In vitro and ex vivo antihydroxyl radical activity of green and roasted coffee

The specific antiradical activity against the hydroxyl radical of the water soluble components in green and dark roasted Coffea arabica and Coffea robusta coffee samples, both in vitro by the chemical deoxiribose assay and ex vivo in a biological cellular system (IMR32 cells), were determined. All the tested coffee solutions showed remarkable antiradical activity. In the deoxiribose assay, all the tested solutions showed similar inhibitory activity (IA%) against the sugar degradation (IA values ranged from 45.2 to 46.9%). In the cell cultures, the survival increase (SI%) ranged from 197.0 to 394.0% with C. robusta roasted coffee being significantly more active than the other samples. The coffee solutions underwent dialysis (3500 Da cutoff membrane) to fraction their components. In both systems, the dialysates (MW < 3500 Da) either from green or roasted coffee, showed antiradical activity, while the only retentates (MW > 3500 Da) from the roasted coffee samples were active. The preparative gel-filtration chromatography of roasted coffee C. robusta dialysate gave three fractions active in the biological system, all containing chlorogenic acid derivatives. The most active fraction was found to be that containing the 5-O-caffeoilquinic acid, which shows a linear relation dose-response ranging from 0.02 to 0.10 mM. The results show that both green and roasted coffee possess antiradical activity, that their more active component is 5-O-caffeoyl-quinic acid, and moreover that roasting process induces high MW components (later Maillard reaction products, i.e., melanoidins), also possessing antiradical activity in coffee. These results could explain the neuroprotective effects found for coffee consumption in recent epidemiological studies.     

In vitro antioxidant and ex vivo protective activities of green and roasted coffee

The antioxidant properties of green and roasted coffee, in relation to species (Coffea arabica and Coffea robusta) and degree of roasting (light, medium, dark), were investigated. These properties were evaluated by determining the reducing substances (RS) of coffee and its antioxidant activity (AA) in vitro (model system beta-carotene-linoleic acid) and ex vivo as protective activity (PA) against rat liver cell microsome lipid peroxidation measured as TBA-reacting substances. RS of C. robustasamples were found to be significantly higher when compared to those of C. arabica samples (p < 0.001). AA for green coffee samples were slightly higher than for the corresponding roasted samples while PA was significantly lower in green coffee compared to that of all roasted samples (p < 0.001). Extraction with three different organic solvents (ethyl acetate, ethyl ether, and dichloromethane) showed that the most protective compounds are extracted from acidified dark roasted coffee solutions with ethyl acetate. The analysis of acidic extract by gel filtration chromatography (GFC) gave five fractions. Higher molecular mass fractions were found to possess antioxidant activity while the lower molecular mass fractions showed protective activity. The small amounts of these acidic, low molecular mass protective fractions isolated indicate that they contain very strong protective agents.     

Cranberries and cranberry products: powerful in vitro, ex vivo, and in vivo sources of antioxidants

Cranberry products and especially cranberry juice (CJ) have been consumed for health reasons primarily due to their effect on urinary tract infections. We investigated the quantity of both free and total (after hydrolysis) phenolic antioxidants in cranberry products using the Folin assay. The order of amount of total polyphenols in cranberry foods on a fresh weight basis was as follows: dried > frozen > sauce > jellied sauce. On a serving size basis for all cranberry products, the order was as follows: frozen > 100% juice > dried > 27% juice > sauce > jellied sauce. High fructose corn syrup (HFCS) is a major source of sugar consumption in the U.S. and contains both glucose and fructose, potential mediators of oxidative stress. We investigated the effect of the consumption of HFCS and ascorbate with CJ antioxidants or without CJ (control) given to 10 normal individuals after an overnight fast. Plasma antioxidant capacity, glucose, triglycerides, and ascorbate were measured 6 times over 7 h after the consumption of a single 240 mL serving of the two different beverages. The control HFCS caused a slight decrease in plasma antioxidant capacity at all time points and thus an oxidative stress in spite of the presence of ascorbate. CJ produced an increase in plasma antioxidant capacity that was significantly greater than control HFCS at all time points. Postprandial triglycerides, due to fructose in the beverages, were mainly responsible for the oxidative stress and were significantly correlated with the oxidative stress as measured by the antioxidant capacity. Cranberries are an excellent source of high quality antioxidants and should be examined in human supplementation studies.     

