Steryl phenolic acid esters in cereals and their milling fractions

The steryl ferulate contents of rye and wheat grains and their milling fractions were analyzed using a reversed-phase high-performance liquid chromatographic (HPLC) method. HPLC-mass spectrometry was used for identification. In addition, steryl ferulates of some selected milling byproducts were determined. The total steryl ferulate contents of rye and wheat grains were 6.0 and 6.3 mg/100 g, respectively. Uneven distribution of steryl ferulates in the grains led to considerable differences in the milling products; their steryl ferulate contents ranged from trace amounts in flours with low ash content to 20 and 34 mg/100 g in rye and wheat brans, respectively. Campestanyl ferulate and sitostanyl ferulate were the main components, followed by campesteryl ferulate and sitosteryl ferulate, whereas sitosterol was the main component in total sterols. Among the other samples, a byproduct of rice milling (pearling dust) was the best source of steryl ferulates, its total steryl ferulate content being 119 mg/100 g, whereas no measurable amounts of steryl ferulates were measured in oat bran or pearling dust of barley. The results indicated that rye and wheat and especially their bran fractions are comparable to corn as steryl ferulate sources.     

Plant Sterols in Cereals and Cereal Products

The total plant sterol contents (free sterols and covalently bound structures) of the main cereals cultivated in Finland were determined. Furthermore, sterol contents were determined for different flour and bran fractions in the milling process of wheat and rye, as well as plant sterol contents in various milling and retail bakery products. The sample preparation procedure included acid and alkaline hydrolysis to liberate sterols from their glycosides and esters, respectively. Free sterols were extracted and, after recovery using solid‐phase extraction, derivatized to trimethylsilyl ethers for gas chromatography (GC) analysis. We used GC with a mass spectrometer (MS) for identification. When two cultivars of rye, wheat, barley, and oats grown in the same year were compared, the highest plant sterol content was observed in rye (mean content 95.5 mg/100 g, wb), whereas the total sterol contents (mg/100 g, wb) of wheat, barley, and oats were 69.0, 76.1, and 44.7, respectively. In addition, the 10 rye cultivars and breeding lines compared had total sterol contents of 70.7–85.6 mg/100 g. In the milling process of rye and wheat, the plant sterols fractionated according to the ash content of the corresponding milling product. In all cereal grain and milling product samples, sitosterol was the main sterol. The level of stanols differed in the different milling process samples; it was lower in the most refined rye and wheat flours (≈15%) than in the bran fractions (≈30% in the bran with 4% ash content). Rye bread with whole meal rye flour as the main or only ingredient was a good source of sterols. Sterol content was higher than that of wheat bread, whereas plant sterol content of other bakery products was affected by the type and amount of fat used in baking.     

Triterpene alcohol and sterol ferulates from rice bran and their anti-inflammatory effects

Six novel feruloyl esters of triterpene alcohols and sterols, viz., two trans-ferulates, cycloeucalenol and 24-methylenecholesterol trans-ferulates, and four cis-ferulates, cycloartenol, 24-methyelenecycloartanol, 24-methylcholesterol, and sitosterol cis-ferulates, besides five known trans-ferulates, cycloartenol (CAR), 24-methylenecycloartanol (24-MCA), 24-methylcholesterol, sitosterol, and stigmastanol trans-ferulates, and one known cis-ferulate, stigmastanol cis-ferulate, were isolated from the methanol extract of edible rice bran. These and eight other synthetic trans- and cis-ferulates of triterpene alcohols and sterols, along with the corresponding free alcohols, were evaluated with respect to their anti-inflammatory activity against 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation (1 microg per ear) in mice. All of the ferulates showed marked inhibitory activity, and their 50% inhibitory dose (ID(50)) was 0. 1-0.8 mg per ear. On the other hand, whereas two free triterpene alcohols, CAR and 24-MCA, showed strong inhibition (ID(50) 0.2-0.3 mg/ear), eight free sterols examined showed weaker activity (ID(50) 0.7-2.7 mg/ear) than their corresponding ferulates.     

