Chemical characterization of Brazilian savannah Byrsonima species (muricis) and their impact on genomic instability and chemopreventive effects.

The identification of new drugs with few or no adverse effects is of great interest worldwide. In cancer therapy, natural products have been used as chemopreventive and chemotherapeutic agents. Plants from the Brazilian savannah belonging to the Byrsonima genus are popularly known as muricis and have attracted much attention due to their various pharmacological activities. However, there are currently no data on these plants concerning their use as chemopreventive or chemotherapeutic agents in human cell lines. The present study assessed the potential of B. correifolia, B. verbascifolia, B. crassifolia, and B. intermedia extracts as natural alternatives in the prevention and/or treatment of cancer. The chemical constituents present in each extract were analyzed by electrospray ionization-mass spectrometry (ESI-MSN). The mutagenic/antimutagenic (micronucleus assay), genotoxic/antigenotoxic (comet assay), apoptotic/necrotic (acridine orange/ethidium bromide uptake), and oxidative/antioxidative (CM-H2DCFDA) effects of the extracts and their influence on gene expression (RTqPCR) were investigated in nonmetabolizing gastric (MNP01) and metabolizing hepatocarcinoma (HepG2) epithelial cells to evaluate the effects of metabolism on the biological activities of the extracts. The genotoxicity, mutagenicity, and apoptotic effects observed in HepG2 cells with B. correifolia and B. verbascifolia extracts are probably associated with the presence of proanthocyanidins and amentoflavone. In MNP01 cells, none of the four extracts showed mutagenic effects. B. crassifolia and B. intermedia extracts exhibited strong antimutagenicity and enhanced detoxification in HepG2 cells and antioxidant capacities in both types of cells, possibly due to the presence of gallic and quinic acids, which possess chemopreventive properties. This study identifies for the first time B. correifolia and B. verbascifolia extracts as potential agents against hepatocarcinoma and B. crassifolia and B. intermedia extracts as putative chemopreventive agents.

Selective anticancer effects of Serjania marginata Casar. extract in gastric cells are mediated by antioxidant response.

Gastric cancer is the fifth most common malignancy worldwide. Serjania marginata Casar. (SM) displays anti-inflammatory properties and has been used to treat gastrointestinal disorders. In the current study, we examined whether the hydroethanolic extract of SM leaves exerted cytotoxic, mutagenic, and protective effects in non-tumor gastric epithelium cells (MNP01) and gastric adenocarcinoma cells (ACP02) in vitro and analyzed whether its action was selective. Initially, cell viability (MTT assay), cell cycle kinetics (flow cytometry), and cell proliferation (total protein content) were analyzed. In addition, genomic instability (cytokinesis-block micronucleus cytome assay), anti/pro-oxidant status (CM-H2 DCFDA probe), and transcriptional expression (RT-qPCR) of genes related to cell cycle, cell death, and antioxidant defense were also evaluated. The SM extract was cytotoxic toward MNP01 and ACP02 cells at concentrations greater than 300 and 100 µg·ml-1 , respectively, and decreased protein content only toward ACP02 cells at 200 µg ml-1 . In ACP02 cells, the SM extract at 100 µg·ml-1 associated with doxorubicin (DXR; 0.2 µg ml-1 ) clearly promoted cell cycle arrest at the G2/M phase. The extract alone was not mutagenic to either cell type and reversed DXR-induced DNA damage and H2 O2 -induced oxidative stress in MNP01 cells. The gene expression experiments showed that SM hydroethanolic extract exerts an antioxidant response via NFE2L2 activation in non-tumor gastric cells, and cell cycle arrest (G2/M) in ACP02 gastric cancer cells via the TP53 pathway. The selective action of SM indicates that it is a promising therapeutic agent to treat gastric diseases and merits further studies.

Pouteria ramiflora (Mart.) Radlk. extract: Flavonoids quantification and chemopreventive effect on HepG2 cells.

