Annona Muricata L. extract restores renal function, oxidative stress, immunohistochemical structure, and gene expression of TNF-α, IL-ß1, and CYP2E1 in the kidney of DMBA-intoxicated rats.

Introduction: 7,12-dimethylbenz (a) anthracene (DMBA) is a harmful polycyclic aromatic hydrocarbon derivative known for its cytotoxic, carcinogenic, and mutagenic effects in mammals and other species. Annona muricata, L. (Graviola; GRV) is a tropical fruit tree traditionally well-documented for its various medicinal benefits. This investigation is the first report on the potential antioxidant and antinfammatory reno-protective impact of GRV against DMBA-induced nephrotoxicity in rats. Methods: Forty male albino rats were allocated into four equal groups (n = 10). The 1st group served as the control, the 2nd group (GRV) was gastro-gavaged with GRV (200 mg/kg b.wt), the 3rd group (DMBA) was treated with a single dose of DMBA (15 mg/kg body weight), and the 4th group (DMBA + GRV) was gastro-gavaged with a single dose of DMBA, followed by GRV (200 mg/kg b.wt). The GRV administration was continued for 8 weeks. Results and Discussion: Results revealed a significant improvement in renal function, represented by a decrease in urea, creatinine, and uric acid (UA) in the DMBA + GRV group. The antioxidant potential of GRV was confirmed in the DMBA + GRV group by a significant decline in malondialdehyde (MDA) and a significant increase in catalase (CAT), superoxide dismutase (SOD), glutathione S transferase (GST), and reduced glutathione (GSH) compared to DMBA-intoxicated rats; however, it was not identical to the control. Additionally, the antiinflammatory role of GRV was suggested by a significant decline in mRNA expression of cytochrome P450, family 2, subfamily e, polypeptide 1 (CYP2E1), tumor necrosis factor-alpha (TNF-α), and interleukin 1 beta (IL-1ß) in the DMBA + GRV group. Moreover, GRV improved the histopathologic and immunohistochemical expression of TNF-α, CYP450, and IL1ß in DMBA-intoxicated kidney tissue. Conclusively, GRV is a natural medicinal product that can alleviate the renal injury resulting from environmental exposure to DMBA. The reno-protective effects of GRV may involve its anti-inflammatory and/or antioxidant properties, which are based on the presence of phytochemical compounds such as acetogenins, alkaloids, and flavonoids.

A Proteomic Study of the Bioactivity of Annona muricata Leaf Extracts in HT-1080 Fibrosarcoma Cells.

Graviola (Annona muricata) is a tropical plant with many traditional ethnobotanic uses and pharmacologic applications. A metabolomic study of both aqueous and DMSO extracts from Annona muricata leaves recently allowed us to identify dozens of bioactive compounds. In the present study, we use a proteomic approach to detect altered patterns in proteins on both conditioned media and extracts of HT-1080 fibrosarcoma cells under treatment conditions, revealing new potential bioactivities of Annona muricata extracts. Our results reveal the complete sets of deregulated proteins after treatment with aqueous and DMSO extracts from Annona muricata leaves. Functional enrichment analysis of proteomic data suggests deregulation of cell cycle and iron metabolism, which are experimentally validated in vitro. Additional experimental data reveal that DMSO extracts protect HT-1080 fibrosarcoma cells and HMEC-1 endothelial cells from ferroptosis. Data from our proteomic study are available via ProteomeXchange with identifier PXD042354.

The Antioxidant Potential of Graviola and Its Potential Medicinal Application.

