Isolation of Polyphenols from Soursop Annona muricata L Leaves Using Green Chemistry Techniques and their Anticancer Effect

HIGHLIGHTS Isolate, fractionate and characterize extracts obtained from soursop leaves. Use of emerging green technologies such as microwave-ultrasound hybridization. The extracts contain kaempferol, procyanidins, catechin, and quercetin. The total ethanolic extract demonstrates cytotoxic effect on HeLa cells.

Abstract Cervical cancer is classified as the fourth most common malignancy in women. Natural compounds are a therapeutic alternative in cancer therapy. The aim of the study is to isolate, fractionate, and characterize extracts obtained from soursop leaves (Annona muricata L.) and determine their cytotoxic effect against HeLa cervical cancer cells and non-carcinogenic fibroblast 3T3 cells. The phytochemicals of soursop leaves were extracted through emerging green technologies such as the novel use of microwave-ultrasound hybridization and the use of environmentally friendly solvents (water and ethanol), in addition to the purification of extracts enriched in polyphenols by liquid chromatography with Amberlite XAD-16. Total aqueous and ethanolic extract were purified, as well as the fraction one of each extract. The extracts recovered from soursop leaves contained kaempferol and its isomers, procyanidins, catechin, and quercetin. The viability of the cells was determined with the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. HeLa and 3T3 cells were exposed to concentrations of 25, 50, 75, 100, 150, 200, and 250 ppm of a solution of soursop leaf extract powder. The MTT assay showed that soursop leaf extracts were toxic to both cell lines in general, however, the ethanolic extract at 25 and 50 ppm demonstrated inhibition in cell viability against the HeLa cancer line and low cytotoxicity for 3T3 fibroblast cells. In conclusion, the novel microwave-ultrasound hybridization technology allows the extraction of polyphenols that may have a potential cytotoxic effect on cancer cells.

Efecto tóxico de los extractos de semillas de Annona muricata potenciados con dimetilsulfóxido sobre larvas IV y pupas de Aedes aegypti / Toxic effect of Annona muricata seed extracts potentiated with dimethyl sulfoxide on IV larvae and pupae of Aedes aegypti

Resumen El objetivo de este estudio fue demostrar que los principios activos de las semillas de Annona muricata combinados con el extracto etanólico y dimetilsulfóxido (EE-DMSO), incrementan la mortalidad de larvas IV y pupas de Aedes aegypti con relación a extractos acuosos (EA) y extractos etanólicos (EE). Las bioactividades se calcularon por comparación de los porcentajes de mortalidad a las 6, 12, 24 y 48 horas in vitro y campo simulado. Los resultados indicaron mortalidad progresiva dependiente de las concentraciones y tiempos de exposición en larvas y reacción knock-down en pupas. In vitro a 5 mg.L-1, EA y EE ejercieron 100% de mortalidad larvaria en 24 horas de exposición (CL50=46.16 y 19.28 mg.L-1 respectivamente), en contraste con EE-DMSO, que inició sobre 62% con 0.5 mg.L-1 a las 6 horas (CL50=20.33 mg.L-1). La acción pupicida de EA y EE reveló 100% de mortalidad desde 24 horas en todas las concentraciones, a diferencia de EE-DMSO que se alcanzó entre 6 y 12 horas. En campo simulado, EA y EE ejercieron 100% de mortalidad a las 24 horas en larvas (16.91 y 21.21 mg.L-1 ), mientras que en pupas (20.44 y 23.03 mg.L-1) ocurrió a las 12 horas, entretanto, la actividad pupicida de EE-DMSO fue 100% en 6 horas. Los efectos comparativos in vitro y campo simulado denotaron patrones similares de respuestas larvicida y pupicida, pero con mayor sensibilidad en pupas. Los principios activos de las semillas de A. muricata combinados con EE-DMSO potencian la respuesta mortal de larvas y pupas de A. aegypti in vitro y campo simulado.

