Mycochemistry, antioxidant activity and anticancer potentiality of ethyl acetate extract of Daldinia eschscholtzii against A549 lung cancer cell line

Recent studies have highlighted the potential of various mushroom or fungal species in promoting health and combating cancer through their bioactive compounds. This growing body of evidence encourages further exploration into their mechanisms of action and possible applications in cancer prevention and treatment. Fruit bodies of wild fungus Daldinia eschscholtzii were extracted in ethyl acetate solvent (purity: 99.9%), then lyophilized to powder known as Daldinia ethyl acetate extract (DEAE). LC-MS (liquid chromatography mass spectrum) was used to detect the bioactive compounds present in DEAE. Antioxidant contents and activity of DEAE were determined by standard methods. The anticancer activity of this extract was evaluated against the A549 cell line (lung cancer) by MTT assay, apoptosis (flow cytometry), and expression of pro- and anti-apoptotic genes; anti-migration was determined by qPCR. The drug likeness of DEAE was evaluated by WebPredictor (SwissADME). Mycochemistry analysis by LC-MS demonstrated that DEAE was a mixture of a total of 28 compounds, among them 8 were flavonoids, 3 polyphenols, and 8 alkaloids. The TPC (total phenolic content), TFC (total flavonoid content), and TTC (total tannin content) of DEAE were 56.7 ± 1.3 mg GAE/g dry weight, 24.0 ± 0.28 mg QE/g dry weight, and 10.39 ± 0.24 mg TAE/g dry weight of the extract, respectively. The total antioxidant capacity (TAC) was 110.2 ± 1.3 mg AAE/g dry weight of the DEAE. The EC₅₀ value for DPPH scavenging was 3.55 ± 0.02 mg/mL. Cytotoxicity assay exhibited anti-proliferation activity with IC₅₀ values of 149.80 ± 0.76, 104.60 ± 1.43, and 87.86 ± 2.29 µg/mL against the A549 lung cancer cell line at 24, 48, and 72 h, respectively. The mechanism of the anticancer effect of DEAE on the cancer cell line (A549) included induction of apoptosis and a change of gene expression levels of Caspase 3, Caspase 9, p53, and Bcl 2 of the cell line. The qPCR study showed that Caspase 3, 9, BAX, and p53 were up-regulated, whereas Bcl 2 was down-regulated after the treatment with DEAE (50, 150, and 200 µg/mL) for 24 h. It had an anti-migration activity, which was justified by the downregulation of MMP 2 & 9 genes. The physicochemical, pharmacokinetic, and medicinal properties of ten compounds in DEAE were screened by web predictors (SwissADME), and among them, skimmianine, lycorine-diacetate, isocorydine, glycyrrhetic acid Me ester, kaempferol-3-O-rhamnoside, and apigenin compounds followed the rules of 5 (Lipinski filter). Skimmianin, glycyrrhetic acid Me ester, and apigenin were found to inhibit some members of the cytochrome family, like CYP1A2, CYP2D6, CYP3A4, CYP2C19, and CYP2C9, indicating their anti-drug-resistant properties. These findings suggest that DEAE may serve as a promising natural therapeutic as an antioxidant and also as an anticancer and anti-metastasis candidate against lung cancer.