Graviola (Annunona muricata L.), a plant growing in tropical regions, has many names and a range of ethnomedicinal uses. The leaves are used to treat insomnia, diabetes, cystitis, and headaches, the crushed seeds have anthelmintic properties, and the fruits are used in the preparation of ice creams, candy, syrups, shakes, and other beverages. The key active components are believed to be annonaceous acetogenins, with more than 100 such compounds having been isolated from A. muricata. The plant is also a source of a range of phenolic compounds, essential oils, alkaloids, flavonol triglycosides, and megastigmanes, together with various minerals, including Mg, Fe, Cu, K, and Ca. Its key phenolic compounds are rutin, kaempferol, and quercetin. This paper provides an overview of the current state of knowledge about the antioxidant properties of various graviola organs and their major constituents, based on a review of various electronic databases. However, few findings have been obtained from clinical trials, and few in vitro and animal studies suggest that graviola preparations have antioxidant properties; as such, the antioxidant potential of graviola, and its safety, remain unclear.

Relationship between the toxicity of raw Annona muricata leaves extract and 7, 12-dimethylbenzathracene (DMBA) with histopathological changes in wistar rats / Relación entre la toxicidad del extracto de hojas crudas de Annona muricata y el 7, 12-dimetilbenz antraceno (DMBA) con cambios histopatológicos en ratas Wistar

Abstract Aims: To identify the histopathological alterations in organs of Wistar rats to evaluate toxic effects of use of Annonamuricata Raw Leaf Extract (AMRLE) alone or in association with DMBA. Settings and Design: Sixty female Wistar rats were used, separated into groups and treated with a single dose of 65 mg/kg of DMBA and/or with 50; 100 and 200 mg/kg of AMRLE. Hematoxylin-Eosin (HE) and 1% methylene blue stains were used in the histopathological analysis and quantification of Aberrant Crypts (ACs) and Aberrant Crypt Focus (ACF). Fischer and Kruskal Wallis tests were used in the statistical analysis. Results: The administration of 65 mg/kg of DMBA and/or 50, 100 and 200 mg/kg of AMRLE did not influence weight development. Some histopathological alterations (hepatic steatosis; inflammatory foci in the liver, kidney and lung; pulmonary lymphoid hyperplasia, ectasia and hyperplasia in mammary gland epithelium) and the development of ACs and ACF in the intestinal colon were observed in all groups, except in the group negative control, with no statistical difference between analysed groups. Conclusions: Histopathological alterations and the formation of ACs and ACF did not show a statistically significant difference between the groups analysed. However, although AMRLE has antioxidant effects due to the presence of phenolic components, there was still the formation of some pathological processes that may be related to the isolated toxic action of DMBA and/or associated with other components of AMRLE, since these changes were not seen in the negative control group.

Resumen Objetivos: Identificar las alteraciones histopatológicas en órganos de ratas Wistar para evaluar los efectos tóxicos del uso del Extracto de Hoja Cruda de Annona muricata (AMRLE) solo o en asociación con DMBA. Configuración y diseño: Se utilizaron sesenta ratas hembras Wistar, se separaron en grupos y se trataron con una dosis única de 65 mg/kg de DMBA y/o con 50, 100 y 200 mg/kg de AMRLE. Se utilizaron tinciones de hematoxilina-eosina (HE) y azul de metileno al 1% en el análisis histopatológico y la cuantificación de criptas aberrantes (CA) y focus de criptas aberrantes (FCA). En el análisis estadístico se utilizaron las pruebas de Fischer y Kruskal Wallis. Resultados: La administración de 65 mg/kg de DMBA y/o 50, 100 y 200 mg/kg de AMRLE no influyó en el desarrollo del peso. Se observaron algunas alteraciones histopatológicas (esteatosis hepática; focus inflamatórios en el hígado, riñón y pulmón; hiperplasia, ectasia en epitelio de la glándula mamaria e hiperplasia linfoide pulmonar) y el desarrollo de CA y FCA en el colon intestinal en todos los grupos, excepto en el grupo control negativo, sin diferencias estadísticas entre los grupos analizados. Conclusiones: Las alteraciones histopatológicas y la formación de CA y FCA no mostraron diferencias estadísticamente significativas entre los grupos analizados. Sin embargo, aunque AMRLE tiene efectos antioxidantes debido a la presencia de componentes fenólicos, aún existe la formación de algunos procesos patológicos que pueden estar relacionados con la acción tóxica aislada del DMBA y/o asociados con otros componentes de AMRLE, ya que estos cambios no fueron observados en el grupo control negativo.