Hydroxytyrosol-rich olive mill wastewater extract protects brain cells in vitro and ex vivo

Elevated oxidative and nitrosative stress both impair the integrity and functioning of brain tissue, especially in aging. As long-term intake of plant foods rich in antioxidant phenolics, such as extra virgin olive oil, positively modulates surrogate markers of many human pathological alterations, the interest in cheap and abundant sources of such phenolics is rapidly growing. Olive mill wastewater is particularly rich in hydroxytyrosol, an o-diphenol with powerful antioxidant, anti-inflammatory, and antithrombotic activities. Due to the deleterious effect of oxidative stress on brain cell survival, the efficacy of a hydroxytyrosol-rich extract to attenuate Fe2+- and nitric oxide (NO)-induced cytotoxicity in murine-dissociated brain cells was investigated. The addition of either Fe2+ or SNP (an NO donor) caused both a severe loss of cellular ATP and a markedly depolarized mitochondrial membrane potential. Preincubation with hydroxytyrosol significantly attenuated the cytotoxic effect of both stressors, although with different efficiencies. Mice feeding studies were performed to assess the brain bioactivity of hydroxytyrosol ex vivo. Subchronic, but not acute, administration of 100 mg of hydroxytyrosol per kilogram body weight for 12 days enhanced resistance of dissociated brain cells to oxidative stress, as shown by reduced basal and stress-induced lipid peroxidation. Also, basal mitochondrial membrane potential was moderately hyperpolarized (P < 0.05), an effect suggestive of cytoprotection. In synthesis, the ex vivo data provide the first evidence of neuroprotective effects of oral hydroxytyrosol intake. Keywords: Hydroxytyrosol; olive mill wastewater; dissociated brain cells; oxidative stress; brain; Mediterranean diet.     

The in vivo and in vitro effects of Ca2+ and Al3+ upon ATPases from barley roots

Experiments were performed to test the effect of Ca²⁺ and Mg²⁺ upon ATPases (E.C. 3.6.1.3) from barley roots (Hordeum distichon L. cv. MULTUM) that had or had not taken up Al³⁺. Furthermore, the effect of the uptake period was examined. With longer duration of the Al³⁺ uptake, the activity of the ATPases dropped, independently of whether they were activated by Ca²⁺ or by Mg²⁺. Mg²⁺ stimulated the activity of the ATPases if no Al³⁺ had been taken up with lacking Ca²⁺ in the assay. If Al³⁺ had been taken up no additional activation by Mg²⁺ to the Ca²⁺ stimulation could be observed, and in some cases Mg²⁺ decreased the Ca²⁺ stimulation.     

In vitro absorption of dietary trans-resveratrol from boiled and roasted peanuts in Caco-2 cells

Previous studies on the transport and absorption of resveratrol (3,5,4-O-trihydroxystilbene) were done using the pure compound. In this study, the absorption of resveratrol in digested peanut micellar from boiled and roasted peanuts was investigated using a human intestinal Caco-2 cell monolayer. The amount transported and rate of transport of both resveratrol glycosides and its hydrolytic product were quantified by a reverse-phase high-performance liquid chromatography method with mass spectrometric detection. Four peaks were identified in the digested peanut micellar of both boiled and roasted peanuts: two resveratrol glycosides, one resveratrol diglycoside, and possibly an acylated resveratrol glycoside. Resveratrol from roasted peanut micellar had a higher transport rate than those from the boiled peanut. This implies that resveratrol from roasted peanut is better absorbed than from boiled peanut. Also, the rate of transport and amount of resveratrol transported were higher for the hydrolytic product than the nonhydrolyzed glycosides. This has strong implications for in vivo absorption as the enzymatic activity of gut microflora could enhance the bioavailability of β-glycosides of dietary polyphenols.     

