Edlayne Gonçalez

Study of the antitumor potential of Bidens pilosa (Asteraceae) used in Brazilian folk medicine.

AIM OF THE STUDY Bidens pilosa (L.) (Asteraceae) is a medicinal plant traditionally used in Brazil for treating conditions that can be related to cancer. Therefore the present study was carried out to evaluate the antitumor activity of extracts obtained from the aerial parts of this plant species. MATERIALS AND METHODS The crude hydroalcoholic extract (HAE) (water:alcohol, 6:4) and solvent fractions (chloroform=CHCl3,ethyl acetate=EtOAc, methanol=MeOH) were assessed for cytotoxicity assay by the brine shrimp and hemolytic, MTT and NRU assays. The antiproliferative potential of the crude extract and fractions was investigated in vivo using the Ehrlich ascites carcinoma (EAC) in isogenic Balb/c mice that were administered intraperitoneally 150 and 300 mg/kg body weight per day for nine days beginning 24 h after tumor inoculation. RESULTS In in vitro cytotoxicity using Ehrlich ascites carcinoma cell line assay CHCl3 extract proved to be more toxic than the crude HAE with an IC(50) of 97+/-7.2 and 83+/-5.2 microg/mL to NRU and MTT, respectively. Histomorphological evaluations indicated that the treatment with CHCl3 and HAE extracts significantly reduced (P<0.05) body weight, abdominal circumference, tumor volume, packed cell volume and viable cell count, when compared to EAC control group. Furthermore, nonviable tumor cell count increased significantly (P<0.01) only under treatment with CHCl3 or HAE, and this was accompanied by a marked percentage increase in life span (54.2 and 41.7%, respectively). Biochemical assays revealed that CHCl3 and HAE extracts were also able to decrease serum LDH activity (39.5 and 30.6%) and GSH concentration (94.6 and 50.7%) in ascitic fluid, respectively. CONCLUSION The chloroform fraction showed the best and methanolic the worst antitumor activity.

Ageratum conyzoides essential oil as aflatoxin suppressor of Aspergillus flavus.

Aflatoxin B(1) (AFB(1)) is a highly toxic and carcinogenic metabolite produced by Aspergillus species on food and agricultural commodities. Inhibitory effects of essential oil of Ageratum conyzoides, on the mycelial growth and aflatoxin B(1) production by Aspergillus flavus were studied. Cultures were incubated in yeast extract-sucrose (YES) broth for days at 25 degrees C at the following different concentrations of the essential oil (from 0.0 to 30mug/mL). The essential oil inhibited fungal growth to different extents depending on the concentration, and completely inhibited aflatoxin production at concentrations above 0.10microg/mL. The analysis of the oil by GC/MS showed that its main components are precocene II (46.35%), precocene I (42.78%), cumarine (5.01%) and Trans-caryophyllene (3.02%). Comparison by transmission electron microscopy of the fungal cells, control and those incubated with different concentrations of essential oil, showed ultra-structural changes which were concentration dependent of the essential oil of A. conyzoides. Such ultra-structural changes were more evident in the endomembrane system, affecting mainly the mitochondria. Degradation was also observed in both surrounding fibrils. The ability to inhibit aflatoxin production as a new biological activity of A.conyzoides L. indicates that it may be considered as a useful tool for a better understanding of the complex pathway of aflatoxin biosynthesis.

Mycobiota and mycotoxins in Brazilian peanut kernels from sowing to harvest

The total mycobiota and the mycotoxin contamination of peanuts were analyzed in plants collected at different stages of the pod maturity sampled in Junqueirópolis, at São Paulo State (Brazil). The prevalent peanut mycobiota were Fusarium spp. and Aspergillus flavus, present in 26% and 17% respectively of the samples analyzed. In soil, the genus Penicillium and Fusarium were most frequently detected, and A. flavus was detected in 8% of the samples. The screening of mycotoxins indicated that aflatoxins and cyclopiazonic acid were present the highest incidence, being detected in 32% of the samples, in concentrations, respectively, from 4.20 microg/kg to 198.84 microg/kg and from 260 microg/kg to 600 microg/kg. Fumonisins were not detected by HPLC. All data were correlated with the occurrence of wind-dispersed fungi and the environmental and soil conditions. Results indicate that good management of the agricultural environment may offer a way to reduce mycotoxins and the toxigenic fungal contamination in peanuts preharvest because the pods are exposed to different environmental conditions during their formation until harvest, and the optimal conditions for mycotoxin production and fungal growth are frequently found in the crop fields.

