Alina Gazizova, Lone Tuchtenhagen, Nadja Engel, Udo Kragl, Christina Oppermann
Malva sylvestris L. is a medicinal plant with reported antioxidant, anti-inflammatory and wound healing properties, as well as potential anticancer properties; however, the relationship between its phytochemical composition and biological activity remains insufficiently understood. This study aimed to combine analytical characterization with functional in vitro assays to assess the biological effects of a Malva sylvestris L. extract on head and neck cancer cell models. A targeted LC-MS/MS method was developed and validated to study selected flavonoids (apigenin-7-O-glucoside, vitexin, genistin and apigenin) in the extract. However, the investigation revealed only trace quantities of the selected flavonoids. Complementary MS/MS-based profiling enabled the tentative identification of additional constituents, including flavone derivatives such as naringenin and naringenin-7-O-glucoside. Biological activity of the extracts was evaluated using metabolic activity assays, flow cytometry and electric cell-substrate impedance sensing (ECIS). The extract induced pronounced concentration-dependent reduction in viability in tumorigenic cell lines, accompanied by impaired proliferation dynamics and an increased Sub G1 cell population. These effects were less pronounced but still observable in non-tumorigenic cells, indicating differential rather than selective cytotoxicity. The combined data from metabolic, impedance-based and cell cycle analyses consistently point to disruption of cellular homeostasis and induction of cell death-associated processes. Importantly, the low abundance of the targeted flavonoids precludes a direct attribution of the observed biological effects to these compounds. Instead, the findings support a model in which the activity arises from other constituents or synergistic interactions within the complex phytochemical extract. In summary, this study integrates validated analytical methodology with complementary biological assays and provides evidence for differential cytotoxic effects of Malva sylvestris L. extracts in vitro . The results underscore the importance of considering the phytochemical complexity of plant extracts and establishing a foundation for future mechanistic investigations.