Ferulic acid as major antioxidant phenolic compound of the Tetragonisca angustula honey collected in Vera Cruz - Itaparica Island, Bahia, Brazil

Abstract The antioxidant activity of Tetragonisca angustula honey (TAH) and its ethanolic extract (TAEE) were investigated. The total levels of phenolic (TPC) and flavonoids (TFC) were also evaluated. The results for TPC were 19.91 ± 0.38 and 29.37 ± 1.82 mg GAE g-1 and for TFC 0.20 ± 0.02 and 0.14 ± 0.01 mg QE g-1 of TAH and TAEE, respectively. Antioxidant activities were 73.29 ± 0.49% and 93.36 ± 0.27% in the DPPH assay and 71.73 ± 4.07% and 97.86 ± 0.35% in ABTS+ for TAH and TAEE, respectively. The total reducing activity was determined by the method of reducing power (PR) and iron ion (Fe III) and the results varied in PR from 151.7 ± 25.7 and 230.7 ± 25.2 mg GAE L-1, for TAH and TAEE respectively and for (Fe III) in EC50 0.284 in TAEE and 0.687 in TAH. Chemical analysis by HPLC-DAD of the ethanolic extract (TAEE) revealed the presence of ferulic acid as majority phenolic component in the extract. The 1H NMR analysis confirmed this structure and showed the also presence of glucose, citric acid, succinic acid, proline and hydrocarbon derivatives. In addition, the botanical origin was also investigated and showed a multifloral characteristic, having found 19 pollen types with a botanical predominance of the Anacardiaceae family, with Tapirira pollen occurring as predominant (42.6%) and Schinus as secondary (25.7%). The results showed that T. angustula honey is an interesting source of antioxidant phenolic compounds due to its floral origin and can act as a protector of human health when consumed.

Resumo A atividade antioxidante do mel de Tetragonisca angustula (TAH) e seu extrato etanólico (TAEE) foram investigados. Os níveis totais de fenólicos (TPC) e flavonóides (TFC) também foram avaliados. Os resultados para TPC foram 19,91 ± 0,38 e 29,37 ± 1,82 mg GAE g-1 e para TFC 0,20 ± 0,02 e 0,14 ± 0,01 mg QE g-1 de TAH e TAEE, respectivamente. As atividades antioxidantes foram 73,29 ± 0,49% e 93,36 ± 0,27% no ensaio DPPH e 71,73 ± 4,07% e 97,86 ± 0,35% no ABTS+ para TAH e TAEE, respectivamente. A atividade redutora total foi determinada pelo método de poder redutor (PR) e íon ferrico (Fe III) e os resultados variaram em PR de 151,7 ± 25,7 e 230,7 ± 25,2 mg GAE L-1, para TAH e TAEE respectivamente e para (Fe III) em EC50 0,284 em TAEE e 0,687 em TAH. A análise química por HPLC-DAD do extrato etanólico (TAEE) revelou a presença de ácido ferúlico como componente majoritário no extrato. A análise de RMN 1H confirmou esta estrutura e mostrou a presença de glicose, ácido cítrico, ácido succínico, prolina e derivados de hidrocarbonetos no TAEE. Além disso, a origem botânica também foi investigada e apresentou característica multifloral, tendo encontrado 19 tipos polínicos com predomínio botânico da família Anacardiaceae, sendo o pólen Tapirira predominante (42,6%) e o Schinus secundário (25,7%). Os resultados mostraram que o mel de T. angustula é uma interessante fonte de compostos fenólicos antioxidantes devido à sua origem floral e pode atuar como protetor da saúde humana quando consumido.

Ferulic acid as major antioxidant phenolic compound of the Tetragonisca angustula honey collected in Vera Cruz - Itaparica Island, Bahia, Brazil / Ácido ferúlico como principal composto fenólico antioxidante do mel de Tetragonisca angustula coletado em Vera Cruz - Ilha de Itaparica, Bahia, Brasil