Antioxidant activity of tocopherols, tocotrienols, and gamma-oryzanol components from rice bran against cholesterol oxidation accelerated by 2,2'-azobis(2-methylpropionamidine) dihydrochloride

The antioxidant activities of vitamin E (alpha-tocopherol, alpha-tocotrienol, gamma-tocopherol, and gamma-tocotrienol) and gamma-oryzanol components (cycloartenyl ferulate, 24-methylenecycloartanyl ferulate, and campesteryl ferulate) purified from rice bran were investigated in a cholesterol oxidation system accelerated by 2,2'-azobis(2-methylpropionamidine) dihydrochloride. All components exhibited significant antioxidant activity in the inhibition of cholesterol oxidation. The highest antioxidant activity was found for 24-methylenecycloartanyl ferulate, and all three gamma-oryzanol components had activities higher than that of any of the four vitamin E components. Because the quantity of gamma-oryzanol is up to 10 times higher than that of vitamin E in rice bran, gamma-oryzanol may be a more important antioxidant of rice bran in the reduction of cholesterol oxidation than vitamin E, which has been considered to be the major antioxidant in rice bran. The antioxidant function of these components against cholesterol oxidation may contribute to the potential hypocholesterolemic property of rice bran.     

Isolation,structural elucidation,and inhibitory effects of terpenoid and lipid constituents from sunflower pollen on Epstein-Barr virus early antigen induced by tumor promoter,TPA

Eight fatty acid esters of triterpene alcohols (1-8), four free triterpene alcohols (9, 12, 17, and 18), four diterpene acids (19-22), two tocopherol-related compounds (23 and 24), four estolides (25-28), three syn-alkane-4,6-diols (29-31), one 1,3-dioxoalkanoic acid (32), and one aliphatic ketone (33), along with the mixture of free fatty acids, were isolated from the diethyl ether extract of the pollen grains of sunflower (Helianthus annuus). Among these compounds, 14 (2-8, 12, 23, 25-28, and 33) were new naturally occurring compounds, and their structures were determined on the basis of spectroscopic methods. Twenty-four terpenoids and lipids (1-4, 6-9, 12, and 19-33) and six free triterpene triols (10, 11, and 13-16), derived from their fatty acid esters (2, 3, and 5-8) by alkaline hydrolysis, were evaluated with respect to their inhibitory effects on the induction of Epstein-Barr virus early antigen (EBV-EA) induced by the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), in Raji cells, which is known to be a primary screening test for antitumor promoters. Among the 30 compounds tested, 21 compounds possessing a di- or a polycyclic ring system in the molecule (1-4, 6-16, and 19-24) showed potent inhibitory effects on EBV-EA induction (91-100% inhibition at 1 x 10(3) mol ratio/TPA).     

Characterization of triterpene alcohol and sterol ferulates in rice bran using LC-MS/MS

Ferulic acid esters of triterpene alcohols and sterols in rice bran oil have been extensively studied and reported to possess important pharmacological actions. Inconsistent results on the numbers and structures of ferulates have been reported, primarily because of the analytical procedures employed. Conventional methods for analysis of phytosterol content in oil are carried out by characterization of trimethylsilylated derivatives (TMS) using GC-EI-MS after saponification of oils or individual compound isolated from oils. This study developed an LC-MS/MS method for the direct analysis of triterpene alcohol and sterol esters in rice bran oil. In addition to verifying the results of previous research, nine new relatively polar triterpene alcohol and sterol esters were characterized by their retention behaviors in LC and ESI-MS data from both negative- and positive-ion mode. This is the first evidence for the presence of hydroxylated ferulate esters and caffeate esters as part of gamma-oryzanol in rice bran. The method enables rapid and direct on-line characterization of triterpene alcohol and sterol esters in oils. LC-MS/MS equipped with reverse-phase LC and ESI-MS should be well-suited for identification and quantification of the polar metabolites of phytosterols in biological fluids after consumption of rice bran oil or other oils.     