Pouteria ramiflora (Mart.) Radlk., popularly known as curriola, is commonly used in Brazil as medicinal plant to treat worm infections, dysentery, pain, inflammation, hyperlipidemia, and obesity. At present the safety of this extract when used therapeutically in human remains to be determined. Thus, the aim of this study was to examine cytotoxicity, antiproliferative, and antimutagenic actions of this extract. The hydroalcoholic extract from P. ramiflora leaves consisted of flavonoids identified and quantified as myricetin-3-O-ß-D-galactopyranoside (13.55 mg/g) and myricetin-3-O-α-L-rhamnopyranoside (9.61 mg/g). The extract exhibited cytotoxicity at concentrations higher than 1.5 µg/ml in human hepatocarcinoma (HepG2)and 2.5 µg/ml in non-tumoral primary gastric (GAS) cells using the MTT assay, and at concentrations higher than 3 µg/ml in HepG2 and 3.5 µg/ml in GAS cells by the neutral red assay. The extract did not show antiproliferative effect as evidenced by the nuclear division index (NDI). However, in the presence of benzo[a]pyrene (BaP) (positive control), an enhanced cytostatic effect in the NDI and flow cytometry was noted. It is of interest that when the extract was co-incubated with BaP a significant decrease in DNA damage was observed indicating an antimutagenic action. This protective effect might be attributed to myricetin and gallic acid found in P. ramiflora extract. The low cytotoxicity action and protective effect observed in the present study encourage further studies regarding other biological effects of P. ramiflora, as well as its potential use as a chemopreventive agent.

Phytochemical study and evaluation of cytotoxicity, mutagenicity, cell cycle kinetics and gene expression of Bauhinia holophylla (Bong.) Steud. in HepG2 cells in vitro.

Bauhinia holophylla (Bong.) Steud. (Fabaceae) is a plant used in Brazilian folk medicine to treat diabetes and inflammation. This study evaluated the phytochemical properties, cytotoxic, apoptotic, mutagenic/antimutagenic effects and alterations in gene expression (RNAm) in HepG2 cells treated with the B. holophylla extract. The phytochemical profile highlight the presence of flavonoids isorhamentin and quercetin derivates. The MTT assay was used to evaluate the cytotoxicity of different concentrations for different treatment times. Three concentrations (7.5, 15, 30 µg/mL) were chosen for assessment of apoptosis (AO/EB), mutagenicity (micronucleus), and cell cycle kinetics (flow cytometry). Thereafter, the concentration of 7.5 µg/mL was chosen to evaluate the protective effects against DNA damage induced by benzo[a]pyrene (B[a]P). At concentrations higher than 7.5 µg/mL (between 10 and 50 µg/mL), the extract was cytotoxic, induced apoptosis, and caused antiproliferative effects. However, it did not induce micronucleus and a reduction of apoptotic and micronucleated cells was observed in treatments that included the extract and B[a]P. The protective effect is attributable to the presence of flavonoids, described as antioxidants, inhibitors of DNA adduct and activators of detoxifying enzymes. The results of the present study such as absence of cytotoxic and mutagenic effects and protective effects against known carcinogens suggest that B. holophylla has potential for use soon as herbal medicine.

LDH, proliferation curves and cell cycle analysis are the most suitable assays to identify and characterize new phytotherapeutic compounds.

Brazilian flora biodiversity has been widely investigated to identify effective and safe phytotherapeutic compounds. Among the investigated plant species, the Byrsonima genus exhibits promising biological activities. This study aimed at evaluating the cytotoxicity of B. correifolia, B. verbascifolia, B. fagifolia and B. intermedia extracts using different assays in two cell lines (primary gastric and HepG2 cells). The different extract concentrations effects on cell viability were assayed using the MTT, aquabluer, neutral red and LDH assays. Non-cytotoxic concentrations were selected to generate cell proliferation curves and to assess cell cycle kinetics by flow cytometry. Byrsonima extracts differentially affected cell viability depending on the metabolic cellular state and the biological parameter evaluated. B. fagifolia and B. intermedia extracts exhibited lower cytotoxic effects than B. correifolia and B. verbascifolia in all assays. The results obtained with LDH and flow cytometry assays were more reliable, suggesting that they can be useful in the screening for herbal medicine and to further characterize these extracts as phytotherapeutic compounds.

Chemical and biological characterisation of Machaerium hirtum (Vell.) Stellfeld: absence of cytotoxicity and mutagenicity and possible chemopreventive potential.

