Antifungal activity of a de novo synthetic peptide and derivatives against fungal food contaminants.

The development of novel solutions to fight microbial food contaminants rests upon two pillars, which are the development of resistant strains and consumers' desire for a reduced consumption of synthetic drugs. Natural antimicrobial peptides possess the qualities to overcome these issues. De novo synthesis of novel antifungal compounds is a major progress that has been facilitated by the identification of parameters involved in the antimicrobial activity. A 14-residue peptide named KK14, with the sequence KKFFRAWWAPRFLK-NH2 , was designed and inhibited conidial germination and fungal growth of food contaminants within the range 6.25 to 50 µg/ml and 6.25 to 100 µg/ml, respectively. The study of three analogues of the peptide highlighted the role of some residues in the structural conformation of the peptide and its antifungal activity. The substitution of a Pro residue with Arg increased the helical content of the peptide not only its antifungal activity but also its cytotoxicity. The insertion of an unnatural bulky residue ß-diphenylalanine or a full d-enantiomerization overall increased the antifungal potency. The four peptides showed similar behaviour towards salt increase, heat treatment, and pH decrease. Interestingly, the denantiomer remained the most active at high pH and after proteolytic digestion. The four peptides did not present haemolytic activity up to 200 µg/ml but had different behaviours of cytotoxicity. These differences could be crucial for potential application as pharmaceutical or food preservatives.

Cereal type significantly affects the composition and reconstitution characteristics of dried fermented milk-cereal composites.

BACKGROUND: Dairy and cereal products are frequently combined to create composites with enhanced nutritional benefits. Commercially available dried dairy-cereal composites are typically reconstituted and cooked to produce porridge or soup. RESULTS: Dried fermented milk-cereal composites (FMCC) with ∼193 g kg-1 protein were prepared by blending fermented milk with parboiled oats (FMCCo), wheat (FMCCw), or barley (FMCCb), incubating the blend, drying, and milling. Cereal type significantly affected the composition of the FMCC and the properties of the reconstituted, cooked FMCC (R-FMCC). The FMCCo had a higher starch and fat content and lower levels of lactose, lactic acid, and amylose than FMCCb. The R-FMCCo had higher viscosity during cooking at 95 °C and cooling to 35 °C, and higher values of yield stress (σ0 ), consistency index (K) and viscosity on shearing from 20 to 120 s-1 at 60 °C than R-FMCCb. The FMCCw had lower levels of fat and ß-glucan than FMCCo or FMCCb, but was otherwise closer to FMMCb with respect to composition, cooking properties and flow behavior. CONCLUSION: Differences in composition and consistency associated with cereal type are likely to affect the nutritional value of the FMCC. © 2018 Society of Chemical Industry.

Antioxidant, immunomodulatory and antiproliferative effects of gelatin hydrolysate from unicorn leatherjacket skin.

BACKGROUND: The in vitro cellular bioactivities including, antioxidant, immunomodulatory and antiproliferative effects of a gelatin hydrolysate (GH) prepared from unicorn leatherjacket skin, using partially purified glycyl endopeptidase, were investigated in order to optimize the use of fish skin waste products as functional food ingredients. RESULTS: GH under the tested concentrations (750-1500 µg mL(-1) ) protected against H2 O2 -induced DNA damage in U937 cells. GH also protected against the H2 O2 -induced reduction in cellular antioxidant enzyme activities, superoxide dismutase and catalase, in HepG2 cells. GH demonstrated immunomodulatory potential by reducing pro-inflammatory cytokine (interleukin-6 (IL-6) and IL-1ß) production and nitric oxide production in lipopolysaccharide-stimulated RAW 264.7 macrophage cells. Cell proliferation in human colon cancer (Caco-2) cells was significantly reduced in a dose-dependent manner following incubation with GH. CONCLUSION: These results indicate that GH has several bioactivities which support its potential as a promising functional food ingredient with various health benefits. © 2015 Society of Chemical Industry.

Antioxidant activities and selected characteristics of gelatin hydrolysates from seabass (Lates calcarifer) skin as affected by production processes.