Abstract The antioxidant activity of Tetragonisca angustula honey (TAH) and its ethanolic extract (TAEE) were investigated. The total levels of phenolic (TPC) and flavonoids (TFC) were also evaluated. The results for TPC were 19.91 ± 0.38 and 29.37 ± 1.82 mg GAE g-1 and for TFC 0.20 ± 0.02 and 0.14 ± 0.01 mg QE g-1 of TAH and TAEE, respectively. Antioxidant activities were 73.29 ± 0.49% and 93.36 ± 0.27% in the DPPH● assay and 71.73 ± 4.07% and 97.86 ± 0.35% in ABTS●+ for TAH and TAEE, respectively. The total reducing activity was determined by the method of reducing power (PR) and iron ion (Fe III) and the results varied in PR from 151.7 ± 25.7 and 230.7 ± 25.2 mg GAE L-1, for TAH and TAEE respectively and for (Fe III) in EC50 0.284 in TAEE and 0.687 in TAH. Chemical analysis by HPLC-DAD of the ethanolic extract (TAEE) revealed the presence of ferulic acid as majority phenolic component in the extract. The 1H NMR analysis confirmed this structure and showed the also presence of glucose, citric acid, succinic acid, proline and hydrocarbon derivatives. In addition, the botanical origin was also investigated and showed a multifloral characteristic, having found 19 pollen types with a botanical predominance of the Anacardiaceae family, with Tapirira pollen occurring as predominant (42.6%) and Schinus as secondary (25.7%). The results showed that T. angustula honey is an interesting source of antioxidant phenolic compounds due to its floral origin and can act as a protector of human health when consumed.

Resumo A atividade antioxidante do mel de Tetragonisca angustula (TAH) e seu extrato etanólico (TAEE) foram investigados. Os níveis totais de fenólicos (TPC) e flavonóides (TFC) também foram avaliados. Os resultados para TPC foram 19,91 ± 0,38 e 29,37 ± 1,82 mg GAE g-1 e para TFC 0,20 ± 0,02 e 0,14 ± 0,01 mg QE g-1 de TAH e TAEE, respectivamente. As atividades antioxidantes foram 73,29 ± 0,49% e 93,36 ± 0,27% no ensaio DPPH● e 71,73 ± 4,07% e 97,86 ± 0,35% no ABTS●+ para TAH e TAEE, respectivamente. A atividade redutora total foi determinada pelo método de poder redutor (PR) e íon ferrico (Fe III) e os resultados variaram em PR de 151,7 ± 25,7 e 230,7 ± 25,2 mg GAE L-1, para TAH e TAEE respectivamente e para (Fe III) em EC50 0,284 em TAEE e 0,687 em TAH. A análise química por HPLC-DAD do extrato etanólico (TAEE) revelou a presença de ácido ferúlico como componente majoritário no extrato. A análise de RMN 1H confirmou esta estrutura e mostrou a presença de glicose, ácido cítrico, ácido succínico, prolina e derivados de hidrocarbonetos no TAEE. Além disso, a origem botânica também foi investigada e apresentou característica multifloral, tendo encontrado 19 tipos polínicos com predomínio botânico da família Anacardiaceae, sendo o pólen Tapirira predominante (42,6%) e o Schinus secundário (25,7%). Os resultados mostraram que o mel de T. angustula é uma interessante fonte de compostos fenólicos antioxidantes devido à sua origem floral e pode atuar como protetor da saúde humana quando consumido.

Analysis of the Hive Yield of Three Species of Bees for Honey Production in Eastern Honduras

Objectives: The analysis of the performance of honey production hives of three species of honeybees was carried out. Study Design: Three harvests per year of honey per hive of each species the first was on March-April, the second was on June-July, and the third was on November -December, with a sampling of ten hives per species, data were evaluated by ANOVA analysis, and Duncan mean separation test developed by SAS/STAT program. Place and Duration of the Study: This research was developed in the department of Para韘o, municipality of Danl�, Honduras, Central America, in Cerro El 羐uila, latitude 13�'59" N, and longitude 86�'59" W, the sampling was carried out in 2020. Methodology: The qualitative variables were evaluated for each species by In Situ observation that characterized the honeycombs of the three species, and the data were subjected to an analysis of variance and Duncan's mean separation, where the dependent variable is the average yield of Kg of honey/hive. Results: According to the ANOVA, there are significant differences in: the harvest of the three species of bees (p<0.0001), for the three annual harvests (p=0.0024), and by hives (p<0.0001) and in the analysis of separation of means the three averages of honey yield observed are not homogeneous (P>0.05), the Apis mellifera produces 63.1 kg/hive-year, the Tetragonisca angustula obtained an average of 0.679 kg / hive-year, and Melipona beecheii obtained 13.19 kg/hive-year. Conclusion: In the three species is observed that they have differences in the structures of the construction of their hives, their entrance, the construction of their cells, production and storage of honey and pollen, birth of queens, and method of defense; and when evaluating the average annual yield by species, by harvest, and by hives, is found that the three variables evaluated of bees are different averages of honey production.

