Eight new cytotoxic annonaceous acetogenins from the seeds of Annona squamosa / 中国天然药物

Eight new annonaceous acetogenins, squamotin A-D (1-4), annosquatin IV-V (5 and 6), muricin O (7) and squamosten B (8), together with four known ones (9-12) were isolated from the seeds of Annona squamosa. Their structures were elucidated by chemical methods and spectral data. The inhibitory activities of compound 1-9 against three multidrug resistance cell lines were evaluated. All tested compounds showed strong cytotoxicity.

Rollinia mucosa (Jacq) Baillon (Annonaceae) active metabolites as alternative biocontrol agents against the lace bug Corythucha gossypii (Fabricius): an insect pest / Metabolitos activos de la semilla de Rollinia mucosa (Jacq) Baillon (Annonaceae) como agentes alternativos de biocontrol sobre el insecto fitopatógeno Corythucha gossypii (Fabricius) / Metabólitos ativos de Rollinia mucosa (Jacq) Baillon (Annonaceae) como agentes de biocontrole alternativos contra Corythucha gossypii (Fabricius): um inseto-praga

Abstract The lace bug, Corythucha gossypii (Fabricius) is a serious pest affecting over 24 wild and commercially important plant species of the families Annonaceae, Passiflorcae, Caricaceae, Euphorbiaceae, and Solanaceae. Thus far, commercial insecticides, such as 0.1% Dimethoate and 0.1% Imidacloprid have shown effectiveness against this insect, but no botanical pesticides are available to control this bug. In the present study, a Rollinia mucosa (Jacq.) Baillon ethanol extract was evaluated as a biological control agent against the lace bug. Through a toxicity assay involving Artemia salina, the median lethal concentration (LC50) of a raw ethanol extract of R. mucosa seeds was determined, as well as that of its Acetogenin (F1) and Alkaloid (F2) fractions; these LC50 were 0.184, 0.082, and 0.0493 μg/mL, respectively. In addition, with an insecticide assay on lace bug nymphs, a mortality percentage of 86.67% at 5 μg/mL after 72h was observed. These data demonstrate that the R. mucosa seed extract is highly active. Further chemical characterization studies revealed that the main active metabolites contributing to extract activity were acetogenins and alkaloids.

Resumen El hemíptero, Corythucha gossypii (Fabricius) es un insecto que causa daño sustancial en cultivos de más de 24 especies de plantas de las familias Annonaceae, Passifloraceae, Caricaceae, Euphorbiaceae y Solanaceae. En su mayoría estas plantas son de interés económico. Aunque insecticidas comerciales como el Dimetoato (0.1%) y el Imidacloprid (0.1%) permiten un manejo eficiente de este insecto-plaga, no se han reportado alternativas botánicas para estos insecticidas sintéticos. En el presente estudio se evaluó el extracto etanólico de la semilla de Rollinia mucosa (Jacq.) Baillon, como un biocontrolador de C. gossypii. A través de un test de toxicidad con Artemia salina se determinó que la concentración del extracto etanólico letal para el 50% de la población bajo estudio (LC50) fue de 0.184 μg/mL. De igual modo se identificó que las fracciones de acetogeninas (F1) y de alcaloides (F2) de este extracto tienen un LC50 de 0.082 y 0.0493 μg/mL, respectivamente. En el ensayo insecticida con ninfas de C. gossypii se observó una mortalidad del 86.67% después de 72 horas de exposición al extracto etanólico a una concentración de 5 μg/mL. Lo anterior demuestra que el extracto es altamente activo. La caracterización química del extracto evidenció que los principales metabolitos activos que contribuyen a su actividad insecticida son las acetogeninas y los alcaloides.