Abstract The objective of this study was to demonstrate that active ingredients of Annona muricata seeds can be enhanced as a result of mixture of both ethanolic extract of A. muricata seeds and Dimethylsulfoxide (EE-DMSO). Percentage mortalities at 6, 12, 24 and 48 hours on fourth instar larvae and pupae of Aedes aegypti were calculated in order to compare bioactivities of aqueous (AE), ethanolic extracts (EE) and EE-DMSO under laboratory and simulated field conditions. Results showed larval mortality concentration- and time-dependent, and knock-down responses in pupae. In laboratory, AE and EE exerted 100% larval mortality at 5 mg.L-1 after 24 hours (LC50= 46.16 and 19.28 mg.L-1). Conversely, EE-DMSO showed between 62 - 100% mortality at 0.5 mg.L-1 for over 6 hours (LC50= 20.33 mg.L-1). Pupicidal effects in AE and EE revealed 100% mortality at 24 hours employing all concentrations, except in EE-DMSO which commenced when individuals were exposed between 6 and 12 hours. In simulated field, AE and EE provoked 100% larval mortality at 24 hours (16.91 y 21.21 mg.L-1) while pupal mortality at 12 hours (20.44 y 23.03 mg.L-1). Percentage mortality of pupae was 100% using EE-DMSO even before 6 hours. Comparative toxic effects of laboratory and simulated-field systems have shown to maintain a similar pattern of larval mortality and more sensitive responses in pupae. Accordingly, larval and pupal mortality responses of A. aegypti were enhanced with the use of EE-DMSO and active ingredients of A. muricata seeds under laboratory and simulated field conditions.

Quality Evaluation of Tea Brewed from Blends of Soursop (Annona muricata) and Moringa (Moringa oleifera) Leaves

Tea is commonly made from the leaves of Camellia sinensis. Production of similar drinks from other plant leaves with potential health benefits would help to prevent diseases. This study examined the chemical composition and antioxidant activity of tea made from blends of dried moringa (Moringa oleifera) and soursop (Annona muricata) leaves. Mature, fresh and green leaves from both plants were washed in water and sun-dried for 10 h. The dried leaves were milled and sieved to obtain the tea powders. Blends of soursop: Moringa tea were formulated as follows: A: 100% Soursop, B: 100% Moringa, and soursop: Moringa blends as C:50:50%; D: 60:40% and E: 40:60%. Ten grams of each blend of tea powder was brewed in 100 ml of hot water (90°C) for 10 min and cooled to room temperature (28 ±- 2°C) before analysis. From the result, 50:50 soursop-moringa tea gave the highest levels of vitamins C and A. Mineral levels were significantly different among the samples (p<0.05) with higher values recorded for calcium (2117.10 mg/100 ml), sodium (146.02 mg/100 ml), magnesium (362.03 mg/100 ml), phosphorous (241 mg/100 ml), zinc (7.13 mg/100 ml) and potassium (1207.20 mg/100 ml) in 50:50 soursop-moringa tea. The pH differed significantly (p<0.05) in all the tea samples and ranged from 7.28–7.81. Total solids gave values ranging from 3.47 mg/l-3.82 mg/l (p˂0.05) and total sugars 1.12–3.07% (p<0.05). The amount of tannin was significantly higher (p<0.05) in all tea blends compared to other antinutrients analyzed in this study and ranged from 8.95-9.84%. Assessment of the antioxidant capacity by Diphenol-2,2picrylhydroxyl (DPPH) and Ferric reducing antioxidant power (FRAP) showed significant differences (p<0.05) among the tea samples with the 50:50 soursop: Moringa blend having the highest antioxidant activity with values up to 89.04% and 531.44 (µM/L) in each case. Overall the soursop-moringa tea blends exhibited good chemical composition and antioxidant activity, with 50:50 formulation showing the best nutritional quality attributes.

Anticancer activity test of ethyl acetate extract of endophytic fungi isolated from soursop leaf (Annona muricata L.)

