Effects of hydrophilic and lipophilic beta-sitosterol derivatives on cholesterol absorption and plasma cholesterol levels in rats

The effects of two phytosterol derivatives of beta-sitosterol, a lipophilic derivative (LPSS) and a hydrophilic derivative (HPSS), on cholesterol uptake and blood lipoprotein levels in rats were compared with those of beta-sitosterol. LPSS and HPSS have solubilities of up to 0.05 g/mL in edible oil and 0.15 g/mL in water at 25 degrees C, respectively. The intragastric administration of either 30 or 50 mg of phytosterols with 10 mg of [4- (14)C]-cholesterol per kg of body weight once a day for 3 consecutive days reduced cholesterol uptake by approximately 30% compared to controls that received cholesterol alone. Feeding a cholesterol-enriched diet containing 1% or 3% beta-sitosterol, LPSS, or HPSS for 2 and 4 weeks resulted in lowered levels of total blood cholesterol and reduced the atherogenic index in all groups. These results indicate that LPSS and HPSS have comparable effects to beta-sitosterol in lowering blood cholesterol levels but they differ from beta-sitosterol in having a solubility advantage.     

Cultivar and year-to-year variation of phytosterol content in rye (Secale cereale L.)

Intake of phytosterols (and -stanols) has been shown to decrease the level of low-density lipoprotein cholesterol and thus protect against development of cardiovascular diseases. Therefore, studies on the cultivar and year-to-year variation in phytosterol content in rye grains have been performed. The phytosterol content and composition of different rye cultivars, grown under identical conditions on the same field in three consecutive years, were analyzed. Both cultivar and year-to-year variation in sterol content were statistically significant (p < 0.0001). The total sterol content varied from 1007 +/- 21 mg/kg in the highest yielding cultivar, Tsulpan 3, to 761 +/- 10 mg/kg in the lowest yielding cultivar (Amando in the 1999 harvest). Because the meteorological conditions varied substantially between the different years, it was possible to deduce the impact of varying weather conditions on phytosterol content in the different cultivars. The studied cultivars had all the lowest phytosterol contents in the dry and warm harvest season of 1999. Although there were statistically significant cultivar and year-to-year variations in the sterol composition (p < 0.0001), these were only between 2 and 4% of the total sterol content.     

Development of an in vitro digestion method to assess carotenoid bioavailability from meals.

The objective of this study was to develop a model for assessing the bioavailability of carotenoids from meals using an in vitro digestion procedure. A meal was prepared using baby food carrots, spinach, and a meat, plus tomato paste. The aqueous fraction was isolated from digesta to determine the quantity of carotenoids transferred from the food to micelles. The micellarization of lutein (25-40%) exceeded (p < 0.01) that of alpha- and beta-carotene (12-18%) and lycopene (<0.5%). Micellarization of carotenoids was not affected by elimination of the gastric phase of the digestive process. The absence of bile extract prevented the transfer of carotenoids from foods to micelles, whereas omission of pancreatin only reduced the micellarization of the carotenes. Differentiated cultures of Caco-2 human intestinal cells accumulated 28-46% of micellarized carotenoids from the medium after 6 h. These results support the usefulness of the in vitro digestion process as a rapid and cost-effective model for screening the bioavailability of carotenoids from meals.     

Evaluation of a commercial enzyme-based serum cholesterol test kit for analysis of phytosterol and phytostanol products

Plant sterols (phytosterols) have been shown to possess serum cholesterol-lowering properties. In recent years, several phytosterol-enriched functional food products have been developed and marketed. Some phytosterol products contain common unsaturated sterols and some contain a subset of phytosterols called phytostanols (saturated sterols, also called plant stanols). Current methods for the quantitative analysis of plant sterols are labor intensive and require sophisticated gas or liquid chromatographs. In this study, a popular commercial spectrophotometric serum cholesterol test kit was evaluated for the analysis of plant sterols. The results indicate that the method could be modified to analyze phytosterols and phytostanols by increasing the incubation time. Both free phytosterols and fatty acyl phytosteryl esters were quantitatively analyzed, but ferulate phytosteryl esters, such as those that are found in corn and other cereals, were not hydrolyzed by the enzymes in the test kit and therefore were not detected.     