Resumo Corythucha gossypii (Fabricius) é uma praga séria que afeta mais de 24 plantas silvestre e de interesse comercial, pertencentes as famílias Annonaceae, Passifloraceae, Caricaceae, Euphorbiaceae e Solanaceae. Até o momento, inseticidas comerciais como Dimetoato (0.1%) e Imidacloprid (0.1%) apresentam um controle eficiente sobre este inseto, entretanto não há reportes de pesticidas de origem vegetal para o seu controle. No presente estudo, o extrato etanólico de Rollinia mucosa (Jacq.) Baillon foi avaliado como um controle biológico contra Corythucha gossypii. Por meio do ensaio de toxicidade com Artemia salina a concentração letal média (LC50) para o extrato etanólico das sementes, suas frações de acetogeninas (F1) e fração de alcaloides (F2) foi de 0.184, 0.082 y 0.0493 μg/mL, respetivamente. Adicionalmente, na avaliação do ensaio inseticida, se obteve uma porcentagem de mortalidade de 86.67% à concentração de 5 μg/mL após de 72 horas de exposição, demonstrando uma alta atividade do extrato de sementes de R. mucosa. Os estudos em relação à caracterização química evidenciaram que os principais metabólitos que aportam à atividade do extrato foram as acetogeninas e alcaloides.

Squamocin, an annonaceous acetogenin, enhances naphthalene degradation mediated by Bacillus atrophaeus CN4 / Squamocin, una acetogenina annonácea, estimula la degradación de naftaleno mediada por Bacillus atrophaeus CN4

Squamocin belongs to a group of compounds called annonaceous acetogenins. They are secondary products of Annonaceae metabolism and can be isolated from Annona cherimolia seeds. This paper deals with the stimulation of biofilm formation of Bacillus atrophaeus CN4 by employing low squamocin concentrations to increase naphthalene degradation. Bacillus atrophaeus CN4, isolated from contaminated soil, has the ability to degrade naphthalene as the only source of carbon and energy. In the absence of additional carbon sources, the strain removed 69% of the initial concentration of naphthalene (approx. 0.2 mmol/l) in the first 12 h of incubation. The addition of squamocin in LB medium stimulated Bacillus atrophaeus CN4 biofilm formation and enhanced naphthalene removal. Squamocin (2.5 pg/ml) does not affect planktonic growth and therefore, the observed increases are solely due to the stimulation of biofilm formation.

Squamocin pertenece a un grupo de compuestos llamados acetogeninas annonáceas (ACG). Las ACG son productos secundarios del metabolismo de plantas de la familia Annonaceae y se pueden aislar a partir de semillas de Annona cherimola. Este artículo trata de la estimulación de la formación de biofilm de Bacillus atrophaeus CN4 mediante el empleo de bajas concentraciones de squamocin para optimizar la degradación de naftaleno. B. atrophaeus CN4, aislado de suelo contaminado, tiene la capacidad de emplear naftaleno como única fuente de carbono y energía. En ausencia de fuentes de carbono adicionales, la cepa degradó el 69% de la concentración inicial de naftaleno (aprox. 0,2 mmol/l) en las primeras 12h de incubación. La adición de squamocin en medio LB estimula la formación de biofilm y la remoción naftaleno de B. atrophaeus CN4. Squamocin (2,5 µg/ml) no afecta al crecimiento planctónico y, por lo tanto, los incrementos observados se deben únicamente a la estimulación de la formación de biofilm.

Anticancer effect of total annonaceous acetogenins on hepatocarcinoma / 中国结合医学杂志

<p><b>OBJECTIVE</b>To confirm the anticancer effect of total annonaceous acetogenins (TAAs) abstracted from Annona squamosa Linn. on human hepatocarcinoma.</p><p><b>METHODS</b>The inhibitory effect of TAAs was demonstrated in H22-bearing mice. The potency of TAAs was confirmed as its 50% inhibiting concentration (IC50) on Bel-7402 cell under Sulfur Rhodamine B staining. Both underlying mechanisms were explored as cellular apoptosis and cell cycle under flow cytometry. Mitochondrial and recipient apoptotic pathways were differentiated as mitochondrial membrane potential under flow cytometry and caspases activities under fluorescence analysis.</p><p><b>RESULTS</b>The inhibitory rate of TAAs in mice was 50.98% at 4 mg/kg dose. The IC50 of TAAs on Bel-7402 was 20.06 µg/mL (15.13-26.61µg/mL). Effective mechanisms of TAAs were confirmed as both of arresting cell cycle at G1 phase and inducing apoptosis dose- and time-dependently. Mitochondrial and recipient pathways involved in apoptotic actions of TAAs.</p><p><b>CONCLUSION</b>TAAs is effective for hepatocarcinoma, via inhibiting proliferation and inducing apoptosis.</p>