Hormetic effect of an Ethanolic Graviola Leaf Extract on HGF-1 cells survival.

Plant-extracted compounds have been used for centuries in traditional pharmacopeia. Some of them have proven to be excellent drug alternatives for cancer treatment as they target metabolic pathways that are key to cancer cells such as apoptosis, energy-producing catabolic pathways, and the response to oxidative stress. Since some anticancer drugs have been shown to produce dose dependent biologically opposite effects, it is crucial to determine the range of doses for which the compounds have maximum therapeutic benefits. Annona muricata or Graviola is a tropical tree that is common in the Puerto Rican landscape. Although a plethora of studies conducted in vitro and in vivo studies have indeed reported that extracts prepared from the Graviola root, fruit, bark, and leaves possess antiproliferative activities in a large variety of cancer cells, the efficiency of Graviola extracts to curb the progression of head and neck cancers has been overlooked. Furthermore, the bioactivity of Graviola extracts on sane/non-cancerous cells has largely been ignored. The present work reports the in vitro antiproliferative/anticancer behavior of an ethanolic Graviola leaf extract on squamous cell carcinoma cell lines 9 and 25 vs. a sane/non-cancerous human gingival fibroblast cell line-1. Our results show that the Graviola extract induces cell death in the squamous cell carcinoma cell lines at all concentrations tested and a dose-dependent biphasic concentration-dependent/hormetic effect on the fibroblastic cells. This suggests that, at low doses, the phytochemicals present in the prepared Graviola extract could offer potential therapeutic avenues for curbing the progression of head and neck cancers.

Natural biomolecules and derivatives as anticancer immunomodulatory agents.

Despite advancements in chemotherapy, the issue of resistance and non-responsiveness to many chemotherapeutic drugs that are currently in clinical use still remains. Recently, cancer immunotherapy has gathered attention as a novel treatment against select cancers. Immunomodulation is also emerging as an effective strategy to improve efficacy. Natural phytochemicals, with known anticancer properties, been reported to mediate their effects by modulating both traditional cancer pathways and immunity. The mechanism of phytochemical mediated-immunomodulatory activity may be attributed to the remodeling of the tumor immunosuppressive microenvironment and the sensitization of the immune system. This allows for improved recognition and targeting of cancer cells by the immune system and synergy with chemotherapeutics. In this review, we will discuss several well-known plant-derived biomolecules and examine their potential as immunomodulators, and therefore, as novel immunotherapies for cancer treatment.

Molecular Targets of Natural Compounds with Anti-Cancer Properties.

Cancer is the second leading cause of death in humans. Despite rapid developments in diagnostic methods and therapies, metastasis and resistance to administrated drugs are the main obstacles to successful treatment. Therefore, the main challenge should be the diagnosis and design of optimal therapeutic strategies for patients to increase their chances of responding positively to treatment and increase their life expectancy. In many types of cancer, a deregulation of multiple pathways has been found. This includes disturbances in cellular metabolism, cell cycle, apoptosis, angiogenesis, or epigenetic modifications. Additionally, signals received from the microenvironment may significantly contribute to cancer development. Chemical agents obtained from natural sources seem to be very attractive alternatives to synthetic compounds. They can exhibit similar anti-cancer potential, usually with reduced side effects. It was reported that natural compounds obtained from fruits and vegetables, e.g., polyphenols, flavonoids, stilbenes, carotenoids and acetogenins, might be effective against cancer cells in vitro and in vivo. Several published results indicate the activity of natural compounds on protein expression by its influence on transcription factors. They could also be involved in alterations in cellular response, cell signaling and epigenetic modifications. Such natural components could be used in our diet for anti-cancer protection. In this review, the activities of natural compounds, including anti-cancer properties, are described. The influence of natural agents on cancer cell metabolism, proliferation, signal transduction and epigenetic modifications is highlighted.