Stability of sterols in phytosterol-enriched milk under different heating conditions

Commercially available phytosterol-enriched milk was subjected to usual and drastic heating conditions to evaluate the stability of the sterols at different treatments. Products showed 422.2 mg of phytosterols/100 g of milk and 132 microg of sterol oxidation products (SOPs)/g of fat (277 microg of SOPs/100 g of milk). Schaal oven conditions (24 h/65 degrees C, equivalent to 1 month of storage at room temperature) reduced the phytosterol content by only 4%. Drastic heating treatments (2 min of microwave heating at 900 W or 15 min of electrical heating at 90 degrees C) led to a 60% decrease of total phytosterol content, with a significant increase of TBARs. The oxysterol amount under those conditions (which was higher in microwave-treated samples) was lower than expected, probably because of the degradation of the oxidation products. Usual heating conditions (1.5 min of microwaves) maintained phytosterol content on physiologically active values (301 mg/100 g of milk) with oxidation percentages around 0.12-0.40% for phytosterols and 1.13% for cholesterol.     

Free and conjugated phytosterols in cured tobacco leaves: influence of genotype, growing region, and stalk position

Although phytosterols in tobacco leaves have specific effects on tobacco quality, there is little research on the distribution of free and conjugated phytosterols in various tobacco leaves. In this paper, we explored the content and composition of phytosterols in representative cured tobacco leaves by gas chromatography of TMS-ether derivatives. We found that phytosterol contents in tobacco leaves ranged from 1.0 to 2.5 mg/g of dried leaf tissue, depending on different types of tobacco leaves. The majority of phytosterols (75-85%) were conjugated as ester and glycosides, with only about 15-25% existing in the free form. Furthermore, the genetic variability gives rise to the significant differences among different tobacco types with phytosterol levels: the contents of phytosterols in tobacco leaves decreased in the order of flue-cured tobacco, Oriental tobacco, Burley tobacco, cigar tobacco, and Maryland tobacco. At the same time, the tobacco curing process leads to a difference in phytosterol existing-form distribution in some variation laws.     

Bioavailability and antioxidant effects of orange juice components in humans

Seven healthy females and six males consumed daily 256 mg of vitamin C, 271 mg of flavanones (mainly as glycosides), 6 mg of carotenoids (mainly xanthophylls and cryptoxanthins), and 0.16 mg of folate by incorporation of daily three times 236 mL of not from concentrate orange juice (OJ) into their habitual diet. At the end of 3 weeks, mean vitamin C, folate, carotenoid, and flavanone plasma concentrations increased significantly relative to baseline by 59% (p < 0.001), 46% (p = 0.018), and 22% (p < 0.001), and 8-fold (p = 0.045), respectively. Flavanones were excreted in urine 9-fold more at the end of the intervention (p = 0.01) but returned to baseline 2 days after study completion. After the 3 week intervention, plasma concentrations of vitamins A and E did not change. 8-Hydroxydeoxyguanosine in white blood cells declined by 16% (p = 0.38; n = 11), and in individuals with high baseline concentrations by 29% (p = 0.36; n = 7), respectively. Low-density lipoprotein (LDL)-/high-density lipoprotein (HDL)-cholesterol ratios decreased but cholesterol (HDL, LDL, total) and thiobarbituric acid reactive substance plasma concentrations did not change significantly. We conclude from this pilot study that OJ is an excellent food source to enhance circulating concentrations of valuable hydrophilic as well as lipophilic phytochemicals.     

Enzymatic determination of cholesterol in egg yolk

The quantitative determination of cholesterol in egg yolk by using an enzymatic test kit is described. Cholesterol in the egg yolk is extracted with other lipid components by methylene chloride-methanol (2 + 1) and is enzymatically determined after saponification of the lipid extract. The method is relatively rapid, simple, and accurate and gives results which agree with those obtained by using a gas-liquid chromatographic (GLC) method. The mean cholesterol content of egg yolk determined by the enzymatic and GLC methods was 1237 and 1240 mg/100 g, respectively.     