Alkylpyridiniums. 2. Isolation and quantification in roasted and ground coffees

Recent model studies on trigonelline decomposition have identified nonvolatile alkylpyridiniums as major reaction products under certain physicochemical conditions. The quaternary base 1-methylpyridinium was isolated from roasted and ground coffee and purified by ion exchange and thin-layer chromatography. The compound was characterized by nuclear magnetic resonance spectroscopy ((1)H, (13)C) and mass spectrometry techniques. A liquid chromatography-electrospray ionization tandem mass spectrometry method was developed to quantify the alkaloid in coffee by isotope dilution mass spectrometry. The formation of alkylpyridiniums is positively correlated to the roasting degree in arabica coffee, and highest levels of 1-methylpyridinium, reaching up to 0.25% on a per weight basis, were found in dark roasted coffee beans. Analyses of coffee extracts also showed the presence of dimethylpyridinium, at concentrations ranging from 5 to 25 mg/kg. This is the first report on the isolation and quantification of alkylpyridiniums in coffee. These compounds, described here in detail for the first time, may have an impact on the flavor/aroma profile of coffee directly (e.g., bitterness), or indirectly as precursors, and potentially open new avenues in the flavor/aroma modulation of coffee.     

Antioxidant and anti-inflammatory potential of hesperetin metabolites obtained from hesperetin-administered rat serum: an ex vivo approach

In recent years much attention has been focused on the pharmaceutical relevance of bioflavonoids, especially hesperidin and its aglycon hesperetin in terms of their antioxidant and anti-inflammatory actions. However, the bioactivity of their metabolites, the real molecules in vivo hesperetin glucuronides/sulfates produced after ingestion, has been poorly understood. Thus, the study using an ex vivo approach is aimed to compare the antioxidant and anti-inflammatory activities of hesperidin/hesperetin or hesperetin metabolites derived from hesperetin-administered rat serum. We found that hesperetin metabolites (2.5-20 μM) showed higher antioxidant activity against various oxidative systems, including superoxide anion scavenging, reducing power, and metal chelating effects, than that of hesperidin or hesperetin. The data also showed that pretreatment of hesperetin metabolites (1-10 μM) within the range of physiological concentrations, compared to hesperetin, significantly inhibited nitric oxide (NO) and prostaglandin E(2) (PGE(2)) production, as evidenced by the inhibition of their precursors, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein levels without appreciable cytotoxicity on LPS-activated RAW264.7 macrophages or A7r5 smooth muscle cells. Concomitantly, hesperetin metabolites dose-dependently inhibited LPS-induced intracellular reactive oxygen species (ROS). Furthermore, hesperetin metabolites significantly downregulate LPS-induced nuclear factor-κB (NF-κB) activation followed by the suppression of inhibitor-κB (I-κB) degradation and phosphorylation of c-Jun N-terminal kinase1/2 (JNK1/2) and p38 MAPKs after challenge with LPS. Hesperetin metabolites ex vivo showed potent antioxidant and anti-inflammatory activity in comparison with hesperidin/hesperetin.     

Inhibition of reactive nitrogen species in vitro and ex vivo by trypsin inhibitor from sweet potato 'Tainong 57' storage roots

Peroxynitrite (ONOO-), formed from a reaction of superoxide and nitric oxide, is one of the most potent cytotoxic species known to oxidize cellular constituents including essential proteins, lipids, and DNA. ONOO- induces cellular and tissue injury, resulting in several human diseases such as Alzheimer's disease, atherosclerosis, and stroke. Due to the lack of endogenous enzymes responsible for ONOO- scavenging activity, finding a specific ONOO- scavenger is of considerable importance. In this study, the ability of trypsin inhibitor (TI), isolated from sweet potato storage roots (SPTI), to scavenge *ON and ONOO- was investigated. The data obtained show that TI generated a dose-dependent inhibition on production of nitrite and superoxide radicals. The IC50 value of TI on superoxide radical was 143.2 +/- 4.29 microg/mL. SOD activity staining was used to confirm SOD activity of SPTI. SPTI also caused a dose-dependent inhibition of the oxidation of dihydrorhodamine 123 (DHR) by peroxynitrite. A calculated IC50 value of 809.1 +/- 32.36 microg/mL was obtained on the inhibition of peroxynitrite radical. Spectrophotometric analyses revealed that TI suppressed the formation of ONOO--mediated tyrosine nitration through an electron donation mechanism. In further studies, TI also showed a significant ability to inhibit nitration of bovine serum albumin (BSA) in a dose-dependent manner. In vivo TI inhibited lipopolysaccharide-induced nitrite production in macrophages in a concentration-dependent manner with an IC50 value of 932.8 +/- 29.85 microg/mL. The present study suggested that TI had an efficient reactive nitrogen species scavenging ability. TI might be a potential effective NO and ONOO- scavenger useful for the prevention of NO- and ONOO--involved diseases.     

