Free and Esterified 4,4′-dimethylsterols in Hazelnut Oil and their Retention During Refining Processes

Free and esterified forms of sterols provide detailed information on the identity and the quality of vegetable oils. In this study, 4,4′-dimethylsterols in free and esterified forms were investigated in hazelnut and virgin olive oils. Moreover, a sample of solvent-extracted hazelnut oil was refined at the laboratory to monitor the effects of processing on the levels of 4,4′-dimethylsterols. Generally, the level of total 4,4′-dimethyslterols was higher in the esterified form (49–68%) compared with that in free form (32–51%) of these compounds in the hazelnut oil. In virgin olive oil samples, cycloartenol and 24-methylenecycloartanol were present in higher amounts in free forms (70–80%) than in esterified forms (20–30%). Among the refining processes, degumming, deodorization, neutralization and bleaching, only neutralization and bleaching considerably reduced 4,4′-dimethylsterols. In fully refined hazelnut oil, 18 and 37% of lupeol and an unknown compound X in the esterified form were lost, respectively. The loss of these two compounds in the free form was considerably higher, 26 and 72%, respectively. GC–MS analysis showed that adulteration of olive oil with a sample of fully refined hazelnut oil could be detected at a level as low as 2% by tracing lupeol in total or only in esterified forms of 4,4′-dimethylsterols. Further studies on the levels of free and esterified 4,4′-dimethylsterols and their retention during refining processes are anticipated in hazelnut cultivars from different origins.     

Sterols,methylsterols, and triterpene alcohols in threeTheaceae and some other vegetable oils

The unsaponifiables from threeTheaceae (Camellia japonica L.,Camellia Sasanqua Thunb., andThea sinensis L.) oils and alfalfa, garden balsam, and spinach seed oils and shea fat were separated into four fractions: sterols, 4-methylsterols, triterpene alcohols, and less polar compounds by thin layer chromatography. While the sterol fraction was the major one for the unsaponifiables from alfalfa and spinach seed oils, the triterpene alcohol fraction was predominant for the unsaponifiables from all other oils. The sterol, 4-methylsterol, and triterpene alcohol fractions were analyzed by gas chromatography. All the sterol fractions were alike in their compositions, consisting exclusively of Δ⁷-sterols, such as α-spinasterol and Δ⁷-stigmastenol as predominant components together with Δ⁷-avenasterol and 24-methylcholest-7-enol. Obtusifoliol, gramisterol (occasionally accompanied with cycloeucalenol), and citrostadienol, together with several other unidentified components, were found in the 4-methylsterol fractions from all of the oils except shea fat. The 4-methylsterol fraction from shea fat showed a characteristic composition containing a large proportion of unidentified components which had relative retention time greater than that of citrostadienol, while no citrostadienol was detected. β-Amyrin, lupeol, and butyospermol were major components of the triterpene alcohol fractions from most of the oils, but the fraction from spinach seed oil contained cycloartenol and 24-methylene-cycloartanol as predominant components. There is a close similarity in the compositions of unsaponifiables (sterols, 4-methylsterols, and triterpene alcohols) of the threeTheaceae oils. Two sterols, α-spinasterol and Δ⁷-stigmastenol, and five triterpene alcohols were isolated from tea seed oil. Moreover, five unidentified components beside parkeol, butyrospermol, α-amyrin, and lupeol were isolated from the triterpene alcohol fraction of shea fat.     

Free,esterified and glucosidic sterols in cocoa butter

Sterol lipids of cocoa butter (cocoa beansLome Tongo) were fractionated into free sterols, steryl esters (SE), steryl glucosides and acylated steryl glucosides (ASG). 4-Desmethyl, 4-methyl and 4,4′-dimethyl sterols or triterpene alcohols, which were isolated as free sterols or which resulted from hydrolysis, were determined by thin layer chromatography-flame ionization detection and identified by gas chromatography and combined gas chromatography-mass spectroscopy. Free sterols comprise the main sterol fraction in cocoa butter. Esterified sterols amount to 11.5% of total sterols and glucosidic sterols to 16.3%. Fatty acids and D-glucose from hydrolysis of esters and glucosides were analyzed. The fatty acids of SE and ASG are richer in unsaturated fatty acids than cocoa butter total fatty acids.     

