DNA damage and DNA protection from digested raw and griddled green pepper (poly)phenols in human colorectal adenocarcinoma cells (HT-29).

PURPOSE: To determine whether (poly)phenols from gastrointestinal-digested green pepper possess genoprotective properties in human colon cells and whether the application of a culinary treatment (griddling) on the vegetable influences the potential genoprotective activity. METHODS: (Poly)phenols of raw and griddled green pepper (Capsicum annuum L.) submitted to in vitro-simulated gastrointestinal digestion were characterized by LC-MS/MS. Cytotoxicity (MTT, trypan blue and cell proliferation assays), DNA damage and DNA protection (standard alkaline and formamidopyrimidine DNA glycosylase (Fpg)-modified comet assay) of different concentrations of (poly)phenolic extracts were assessed in colon HT-29 cells. RESULTS: A total of 32 (poly)phenolic compounds were identified and quantified in digested raw and griddled green pepper. Twenty of them were flavonoids and 12 were phenolic acids. Griddled pepper doubled the (poly)phenol concentration compared to raw; luteolin 7-O-(2-apiosyl)-glucoside and quercitrin constituted the major (poly)phenols in both extracts. Raw and griddled pepper (poly)phenolic extracts impaired cell proliferation and induced low levels of Fpg-sensitive sites, in a dose-dependent manner, even at a non-cytotoxic concentration. None of the concentrations tested induced DNA strand breaks or alkaline labile sites. Nor did they show significant genoprotection against the DNA damage induced by H2O2 or KBrO3. CONCLUSIONS: Green pepper (poly)phenols did not show genoprotection against oxidatively generated damage in HT-29 cells at simulated physiological concentrations, regardless of the application, or not, of a culinary treatment (griddling). Furthermore, high concentrations of (poly)phenolic extracts induced a slight pro-oxidant effect, even at a non-cytotoxic concentration.

Digestion and Colonic Fermentation of Raw and Cooked Opuntia ficus-indica Cladodes Impacts Bioaccessibility and Bioactivity.

The bioactivity of (poly)phenols from a food is an interplay between the cooking methods applied and the interaction of the food with the gastrointestinal tract. The (poly)phenolic profile and biological activity of raw and cooked cactus ( Opuntia ficus-indica Mill.) cladodes following in vitro digestion and colonic fermentation were evaluated. Twenty-seven (poly)phenols were identified and quantified by HPLC-ESI-MS, with piscidic acid being the most abundant. Throughout the colonic fermentation, flavonoids showed more degradation than phenolic acids, and eucomic acid remained the most relevant after 24 h. The catabolite 3-(4-hydroxyphenyl)propionic acid was generated after 24 h of fermentation. Cytotoxicity, genotoxicity, and cell cycle analyses were performed in HT29 cells. Cactus colonic fermentates showed higher cell viability (≥80%) in comparison to the control fermentation with no cactus and significantly ( p < 0.05) reduced H2O2-induced DNA damage in HT29 cells. Results suggest that, although phenolic compounds were degraded during the colonic fermentation, the biological activity is retained in colon cells.

Development of a Novel High-Density [3H]Hypoxanthine Scintillation Proximity Assay To Assess Plasmodium falciparum Growth.

The discovery and development of new antimalarial drugs are becoming imperative because of the spread of resistance to current clinical treatments. The lack of robustly validated antimalarial targets and the difficulties with the building in of whole-cell activity in screening hits are hampering target-based approaches. However, phenotypic screens of structurally diverse molecule libraries are offering new opportunities for the identification of novel antimalarials. Several methodologies can be used to determine the whole-cell in vitro potencies of antimalarial hits. The [(3)H]hypoxanthine incorporation assay is considered the "gold standard" assay for measurement of the activity of antimalarial compounds against intraerythrocytic forms of Plasmodium falciparum However, the method has important limitations, as the assay is not amenable for high-throughput screening since it remains associated with the 96-well plate format. We have overcome this drawback by adapting the [(3)H]hypoxanthine incorporation method to a 384-well high-density format by coupling a homogeneous scintillation proximity assay (SPA) and thus eliminating the limiting filtration step. This SPA has been validated using a diverse set of 1,000 molecules, including both a representative set from the Tres Cantos Antimalarial Set (TCAMS) of compounds and molecules inactive against whole cells. The results were compared with those from the P. falciparum lactate dehydrogenase whole-cell assay, another method that is well established as a surrogate for parasite growth and is amenable for high-throughput screening. The results obtained demonstrate that the SPA-based [(3)H]hypoxanthine incorporation assay is a suitable design that is adaptable to high-throughput antimalarial drug screening and that maintains the features, robustness, and reliability of the standard filtration hypoxanthine incorporation method.