Isolation, identification, and quantification of roasted coffee antibacterial compounds

Coffee brew is a widely consumed beverage with multiple biological activities due both to naturally occurring components and to the hundreds of chemicals that are formed during the roasting process. Roasted coffee extract possesses antibacterial activity against a wide range of microorganisms, including Staphylococcus aureus and Streptococcus mutans, whereas green coffee extract exhibits no such activity. The naturally occurring coffee compounds, such as chlorogenic acids and caffeine, cannot therefore be responsible for the significant antibacterial activity exerted by coffee beverages against both bacteria. The very low minimum inhibitory concentration (MIC) found for standard glyoxal, methylglyoxal, and diacetyl compounds formed during the roasting process points to these alpha-dicarbonyl compounds as the main agents responsible for the antibacterial activity of brewed coffee against Sa. aureus and St. mutans. However, their low concentrations determined in the beverage account for only 50% of its antibacterial activity. The addition of caffeine, which has weak intrinsic antibacterial activity, to a mixture of alpha-dicarbonyl compounds at the concentrations found in coffee demonstrated that caffeine synergistically enhances the antibacterial activity of alpha-dicarbonyl compounds and that glyoxal, methylglyoxal, and diacetyl in the presence of caffeine account for the whole antibacterial activity of roasted coffee.     

Isolation and determination of alpha-dicarbonyl compounds by RP-HPLC-DAD in green and roasted coffee

Glyoxal, methylglyoxal, and diacetyl formed as Maillard reaction products in heat-treated food were determined in coffee extracts (coffee brews) obtained from green beans and beans with different degrees of roast. The compounds have been reported to be mutagenic in vitro and genotoxic in experimental animals in a number of papers. More recently, alpha-dicarbonyl compounds have been implicated in the glycation process. Our data show that small amounts of glyoxal and methylglyoxal occur naturally in green coffee beans. Their concentrations increase in the early phases of the roasting process and then decline. Conversely, diacetyl is not found in green beans and forms later in the roasting process. Therefore, light and medium roasted coffees had the highest glyoxal and methylglyoxal content, whereas dark roasted coffee contained smaller amounts of glyoxal, methylglyoxal, and diacetyl. For the determination of coffee alpha-dicarbonyl compounds, a reversed-phase high performance liquid chromatography with a diode array detector (RP-HPLC-DAD) method was devised that involved the elimination of interfering compounds, such as chlorogenic acids, by solid phase extraction (SPE) and their derivatization with 1,2-diaminobenzene to give quinoxaline derivatives. Checks of SPE and derivatization conditions to verify recovery and yield, respectively, resulted in rates of 100%. The results of the validation procedure showed that the proposed method is selective, precise, accurate, and sensitive.     

Isolation and identification of flavonoids accumulated in proanthocyanidin-free barley

Flavonoids accumulated in proanthocyanidin-free near-isogenic lines iso ant 13, iso ant 17, and iso ant 22 of Nishinohoshi, developed by backcross breeding using a leading cultivar, Nishinohoshi, as a recurrent parent and a proanthocyanidin-free mutant as a nonrecurrent parent in Japan, were examined. A new flavanone, (2RS)-dihydrotricin 7-O-β-D-glucopyranoside (1), known flavanones (2RS)-dihydrotricin (2) and (2RS)-homoeriodictyol (3), and known flavones chrysoeriol 7-O-[α-L-rhamnopyranosyl-(1→6)-β-D-glucopyranoside] (4), chrysoeriol 7-O-β-D-glucopyranoside (5), tricin (6), and chrysoeriol (7) were isolated from iso ant 17 of Nishinohoshi. The structures and stereochemistries of the isolated flavonoids (1-7) were elucidated on the basis of spectroscopic analyses. The concentrations of the isolated flavonoids (1-7) in iso ant 13, iso ant 17, and iso ant 22 of Nishinohoshi were similar to each other, whereas the flavonoids 1-5 and 7 were not detected in Nishinohoshi, an old Japanese cultivar, Amaginijo, and North American cultivar Harrington. The concentration of tricin (6) in Nishinohoshi was a half those in iso ant 13, iso ant 17, and iso ant 22 of Nishinohoshi. Except for iso ant 13, iso ant 17, and iso ant 22 of Nishinohoshi, the concentration of tricin (6) was highest in Nishinohoshi, followed by Amaginijo and Harrington. Thus, tricin (6), its precursor dihydrotricin (2), and its glucopyranoside, dihydrotricin 7-O-β-D-glucopyranoside (1), as well as chrysoeriol (7) and homoeriodictyol (3) were accumulated in iso ant 13, iso ant 17, and iso ant 22 of Nishinohoshi probably by blocking at the step of flavanone 3-hydroxylase in the procyanidin biogenetic pathway, resulting in enhancement of the alternative biogenetic pathway.     

