Development and characterisation of EPP-AF(R) propolis glycolic extract

Propolis is produced and used by Apis mellifera bees. Several biological activities have been demonstrated, mainly with the most traditional extraction process using an alcohol solution as a solvent. Although this type of solvent commonly offers the most useful chemical profile and high biomarker values, the alcohol content is considered inappropriate. Several alternative solvents have been proposed. However, the challenge is to equate the chemical profile, efficacy, and safety with that of the traditional alcohol version. An EPP-AF® propolis glycolic extract was obtained by evaporating the alcohol solvent from the EPP-AF® alcoholic extract, using controlled temperature and reduced pressure, with propylene glycol as a solvent. Total phenolics and flavonoids were determined according to colorimetric methods using gallic acid and quercetin as biomarkers, with procedures validated according to the Brazilian agency ANVISA’s guidelines. The chemical profile was also compared by HPLC and TLC procedures. Antimicrobial activity was determined using the inhibition zone model, and antioxidant activity was measured by FRAP and DPPH methodologies. Also, anti-inflammatory action was evaluated by measuring IL-6, IL-10, and TNF-alpha in narrow bone macrophages stimulated with LPS. The validation covered selectivity, accuracy, intra-day and inter-day precision, linearity, range, quantification, detection limits, and robustness. There was equivalent antimicrobial action against S. aureus and S. epidermidis and antioxidant activity. IL-6 and TNF-alpha, inflammatory cytokines, were reduced in a dose-dependent manner for both extracts, in the range of 16.7, 50, and 150 µg/mL, which were not cytotoxic for macrophage cells. The stability studies demonstrated a shelf-life of 24 months, as the biomarkers were below the maximum limit of degradation of 15%, following ANVISA regulations.