Influence of heat treatment on antioxidant capacity and (poly)phenolic compounds of selected vegetables.

The impact of cooking heat treatments (frying in olive oil, frying in sunflower oil and griddled) on the antioxidant capacity and (poly)phenolic compounds of onion, green pepper and cardoon, was evaluated. The main compounds were quercetin and isorhamnetin derivates in onion, quercetin and luteolin derivates in green pepper samples, and chlorogenic acids in cardoon. All heat treatments tended to increase the concentration of phenolic compounds in vegetables suggesting a thermal destruction of cell walls and sub cellular compartments during the cooking process that favor the release of these compounds. This increase, specially that observed for chlorogenic acids, was significantly correlated with an increase in the antioxidant capacity measured by DPPH (r=0.70). Griddled vegetables, because of the higher temperature applied during treatment in comparison with frying processes, showed the highest amounts of phenolic compounds with increments of 57.35%, 25.55% and 203.06% compared to raw onion, pepper and cardoon, respectively.

Assessment of total (free and bound) phenolic compounds in spent coffee extracts.

Spent coffee is the main byproduct of the brewing process and a potential source of bioactive compounds, mainly phenolic acids easily extracted with water. Free and bound caffeoylquinic (3-CQA, 4-CQA, 5-CQA), dicaffeoylquinic (3,4-diCQA, 3,5-diCQA, 4,5-diCQA), caffeic, ferulic, p-coumaric, sinapic, and 4-hydroxybenzoic acids were measured by HPLC, after the application of three treatments (alkaline, acid, saline) to spent coffee extracts. Around 2-fold higher content of total phenolics has been estimated in comparison to free compounds. Phenolic compounds with one or more caffeic acid molecules were approximately 54% linked to macromolecules such as melanoidins, mainly by noncovalent interactions (up to 81% of bound phenolic compounds). The rest of the quantitated phenolic acids were mainly attached to other structures by covalent bonds (62-97% of total bound compounds). Alkaline hydrolysis and saline treatment were suitable to estimate total bound and ionically bound phenolic acids, respectively, whereas acid hydrolysis is an inadequate method to quantitate coffee phenolic acids.

Variations in caffeine and chlorogenic acid contents of coffees: what are we drinking?

The effect of roasting of coffee beans and the extraction of ground coffee with different volumes of hot pressurised water on the caffeine and the total caffeoylquinic acids (CQAs) content of the resultant beverages was investigated. While caffeine was stable higher roasting temperatures resulted in a loss of CQAs so that the caffeine/CQA ratio was a good marker of the degree of roasting. The caffeine and CQA content and volume was determined for 104 espresso coffees obtained from coffee shops in Scotland, Italy and Spain, limited numbers of cappuccino coffees from commercial outlets and several instant coffees. The caffeine content ranged from 48-317 mg per serving and CQAs from 6-188 mg. It is evident that the ingestion of 200 mg of caffeine per day can be readily and unwittingly exceeded by regular coffee drinkers. This is the upper limit of caffeine intake from all sources recommended by US and UK health agencies for pregnant women. In view of the variable volume of serving sizes, it is also clear that the term "one cup of coffee" is not a reproducible measurement for consumption, yet it is the prevailing unit used in epidemiology to assess coffee consumption and to link the potential effects of the beverage and its components on the outcome of diseases. More accurate measurement of the intake of coffee and its potentially bioactive components are required if epidemiological studies are to produce more reliable information.

Antioxidant and genoprotective effects of spent coffee extracts in human cells.