Ameliorative Effect of Annona muricata (Graviola) Extract on Hyperglycemia Induced Hepatic Damage in Type 2 Diabetic Mice.

Annona muricata (AM) is evergreen plant of the Annonaceae family and known to have anticancer and antidiabetic effects. However, anti-diabetic mechanisms of AM extracts (AME) associated with hepatic glucose regulation and lipid metabolism remain unclear. In this study, we investigated the protective effect of AME extracted on hepatic damage in diabetic mice. Diabetes was induced by a high-fat diet with two-times streptozotocin (STZ) injection (60 mg/kg BW) in C57BL/6 male mice. The diabetic mice were daily administered with AME (50 or 100 mg/kg BW) by gavage for 9 weeks. Biomarkers related to energy metabolism and insulin signaling were examined to identify the effect of AME on hyperglycemia induced hepatic damage. AME supplementation reduced levels of FBG, HbA1c, HOMA-IR and hepatic lipid profiles as well as enhanced insulin signaling by increased the protein levels of IRS-1 accompanied GLUT2 in diabetic mice. Especially low dose of AME showed the beneficial effect of reducing oxidative stress (4-HNE, protein carbonyls, Nrf2, NQO1) and improved hepatic morphology demonstrated by lipid droplets along with upregulation of lipophagy (pAMPK, p-mTOR/mTOR, LC3-2/LC3-1) in diabetic mice. Moreover, AME supplementation ameliorated hepatic lipid metabolism (FAS, SREBP1c, C/EBPα, PPARγ, CPT1A, PPARα) and energy metabolism (pAMPK, PGC1α) in diabetic mice. Taken together, this study suggested that AME could be helpful to prevent hepatic abnormality by regulation of insulin signaling associated with energy metabolism and autophagy in diabetes.

Non-targeted metabolomics characterization of Annona muricata leaf extracts with anti-angiogenic activity.

The tropical plant Annona muricata has been widely used for traditional ethnobotanic and pharmacologic applications. Extracts from different parts of this plant have been shown to have a wide range of biological activities. In the present study, we carry out a metabolomic study of both aqueous and DMSO extracts from Annona muricata leaves that has allowed us to identify 33 bioactive compounds. Furthermore, we have shown that aqueous extracts are able to inhibit endothelial cell migration and both aqueous and DMSO extracts inhibit the formation of tubule-like structures by endothelial cells cultured on Matrigel. We conclude that extracts of Annona muricata leaves have great potential as anti-angiogenic natural combinations of bioactive compounds.

Evaluation of Annona muricata (Graviola) leaves activity against experimental trichinellosis: in vitro and in vivo studies.

Our work aimed to evaluate the possible effect of Annona muricata (Graviola) leaf extract on Trichinella spiralis in in vitro and in vivo studies. Trichinella spiralis worms were isolated from infected mice and transferred to three culture media - group I (with no drugs), group II (contained Graviola) and group III (contained albendazole) - then they were examined using the electron microscope. In the in vivo study, mice were divided into five groups: GI (infected untreated), GII (prophylactically treated with Graviola for seven days before infection), GIII (infected and treated with Graviola), GIV (infected and treated with albendazole) and GV (infected and treated with a combination of Graviola plus albendazole in half doses). Drug effects were assessed by adults and larvae load beside the histopathological small intestinal and muscular changes. A significant reduction of adult and larval counts occurred in treated groups in comparison to the control group. Histopathologically, marked improvement in the small intestinal and muscular changes was observed in treated groups. Also, massive destruction of the cultured adults' cuticle was detected in both drugs. This study revealed that Graviola leaves have potential activity against trichinellosis, especially in combination with albendazole, and could serve as an adjuvant to anti-trichinellosis drug therapy.