Avaliação da atividade citotóxica das sementes de Annona cornifolia A. St.-Hil. (Annonaceae) / Evaluation of the cytotoxic activity of seeds of Annona cornifolia A. St.-Hil. (Annonaceae)

Na família Annonaceae, especialmente o gênero Annona é muito apreciado por fornecer frutos comestíveis. Espécies desse gênero são utilizadas na medicina popular contra diabetes, malária e infecções. Muitas dessas atividades biológicas têm sido relacionadas às acetogeninas de anonáceas. O objetivo deste estudo foi avaliar a atividade citotóxica dos grupos e de uma acetogenina pura (cornifolina) obtidos a partir do extrato etanólico das sementes de Annona cornifolia A. St.-Hil. (Annonaceae). Esta atividade foi avaliada pelo ensaio colorimétrico MTT. Cornifolina (1), a única substância pura testada, apresentou citotoxicidade positiva sobre todas as linhagens tumorais avaliadas. Os grupos testados, todos caracterizados por espectroscopia no infravermelho (IV), apresentaram 68,7% dos valores de CI50 menores que 20,0 µg mL-1, sendo também considerados citotóxicos. As amostras testadas foram mais ativas que o taxol sobre melanoma humano (MeWo) e, ainda, o grupo G10-5 apresentou melhor atividade sobre fibroblasto tumorigênico de camundongo (L929). Além disso, os grupos mostraram menor citotoxidade do que o taxol sobre a linhagem normal (CHO).

The family Annonaceae, especially the genus Annona, is greatly appreciated for providing edible fruits. Species of this genus are used in folk medicine against diabetes, malaria and infections. Many of these biological activities have been related to annonaceous acetogenins. The aim of this study was to evaluate the cytotoxic activity of groups and a pure acetogenin (cornifolin) obtained from the ethanol extract of the seeds of Annona cornifolia A. St.-Hil. (Annonaceae). This activity was evaluated by using MTT colorimetric assay. Cornifolin (1), the only tested substance that was pure, showed positive cytotoxicity on all evaluated tumor cell lines. The tested groups, all characterized by infrared spectroscopy (IR), showed 68.7% of the IC50 values lower than 20.0 µg mL-1, also considered cytotoxic. The tested samples were more active than taxol on human melanoma (MeWo) and the group G10-5 showed better activity on mouse tumorigenic fibroblast (L929). In addition, the tested groups showed less cytotoxicity than taxol on the normal line (CHO).

Effect of annonaceous acetogenin mimic AA005 on proliferative inhibition of leukemia cells in vitro and its possible mechanisms / 中国实验血液学杂志

This study was aimed to investigate the biological behavior of annonaceous acetogenin mimic AA005 in various kinds of leukemia cells and further elaborated its possible mechanisms in acute promyelocytic leukemia (APL) cell line NB4. The proliferative inhibition of leukemia cells was measured by CCK-8 method. Cell death was determined by trypan blue. Cell morphological features of NB4 treated with AA005 were examined by microscopy after Wright's staining. The form of cell death was measured by flow cytometry. Proteins PARP-1 and caspase-3 were detected by Western blot. Flow cytometry was used to detect the cell cycle arrest induced by AA005 of low concentration. The results showed that AA005 (> 200 nmol/L) significantly inhibited proliferation of all tested leukemia cell lines in a concentration-dependent manner. The vast majority of cells went to die after leukemia cell lines of NB4, U937 and K562 were treated with different concentration of AA005 for 48 h. Typical morphologic changes significantly appeared in NB4 cells after AA005 treatment. AA005 almost simultaneously induced early apoptosis and late apoptosis. The little cleavage of PARP-1 and activation of caspase-3 happened in AA005-induced cell death, and caspase-3 inhibitor Z-VAD-fmk could not block the cell death. The non-toxic concentrations of AA005 (< 200 nmol/L) caused NB4 cells G(2)/M-phase arrest. It is concluded that annonaceous acetogenin mimic AA005 induces significant proliferative inhibition of various leukemia cell lines in a concentration-dependent manner, which may be associated with cell death and G(2)/M-phase arrest induced by AA005.

