Bioactive Fraction of Geopropolis from Melipona scutellaris Decreases Neutrophils Migration in the Inflammatory Process: Involvement of Nitric Oxide Pathway

Abstract

The aim of this study was to evaluate the activity of the ethanolic extract of geopropolis (EEGP) from Melipona scutellaris and its fractions on the modulation of neutrophil migration in the inflammatory process, and the participation of nitric oxide (NO) pathway, as well as to check the chemical profile of the bioactive fraction. EEGP and its aqueous fraction decreased neutrophil migration in the peritoneal cavity and also the interaction of leukocytes (rolling and adhesion) with endothelial cells. The levels of chemokines CXCL1/KC and CXCL2/MIP-2 were not altered after treatment with EEGP and the aqueous fraction. It was found that the injection of NO pathway antagonists abolished the EEGP and the aqueous fraction inhibitory activity on the neutrophil migration. The expression of intercellular adhesion molecule type 1 (ICAM-1) was reduced, and nitrite levels increased after treatment with EEGP and aqueous fraction. In the carrageenan-induced paw edema model, EEGP and the aqueous fraction showed antiedema activity. No pattern of flavonoid and phenolic acid commonly found in propolis samples of Apis mellifera could be detected in the aqueous fraction samples. These data indicate that the aqueous fraction found has promising bioactive substances with anti-inflammatory activity.

Figure 1

Inhibitory effect of the ethanolic extract of geopropolis (EEGP) and aqueous fraction (AF) on neutrophils migration into the peritoneal cavity induced by carrageenan. Neutrophil migration was determined 4 h after the injection of carrageenan 500 μg/cavity. Mice previously treated with vehicle (saline and carrageenan), EEGP (a), hexane fraction (HF-(b)), chloroform (CF-(c)), ethyl acetate (EAF-(d)), or aqueous (AF-(e)). The data are expressed as mean ± SEM, n = 6. Symbols indicate statistical difference (P < 0.05, Tukey test). ^#Compared to the saline group; *compared to the carrageenan group.

Figure 2

Inhibitory effect of the ethanolic extract of geopropolis (EEGP) and aqueous fraction (AF) on rolling (a) and adhesion (b) of leukocytes assessed by intravital microscopy in mesenteric tissue of mice, 2 and 4 h after IP injection of carrageenan 500 μg/cavity. Mice were pretreated with vehicle (saline and carrageenan), EEGP, or aqueous fraction (10 mg/kg). The data are expressed as mean ± SEM, n = 5. Symbols indicate statistical difference (P < 0.05, Tukey test). ^#Compared to the saline group; *compared to the carrageenan group.

Figure 3

Effect of the ethanolic extract of geopropolis (EEGP) and aqueous fraction (AF) on the levels of CXCL1/KC (a) and CXCL2/MIP-2 (b) 3 h after the injection of 500 μg/cavity of carrageenan in the peritoneal cavity. Mice were pretreated with vehicle (saline and carrageenan), EEGP, or aqueous fraction (10 mg/kg, s.c.). The data are expressed as mean ± SEM, n = 5. Symbols indicate statistical difference (P < 0.05, Tukey test). ^#Compared to the saline group.

Figure 4

Inhibitory effect of the ethanolic extract of geopropolis (EEGP) and aqueous fraction (AF) on neutrophils migration into the peritoneal cavity induced by carrageenan. Neutrophil migration was determined 4 h after the injection of carrageenan 500 μg/cavity. Mice were pretreated with aminoguanidine (AG—50 mg/kg, (a)) and ODQ (5 μmol/kg, (b)) antagonists. After 30 min, the animals were pretreated with vehicle (saline and carrageenan), EEGP, or aqueous fraction (10 mg/kg). The data are expressed as mean ± SEM, n = 6. Symbols indicate statistical difference (P < 0.05, Tukey test). ^#Compared to the saline group; *compared to the carrageenan group; **compared to the groups that received only EEGP and the aqueous fraction.

Figure 5

Effect of the ethanolic extract of geopropolis (EEGP) and aqueous fraction (AF) on the expression of ICAM-1 (a) and nitrite levels (b) 4 h after the injection of 500 μg/cavity of carrageenan (Cg) in the peritoneal cavity. Mice were pretreated with vehicle (saline and carrageenan), EEGP, or aqueous fraction (10 mg/kg, s.c.). The data are expressed as mean ± SEM, n = 5. Symbols indicate statistical difference (P < 0.05, Tukey test). ^#Compared to the saline group; *compared to the carrageenan group.

Figure 6

Chromatograms obtained by HPLC-RP of the aqueous fraction. 1: UV λ 240 nm, RT = 3.71 min; 2: UV λ 238 nm, RT = 32.36 min.