Comparative effects of phytosterol oxides and cholesterol oxides in cultured macrophage-derived cell lines

The cytotoxicity of cholesterol and a mixture of beta-sitosterol/campesterol (50%/40%) and their oxides was examined in a cultured-derived macrophage cell line, C57BL/6. Cell numbers, lactate dehydrogenase (LDH) leakage, protein content, lipid uptake, and mitochondria dehydrogenase activity were determined after exposure of cell mononlayers to sterols and sterol oxides at a concentration of 200 microg/mL for up to 120 h. Results indicate that the oxides of cholesterol, beta-sitosterol, and campesterol exhibited similar patterns of toxicity as indicated by LDH leakage, cell viability, and mitochondria dehydrogenase activity. Greatest cell damage was associated with treatments containing 5 alpha,6 alpha-epoxide or cholesterol oxides, followed by beta-sitosterol/campesterol oxides, cholesterol, and beta-sitosterol. The oxides of beta-sitosterol/campesterol caused less LDH leakage and less of an effect on protein content. Results of this study demonstrate that phytosterols contained in vegetable oils, when subjected to frying conditions, do oxidize and may cause cellular damage in an in vitro cell line similar to cholesterol oxides, although less severe.     

The dietary hydroxycinnamate caffeic acid and its conjugate chlorogenic acid increase vitamin e and cholesterol concentrations in Sprague-Dawley rats

Vegetarian diets are correlated with a reduced risk of developing cardiovascular disease and comprise a great variety of bioactive compounds, including hydroxycinnamic acid derivatives. Therefore, this study aimed to identify dietary hydroxycinnamic acid derivatives that may alter two important factors related to the development of cardiovascular disease, namely, tocopherol (T) and cholesterol (C) concentrations in the body. The effects of caffeic acid (CA), chlorogenic acid (CGA), and ferulic acid (FA) on alpha-T, gamma-T, and C levels in blood plasma, liver, and lungs were investigated after these compounds had been fed to rats for 4 weeks at concentrations of 2 g/kg in semisynthetic diets. None of the regimens affected weight gain, feed intake, or absolute weights of livers and lungs, although CA increased the liver weight relative to the body weight (P < 0.05). CA- and CGA-fed animals showed a tendency toward sparing vitamin E in all tissues, but statistical significance was obtained only for gamma-T in the liver of CA-fed animals (P < 0.005) and for alpha-T in the lungs of CGA-treated rats (P < 0.05). CGA supplementation reduced concentrations of lipids in the lung tissue (P < 0.05). CA and CGA elevated the concentrations of C in liver tissue and lipids to a similar extent, but only CA decreased the ratio of high-density lipoprotein C to total C in blood plasma (P < 0.05 for all effects). Animals eating FA showed T and C values comparable to those in the control group. In conclusion, this study demonstrates that dietary caffeic and chlorogenic acid may elevate tocopherols and cholesterol in vivo.     

Cholesterol oxides,cholesterol, total lipid,and fatty acid composition in turkey meat

The contents of cholesterol oxides, cholesterol, and total lipid, and the fatty acid composition were determined in frozen turkey meat. The 7-ketocholesterol content varied from 33 microg/100 g in the breast to 765 microg/100 g in the skin, and the levels of 7 beta-hydroxycholesterol varied from not detected in the leg, breast, and skin to 370 microg/100 g in the skin. The values for total lipid (g/100 g) in the wings, legs, breast, and skin were 0.9 +/- 0.4, 1.1 +/- 0.2, 0.5 +/- 0.1, and 12 +/- 3, respectively. The contents for cholesterol (mg/100 g) were 46 +/- 5, 35 +/- 2, 27 +/- 3, and 81 +/- 6 in the wing, legs, breast, and skin, respectively. The main fatty acids identified in all cuts were C18:2n6, C18:1n9, C16:0, C18:0, and C20:4n6.     