Propolis from native Stingless Bees: ultrasound-assisted extraction / Propóleos de abejas nativas sin aguijón: extracción asistida por ultrasonido

BACKGROUND: Propolis has been considered a highly valuable material due to its therapeutic properties. However, in Colombia, the commercialization of propolis is limited not only by low production but also by the little knowledge about its efficient extraction. Therefore, finding an optimal and economical extraction method to obtain propolis is a necessity for beekeepers that would open new possibilities for industrial use and, therefore, for the market. OBJECTIVES:The objective of this study was to evaluate a conventional and ultrasound-assisted extraction method, seeking to obtain the highest yield and a high amount of content of bioactive compounds in propolis extracts. METHODS: The extraction was carried out for three crude propolis from different types of bees: Tetragoniscaangustula or Angelita (ANG), Meliponaeburnea or Melipona(MEL), and Scaptotrigonaspp (SCT). The extracts were characterized by color, pH, visual appearance, solid content, antioxidant capacity, total polyphenol content, and bacterial inhibition capacity. RESULTS: The highest extraction performance was obtained when the ultrasound-assisted method was used, especially for the ANG extract, which in addition to presenting inhibition for gram-negative (E. coli) and gram-positive (S. Aureus) bacteria, had the best antioxidant activity with a value of 545 mg GAE / 100 g of sample and total polyphenol content of 1,884 mg GAE / 100 g of sample. CONCLUSIONS: Ultrasound-assisted extraction can be considered a low-cost alternative to increase the extraction performance of crude propolis, together with its total polyphenol content and antioxidant capacity, without altering its physical properties

ANTECEDENTES: El propóleos ha sido considerado un material de alto valor por sus propiedades terapéuticas. Sin embargo, en Colombia la comercialización de propóleos está limitada no solo por la baja producción sino también por el incipiente conocimiento sobre la extracción eficiente de este. Por ello, encontrar un método de extracción óptimo y económico para la obtención de propóleos es una necesidad para los apicultores que abriría nuevas posibilidades para el uso industrial y por tanto para el mercado. OBJETIVOS: El objetivo de este estudio fue evaluar un método de extracción convencional y asistido por ultrasonido (US) buscando el mayor rendimiento y alto contenido de compuestos bioactivos en extractos de propóleos. MÉTODOS: La extracción se realizó para tres propóleos crudos de diferentes tipos de abejas Tetragonisca angustula o Angelita(ANG), Melipona eburnea o Melipona (MEL) y Scaptotrigona spp (SCT). Todos los extractos se caracterizaron por su color, pH, apariencia visual, contenido de sólidos, capacidad antioxidante, contenido total de polifenoles y capacidad de inhibición bacteriana. RESULTADOS: El mayor rendimiento de extracción se obtuvo cuando se usó el método asistido por ultrasonido y específicamente para el extracto ANG, que además de presentar inhibición para bacterias gram negativas (E. coli) y gram positivas (S. Aureus), tuvo la mejor actividad antioxidante con un valor de 545 mg GAE / 100 g de muestra y contenido total de polifenoles de 1884 mg GAE / 100 g de muestra. CONCLUSIONES: La extracción asistida por ultrasonido puede considerarse una alternativa de bajo costo para aumentar el rendimiento de extracción del propóleos crudo, así como su contenido total de polifenoles y capacidad antioxidante sin alterar sus propiedades físicas

Atividade antiviral e virucida de extratos hidroalcoólicos de própolis marrom, verde e de abelhas Jataí (Tetragonisca angustula) frente ao herpersvírus bovino tipo 1 (BoHV-1) e ao vírus da diarreia viral bovina (BVDV) / Antiviral and virucidal activity of hydroalcoholic extracts of propolis brown, green and jataí bees (Tetragonisca angustula) against Bovine Herpesvirus Type-1 (BoHV-1) and Bovine Viral Diarrhea Virus (BVDV)