Leishmanicidal activity and cytotoxicity of compounds from two Annonacea species cultivated in Northeastern Brazil / Atividade leishmanicida e citotoxicidade de constituintes químicos de duas espécies de Annonaceae cultivadas no nordeste do Brasil

INTRODUCTION: Visceral leishmaniasis is endemic in 88 countries, with a total of 12 million people infected and 350 million at risk. In the search for new leishmanicidal agents, alkaloids and acetogenins isolated from leaves of Annona squamosa and seeds of Annona muricata were tested against promastigote and amastigote forms of Leishmania chagasi. METHODS: Methanol-water (80:20) extracts of A. squamosa leaves and A. muricata seeds were extracted with 10 percent phosphoric acid and organic solvents to obtain the alkaloid and acetogenin-rich extracts. These extracts were chromatographed on a silica gel column and eluted with a mixture of several solvents in crescent order of polarity. The compounds were identified by spectroscopic analysis. The isolated compounds were tested against Leishmania chagasi, which is responsible for American visceral leishmaniasis, using the MTT test assay. The cytotoxicity assay was evaluated for all isolated compounds, and for this assay, RAW 264.7 cells were used. RESULTS: O-methylarmepavine, a benzylisoquinolinic alkaloid, and a C37 trihydroxy adjacent bistetrahydrofuran acetogenin were isolated from A. squamosa, while two acetogenins, annonacinone and corossolone, were isolated from A. muricata. Against promastigotes, the alkaloid showed an IC50 of 23.3 µg/mL, and the acetogenins showed an IC50 ranging from 25.9 to 37.6 µg/mL; in the amastigote assay, the IC50 values ranged from 13.5 to 28.7 µg/mL. The cytotoxicity assay showed results ranging from 43.5 to 79.9 µg/mL. CONCLUSIONS: These results characterize A. squamosa and A. muricata as potential sources of leishmanicidal agents. Plants from Annonaceae are rich sources of natural compounds and an important tool in the search for new leishmanicidal therapies.

INTRODUÇÃO: A leishmaniose visceral é uma enfermidade endêmica em 88 países, com um total de 12 milhões de pessoas infectadas e 350 milhões em risco. Na procura de novos agentes com ação leishmanicida, alcalóides e acetogeninas isoladas de Annona squamosa e Annona muricata, foram testados contra as formas promastigotas e amastigotas de Leishmania chagasi. MÉTODOS: Foram preparados extratos com metanol: água (80: 20) das folhas de A. squamosa e sementes de A. muricata que foram extraídos com solução de ácido fosfórico 10 por cento e solventes orgânicos, para obter extratos ricos em alcalóides e acetogeninas. Estes extratos foram cromatografados em coluna de sílica gel sendo eluídos com solventes de diferentes polaridades para o isolamento dos constituintes, e feita a determinação estrutural por análise espectroscópica. Os constituintes isolados foram testados contra Leishmania chagasi, responsável pela leishmaniose visceral, utilizando o teste MTT. Testes de toxicidade foram realizados em todos os compostos isolados, sendo utilizadas células RAW 264.7. RESULTADOS: Um alcalóide benzilisoquinolínico, O-metilarmepavina, e uma C37-triidróxi-acetogenina com anel bistetrahidrofurânico adjacente foram isolados de A. squamosa e duas acetogeninas annonacinona e corossolona da A. muricata. O alcalóide mostrou um índice de inibição médio (IC50) de 23,3µg/mL e as acetogeninas apresentaram IC50 variando entre 25,9 a 37,6µg/mL contra promastigotas, e no ensaio de amastigotas, o IC50 valores variaram entre 13,5 a 28,7 µg/mL. A toxicidade mostrou resultados que variaram entre 43,5 a 79,9µg/mL. CONCLUSÕES: Estes resultados caracterizam A. squamosa e A. muricata como fontes potenciais de agentes leishmanicidas.