Effects of processing on availability of total plant sterols, steryl ferulates and steryl glycosides from wheat and rye bran

Rye and wheat bran are excellent natural sources of plant sterols in the diet. Their content, however, may vary according to processing. Thermal (roasting and heating in a microwave oven), mechanical (milling and cryogenic grinding), and enzymatic treatments (hydrolysis with xylanase or beta-glucanase or a mixture of these two enzymes) were performed, and their effect on sterol content, extractability of sterols and the characteristic steryl conjugates of cereals (steryl ferulates, steryl glycosides, and acylated steryl glycosides) were studied. Mechanical and enzymatic treatments increased the apparent sterol content, whereas aqueous processing without enzymes hindered the availability of total sterols, especially from rye bran. Changes were also seen in the amounts of steryl conjugates caused by the enzymatic treatments. On the basis of the results of this study, it can be speculated that a combination of fine particle size and enzymatic processing results in optimal sterol availability in cereal processing.     

Purification and identification of components of gamma-oryzanol in rice bran Oil.

High-purity gamma-oryzanol was obtained from crude rice bran oil using a normal-phase preparative scale HPLC. A reverse-phase HPLC method was used for separating the individual components of gamma-oryzanol present in rice bran oil. Ten fractions were isolated and collected using the reverse-phase HPLC method, and their structures were identified. Identification was accomplished using GC/MS with an electron impact mass spectrum after components were transformed into trimethylsilyl ether derivatives. The 10 components of gamma-oryzanol were identified as Delta(7)-stigmastenyl ferulate, stigmasteryl ferulate, cycloartenyl ferulate, 24-methylenecycloartanyl ferulate, Delta(7)-campestenyl ferulate, campesteryl ferulate, Delta(7)-sitostenyl ferulate, sitosteryl ferulate, compestanyl ferulate, and sitostanyl ferulate. Three of these, cycloartenyl ferulate, 24-methylenecycloartanyl ferulate, and campesteryl ferulate, were major components of gamma-oryzanol.     

Inhibitory effect of natural phenolic lipids upon NAD-dependent dehydrogenases and on triglyceride accumulation in 3T3-L1 cells in culture

Alkylresorcinols are phenolic lipids present at levels of 0.03-0.15% in wheat and rye grains and almost 10 times higher in respective bran products. Despite numerous studies on the influence of dietary fibers on the regulation of energy metabolism, this issue still remains controversial. The objective of our current studies was to investigate whether 5-n-alk(en)ylresorcinols, natural phenolic components of high fiber human diets, may be considered as natural regulators of excessive fat accumulation. Our studies revealed that 5-n-alk(en)ylresorcinols isolated from wheat and rye bran inhibit glycerol-3-phosphate dehydrogenase, the key enzyme in triglyceride synthesis in adipocytes, specifically and effectively. Further in vitro studies showed that these compounds also prevent triglyceride accumulation in 3T3-L1 cells. The most effective compound in both systems was 5-n-heneicosylresorcinol. The results indicate that the potential to prevent triglyceride accumulation increases with the hydrophobicity of the phenolic inhibitor.     

The effect of in vitro digestion on steryl ferulates from rice (Oryza sativa L.) and other grains

Polished and cargo rice, wild rice, rice bran, corn bran, and wheat bran were subjected to a static in vitro digestion model, to monitor changes in their steryl ferulate content and composition. Free sterols, possible hydrolysis products of steryl ferulates, were also measured. Additionally, steryl ferulate bioaccessibility was calculated as the percentage of steryl ferulates liberated from the grain matrix into the digestive juice. Steryl ferulate content ranged between 6.1 and 3900 μg/g and decreased by 1-63% due to digestion. A parallel increase in free sterols of more than 70% was observed in all samples. Additionally, bioaccessibility of steryl ferulates was found to be almost negligible. These findings suggest that intestinal enzymes immediately hydrolyze steryl ferulates, which are liberated from the grain matrix, and thus they are practically unavailable for absorption in the small intestine. This further indicates that the hydrolysis products of steryl ferulates could be bioactive in the gut.     