Antioxidant activities and selected characteristics of gelatin hydrolysates from seabass skin as affected by production processes were investigated. Hydrolysates were prepared using different processes, including hydrolysis during and after gelatin extraction. Samples hydrolysed during gelatin extraction showed a higher degree of hydrolysis (DH) and yield compared with those hydrolysed after gelatin extraction (p < 0.05). All hydrolysates had a creamy yellowish colour. A lower abundance of volatile compounds was found in the hydrolysates produced during gelatin extraction, in comparison with those obtained after gelatin extraction. Hydrolysates prepared during gelatin extraction had higher 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, ferric reducing antioxidative power (FRAP) and ferrous ion chelating activity (p < 0.05). Following a simulated in vitro gastrointestinal digestion, the DPPH radical scavenging activity and FRAP of the hydrolysates was retained, whilst ferrous ion chelating activity increased. The most appropriate conditions for the generation of antioxidant hydrolysates from seabass skin were identified.

A study of the ability of bioactive extracts from brewers' spent grain to enhance the antioxidant and immunomodulatory potential of food formulations following in vitro digestion.

Bioactivity of a snack-bar, chocolate-drink and yogurt fortified with brewers' spent grain (BSG) phenolic extracts (P2 or B2) or protein hydrolysates (barley protein hydrolysate (BPH), BPH < 3 kDa, BPH < 5 kDa, BPH > 5 kDa) was measured following gastrointestinal in vitro digestion. Concentrations of 0.5 and 0.1% (v/v) digestates were chosen for addition to Caco-2 and Jurkat T cells, respectively. Yogurt and B2 digestate protected against H2O2-induced DNA damage in Caco-2 cells (p < 0.05), by the comet assay. Snack-bar digestates possessed significant (p < 0.05) immunomodulatory effects, measured by ELISA in concanavalin-A stimulated Jurkat T cells. Addition of BPH enhanced (p < 0.05) the IFN-γ reducing capacity of the snack-bar while addition of BPH < 3 and < 5 kDa reduced IL-2 production to a greater extent than unfortified yogurt (p < 0.05). Selected BSG components can enhance the antioxidant and anti-inflammatory potential of foods.

Immunomodulatory potential of a brewers' spent grain protein hydrolysate incorporated into low-fat milk following in vitro gastrointestinal digestion.

Brewers' spent grain (BSG) protein rich fraction was previously hydrolysed using Alcalase (U) and three additional fractions were prepared by membrane fractionation; a 5-kDa retentate (U > 5), a 5-kDa permeate (U < 5) and a 3-kDa permeate (U < 3). In the present study, these fractions were added to milk, subjected to simulated gastrointestinal digestion (SGID) and their anti-inflammatory potential was investigated. The digestates caused a significant reduction (p < 0.05) in interleukin-6 (IL-6) production in Concanavalin-A (ConA)-stimulated Jurkat T cells. The samples did not significantly alter the production of IL-6 in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. IL-2 and interferon-γ (IFN-γ) production in stimulated Jurkat T cells and IL-1ß and tumor necrosis factor-α (TNF-α) production in stimulated RAW 264.7 cells were not affected in the presence of the digestates. Results show that a SGID milk product supplemented with BSG hydrolysate and its associated ultrafiltered fractions can confer anti-inflammatory effects in Jurkat T cells.

Recent advances in Phytosterol Oxidation Products.

Phytosterols and their oxidation products have become increasingly investigated in recent years with respect to their roles in diet and nutrition. We present a comprehensive review of recent literature on Phytosterol Oxidation Products (POP) identifying critical areas for future investigation. It is evident that POP are formed on food storage/preparation; are absorbed and found in human serum; do not directly affect cholesterol absorption; have evidence of atherogenicity and inflammation; have distinct levels of cytotoxicity; are implicated with high levels of oxidative stress, glutathione depletion, mitochondrial dysfunction and elevated caspase activity.

In vitro investigation of the bioaccessibility of carotenoids from raw, frozen and boiled red chili peppers (Capsicum annuum).