Identification and quantification of cholesterol and cholesterol oxidation products in different types of Iberian hams

Cholesterol and cholesterol oxidation products (COPs) were determined in four different groups of dry-cured Iberian hams, based on the feeding received by pigs and their degree of crossbreeding. After lipid extraction, GC-FID for cholesterol determination and GC-MS to analyze COPs were used. Cholesterol content ranged from 30 to 34 mg/100 g of muscle. Some of the COPs analyzed, such as 7alpha-hydroxycholesterol, 7beta-hydroxycholesterol, and 7-ketocholesterol, were detected in all of the samples. The major cholesterol oxide was 7-ketocholesterol; its concentration ranged from 57 to 71 microg/100 g of muscle. The content of cholesterol and cholesterol oxides in intramuscular lipids of hams was not affected by diet or crossbreeding of Iberian pigs.     

Optimization of bioactive compounds in buckwheat sprouts and their effect on blood cholesterol in hamsters

Nutrient levels in buckwheats that were maximized in day 8 sprouts (D8SP) included total phenolics, quercetin, and l-ascorbic acid, whereas those of oxalic, malic, tartaric, and citric acids, rutin, and gamma-aminobutyric acid (GABA) were found to reach maximum levels on day 10. Ethanolic extract of D8SP (2.5 mg/mL) revealed potent free-radical scavenging (FRS) and antioxidative (ANO) capabilities. However, its Fe2+-chelating capability was only moderate. To further study the hypolipidemic activity of D8SP, 36 Syrian hamsters were grouped into six groups and fed for 28 days, respectively, with (i) control meal, (ii) high fat plus high cholesterol meal, (iii) high fat plus high cholesterol plus 2.5% of buckwheat seeds, (iv) high fat plus high cholesterol plus 25% of buckwheat seeds, (v) high fat plus high cholesterol plus 2.5% of D8SP, and (vi) high fat plus high cholesterol plus 25% of D8SP. High seed meal prominently enhanced body weight gain, whereas high sprout meal exhibited the highest feed efficiency. Ratios of liver/body weight (L/B) were significantly lowered by all BS meals. Although low seed meal reduced serum total cholesterol (TC) levels (p<0.05), its effect was still inferior to the high seed and sprout meals (p<0.01). In contrast, serum triglyceride (TG) levels were lowered only by the high seed and sprout meals (p<0.05). Alternatively, levels of serum low-density lipoprotein cholesterol (LDL-C) were significantly suppressed by all buckwheat meals (p<0.01). Serum high-density lipoprotein cholesterol (HDL-C) levels were increased, however, insignificantly. Nutraceutically more meaningful is that both LDL-C/HDL-C and TC/HDL-C ratios were significantly lowered (p<0.01). Apparently, hepatic TC levels were significantly reduced, whereas hepatic TG levels were totally unaffected. Conclusively, sprouting triggers a variety of nutritional changes in buckwheats. Day 8 sprouts, consisting of high polyphenolic and moderate quercetin contents, are nutraceutically maximized when hypocholesterolemic, hypotriglyceridemic, and antioxidative activities are concerned.     

Insoluble fraction of buckwheat (Fagopyrum esculentum Moench) protein possessing cholesterol-binding properties that reduce micelle cholesterol solubility and uptake by Caco-2 cells

Buckwheat (Fagopyrum esculentum Moench) protein (BWP) exhibits hypocholesterolemic activity in several animal models by increasing fecal excretion of neutral and acidic sterols. In the current study, the ability of BWP to disrupt micelle cholesterol solubility by sequestration of cholesterol was investigated. When BWP (0.2%) was incubated with cholesterol and micelle lipid components prior to micelle formation, cholesterol solubility was reduced 40%. In contrast, cholesterol solubility was not decreased when BWP (0.2%) was incubated after micelle formation and incorporation of soluble cholesterol. Buckwheat flour, from which BWP was derived, had no significant effect on cholesterol solubility. Cholesterol uptake in Caco-2 cells from micelles made in the presence of BWP (0.2%) was reduced by 47, 36, 35, and 33% when compared with buckwheat flour, bovine serum albumin, casein, and gelatin, respectively. Reduction in cholesterol uptake in Caco-2 cells was dose-dependent, with maximum reductions at 0.1-0.4% BWP. In cholesterol-binding experiments, 83% of the cholesterol was associated with an insoluble BWP fraction, indicating strong cholesterol-binding capacity that disrupts solubility and uptake by Caco-2 cells.     

