Maria Helena Rossi

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.

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.

Biological activities of extracts from Chenopodium ambrosioides Lineu and Kielmeyera neglecta Saddi

BackgroundChenopodium ambrosioides and Kielmeyera neglecta are plants traditionally used in Brazil to treat various infectious diseases. The study of the biological activities of these plants is of great importance for the detection of biologically active compounds.MethodsExtracts from these plants were extracted with hexane (Hex), dichloromethane (DCM), ethyl acetate (EtOAc) and ethanol (EtOH) and assessed for their antimicrobial properties, bioactivity against Artemia salina Leach and antifungal action on the cell wall of Neurospora crassa.ResultsExtracts from C. ambrosioides (Hex, DCM and EtOH) and K. neglecta (EtOAc and EtOH) showed high bioactivity against A. salina (LD50 < 1000 μg/mL), which might be associated with cytotoxic activity against cancer cells. C. ambrosioides Hex and DCM showed specific activity against yeasts, highlighting the activity of hexanic extract against Candida krusei (MIC = 100 μg/mL). By comparing the inhibitory concentration of 50% growth (IC 50%) with the growth control, extracts from K. neglecta EtOAc and EtOH have shown activities against multidrug-resistant bacteria (Enterococcus faecalis ATCC 51299 and Staphylococcus aureus ATCC 43300), with IC 50% of 12.5 μg/mL The assay carried out on N. crassa allowed defining that extracts with antifungal activity do not have action through inhibition of cell wall synthesis.ConclusionsGenerally speaking, extracts from C. ambrosioides and K. neglecta showed biological activities that have made the search for bioactive substances in these plants more attractive, illustrating the success of their use in the Brazilian folk medicine.

Brazilian Bidens pilosa Linné yields fraction containing quercetin-derived flavonoid with free radical scavenger activity and hepatoprotective effects

Bidens pilosa is a plant used by Amazonian and Asian folks for some hepatopathies. The hydroethanol crude extract and three fractions were assessed for antioxidant and hepatoprotective effects. Higher levels of scavenger activity on the 1,1-diphenyl-2-picrylhydrazyl radical, inhibition of deoxyribose oxidation and lipid peroxidation in vitro were detected for the ethyl acetate fraction (IC50∼4.3–32.3 µg/ml) followed by the crude extract (IC50∼14.2–98.0 µg/ml). The ethyl acetate fraction, again followed by the crude extract, showed high contents of total soluble polyphenols (3.6±0.2 and 2.1±0.2 GAE/mg, respectively) and presence of a quercetin-derived flavonoid identified as quercetin 3,3′-dimethyl ether 7-O-β-d-glycopyranoside. Both products were assayed for hepatoprotector effects against CCl4-induced liver injury in mice. Markers of oxidative stress and hepatic injury were evaluated. The results showed that the 10-day pretreatments (15 mg/kg, p.o.) protected the livers against injury by blocking CCl4-induced lipid peroxidation and protein carbonylation and the DNA fragmentation was decreased (∼60%). The pretreatments avoided the loss of the plasma ferric reducing/antioxidant power and the elevation of serum transaminases and lactate dehydrogenase activities. The results suggest that the main constituents responsible for the hepatoprotective effects with free radical scavenger power associated are well extracted by performing fractionation with ethyl acetate. The findings support the Brazilian traditional use of this plant and justify further evaluations for the therapeutic efficacy and safety of the constituents of the ethyl acetate fraction to treat some liver diseases.

Genipa americana (Rubiaceae) Fruit Extract Affects Mitogen-Activated Protein Kinase Cell Pathways in Human Trophoblast–Derived BeWo Cells: Implications for Placental Development

Genipa americana L. (Rubiaceae) is a fruit tree and a traditional medicine used to treat anemia, icterus, asthma, and liver and spleen problems. The aim of the present study was to verify the effect of G. americana fruit ethanolic extract on the mechanism for proliferation and differentiation of trophoblast-like cells. Qualitative analysis of G. americana fruit extract was performed, and BeWo cells, a well-established placental choriocarcinoma cell line that can undergo differentiation, were used to analyze cell viability and proliferation. Methods consisted of cytotoxic and proliferation measurement, detection of release of human chorionic gonadotrophins, cell fusion observation, and evaluation of cell-signaling pathways (production of cyclic adenosine monophosphate and phosphorylation of mitogen-activated protein kinases [MAPKs]). A stock solution of the extract was diluted in Hams F-12 medium with 10% fetal bovine serum at concentrations ranging from 50 to 1000 μg/mL. Cells treated with dimethylsulfoxide, forskoline, and MAPK inhibitors (PD98059 or SB203580) were used as a control. Forskoline was used to induce the differentiation state in BeWo cells. Phytoanalysis indicated the presence of steroids only. Results showed that the G. americana fruit extract did not cause any cytotoxicity or interference in cell differentiation. However, a significant antiproliferative state related to inhibition and reactivation of ERK1/2 and p38 MAPK in BeWo cells was seen. These results suggest that steroids from G. americana may affect placental cell regulation.

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.

