The past decade has witnessed the overwhelming interest in isolation and characterization of bioactive compounds from medicinal mushrooms due to the fact that natural products of mushroom origin are found to possess a remarkably high pharmacological index [
26,
27]. The mounting evidences from various scientific studies regarding the applications of mushroom extracts and substances in the development of anti-cancer therapeutics unarguably make it a fast-track research area worth mass attention. Consequently, mushroom consumption has been shown to prevent the development of cancer later in life with studies reporting an inverse correlation between mushroom intake and the risk of developing cancer [
28,
29]. In this perspective, the current study has been succeeded in the isolation of a potent cytotoxic compound namely ergone which is derived from a Sri Lankan mushroom. Ergone is an essential steroid, which belongs to ergosterol group [
30]. Since it is necessary to expand the current extraction protocols with the increasing interest in mushroom metabolites, a novel and easy isolation method have been optimized in the current invention to isolate ergone with high reproducibility
. Specifically, the extraction and isolation process of ergone is rapid, simple, inexpensive and comprehensive with respect to the constituents to be isolated.
In vitro cytotoxic activity of isolated product ergone against RD and HepG-2 cells were determined by MTT cell viability assay, which is a well-established colorimetric method to assess the cell proliferation and cell viability. It has been revealed that ergone isolated from F. fastuosus has promising cytotoxic properties against RD cells with less cytotoxicity effect on normal CC-1 cells (SI = 15.44). Moreover, ergone also showed strong cytotoxic activity against HepG-2 cells being less toxic to normal CC-1 cells. However, cytotoxic activity of ergone against RD cell line is significantly high compared to HepG-2 cells (p < 0.05). The apoptotic features observed via microscopic examination of treated cells and AO/EB fluorescent staining evidenced the apoptosis induced by ergone against RD and HepG-2 cells. Morphological characterization and ethidium bromide/acridine orange staining of treated cells revealed that the cell death induced by ergone has mediated through apoptosis. It strengthens the results obtained via MTT cell viability assay.
Our previous study has revealed that the crude methanolic extract of
F. Fastuosus possess cytotoxic activity against RD cell line with an IC
50 of 14.72 ± 1.56 μg/mL [
31]. Ergone’s cytotoxic activity against RD cell line (584.01 ± 2.74 ng/mL) has become twenty five times greater than the cytotoxic activity shown by the crude extract of
F. fastuosus in terms of IC
50 against RD cell line. Hence, it can be concluded that the promising cytotoxic activity of
F. fastuosus could be largely contributed by the remarkably high cytotoxic activity of ergone. In addition, mangiferin, a strong anticancer compound isolated from
Mangifera indica has shown an inhibitory concentration (IC
50) of 33.40 μg/mL against RD cells [
32]. Interestingly, antiproliferative activity shown by ergone was found to be significantly greater (
p < 0.05) than the anticancer activity of mangiferin. Importantly, the present discovery has become the first study of isolating ergone from
F. fastuosus and it is appeared to be an essential anti-tumor drug lead against rhabdomyosarcoma. Currently, the treatment regime of rhabdomyosarcoma comprises of chemotherapy and radiation along with surgery while producing adverse side effects including neurotoxicity, hepatotoxicity and cardiotoxicity [
33]. Hence, the use of afore kind of natural products against RD is an extremely promising strategy for remedy of rhabdomyosarcoma. Intriguingly, most of the mushrooms belong to the family of Hymenochaetaceae have long been used as edible forms [
34]. Therefore, ergone, which is isolated from family Hymenochaetaceae, has a greater potential to act safely in human body without creating any complications. In addition, this fact was supported by the high selectivity index shown by the ergone against RD sarcoma cells.