Objective To analyze anticancer activity of an ethyl acetate extract of endophytic fungi isolated from soursop leaf (Annona muricata L.). Methods Anticancer activity of fungal extracts was determined by observing its toxicity against MCF-7 (Michigan Cancer Foundation-7) cells in vitro by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay method. At an extract concentration of 100 μg/mL, 4 isolates out of 12 showed high activity against the cancer cell growth. The four isolates were then selected for further IC

Experimental Lachesis muta rhombeata envenomation and effects of soursop (Annona muricata) as natural antivenom

In the Atlantic forest of the North and Northeast regions of Brazil, local population often uses the fruit juice and the aqueous extract of leaves of soursop (Annona muricata L.) to treat Lachesis muta rhombeata envenomation. Envenomation is a relevant health issue in these areas, especially due to its severity and because the production and distribution of antivenom is limited in these regions. The aim of the present study was to evaluate the relevance of the use of soursop leaf extract and its juice against envenomation by Lachesis muta rhombeata. Methods We evaluated the biochemical, hematological and hemostatic parameters, the blood pressure, the inflammation process and the lethality induced by Lachesis muta rhombeata snake venom. We also assessed the action of the aqueous extract of leaves (AmL) and juice (AmJ) from A. muricata on the animal organism injected with L. m. rhombeata venom (LmrV) in the laboratory environment. Results LmrV induced a decrease of total protein, albumin and glucose; and increase of creatine kinase, aspartate aminotransferase, and urea concentrations. It provoked hemoconcentration followed by reduction of hematocrit, an increase in prothrombin time and partial thromboplastin time and a decrease of the blood pressure. LmrV induced the release of interleukin-6, an increase in neutrophils and changes in the serum protein profile, characteristic of the acute inflammatory process. LD50 values were similar for the groups injected with LmrV and treated or untreated with AmJ and AmL. Both treatments play a role on the maintenance of blood glucose, urea and coagulation parameters and exert a protective action against the myotoxicity. However, they seem to worsen the hypotension caused by LmrV. Conclusion The treatments with AmJ and AmL present some beneficial actions, but they might intensify some effects of the venom. Therefore, additional studies on A. muricata are necessary to enable its use as natural antivenom for bushmaster snakebite.

Experimental Lachesis muta rhombeata envenomation and effects of soursop (Annona muricata) as natural antivenom

Background In the Atlantic forest of the North and Northeast regions of Brazil, local population often uses the fruit juice and the aqueous extract of leaves of soursop (Annona muricata L.) to treat Lachesis muta rhombeata envenomation. Envenomation is a relevant health issue in these areas, especially due to its severity and because the production and distribution of antivenom is limited in these regions. The aim of the present study was to evaluate the relevance of the use of soursop leaf extract and its juice against envenomation by Lachesis muta rhombeata. Methods We evaluated the biochemical, hematological and hemostatic parameters, the blood pressure, the inflammation process and the lethality induced by Lachesis muta rhombeata snake venom. We also assessed the action of the aqueous extract of leaves (AmL) and juice (AmJ) from A. muricata on the animal organism injected with L. m. rhombeata venom (LmrV) in the laboratory environment. Results LmrV induced a decrease of total protein, albumin and glucose; and increase of creatine kinase, aspartate aminotransferase, and urea concentrations. It provoked hemoconcentration followed by reduction of hematocrit, an increase in prothrombin time and partial thromboplastin time and a decrease of the blood pressure. LmrV induced the release of interleukin-6, an increase in neutrophils and changes in the serum protein profile, characteristic of the acute inflammatory process. LD50 values were similar for the groups injected with LmrV and treated or untreated with AmJ and AmL. Both treatments play a role on the maintenance of blood glucose, urea and coagulation parameters and exert a protective action against the myotoxicity. However, they seem to worsen the hypotension caused by LmrV. Conclusion The treatments with AmJ and AmL present some beneficial actions, but they might intensify some effects of the venom. Therefore, additional studies on A. muricata are necessary to enable its use as natural antivenom for bushmaster snakebite.(AU)

Aproveitamento de soro lácteo na formulação de bebidas com frutas e hortaliças / The use of whey in the formulation of beverages with fruits and vegetables