BACKGROUND: Carotenoid-rich foods are associated with antioxidant activity and the ability to alleviate chronic diseases. PURPOSE: The present study investigated the effect of processing on the content and bioaccessibility of carotenoids from 13 cultivars of red chili pepper (Capsicum annuum). METHODS: Carotenoids in chili peppers were analyzed before an in vitro digestion process. The portion of carotenoid transferred to the micelle fraction (bioaccessibility) was also quantified. RESULTS: ß-Carotene, ß-cryptoxanthin, capsanthin and antheraxanthin were the most abundant carotenoids. Zeaxanthin, violaxanthin, neoxanthin and lutein were detected at lower concentrations. In general, freezing and boiling reduced carotenoid contents. Capsanthin and zeaxanthin had the highest bioaccessibility at an average value from 36 to 40%, followed by antheraxanthin (26%). Bioaccessibility of ß-cryptoxanthin, violaxanthin and ß-carotene was lower, averaging 6.1, 4.8 and 4.0%, respectively. Neoxanthin and lutein were not detected in micelles. Freezing increased the bioaccessibility of capsanthin, zeaxanthin, antheraxanthin, ß-cryptoxanthin and violaxanthin; ß-cryptoxanthin bioaccessibility increased and capsanthin and zeaxanthin bioaccessibility decreased following boiling. CONCLUSIONS: Differences in the contents and bioaccessibility of carotenoids in 13 C. annuum cultivars and between the processed methods were herein evidenced.

Phenolic-enriched fractions from brewers' spent grain possess cellular antioxidant and immunomodulatory effects in cell culture model systems.

BACKGROUND: Large quantities of brewers' spent grain (BSG), a co-product of the brewing industry, are produced annually. BSG contains hydroxycinnamic acids, and phenolic-rich extracts from BSG have previously demonstrated the ability to protect against oxidant-induced DNA damage. The present study investigated the anti-inflammatory potential of eight phenolic extracts from BSG: four pale (P1-P4) and four black (B1-B4) extracts. RESULTS: BSG extracts were more cytotoxic in Jurkat T than U937 cells, with lower IC50 values in Jurkat T cells, measured using the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Pale BSG extracts P2 and P3 showed the greatest anti-inflammatory potential, significantly (P < 0.05) reducing interleukin-2 (IL-2), interleukin-4 (IL-4, P2 only), interleukin-10 (IL-10) and interferon-γ (IFN-γ) production. In addition, extracts P1-P3 and B2-B4 showed significant (P < 0.05) antioxidant effects, determined by the cellular antioxidant activity assays superoxide dismutase, catalase and glutathione content (GSH). CONCLUSION: Phenolic extracts from BSG, particularly the pale BSG extracts, have the ability to reduce a stimulated cytokine production and may also protect against cellular oxidative stress. Results of the present study highlight the potential of BSG phenolic extracts to act as functional food ingredients, providing an alternative use and improving the value of this brewing industry co-product.

In vitro assessment of the bioaccessibility of carotenoids from sun-dried chilli peppers.

Chilli peppers have been recognized as an excellent source of antioxidants as they are rich in bioactive phytochemicals such as carotenoids which are known to exert various beneficial effects in vivo. Absorption is an important factor in the determination of the potential biological effects of carotenoids. The bioaccessibility of a food constituent such as a carotenoid represents its potential to be absorbed in humans. There is very limited information in the literature regarding the content and bioaccessibility of carotenoids from dried peppers. Therefore, the objectives of the present study were: first, to determine the carotenoid content of 20 varieties of red, orange or yellow coloured sun-dried chilli peppers belonging to either of four Capsicum species (annuum, baccatum, chinense and chacoense); and second, to quantify the carotenoid micellarization (bioaccessibility) following an in vitro digestion procedure. Red peppers had a higher carotenoid content and bioaccessibility than either the orange peppers or yellow pepper. Xanthophylls showed greater bioaccessibility than carotenes. Our findings confirm that dried chilli peppers are a good source of carotenoids.

Development of a dehydrated fortified food base from fermented milk and parboiled wheat, and comparison of its composition and reconstitution behavior with those of commercial dried dairy-cereal blends.

