TY  - EJOU
AU  - Pilipović, Kristina 
AU  - Fabijančić, Lucia 
AU  - Kristić, Iva 
AU  - Martinović, Lara Saftić 

TI  - Cytotoxicity Thresholds and Limited Modulation of LPS-Induced Microglial Activation by Croatian Olive Leaf Extracts: A Preliminary Study in a BV-2 Washout Model
T2  - BIOCELL

PY  - 
VL  - 
IS  - 
SN  - 1667-5746

AB  - <b>Objectives:</b> Neuroinflammation, largely mediated by microglial activation, plays a central role in the pathogenesis of neurodegenerative diseases. In this study, we aimed to evaluate olive leaf extracts (OLEs) from three Croatian cultivars (Buža, Oblica, and Leccino) in the context of lipopolysaccharide (LPS)-induced activation of BV-2 microglia. <b>Methods:</b> Extracts were prepared by aqueous maceration and ethanol extraction under conditions compatible with downstream cell-culture use. Total phenolic and flavonoid contents were determined for both extract types, alongside targeted liquid chromatography–mass spectrometry (LC–MS/MS) quantification of major phenolic compounds. Based on compositional analysis, ethanolic extracts were selected for further evaluation, including antioxidant capacity assays [2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS)] and assessment of BV-2 microglial responses to LPS, i.e., measurements of cell viability, activation markers (CD86, CD206, iNOS), and selected markers of inflammatory (phospho-p65, Sirt1) and cytoprotective pathways (Nrf2, HSP70). <b>Results:</b> Ethanolic extracts exhibited higher total phenolic and flavonoid contents than aqueous macerates, supported by LC–MS/MS analysis showing increased levels of key constituents such as oleuropein and other flavonoids. Antioxidant activity varied depending on the assay: DPPH activity was comparable across cultivars (<i>p</i> = 0.858), whereas ABTS activity was highest for the Oblica extract (<i>p</i> = 0.0002). Ethanolic extracts were non-cytotoxic at 10 μg/mL, while higher concentrations induced dose-dependent cytotoxicity (<i>p</i> = 0.0001). In BV-2 cells, LPS (1 μg/mL, 3 h) increased CD86 (<i>p</i> = 0.017) and CD206 expression (<i>p</i> &lt; 0.0001), with no change in iNOS levels (<i>p</i> = 0.0697). Short-term, non-cytotoxic pretreatment with ethanolic extracts (10 μg/mL, 3 h) did not significantly alter microglial activation markers or the examined inflammatory and cytoprotective pathways. <b>Conclusion:</b> These findings define the experimental limits under the applied conditions and indicate that compositional richness alone does not necessarily translate into measurable biological effects, supporting further studies with extended exposure and refined dosing.
KW  - Olive leaf extracts; BV-2 microglia; microglial activation; lipopolysaccharide; Croatian olive cultivars

DO  - 10.32604/biocell.2026.079918