Edilson Divino de Araújo

Antinociceptive and anti-inflammatory effects of Brazilian red propolis extract and formononetin in rodents

ETHNOPHARMACOLOGICAL RELEVANCE Propolis has been used as a folk medicine for centuries around the world due to its wide spectrum of biological activities. The red propolis, a new Brazilian variety of this apimaterial, has presented an unusual chemical composition, including isoflavones such as formononetin and biochanin A. Since both the green and red varieties of propolis are traditionally used as medicine and commercialized with no label differentiation, the study of the activities of red propolis extract has become important in order to clarify whether this product has the same activities as commercial ones. In this work, we demonstrated the potential action of the hydroalcoholic extract of red propolis (HERP) and its biomarker, formononetin, as antinociceptive and anti-inflammatory drugs on experimental models. MATERIALS AND METHODS The HERP was chemically characterised by HPLC/DAD analyses. The biological activities of the HERP (3, 10, and 30mg/kg) and formononetin (10mg/kg) were evaluated using the antinociceptive (acetic acid, formalin, and glutamate injections) and anti-inflammatory (carrageenan-induced hindpaw oedema and peritonitis) models in mice after oral administration. The open field test was also performed. RESULTS Formononetin, one of the main biomarker of red propolis, was identified in the HERP (21.62mg/g). Pretreatment with the HERP (10 and 30mg/kg) and formononetin (10mg/kg) produced reduction (P<0.001) in the number of abdominal writhes, but the HERP was more effective (P<0.001) than formononetin. In the formalin test, all HERP doses (3, 10, and 30mg/kg, P<0.001) inhibited the late phase (inflammatory pain) of formalin-induced licking, but the inhibition of neurogenic pain was observed only when the higher doses (10 and 30mg/kg; P<0.05) were used. Formononetin caused inhibition (P<0.001) only in the second phase of formalin-induced nociception similarly at all HERP doses in the same phase of the test. The responses in glutamate-induced model presented crescent inhibition (P<0.05) with 10 and 30mg/kg of HERP. Also, formononetin inhibited (P<0.001) the nociception induced by glutamate similarly to 30mg/kg of HERP. There were no significant differences in the open field test after HERP administration, but formononetin decrease the spontaneous motor behaviour. Regarding the anti-inflammatory assessment, the HERP (10 and 30mg/kg, P<0.05) and formononetin (P<0.001) treatments caused a significant inhibition of the oedema response. All doses of HERP (3, 10, and 30mg/kg, P<0.05) and formononetin (P<0.001) also inhibited the carrageenan-induced leukocyte migration. In both cases, the results for the HERP at 30mg/kg and formononetin were similar. CONCLUSIONS The HERP and formononetin presented significant anti-inflammatory activity. Moreover, the HERP presented antinociceptive action on inflammatory and neurogenic pain without motor side effects, possibly due to the action of other constituents present in the extract. These results, together, support the popular usage of this natural product.

Extraction of DNA from honey and its amplification by PCR for botanical identification

The physiochemical and biological properties of honey are directly associated to its floral origin. Some current commonly used methods for identification of botanical origin of honey involve palynological analysis, chromatographic methods, or direct observation of the bee behavior. However, these methods can be less sensitive and time consuming. DNA-based methods have become popular due to their simplicity, quickness, and reliability. The main objective of this research is to introduce a protocol for the extraction of DNA from honey and demonstrate that the molecular analysis of the extracted DNA can be used for its botanical identification. The original CTAB-based protocol for the extraction of DNA from plants was modified and used in the DNA extraction from honey. DNA extraction was carried out from different honey samples with similar results in each replication. The extracted DNA was amplified by PCR using plant specific primers, confirming that the DNA extracted using the modified protocol is of plant origin and has good quality for analysis of PCR products and that it can be used for botanical identification of honey.