Quantification of melanoidin concentration in sugar-casein systems

Melanoidins are the final, brown, high molecular weight products of the Maillard reaction. The aim of the present study was to determine the average molar extinction coefficients of melanoidins formed in heated glucose-casein and fructose-casein systems. The value of the extinction coefficient can be used to translate spectrophotometrically measured browning (absorbance values) into melanoidin concentration. In the present study the melanoidins were quantified by measuring the concentration of sugar incorporated into the melanoidins, using (14)C-labeled sugar. The extinction coefficient of the melanoidins remained constant during the observation period as the absorbance at 420 nm increased to approximately 8 units, and it was calculated to be 477 (+/- 50) L mol(-1) cm(-1) in the glucose-casein reaction and 527 (+/- 35) L mol(-1) cm(-1) in the fructose-casein reaction. This difference is not significant. An increase of the number of sugar molecules per reactive amino group during the heating of glucose-casein and the fructose-casein mixtures was observed by the radiochemical method as well as by microanalysis of the high molecular weight fraction.     

Volatiles from roasted byproducts of the poultry-processing industry

Volatiles of roasted chicken breast muscle and byproducts, such as backbones, breastbones, spent bones, and skin, were investigated. Total volatile concentrations ranged from 2030 ppb in the roasted backbones to 4049 ppb in the roasted skin. The major classes of volatile compounds detected in roasted samples were aldehydes (648-1532 ppb) and alcohols (336-1006 ppb). Nitrogen- and/or sulfur-containing compounds were also detected in appreciable quantities (161-706 ppb) in all samples. For all samples, hexanal and 2-methyl-2-buten-1-ol were dominant among the aldehydes and alcohols, respectively. Among the nitrogen- and sulfur-containing compounds, Maillard reaction products, such as tetrahydropyridazines, piperidines, and thiazoles, were the major contributors to the total volatile content in all samples. The composition of volatiles observed in roasted byproducts was markedly different from that of the roasted breast muscle. Therefore, the blending of the byproducts in appropriate proportions or blending of volatile flavor extracts from different byproducts may be necessary to obtain an aroma that mimics roasted chicken aroma.     

In vitro and in vivo release of aroma compounds from yellow-fleshed kiwifruit

Comparisons were made between the aroma volatiles of the yellow-fleshed kiwifruit, "Hort16A", at two different stages of eating ripeness: firm and soft. The firm fruit contained a small number of aroma compounds that the soft fruit did not contain. In general, however, the largest difference between the two firmness categories was in the levels of esters, with the soft fruit containing higher concentrations and a larger number of esters than the firm fruit. In vitro analysis directly after maceration using atmospheric pressure chemical ionization mass spectrometry (APCI-MS) showed the relative importance of the most intense aromas between fruit at the two different firmness stages and was used to compare the release rates of aromas. A comparison of the aroma concentrations from gas chromatography mass spectrometry (GC-MS) and APCI-MS headspace analyses showed that the APCI-MS headspace showed less bias toward enzymatically generated lipid degradation compounds. A GC-sniffing study showed that many of the most intense compounds, acetaldehyde, hexanal, ethyl butanoate, and (E)-2-hexenal but not ethanol, showed odor activity in macerated fruit. In addition, dimethyl sulfide (DMS), a volatile present at very low levels in the fruit, also appeared to be an important contributor to the odor. In vivo analyses also showed much higher levels of aroma compounds in the soft fruit compared to the firm fruit, with evidence of persistence of some compounds, including DMS. There were a number of similarities between the breath profiles of the two panelists, which confirmed the importance of DMS in "Hort16A" aroma.     