Therapeutic Role of Annona muricata Fruit and Bee Venom Against MNU-Induced Breast Cancer in Pregnant Rats and its Complications on the Ovaries.

AIM: To evaluate the potential therapeutic role of Annona muricata (graviola) fruit and bee venom (BV) against N-methylnitrosourea (MNU)-induced breast cancer in pregnant female rats and complications in the ovaries. METHODS: A total of 24 female rats were induced with a single dose of MNU (50 mg/kg body weight). After confirmation of positive tumor marker, female rats were placed with the males for mating. The pregnant rats were randomly divided into four groups (n=6): MNU-induced only (group 1), MNU-induced rats and supplemented with A. muricata 200 mg/kg diet (group 2), MNU-induced and treated with two doses of BV 75 µg/kg (group 3), and MNU-induced and treated with both A. muricata and BV (group 4). RESULTS: In group 1, the breast tissue of mothers revealed pronounced cellular hyperplasia and histopathological signs. Also, the ovarian tissue of mothers and their offspring displayed deleterious histological changes. In groups 2 and 4, histopathological signs and cellular hyperplasia markedly disappeared in breast tissue. However, the histopathological signs induced by MNU in the ovarian tissue reversed to normal in groups 2-4. Also in groups 2-4, levels of serum MMP1, NFκB, and TNFα significantly decreased, and serum caspase 3 significantly increased either in mother rats or their offspring compared to the MNU-alone group. Levels of serum MDA significantly decreased; however, levels of serum antioxidants (CAT and SOD) significantly increased in all groups 2-4 compared to MNU-alone group. CONCLUSION: A. muricata has a more powerful therapeutic role than BV against MNU-induced breast cancer in rats; however, both have a powerful ameliorative role against ovarian histopathological alterations induced by MNU. Such ameliorative effects of A. muricata and BV are mainly attributed to their antioxidant, anti-inflammatory, and antiproliferative constituents.

Graviola mitigates acetic acid-induced ulcerative colitis in rats: insight on apoptosis and Wnt/Hh signaling crosstalk.

In this study, we elucidated the potential protective effects of graviola leaves, compared with sulfasalazine, against acetic acid (AA)-induced ulcerative colitis (UC) in rats. Twenty-eight mature male rats were divided into four groups, Sham, Colitis, Colitis/Sulfa, and Colitis/Graviola, and were treated orally with either saline, saline, sulfasalazine (100 mg/kg/day), or graviola (100 mg/kg/day), respectively, for 7 days. On the 4th day, UC was induced by transrectal administration of 4% AA. Colon tissues were excised for macroscopic and histopathological evaluation and immunohistochemical analysis of caspase-3, B-cell lymphoma 2 (Bcl-2), and Bcl-2-associated X protein (Bax). Also, levels of oxidative mediators, Wnt family member1 (Wnt1), smoothened (Smo), and glioblastoma-1 (Gli1) were evaluated. Macroscopic and histopathological examination revealed that both graviola and sulfasalazine significantly mitigated colonic damage. Besides, both treatments significantly alleviated AA-induced oxidative stress, as evidenced by reduced nitric oxide (No) and malondialdehyde (MDA) levels and myeloperoxidase (MPO) activity and raised reduced glutathione (GSH) content. Both treatments significantly attenuated AA-induced apoptosis via downregulating the expression of Bax and caspase-3 and upregulating the expression of the anti-apoptotic protein, Bcl-2. Furthermore, downregulation of mRNA expression of Wnt1 with concomitant upregulation of Smo and Gli1 was observed in rats treated with either sulfasalazine or graviola. Based on these observations, graviola may attenuate AA-induced UC, at least partially, by modulating apoptosis and Wingless/Int1 (Wnt) and hedgehog (Hh) signaling crosstalk.

Evaluation of metabolomics behavior of human colon cancer HT29 cell lines treated with ionic liquid graviola fruit pulp extract.