Spelt (Triticum spelta L.) and winter wheat (Triticum aestivum L.) wholemeals have similar sterol profiles, as determined by quantitative liquid chromatography and mass spectrometry analysis

From a nutritional point of view, cereal lipids include valuable molecules, such as essential fatty acids, phytosterols, and fat-soluble vitamins. Spelt (Triticum spelta L.) is an alternative hulled bread cereal mostly grown in Belgium, where it is mainly intended for animal feed but should increasingly be used for human consumption. The present research focused on phytosterol quantification by LC/APCI-MS2 in saponified wholemeal extracts of 16 dehulled spelt and 5 winter wheat (Triticum aestivum L.) varieties grown in Belgium during 2001-2002 at the same location. Glycosylated sterols and free and formerly esterified sterols could be determined in saponified extracts. Results show that the mean phytosterol content is comparable in both cereals (whereas other lipids, such as oleic and linoleic acids, are increased in spelt wholemeal): spelt extract has, on average, 527.7 microg of free and esterified sterols g(-1) of wholemeal and 123.8 microg of glycosylated sterols g(-1) of wholemeal versus 528.5 and 112.6 microg x g(-1) in winter wheat (values not corrected for recoveries). This is the first report on the application and validation of an LC/MS2 method for the quantification of phytosterols in spelt and winter wheat.     

Phytosterol and tocopherol components in extracts of corn distiller's dried grain

As the ethanol industry continues to grow, it will become very important to develop value-added markets for its coproducts in order for the industry to remain profitable. Corn distiller's dried grain (DDG) is a major coproduct of ethanol fermentation from corn processed by dry-milling and is primarily sold as livestock feed. The objective of this research was to determine if valuable phytochemicals found in corn oil and corn fiber oil, such as phytosterols and their saturated equivalents, phytostanols, ferulate phytosterol esters (FPE), tocopherols, and tocotrienols, are retained in DDG. Hexane and supercritical carbon dioxide (CO2) extracts of DDG were similar in their concentrations of total phytosterols (15.8-17.3 mg/g of extract), FPE (3.75-3.99 mg/g of extract), and tocols (1.7-1.8 mg/g of extract). Ethanol extracts were slightly lower in concentration of phytosterols (8.9-11.4 mg/g of extract), FPE (1.62-1.98 mg/g of extract), and tocols (0.73-0.76 mg/g of extract).     

Impact of fatty acyl composition and quantity of triglycerides on bioaccessibility of dietary carotenoids

A carotenoid-rich salad meal with varying amounts and types of triglycerides (TG) was digested using simulated gastric and small intestinal conditions. Xanthophylls (lutein and zeaxanthin) and carotenes (alpha-carotene, beta-carotene, and lycopene) in chyme and micelle fraction were quantified to determine digestive stability and efficiency of micellarization (bioaccessibility). Micellarization of lutein (+zeaxanthin) exceeded that of alpha- and beta-carotenes, which was greater than that of lycopene for all test conditions. Micellarization of carotenes, but not lutein (+zeaxanthin), was enhanced (P < 0.05) by addition of TG (2.5% v/w) to the meal and was dependent on fatty acyl chain length in structured TG (c18:1 > c8:0 > c4:0). The degree of unsaturation of c18 fatty acyl chains in TG added to the salad purée did not significantly alter the efficiency of micellarization of carotenoids. Relatively low amounts of triolein and canola oil (0.5-1%) were required for maximum micellarization of carotenes, but more oil (approximately 2.5%) was required when TG with medium chain saturated fatty acyl groups (e.g., trioctanoin and coconut oil) was added to the salad. Uptake of lutein and beta-carotene by Caco-2 cells also was examined by exposing cells to micelles generated during the simulated digestion of salad purée with either triolein or trioctanoin. Cell accumulation of beta-carotene was independent of fatty acyl composition of micelles, whereas lutein uptake was slightly, but significantly, increased from samples with digested triolein compared to trioctanoin. The results show that the in vitro transfer of alpha-carotene, beta-carotene, and lycopene from chyme to mixed micelles during digestion requires minimal (0.5-1%) lipid content in the meal and is affected by the length of fatty acyl chains but not the degree of unsaturation in TG. In contrast, fatty acyl chain length has limited if any impact on carotenoid uptake by small intestinal epithelial cells. These data suggest that the amount of TG in a typical meal does not limit the bioaccessibility of carotenoids.     

