Two pure fungal strains were isolated and identified from Ficus sycomorus and Morus nigra, namely, Penicillium oxalicum (OR673586) and Phoma herbarum (OR673589), respectively. The extract and fractions of secondary metabolites of each fungus were evaluated for antioxidant, anti-inflammatory, antimicrobial, antibiofilm, antidiabetic, and cytotoxic activities. The chloroform fraction of P. oxalicum showed potent cytotoxic activity (IC50 = 7.695 μg mL-1) against Hep-G2 cell line, alongside moderate antioxidant and anti-inflammatory activities. On the other hand, the P. herbarum chloroform fraction showed potent antioxidant (DPPH IC50 = 5.649 μg mL-1) and antidiabetic activities (IC50 = 14.91 μg mL-1) against inhibition of α-glucosidase, in addition to moderate cytotoxicity, anti-inflammatory, and antimicrobial activities. Guided cytotoxic fractionation leads to identifying bioactive compounds using hyphenated techniques. LC-MS identified fourteen compounds for P. herbarum and thirteen compounds for P. oxalicum. Three known compounds, mevalolactone (1), glycerol monolinoleate (3), and ergosterol (7) in addition to one new compound, barcelonyl acetate (2), were isolated from P. herbarum. On the other hand, four known compounds, 4-hydroxyphenyl acetic acid (4), secalonic acid D (5), altersolanol A (6), and ergosterol (7), were isolated from P. oxalicum. Altersolanol A (6) and secalonic acid D (7) exhibited outstanding cytotoxic activity against Hep-G2 and Caco-2 cell lines, with IC50 values ranging from 0.00038 to 0.208 μM. In silico study findings showed altersolanol A (6), 4-hydroxyphenyl acetic acid (4), glycerol monolinoleate (3), and barcelonyl acetate (2) displayed significant potential but may benefit from further optimization as lead for developing potent c-Jun N-terminal kinase 2 (JNK2, PDB: 3NPC) inhibitors, potentially leading to novel therapeutic strategies targeting cancer therapy.
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