Dentre as propriedades biológicas da própolis, a atividade antimicrobiana tem merecido destacada atenção. No presente trabalho, descreve-se a ação antiviral e virucida de três extratos hidroalcoólicos de própolis (marrom, verde e de abelhas jataí (Tetragonisca angustula), frente ao Herpesvírus Bovino tipo (BoHV-1) e ao Vírus da Diarreia Viral Bovina (BVDV). Os três extratos hidroalcoólicos foram obtidos de extração etanólica e são oriundos do sul do Brasil. A composição química dos extratos de própolis foi determinada pela cromatografia líquida de alta eficiência acoplada a espectrômetro de massas (UFLC-PDA-ESI-TOF/MS) que identificou e quantificou compostos como: ácido cafeico e ácido p-cumárico, ácido clorogênico, ácido ferúlico, além de flavonoides como a rutina. A toxicidade celular bem como a atividade antiviral dos extratos de própolis em monocamadas de células MDBK (Madin-Darby Bovine Kidney) foi avaliada através de observação microscópica e quantificada pelo teste de MTT (3-(4,5 dimetiltiazol-2yl)-2-5-difenil-2H tetrazolato de bromo). O extrato de própolis de abelhas jataí demonstrou ser menos citotóxico (1,57µg/mL), quando comparado aos extratos verde (0,78µg/mL) e marrom (0,39µg/mL). Quanto a atividade antiviral, a própolis verde demostrou maior eficácia em ambos os tratamentos celulares (pós e pré-exposição) frente ao BoHV-1 em relação aos outros extratos, ou seja, houve maior viabilidade celular quando comparada aos controles de células e vírus. Já a de jataí apresentou atividade frente aos dois vírus (BoHV-1 e BVDV) no método pré-infecção, enquanto a própolis marrom demonstrou ação apenas frente ao BoHV-1 também no método pré-infecção. Para determinação da atividade virucida foram utilizadas diferentes diluições dos vírus, bem como temperaturas e tempos distintos de incubação. A própolis verde a 37°C propiciou a maior redução no título viral (4,33log) em relação a marrom (log = 3,5log) e de jataí (log = 3,24log). No entanto, frente ao BVDV a própolis jataí apresentou os melhores resultados em ambas as temperaturas (22oC e 37oC). Portanto, os extratos avaliados apresentaram atividade antiviral e virucida frente ao BoHV-1 e BVDV, o que os torna alvo para o desenvolvimento de novos biofármacos como alternativa ao uso de antivirais comerciais em Medicina Veterinária.(AU)

Among the biological properties of propolis, the antimicrobial activity has received prominent attention. In this paper, we describe the antiviral and virucidal effect of three hydroalcoholic extracts of propolis (brown, green and jataí bees (Tetragonisca angustula), against bovine herpesvirus type-1 (BoHV-1) and bovine viral diarrhea Virus (BVDV). All hydroalcoholic extracts were obtained from ethanol extraction. The chemical composition of propolis extracts was determined by high-performance liquid chromatography coupled to mass spectrometer (UFLC-PDA-ESI-TOF/MS) to identify and quantify compounds such as caffeic acid and p-coumaric acid, chlorogenic acid, ferulic, and flavonoids such as rutin. Cell toxicity and antiviral activity of propolis extracts in monolayers of MDBK cells (Madin-Darby Bovine Kidney) were assessed by microscopic observation and quantified by the MTT assay (3- (4.5 dimethylthiazol-2yl) -2- 5-diphenyl-2H-tetrazolato bromine). Propolis extract from Jataí bees proved to be less cytotoxic (1.57mg / ml) when compared to green extracts (0.78mg / ml) and brown (0.39mg/mL). Regarding antiviral activity, propolis has shown greater efficacy in both cellular treatments (post and pre-exposure) against BoHV-1 when compared to other extracts, ie, there was increased cell viability compared to cell and virus controls. Extracts from Jataí showed activity against both viruses (BoHV-1 and BVDV) infection in the pre-test, whereas brown propolis demonstrated action only against the BoHV-1 in the pre-infection method. To determine the virucidal activity, it were used different dilutions of virus, as well as different temperatures and incubation times. The green propolis at 37°C led to a greater reduction in viral titer (4.33log) compared to brown (3.5log) and jataí (3.24log). Jataí propolis showed the best results in both temperatures (22oC and 37oC) when tested against BVDV. In summary, the evaluated extracts showed antiviral and virucidal activity against BoHV-1 and BVDV, and may be important targets for the development of new compounds as an alternative to commercial antivirals.(AU)

PODER ANTIBACTERIAL DE MIELES DE Tetragonisca angustula, VALORADA POR CONCENTRACIÓN MÍNIMA INHIBITORIA / Antibacterial Power Of Honeys From Tetragonisca angustula Assessed By Minimum Inhibitory Concentration

