| Summary: | Liquid chromatography electrospray ionization tandem mass spectrometric (LC-ESI-MS/MS) qualitative and quantitative analysis of different extracts from the aerial parts and roots of <i>Alchemilla acutiloba</i> led to the identification of phenolic acids and flavonoids. To the best of our knowledge, isorhamnetin 3-glucoside, kaempferol 3-rutinoside, narcissoside, naringenin 7-glucoside, 3-<i>O</i>-methylquercetin, naringenin, eriodictyol, rhamnetin, and isorhamnetin were described for the first time in <i>Alchemilla</i> genus. In addition, the antioxidant, anti-inflammatory and cytotoxic activity of all extracts were evaluated. The results clearly showed that among analyzed extracts, the butanol extract of the aerial parts exhibited the highest biological activity comparable with the positive controls used.
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