Phytochemical Characterization of Astragalus aureus Willd. Root Extracts by LC–HRMS and HPTLC with Evaluation of Their Biological Activities

In this study, it is aimed to evaluate the phytochemical properties and antioxidant and anti-inflammatory activities of the methanol extract obtained from the roots of Astragalus aureus Willd., as well as the hexane, chloroform, ethyl acetate, and aqueous methanol fractions. Additionally, the phenolic components of the most active fraction were characterized using the bioactivity-guided approach with LC–MS/MS, and the amount of Astragaloside IV was determined using the HPTLC method. The methanol extract was prepared from root samples using the maceration method, and then fractions were obtained through liquid-liquid extraction. Total phenolic content (TPC) and total flavonoid content (TFC) were determined using spectrophotometric methods. Antioxidant activity was evaluated using DPPH and ABTS radical scavenging tests, while anti-inflammatory activity was assessed using the 5-lipoxygenase (5-LOX) inhibition test. The extract showing the highest biological activity was analyzed by LC–MS/MS, and the amount of Astragaloside IV was determined by the HPTLC method. The ethyl acetate fraction showed the highest total phenolic (102.7 ± 0.21 mg GAE/g) and total flavonoid (70.5 ± 0.21 mg QE/g) content. In terms of antioxidant activity, the ethyl acetate fraction exhibited higher radical scavenging activity compared to other fractions, while the strongest 5-LOX inhibitory activity was determined in the methanol extract (IC₅₀ = 37.47 µg/mL). In the LC–MS/MS analysis, leucoside, salicylic acid, liquiritigenin, diosmetin, benzoic acid, and syringic acid were identified as the main phenolic compounds. In the HPTLC analysis, the amount of Astragaloside IV in the ethyl acetate fraction was determined to be 94.32 ± 6.52 µg/g. The findings indicate that the A. aureus roots possess a phytochemical profile rich in phenolic compounds and Astragaloside IV, and exhibit significant antioxidant and anti-inflammatory potential. The study is one of the comprehensive studies that reveal the phytochemical content and biological activities of A. aureus together and report the amount of Astragaloside IV for the first time, contributing to the evaluation of the species as a natural source of bioactive compounds.