Anti-inflammatory activity of flavonoid rich fractions of Nectandra angustifolia ethanolic extract

Abstract

We have previously shown that the ethanolic extract of Nectandra angustifolia (NaE), from its aerial parts (leaves and stems), has notable anti-inflammatory effects in both in vitro and in vivo studies. These effects were demonstrated in LPS-stimulated macrophage cultures (RAW 264.7) and the BV2 microglial cell line. This study further explores NaE’s anti-inflammatory properties, focusing on its mechanisms in microglia using an in vitro approach.NaE was fractionated by flash chromatography and thin layer chromatography (TLC). Selected fractions (F4, F5, F6, F9) were tested for their activity. Concentrations were based on previous work with NaE. Screening involved measuring nitric oxide production (Griess reaction), IL-6, TNFα (ELISAs), and iNOS and COX-2 expression (western blot). No significant differences were observed compared to NaE at 50 μg/mL. Thus, fractions F5 and F6 were further tested at 25 μg/mL, showing improved results over the whole extract, with F5 selected for deeper analysis.Fraction F5 inhibited p65 subunit translocation to the nucleus, as seen by confocal microscopy, and reduced phosphorylation of p65 and IκBα. Western blot analysis also showed decreased phosphorylation of c-JUN compared to LPS. This suggests an upstream inhibition mechanism. Inhibitors of laminin receptor 67 and TGR5 were also tested but did not affect the observed bioactivity.We examined IL-4 polarization effects, finding that F5 did not induce arginase-1 expression nor alter it after IL-4 stimulation. Additionally, we investigated flavonoid localization within treated cells. Staining showed flavonoids present inside cells, with increased fluorescence after 24 hours.In summary, our study highlights the anti-inflammatory activity of a fraction of NaE. The localization of flavonoids and further mechanistic insights will guide future research on their interactions with upstream targets in LPS-triggered inflammatory pathways.