Apples prevent mammary tumors in rats

Regular consumption of fruits and vegetables has been consistently shown to be associated with reduced risk of developing chronic diseases such as cancer and cardiovascular disease. Apples are commonly consumed and are the major contributors of phytochemicals in human diets. It was previously reported that apple extracts exhibit strong antioxidant and antiproliferative activities and that the major part of total antioxidant activity is from the combination of phytochemicals. Phytochemicals, including phenolics and flavonoids, are suggested to be the bioactive compounds contributing to the health benefits of apples. Here it is shown that whole apple extracts prevent mammary cancer in a rat model in a dose-dependent manner at doses comparable to human consumption of one, three, and six apples a day. This study demonstrated that whole apple extracts effectively inhibited mammary cancer growth in the rat model; thus, consumption of apples may be an effective strategy for cancer protection.     

Hazard identification of contaminated sites—ranking potential toxicity of organic sediment extracts in crustacean and fish

Background, aim, and scope  It is well known that contaminated sediments represent a potential long-term source of pollutants to the aquatic environment. To protect human and ecosystem health, it is becoming common to remediate contaminated sites. However, the great cost associated with, e.g., dredging in combination with the large numbers of contaminated sites makes it crucial to pinpoint those sites that are in greatest need of remediation. In most European countries, this prioritization process has almost exclusively been based on chemical analyses of known substances; only seldom toxicity data has been considered. The main objective of the current study was therefore to develop a tool for hazard identification of sediment by ranking potential toxicity of organic sediment extracts in a crustacean and a fish. A secondary objective was to investigate the difference in potential toxicity between compounds with different polarities. Materials and methods  Early life stages of the crustacean Nitocra spinipes and the fish Oncorhynchus mykiss, which represent organisms from different trophic levels (primary and secondary consumer) and with different routes of exposure (i.e., ingestion through food, diffusive uptake, and maternal transfer), were exposed to hexane and acetone fractions (semi-polar compounds) of sediment from five locations, ranging from heavily to low contaminated. Preliminary tests showed that the extracts were non-bioavailable to the crustacean when exposed via water, and the extracts were therefore loaded on silica gel. Rainbow trout embryos were exposed using nano-injection technique. Results and discussion  Clear concentration–response relationships of both mortality and larval development were observed in all tests with N. spinipes. Also for rainbow trout, the observed effects (e.g., abnormality, hemorrhage, asymmetric yolk sac) followed a dose-related pattern. Interestingly, our results indicate that some of the locations contained toxic semi-polar compounds, which are normally not considered in risk assessment of sediment since they are focused on compounds isolated in the hexane fraction. Conclusions  The ranking of the five sediments followed the expected pattern of potential toxicity in both organisms, i.e., sediments with known pollution history caused major effects while reference sediments caused minor effects in the two test systems. Silica gel turned out to be an excellent carrier for exposure of N. spinipes to very hydrophobic and otherwise non-bioavailable sediment extracts. Recommendations and perspectives  Since both test systems demonstrated that a substantial part of the potential toxicity was caused by semi-polar compounds in the acetone fractions, this study enlightens our poor understanding of which compounds are causing adverse effects in environmental samples. Therefore, by investigating potential toxicity (i.e., hazard identification) as a first screening step in prioritizing processes, these implications could be avoided. For proper sediment risk assessment, we however recommend whole sediment toxicity tests to be used for selected sites at following tiers.     

Neuroprotective effects of citrus flavonoids

Recent attention has been given to the influence of dietary factors on health and mental well-being. Oxidative stress is associated with many diseases including neurodegenerative disorders. Dietary flavonoids exert cardioprotective, chemopreventive, and neuroprotective effects. The biological activities of flavonoids have been attributed to their antioxidant, anti-inflammatory, and signaling properties. A clear understanding of the mechanisms of action, as either antioxidants or signaling molecules, is crucial for the application of flavonoids as interventions in neurodegeneration and as brain foods. Citrus flavonoids exert little adverse effect and have low or no cytotoxicity to healthy, normal cells. The main citrus flavonoids can also traverse the blood-brain barrier; hence, they are promising candidates for intervention in neurodegeneration and as constituents in brain foods. In this review, we discuss the bioactivity, multiple neuroprotection mechanisms, and antioxidant and signaling properties of citrus flavonoids. Receptor-mediated neuroprotective actions and parallel signaling pathways are also explored. Finally, the induction of cellular defense proteins against oxidative stress and neurotoxicity by hesperetin, a main and widespread citrus flavonoid, are also discussed. It is suggested that citrus fruits, which are rich in abundant sources of hesperetin and other flavonoids, are promising for the development of general food-based neuroprotection and brain foods.     