Azaphilones, furanoisophthalides, and amino acids from the extracts of Monascus pilosus-fermented rice (red-mold rice) and their chemopreventive effects

Six azaphilones, monascin (1), ankaflavin (2), rubropunctatin (3), monascorburin (4), rubropunctamine (5), and monascorburamine (6), two furanoisophthalides, xanthomonasin A (7) and xanthomonasin B (8), and two amino acids, (+)-monascumic acid (9) and (-)-monascumic acid (10), isolated from the extracts of Monascus pilosus-fermented rice (red-mold rice) were evaluated for their inhibitory effects on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation in mice, on the induction of Epstein-Barr virus early antigen (EBV-EA) by TPA in Raji cells, and on the activation of (+/-)-(E)-methyl-2[(E)-hydroxy-imino]-5-nitro-6-methoxy-3-hexemide (NOR 1), a nitric oxide (NO) donor. Among the compounds tested, seven compounds (1-6 and 10) on TPA-induced inflammation, and six compounds (1, 3-5, 9, and 10) on EBV-EA activation, exhibited potent inhibitory effects. All of the compounds tested showed moderate inhibitory effects on NOR 1 activation.     

Comparison of oil and phytosterol levels in germplasm accessions of corn, teosinte, and Job's tears

Seeds of 49 accessions of corn (Zea mays ssp. mays), 9 accessions of teosinte (Zea species that are thought to be ancestors and probable progenitors to corn), and 3 accessions of Job's tears (Coix lacryma), obtained from a germplasm repository, were ground and extracted with hexane. Whole kernel oil yields and levels of four phytonutrients (free phytosterols, fatty acyl phytosterol esters, ferulate phytosterol esters, and gamma-tocopherol) in the oils were measured. Among the seeds tested, oil yields ranged from 2.19 to 4.83 wt %, the levels of ferulate phytosterol esters in the oil ranged from 0.047 to 0.839 wt %, the levels of free phytosterols in the oil ranged from 0.54 to 1.28 wt %, the levels of phytosterol fatty acyl esters in the oil ranged from 0.76 to 3.09 wt %, the levels of total phytosterols in the oil ranged from 1.40 to 4.38 wt %, and the levels of gamma-tocopherol in the oil ranged from 0.023 to 0.127 wt %. In general, higher levels of all three phytosterol classes were observed in seed oils from accessions of Zea mays ssp. mays than in seed oils from accessions of the other taxonomic groups. The highest levels of gamma-tocopherol were observed in teosinte accessions.     

Enzymatic solubilization of arabinoxylans from native, extruded, and high-shear-treated rye bran by different endo-xylanases and other hydrolyzing enzymes

The overall objective of this research was to find a new way to valorize rye bran, by producing a gellifier from the enzymatic solubilization of arabinoxylans (AX). The effects of three pure endo-xylanases from Aspergillus niger (Xyl-1), Talaromyces emersonii (Xyl-2), and Bacillus subtilis (Xyl-3) and of Grindamyl S100 (GS100), a commercial enzyme preparation containing a Xyl-1 type endo-xylanase, were tested on rye bran to study the solubilization of water-unextractable arabinoxylans (WUAX). Eight different extrusion-treated rye brans were also used as substrates to find the best physical treatment to facilitate enzymatic arabinoxylan (AX) solubilization. Arabinoxylans were better solubilized from the bran extruded at high temperature using Xyl-3. This enzyme was then tested in combination with pure (1,4)-beta-d-arabinoxylan arabinofuranohydrolase (AXH) and endo-beta-d-glucanase or ferulic acid esterase (FAE), from A. niger. Only beta-glucanase in combination with Xyl-3 improved the AX extraction, but it did not have a marked effect on the viscosity of the extracts. Xyl-3 was then tested on a high-shear-treated rye bran, and results were compared to those obtained with the high-temperature-extruded rye bran. The high-shear treatment did not improve the bran AX enzymatic solubilization. The combination of FAE with Xyl-1 or Xyl-3 did not improve the AX extraction from untreated and high-shear-treated rye bran. Finally, to study the gelation capacity of the enzymatically solubilized AX, the effect of the hydrogen peroxide/horseradish peroxidase (H(2)O(2)/POD) was tested on the Xyl-3 high-temperature-extruded bran extracts. Solubilized AX did not gel in the presence of the oxidizing system.     