Dehydrated blends of milk and cereal are reconstituted and consumed as a nutritious soup or porridge in many regions; the composition and reconstitution behavior of the blends are likely to impact on nutritional quality and consumer acceptability of the soup/porridge. Experimental samples of dried fermented milk-bulgur wheat blend (FMBW) and commercial samples of dried dairy-cereal blends, namely kishk, tarhana, and super cereal plus corn-soy blend (SCpCSB) were compared for composition, color, water sorption, and reconstitution characteristics. FMBW blends had higher contents of protein, Ca, lactose and lactic acid, lower levels of salt (NaCl) and Fe, and a lighter, more-yellow color (higher L* and b*-color co-ordinates) than tarhana or kishk. Compared with SCpCSB, FMBW had numerically higher levels of protein, lactose, and lactic acid, lower levels of Ca, Fe, Zn, and Mg, and lower pH. Tarhana had highest mean levels of starch, and on reconstitution (133 g/kg) had highest water holding capacity, viscosity during pasting and cooling, yield stress (σ 0), consistency coefficient (K), and viscosity on shearing from 20 to 120 s-1 at 60°C. Reconstituted FMBW, kishk, and SCpCSB had similar pasting and flow behavior properties. Overall, the composition (starch, protein, Ca, Mg), pasting and flow behavior characteristics of FMBW were closer to those SCpCSB and kishk than to tarhana. The results suggest that the FMBW powder, on appropriate supplementation with Ca, Fe, Zn and Mg, could be used for the development of customized fortified blended foods for specific groups.

Comparison of the nutritional composition of experimental fermented milk:wheat bulgur blends and commercially available kishk and tarhana products.

Dried, fermented blends of dairy products and cereals, such as kishk and tarhana, are foodstuffs traditionally consumed in many regions as they possess good nutritional qualities and extended storage stability. This study examined the nutritional composition of kishk or tarhana type products and compared with experimental blends of fermented milk and wheat bulgur containing 60-80% milk. The blends with higher milk contents had levels of protein (18.9%) and fat (5.8%) at the concentrations specified in fortified blended foods as outlined by the World Food Program. Higher milk contents were also associated with higher contents of calcium (323.2 mg/100 g), phosphorus (335.3 mg/100 g), vitamin A (486.7 µg/100 g) and α-tocopherol (174.5 µg/100 g). The nutritional content of the experimental fermented milk:wheat bulgur blends compared favourably with that of the commercial samples. These blends may be suitable as base products, to be fortified with micronutrients, for the development of fortified blended foods (FBFs) for humanitarian distribution.

Characterisation of the in vitro bioactive properties of alkaline and enzyme extracted brewers' spent grain protein hydrolysates.

Brewer's spent grain (BSG) is a co-product of the brewing industry that has been shown to contain a range of bioactive peptides encrypted within its protein sequences. Two methods were evaluated herein to generate bioactive peptides; (i) an alkaline extracted BSG protein rich fraction (BSG-PI) was hydrolysed using different combinations of proteolytic enzymes and (ii) BSG was pre-treated with carbohydrases followed by direct hydrolysis using proteolytic enzymes (BSG-DH). BSG-DH with Alcalase/Flavourzyme resulted in significantly higher (p < .05) protein yield when compared to BSG-PI (63.09 ±â€¯0.27 and 58.90 ±â€¯1.45%, respectively). The antioxidant activities (ORAC, FRAP and ABTS) of the BSG-PI and -DH hydrolysates differed depending on the assay and proteolytic enzyme combination preparations used for hydrolysis. Inhibition of DPP-IV by the BSG-PI hydrolysates ranged from 87.01 ±â€¯0.15 to 89.61 ±â€¯0.12% while inhibition by the BSG-DH hydrolysates ranged from 35.71 ±â€¯0.72 to 85.06 ±â€¯0.17%. A significant reduction in the release of interleukin-6 in lipopolysaccharide-stimulated RAW 264.7 cells was observed following treatment with BSG-PI hydrolysates generated with Prolyve/Protease P (58.30 ±â€¯13.76%) and Corolase PP/Flavourzyme (48.02 ±â€¯10.82%) when compared to untreated LPS stimulated control cells (100%). BSG-DH hydrolysates were subjected to in vitro simulated gastrointestinal digestion (SGID) which resulted in a reduction in antioxidant activity, an increase in DPP-IV inhibition and no change in the immunomodulatory activity. Ultrafiltration of selected BSG-DH hydrolysates (through 30 and 10 kDa membranes) gave some permeates with enhanced bioactivities. The results demonstrate that direct enzymatic hydrolysis of BSG is a feasible approach for the generation of bioactive peptides without the prior use of an alkali protein extraction step.