Protective effects of quercetin and vitamin C against oxidative stress-induced neurodegeneration

Clinical trials of several neurodegenerative diseases have increasingly targeted the evaluation of various antioxidants' effectiveness. The human diet contains several thousand phytochemicals, many of which have significant bioactivities. Vitamin C, a naturally occurring antioxidant, is known to reduce the risk of neurodegenerative disorders such as Alzheimer's disease. Quercetin, one of the major flavonoids in some fruits and vegetables, has much stronger antioxidative and anticarcinogenic activities than vitamin C. Therefore, we investigated the protective effects of quercetin on hydroxy peroxide-induced neurodegeneration. To determine the protective effects, PC12 cells were preincubated with quercetin and vitamin C before H(2)O(2) treatment for 2 h. Results showed that cell viability was clearly improved with quercetin, and quercetin showed a higher protective effect than vitamin C. Because oxidative stress is known to increase neuronal cell membrane breakdown, we further investigated lactate dehydrogenase and trypan blue exclusion assays. We observed that quercetin decreased oxidative stress-induced neuronal cell membrane damage more than vitamin C. These results suggest that quercetin, in addition to many other biological benefits, contributes significantly to the protective effects of neuronal cells from oxidative stress-induced neurotoxicity, such as Alzheimer disease.     

In vitro inhibition of human cGMP-specific phosphodiesterase-5 by polyphenols from red grapes

A moderate consumption of red wine may reduce the risk of cardiovascular diseases via wine-derived phenolic compounds. A variety of biological mechanisms have been proposed for wine-derived phenolic compounds including nitric oxide-mediated vasorelaxation. This study examined whether the vasodilating effect of wine-derived phenolic compounds was associated with the inhibition of phosphodiesterases (PDEs) and, in particular, PDE5. For this purpose, human recombinant PDE5A1 isoform was prepared by expression of the full-length cDNA of PDE5A1 into COS-7 cells. Red wine and the extracts from grape skin inhibited PDE5A1 activity, whereas the seed extracts had a negligible effect. The mixture of anthocyanins inhibited the enzyme activity (IC50 = 11.6 microM), with malvidin-3-O-beta-glucoside (IC50 = 35.4 microM) and malvidin (IC50 = 24.9 microM) the most potent among the monoglucosides and aglycons, respectively. trans-Resveratrol and trans-piceid exhibited negligible effects, whereas hydroxycinnamates were completely inactive. These results indicate that polyphenols-induced vasorelaxation may also be sustained by smooth muscle PDE inhibition by anthocyanins present in red wines and grapes.     

Simplified electrophoretic assay for human salivary alpha-amylase inhibitor detection in cereal seed flours

Alpha-amylase inhibitors are antinutritional proteins largely found in cereal seeds. An in-gel assay was developed that allowed the rapid screening of these compounds in complex seed extracts. The assay was based on the electrophoretic separation of the extract proteins on starch-containing gels, followed by the detection of alpha-amylase-inhibiting proteins after incubation of the gel in an alpha-amylase solution; inhibitors were revealed by a staining method based on iodine binding to nondigested starch. The one-dimensional method can be useful to test inhibitory activity of purified proteins or to assay fractions recovered during a purification procedure. A two-dimensional (IEF x PAGE) non-denaturing system with second-dimension separation on starch-PAGE was also developed; the technique allowed the screening of complex protein mixtures for multiple inhibitory proteins. The newly developed assay method was used to test the presence of inhibitory activity in a crude extract from wheat flour, and it was validated by comparing in-gel and in-solution assays of commercially available alpha-amylase inhibitors.     

Isolation and identification of strawberry phenolics with antioxidant and human cancer cell antiproliferative properties