Machaerium hirtum (Vell.) Stellfeld (M.hirtum) is a plant known as 'jacarandá-bico-de-pato' whose bark is commonly used against diarrhea, cough and cancer. The aim of this study was to phytochemically characterise the hydroethanolic extract of this plant, investigate its antimutagenic activities using the Ames test and evaluate its effects on cell viability, genomic instability, gene expression and cell protection in human hepatocellular carcinoma cells (HepG2). Antimutagenic activity was assessed by simultaneous pre- and post-treatment with direct and indirect mutagens, such as 4-nitro-o-phenylenediamine (NPD), mitomycin C (MMC), benzo[a]pyrene (B[a]P) and aflatoxin B1 (AFB1), using the Ames test, cytokinesis blocking micronucleus and apoptosis assays. Only 3 of the 10 concentrations evaluated in the MTT assay were cytotoxic in HepG2 cells. Micronucleated or apoptotic cells were not observed with any of the tested concentrations, and there were no mutagenic effects in the bacterial system. However, the Nuclear Division Index and flow cytometry data showed a decrease in cell proliferation. The extract showed an inhibitory effect against direct (NPD) and indirect mutagens (B[a]P and AFB1). Furthermore, pre- and post-treated cells showed significant reduction in the number of apoptotic and micronucleated cells. This effect is not likely to be associated with the modulation of antioxidant genes, as shown by the RT-qPCR results. Six known flavonoids were identified in the hydroethanolic extract of Machaerium hirtum leaves, and their structures were elucidated by spectroscopic and spectrophotometric methods. The presence of the antioxidants apigenin and luteolin may explain these protective effects, because these components can inhibit the formation of reactive species and prevent apoptosis and DNA damage. In conclusion, the M.hirtum extract showed chemopreventive potential and was not hazardous at the tested concentrations in the experiments presented here. Moreover, this extract should be investigated further as a chemopreventive agent.

Antimutagenicity and induction of antioxidant defense by flavonoid rich extract of Myrcia bella Cambess. in normal and tumor gastric cells.

ETHNOPHARMACOLOGICAL RELEVANCE: The Brazilian "Cerrado" is an important source of natural products, such as Myrcia bella Cambess (MB, also known as "mercurinho"). MB leaves are popularly used for the treatment of diabetes and gastrointestinal disorders; however, only its hypoglycemic activity has been experimentally described. AIM OF THE STUDY: Because MB is used to treat gastrointestinal disorders, the present study characterized biological activities of hydroalcoholic MB extract in human normal and tumor gastric cells. MATERIALS AND METHODS: Cytotoxic, antiproliferative, genotoxic and protective effects were evaluated, as well as the effects of the MB extract on gene expression. RESULTS: The MB extract induced cytotoxicity in tumor cells at lower concentrations compared with normal cells as assessed by the MTT assay. Moreover, the MB extract induced necrosis based on acridine orange/ethidium bromide staining. An antiproliferative effect was evidenced through an arrest in the G2/M phase detected by flow cytometry and a decrease in the nuclear division index using the cytokinesis-block micronucleus cytome assay. Cells treated with MB extract combined with doxorubicin (DXR) showed increased NUBDs, which may be related to the gene amplification of CCND1. Antimutagenic effects were also observed and may be associated with the antioxidant activities detected using the CM-H2DCFDA probe. CONCLUSIONS: Our findings showed the following: (a) high concentrations of MB induced cytotoxicity and cell death by necrosis; (b) its antiproliferative effect was associated with G2/M arrest; and (c) its antioxidant activity could be responsible for the observed antimutagenic effects and for protective effects against gastrointestinal disorders previously described to MB. Although these effects are not specific to normal or tumor cells, they provide a panel of biological activities for further exploration.

Interaction of Candida parapsilosis isolates with human hair and nail surfaces revealed by scanning electron microscopy analysis.

Candida parapsilosis is found frequently as commensal organism on epithelial tissues, and is also an increasing cause of nosocomial infection. Scanning electron microscope (SEM) observations were used to analyse the capability of C. parapsilosis cells to adhere and grow as biofilm on human natural substrates and to compare the adherence pattern of isolates exhibiting distinct phenotypes. Cells from the crepe phenotype are predominantly elongated and form pseudohyphae whereas cells from the smooth phenotype are yeast-shaped, either in liquid cultures or on human nail and hair surfaces. The electron micrographs revealed that C. parapsilosis cells from the smooth phenotype adhered in higher number to both surfaces compared to the observed for the crepe phenotype. SEM analysis of human hair surface revealed that cells from the smooth phenotype appear as clumped blastoconidia of uniform morphology embedded in a flocculent extracellular material forming biofilm. The extracellular material and biofilm were seeing in a less extension in the crepe phenotype. A distinct adherence pattern was observed when human nail was used as substrate. Here C. parapsilosis cells seem to be linked to surface structures of human nail plate. Fibrillar extracellular material was observed connecting neighbouring cells as well as nail surface.