Fortified Blended Food Base: Effect of Co-Fermentation Time on Composition, Phytic Acid Content and Reconstitution Properties.

Dehydrated blends of dairy-cereal combine the functional and nutritional properties of two major food groups. Fortified blended food base (FBFB) was prepared by blending fermented milk with parboiled wheat, co-fermenting the blend at 35 °C, shelf-drying and milling. Increasing co-fermentation time from 0 to 72 h resulted in powder with lower lactose, phytic acid and pH, and higher contents of lactic acid and galactose. Simultaneously, the pasting viscosity of the reconstituted base (16.7%, w/w, total solids) and its yield stress (σ0), consistency index (K) and viscosity on shearing decreased significantly. The changes in some characteristics (pH, phytic acid, η120) were essentially complete after 24 h co-fermentation while others (lactose, galactose and lactic acid, pasting viscosities, flowability) proceeded more gradually over 72 h. The reduction in phytic acid varied from 40 to 58% depending on the pH of the fermented milk prior to blending with the parboiled cereal. The reduction in phytic acid content of milk (fermented milk)-cereal blends with co-fermentation time is nutritionally desirable as it is conducive to an enhanced bioavailability of elements, such as Ca, Mg, Fe and Zn in milk-cereal blends, and is especially important where such blends serve as a base for fortified-blended foods supplied to food-insecure regions.

Optimisation of the antifungal potency of the amidated peptide H-Orn-Orn-Trp-Trp-NH2 against food contaminants.

The design of novel efficient antimicrobial peptides (AMPs) faces several issues, such as cost of synthesis, proteolytic stability or cytotoxicity. The identification of key determinants involved in the activity of AMPs, such as cationicity and amphipathicity, allowed the synthesis of short peptides with optimized properties. An ultrashort peptide made of the sequence H-Orn-Orn-Trp-Trp-NH2 (O3TR) showed antifungal activity against several contaminants from food products. This peptide inhibited the growth of the filamentous fungi Fusarium culmorum, Penicillium expansum and Aspergillus niger within a range of concentration of 12.5-50µg/ml. In addition, O3TR inhibited the growth of the yeast Saccharomyces cerevisiae, Zygosaccharomyces bailii, Zygosaccharomyces rouxii, Debaryomyces hansenii and Kluyveromyces lactis within the range 12.5-50µg/ml. A derivative peptide, called C12O3TR, made by the addition of lauric acid at the N-terminus of O3TR was 2- to 8-fold more active than O3TR against every species. In addition to the inhibition of conidial germination, O3TR and C12O3TR killed F. culmorum hyphae at 100 and 50µg/ml respectively. The MIC of the two peptides against F. culmorum and Z. bailii after heat treatment at 100°C for 60 min and within the pH range 3-10, were not changed. However, the activity of O3TR against F.culmorum and Z. bailii was strongly reduced in salt solutions, whereas the lauric acid peptide kept its antifungal activity and resistance to proteolytic digestion. The conjugation with lauric acid reduced the random coiled structure and increased the α-helical content of O3TR. After conjugation with the dye tetramethylrhodamine (TMR), both peptides entered F. culmorum spores. They also both induced permeabilization of F. culmorum hyphae but only C12O3TR permeabilized Z. bailii membrane. In contrast to the lipopeptide, O3TR did not show haemolytic or cytotoxic activity when applied at the concentrations that exhibited antifungal potency. The two peptides were challenged against a yeast cocktail of S. cerevisiae and Z. bailii, and A. niger in different commercial beverages. After 7 days, O3TR was able to inhibit the yeast cocktail in a commercial lager and carbonated drink. Due to its antifungal potency, high stability and low cytotoxicity, the tetrapeptide could represent a promising starting point of a novel food preservative.