Study of syntrophic anaerobic digestion of volatile fatty acids using enriched cultures at mesophilic conditions

Volatile fatty acids are the most important intermediates in anaerobic digestion, and their degradations are extremely complicated thermodynamically. In this research, syntrophic anaerobic digestion of volatile fatty acids using enriched acetogenic and methanogenic cultures in a batch reactor at mesophilic conditions was investigated. Interactive effects of key microbiological and operating variables [propionic, butyric and acetic acids, retention time and methanogen to acetogen populations ratio] on the anaerobic degradation of volatile fatty acids were analyzed. Acetogenic and methanogenic anaerobes in the granular sludge from an up-flow anaerobic sludge blanket reactor were enriched at mesophilic conditions within a period of four weeks, separately. Enriched cultures were mixed with known proportions and then used in the bioreactor. Experiments were carried out based on central composite design and analyzed using response surface methodology. Four parameters [final concentrations of propionic, butyric and acetic acids and biogas production] were directly measured as response. Also, the optimum conditions for volatile fatty acid degradation were found to be 937.5 mg/L, 3275.5 mg/L, 2319.5 mg/L, 45 h and 2.2 proportions for propionic acid, butyric acid, acetic acid, retention time and methanogen to acetogen populations ratio, respectively [corresponding to maximum volatile fatty acid removal efficiencies and biogas production]. The results of the verification experiment and the predicted values from the fitted correlations at the optimum conditions were in close agreement at a 95% confidence interval. The present study provides valuable information about the interrelations of quality and process parameters at different values of microbiological and operating variables

Anthelmintic acetogenin from Annona squamosa L. Seeds

Annona squamosa seeds extracts showed anthelmintic activity against Haemonchus contortus, the main nematode of sheep and goat in Northeastern Brazil. A compound 1 was isolated from ethyl acetate extract and inhibited the egg hatching of H. contortus at 25 mg ml-1. The structure of 1 was determined as a C37 trihydroxy adjacent bistetrahydrofuran acetogenin based on spectroscopic analysis.

Extratos das sementes de Annona squamosa demonstraram atividade anti-helmíntica contra Haemonchus contortus, o principal nematódeo de ovinos e caprinos no Nordeste do Brasil. O composto 1 foi isolado do extrato acetato de etila e inibiu a eclosão dos ovos de H. contortus a 25 mg ml-1. A estrutura de 1 foi determinada como uma acetogenina C37 tri-hidroxi bis-tetrahydrofurano adjacente, baseando-se nos dados espectrais.

Differential cytotoxic effects of Annona squamosa seed extracts on human tumour cell lines: role of reactive oxygen species and glutathione.

Annonaceous acetogenins are a new class of compounds that have been reported to have potent pesticidal, parasiticidal, anti-microbial, cell growth inhibitory activities. In this study, organic and aqueous extracts from the defatted seeds of Annona squamosa (custard apple) were tested on different human tumour cell lines for antitumoural activity. While organic and aqueous extracts induced apoptosis in MCF-7 and K-562 cells, they failed to do so in COLO-205 cells. Treatment of MCF-7 and K-562 cells with organic and aqueous extracts resulted in nuclear condensation, DNA fragmentation, induction of reactive oxygen species (ROS) generation and reduced intracellular glutathione levels. In addition downregulation of Bcl-2 and PS externalization by Annexin-V staining suggested induction of apoptosis in MCF-7 and K-562 cells by both the extracts through oxidative stress. On the contrary, COLO-205 cells showed only PS externalization but no change in ROS and glutathione levels. These observations suggest that the induction of apoptosis by A. squamosa extracts can be selective for certain types of cancerous cells.

Inhibition of oxygen consumption by annonaceous acetogenins in liver cell respiration and their structure-activity relationship / 药学学报

<p><b>AIM</b>To study the inhibition of oxygen consumption by annonaceous acetogenins (ACG) and their structure-activity relationship (SAR).</p><p><b>METHODS</b>The inhibition of oxygen consumption in chicken liver cell respiration by different structural ACG was studied by using oxygen electrode technique.</p><p><b>RESULTS</b>Six ACG showed potent inhibitory effects like rotenone which was a classical inhibitor of mitochondrial complex I, and was more potent than complex IV inhibitor KCN. The IC50 values of six ACG for inhibiting oxygen consumption suggested that bistetrahydrofuran (THF) ACG was 7-11 times more active than non-THF ACG, and A1-type ACG was more potent than A2-type ACG.</p><p><b>CONCLUSION</b>The terminal gamma-lactone was crucial for the inhibition of oxygen consumption. The distance between THF and gamma-lactone, the hydroxyl groups in the alkyl chain, were the important factors of SAR, but the 4-OH group possibly played some negative role in the exhibit of potent activity.</p>