ETHNOPHARMACOLOGICAL RELEVANCE: Medicinal plants have been used by indigenous people across the world for centuries to help individuals preserve their wellbeing and cure diseases. Annona muricata L. (Graviola) which is belonging to the Annonaceae family has been traditionally used due to its medicinal abilities including antimicrobial, anti-inflammatory, antioxidant and cancer cell growth inhibition. Graviola is claimed to be a potential antitumor due to its selective cytotoxicity against several cancer cell lines. However, the metabolic mechanism information underlying the anticancer activity remains limited. AIM OF THE STUDY: This study aimed to investigate the effect of ionic liquid-Graviola fruit pulp extract (IL-GPE) on the metabolomics behavior of colon cancer (HT29) by using an untargeted GC-TOFMS-based metabolic profiling. MATERIALS AND METHODS: Multivariate data analysis was used to determine the metabolic profiling, and the ingenuity pathway analysis (IPA) was used to predict the altered canonical pathways after treating the HT29 cells with crude IL-GPE and Taxol (positive control). RESULTS: The principal components analysis (PCA) identified 44 metabolites with the most reliable factor loading, and the cluster analysis (CA) separated three groups of metabolites: metabolites specific to the non-treated HT29 cells, metabolites specific to the treated HT29 cells with the crude IL-GPE and metabolites specific to Taxol treatment. Pathway analysis of metabolomic profiles revealed an alteration of many metabolic pathways, including amino acid metabolism, aerobic glycolysis, urea cycle and ketone bodies metabolism that contribute to energy metabolism and cancer cell proliferation. CONCLUSION: The crude IL-GPE can be one of the promising anticancer agents due to its selective inhibition of energy metabolism and cancer cell proliferation.

Ameliorative Effect of Graviola (Annona muricata) on Mono Sodium Glutamate-Induced Hepatic Injury in Rats: Antioxidant, Apoptotic, Anti-inflammatory, Lipogenesis Markers, and Histopathological Studies.

Monosodium glutamate (MSG) is a widely used food additive, and there is a trepidation that MSG plays a critical role in multiple hepatic disorders. This study was planned to investigate Graviola extract (GE) effects on hepatic and cellular alterations induced by MSG. Fifty Wistar rats were randomly allocated into five groups: control (received normal saline), Graviola (received 200 mg/kg body weight), MSG (received 2.4 gm MSG/kg, 15% of Lethal dose (LD50) of MSG), Graviola + monosodium glutamate (MSG + GE; received GE, 200 mg/kg/day and MSG 2.4 gm/kg body weight (BW) for the next four weeks), and monosodium glutamate + Graviola (received MSG only (2.4 gm/kg BW) daily for four weeks, then concomitant with Graviola (200 mg/kg BW) daily for the next four weeks. MSG and GR were administered orally for eight weeks. Our results showed that MSG caused a significant increase in oxidative stress markers malondialdehyde (MDA), reactive oxygen species (ROS), nitric oxide (NO), hydrogen peroxide (H2O2), proinflammatory cytokines interleukin 6 (IL-6) level, a tumor protein (P53), hepatic cellular damage, as well as proapoptotic markers caspase-3, and B-cell lymphoma 2 (BCL-2)-like protein 4 (Bax). A significant decrease in superoxide dismutase (SOD), catalase (CAT), glutathione S transferase (GST), reduced glutathione (GSH), and an antiapoptotic agent B-cell lymphoma 2 (BCl-2) was observed. The detected MSG effects were normalized by Graviola administration, either a prophylactic or protecting dose. Besides, Graviola reduced the expression of inducible nitric oxide synthase (iNOS) and hepatic fatty acid synthase (FAS) and led to the upregulation of the silent information regulator protein one gene expression gene (SIRT1).In conclusion, the results suggest that Gaviola's interrelated antiapoptotic, antioxidant, and anti-inflammatory properties are potential mechanisms to enhance hepatic deficits and protect the liver. Graviola can, therefore, be considered a promising hepatoprotective supplement. Additionally, further human clinical trials are also necessary to validate the present research.