The Proportion of Fermented Milk in Dehydrated Fermented Milk⁻Parboiled Wheat Composites Significantly Affects Their Composition, Pasting Behaviour, and Flow Properties on Reconstitution.

Dairy and cereal are frequently combined to create composite foods with enhanced nutritional benefits. Dehydrated fermented milk⁻wheat composites (FMWC) were prepared by blending fermented milk (FM) and parboiled wheat (W), incubating at 35 °C for 24 h, drying at 46 °C for 48 h, and milling to 1 mm. Increasing the weight ratio of FM to W from 1.5 to 4.0 resulted in reductions in total solids (from 96 to 92%) and starch (from 52 to 39%), and increases in protein (15.2⁻18.9%), fat (3.7⁻5.9%), lactose (6.4⁻11.4%), and lactic acid (2.7⁻4.2%). FMWC need to be reconstituted prior to consumption. The water-holding capacity, pasting viscosity, and setback viscosity of the reconstituted FMWC (16.7% total solids) decreased with the ratio of FM to W. The reconstituted FMWC exhibited pseudoplastic flow behaviour on shearing from 18 to 120 s-1. Increasing the FM:W ratio coincided with a lower yield stress, consistency index, and viscosity at 120 s-1. The results demonstrate the critical impact of the FM:W ratio on the composition, pasting behavior, and consistency of the reconstituted FMWC. The difference in consistency associated with varying the FM:W ratio is likely to impact on satiety and nutrient value of the FMWCs.

Modulatory effects of resveratrol, citroflavan-3-ol, and plant-derived extracts on oxidative stress in U937 cells.

Phytochemicals and plant extracts, present in fruit, vegetables, plants, herbs, and beverages, have been shown to have antioxidant potential that may modulate the etiology of certain chronic diseases. The objective of the present study was to determine the concentration of compound that inhibited cell growth by 50% (IC(50)) of a range of phytochemicals and plant extracts and to investigate their antioxidant and genoprotective effects under conditions of oxidative stress in U937 cells. Two phytochemicals-resveratrol and citroflavan-3-ol-and four plant extracts-grapeseed polyphenols, olive leaf extract, bearberry, and Echinacea purpurea-were examined. Viability was assessed by the fluorescein diacetate/ethidium bromide assay. The IC(50) was calculated. To examine their antioxidant and genoprotective effects, U937 cells were pretreated with the test compounds at levels below the IC(50) and then exposed to oxidants: 0.5 microM etoposide or 100 microM hydrogen peroxide (H(2)O(2)) or 400 microM tert-butylhydroperoxide (tBOOH). Cellular reduced glutathione levels were measured as an indicator of oxidative stress. DNA damage was assessed by the alkaline single-cell gel electrophoresis assay or comet assay. Resveratrol demonstrated the highest IC50 value of 13.7 microg/mL, with Echinacea the lowest at 9,400 microg/mL. Etoposide-induced oxidative stress was strongly reduced by olive leaf extract and bearberry. Grapeseed polyphenols and bearberry strongly protected against H2O2- and tBOOH-induced DNA damage. In conclusion, these results provide evidence that non-nutrient dietary constituents may act as significant bioactive compounds and that plant extracts, such as bearberry, grapeseed polyphenols, and olive leaf extract, strongly protect against oxidative stress.

Cellular Transport and Bioactivity of a Major Saffron Apocarotenoid, Picrocrocin (4-(ß-D-Glucopyranosyloxy)-2,6,6-trimethyl-1-cyclohexene-1-carboxaldehyde).