Spent coffee has been shown as a good source of hydrophilic antioxidant compounds. The ability of two spent coffee extracts rich in caffeoylquinic acids, mainly dicaffeoylquinic acids, and caffeine (Arabica filter and Robusta espresso) to protect against oxidation and DNA damage in human cells (HeLa) was evaluated at short (2 h) and long (24 h) exposure times. Cell viability (MTT) was not affected by spent coffee extracts (>80%) up to 1000 µg/mL after 2 h. Both spent coffee extracts significantly reduced the increase of ROS level and DNA strand breaks (29-73% protection by comet assay) induced by H2O2. Pretreatment of cells with robusta spent coffee extract also decreased Ro photosensitizer-induced oxidative DNA damage after 24 h exposure. The higher effectiveness of Robusta spent coffee extract, with less caffeoylquinic acids and melanoidins, might be due to other antioxidant compounds, such as caffeine and other Maillard reaction products. This work evidences the potential antioxidant and genoprotective properties of spent coffee in human cells.

Evaluation of spent coffee obtained from the most common coffeemakers as a source of hydrophilic bioactive compounds.

The main hydrophilic antioxidant compounds (3-, 4-, and 5-monocaffeoylquinic and 3,4-, 3,5-, and 4,5-dicaffeoylquinic acids, caffeine, and browned compounds, including melanoidins) and the antioxidant capacity (Folin-Ciocalteu, ABTS, DPPH, Fremy's salt, and TEMPO) were evaluated in Arabica and Robusta spent coffee obtained from the preparation of coffee brews with the most common coffeemakers (filter, espresso, plunger, and mocha). All spent coffee grounds, with the exception of those from the mocha coffeemaker, had relevant amounts of total caffeoylquinic acids (6.22-13.24 mg/g of spent coffee), mainly dicaffeoylquinic acids (3.31-5.79 mg/g of spent coffee), which were 4-7-fold higher than in their respective coffee brews. Caffeine ranged from 3.59 to 8.09 mg/g of spent coffee. The antioxidant capacities of the aqueous spent coffee extracts were 46.0-102.3% (filter), 59.2-85.6% (espresso), and <42% (plunger) in comparison to their respective coffee brews. This study obtained spent coffee extracts with antioxidant properties that can be used as a good source of hydrophilic bioactive compounds.

Influence of brewing method and acidity regulators on the antioxidant capacity of coffee brews.

The antioxidant capacity of coffee brews prepared with different coffeemakers (filter, plunger, mocha, and espresso) was measured by colorimetric (total phenolic compounds and ABTS) and electron spin resonance (ESR) spectroscopy techniques (Fremy's salt and TEMPO). The mocha coffeemaker had the highest yield in coffee antioxidant extraction per gram of ground roasted coffee, but espresso coffee was richest in terms of antioxidant intake (per milliliter of coffee brew) followed by mocha, plunger, and filter. Both Folin-Ciocalteu (total phenolic compounds) and ABTS assays reacted with standard solutions of chlorogenic acids (CGA) and melanoidins (MO-Ala and MO-Gly). However, Fremy's salt was mainly scavenged by chlorogenic acids, whereas the stabilized radical TEMPO was effectively scavenged by melanoidins, but not by chlorogenic acids. Thus, ESR spectroscopy allows distinguishing between phenolic and nonphenolic antioxidants. Moreover, the addition of pH-regulator agents to coffee, such as sodium carbonate (75 ppm) and bicarbonate (75 ppm), to extend its shelf life, slightly increases the pH, modifying the antioxidant capacity in those coffee brews with the highest capacity (mocha and espresso).

Application of multivariate analysis to the effects of additives on chemical and sensory quality of stored coffee brew.

The aim of this work was to obtain a black coffee brew to be consumed hot by extension of its shelf life, by addition of additives. Four pH-regulator agents (sodium and potassium carbonates and bicarbonates), one pH regulator and antioxidant (sodium citrate), three antioxidants [sodium ascorbate, ethylenediaminetetracetic acid (EDTA), and sodium sulfite], and lactoserum were tested by sensory analysis. Sodium carbonate and bicarbonate were selected for a study of the physicochemical (soluble and volatile compounds related to the sensory properties) and sensorial quality of coffee brew stored for 90 days at 4 degrees C. Although both additives extended the shelf life of the coffee brew up to 60 days, sodium carbonate was the chosen additive because it was the most useful in limiting the pH decrease and perception of sourness, which are some of the main factors involved in the rejection of stored coffee brews, and it better maintained the aroma and taste/flavor. Moreover, the application of multivariate analysis facilitated first the description of the global changes of the coffee brews with or without additives throughout the storage using principal component analysis and second the obtainment of a simple equation only with pH and caffeic acid parameters to discriminate the three types of coffee brews and simplify the analytical process, by means of the stepwise discriminant analysis.

