were found to be acacetin, alizarin, anthragallol, 2-hydroxyanthraquinone, and xanthopurpurin. The most enriched cancer-linked target genes were PLCG1, BCL2, CYP1B1, NSD2, and ESR2. The pathways that were mostly involved in the anticancer mechanism of R. tinctorum metabolites were found to be metabolic pathways as well as pathways in cancer and apoptosis. Molecular docking of the identified hit anticancer constituents on the active sites of the most enriched genes unveiled that acacetin and alizarin possessed the lowest binding energies on the active sites of NSD2 and BCL2, respectively. While anthragallol showed the most stabilized interaction on the active sites of PLCG1, CYP1B1, and ESR2. Consequently, R. tinctorum extracts were evaluated for their in vitro cytotoxicity on a panel of cancerous cells. Among the tested R. tinctorum extracts, the chloroform extract was the strongest one with an IC₅₀ = 3.987 μg/mL on the MCF-7 breast cancer cell line. Consequently, it was subjected to chromatographic separation and purification to isolate its major components with reported anticancer activity (scopoletin, rubiadin, chrysophanic acid, alizarin, purpurin, nor-damnacanthal, emodin, and rutin). Alizarin and purpurin constituted the main anthraquinones in R. tinctorum . Thus, they were quantified using LC/MS analysis. Moreover, a semisynthetic approach of alizarin toward the enhancement of its anticancer effect on the tested cancer cells was attained. Among the synthesized compounds, 2-methyl alizarin was the most active one with an IC₅₀ = 8.878 μg/mL against the HepG2 cell line. This study provides deep insights into the anticancer mechanisms of R. tinctorum metabolites for the first time using network pharmacology and valorizes their significance as valuable anticancer agents.

Scheme 1. Overview Workflow of the Study

Figure 1

Distributions % of the compound–target gene (C–T) interactions on R. tinctorum constituents (A) and the identified cancer-related genes (B).

Figure 2

Compound–target–pathway network (compounds are represented in purple color, targets are presented in blue color, and pathways are presented in green color).

Figure 3

BIOCARTA (green) and KEGG (orange) pathways involved in cancer and generated by DAVID database (A). GO enrichment analysis of identified cancer targets. Biological processes are colored orange, cellular components are yellow, and molecular functions are green (B). The order of importance was ranked by −log 10 (adjusted P-value) with a bar chart. The number of targets stick into each term with a line chart.

Figure 4

2D and 3D interaction diagrams of (A) anthragallol in the active site of 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase gamma-1 (PDB ID 7NXE); (B) anthragallol in the active site of cytochrome P450 1B1 (PDB ID 6IQ5); and (C) anthragallol in the active site of estrogen receptor beta (PDB ID 2GIU).

Figure 5

2D and 3D interaction diagrams of (A) alizarin in the active site of the apoptosis regulator Bcl-2 (PDB ID 2Y6W); (B) acacetin in the active site of histone-lysine N-methyltransferase NSD2 (PDB ID 7VLN).

Figure 6

Chemical structures of the hit anticancer constituents isolated from R. tinctorum L.

Figure 7

Intramolecular H-bonding in the alizarin molecule.

Figure 8

Scheme for the synthesis of alizarin derivatives.

Figure 9

In vitro cytotoxic activity of R. tinctorum crude extracts, alizarin, purpurin, and alizarin derivatives using the MTT assay on normal cell line, HSF (A), breast cancer cell lines, MDA-MB-231 (B) and MCF-7 (C), and liver cancer cell line, HepG2 (D).