UNTARGETED METABOLOMICS AND BIOLOGICAL CHARACTERIZATION OF BLUE GEOPROPOLIS FROM "Melipona quadrifasciata quadrifasciata"

Geopropolis is a product made by stingless bees, consisting of resins, waxes and soil, traditionally used in folk medicine. Despite its bioactive potential, its chemical and biological properties remain poorly explored, which limits its added value. In this study, blue geopropolis from Melipona quadrifasciata quadrifasciata, collected in Paranaguá (Paraná, Brazil), was characterized in terms of its metabolomic profile, total phenolic content, antioxidant capacity, anti-inflammatory potential, and in vitro cytotoxicity. An ethanolic extract was prepared, designated as EEBP, and its total phenolic content was quantified by the Folin-Ciocalteu method. The chemical profile was determined by untargeted metabolomics (UHPLC-ESI-QTOF-MS/MS), with molecular networks built in the Global Natural Products Social Molecular Networking (GNPS) platform and complementary in silico annotation using SIRIUS software. Antioxidant capacity was performed by scavenging of peroxyl radicals (ORAC), superoxide anion, and hypochlorous acid (HOCl). Anti-inflammatory potential was investigated in RAW-Luc 264.7 macrophages stimulated with lipopolysaccharide (LPS), by evaluating NF-κB pathway activation, and TNF-α levels. Cytotoxicity was determined by the MTT assay in macrophages and in Caco-2, HepG2, RAW-Luc 264.7, and HaCaT cells. Twenty-seven compounds were putatively annotated at MSI level 2, predominantly pyranocoumarins, along with diterpenoids, flavonoids, pyranochromones, lignans, and xanthones. Total phenolic content was 49.28 mg GAE/g of dry extract, lower than that of conventional propolis, possibly due to the incorporation of soil, but comparable to that of other geopropolis samples. EEBP showed selective antioxidant activity, with pronounced HOCl scavenging (IC₅₀ = 17.08 µg/mL) and an ORAC value of 342.92 ± 42.06 µmol TE/g, with no inhibition of superoxide anion observed. In macrophages, EEBP was not cytotoxic up to 30 µg/mL and, within this range, reduced NF-κB activation by up to 49% and TNF-α levels by up to 23%. Cytotoxicity varied according to cell type, with cell mortality rate of 74.5% in Caco-2 (1000 µg/mL), 80.02% in HaCaT (125 µg/mL), and 29.18% in HepG2 (1000 µg/mL). These effects occurred at concentrations higher than those showing anti-inflammatory activity, indicating a margin between effect and toxicity, wider for intestinal and hepatic cells and narrower for keratinocytes. Overall, the results indicate that blue geopropolis from M. q. quadrifasciata is a promising source of bioactive compounds, with selective antioxidant activity against HOCl, whose attenuation may help reduce oxidative damage associated with inflammation, as well as promising anti-inflammatory potential. Classes such as pyranocoumarins, flavonoids, and xanthones are candidates to contribute to these activities. The findings also provide a first characterization of this previously unstudied and novel type of geopropolis, supporting the identification of chemical markers for product standardization and the safety assessment of nutraceutical applications, while adding value to meliponiculture.
This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001.