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Background/Objectives: Astragalus L. is a genus of the Fabaceae family, encompassing over 3000 species globally, with 380 species found in Turkey. This is the inaugural examination of the phytochemical, antioxidant, antibacterial, and cytotoxic properties of Astragalus pisidicus. Methods: The water and methanolic fractions of four parts (stems, flowers, leaves, root) as well as the whole plant were quantified and identified by Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometry (LC–ESI–MS/MS) analysis. Cell death was assessed using the WST-1 assay, while apoptosis was identified by colorimetric protease assay for caspase 2, -3, -6, -8, and -9, as well as cellular DNA fragmentation assay. Antioxidant activity of A. pisidicus water and methanolic extracts was investigated with eight different assays. Antimicrobial activities of the extracts were evaluated against 16 bacterial strains by disc diffusion and broth microdilution methods. Results: A total of 13 phytochemicals were detected in the extracts at various concentrations. Hesperidin (147–40,174 µg/g extract) and hyperoside (363–2677 µg/g extract) comprised the principal constituents among the extracts. Fm (IC50 = 9.57 µg/mL), Rm (IC50 = 14.89 µg/mL), and Sm (IC50 = 9.57 µg/mL) were evaluated as active crude extracts on H1299, HT-29, and Panc-1 cells, while Rm (IC50 = 32.057 µg/mL) and Fm (IC50 = 64.25 µg/mL) were assessed as moderately active on MCF-7 and 22RV1 cells, respectively. The elevation of caspase 2, 3, 6, 8, and 9 enzyme activities, along with DNA fragmentation, signifies that the mode of cell death is apoptosis. According to the disc diffusion test results, Fm, Lm, Sm, and WPm extracts exhibited antimicrobial activity against gram (+) bacteria. Conclusions: A. pisidicus elicited apoptotic cell death in cancer cells selectively by the activation of caspases and subsequent DNA fragmentation and may serve as a novel source of an apoptosis-inducing anticancer drug.

Since Astragalus is a genus with many important medicinal plant species, the present work aimed to investigate the phytochemical composition and some biological activities of Astragalus gymnolobus. The methanolic fractions of four organs (stems, flowers, leaves, root and whole plant) were quantified and identified by Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometry (LC–ESI–MS/MS) analysis. Hesperidin, hyperoside, p-hydroxybenzoic acid, protocatechuic acid and p-coumaric acid were identified as main compounds among the extracts. Among all cells, leaf methanol (Lm) extract had the highest cytotoxic effect on HeLa cells (IC50 = 0.069 μg/mL). Hesperidin, the most abundant compound in A. gymnolobus extract, was found to show a strong negative correlation with the cytotoxic effect observed in HeLa cells according to Pearson correlation test results and to have the best binding affinity to targeted proteins by docking studies. The antimicrobial activity results indicated that the most susceptible bacterium against all extracts was identified as Streptococcus pyogenes with 9–11 mm inhibition zone and 8192 mg/mL MIC value. As a result of the research, it was suggested that A. gymnolobus could be considered as a promising source that contributes to the fight against cancer.