Antioxidant and cytotoxic activities of extracts from rare saprobic fungi, Melanographium smilacis, Helminthosporium chiangraiense, Pleopunctum thailandicum, and Pseudochaetosphaeronema chiangraiense.

Saprobic fungi remain underexplored sources of bioactive secondary metabolites with pharmaceutical potential. This study presents the first biological evaluation of four rarely studied species, Melanographium smilacis, Helminthosporium chiangraiense, Pleopunctum thailandicum, and Pseudochaetosphaeronema chiangraiense, isolated from submerged plant material in northern Thailand. Crude ethyl acetate extracts were examined for antioxidant capacity, cytotoxicity, oxidative stress, and apoptosis-related effects. All extracts exhibited dose-dependent DPPH scavenging ranging from 47 to 89%. The strongest activity was recorded for M. smilacis and P. thailandicum, both approaching the inhibition level of ascorbic acid. Cytotoxicity assays revealed selective viability reduction in A549 lung carcinoma cells, with up to 40% inhibition at higher concentrations, while NIH3T3 fibroblasts were largely unaffected, indicating limited toxicity toward non-malignant cells. Acridine orange/ethidium bromide staining and ROS assays demonstrated oxidative stress and apoptotic features in A549 cells, particularly after treatment with M. smilacis and H. chiangraiense. Nevertheless, apoptosis induction remained quantitatively weak compared with the positive control, suggesting only preliminary pro-apoptotic potential. These findings suggest that the studied fungi harbor metabolites associated with antioxidant activity and selective cytotoxic effects. This work establishes a biological baseline for these taxa and highlights the need for bioactivity-guided fractionation, and mechanistic validation to determine their pharmacological relevance.