Studies suggest that consumption of berry fruits, including strawberries ( Fragaria x ananassa Duch.), may have beneficial effects against oxidative stress mediated diseases such as cancer. Berries contain multiple phenolic compounds, which are thought to contribute to their biological properties. Comprehensive profiling of phenolics from strawberries was previously reported using high-performance liquid chromatography with mass spectrometry (HPLC-MS) detection. The current study reports the isolation and structural characterization of 10 phenolic compounds from strawberry extracts using a combination of Amberlite XAD16-resin and C18 columns, HPLC-UV, and nuclear magnetic resonance (NMR) spectroscopy methods. The phenolics were cyanidin-3-glucoside ( 1), pelargonidin (2), pelargonidin-3-glucoside (3), pelargonidin-3-rutinoside (4), kaempferol (5), quercetin (6), kaempferol-3-(6'-coumaroyl)glucoside) (7), 3,4,5-trihydroxyphenyl-acrylic acid (8), glucose ester of ( E)- p-coumaric acid (9), and ellagic acid . Strawberry crude extracts and purified compounds 1- 10 were evaluated for antioxidant and human cancer cell antiproliferative activities by the Trolox equivalent antioxidant capacity (TEAC) and luminescent ATP cell viability assays, respectively. Among the pure compounds, the anthocyanins 1 (7156 microM Trolox/mg), 2 (4922 microM Trolox/mg), and 4 (5514 microM Trolox/mg) were the most potent antioxidants. Crude extracts (250 microg/mL) and pure compounds (100 microg/mL) inhibited the growth of human oral (CAL-27, KB), colon (HT29, HCT-116), and prostate (LNCaP, DU145) cancer cells with different sensitivities observed between cell lines. This study adds to the growing body of data supporting the bioactivities of berry fruit phenolics and their potential impact on human health.     

Development of a cell culture/ELISA assay to detect anticoagulant rodenticides and its application to analysis of rodenticide treated grain

This study describes a generic biological screening assay designed to detect anticoagulant rodenticides based on their inhibitory action on the vitamin K epoxide reductase protein complex, resulting in an accumulation of under-carboxylated prothrombin or proteins induced by vitamin K antagonism (PIVKA-II). A combined cell culture/ELISA assay was optimized to measure PIVKA-II production by the human hepatoma HepG2 cell line cultured in the presence of anticoagulant rodenticides. The specificity and sensitivity of the assay was validated using 41 grain extracts containing representative concentrations of rodenticide or appropriate nonrodenticide control compounds. In all cases, PIVKA-II produced by HepG2 cells in response to grain extracts spiked with rodenticides was detected by ELISA, while PIVKA-II was not detected in supernatants collected from cells exposed to nonrodenticide controls. This represents a novel, class-specific biological assay for the detection of anticoagulant rodenticides present in contaminated grain.     

Antioxidant and iron-binding properties of curcumin, capsaicin, and S-allylcysteine reduce oxidative stress in rat brain homogenate

Research demonstrates that antioxidants and metal chelators may be of beneficial use in the treatment of neurodegenerative diseases, such as Alzheimer's disease (AD). This study investigated the antioxidant and metal-binding properties of curcumin, capsaicin, and S-allylcysteine, which are major components found in commonly used dietary spice ingredients turmeric, chilli, and garlic, respectively. The DPPH assay demonstrates that these compounds readily scavenge free radicals. These compounds significantly curtail iron- (Fe2+) and quinolinic acid (QA)-induced lipid peroxidation and potently scavenge the superoxide anion generated by 1 mM cyanide in rat brain homogenate. The ferrozine assay was used to measure the extent of Fe2+ chelation, and electrochemistry was employed to measure the Fe3+ binding activity of curcumin, capsaicin, and S-allylcysteine. Both assays demonstrate that these compounds bind Fe2+ and Fe3+ and prevent the redox cycling of iron, suggesting that this may be an additional method through which these agents reduce Fe2+-induced lipid peroxidation. This study demonstrates the antioxidant and metal-binding properties of these spice ingredients, and it is hereby postulate that these compounds have important implications in the prevention or treatment of neurodegenerative diseases such as AD.     

Toxicity and antioxidant activity in vitro and in vivo of two Fucus vesiculosus extracts

The consumption of seaweeds has increased in recent years. However, their adverse and beneficial effects have scarcely been studied. Two extracts from the brown seaweed Fucus vesiculosus containing 28.8% polyphenols or 18% polyphenols plus 0.0012% fucoxanthin have been obtained and studied to determine their toxicity in mice and rats and also their antioxidant activity. Both extracts were shown to lack any relevant toxic effects in an acute toxicity test following a 4 week daily treatment in rats. The extracts exhibited antioxidant activity in noncellular systems and in activated RAW 264.7 macrophages, as well as in ex vivo assays in plasma and erythrocytes, after the 4 week treatment in rats. Our ex vivo results indicated that compounds from extract 2 may be more easily absorbed and that the antioxidants in their parent or metabolized form are more active. These findings support the view that the daily consumption of F. vesiculosus extract 2 (Healsea) would have potential benefits to humans.     