Nonaqueous reverse phase liquid chromatographic analysis for cholesterol in milk chocolate

A method using liquid chromatography was developed for the analysis of cholesterol in milk chocolate products. The method involves saponification of the sample with methanolic KOH followed by extraction with ether. Potentially interfering components are eliminated through the use of a silica Sep-Pak cleanup step before injection. The nonaqueous reverse phase LC system consists of a C18 column and an isopropanol-hexane mobile phase with direct detection at 205 nm. Recoveries of 1, 3, and 5 mg cholesterol added to 1 g sample of milk chocolate were 88.6, 102.8, and 110.1%, respectively. Studies conducted with [4-14C]-cholesterol were undertaken to further document the accuracy of the method.     

Antioxidative and antiproliferative properties of selected barley (Hordeum vulgarae L.) cultivars and their potential for inhibition of low-density lipoprotein (LDL) cholesterol oxidation

Aqueous methanolic extracts of whole kernels from six different barley cultivars, namely, Falcon, AC Metcalfe, Tyto, Tercel, Phoenix, and Peregrine, were examined for their total phenolic content (TPC), oxygen radical scavenging capacity (ORACFL), hydroxyl radical scavenging capacity (HORACFL), potency in prevention of lipid oxidation using the Rancimat method, efficacy in inhibition of Cu(II)-induced human LDL cholesterol oxidation, and antiproliferative activities using Caco-2 colorectal adenocarcinoma cell line. Total phenolic content as measured by Folin-Ciocalteu's method ranged from 0.68 to 1.19 mg of ferulic acid equiv/g of defatted material, whereas ORACFL and HORACFL values were 11.28-19.10 and 9.06-12.99 micromol of Trolox equiv/g of defatted material, respectively. Protection factor (PF), a measure of the effect of extracts on the prevention of oxidation of stripped corn oil as measured by Rancimat, ranged from 0.97 to 1.59. Furthermore, barley extracts showed 19.64-33.93% inhibition against Cu(II)-induced human LDL cholesterol oxidation at a final concentration of 0.02 mg/mL. The proliferation of Caco-2 colon cancer cells was significantly (p < 0.05) inhibited in a dose-dependent fashion in the presence of all barley extracts tested at the end of the day 4 of incubation. At the end of day 4, barley extracts rendered 29.3-51.2 and 9.3-15.9% inhibition of cell proliferation at 0.5 and 0.05 mg/mL, respectively. Phenolic extracts from whole barley kernel tested possessed high antioxidant, antiradical, and antiproliferative potentials. Therefore, inclusion of whole barley into the daily diet may render beneficial health benefits.     

Red and white wines inhibit cholesterol oxidation induced by free radicals

The capabilities of two red (RW) and two white wines (WW) in inhibiting cholesterol oxidation were evaluated using a cholesterol emulsion (CE) system. Each RW or WW was mixed with CE at different (v/v) ratios. Cholesterol oxidation was accelerated by a free radical generator, 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AAPH), at 37 °C. The major oxidation product, 7-ketocholesterol, was monitored to determine cholesterol stability in the CE system. At a ratio of 1:250 (RW/CE), 7-ketocholesterol production was not detected during 72 h of oxidation. At a 1:1000 ratio, the inhibition rate of each RW was maintained at 100% at 24 h but decreased afterward. Both WWs had 100% inhibition rate within 48 h at a ratio of 1:10. Also, the capabilities of catechin and resveratrol solutions (1 mg/mL) in inhibiting cholesterol oxidation were studied. Each of the wine polyphenolics showed a 100% of 7-ketocholesterol inhibition rate in 24 h at a ratio of 1:500 (solution/CE). However, the inhibition rate of resveratrol was lower than that of catechin at 48 or 72 h. The results demonstrated that red wine possesses great anti-cholesterol-oxidation capability, which may contribute to health benefits in preventing cardiovascular diseases. Catechin may play a more important role than resveratrol in inhibiting cholesterol oxidation.     