Anticancer and Apoptogenic Effect of Graviola and Low-Dose Radiation in Tumor Xenograft in Mice.

Background:Annona muricata (graviola) has been claimed for its potential against various diseases including cancer. Objective: The present study aimed to investigate the anticancer effect of graviola extract on Ehrlich solid tumor (EST) mice along with or without a low dose of γ radiation (LDR). Methods: Mice were treated with graviola 50 mg/kg body weight orally for 30 days after EST induction and exposed to γ-ray (2 Gy/week for 3 weeks). Cell cycle, CD44, TGF-ß, Bcl-2, and annexin V were determined in tumor tissue. Results: The result obtained showed a significant decrease (P < .05) of tumor size in 28 graviola-treated EST-bearing mice group (EG) or graviola-treated and irradiated EST-30-bearing mice (EGR) groups versus the EST group. The large number of cells in the sub-G0/G1 population and low cell number at S and M phases signify tumor cell apoptosis and inhibition of cell division in EG or EGR groups. Additionally, significant increases in the expression of CD44 and TGF-ß were recorded in EST mice as compared with EG or EGR mice. Furthermore, EST mice exhibited a decrease in the apoptotic marker annexin v and increase in antiapoptotic Bcl-2 compared with EG and EGR mice. Conclusion: It could be suggested that graviola exerts its antitumor effect throughout the regulation of the tumor cell cycle as well as inducing apoptotic signals. The combined treatment of graviola and LDR augments their effect on tumor proliferation.

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.

Phenolic Compounds and Metals in Some Edible Annonaceae Fruits.

The concentrations of 3,4-dihydroxybenzoic acid, caffeic acid, catechin, chlorogenic acid, epicatechin, gallic acid, p-coumaric acid, quercetin, rutin, ferulic acid, and the major metals in graviola (Annona muricata), atemoya (A. squamosa x A. cherimola), fruta do conde (A. squamosa), biribá (Rollinia mucosa), and the North American pawpaw (Asimina triloba) were determined by UPLC-ESI (-)-MS/MS. It enabled the identification and quantification of phenolic compounds. Catechin was only found in atemoya, at a concentration of 38.6 µg/g-dw. Only 3,4-dihydroxybenzoic acid was found in the fruit pulps of all five fruits analyzed. Atemoya stands out for not only having catechin but also for having much more epicatechin (239 µg/g-dw) than the other fruits. At the same time, graviola had more p-coumaric acid (62.6 µg/g-dw), and the North American pawpaw had more chlorogenic acid (48.1 µg/g-dw) than the other fruits. Metals were determined by ICP equipped with axially viewed plasma. All five fruit pulps had relatively high levels of potassium, with concentrations ranging from 7640 to 15,000 µg/g-dw, with pawpaw being the lowest and atemoya being the highest. The concentrations of other metals ranged from Ca 547 to 1110, Na 14.3 to 123, P 1210 to 1690, Mg 472 to 980, Mn 1.86 to 5.27, and Zn 5.55 to 7.32 µg/g-dw. All five fruits in the Annonaceae family that were analyzed in this study have several phenolic compounds in them and were good sources of potassium, calcium, phosphorus, and magnesium.

ANTIPROLIFERATIVE PROPERTIES OF ETHANOLIC AND AQUEOUS GRAVIOLA LEAF EXTRACTS ON TONGUE SQUAMOUS CELL CARCINOMA CELL LINE-25.