Genotoxicity of melanoidin fractions derived from a standard glucose/glycine model

Genotoxic compounds can act at various levels in the cell (causing gene, chromosome, or genome mutations), necessitating the use of a range of genotoxicity assays designed to detect these different types of mutations. The production of melanoidins during the processing and cooking of foods is associated with changes in their nutritional character, and the discovery of mutagenic substances in pyrolyzed protein and amino acids has raised concern about the safety of these foods. The aim of this work was to test melanoidin fractions in three different in vitro assays (Ames test, Vitotox test, and micronucleus test). These melanoidin fractions were produced from the condensation of glucose with glycine and their separation was conducted by dialysis. The crude reaction mixture (before dialysis) and both the LMW and HMW fractions obtained by dialysis showed no genotoxicity in these assays, despite being tested at concentrations much higher than those naturally found in food products. The LMW fraction, however, showed toxicity at these high concentrations. The volatile fraction produced in this reaction showed genotoxicity only in the Vitotox test, at high concentrations.     

Chocolate is a powerful ex vivo and in vivo antioxidant, an antiatherosclerotic agent in an animal model, and a significant contributor to antioxidants in the European and American Diets

Chocolate today is often viewed as a food or snack with little nutritional value. The high saturated fat content of chocolate has also contributed to the belief that its consumption increases the risk of heart disease. However, recent human studies have proven that chocolate has beneficial effects on some pathogenic mechanisms of heart disease such as endothelial function and blood pressure. Although the antioxidant properties of chocolate have been known for some time, there has been no examination of its place in the U.S. diet as a source of antioxidants. This paper demonstrates that chocolate makes a significant contribution to U.S. per capita dietary antioxidants and by inference the European Community's. In the U.S. diet chocolate is the third highest daily per capita antioxidant source. An ex vivo study shows that epicatechin, a major polyphenol in chocolate and chocolate extracts, is a powerful inhibitor of plasma lipid oxidation due to polyphenols' ability to bind to lower density lipoproteins. Conversely, the fat from chocolate alone is a pro-oxidant in this model. This is also demonstrated in an in vivo human study. After consumption of dark chocolate and cocoa powder, the lower density lipoproteins isolated from plasma were protected from oxidation compared to the lipoproteins isolated after cocoa butter consumption, which were put under oxidative stress. In an animal model of atherosclerosis, cocoa powder at a human dose equivalent of two dark chocolate bars per day significantly inhibited atherosclerosis, lowered cholesterol, low-density lipoprotein, and triglycerides, raised high-density lipoprotein, and protected the lower density lipoproteins from oxidation. Chocolate has thus been shown to have potential beneficial effects with respect to heart disease.     

一株百合内生细菌 Burkholderia sp. FJb-2的分离鉴定及其体外抑菌促生效应

从北京市房山区百合资源圃采集红芯百合(Fangio)植株样品,通过组织块分离法分离内生细菌;进一步以百合灰霉病菌—灰葡萄孢菌( Botrytis cinerea)ACCC36423、百合叶尖干枯病菌—葡萄座腔菌( Botryosphaeria dothidea)ACCC37263和百合枯萎病菌—尖孢镰刀菌( Fusarium oxysporum)F01139等几种主要百合病原真菌为指示菌,采用平板对峙法筛选具有拮抗活性的拮抗菌株;并对拮抗菌株进行产 1-氨基环丙烷 -1-羧酸(ACC)脱氨酶活性、产生长素(IAA)、溶磷、产铁载体等多种植物促生特性测定;最后通过形态观察及 16S rRNA基因序列分析,初步鉴定其种属。结果从百合茎部分离获得一株内生细菌菌株 FJb-2;该菌株对灰葡萄孢菌、葡萄座腔菌和尖孢镰刀菌均有较高拮抗活性,对病原真菌生长的抑制率范围为 60%～ 81%;该菌株产 ACC脱氨酶活性为