Changes in volatile compounds and overall aroma profile during storage of coffee brews at 4 and 25 degrees C.

In this work, the chemical changes occurring in the volatile fraction of Arabica coffee brews during storage at 4 and 25 degrees C for 30 days have been characterized for the first time by means of HS-GC-MS. A total of 47 compounds were identified and quantified: 2 sulfur compounds, 7 aldehydes, 3 esters, 15 furans, 5 ketones, 1 alcohol, 2 thiophenes, 4 pyrroles, 1 pyridine, 5 pyrazines, 1 alkene, and 1 acid. No new volatile compounds were detected at the end of the storage time. The changes observed are, in general, slower and less pronounced at refrigeration temperature. Storage also affects the sensory characteristics of the stored coffee brews, which lose part of their aroma intensity and freshness, acquiring some nondesirable notes such as rancid aroma, mainly during storage at 25 degrees C. Furthermore, seven aroma indices have been proposed as indicators of coffee brew staling, which show a good correlation with some sensory attributes, not only for aroma but also overall sensory quality. Consequently, they could be considered useful to monitor both the "age" and the sensory quality of stored coffee brews.

Correlation of selected constituents with the total antioxidant capacity of coffee beverages: influence of the brewing procedure.

Relationships between volatile and nonvolatile compounds and the antioxidant capacity of coffee brews prepared from commercial conventional and torrefacto roasted coffees, employing commonly used doses and prepared by four brewing procedures (filter, plunger, mocha, and espresso machine) were assessed. Significant correlations between volatile Maillard reaction products and antioxidant capacity (measured by both 2,2-diphenyl-1-picrylhydrazyl radical and redox potential methods) were not observed. Highly positive correlations between browned compounds and caffeine with both antioxidant capacity parameters were reported. Principal component analysis allowed coffee brews separation according to coffee roasting processes (PC1) and brewing procedures (PC2), showing that in all cases coffee brews from torrefacto roasted coffee were more antioxidant that those extracted from conventional ones; also, coffee brews extracted by an espresso machine were more antioxidant than those extracted by mocha, plunger, and filter machines.

Changes in headspace volatile concentrations of coffee brews caused by the roasting process and the brewing procedure.

Headspace-solid-phase microextraction technique (HS-SPME) coupled with gas chromatography-mass spectrometry (GC-MS) and gas chromatography-olfactometry (GC-O) were used to characterize the aroma compounds of coffee brews from commercial conventional and torrefacto roasted coffee prepared by filter coffeemaker and espresso machine. A total of 47 volatile compounds were identified and quantified. Principal component analysis (PCA) was applied to differentiate coffee brew samples by volatile compounds. Conventional and torrefacto roasted coffee brews were separated successfully by principal component 1 (68.5% of variance), and filter and espresso ones were separated by principal component 2 (19.5% of variance). By GC olfactometry, a total of 34 aroma compounds have been perceived at least in half of the coffee extracts and among them 28 were identified, among which octanal was identified for the first time as a contributor to coffee brew aroma.

Chemical and sensorial characteristics of espresso coffee as affected by grinding and torrefacto roast.

Grinding is a critical step in the preparation of espresso coffee (EC). The addition of sugar during the torrefacto roasting process could influence the degree of brittleness and grinding. The aim of this work was to study the influence of the grinding grades (coarse, fine, and very fine) in Arabica/Robusta 20:80, natural roasted (A20:R80), and Arabica/Robusta 20:80 with 50% Robusta torrefacto roasted (A20:R80 50% torrefacto) on the chemical and sensorial characteristics of EC in order to select the optimal espresso grinding grade. A higher percentage of coarse particles was found in A20:R80 ground coffee. In both ECs, the extraction of solids and soluble and aroma compounds increased inversely with particle size. Higher foam indices and extraction yields were found in A20:R80 50% torrefacto ECs probably due to the solubilization of caramelized sugar and melanoidins. It has been suggested that the range of an acceptable extraction yield could be extended to 25% in A20:R80 50% torrefacto ECs. In conclusion, the optimal grinding grade for the obtainment of an EC with A20:R80 was fine and that for A20:R80 50% torrefacto was coarse.