Sandfly fauna in an area endemic for visceral leishmaniasis in Aracaju, State of Sergipe, Northeast Brazil

INTRODUCTION In recent years, visceral leishmaniasis, a major public health problem, has been spreading from the rural to urban areas in many areas of Brazil, including Aracaju, the capital of the State of Sergipe. However, there are no studies of the sandfly fauna in this municipality or its variation over the year. METHODS Phlebotomine sandflies were collected from a rural area of Aracaju from September 2007 to July 2009. Modified CDC ultra-violet (UV) light traps were used to evaluate sandfly monthly distribution and their presence in the domestic and peridomestic environments. RESULTS The most abundant species was Lutzomyia longipalpis (90.4%) followed by Evandromyia lenti (9.6%). A chicken shed trap site had the highest proportion of L. longipalpis (51.1%) and large numbers of L. longipalpis were also collected in the houses closest to the chicken shed. There was a positive correlation between monthly rainfall and L. longipalpis abundance. CONCLUSIONS Lutzomyia longipalpis is the most abundant species and is probably the main vector of the visceral leishmaniasis agent in the rural area of Aracaju. An increase in L. longipalpis frequency was observed during the rainy season. The peridomicile-intradomicile observations corroborate the importance of chicken sheds for the presence of L. longipalpis in the peridomestic environment. The great numbers of L. longipalpis inside the houses confirm the endophilic behaviour of this species and the possibility of visceral transmission in the intradomicile.

Botanical Origin of the Brazilian Red Propolis: a New Approach Using DNA analysis

Abstract Propolis is produced by the honeybees by using resin and other plant secretions. Propolis from different geographical regions have different chemical compositions. This is because the chemical constituents of propolis depend on the vegetation surrounding the apiary. In this report we present a new approach using DNA barcoding for the identification of the botanical origin of propolis. Red propolis samples were collected at different times of the year from the state of Sergipe situated in Northeast Brazil. Extraction of the DNA from propolis was made using a CTA B method. Amplification was done using ITS 2 universal primers, followed by DNA sequencing. Sequence analysis confirmed the presence of Dalbergia ecastaphyllum in the Brazilian red propolis. Formononetin is a chemical marker for the Brazillian red propolis and D. ecastaphyllum. Propolis samples analysed by DNA sequencing, were also checked by Ultra-Fast Liquid Chromatography for the presence of formononetin. Peaks corresponding to formononetin were observed in all the analysed propolis samples. This is the first report of the botanical origin of propolis using DNA technology.

Chemical markers and antifungal activity of red propolis from Sergipe, Brazil

The aim of this study was to analyze the physicochemical properties and antifungal activities of the red propolis samples from Sergipe, Brazil, and also evaluate their variability throughout the year. The characterization of the hydroalcoholic extract (HPE) of the red propolis samples was performed monthly from October 2009 to September 2010. The concentrations of the bioactive compounds varied during the year, but their chromatographic profiles were similar. Four compounds were identified by comparison with authentic standards. Formononetin was one of the predominant compounds in all propolis extracts. In our study, it was observed that all the propolis samples inhibited the growth of Candida species. Multivariate analysis confirmed the variations in chemical composition and color of the HPEs throughout the year. The biological activities of the HPEs were statistically significant (p<0.05), and all samples exhibited antifungal properties.

Opinions concerning pre-implantation genetic diagnosis and sex selection among gynecologist-obstetricians in Brazil

OBJECTIVE The aim of this study was to assess a sample of obstetrician-gynecologists on their technical knowledge of PGD as well as their views of PGD and sex selection (SS). STUDY DESIGN Cross-sectional observational study, carried out during a Congress on Gynecology and Obstetrics. A self-report questionnaire was offered to all professionals registered to attend the Congress and answers of 723 physicians were analyzed. RESULTS Of the participants, 436 (60.3%) were female and 287 (39.7%) were male; 517 (71.5%) had children. Regarding knowledge of PGD techniques, 63.2% had heard something about PGD and/or preconception SS. Concerning SS, physicians believed that SS is always the couples choice (36.4%), should be the couples choice only in specific situations (42.6%), or did not agree that SS should be a couples choice (17.4%). A majority of the respondents were in favor of PGD and believed that pre-embryos with 6-8 cells are human life in potency. CONCLUSIONS In spite of a small percentage of gynecologists-obstetricians in Brazil had knowledge of PGD techniques, many of them recognized it to be a method to prevent genetic diseases and agreed with its use. The SS appears to be highly accepted for use in specific situations.