The cellular transport and bioactivity of the second major saffron apocarotenoid, picrocrocin, was examined in parallel to that of the major group, crocetin sugar esters, in aqueous extracts. The transport of pure picrocrocin was investigated in comparison to that of other saffron apocarotenoids, trans-crocetin (di-ß-D-gentiobiosyl) ester and crocetin using the Caco-2 cell model coupled with an in vitro digestion procedure. RP-HPLC-DAD was employed to quantify the bioaccessible and bioavailable amounts of individual apocarotenoids. Picrocrocin and crocetin sugar esters though highly bioaccessible (75% and 60%, respectively) were transported at minute quantities (0.2% and 0.5%, respectively; 10-fold lower than crocetin). Picrocrocin did not protect against oxidant-induced DNA damage in U937, human monocytic blood cells at the concentration investigated, however, it reduced the proliferation of human adenocarcinoma and hepatocarcinoma cells. Our findings may be useful for the requirements of food legislation regarding saffron preparations, in which both apocarotenoid groups coexist.

Effects of cholesterol oxides on cell death induction and calcium increase in human neuronal cells (SK-N-BE) and evaluation of the protective effects of docosahexaenoic acid (DHA; C22:6 n-3).

Some oxysterols are associated with neurodegenerative diseases. Their lipotoxicity is characterized by an oxidative stress and induction of apoptosis. To evaluate the capacity of these molecules to trigger cellular modifications involved in neurodegeneration, human neuronal cells SK-N-BE were treated with 7-ketocholesterol, 7α- and 7ß-hydroxycholesterol, 6α- and 6ß-hydroxycholesterol, 4α- and 4ß-hydroxycholesterol, 24(S)-hydroxycholesterol and 27-hydroxycholesterol (50-100µM, 24h) without or with docosahexaenoic acid (50µM). The effects of these compounds on mitochondrial activity, cell growth, production of reactive oxygen species (ROS) and superoxide anions (O2(-)), catalase and superoxide dismutase activities were determined. The ability of the oxysterols to induce increases in Ca(2+) was measured after 10min and 24h of treatment using fura-2 videomicroscopy and Von Kossa staining, respectively. Cholesterol, 7-ketocholesterol, 7ß-hydroxycholesterol, and 24(S)-hydroxycholesterol (100µM) induced mitochondrial dysfunction, cell growth inhibition, ROS overproduction and cell death. A slight increase in the percentage of cells with condensed and/or fragmented nuclei, characteristic of apoptotic cells, was detected. With 27-hydroxycholesterol, a marked increase of O2(-) was observed. Increases in intracellular Ca(2+) were only found with 7-ketocholesterol, 7ß-hydroxycholesterol, 24(S)-hydroxycholesterol and 27-hydroxycholesterol. Pre-treatment with docosahexaenoic acid showed some protective effects depending on the oxysterol considered. According to the present data, 7-ketocholesterol, 7ß-hydroxycholesterol, 24(S)-hydroxycholesterol and 27-hydroxycholesterol could favor neurodegeneration by their abilities to induce mitochondrial dysfunctions, oxidative stress and/or cell death associated or not with increases in cytosolic calcium levels.

Anti-inflammatory effects of wild Irish mushroom extracts in RAW264.7 mouse macrophage cells.

Mushrooms and mushroom extracts have traditionally been used as therapies for a wide variety of ailments, including allergy, arthritis, and other inflammatory disorders. However, more evidence is required on the mechanism by which mushrooms exert these effects. In the present study, the anti-inflammatory properties of ethanol and hot water extracts prepared from 27 fungal samples collected between October and November 2011 at various forest locations in the southwest of Ireland were investigated using the lipopolysaccharide (LPS)-stimulated mouse macrophage (RAW264.7 cells) model of inflammation. LPS-stimulated cells were incubated in the presence of mushroom extracts at nontoxic concentrations for 24 h and the production of interleukin-6 (IL-6) was quantified by ELISA. Seven ethanolic and one hot water extract that decreased IL-6 production were selected for further study. The extracts were then incubated with LPS-stimulated cells for 24 h and the production of IL-6, tumor necrosis factor-alpha (TNF-α), and nitric oxide (NO) was measured. Ethanolic extracts prepared from Russula mairei, Lactarius blennius, Craterellus tubaeformis, Russula fellea, and Craterellus cornucopioides demonstrated selective anti-inflammatory activity by decreasing the production of NO and IL-6 but not TNF-α in LPS-stimulated RAW264.7 cells. These findings support existing evidence of the anti-inflammatory potential of mushroom extracts.