Brazilian Green Propolis‐Based Nanostructured System: Development, Characterization and its Role in the Modulation of Cytokines in In Vitro and In Vivo Experimental Models

Abstract

Brazilian green propolis from Baccharis dracunculifolia (“alecrim do campo”), stands out for the majority presence of phenolic acids with therapeutic properties well‐described in the literature, such as anti‐inflammatory, antioxidant, and antitumor. However, its direct application, like many natural products, is limited due to its unfavorable physicochemical properties. Thus, we hypothesize that the encapsulation of these compounds in nanostructured systems is a suitable instrument to overcome these limitations. In this work, an oil/water (O/W) nanoemulsion containing Brazilian green propolis extract (NE‐BGP) was developed, and its bioactivity was evaluated in vivo and in vitro experimental models. Quantitative analysis by HPLC showed the presence of coumaric acid, artepelin C and baccharin in the standardized extract obtained, corresponding to 3.2%, 16.8% and 2.3% (w/w), respectively. The developed nanoemulsion showed high stability evaluated for 90 days, with the size around 130 nm, PdI less than 0.3, and negatively charged zeta potential (‐20 mV). The encapsulation efficiency of the markers evaluated in NE‐BGP determined by the validated UPLC‐MS method was superior to 97% for 90 days. Morphological analysis using transmission electron microscopy (TEM) showed globular and spherical shapes. The cytotoxicity was evaluated in 3T3 (murine fibroblasts) and AMJ2‐C11 (murine alveolar macrophages) cells and showed IC50 between 20.88 and 49.32 µg/mL after 24 hours of treatment by the neutral red method, respectively. The in vitro anti‐inflammatory activity in AMJ2‐C11 cells stimulated with LPS 5 µg/mL showed a significant reduction in the levels of nitric oxide (NO), as well as cytokines quantified by CBA (Cytometric Bead Array), IFN‐γ, IL‐4, and IL‐6 in cells treated with NE‐BGP (15 µg/ml) when compared to the control group. Male Balb/C mice (n=5) were used to evaluate the in vivo anti‐inflammatory activity. NE‐BGP administered orally at 100mg/Kg showed a reduction in edema, in the number of inflammatory cells in the joints, and an increase in the mechanical threshold reducing nociceptive stimuli in zymosan‐induced mice arthritis. These results indicate that the developed formulation is an exciting system to encapsulate green propolis extract with high physicochemical stability, anti‐inflammatory and analgesic effects, suggesting NE‐BGP is a possible candidate for the arthritis treatment.