Supplementation with Phaseolus vulgaris Leaves Improves Metabolic Alterations Induced by High-Fat/Fructose Diet in Rats Under Time-Restricted Feeding.

Time-restricted feeding and food enriched in polyphenols are strategies to prevent or reduce metabolic disorders. Bean leaves (Phaseolus vulgaris L.) are a recognized source of polyphenolic compounds, whose effects on metabolic pathways are not well studied. We evaluated the combined effects of dietary supplementation with Phaseolus vulgaris leaves (10% w/w) (BL) and a 7-h daytime-restricted feeding protocol (RF) under a hypercaloric diet (high fat + high fructose) (HFFD) on the metabolic parameters related to glucose and lipid handling. Adult male Wistar rats were treated for 8 weeks with standard and HFFD diets with or without BL. The results showed that RF improved metabolic alterations induced by HFFD (e.g., hepatic steatosis, increased triacylglycerols, and serum lipoproteins). Supplementation with BL significantly enhanced this effect and downregulated the mRNA expression of carbohydrate and lipid metabolism genes in the liver. These results indicate that dietary supplementation with BL enhances the benefits elicited by RF.

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.

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.

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.

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.