Application of effect-directed analysis using TLC-bioautography for rapid isolation and identification of antidiabetic compounds from the leaves of Annona cherimola Mill.

INTRODUCTION: Type 2 diabetes mellitus is a globally prevalent chronic disease characterised by hyperglycaemia and oxidative stress. The search for new natural bioactive compounds that contribute to controlling this condition and the application of analytical methodologies that facilitate rapid detection and identification are important challenges for science. Annona cherimola Mill. is an important source of aporphine alkaloids with many bioactivities. OBJECTIVE: The aim of this study is to isolate and identify antidiabetic compounds from alkaloid extracts with α-glucosidase and α-amylase inhibitory activity from A. cherimola Mill. leaves using an effect-directed analysis by thin-layer chromatography (TLC)-bioautography. METHODOLOGY: Guided fractionation for α-glucosidase and α-amylase inhibitors in leaf extracts was done using TLC-bioassays. The micro-preparative TLC was used to isolate the active compounds, and the identification was performed by mass spectrometry associated with web-based molecular networks. Additionally, in vitro estimation of the inhibitory activity and antioxidant capacity was performed in the isolated compounds. RESULTS: Five alkaloids (liriodenine, dicentrinone, N-methylnuciferine, anonaine, and moupinamide) and two non-alkaloid compounds (3-methoxybenzenepropanoic acid and methylferulate) with inhibitory activity were isolated and identified using a combination of simple methodologies. Anonaine, moupinamide, and methylferulate showed promising results with an outstanding inhibitory activity against both enzymes and antioxidant capacity that could contribute to controlling redox imbalance. CONCLUSIONS: These high-throughput methodologies enabled a rapid isolation and identification of seven compounds with potential antidiabetic activity. To our knowledge, the estimated inhibitory activity of dicentrinone, N-methylnuciferine, and anonaine against α-glucosidase and α-amylase is reported here for the first time.

Functional fermented cherimoya (Annona cherimola Mill.) juice using autochthonous lactic acid bacteria.

The biochemical and functional properties of fermented Annona cherimola Mill. (cherimoya) juice using five lactic acid bacteria (LAB) isolated from autochthonous fruits from Northwestern Argentina were studied in this work. Fermentation was carried out at 30 °C for 48 h followed by a 21 day-storage period at 4 °C. The assayed LAB grew well during fermentation (final count of 108 CFU/mL, ΔpH ca. 1 U) and survived after the storage period. All strains consumed fructose and glucose present in cherimoya juice as energy sources, with the consequent synthesis of lactic and/or acetic acids as final metabolic products. However, only two of the five evaluated strains were capable to produce fermented cherimoya juices with a perceptible color change. Due to lactic acid fermentation, a moderate reduction in the total phenolic content (between 13% and 43%) was observed in the majority of the samples, although no change in the antioxidant capacity was detected. The fermented cherimoya juices showed a weak antiplatelet activity when adenosine diphosphate agonist was used. The findings of this study evidenced the potential use of Annona cherimola Mill. fermented juice as a novel matrix for the formulation of stable functional beverages with appealing nutritional and functional properties.

An improved method for a fast screening of α-glucosidase inhibitors in cherimoya fruit (Annona cherimola Mill.) applying effect-directed analysis via high-performance thin-layer chromatography-bioassay-mass spectrometry.

α-glucosidase inhibitors (AGIs) are very attractive bioactive compounds due to their therapeutic profile that includes beneficial effects over glycemic control in type 2 diabetes mellitus and viral infections. Its detection and identification in plants and fruits has gained growing attention, and certainly requires efficient screening methodologies. The objective of the present work was to develop a fast methodology to detect and identify AGIs in cherimoya fruit (Annona cherimola Mill.) applying effect-directed analysis via high-performance thin layer-chromatography (HPTLC) linked with bioassay and mass spectrometry (MS). Both, HPTLC and bioassay conditions, were optimized accomplishing 50% and 83% reduction on enzyme concentration and incubation time respectively, compared to the original method. Additionally, the contrast between inhibitory bands and purple background was also enhanced by enzyme substrate impregnation on HPTLC plate. The optimized detection conditions established were the following: 5.0 U mL-1 of enzyme solution, 1.0 mg mL-1 of 2-naphthyl-α-D-glucopyranoside substrate, 1.0 mg mL-1 of Fast Blue B salt solution and 10 min as incubation time. Applying this methodology, coupled to HPTLC-MS and ultra-high-performance liquid chromatography (UHPLC)-diode array detector (DAD)-MS/MS, it was possible for the first time to detect and identify three AGIs in cherimoya peel and seeds. Compounds were tentatively assigned as phenolamides (phenylethyl cinnamides): N-trans-feruloyl tyramine (m/z 314 [M+H]+; UV λmax 293 and 316 nm), N-trans-p-coumaroyl tyramine (m/z 284 [M+H]+; UV λmax 296 nm) and N-trans-feruloyl phenethylamine (m/z 298 [M+H]+; UV λmax 288 nm). To the best of our knowledge, the presence of latter compound is reported for the first time in cherimoya.