Variation morphogeometrics of Africanized honey bees (Apis mellifera) in Brazil

The morphometrics of the honey bee Apis mellifera L., 1758 has been widely studied mainly because this species has great ecological importance, high adaptation capacity, wide distribution and capacity to effectively adapt to different regions. The current study aimed to investigate the morphometric variations of wings and pollen baskets of honey bees Apis mellifera scutellata Lepeletier, 1836 from the five regions in Brazil. We used geometric morphometrics to identify the existence of patterns of variations of shape and size in Africanized honey bees in Brazil 16 years after the classic study with this species, allowing a temporal and spatial comparative analysis using new technological resources to assess morphometrical data. Samples were collected in 14 locations in Brazil, covering the five geographical regions of the country. The shape analysis and multivariate analyses of the wing allowed to observe that there is a geographical pattern among the population of Apis mellifera in Brazil. The geographical variations may be attributed to the large territorial extension of the country in addition to the differences between the bioregions.

Phenolic Composition and Leishmanicidal Activity of Red Propolis and Dalbergia ecastaphyllum (L.) Taub (Fabaceae) Extracts from Sergipe, Brazil

Leishmaniasis is a parasitic disease caused by protozoa of the Leishmania genus. It may manifest in visceral and tegumentary forms, and pentavalent antimonials are the first choice drugs used for the treatment. Frequently these drugs show low efficiency and high toxicity to mammalian host. The present study describes the chemical profile and the in vitro leishmanicidal effects of red propolis and Dalbergia ecastaphyllum extracts from Sergipe, Brazil, in Leishmania chagasi and Leishmania amazonensis promastigotes. The phenolic composition of the extracts was evaluated by direct infusion electrospray ionization mass spectrometry (ESI-MS) fingerprinting. The leishmanicidal effect was evaluated by the Resazurin colorimetric method. Similar composition profiles have been found for D. ecastaphyllum and propolis samples. The isoflavones formononetin, biochanin A, daidzein and pinocembrin were identified in both extracts. Propolis extract showed leishmanicidal activity in both L. chagasi and L. amazonensis, with IC50 values of 21.54 and 9.73 μg/mL, respectively. The D. ecastaphyllum extract presented activity only in L. amazonensis, with IC50 of 53.42 μg/mL. These results suggest that red propolis extract from Sergipe has the leguminosae D. ecastaphyllum as botanical origin, and that it presents potential leishmanicidal activity, which may be associated with the presence of the phenolic compounds found in its composition.

Chemical and genetic similarity between Dalbergia ecastaphyllum and red propolis from the Northeastern Brazil

Dalbergia ecastaphyllum was found to be the botanical origin of Brazilian red propolis by chemical and molecular studies increasing the interest in research with this plant species. The National Institute of Industrial Property (INPI), Brazil granted the designation of origin to red propolis and red propolis extract from Alagoas state whose composition includes, among other chemical compounds, formononetin and daidzein. However, several studies have identified the chemical markers of this product in samples of D. ecastaphyllum and propolis of Sergipe state. The objective of this study was to compare the chemical and genetic identity between D. ecastaphyllum populations and propolis samples collected from the north (Alagoas) and south (Sergipe) banks of the São Francisco river also known as the lower São Francisco region. D. ecastaphyllum samples and propolis from this region showed similar chromatographic profiles and ESI (−) – MS fingerprints. The markers formononetin, biochanin A and daidzein were found in D. ecastaphyllum and propolis collected from Sergipe and Alagoas. DNA sequencing demonstrated that Dalbergia population from Alagoas and Sergipe are highly homogeneous, having exactly the same haplotype. Thus D. ecastaphyllum from the lower São Francisco have the same genetic characteristics and chemical profiles including the chemical markers considered for propolis from Alagoas, asking for more comparative studies between propolis produced in the northeastern region of Brazil with the possible extension of geographical indication to other states as well.