BACKGROUND: Annona muricata, commonly known as Graviola, soursop or guanabana, is an evergreen tree native to the tropics with a long history of use in ethnomedicine in indigenous communities in Africa and South America. Its active phytoconstituents have provided medicinal benefits against various ailments and diseases such as arthritis, parasitic infection, hypertension, fever, or diabetes. Studies conducted in vitro and in vivo have concluded that Graviola phytocomponents have anti-cancer and anti-tumor properties. One of the characteristics of cancer cells is their uncontrolled proliferation rate. In that sense, molecules that inhibit cell proliferation offer potential therapeutical benefits. METHODS: We prepared ethanolic and aqueous extracts from dried Graviola leaves and tested their respective antiproliferative activities on tongue Squamous Cell Carcinoma cell line-25. We treated the cells with increasing concentrations of the extracts for 24 h. The respective doses leading to a 50% inhibition of cells growth (GI50) were determined. RESULTS: Our results showed that the ethanolic extract was 4 times more active in inhibiting the growth of Squamous Cell Carcinoma cell line-25 than the aqueous extract (respective GI50 of 61.7 µg/mL, and 274.6 µg/mL). CONCLUSION: We hypothesize that some organic compounds involved in the antiproliferative/cytotoxicity of Graviola leaves were selectively extracted by Ethanol. Future plans include characterizing those bioactive compounds and assessing their activity on Squamous Cell Carcinoma cell line-25 vs. non-cancerous oral cells. Our hope is to discover natural molecules to be used as alternative treatments for oral Squamous Cell Carcinomas.

Use of an Extract of Annona muricata Linn to Prevent High-Fat Diet Induced Metabolic Disorders in C57BL/6 Mice.

Annona muricata Linn, commonly known as graviola, is one of the most popular plants used in Brazil for weight loss. The aim of this study is to evaluate the therapeutic effects of three different doses (50 mg/kg, 100 mg/kg, and 150 mg/kg) of aqueous graviola leaf extract (AGE) supplemented by oral gavage, on obese C57BL/6 mice. Food intake, body weight, an oral glucose tolerance test (OGTT), an insulin sensitivity test, quantification of adipose tissue cytokines, weight of fat pads, and serum biochemical and histological analyses of the liver, pancreas, and epididymal adipose tissue were measured. AGE had an anti-inflammatory effect by increasing IL-10 at doses of 50 and 100 mg/kg. Regarding the cholesterol profile, there was a significant decrease in LDL-cholesterol levels in the AGE 150 group, and VLDL-cholesterol and triglycerides in the AGE 100 and 150 groups. There was an increase in HDL cholesterol in the AGE 150 group. The extract was able to reduce the adipocyte area of the epididymal adipose tissue in the AGE 100 and 150 groups. According to the histological analysis of the liver and pancreas, no significant difference was found among the groups. There were no significant effects of AGE on OGTT and serum fasting glucose concentration. However, the extract was effective in improving glucose tolerance in the AGE 150 group.

Increased Intestinal Absorption of Vitamin U in Steamed Graviola Leaf Extract Activates Nicotine Detoxification.

Graviola leaves contain much vitamin U (vit U), but their sensory quality is not good enough for them to be developed as food ingredients. Addition of excipient natural ingredients formulated alongside vit U as active ingredients could enhance not only its sensory quality but also its bioavailability. The objectives of this study were to measure the bioaccessibility and intestinal cellular uptake of bioactive components, including rutin, kaempferol-rutinoside, and vit U, from steamed extract of graviola leaves (SGV) and SGV enriched with kale extract (SGK), and to examine how much they can detoxify nicotine in HepG2 cells. The bioaccessibility of vit U from SGV and SGK was 82.40% and 68.03%, respectively. The cellular uptake of vit U in SGK by Caco-2 cells was higher than that in SGV. Cotinine content converted from nicotine in HepG2 cells for 120 min was 0.22 and 0.25 µg/mg protein in 50 µg/mL of SGV and SGK, respectively, which were 2.86 and 3.57 times higher than the no-treatment control. SGK treatment of HepG2 cells upregulated CYP2A6 three times as much as did that of SGV. Our results suggest that graviola leaf extract enriched with excipient ingredients such as kale could improve vit U absorption and provide a natural therapy for detoxifying nicotine.