Isolation, structure elucidation and increasing anticancer efficacy of an anti-cancer secondary metabolite from a marine-derived endophytic fungus, Aspergillus species

Abstract

Cancer is the leading cause of mortality globally, contributing to ~10 million fatalities in 2020, or roughly one in every six deaths, according to global cancer statistics 2020. Around 50% of all small molecules approved for treatment of cancer between the 1940s and the end of 2014 are comprised of either naturally occurring substances or compounds that were synthesized using those substances. The majority of bioactive chemicals are obtained from terrestrial microorganisms, and while the terrestrial environment is abundant in bioactive producers, the finding of new metabolites is withering. Marine microorganisms are a possible sustainable source of new physiologically bioactive chemicals since the biodiversity of the oceans makes up 50% of the world's total biodiversity. The endophytic fungal community in the marine ecosystem has been demonstrated to be a relevant source of novel and pharmacologically active secondary metabolites. This study aimed to identify potential lead compounds for therapeutic development by evaluating natural compounds produced by endophytic fungi of marine algae, with an emphasis on chemotherapeutic leads. The seaweed S. muticum, a brown alga was collected from the Kovalam Coast, Thiruvananthapuram, Kerala, India. To accomplish this, five fungal species associated sargassum muticum were isolated, grown, and their extracts evaluated for anticancer potential. Two fungal species out of five were chosen as they exhibit the best cytotoxicity on HeLa (based on IC50). The two fungi, Aspergillus sp. and Talaromyces purpureogenus, were grown in liquid media, extracted with ethyl acetate, and their ethyl acetate extracts were tested for their potential to induce cell death on the HeLa cell line. Aspergillus sp, which showed the best cytotoxicity was used for purifying specific compounds responsible for cytotoxicity. Aspergillus sp, ethyl acetate extract as an organic solvent was used for the bioactivity-guided fractionations that resulted in the identification of an anticancer compound. The compound was successfully isolated in significant proportions using silica gel column chromatography, followed by crystallization of the Aspergillus sp ethyl acetate extract. Single crystal XRD, 1D NMR (1H, 13C, DEPT), FT-IR and HRMS analysis revealed that the structure of the isolated molecule is kojic acid (5-Hydroxy-2-(hydroxymethyl)-4H-pyran-4-one). The cytotoxic potential of the kojic acid was studied in vitro against A-431 (human skin carcinoma). Kojic acid-induced cell death on the A431 cell line in a concentration-dependent and Reactive oxygen species (ROS)-independent manner. To enhance the efficacy of kojic acid as a potent anti-cancer compound and solve its limitations. Carbon dots (CDs) are photoluminescent nanostructures with numerous applications. Carbon dots derived from kojic acid were synthesized hydrothermal method and examined using XPS, TEM, and FT-IR. The optical properties were analysed by spectrophotometer and spectrofluorometer. The CDs were tested for cytotoxicity on HeLa, A549, A375, and MDA MB 231 cancer cell lines. The CDs showed best cytotoxicity on MDA MB 231 cell line. Cell death induced by CDs in MDA MB 231 cells is independent of ROS, results in increased mitochondrial membrane depolarization, and arrests cell cycle progression at the sub G1 phase. This opens new avenues for investigating their potential use as anticancer drugs in the treatment of cancer development.