Formation of phenolic microbial metabolites and short-chain Fatty acids from rye, wheat, and oat bran and their fractions in the metabolical in vitro colon model

Rye bran and aleurone, wheat bran and aleurone, and oat bran and cell wall concentrate were compared in their in vitro gut fermentation patterns of individual phenolic acids and short-chain fatty acids, preceded by enzymatic in vitro digestion mimicking small intestinal events. The formation of phenolic metabolites was the most pronounced from the wheat aleurone fraction. Phenylpropionic acids, presumably derived from ferulic acid (FA), were the major phenyl metabolites formed from all bran preparations. The processed rye, wheat, and oat bran fractions contained more water-extractable dietary fiber (DF) and had smaller particle sizes and were thus more easily fermentable than the corresponding brans. Rye aleurone and bran had the highest fermentation rate and extent probably due to high fructan and water-extractable arabinoxylan content. Oat samples also had a high content of water-extractable DF, β-glucan, but their fermentation rate was lower. Enzymatic digestion prior to in vitro colon fermentation changed the structure of oat cell walls as visualized by microscopy and increased the particle size, which is suggested to have retarded the fermentability of oat samples. Wheat bran was the most slowly fermentable among the studied samples, presumably due to the high proportion of water-unextractable DF. The in vitro digestion reduced the fructan content of wheat samples, thus also decreasing their fermentability. Among the studied short-chain fatty acids, acetate dominated the profiles. The highest and lowest production of propionate was from the oat and wheat samples, respectively. Interestingly, wheat aleurone generated similar amounts of butyrate as the rye fractions even without rapid gas production.     

Alkylresorcinols in cereals and cereal products

The alkylresorcinol (AR) content of 8 commonly consumed cereals, 125 Triticum cultivars, milling fractions of wheat and rye, bread, and other cereal products was analyzed. ARs were found in wheat (489-1429 microgram/g), rye (720-761 microgram/g), triticale (439-647 microgram/g), and barley (42-51 microgram/g), but not in rice, oats, maize, sorghum, or millet. One durum wheat variety was found to have an exceptionally low level of ARs (54 microgram/g) compared to other durum wheat varieties (589-751 microgram/g) and Triticumspecies analyzed. The AR content of milling fractions closely followed the ash content and could be used as a marker of the presence of bran in flour. Using hot 1-propanol extraction, all ARs could be extracted from bread, contrary to previous studies which suggested that ARs were destroyed during baking. Cereal products varied greatly in AR content, with those containing wheat bran or whole rye having the highest content.     

Alkylresorcinols in selected Polish rye and wheat cereals and whole-grain cereal products

The alkylresorcinol content and homologue composition in selected Polish rye and wheat cultivars and selected whole-grain cereal products were determined in this study. Cereal grains and whole-grain cereal products were extracted with acetone, whereas bread types were extracted with hot 1-propanol. The average alkylresorcinol content in tested rye (approximately 1100 mg/kg DM) and wheat (approximately 800 mg/kg DM) grains harvested in Poland was within the range previously reported in Swedish and Finnish samples. The total alkylresorcinol content in tested cereal products available on the Polish market varied from very low levels in barley grain-based foods up to 3000 mg/kg DM in wheat bran. The total alkylresorcinol content in 14 bread samples extracted with hot 1-propanol varied from approximately 100 mg/kg DM in whole bread made with honey up to approximately 650 mg/kg DM in whole-rye bread. Calculated ratios of C17:0 to C21:0 homologues, a useful parameter previously used to distinguish between rye and wheat cereals and their derived products, was about 1.2-1.4 in rye products, about 0.2 in wheat products, and varied between 0.2 and 0.6 in cereal-derived products containing a mixture of whole rye and/or wheat. The data set obtained were subsequently compared using cluster and principal component analysis, which allowed the tested cereal products to be classified into two major groups consisting of whole-rye or whole-wheat products, respectively. On the basis of that approach, mixed cereal products containing rye and wheat bran or whole rye and wheat flour were grouped between those two well-defined clusters. Our work not only provides a detailed examination of alkylresorcinols in selected Polish rye and wheat cultivars and selected whole-grain cereal products, but also demonstrates that this type of analysis accompanied by the use of proper statistical algorithms offers an objective way to evaluate the quality of whole-grain rye and/or wheat and their derived products.     