Mediante pruebas in vitro se determinó la capacidad antibacteriana, de 37 mieles de la especie Tetragonisca angustula de siete regiones de Colombia, con la técnica de Concentración Mínima Inhibitoria (CMI) frente a tres especies de bacterias Gram positivas y tres Gram negativas. La técnica CMI se evaluó en microdiluciones (v/v) al 90%, 45%, 22,5%, 12,3%, 5,6% de miel, contra las cepas Gram negativas Salmonella enterica sp. enterica serovar Typhimurium, ATCC 14028; Escherichia coli, ATCC 31617 y Klebsiella pneumoniae sp. pneumoniae, ATCC 700603 y Gram positivas Bacillus subtilis sp. spizizenii, ATCC 6633; Staphylococcus aureus sp. aureus Rosenbach, ATCC 6538 y Micrococcus luteus Kocuria rhizophila, ATCC 9341. Las muestras de miel correspondieron a los departamentos de Antioquia, Cauca, Cundinamarca, Magdalena, Santander, Tolima y Valle del Cauca. Las mieles analizadas por prueba binomial presentaron efecto bactericida contra todas las cepas bacterianas del ensayo con mayor efectividad para E. coli, M. luteus y S. enterica, con una probabilidad de p(x)=1 a una dilución de 90%; al ser diluidas el mayor efecto se evidenció frente a M. luteus donde aún en dilución 5,6% presentaban probabilidades de p(x)=0,62 de actividad inhibitoria por parte de las mieles. En dilución de miel al 90%, el efecto fue solo cercano a p(x)=0,5 para K. pneumoniae, B. subtilis y S. aureus. La región de Antioquia, la cual hace parte de las mieles con mayor número de muestras dentro del ensayo, exhibe la mayor acción bactericida.

The in vitro antibacterial capacity was determined for 37 honeys of Tetragonisca angustula from seven regions of Colombia, by minimum inhibitory concentration (CMI) against three positive Gram bacteria and three negative Gram bacteria. The CMI technique was evaluated by microdilutions (v/v) to 90%, 45%, 22.5%, 12.3%, 5.6% of honey against the negative Gram bacteria, Salmonella enterica sp. enteric serovar Typhimurium, ATCC 1402; Escherichia coli, 31617 ATCC and Klebsiella pneumoniae subsp. pneumoniae, ATCC 700603 and to positive Gram bacteria, Bacillus subtilis sp. spizizenii, ATCC 6633; Staphylococcus aureus sp. aureus Rosenbach, 6538 ATCC and Micrococcus luteus Kocuria rhizophila, ATCC 9341. The honey samples corresponded to Colombia regions: Antioquia, Cauca, Cundinamarca, Magdalena, Santander, Tolima and Valle del Cauca. The honeys analyzed by binomial test showed bactericidal effect against the tested bacteria; the greater effectiveness was detected in E. coli, M. luteus and S. enterica, with probability or p(x)=1 in a 90% dilution; for followed up dilutions, the effect was still demonstrated against M. luteus in a 5.6% dilution with an inhibition activity from honeys at p(x)=0.62. In a 90% dilution, the effect was only closed to p(x)=0.5, for K. pneumoniae, B. subtilis and S. aureus. The region of Antioquia, exhibits honeys with superior bactericidal action between the sampling areas.

Characterization of esterases in tetragonisca angustula and tetragona clavipes (Hymenoptera Meliponinae)

Esterases form a large, diverse group of enzymes with wide, overlapping substrate specificities and patternsof inhibition. These enzymes occur in a large variety of isoforms encoded by distinct gene loci with ahigh genetic variability and temporal differences in expression that make them appropriate for studyingpopulation structure. In this work, we investigated the substrate specificity and pattern of esterase expressionin body parts of the stingless bees Tetragonisca angustula and Tetragona clavipes. Fourteen hives of T.angustula were collected in Cianorte and T. clavipes were collected from three colonies in Maringá, twocities in the southern Brazilian state of Paraná. The esterase electrophoretic patterns were determinedusing polyacrylamide gels. Seven bands of esterase activity were detected in T. clavipes (EST-1 to EST-7)and two bands in T. angustula (EST-1 and EST-2). There was variation in the tissue esterase activity of T.clavipes, with EST-6 occurring in the abdomen of workers and EST-7 occurring in cephalic/thoracic extracts.The differences in the number of esterase bands and substrate specificity were attributed to the number ofesterase loci involved in each species, and/or variation in substrates specificities. The variation seen hereshould be useful for determining the role of esterases in intermediate metabolism in the Trigonini, as wellas to use esterases as a genetic marker for this stingless bee.