Evaluation of antifungal activity of seaweed extracts

Seaweeds are subject to numerous biological interactions and sometimes to extreme abiotic conditions, so they have developed among other defense mechanisms, the ability to produce biologically active substances. Thus, these organisms produce mainly terpenes and phenols. Among others, the antifungal activity, due to its importance in human and animal health and the production of agricultural products, has been the subject of several studies. In the present work, this activity was investigated in ten seaweeds extracts, by direct bioautography assays, compared to Colletotrichum lagenarium and disk diffusion assay, compared to Aspergillus flavus. The organisms studied were: Stypopodium zonale, Laurencia dendroidea, Ascophyllum nodosum, Sargassum muticum, Pelvetia canaliculata, Fucus spiralis, Sargassum filipendula, Sargassum stenophyllum, Laminaria hyperborea and Gracilaria edulis. S. zonale, L. dendroidea, P. canaliculata, S. muticum, A. nodosum and F. spiralis extracts significantly inhibited the C. lagenarium growth, but not inhibited significantly the A. flavus growth. The presence of terpenes in all of these extracts was confirmed by thin layer chromatography whereas the presence of phenolic compounds was confirmed only in extracts of P. canaliculata, A. nodosum and S. muticum. In chemical study by column chromatography, followed by gas chromatography/mass spectrometry analysis, the terpenes neophytadiene, cartilagineol, obtusol elatol; and the ester ethyl hexadecanoate were identified in the L. dendroidea extract. This is the first report on the activity of seaweed extracts against C. lagenarium, a fungus bearing agricultural importance.

ACTIVITY OF THE AQUEOUS EXTRACT FROM POLYMNIA SONCHIFOLIA LEAVES ON GROWTH AND PRODUCTION OF AFLATOXIN B1 BY ASPERGILLUS FLAVUS

The aqueous extract from Polymnia sonchifolia leaves (AE) was tested for inhibitory activity on aflatoxin B1(AFB1) production and growth of Aspergillus flavus. The cytotoxicity of AE on Vero cells was also performed. Suspensions of A. flavus spores were inoculated into 50 mL of YES medium together with different concentrations of the AE. The aflatoxin B1 was extracted, analyzed by thin layer chromatography and quantified by photodensitometry. All the concentrations of AE induced inhibition of AFB1 production. The aqueous extract showed in vitro cytotoxicity to Vero cells only at concentrations above 500 µg/mL.

Potential of essential oils for protection of grains contaminated by aflatoxin produced by Aspergillus flavus

Aflatoxin B1 (AFB1) is a highly toxic and carcinogenic metabolite produced by Aspergillus species on food and agricultural commodities. Inhibitory effects of essential oils of Ageratum conyzoides (mentrasto) and Origanum vulgare (oregano) on the mycelial growth and aflatoxin B1 production by Aspergillus flavus have been studied previously in culture medium. The aim of this study was to evaluate aflatoxin B1 production by Aspergillus flavus in real food systems (corn and soybean) treated with Ageratum conyzoides (mentrasto) and Origanum vulgare (oregano) essential oils. Samples with 60 g of the grains were treated with different volumes of essential oils, 200, 100, 50, and 10 μL for oregano and 50, 30, 15, and 10 μL for mentrasto. Fungal growth was evaluated by disk diffusion method. Aflatoxin B1 production was evaluated inoculating suspensions of A. flavus containing 1.3 × 105 spores/mL in 60 g of grains (corn and soybeans) after adjusting the water activity at 0.94. Aflatoxin was quantified by photodensitometry. Fungal growth and aflatoxin production were inhibited by essential oils, but the mentrasto oil was more effective in soybeans than that of oregano. On the other hand, in corn samples, the oregano essential oil was more effective than that of mentrasto. Chemical compositions of the essential oils were also investigated. The GC/MS oils analysis showed that the main component of mentrasto essential oil is precocene I and of the main component of oregano essential oil is 4-terpineol. The results indicate that both essential oils can become an alternative for the control of aflatoxins in corn and soybeans.

