Herbal Extract-Induced DNA Damage, Apoptosis, and Antioxidant Effects in <em>C. elegans</em>: A Comparative Study of <em>M. longifolia</em>,<em> S. orientalis</em>, and <em>E. biebersteinii</em>

Background: Medicinal herbs are increasingly used as functional foods and therapeutics, yet their biological effects and potential toxicity remain incompletely understood. Using Caenorhabditis elegans, we previously identified several herbal extracts with potent cytotoxic effects. Methods: We focused on three herbal extracts—Mentha longifolia, Scrophularia orientalis, and Echium biebersteinii—to investigate their impact on germline development and fertility. Phenotypic analyses, apoptosis quantification, qRT-PCR, and LC-MS profiling were performed to assess cytotoxicity, meiotic defects, DNA damage responses, and chemical composition. Results: All three extracts significantly reduced worm survival, induced larval arrest, and increased the high incidence of males phenotype, suggesting chromosomal mis-segregation. Germline defects included disorganized nuclei, altered meiotic progression, and reduced bivalent formation. These were accompanied by upregulation of DNA damage checkpoint genes, increased pCHK-1 foci, and elevated apoptosis in the pachytene region. We identified 21 major compounds in three herb extracts, with four shared across all extracts. Among them, thymol and carvyl acetate activated checkpoint responses and apoptosis, while thymol and luteolin-7-O-rutinoside exhibited antioxidant activity. Conclusion: These findings emphasize the value of dissecting complex herbal mixtures to uncover specific bioactive compounds, which is essential for evaluating both the therapeutic potential and safety risks of medicinal plants. Also, this study underscores the need to analyze individual phytochemicals within herbal mixtures to understand their distinct biological effects. Such insight is essential for evaluating both the therapeutic potential and safety risks of medicinal plants used in food and supplements.