Inhibition of tumor proliferation associated with cell cycle arrest caused by extract and fraction from Casearia sylvestris (Salicaceae)

ETHNOPHARMACOLOGICAL RELEVANCE Casearia sylvestris is a tree found in tropical America. In Brazil it is known mainly as Guaçatonga. Literature reports suggest that the leaves and other plant parts have been used by indigenous populations from South America in preparations, mainly aqueous or hydroethanolic macerations or decoctions, most times taken orally for the primary treatment of several diseases, including cancer. AIM OF THE STUDY This article reports the results of an investigation about the antiproliferative effects of Casearia sylvestris on tumor cells in vitro and in vivo. MATERIAL AND METHODS Aqueous ethanolic maceration and column chromatography were done to obtain a crude aqueous ethanolic extract (CAE) and a chloroform fraction (f-CHCl3). The human breast cancer cell line MCF-7 was used in culture. In vitro, non-cytotoxic concentrations were determined by MTT assay and the antiproliferative effect was assessed by the colony forming unit assay using non-cytotoxic concentrations. Effects on the cell cycle were observed through flow cytometry using a propidium iodide kit. Casearin C was identified in f-CHCl3 by chromatography and H(1) nuclear magnetic resonance. The effect on some key proteins of DNA damage (phosphorylation on the histone H2AX) and cell cycle control (p53, p16, cdk2) was evaluated through immunoblot. Antiproliferative effects in vivo were measured in tumor tissue from Ehrlich ascites-bearing mice through the (3)H-thymidine uptake assay and the trypan blue exclusion method. RESULTS In vitro, EC50 values found at 24 h on MCF-7 cells were 141 µg/mL for CAE and 66 µg/mL for f-CHCl3. Inhibition on proliferation was recorded at concentrations as low as 4 µg/mL in the case of the f-CHCl3 (up to 40%) and up to 50% when CAE was added at 9 µg/mL. The cell cycle arrest was demonstrated by the reduction in terms of number of cells in phases G2/M and S, up to 38.9% and 51.9% when cells were treated with CAE, and 53.9% and 66.2%, respectively, when cells were treated with f-CHCl3. The number of cells in G1 was increased when the cells were treated with CAE (21.4%) or f-CHCl3 (27.8%). Key proteins of cell cycle control were affected. The treatments caused activation of p53, p16 and DNA damage found by the appearance of bands corresponding to γ-H2AX. The treatments caused inhibition of cdk2. CAE and particularly f-CHCl3 caused significant inhibition on tumor growth in mice (40% and 60%, respectively). Uptake of (3)H-thymidine, thus proliferation was reduced in tumor cells from mice treated with CAE (>30%) or f-CHCl3 (up to 50%) compared to cells from control animals. Data from the trypan blue assay indicating a lower number of tumor cells in treated animals. From the overall, data from this study are in line with the traditional claims for the antitumor effect of Casearia sylvestris. CONCLUSIONS This investigation suggests that whether the extracts from Casearia sylvestris are cytotoxic at high concentrations, lower concentrations have antiproliferative effect and could be useful to complement conventional cytotoxic chemotherapy, and should be evaluated further.

Free-radical scavenging by Ouratea parviflora in experimentally-induced liver injuries

Abstract The antioxidant potential of crude extracts and fractions from leaves of Ouratea parviflora, a Brazilian medicinal plant used for the treatment of inflammatory diseases, was investigated in vitro through the scavenging of radicals 2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH), hydroxyl radical (HO•), superoxide anion (O2•−), and lipid peroxidation in rat liver homogenate. The crude extract (CEOP) and hydro-alcoholic fraction (OP4) showed strong inhibitory activity toward lipid peroxidation induced by tert-butyl peroxide (IC50 = 2.3 ± 0.2 and 1.9 ± 0.1 μg/ml, respectively). The same products exhibited a strong concentration-dependent inhibition of deoxyribose oxidation (14.9 ± 0.2 and 0.2 ± 0.1 μg/ml, respectively), and also showed a considerable antioxidant activity against O2•−(87.3 ± 0.1 and 73.1 ± 0.4 μg/ml, respectively) and DPPH radicals (55.4 ± 0.3 and 38.3 ± 0.4 μg/ml, respectively). The protective effects of CEOP and OP4 were also studied in mouse liver. CCl4 significantly increased (by 90%) levels of lipid hydroperoxides, carbonyl protein content (64%), DNA damage index (133%), aspartate aminotransferase (261%), alanine aminotransferase (212%), catalase activity (23%), and also caused a decrease of 60% in GSH content. The results showed that CEOP and OP4 exerted cytoprotective effects against oxidative injury caused by CCl4 in rat liver, probably related to the antioxidant activity showed by the in vitro free radical scavenging property.

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.

Cytotoxicity of subfractions and compounds from Polymnia sonchifolia

Our previous works showed that extracts and fractions from Polymnia sonchifolia had presented an inhibitory activity on growth and aflatoxin production by Aspergillus flavus in non-cytotoxic concentrations. In this work, report results on the in vitro cytotoxicity of subfractions and of a mixture of sesquiterpenes lactones from Polymnia sonchifolia on Vero cells. The cytotoxicity was assayed through crystal violet staining (CVS) method and the 50% inhibitory concentration for cell growth (IC50) was calculated. Both subfractions and the mixture of sesquiterpenes lactones did not show in vitro cytotoxicity to Vero cells on active biologically concentrations against production of aflatoxin B1 by Aspergillus flavus.