Inhibitory effects of anthocyanins and other phenolic compounds on nitric oxide production in LPS/IFN-gamma-activated RAW 264.7 macrophages

Flavonoids have been reported to lower oxidative stress and possess beneficial effects on cardiovascular diseases and chronic inflammatory diseases associated with nitric oxide (NO). Common phenolic compounds, including phenolic acids, flavonols, isoflavones, and anthocyanins, present in fruits were investigated for their effects on NO production in LPS/IFN-gamma-activated RAW 264.7 macrophages. Phenolic compounds at the range of 16-500 microM that inhibited NO production by > 50% without showing cytotoxicity were the flavonols quercetin and myricetin, the isoflavone daidzein, and the anthocyanins/anthocyanidins pelargonidin, cyanidin, delphinidin, peonidin, malvidin, malvidin 3-glucoside, and malvidin 3,5-diglucosides. Anthocyanins had strong inhibitory effects on NO production. Anthocyanin-rich crude extracts and concentrates of selected berries were also assayed, and their inhibitory effects on NO production were significantly correlated with total phenolic and anthocyanin contents. This is the first study to report the inhibitory effects of anthocyanins and berry phenolic compounds on NO production.     

Biomedical applications of poisonous plant research

Research designed to isolate and identify the bioactive compounds responsible for the toxicity of plants to livestock that graze them has been extremely successful. The knowledge gained has been used to design management techniques to prevent economic losses, predict potential outbreaks of poisoning, and treat affected animals. The availability of these compounds in pure form has now provided scientists with tools to develop animal models for human diseases, study modes of action at the molecular level, and apply such knowledge to the development of potential drug candidates for the treatment of a number of genetic and infectious conditions. These advances are illustrated by specific examples of biomedical applications of the toxins of Veratrum californicum (western false hellebore), Lupinus species (lupines), and Astragalus and Oxytropis species (locoweeds).     

Production and characterization of human extracellular superoxide dismutase in the methylotrophic yeast Pichia pastoris

Reactive oxygen species are associated with various diseases including cardiovascular diseases, neurological disorders, and pulmonary diseases. Extracellular superoxide dismutase (ECSOD) is an antioxidant enzyme secreted by cells to prevent overproduction of reactive oxygen species. We expressed an ECSOD gene isolated from a human aortic smooth muscle cDNA library in the methylotrophic yeast Pichia pastoris. A synthetic secretion cassette was constructed with the inducible promoter of the alcohol oxidase 1 gene (AOX1) and the yeast alpha-mating factor signal peptide. As much as 25% of the total protein was ECSOD in some transformants grown under inducing conditions. After 36 h of methanol induction, ECSOD was exported into the culture medium at a concentration of approximately 440 mg/L with an antioxidative activity of 760 +/- 20 U/mg ECSOD. Transformed yeast cells were more resistant to heat shock and H(2)O(2) oxidative stress, indicating that the human ECSOD expressed by P. pastoris had multiple biological functions. Our data suggest that the methylotrophic yeast inducible system is suitable for large-scale production of enzymatically active human ECSOD.     

Comparative effects of food-derived polyphenols on the viability and apoptosis of a human hepatoma cell line (HepG2)

Consumption of fruits and vegetables, which are rich in polyphenols, has been associated with a reduced risk of chronic diseases such as cancer. Dietary polyphenols have antioxidant and antiproliferative properties that might explain their beneficial effect on cancer prevention. The aim of this study was to investigate the effects of different pure polyphenols [quercetin, chlorogenic acid, and (-)-epicatechin] and natural fruit extracts (strawberry and plum) on viability or apoptosis of human hepatoma HepG2 cells. The treatment of cells for 18 h with quercetin and fruit extracts reduced cell viability in a dose-dependent manner; however, chlorogenic acid and (-)-epicatechin had no prominent effects on the cell death rate. Similarly, quercetin and strawberry and plum extracts, rather than chlorogenic acid and (-)-epicatechin, induced apoptosis in HepG2 cells. Moreover, quercetin and fruit extracts arrested the G1 phase in the cell cycle progression prior to apoptosis. Quercetin and strawberry and plum extracts may induce apoptosis and contribute to a reduced cell viability in HepG2 cells.     

