Isolation and characterization of a bioactive compound from Sphingomonas sanguinis DM with cytotoxic and molecular...

Datura metel, a common plant in the Solanaceae family, is known for its valuable medicinal properties. The metabolites created by its rhizosphere bacterium, Sphingomonas sanguinis DM, have garnered interest for their potential biological effects. This study will discuss the steps involved in fermenting and processing a bacterial strain to extract potent secondary metabolites. The ethyl acetate extract of the propagated strain was subjected to fractionation and purification through various chromatographic techniques. The purified compound was characterized through multiple spectroscopic methods for structure elucidation, including UV, MS, 1D, and 2D-NMR. Its cytotoxic activity was assessed on malignant skin cells (A-431) using the MTT test compared with normal melanocytes (HFB 4). Furthermore, A-431 cells were double-stained with PI and annexin V-FITC and analyzed by flow cytometry to detect Apoptosis. Molecular investigations include PCR screening to detect genes related to the biosynthesis of bioactive metabolites, such as NRPS and lipopeptide ItuD genes. A prospective effective strategy to overcome tumor plasticity in melanoma is to target the Wnt signaling pathways. Molecular docking studies were conducted in the different proteins (Fz4-CRD, LRP6, GSK3β) of the Wnt signaling pathway and Protein Kinase B/Akt for the isolated compound to investigate the possible pathway to inhibit melanoma. Sphingomonas sanguinis DM produced bis (2-methylheptyl) benzene-1,4-dicarboxylate isolated for the first time from a natural source. It was cytotoxic against the A-431 human skin carcinoma cell line (IC₅₀ = 191.61 µg/mL) but less effective against HFB 4 human normal melanocytes (IC₅₀ = 416.23 µg/mL; selectivity index = 2.17). The A-431 cells showed a significant increase in early Apoptosis and a moderate rise in late Apoptosis. PCR amplification confirmed genes encoding A domain and Iturin A. Bacterial sequences are available in NCBI GenBank with accession codes OR597597 and OR597598. Consequently, Sphingomonas sanguinis DM synthesized a cytotoxic natural terephthalate diester derivative, along with the host specificity of the strain.