Compuestos fenólicos y actividad antioxidante de los extractos de la hoja de chirimoya (Annona cherimola Mill) / Phenolic compounds and antioxidant activity of extracts from chirimoya (Annona cherimola Mill) leaf / Compostos fenólicos e atividade antioxidante dos extratos da folha de cherimoya (Annona cherimola Mill)

Resumen Después de la cosecha del fruto chirimoya, los árboles liberan enormes cantidades de hojas, las cuales son quemadas por los agricultores. Este trabajo muestra que las hojas pueden ser usadas para nuestro beneficio como antioxidante. Se determinaron los compuestos fenólicos (CF) y la actividad antioxidante (AA) de los extractos de la hoja seca de Annona cherimola Mill en etanol al 70% v/v, agua a 80 °C, y agua subcrítica (AS) a 110, 120 y 130 °C, siguiendo un diseño factorial con el programa Minitab. Los CF se cuantificaron con la metodología de Folin Ciocalteu y la AA con el 2,2-difenil-1-picrilhidrazilo (DPPH) y con el poder antioxidante reductor del hierro (FRAP). Los resultados indicaron que el tipo de solvente y el tiempo de extracción presentaron un efecto significativo en el contenido de CF y AA de los extractos. Se concluyó que el extracto de hoja de chirimoya es un potencial antioxidante. El extracto de AS a 130 °C presentó el mayor contenido de CF (5,6 g EAG/100 g de hoja seca) y el extracto etanólico presentó mayor AA (0,86 mg equivalente trolox/mg extracto seco; IC50=0,020 mg de extracto seco/mL de extracto de hoja seca y FRAP de 1710,14 μmol equivalente trolox /g de hoja seca) y los extractos obtenidos con AS a menor temperatura presentaron mayores valores de AA.

Abstract After harvesting cherimoya fruit, the trees release huge amounts of leaves, which are burned by farmers. This work shows that the leaves can be used as a source of antioxidants. Total phenolic compounds (TPC) and antioxidant activity (AA) of extracts of the dry leaf of Annona cherimola Mill in 70% v/v ethanol, water at 80 °C, subcritical water (SW) at 110 °C, 120 °C and 130 °C were determined, following a factorial design with the Minitab program. The TPC was quantified with the Folin Ciocalteu methodology, and the AA with 2,2-diphenyl-1-picrilhidrazil (DPPH) and Ferric Reducing Antioxidant Power (FRAP). Results indicate that the type of solvent and the extraction time had a significant effect on the TPC and AA of the extracts. Extracts of cherimoya leaves were found to be a potential antioxidant. The extract of SW at 130 °C presented the highest content of TPC (5.6 g EAG/100 g dry leaves) and the ethanolic extract had the highest AA (0.86 mg trolox equivalent/mg dry extract, IC50 = 0.020 mg dry extract/mL extract of dry leaves and FRAP of 1710.14 μmol ET/g dry leaves) and the extracts obtained with SW at a lower temperature presented a higher AA value.

