Teresa García-Gasca

Human Gut Flora-Fermented Nondigestible Fraction from Cooked Bean (Phaseolus vulgaris L.) Modifies Protein Expression Associated with Apoptosis, Cell Cycle Arrest, and Proliferation in Human Adenocarcinoma Colon Cancer Cells

Metabolism of the nondigested fraction (NDF) from common bean ( Phaseolus vulgaris L.) by the human gut flora (hgf) produces short-chain fatty acids (SCFAs) that may benefit cancer by reducing colorectal tumor risks. This paper reports the effect of fermentation products (FP) by hgf (FP-hgf) from NDF of cooked beans on survival and protein expression associated with apoptosis, cell cycle arrest, and proliferation in human adenocarcinoma colon cancer cells. FP-hgf was the only inoculum eliciting butyrate production after 24 h of NDF fermentation using different bacterial sources. FP-hgf inhibited HT-29 cell growth and modulated protein expression associated with apoptosis, cell cycle arrest, and proliferation, as well as morphological changes linked to apoptosis evaluated by TUNEL and hematoxylin and eosin stains, confirming previous results on gene expression. The current results suggest that fermentation of NDF from common beans can elicit beneficial chemoprotective effects in colon cancer by modulating protein expression in HT-29 cells.

Fermented nondigestible fraction from common bean (Phaseolus vulgaris L.) cultivar Negro 8025 modulates HT-29 cell behavior.

The aim of the study was to evaluate the effect of a fermented nondigestible fraction (FNDF) of cooked bean (Phaseolus vulgaris L.) cultivar Negro 8025 on human colon adenocarcinoma HT-29 cell survival. Negro 8025 was chosen for in vitro fermentation based on comparison of chemical composition with 2 other cultivars: Azufrado Higuera and Pinto Durango. Negro 8025 had 58% total dietary fiber, 27% resistant starch, and 20 mg of (+)-catechin equivalents per gram of sample. Short-chain fatty acids (SCFAs) production and pH of the medium were measured after fermentation as indicators of colon protection through induced arrest on cell culture and apoptosis. Butyrate and pH of FNDF of Negro 8025 were higher than the control fermented raffinose extract. The FNDF inhibited HT-29 cell survival in a time- and concentration-dependent manner. The lethal concentration 50 (LC(50)) was 13.63% FNDF (equivalent to 7.36, 0.33, and 3.31 mmol of acetic, propionic, and butyric acids, respectively). DNA fragmentation, an apoptosis indicator, was detected by the TdT-mediated dUTP nick end labeling method in cells treated with the LC(50)-FNDF and a synthetic mixture of SCFAs mimicking LC(50)-FNDF. Our results suggest that common bean is a reliable source of fermentable substrates in colon, producing compounds with potential chemoprotective effect on HT-29 colon adenocarcinoma cells, so it may present an effective alternative to mitigate colon cancer development.

The effects of a protease inhibitor fraction from tepary bean (Phaseolus acutifolius) on in vitro cell proliferation and cell adhesion of transformed cells.

Some protease inhibitors (PI), such as the soybean Bowman-Birk protease inhibitor (SBBI), have been described as anticarcinogenic agents. Although PI are ubiquitous compounds in living organisms, the anticarcinogenic potential of PIs other than SBBI remain poorly explored. We evaluated the antiproliferative effect of a protein fraction from tepary bean (Phaseolus acutifolius) seeds with protease inhibitor activity (TPIF), on normal and on malignant cells. TPIF was obtained after precipitation with ammonium sulfate and gel filtration, and its bioactivity was assayed in vitro on HeLa cells, normal 3T3 fibroblasts and 3T3/v-mos transformed fibroblasts. TPIF showed antiproliferative and cytotoxic effects on 3T3/v-mos transformed fibroblasts in a dose-dependent way. On the contrary, TPIF was only cytostatic for normal 3T3 cells at the highest doses assayed, and had no effect on epithelial HeLa cells proliferation. Sublethal TPIF doses also stimulated cell adhesion of poorly adherent 3T3/v-mos cell line.

Effects of Tepary Bean (Phaseolus acutifolius) Protease Inhibitor and Semipure Lectin Fractions on Cancer Cells

Some natural and synthetic protease inhibitors (PI), such as the Bowman-Birk PI from soybean, have anticancer effects. We previously purified and characterized a Bowman-Birk-type PI from Tepary bean (Phaseolus acutifolius) seeds (TBPI). A semipure protein fraction containing this inhibitor, when tested its in vitro effect on transformed cells, showed a differential cytotoxic effect, as well as an increase in cell attachment to culture dishes. In this article we report that lectins were responsible for the cytotoxic effect previously observed, exhibiting a differential, antiproliferative effect on nontransformed cells and on different lineages of cancer cells. Although the purified TBPI lacked cytotoxicity, it was found to be responsible for the increase in cell adhesion, decreasing culture dishes’ extracellular matrix degradation, leading to a decrease of the in vitro cell invasion capacity. This effect coincided with the suppression of Matrix Metalloproteinase-9 activity. These results indicate that Tepary bean seeds contain at least 2 different groups of bioactive proteins with distinct effects on cancer cells.

Total phenolic compounds in milk from different species. Design of an extraction technique for quantification using the Folin-Ciocalteu method.

