Background: Inflammation is a natural body’s defense mechanism against harmful stimuli such as pathogens, chemicals, or irradiation. But when the inflammatory response becomes permanent, it can lead to serious health problems. In the present study, the antioxidant and anti-inflammatory potentials of the Eriosema montanum methanolic extract (EMME), as well as its isolated fractions (FA-FJ) and compounds (1–7), were evaluated by using in vitro and cellular models. Methods: The total phenolic and flavonoid contents were determined using, respectively, Folin–Ciocalteu and aluminum chloride colorimetric methods, while 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS), 2,2′-diphenyl-1-picrylhy-drazyl (DPPH), and ferric ion reducing antioxidant power (FRAP) were used to determine the antioxidant activity. Thin Layer Chromatography (TLC) and column chromatography (CC) were used to isolate and purify the compounds and their elucidation using their NMR spectroscopic data. Results: EMME had moderate antioxidant and anti-inflammatory activities, while fraction FF showed much higher efficacy with IC50 values of 34.64, 30.60, 16.43, and 77.29 μg/mL against DPPH, ABTS, NO, and 15-LOX inhibitory activities, respectively. The EMME fraction was found to be very rich in flavonoids and phenolic compounds, with 82.11 mgQE/g and 86.77 mgGAE/g of dry extract, respectively. Its LC-MS profiling allowed us to identify genistin (5) as the most concentrated constituent in this plant species, which was further isolated together with six other known compounds, namely, n-hexadecane (1), heptacosanoic acid (2), tricosan-1-ol (3), lupinalbin A (4), d-pinitol (6), and stigmasterol glucoside (7). Given these compounds, genistin (5) showed moderate activity against reactive oxygen species (ROS) and NO production in LPS-stimulated RAW264.7 cells compared to EMME, which suggested a synergy of (5) with other compounds. To the best of our knowledge, compounds (1), (2), and (3) were isolated for the first time from this plant species.
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