Low-density lipoprotein, collagen, and thrombin models reveal that Rosemarinus officinalis L. exhibits potent antiglycative effects

Using the low-density lipoprotein (LDL), collagen, and thrombin models, we report here that the rosemary extracts (REs), either the aqueous (REw) or the acetonic (REA), all possessed many antiglycation-related features, and the effective concentrations required were as follows: 0.1 mg/mL for suppressing the relative electrophoretic mobility, 1.3 microg/mL for anticonjugated diene induction, 0.5 mg/mL for inhibition of thiobarbituric acid reactive substances production, 0.1 mg/mL for AGEs (advanced glycation end products) formation, 0.1 mg/mL to block glucose incorporation, and 0.05 mg/mL as an effective anti-antithrombin III. Using high-performance liquid chromatography/mass spectrometry, we identified five major constituents among eight major peaks, including rosmarinic acid, carnosol, 12-methoxycarnosic acid, carnosic acid, and methyl carnosate. In the LDL model, REA was proven to be more efficient than REw; yet, the reverse is true for the collagen and the thrombin III models, the reason of which was ascribed to the higher lipid-soluble antioxidant content (such as rosmarinic acid, carnosol, carnosic acid, 12-methoxycarnosic acid and methyl carnosate) in REA than in REw and the different surface lipid characteristics between LDL and collagen; although to act as anti-AGEs, both extracts were comparable. To assist the evidence, a larger 2,2-diphenyl-1-picrylhydrazyl radical scavenging capability with less total polyphenolic content was found in REA. We conclude that rosemary is an excellent multifunctional therapeutic herb; by looking at its potential potent antiglycative bioactivity, it may become a good adjuvant medicine for the prevention and treatment of diabetic, cardiovascular, and other neurodegenerative diseases.     

Evaluation of the light-sensitive cytotoxicity of Hypericum perforatum extracts, fractions, and pure compounds

Hypericum perforatum (Hp) is known for possessing antidepressant and antiviral activities. Despite its use as an alternative to conventional antidepressants, the identification of the cytotoxic chemicals derived from this herb is incomplete. In this study, the cytotoxicity of Hp extracts prepared in solvents ranging in polarity, fractions of one extract, and purified compounds were examined in three cell lines. All extracts exhibited significant cytotoxicity; those prepared in ethanol (no hyperforin, 3.6 microM hypericin, and 134.6 microM flavonoids) showed between 7.7 and 77.4% cell survival (p < 0.0001 and 0.01), whereas the chloroform and hexane extracts (hyperforin, hypericin, and flavonoids not detected) showed approximately 9.0 (p < 0.0001) and 4.0% (p < 0.0001) survival. Light-sensitive toxicity was observed primarily with the ethanol extracts sequentially extracted following removal of material extracted in either chloroform or hexane. The absence of light-sensitive toxicity with the Hp extracts suggests that the hypericins were not playing a prominent role in the toxicity of the extracts.     

Effect of reddening-ripening on the antioxidant activity of polyphenol extracts from cv. 'Annurca' apple fruits

Apple is among the most consumed fruits worldwide, and several studies suggest that apple polyphenols could play a role in the prevention of degenerative diseases. 'Annurca' apple fruit undergoes, after harvest, a typical reddening treatment to turn the apples' skin red, and it is noted for its high firmness. This paper reports the effect of reddening-ripening treatment on polyphenol concentration and antioxidant activity of both peel and flesh extracts. The in vitro antioxidant properties have been compared with the protective effect against the cytotoxic effects of reactive oxygen species using Caco-2 cells as model system. Pretreatment of cells with different polyphenolic apple extracts provides a remarkable protection against oxidative damage. This effect seems to be associated with the antioxidant activity of 'Annurca' apple polyphenolic compounds. The flesh has antioxidant properties comparable to those possessed by the peel. Neither the reddening nor the fruit conservation causes changes in the antioxidant properties possessed by this apple variety. The data indicate that polyphenolic compounds in 'Annurca' apples are relatively stable in the peel and also in the flesh; therefore, the health benefits of polyphenols should be maintained during long-term storage. Finally, a diet rich in apple antioxidants could exert a beneficial effect in the prevention of intestinal pathologies related to the production of reactive oxygen species.     

Investigation of Australian olive mill waste for recovery of biophenols

Olive mill waste is a potential source for the recovery of phytochemicals with a wide array of biological activities. Phytochemical screening of hexane, methanol, and water extracts revealed a diversity of compounds, perhaps overlooked in previous studies through intensive cleanup procedures. Methanol and water extracts contained large amounts of biophenols, and further testing of polar extraction solvents, including ethyl acetate, ethanol, propanol, acetone, acetonitrile, and water/methanol mixtures, highlighted the latter as the solvent of choice for extraction of the widest array of phenolic compounds. Stabilization of the resulting extract was best achieved by addition of 2% (w/w) sodium metabisulfite. Quantitative data are reported for nine biophenols extracted using 60% (v/v) methanol in water with 2% (w/w) sodium metabisulfite. Six compounds had recoveries of greater than 1 g/kg of freeze-dried waste: hydroxytyrosol glucoside, hydroxytyrosol, tyrosol, verbascoside, and a derivative of oleuropein.