O uso do soro lácteo como complemento na formulação de alimentos visando ao seu aproveitamento é uma alternativa para a obtenção de novos produtos. Neste trabalho, desenvolveram-se bebidas à base de soro oriundo de queijo coalho tipo A com frutas e hortaliças. Na formulação das bebidas, utilizaram-se diferentes proporções entre soro lácteo, frutas e hortaliças, com adição de 10% de açúcar. Seis bebidas foram formuladas e submetidas a um teste de preferência, e as três primeiras colocadas foram submetidas a um teste de aceitação sensorial. As três preferidas foram as bebidas sabor graviola, morango e goiaba. A bebida sabor graviola obteve aceitação com as melhores notas nos atributos sensoriais cor, sabor, aparência e qualidade global, seguida pela formulação de sabor morango, mas sem diferença significativa (P>0,05). A bebida sabor goiaba teve o diferencial no atributo odor e superou as bebidas sabor graviola e morango (P<0,05). Conclui-se que é viável a elaboração de bebidas à base de soro de queijo coalho com frutas.

The use of whey as complement in food formulation aiming it's an alternative for the attainment of new products. The aim of this work was developed a type A curd cheese whey derived drink with fruits and vegetables. On drink's formulation was used different ratios of whey, fruits and vegetables, with 10% of sugar. Six drinks were formulated and submited to a preference test. The three best drinks were submitted to a acceptance test. The three best drinks were graviola, strawberry and guava flavours. The graviola one had the greater acceptability with best grades in color's sensory attributes, flavor, appearance and overall quality, following the strawberry flavor, but without significant difference (P<0,05). The guava dink had it's differential on scent character, overcoming the graviola and strawberry drinks (P<0,05). In conclusion we can say that whey-based drink with fruits is viable.

Plant-based Complementary and alternative medicine used by breast cancerpatients at the Hospital Universitario San Ignacio in Bogotá, Colombia / Uso de medicina alternativa y complementaria basada en plantas, por pacientes con cáncer de seno en el Hospital Universitario San Ignacio en Bogotá, Colombia

Durante este estudio se buscó determinar la frecuencia de uso demedicina alternativa y complementaria (CAM) basada en plantas, enpacientes con cáncer de seno. Se realizó por medio de encuestas enpacientes con cáncer de seno que asistieron a consulta externa al CentroJaveriano de Oncología del Hospital San Ignacio en Bogotá en losmeses de Junio a Diciembre del 2011. De las 404 encuestas aplicadasen mujeres con cáncer de seno, el 57% consumieron algún tipo deCAM para el cáncer de seno y de estos el 76% consumieron plantasmedicinales como el anamú, la sábila, los frutos rojos y la guanábana,entre otros. El 65% de los pacientes tuvieron una percepción positivafrente al consumo de las plantas medicinales y el 57% de los usuariosde terapias basada en plantas, la uso simultáneamente al tratamientoalopático recomendado por el médico oncólogo. Concluimos quela frecuencia de uso de CAM en pacientes con cáncer de seno delCentro Javeriano de Oncología en Bogotá, esta dentro del rangode prevalencia reportado mundialmente, aunque existen diferenciasmarcadas en los tipos y frecuencias de CAM consumidas. La altaproporción de pacientes que usan CAM basada en plantas sindiscutirlo con el médico oncólogo, tiene como consecuencia la faltade evaluación con respecto a los efectos sinérgicos o antagónicos deestas terapias frente al tratamiento alopático del cáncer de mama; asícomo el potencial antitumoral y inmunomodulador real de las plantasusadas de manera tradicional por lo pacientes oncológicos...

The present study estimates the frequencyof the use of plant-based Complementary andAlternative Medicine (CAM) by breast cancer patients.From June to December of 2011, a self-administeredquestionnaire was given to 404 breast cancer patientsreceiving outpatient therapy at the Javeriana OncologyCenter of the Hospital Universitario San Ignacio inBogotá. The prevalence of patient CAM use was 57%,out of which 76% was based on plants like anamú,aloe, red fruits and soursop. Sixty-five percent of thepatients had a positive perception of using medicinalplants and 57% used them simultaneously with theoncologist recommended allopathic treatment. Weconcluded that the frequency of CAM use in breastcancer patients at the Javeriana Oncology Centeris within the prevalence range reported worldwide,despite differences in CAM types and frequencies. Thehigh rates of plant-based CAM use without physicianconsent, brings about the lack of assessment of thesynergic or antagonistic effects of CAM therapieson the allopathic treatment of breast cancer andevaluation of the antitumor and immunomodulatorypotential of the traditionally used plants...