Turkish Tombul hazelnut (Corylus avellana L.). 2. Lipid characteristics and oxidative stability

The quality of crude hazelnut oil extracted from Tombul (Round) hazelnut, grown in the Giresun province of Turkey, was determined by measuring lipid classes, fatty acids, and fat soluble bioactives (tocopherols and phytosterols). Oxygen uptake, peroxide value, thiobarbituric acid reactive substances, and alpha-tocopherol levels of stripped and crude hazelnut oils in bulk and oil-in-water (o/w) emulsion systems were also evaluated as indices of lipid oxidation over a 21 day storage period at 60 degrees C in the dark. The total lipid content of Tombul hazelnut was 61.2%, of which 98.8% were nonpolar and 1.2% polar constituents. Triacylglycerols were the major nonpolar lipid class and contributed nearly 100% to the total amount. Phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol were the most abundant polar lipids, respectively. Sixteen fatty acids were identified, among which oleic acid contributed 82.7% to the total, followed by linoleic, palmitic, and stearic acids. Unsaturated fatty acids accounted for 92.2% of the total fatty acids present. Among oil soluble bioactives, alpha-tocopherol (38.2 mg/100 g) and beta-sitosterol (105.5 mg/100 g) were predominant in hazelnut oil and comprised 88 and 93% of the total tocopherols and phytosterols present, respectively. The results also showed that both stripped and crude hazelnut oils were more stable in terms of lipid oxidation in the bulk oil as compared to those in an o/w emulsion.     

Differential scanning calorimetry evidence of the enhancement of beta-sitosterol absorption across biological membranes mediated by beta-cyclodextrins

beta-Sitosterol is a plant sterol that has received much attention because of its effectiveness in reducing the absorption of dietary cholesterol, as well as in offering protection from cardiovascular diseases and cancer development. Thus, the knowledge of the interaction of beta-sitosterol with biological membranes can help in understanding its mechanism of action. In the present paper, the differential scanning calorimetry technique has been used to study the interaction of beta-sitosterol with a biomembrane model constituted by dimyristoylphosphatidylcholine multilamellar vesicles. Furthermore, kinetic experiments have been carried out to follow the uptake of beta-sitosterol by biomembranes and the effect of beta-cyclodextrins on such a process. Our results indicate that opportune concentrations of beta-cyclodextrins improve the uptake of beta-sitosterol by phospholipid membranes.     

Sterol oxidation in ready-to-eat infant foods during storage

The effect of storage on sterol oxidation of ready-to-eat infant foods was evaluated. Two different liquid infant foods (honey or fruits flavors), prepared with milk and cereals, were stored for 0, 2, 4, 7 and 9 months at 25 degrees C. Sterol oxidation products (SOP) were isolated by cold saponification, purified by silica solid-phase extraction, and analyzed by gas chromatography (GC) and GC-mass spectrometry. beta-Sitosterol was the most representative sterol, followed by cholesterol and campesterol. No significant differences in the total and single SOP content (0.8-1 mg/kg of product) were observed with respect to storage time and type of sample; the main SOP found was 7-ketositosterol (<0.2 mg/kg of product). The extent of stigmasterol oxidation (2.9%) was higher than that of cholesterol (1.9%) and beta-sitosterol (1.4%). The type and quality of raw materials, as well as the processing conditions, seem to greatly influence SOP formation and accumulation in infant foods.     

Effect of Cattle Slurry on the Growth of Spinach Plants in Cd-contaminated Soil

ABSTRACT

In this work the effect of the addition of different amounts of cattle slurry (CS) to a Cd contaminated soil, was studied regarding its effect in spinach plants. Two levels of Cd contamination (2 and 10 mg/kg) and three levels of CS addition were evaluated (2.5, 5 and 10 g CS/100 g soil). Spinach was shown to be a tolerant species, able to accumulate relatively high amounts of Cd (up to 367.7 mg/kg in the leaves), exceeding the limits established by European regulations for leaf vegetables. The addition of 2.5 and 5 g CS/100 g to soil containing 2 mg/kg Cd did not reduce the uptake of this metal but allowed the plants to grow as much as the control. The addition of 10 g CS/100 g lead to a reduced Cd uptake but also to a lower plant growth compared to the lower CS levels. The combined effects of Cd and CS changed element content in the plant, but without causing severe toxicity or deficiency effects.     