Resumo Depois da colheita da fruta Cherimoya, as árvores liberam grandes quantidades de folhas, que são queimadas pelos agricultores. Este trabalho mostra que as folhas podem ser usadas para nosso benefício como um antioxidante. Compostos fenólicos (FC) e a actividade antioxidante (AA) de extractos de folha seca Annona cherimola Mill em etanol a 70% v/v água a 80 °C, água subcrítica (AS) 110, 120, 130 °C, foram determinados seguindo um planejamento fatorial com o programa Minitab. Os FC foram quantificados com a metodologia Folin Ciocalteu; e a capacidade antioxidante (AA) com 2,2-difenil-1-picrilhidrazil (DPPH) e poder antioxidante redutor de ferro (FRAP). Os resultados indicaram que o tipo de solvente e o tempo de extração tiveram um efeito significativo no conteúdo deCFeAA dos extratos. Conclui-se que o extracto de folha de cherimoya é um potencial antioxidante, o extracto de AS 130 °C tinha o maior conteúdo de CF (5,6 g EAG/100 g folha seca) e o extracto etanólico mostraram maior AA (0,86 mg equivalente Trolox/mg extrato seco, IC50 = 0,020 mg extrato seco/mL extracto de folha seca e FRAP de 1710,14 μmol ET/g folha seca). Os extratos obtido com AS a uma temperatura mais baixa apresentaram um maior valor de AA.

A Mesoamerican origin of cherimoya (Annona cherimola Mill.): Implications for the conservation of plant genetic resources.

Knowledge on the structure and distribution of genetic diversity is a key aspect to plan and execute an efficient conservation and utilization of the genetic resources of any crop as well as for determining historical demographic inferences. In this work, a large data set of 1,765 accessions of cherimoya (Annona cherimola Mill, Annonaceae), an underutilized fruit tree crop native to the Neotropics and used as a food source by pre-Columbian cultures, was collected from six different countries across the American continent and amplified with nine highly informative microsatellite markers. The structure analyses, fine representation of the genetic diversity and an ABC approach suggest a Mesoamerican origin of the crop, contrary to previous reports, with clear implications for the dispersion of plant germplasm between Central and South America in pre-Columbian times. These results together with the potential distribution of the species in a climatic change context using two different climate models provide new insights for the history and conservation of extant genetic resources of cherimoya that can be applied to other currently underutilized woody perennial crops.

Desenvolvimento da cochonilha-branca planococcus citri (risso, 1813) (hemiptera: pseudococcidae) em frutíferas

ABSTRACT The citrus mealybug, Planococcus citri is an important pest attacking several crops. Biotic and abiotic factors, as well as the substrate they feed on influence its population. The present study was aimed to study the effect of some feeding substrates on mealybug development in laboratory conditions. Leaf disks of guava, fig and cherimoya were placed individually on an agar film inside a Petri dish. One egg of the insect was confined inside each container, replicated 60 times for each treatment. Containers were kept in a climatized chamber at 25º C, 70 ± 10% RH and 12 hours photophase. Insects reared on guava leaves showed a longer nymphal development, while the female longevity was longer on fig leaves. However, no statistical differences were found in the survival index, which showed values higher than 83%, suggesting that all substrates studied here were adequate for the development of P. citri.

RESUMO A cochonilha-branca, Planococcus citri, constitui-se em uma importante praga em diversas culturas, sendo suas populações influenciadas por vários fatores bióticos e abióticos. Considerando-se a diversidade de hospedeiros que podem ser infestados, bem como a escassez de informações sobre seu desenvolvimento, esse trabalho objetivou estudar alguns aspectos da biologia de P. citri criada em três substratos alimentares. Ovos de P. citri, em número de 60, foram individualizados em placas de Petri contendo discos foliares de goiabeira (Psidium guajava L.) variedade Paluma, figueira (Ficus carica L.) variedade Roxo de Valinhos e gravioleira (Annona muricata L.) variedade Lisa, dispostos sobre uma lâmina de ágar-água a 1%. As placas foram vedadas com filme plástico PVC e mantidas em câmara climatizada a 25ºC, 70±10% UR e 12 horas de fotofase. Os substratos alimentares influenciaram a duração do período ninfal e longevidade de fêmeas e machos. A goiabeira proporcionou maior duração do período ninfal e a figueira maior longevidade das fêmeas. A sobrevivência em cada ínstar e no período ninfal não diferiu em função dos substratos avaliados, os quais proporcionaram viabilidades superiores a 83%, constituindo-se em hospedeiros adequados para o desenvolvimento de P. citri.