In vitro antioxidant and anti-inflammatory effects of Plantago major, Cannabis sativa and Ranunculus multifidus on LPS-stimulated RAW 264.7 macrophages

Chronic inflammation is a major contributor to cancer progression, driven by persistent pro-inflammatory signalling and oxidative stress. Plant-derived bioactive compounds offer therapeutic potential by modulating these pathways. This study evaluated the antioxidant and anti-inflammatory effects of leaf extracts from Cannabis sativa L., Plantago major L., and Ranunculus multifidus Forssk. in LPS-stimulated RAW 264.7 macrophages. Extracts were tested for cytotoxicity (MTT assay), nitric oxide (NO) inhibition (Griess assay), cytokine modulation (ELISA), antioxidant activity (DPPH and ABTS assays), and 15-lipoxygenase (15-LOX) inhibition. UPLC-MS analysis was used to identify potential phytochemical constituents. All extracts were non-cytotoxic at 5 - 15 µg/mL concentrations. The extract from R. multifidus had the highest NO inhibition (20%), followed by P. major (17%), while C. sativa exhibited no significant effect. The ELISA assay revealed extract-specific cytokine modulation. P. major strongly suppressed IL-6 and IL-1β and enhanced IL-10 secretion, while C. sativa inhibited IL-6, PTGS2 and moderately reduced iNOS. In contrast, R. multifidus showed variable effects and did not inhibit iNOS and IL-1β. However, it induced variable effects on IL-6 inhibition and IL-10 secretion. The antioxidant assays revealed potent radical scavenging: C. sativa achieved 92.5% DPPH scavenging comparable to ascorbic acid, while ABTS scavenging exceeded 90% for P. major and R. multifidus, closely matching Trolox. All extracts inhibited 15-LOX, with C. sativa showing the highest inhibition (96.2%). UPLC-MS identified the presence of cannabigerolic acid (CBGA) and Δ⁹-THC in C. sativa and verbascoside in P. major , while no major compounds were conclusively identified in R. multifidus . These findings provide the first evidence of cytokine modulation and anti-inflammatory activity by R. multifidus. Moreover, they highlight the complementary antioxidant and anti-inflammatory potential of C. sativa and P. major , supported by phytochemical evidence. These extracts represent promising natural immunomodulatory agents.