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.     

Digestive Stability, micellarization, and uptake of beta-carotene isomers by Caco-2 human intestinal cells

While isomeric profiles of carotenoids found in food often differ from those in body fluids and tissues, insights about the basis for these differences remain limited. We investigated the digestive stability, relative efficiency of micellarization, and cellular accumulation of trans and cis isomers of beta-carotene (BC) using an in vitro digestion procedure coupled with human intestinal (Caco-2) cells. A meal containing applesauce, corn oil, and either water-soluble beadlets (WSB) or Dunaliella salina (DS) as a BC source was subjected to simulated gastric and small intestinal digestion. BC isomers were stable during digestion, and the efficiency of micellarization of cis-BC isomers exceeded that of all-trans-BC isomers. The cellular profile of carotenoids generally reflected that in micelles generated during digestion, and intracellular isomerization was minimal. These data suggest that cis isomers of BC are preferentially micellarized during digestion and transferred across the brush-border surface of the enterocyte from mixed micelles with similar efficiency as all-trans-BC at the concentrations of the carotenoids utilized in this study.     

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.     

Yield and composition of grape seed oils extracted by supercritical carbon dioxide and petroleum ether: varietal effects

Grape seed has a well-known potential for production of oil as a byproduct of winemaking and is currently produced as a specialty oil byproduct of wine manufacture. Seed oils from eight varieties of grapes crushed for wine production in British Columbia were extracted by supercritical carbon dioxide (SCE) and petroleum ether (PE). Oil yields by SCE ranged from 5.85 +/- 0.33 to 13.6 +/- 0.46% (w/w), whereas PE yields ranged from 6.64 +/- 0.16 to 11.17 +/- 0.05% (+/- is standard deviation). The oils contained alpha-, beta-, and gamma-tocopherols and alpha- and gamma-tocotrienols, with gamma-tocotrienol being most important quantitatively. In both SCE- and PE-extracted oils, phytosterols were a prominent feature of the unsaponifiable fraction, with beta-sitosterol quantitatively most important with both extractants. Total phytosterol extraction was higher with SCE than with PE in seven of eight variety extractions. Fatty acid composition of oils from all varieties tested, and from both extraction methods, indicated linoleic acid as the major component ranging from 67.56 to 73.23% of the fatty acids present, in agreement with literature reports.     

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.     

Effect of red mold rice supplements on serum and egg yolk cholesterol levels of laying hens

Monacolin K is a secondary metabolite of Monascus species and reduces cholesterol levels. This research focuses on the effect of adding red mold rice to hens' diet on cholesterol level in egg yolk and on cholesterol, triglyceride, high-density lipoprotein (HDL), and low-density lipoprotein (LDL) levels in serum. Forty-eight Hy-line laying hens of 48 weeks of age were studied by dividing them into four groups. Except for the control group, the feed for three other groups contained 2.0, 5.0, and 8.0% red mold rice (monacolin K concentrations were 0.0145, 0.035, and 0.056%, respectively). The experiment lasted 6 weeks. During this period, egg weight and egg production were recorded every day, and cholesterol, triglyceride, HDL, and LDL in serum were measured weekly as well. The result showed that the cholesterol in eggs produced by experimental groups was lower than that of the control group (0%, 194.14 +/- 8.30; 2%, 167.17 +/- 4.34; 5%, 168.93 +/- 9.38; 8%, 183.02 +/- 7.63 mg/egg; p < 0.05), and the triglyceride (0%, 1494 +/- 178; 2%, 1280 +/- 174; 5%, 1189 +/- 248; 8%, 1381 +/- 218 mg/dL; p < 0.05) and LDL levels (0%, 36.81 +/- 5.53; 2%, 32.25 +/- 7.93; 5%, 30.06 +/- 4.39; 8%, 28.81 +/- 4.16 mg/dL; p < 0.05) were also significantly lowered in the experimental groups. However, the HDL level did not show significant change for either control or experimental groups (0%, 36.06 +/- 3.96; 2%, 36.25 +/- 5.39; 5%, 33.13 +/- 3.68; 8%, 31.44 +/- 4.29 mg/dL; p > 0.05). Besides, the addition of red mold rice also helps to inhibit production of malondialdehyde (MDA) in serum lipid oxidation (0%, 27.42 +/- 0.53; 2%, 25.62 +/- 0.76; 5%, 24.35 +/- 0.59; 8%, 23.63 +/- 0.48 microM; p < 0.05).     