O presente estudo se propôs a avaliar a frequência dautilização de Medicina Alternativa e Complementar (CAM) baseadaem plantas por pacientes com câncer de mama. Um questionário autoadministradofoi dado a 404 pacientes com câncer que frequentarama terapia ambulatória no Centro Javeriano de Oncologia do HospitalUniversitário San Ignacio, em Bogotá entre junho e dezembro de2011. O consumo de algum tipo de CAM por parte dos pacientesfoi de 56%. 76% selecionaram plantas como anamú, aloé , frutosvermelhos e graviola. Os resultados foram que 65% dos pacientestiveram uma percepção positiva de usar plantas medicinais. 57%dos pacientes utilizaram o tratamento alopático simultaneamente.Conclui-se que a freqüência do uso de CAM em pacientes com câncerde mama no Centro Javeriano de Oncologia está dentro da faixade prevalência registrada no mundo, embora com diferenças nostipos de CAM e freqüências. A elevada taxa de pacientes que usamCAM baseada em plantas sem consentimento médico é o resultadoda falta de respeito ou avaliação dos efeitos sinérgicos o antagônicosde terapias CAM contra o câncer da mama, bem como o potencialtratamento alopático antitumoral e imunomoduladora das plantasreais utilizados tradicionalmente...

Biología y hábitos del barrenador de la semilla Bephratelloides cubensis ashmead (Hymenoptera: Eurytomidae) / Biology and behavior of the seed borer wasp Bephratelloides cubensis ashmead (Hymenoptera: Eurytomidae)

The soursop Annona muricata is an important fruit for national market, and for exportation, but the crop is affected by pests and diseases. The seed borer wasp Bephratelloides cubensis Ashmead is the pest that produces the highest damage to the crop in Mexico. Sixty percent of damaged fruits and 5-50 seeds per fruit have been registered, with 25 percent reduction in yield. In Nayarit, Mexico, 100 percent of damaged fruits were recorded. In this State, an experiment with soursop was conducted to study the life cycle under field conditions and to determine diurnal behavior of the female of B. cubensis. The highest activity of the wasp was observed between 12:00h and 13:00h (35ºC, 54 percent RH and 409.34 luxes). Females oviposited in fruits with a diameter of 3.1-7.6 cm. Larvae of B. cubensis developed five instars, adults survived no longer than 22 days, and female survived longer than males; they lived 22 and 15 days, respectively. Life cycle of B. cubensis varied from 69 to 122 days.

Insetos associados á gravioleira (Annona muricata L., Annonaceae) na região de Manaus, Amazonas, Brasil

Insects collected on soursop (Annonaceae: Annona muricata L.) nearby Manaus, Amazonas, Brazil, are reported. Thirty-seven species were found, but only six were considered injurious to the plant: Bephratelloides pomorum V. (Eurytomidae), Cerconota anonella Sepp (Stenomatidae), Membracis suctifructus Boulard & Couturier (Membracidade) and Pinnaspis aspidistrae Signoret (Diaspididae) attacking fruits; Aphis spiraecola Patch and Aphis gossypii Glover (Aphididae) feeding on leaves, and Cratosomus bombina F. (Curculionidae) boring trunks and branches. Twenty four species are recorded for the first time.

Os insetos da graviola (Annonaceae: Annona muricata L.) foram estudados na região de Manaus, Amazonas, Brasil. Encontram-se 37 espécies, entre os quais, seis foram consideradas prejudiciais, quatro atacando o fruto (Bephratelloides pomorum F. (Eurytomidae), Cerconota anonella Sepp (Stenomatidae), Membracis suctifructus Boulard & Couturier (Membracidae) e Pinnaspis aspidistrae Signoret (Diaspididae); duas nas folhas jovens; (Aphis spiraecola Patch e A. gossypii Glover (Aphididae); e uma perfurando tronco e ramos, Cratosomus bombina (Curculionidae). Trinta e quatro espécies são registradas pela primeira vez.