Mulberry leaf polyphenols possess antiatherogenesis effect via inhibiting LDL oxidation and foam cell formation

Oxidized low-density lipoprotein (ox-LDL) and its uptake by machrophage are the hallmark in atherogenesis. In the present study, we aimed to investigate the antiatherogenic effect of mulberry leaf extracts (MLE) and the polyphenolic extracts (MLPE), which contained polyphenols including quercetin (11.70%), naringenin (9.01%) and gallocatechin gallate (10.02%). Both MLE and MLPE inhibited the oxidation and lipid peroxidation of LDL, while MLPE was shown to be more potent. As 1.0 mg/mL MLE reduced 30% of ox-LDL-generated ROS, 0.5 mg/mL MLPE decreased 46% of the ROS and was shown to be more potent on elevating SOD-1 and GPx in macrophages. At the same dose of 0.5 mg/mL, MLPE exhibited 1.5-fold potency than MLE in decreasing the formation of foam cells. Both MLE and MLPE reduced the expression of PPARγ, CD36 and SR-A, implicating the molecular regulation on ox-LDL uptake. These results suggested that MLPE potentially could be developed as an antiatherogenic agent and deserve further investigation.     

生物质炭和氮肥配施对菠菜产量和硝酸盐含量的影响

施用生物质炭是提高作物产量和氮肥利用效率的潜在有效措施。以菠菜为供试作物开展盆栽试验,研究了生物质炭与氮肥配施对菠菜产量、组织中硝酸盐含量及养分（氮磷钾）含量的影响。生物质炭设3个水平：C0（0g·kg-1）、C5（5g·kg-1）和C10（10g·kg-1）,氮素3个水平分别为N0（0mg·kg-1）、N1（90mg·kg-1）和N2（120mg·kg-1）。试验结果表明,在N0和N1水平下,施用生物质炭显著提高了菠菜产量,增幅为16.6%～57.3%,而在N2水平下,生物质炭对菠菜产量无显著影响（P〉0.05）。同时,在N1水平下,与C0处理相比,C5和C10处理菠菜组织中硝酸盐含量分别增加了198.7%和233.4%;而在N2水平下,C5和C10处理的硝酸盐增幅分别为8.8%和46.3%。在不同氮素水平下,生物质炭的施用增加了菠菜对氮和钾的吸收,而对磷素吸收的影响不明显。总之,生物质炭与氮肥配施可以提高菠菜产量,明显增加氮肥当季利用效率。     

Barley intake induces bile acid excretion by reduced expression of intestinal ASBT and NPC1L1 in C57BL/6J mice

To investigate the hypocholesterolemic mechanism of barley in vivo, six-week-old C57BL/6J mice were fed a high-fat diet (HFD) or high-fat diet containing barley (HFD-B) for seven weeks. Total and LDL cholesterol concentrations were significantly reduced in the HFD-B group while fecal cholesterol and bile acid was increased. Real-time PCR and immunoblot analysis revealed the induction of FXR expression, which in turn suppressed the expression of ASBT and NPC1L1 in the HFD-B group compared with the controls. In the liver, the expression of HMG-CoA reductase was significantly reduced while LDL receptor expression was unaltered in the HFD-B group compared with the controls. Our data suggest that the hypocholesterolemic effects of barley are primarily the result of reduced dietary cholesterol uptake and bile acid resorption. Reduced expression of intestinal ASBT and NPC1L1 may play a key role in the regulation of dietary cholesterol and bile acid metabolism in mice consuming a diet containing barley.     

Evaluation of direct saponification method for determination of cholesterol in meats

A gas chromatographic (GC) method has been developed for determination of cholesterol in meats. The method involves ethanolic KOH saponification of the sample material, homogeneous-phase toluene extraction of the unsaponifiables, derivatization of cholesterol to its trimethylsilylether, and quantitation by GC-flame ionization detection using 5-alpha-cholestane as internal standard. This direct saponification method is compared with the current AOAC official method for determination of cholesterol in 20 different meat products. The direct saponification method eliminates the need for initial lipid extraction, thus offering a 30% savings in labor, and requires fewer solvents than the AOAC method. It produced comparable or slightly higher cholesterol results than the AOAC method in all meat samples examined. Precision, determined by assaying a turkey meat sample 16 times over 4 days, was excellent (CV = 1.74%). Average recovery of cholesterol added to meat samples was 99.8%.     