Self-diffusion nuclear magnetic resonance,microstructure transitions,and solubilization capacity of phytosterols and cholesterol in Winsor IV food-grade microemulsions

Microemulsions are of growing interest to the food industry as vehicles for delivering and enhancing solubilization of natural food supplements with nutritional and health benefits. The incorporation of molecular phytosterols, cholesterol-lowering agents, in food products is of great interest to the food industry. In this work is demonstrated the use of water dilutable food-grade microemulsions consisting of ethoxylated sorbitan ester (Tween 60), water, R-(+)-limonene, ethanol, and propylene glycol as vehicles for enhancing the phytosterols solubilization. Phytosterols were solubilized up to 12 times more than the dissolution capacity of the oil [R-(+)-limonene] for the same compounds. The solubilization capacity of phytosterols and cholesterol along a dilution line in a pseudo-ternary phase diagram [on this dilution line the weight ratio of R-(+)-limonene/ethanol/Tween 60 is constant at 1:1:3] was correlated to the microstructure transitions along the dilution line. Structural aspects were studied by self-diffusion NMR spectroscopy. The ability of phytosterols to compete with cholesterol for penetration into bile salt micelles in the gut may be limited to rich aqueous systems (O/W microemulsion).     

Phytosterols in wheat genotypes in the HEALTHGRAIN Diversity Screen

The phytosterol contents of 130 winter wheat, 20 spring wheat, 10 durum wheat, 5 spelt, 5 einkorn, and 5 emmer wheat genotypes, grown at the same location in the same year, were analyzed with gas chromatography. Considerable variation was observed in total phytosterol contents in all wheat types. The total sterol contents ranged from 670 to 959 microg/g of dm in winter wheat and from 797 to 949 microg/g of dm in spring wheat. The highest sterol contents were found in spelt, durum wheat, and einkorn wheat. The proportions of the main phytosterols also varied substantially among the different genotypes. The most abundant phytosterol in all wheat genotypes was sitosterol (40-61% of total sterols), whereas the highest variation was seen in total stanols (7-31% of total sterols). The comprehensive data set produced in this study constitutes a valuable basis for plant breeding and selection of phytosterol-rich genotypes.     

Functional lipid characteristics of Turkish Tombul hazelnut (Corylus avellana L.)

The quality of crude oil extracted from Tombul (Round) hazelnut, grown in the Giresun province of Turkey, was evaluated for its fatty acid, triacylglycerol (TAG), tocol, and phytosterol compositions. Oleic acid contributed 82.78% to the total fatty acids, followed by linoleic, palmitic, and stearic acids. Among 12 TAGs separated, 11 were identified (including one unknown): LLL, OLL, PLL, OOL, POL, PPL, OOO, POO, PPO, SOO, and PSO (where P, palmitoyl; S, stearoyl; O, oleoyl; and L, linoleoyl). The main components were OOO (71.31%), OOL (12.26%), and POO (9.45%), reflecting the high content of oleic acid present in hazelnut oil. Seven tocol isoforms (four tocopherols and three tocotrienols) and eight phytosterols as well as cholesterol were positively identified and quantified; among these, alpha-tocopherol (40.40 mg/100 g) and beta-sitosterol (134.05 mg/100 g) were predominant in hazelnut oil and contributed 78.74 and 81.28% to the total tocols and phytosterols present, respectively. Tocotrienols were detected in small amounts (1.02% to the total tocols). The crude hazelnut oil extracted from Turkish Tombul hazelnut, thus, serves as a good source of nutrients, bioactives, and health-promoting components.