Quantification of a broad spectrum of lignans in cereals, oilseeds, and nuts

Twenty-four plant lignans were analyzed by high-performance liquid chromatography-tandem mass spectrometry in bran extracts of 16 cereal species, in four nut species, and in two oilseed species (sesame seeds and linseeds). Eighteen of these were lignans previously unidentified in these species, and of these, 16 were identified in the analyzed samples. Four different extraction methods were applied as follows: alkaline extraction, mild acid extraction, a combination of alkaline and mild acid extraction, or accelerated solvent extraction. The extraction method was of great importance for the lignan yield. 7-Hydroxymatairesinol, which has not previously been detected in cereals because of destructive extraction methods, was the dominant lignan in wheat, triticale, oat, barley, millet, corn bran, and amaranth whole grain. Syringaresinol was the other dominant cereal lignan. Wheat and rye bran had the highest lignan content of all cereals; however, linseeds and sesame seeds were by far the most lignan-rich of the studied species.     

Phytosterol composition of hybrid Hibiscus seed oils

The seed oils from fifteen hybrid Hibiscus varieties were analyzed for desmethyl sterol content to identify bioactive compounds that could promote the use of these oils for edible applications. Hibiscusis being developed as a new crop with edible and nutraceutical applications for the component tissues and tissue extracts. Previously, hybrid varieties were developed for ornamental purposes on the basis of flower morphology and color. Currently, the effects of selective breeding on seed oil components are of interest as these represent potential natural products with bioactive properties. In the present study, sterol structures were identified as the corresponding trimethyl silyl ether derivatives obtained from the unsaponifiable fraction of the seed oils. This material contained an average of 32 wt % sterols and exhibited a relative composition of sitosterol, 76.3%; campesterol, 10.3%; stigmasterol, 7.3%; 5-avenasterol, 4.4%; and cholesterol, 0.6%. The content of 5-avenasterol showed statistically significant variation among the hybrid varieties with a range of 1.2-5.8%.     

Effect of Extrusion on Hypocholesterolemic Properties of Rice,Oat, Corn,and Wheat Bran Diets in Hamsters

Brans from rice, oats, corn, and wheat were cooked in a twin-screw extruder at either high or low energy input, and their cholesterol-lowering effects were compared with those of unprocessed brans when fed to four-week-old male golden Syrian hamsters (n = 10 per treatment) for three weeks. Peanut oil was added to oat, corn, and wheat bran during the extrusion process to match the oil content of rice bran. Diets contained 10% total dietary fiber, 10.3% fat, 3% nitrogen, and 0.3% cholesterol. Plasma and liver cholesterol and total liver lipids were significantly lower with low-energy extruded wheat bran compared with unprocessed wheat bran. Extrusion did not alter the hypocholesterolemic effects of rice, oat, or corn brans. Plasma and liver cholesterol levels with corn bran were similar to those with oat bran. Relative cholesterol-lowering effects of the brans, determined with pooled (extruded and unextruded) bran data, were rice bran > oat bran > corn bran > wheat bran. Rice bran diets resulted in significantly lower levels of total plasma cholesterol and very low density lipoprotein cholesterol compared with all other brans. Total liver cholesterol and liver cholesterol concentrations (mg/g) were significantly lower with high-energy extruded rice bran compared with the cellulose control group. Plasma cholesterol and total liver cholesterol values with low-energy extruded wheat bran were similar to those with rice bran (unextruded or extruded) diets. Lowered cholesterol with rice bran diets may result in part from greater lipid and sterol excretion with these diets. Results with low-energy extruded wheat bran suggest that this type of processing may improve the potential for lowering cholesterol with wheat bran products.     

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.