Drug resistance to the classically used chemotherapeutic drugs is the major challenge in their treatment of cancer needing the discovery of novel anticancer drugs. In terms of finding novel therapeutics, endophytes are quite promising as they are an excellent source of novel structures, which exhibit bioactivity. The present study demonstrated a dose-dependent antiproliferative activity of mycelial-derived secondary metabolites from a macroalgae associated endophyte Aspergillus unguis AG 1.1 (G). The antiproliferative activity of A. unguis mycelial extract (AUME) was observed on different human cancer cell lines. PI live/dead assay further confirmed the cytotoxic potential of the mycelial extract. Furthermore, AUME caused mitochondrial membrane aberration and generated ROS production as well indicating its potential to induce cell death by apoptosis. The metabolic profiling of the mycelial extract using GC-MS and LC-MS/MS revealed the presence of fatty acids, a benzoquinolinone derivative, imidazolidinedione derivative, diethyl phthalate and phthalate acid ester, a difuraxanthone, two prenylxanthone analogs and a phthalide derivative and some unknown metabolites. Presence of 4-(4-Hydroxy-3,5-dimethoxy-phenyl)-3,4-dihydro-1H-benzo[h]quinolin-2-one, 1-hydroxy-3,5-dimethoxy-2-prenylxanthone, 1,6-dihydroxy-3-methoxy-2-prenylxanthone and 3-butylidene-7-hydroxyphthalide in AUME could be correlated to the notable cytotoxicity exhibited by the endophyte. The additional presence of many unidentified compounds heightened the prospects of finding some novel bioactive metabolites. Our results indicated that secondary metabolites produced by A. unguis AG 1.1 (G) have therapeutic potential as anticancer agents.

The Western Ghats, India, is a hotspot for lichen diversity. However, the pharmacological importance of lichen-associated metabolites remains untapped. This study aimed to evaluate the cytotoxic potential of lichens of this region. For this, sixteen macrolichens were collected and identified from two locations in the Western Ghats. The acetone extract of Usnea cornuta (UC2A) showed significant cytotoxicity towards multiple human cancer cell lines. Interestingly, co-treatment with chloroquine (CQ), an autophagy inhibitor, increased the cytotoxic potential of the UC2A extract. A gas chromatography mass spectrometry (GCMS) study revealed usnic acid (UA), atraric acid and barbatic acid as the dominant cytotoxic compounds in the UC2A extract. Further, UA was purified and identified from the UC2A extract and evaluated for cytotoxicity in HeLa cells. The monodansyl cadaverine and mitotracker red double staining revealed the autophagy-inducing activities of UA, and the inhibition of autophagy was confirmed via CQ treatment. Autophagy inhibition increased the cytotoxicity of UA by 12–16% in a concentration-dependent manner. It also increased lipid peroxidation, ROS levels and mitochondrial depolarization and decreased glutathione availability. A decrease in zeta potential and a 40% increase in caspase 3/7 activity were also noted after CQ treatment of UA-treated cells. Thus, cytotoxicity of UA can be increased by inhibiting autophagy.

Flavonoids encompass a heterogeneous group of secondary metabolites with exceptional health benefits. Chrysin, a natural dihydroxyflavone, possesses numerous bioactive properties, such as anticancer, antioxidative, antidiabetic, anti-inflammatory, etc. However, using traditional sources of chrysin involves extracting honey from plants, which is non-scalable, unsustainable, and depends on several factors, including geography, climatic conditions, and the season, which limits its production at a larger scale. Recently, microbial production of desirable metabolites has garnered attention due to the cost-effectiveness, easy scale-up, sustainability, and low emission of waste. We previously reported for the first time the chrysin-producing marine endophytic fungus Chaetomium globosum, associated with a marine green alga. To extend our understanding of chrysin biosynthesis in C. globosum, in the present study, we have assessed the presence of flavonoid pathway intermediates in C. globosum extracts using LC-MS/MS. The presence of several key metabolites, such as dihydrokaempferol, chalcone, galangin, baicalein, chrysin, p-Coumaroyl-CoA, and p-Cinnamoyl-CoA, indicates the role of flavonoid biosynthesis machinery in the marine fungus. Further, we have aimed to enhance the production of chrysin with three different strategies: (1) optimizing the fermentation parameters, namely, growth medium, incubation time, pH, and temperature; (2) feeding key flavonoid pathway intermediates, i.e., phenylalanine and cinnamic acid; (3) elicitation with biotic elicitors, such as polysaccharide, yeast extract, and abiotic elicitors that include UV radiation, salinity, and metal stress. The combined effect of the optimized parameters resulted in a 97-fold increase in the chrysin yield, resulting in a fungal cell factory. This work reports the first approach for enhanced production of chrysin and can serve as a template for flavonoid production enhancement using marine endophytic fungi.