Evaluation of antifungal activity of Pittosporum undulatum L. essential oil against Aspergillus flavus and aflatoxin production

A presenca de micotoxinas como resultado do ataque fungico pode ocorrer antes, apos e durante a colheita e tambem no armazenamento de graos e alimentos. Considerando as propriedades inibitorias dos oleos essenciais de plantas no desenvolvimento do micelio dos fungos e a importâncias do Aspergillus flavus, principal produtor de aflatoxinas, relatou-se neste trabalho, a atividade toxica do oleo essencial do Pittosporum undulatum em cultura de A. flavus. Os oleos essenciais de P. undulatum foram obtidos a partir de folhas coletadas em diferentes meses e analisado por CG/EM. Os oleos se mostraram ricos em hidrocarbonetos, monoterpenos e sesquiterpenos e foi observada uma significante variacao na composicao quimica destes oleos nos diferentes meses de coleta. Os oleos essenciais mostraram diferentes espectros de inibicao do crescimento de A. flavus, porem todos foram capazes de inibir a producao de aflatoxina B1.

Evaluation of fungal burden and aflatoxin presence in packed medicinal plants treated by gamma radiation.

This study was developed to evaluate the fungal burden, toxigenic molds, and mycotoxin contamination and to verify the effects of gamma radiation in four kinds of medicinal plants stored before and after 30 days of irradiation treatment. Eighty samples of medicinal plants (Peumus boldus, Camellia sinensis, Maytenus ilicifolia, and Cassia angustifolia) purchased from drugstores, wholesale, and open-air markets in São Paulo city, Brazil, were analyzed. The samples were treated using a (60)Co gamma ray source (Gammacell) with doses of 5 and 10 kGy. Nonirradiated samples were used as controls of fungal isolates. For enumeration of fungi on medicinal plants, serial dilutions of the samples were plated in duplicate onto dichloran 18% glycerol agar. The control samples revealed a high burden of molds, including toxigenic fungi. The process of gamma radiation was effective in reducing the number of CFU per gram in all irradiated samples of medicinal plants after 30 days of storage, using a dose of 10 kGy and maintaining samples in a protective package. No aflatoxins were detected. Gamma radiation treatment can be used as an effective method for preventing fungal deterioration of medicinal plants subject to long-term storage.

Avaliação da micoflora e ocorrência de micotoxinas em cascas de amendoim em diferentes estágios de maturação da vagem

As cascas de amendoim (Arachis hypogaea L.) sao de grande importância para confeccao de cama de frangos, de gado de leite e como fonte de fibras para ruminantes, portanto a elucidacao dos mecanismos de contaminacao por fungos toxigenicos e por micotoxinas em amendoim e imprescindivel, especialmente para que medidas preventivas possam ser tomadas. Realizou-se, este trabalho, em Junqueiropolis, Estado de Sao Paulo, Brasil. Os principais fungos isolados nas cascas de amendoim foram Fusarium ssp. (78,75 %), Rhizopus ssp. (14,1 %) e A. flavus (11,75 %). No solo foram isolados Penicillium spp., Fusarium spp. e Aspergillus flavus, entre outros. Aflatoxinas foram detectadas em amostras de cascas de amendoim a partir do estagio de granacao em concentracoes que variaram de 5,42 μg/kg a 218,52 μg/kg. Acido ciclopiazonico e fumonisinas B1 e B2 nao foram detectadas. A presenca de A. flavus e aflatoxinas nas amostras, revela a importância de um controle das cascas de amendoim antes de sua utilizacao. Boas praticas agricolas sao indicadas para regiao, uma vez que a contaminacao das vagens ocorreu antes da colheita.

Intoxicação de vacas leiteiras por farelo de algodão naturalmente contaminado com aflatoxinas

Aflatoxina M1 e um dos metabolitos toxico da aflatoxina B1, excretada pelo leite de animais que ingerem alimentos contaminados. O presente trabalho descreve o caso de uma fazenda produtora de leite na regiao de Itu, Sao Paulo, onde os animais foram alimentados com farelo de algodao naturalmente contaminado com as aflatoxinas. As aflatoxinas B1, B2, G1 e G2 foram identificadas por CCD e quantificadas por fotodensitometria nas concentracoes de 43,5; 15,2; 9,1 e 8,6ng.g-1 respectivamente. O leite destes animais foi analisado por cromatografia liquida de alta eficiencia (CLAE), e a aflatoxina M1 foi identificada e quantificada (0,64ng.mL-1). As concentracoes encontradas para as aflatoxinas no farelo de algodao e no leite estao acima dos valores permitidos pela legislacao brasileira, representando um risco a saude publica.