Regulation effects of Crataegus pinnatifida leaf on glucose and lipids metabolism

The leaf of Crataegus pinnatifida (Rosaceae) is commonly consumed either raw or cooked to improve digestion and promote blood circulation in China. To investigate the regulation effects of it on glucose and lipid metabolism, the flavonoids fraction was prepared and analyzed by HPLC and LC-MS. In vivo, at doses of 250 and 500 mg/kg, the flavonoids fraction showed inhibitory effects on TG and glucose absorption, accelerating effects on gastrointestinal transit but no effect on gastric emptying. In vitro, treatment of 3T3-L1 preadipocytes with 30 μg/mL flavonoids fraction significantly suppressed the accumulation of TG and free fatty acid. It also suppressed the gene expressions of C/EBPα, PPARγ, SREBP 1c, aP2 and adiponectin but did not affect that of leptin. C. pinnatifida leaf may be useful for type 2 diabetics and hyperlipidemics as a foodstuff.     

Supplementation of test meals with fat-free phytosterol products can reduce cholesterol micellarization during simulated digestion and cholesterol accumulation by Caco-2 cells

Phytosterols have been shown to reduce cholesterol absorption in humans. Supplementing phytosterols in fat-free formulations, however, has yielded controversial results. In the present study, we investigated the effect of supplementing test meals with different fat-free phytosterol products on cholesterol incorporation into mixed micelles during simulated digestion and accumulation of micellar cholesterol by Caco-2 cells: control orange juice (OJ), orange juice supplemented with either multivitamin/multimineral tablets (MVT), multivitamin/multimineral tablets containing phytosterols (MVT+P), and phytosterol powder (PP). These combinations were added to Ensure-based test meals and spiked with cholesterol of natural isotopic composition or 13C2-cholesterol to differentiate external from endogenous cholesterol. After simulated gastric/small intestinal digestion, micelle fractions were analyzed for cholesterol enzymatically (n = 6-20/product) and by high-performance liquid chromatography-tandem mass spectrometry (n = 12/product) and added to Caco-2 cells to determine the accumulation of 13C2-cholesterol (n = 10-24/product). As compared to OJ, PP and MVT+P significantly decreased cholesterol micellarization (determined enzymatically) by 70 +/- 39 (mean +/- SD) and 70 +/- 39%, respectively (P < 0.001, Bonferroni). The stable isotope experiments revealed that both PP and MVT+P reduced cholesterol micellarization [by 25 +/- 12 (P = 0.055) and 21 +/- 8% (P = 0.020), respectively, Fisher's protected LSD test] and Caco-2 cell accumulation (by 28 +/- 8 and 10 +/- 8%, respectively; P < 0.010, Bonferroni). OJ+P did not inhibit micellarization or accumulation of cholesterol by Caco-2 cells. This study shows that fat-free phytosterol-containing products can significantly inhibit cholesterol micellarization and Caco-2 cell bioaccessibility, albeit to different extents depending on individual formulations. This is most likely explained by inhibition of cholesterol micellarization.     

Rapid determination of total cholesterol in homogenized milk

A rapid method that is amenable to automation has been developed for the determination of total cholesterol in homogenized milk. The milk sample is saponified in ethanolic KOH in the presence of an internal standard, cholestane. Cholesterol and the internal standard are then isolated by solid-phase extraction on a nonpolar adsorbent and eluted with organic solvent. The evaporated extract is derivatized and analyzed by capillary gas chromatography. Average recovery of cholesterol acetate added to milk prior to saponification was 95%. The average relative standard deviation for repeated analyses was 2%. The limit of detection for this method is 2 mg/100 g. Twenty samples can be analyzed by one analyst in a normal work day if the gas chromatograph is equipped with an autosampler. This method has been compared with a modified AOAC method for the determination of total cholesterol. At a confidence level of 95%, no difference was observed between the 2 methods.