Bioprospecção anti-helmíntica de geoprópolis de Melipona fasciculata Smith em testes in vitro com ovos e larvas de Haemochus contortus de pequenos ruminantes

Abstract

One of the main barriers to creation of goats and sheep are gastrointestinal parasites, which represent the largest and most serious health problem, reaching economically derail the creation, due to the resistance of parasites to anthelmintic drugs. Natural products, especially geopropolis produced by stingless bees of the genus Melipona, especially Melipona fasciculata Smith (tiúba) is an alternative for treatment of these diseases. Data in the literature on anthelmintic activity with geopropolis are scarce in this way, we aimed to evaluate the chemical composition of geopropolis of different phytogeographical regions, antioxidant activity and anthelmintic activity in sheep and goats, in order to obtain an anthelmintic product. The samples of geopropolis were collected in the municipalities of Palmeirândia and Fernando Falãao, MA and subjected to extraction to obtain the hydroalcoholic extracts of geopropolis. Geopropolis extracts obtained from the municipalities of Palmeirândia and Fernando Falcão, MA, were determined the total phenolic content by the Folin-Ciocalteu and antioxidant activity using the in vitro assay with 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP). Extracts were derivatized and analyzed by gas chromatography-mass spectrometer (GC/MS) to identify the chemical constituents. Samples of geopropolis of different phytogeographical regions showed variations in the total phenolic content (126.60 – 847.50 mgGAE/g), and antioxidant activity antioxidante for DPPH (IC50 4.24 – 44.44 µg/mL) and FRAP (1.29 – 18.42 mmol FeII/g), the geopropolis from Fernando Falcao, showed better levels. On geopropolis from municipality of Palmeirândia were identified triterpenes cicloartane type, ursane and oleanane and phenolic acids (gallic acid and protocatechuic acid), whereas in geopropolis from Fernando Falcão, phenolic acids, gallic acid and ellagic were the main constituents. Besides the chemical identification extracts were subjected to anthelmintics in vitro larval exsheathment inhibition assay with Haemonchus contortus, with activity only the extract originating from the municipality of Fernando Falcão, and gotten a bioproduct (patent filed) when tested under the same conditions also presents action anthelmintic. This same sample from municipality of Fernando Falcão was subjected to a new extraction to obtain a higher yield, fractioned and, biomonitoring by liquid/liquid partition with solvents of different polarities, yielding fractions: hexane (HFG), chloroform (CFG), ethyl acetate (EAFG) and hydroalcoholic (HAFG). The extract and fractions were evaluated with anthelmintics in vitro assays larval exsheathment inhibition and egg hatch of Haemonchus contortus. New spectrophotometric analyzes were conducted to determine the polyphenol contents, in vitro antioxidant activity on DPPH and FRAP and chromatographic profile by high-performance liquid chromatography coupled with UV detector (HPLC/UV) and identification of the chemical components of the active fractions was performed by highperformance liquid chromatography coupled to mass spectrometry (HPLC/MS). For the egg hatch test EC50% ranged from 2.01 to 3.73 mg/ml to extract and fractions being the most active fraction EAFG with EC50 2.01 mg/ml, and exsheathment larval varied with EC50 0.12 - 0.55 mg/ml, most active fraction HAFG EC50 0.12 mg/ml. The extract and fractions showed high levels of polyphenols (64.50 - 650.19 mgGAE/g) and antioxidant activity (IC50 6.50 – 29.80 µg/mL) and FRAP (6.91 -17.19 mM Fe2+/g). The hexane fraction had low income in this way was not subject to anthelmintics tests did not show antioxidant and had the lowest concentrations of polyphenols. The active fractions were identified phenolic acid (ellagic acid and derivatives, and brevifolin carboxilate, caftaric acid), hydrolyzable tannins (gallotannins and ellagitannins). The anthelmintic activity of geopropolis of Melipona fasciculata (tiúba) is probably related to the antioxidant activity and the presence of phenolic compounds mainly phenolic acids and hydrolyzable tannins, also plant sources used by bees to produce the geopropolis contribute to the composition chemical and bioactivity of the product.