Milk protects the health of newborns because it contains essential compounds that perform metabolic activities. Despite these benefits, the study of phenolic compounds in milk has been poorly explored. The objective of this study was to develop and validate a technique for extracting total phenolic compounds (TPCs) from a milk matrix and then analyzing them using the Folin-Ciocalteu method. The extraction technique was applied to goat milk and involved the addition of methanol, acetonitrile, and Carrez I and II reagents, after which protein was separated from fat through centrifugation. Subsequently, the technique was applied to goat (69.03±6.23mg GAE/L), cow (49.00±10.77mg GAE/L), sheep (167.6±58.77mg GAE/L) and human milk (82.45±12.3mg GAE/L). The technique showed an acceptable linearity (R(2)=0.9998), limit of detection (6.03mg GAE/L) and quantification (16.2mg GAE/L), repeatability (RSD=4%), reproducibility (RSD=6.8%) and recovery (>85.41%); it is thus effective and can be used in the routine analysis of milk. TPCs obtained from each type of milk indicate a high variability among species and among members of the same species.

Bean seeds: leading nutraceutical source for human health

Phaseolus is the most important food legume for human consumption in the world. Its seeds consist mainly of carbohydrates and are a good source of nitrogen and protein. It also contains calcium, magnesium, potassium, phosphorus, copper, iron, zinc, manganese and sulfur. This legume is rich in bioactive components such as enzyme inhibitors, lectins, phytates, oligosaccharides and phenolics, which exhibit metabolic roles in humans and animals. Among the observed biological activities are the antioxidant capacity, the reduction of cholesterol and reduction of low-density lipoproteins, thus Phaseolus has a protective effect against cardiovascular diseases. Also it has shown favourable effects against cancer because of the antimutagenic and antiproliferative properties of their phenolics, lectins and protease inhibitors. Additionally, it has showed effects on obesity and diabetes due to its content of resistant starch and α-amylase inhibitor. Here we present a review of the beneficial properties of beans as a nutraceutical food.

Content and evolution of potential furfural compounds in commercial milk-based infant formula powder after opening the packet

Potential furfural compounds were examined by RP-HPLC-DAD in 20 commercial milk-based powdered infant formula (IF) brands from local markets from Paris, France; DF, Mexico; Copenhagen, Denmark; England, UK; and Barcelona, Spain. We traced the evolution of these compounds after the packets had been opened at 0, 30 and 70 days of storage at room temperature (≈25 °C; minimum 23 °C and maximum 25.5 °C). All formula brands were analysed during the first 3-5 months of their shelf life. The mean values of all IFs for potential 5-hydroxymethyl-2-furaldehyde (HMF)+2-furaldehyde (F) were 1115.2 μg/100 g (just opened), 1157.6 μg/100 g (30 days) and 1344.5 μg/100 g of product (70 days). In general, slight increases of potential furfural contents were observed in most of the studied IFs, which suggests that the Maillard reaction increases after opening the packets. The main furfural compound found was HMF, as expected. The range of potential HMF consumed for an infant about 6 months old feeding only on formula was estimated between 0.63 mg and 3.25 mg per day.

Colorimetric quantitation of in vitro cell density using carmine, a chromosome-specific stain.

Cell number is usually evaluated during in vitro studies to estimate metabolic or pharmacological effects of specific compounds. However, estimation of in vitro cell density by direct cell counting is a laborious and time-consuming task, whereas indirect methods for cell quantitation have serious disadvantages such as environmental costs or inaccuracies derived from non-specific interferences. We developed a new method for in vitro cell density quantitation which employs carmine, a natural dye widely used for chromosome staining in cytological studies. Normal or transformed murine fibroblasts, avian normal fibroblasts, human epithelial HeLa cells, and insect cells, inoculated at a range of cell densities, were fixed with 4% formaldehyde/PBS and stained with 0.4% alcoholic-HCl carmine. The stain retained in cell monolayers was extracted with 0.01 M NaOH and spectrophotometrically measured at 531 nm. Invariably, high correlation coefficients between cell number and absorbance were obtained for each cell type, within a range of 5 x 10(3) to 5 x 10(5) cells. Moreover, identical cell growth curves were obtained when cell number was estimated over several days of culture by both direct cell counting and carmine staining methods. Our results show that the carmine staining method represents an easy, precise and reliable alternative for in vitro cell quantitation, avoiding interferences caused by cell components modulable by culture treatments, and over a wide range of cell types and cell densities.

Effect of carotenoids against genotoxicity of diethylnitrosamine on rat hepatocytes.

Carotenoids have been considered as special nutrients due to their biological activity as provitamin A compounds, and because of their natural antioxidant and anticarcinogenic properties. The main objective of this study was to evaluate the protective effect of carotenoids against the genotoxic cellular damage induced by diethylnitrosamine (DEN), a potent hepatocarcinogen. Normal and freshly isolated hepatocytes were cultured as the biological system. Concentrations of 2.5 and 5 mum DEN caused 1.3 and 2.0 times more DNA T(3)H incorporation, respectively, when compared with control cells. Pure carotenoids, beta-carotene (50 mum), lutein (1 mum) and a carotenoid extract from green peppers (1 mum eq. lutein) were used as functional nutrients to protect the cells. All the carotenoids studied prevented the genotoxic damage caused by 2.5 mum DEN. When 5 mum DEN was used, only beta-carotene and the pepper extract inhibited the damage up to 30-40%. Carotenoids provide a dose-dependent protective effect against DNA damage induced by DEN in isolated hepatocytes.

Tolerability assessment of a lectin fraction from Tepary bean seeds (Phaseolus acutifolius) orally administered to rats

Highlights • We examine the toxicological profile of Tepary Bean lectins by oral route.• Tepary bean lectins showed digestion resistance up to 72 h.• Tepary bean lectins induce granulocyte increase after 24 h treatment.• A reduction in body weight gain was observed after 6 weeks treatment.• No toxicity was observed for Tepary bean lectins after 6 weeks.