In vitro evaluation of putative virulence attributes of oral isolates of Candida spp. obtained from elderly healthy individuals.

Identification of Candida isolates obtained from oral cavity of elderly healthy individuals revealed the predominance of non-albicans Candida species (88.9%) compared to Candida albicans (11%). CHROMagar Candida differential medium and PCR revealed the presence of Candida tropicalis (33.3%), Candida glabrata (27.8%), and Candida krusei (16.7%). We investigated the presence of virulence attributes in a total of 18 isolates, including acid protease and phospholipase production, hemolytic activity, and biofilm production. Extracellular protease was found in five isolates (27.8%) whereas extracellular phospholipase was found in three isolates (17%). All isolates showed hemolytic activity. About 56% of the isolates were weakly positive for biofilm formation (score +) whereas a minority (5.6%) of them showed strong biofilm formation (score 4+). Susceptibility in vitro of the isolates to fluconazole was carried out by microdilution method. Fluconazole showed a strong inhibition against most buccal isolates. The resistant isolates were 2 C. tropicalis, 2 C. glabrata, and 1 C. krusei.

Estudo da incidência de amostras clínicas do gênero Candida Estudo da incidência de amostras clínicas do gênero Candida isoladas de diversos sítios anatômicos / Study of the incidence of clinical samples belonging to the genus Candida obtained from different anatomic sites

A candidíase, principal infecção fúngica do ser humano, é provocada por leveduras do gênero Candida, que fazem parte da microbiota endógena e exógena do corpo humano. O objetivo deste trabalho foi avaliar a presença das espécies de Candida spp, em diversos sítios anatômicos. Um total de 90 amostras clínicas foram isoladas em ágar Sabouraud contendo antibióticos e identificadas em meio cromogênico CHROMagar Candida ®. Deste total, 58 amostras foram provenientes de secreção vaginal, 17 de raspado de unha, 8 de escarro e 7 de raspado de pele. Das amostras de secreção vaginal, 89% corresponderam a espécie C. albicans, seguido de espécies de Candida não-albicans, sendo 5,1% de C. krusei e 1,7% de C. glabrata, C. tropicalis e Cândida sp. , individualmente. Para as amostras isoladas de raspado de pele não foi observada prevalência de C. albicans (28%). As espécies Candida não-albicans corresponderam a 70% das amostras, sendo distribuídas em C. glabrata (14%), C. krusei (28%) e demais espécies de Candida (28%). A prevalência de espécies Candida não-albicans foi também observada para as amostras isoladas de raspado de unha, sendo que 29% foram caracterizadas como C. glabrata, 23% C. krusei e Cândida sp. , individualmente e 11% de C. tropicalis. Amostras de C. albicans, isoladas neste sítio anatômico, representaram somente 11%. Em amostras obtidas de escarro foi observada somente duas espécies, com prevalência de C. albicans (75%) seguida de C. tropicalis (25%). As amostras identificadas como C. albicans em meio CHROMagar Candida ® , foram confirmadas quanto ao crescimento a 42ºC e pelo emprego da técnica de seminested PCR.

Candidiasis is the main human fungal infection caused by yeasts of the genus Candida which are part of endogenous microflora of the human body. The aim of this study was to analyze the incidence of Candida species obtained from different infection processes. A total of 90 clinical samples were isolated in Sabouraud agar supplemented with antibiotics, and identified by CHROMagar Candida ®. Among them, 58 samples were isolated from vaginal secretion, 17 samples were from nail, 8 were from sputum and 7 were from skin. From the former, 89% were identified as C. albicans, followed by non albicans species, being 5.1% C. krusei, 1.7% C. glabrata, 1.7% C. tropicalis and 1.7% Candida sp. For samples isolated from nail it was not observed prevalence of C. albicans (28%); species nonalbicans corresponded to 70% of the samples, being 14% C. glabrata, 528% C. krusei and 28% Candida sp. The prevalence of non albicans species was also observed from samples isolated from nail, being 29% C. glabrata, 23% C. krusei, 23% Candida sp and 11% C. tropicalis. C. albicans in this infection site represented only 11%. Only two species were present in samples from sputum, being 75% C. albicans and 25% C. tropicalis. Samples identified as C. albicans in CHROMagar Candida ® agar were confirmed by growth at 42ºC and by seminested PCR approach.