LC–MS/MS and GC–MS Analysis‐Based Novel Bioactive Compounds From Endophyte Acremonium sclerotigenum CBS 124.42 Isolated From Stevia rebaudiana (Bertoni) Bertoni

ABSTRACT Endophytes are microorganisms that reside inside the living tissues of leaves and possess antimicrobial, antioxidant, anticancer and anti‐inflammatory activity etc. Liquid chromatography–tandem mass spectrometry (LC–MS/MS) represents a powerful analytical platform for comprehensive metabolomic profiling of endophytic fungi, offering superior sensitivity, selectivity and structural elucidation capabilities compared to conventional methods. This study employed LC–MS/MS and GC–MS analyses to characterise bioactive compounds from the methanolic extract of endophytic fungus Acremonium sclerotigenum CBS 124.42 isolated from Stevia rebaudiana leaves. LC–MS/MS analysis identified nine major bioactive compounds, including flavonoid glycosides (928.4 m/z), rutin derivatives (1454.7 m/z), cyclic depsipeptides (1445.4 m/z), cyanidin‐3‐O‐rutinoside (595.3 m/z), macrolactams (1394.2 m/z), proanthocyanins (1232.8 m/z), oligomeric imperatorin (676.8 m/z) and cyanidin‐3‐O‐glucoside (1232.8 m/z). Stevioside was detected at 9.02 min with characteristic fragments at 317.9 m/z. GC–MS analysis revealed 18 distinct compounds, with benzene (azidomethyl) being most abundant (100% relative area), followed by hydrazine carbothioamide (71.3%) and benzyl(1,2,3‐thiadiazol‐4‐yl) carbamate (43.22%). Significantly, this study reports the first identification of hydrazine carbothioamide and benzyl(1,2,3‐thiadiazol‐4‐yl) carbamate from natural sources, compounds previously known only from chemical synthesis. These compounds demonstrated significant bioactivities including antioxidant (IC 50 = 28.1 ± 6.96 mg/g), antimicrobial (zone of inhibition: 10.3–23.6 mm) and anticancer properties, establishing A. sclerotigenum CBS 124.42 as a novel source of therapeutically relevant secondary metabolites.