Sterol fractions in hazelnut and virgin olive oils and 4,4′-dimethylsterols as possible markers for detection of adulteration of virgin olive oil

Reports on the methylsterol fractions of hazelnut oils are scarce. The objectives of this study were to characterize methylsterols in hazelnut and virgin olive oils and to study the possibility of detection of adulteration of virgin olive oils. In hazelnut oils, 4-desmethylsterols were present in higher proportions (86 to 91%) than in virgin olive oils where this fraction was ca. 50% of the total sterol. In the 4-monomethylsterol fraction, citrostadienol was the major component in both kinds of oils followed by cycloeucalenol and obtusifoliol in virgin olive oils, and obtusifoliol in hazelnut oils. 24-Methylenecycloartanol was predominant in both kinds of oils in the 4,4′-dimethylsterols. For the first time, δ-amyrin was tentatively identified by comparing published mass spectral data in the analyzed samples of both kinds of oils. An unknown compound X (containing a lupane skeleton) and lupeol were detected only in the 4,4′-dimethylsterols fraction of hazelnut oils at a level of 2–8 and 6–10%, respectively. GC-MS analysis showed that adulteration of virgin olive oil by hazelnut oil could be detected at a level less than 4% by using these two compounds as possible potential markers.     

Rapid Separating and Enrichment of 4,4′-Dimethylsterols of Vegetable Oils by Solid-Phase Extraction

Phytosterols are separated into three classes: 4-desmethylsterols, 4-monomethylsterols and 4,4′-dimethylsterols. 4,4′-Dimethylsterols are used to detect vegetable oil adulteration and some compounds from this class can have anti-inflammatory and anticancer properties. There are methods such as thin layer chromatography (TLC) and solid phase extraction (SPE) used to separate phytosterol classes from each other. However, in some cases, separation of all three classes is not required. In addition, TLC has some drawbacks such as low recovery and it is time consuming. An SPE method has previously been used, but it was necessary to use high volume of solvents with this method to avoid coelution of phytosterol classes. In this study, an SPE (silica, 1 g) method was developed to separate and enrich only 4,4′-dimethylsterols from unsaponifiables of vegetable oil samples using 25 mL n-hexane and diethyl ether (95:5, v:v). This method was applied to hazelnut and olive oils and results were compared with those of TLC and the previously developed SPE method. Recovery of 4,4′-dimethylsterols was two times higher with the new SPE method compared with the TLC method. The newly developed SPE method generally gave a similar recovery compared with the previously developed SPE method. Moreover, the SPE method developed in this study has the advantage of using a 3.5 times lower volume of solvent than previously developed SPE methods. Because the newly developed SPE method has a single step requiring a low volume of solvents, it is rapid and simple, and can easily be used to detect olive oil adulteration with hazelnut oil and to analyze and quantify effective nutritional compounds in the 4,4′-dimethylsterols class.     

Fish oil fatty acid esters of phytosterols alter plasma lipids but not red blood cell fragility in hamsters

In an attempt to combine the hypocholesterolemic properties of plant sterols with the hypotriglyceridemic action of fish oil FA, plant sterols have recently been esterified to fish oil n−3 PUFA. The objective of this study was to determine the effects of plant sterols esterified to n−3 PUFA on plasma lipid levels and erythrocyte fragility. For 5 wk, male Golden Syrian hamsters were fed diets varying in cholesterol and plant sterol content: (i) Noncholesterol (semipurified diet with no added cholesterol or plant sterols) (ii), Cholesterol (0.25% cholesterol) (iii), Sterols (0.25% cholesterol plus 1% nonesterified plant sterols), or (iv) Fish oil esters of plant sterols (0.25% cholesterol plus 1.76% EPA and DHA sterol esters, providing 1% plant sterols). The addition of fish oil esters of plant sterols to the cholesterol diet decreased (P=0.001) plasma total cholesterol levels by 20%, but nonesterified plant sterols did not have such a beneficial impact. In addition, non-HDL cholesterol concentrations were 29% lower in hamsters fed fish oil esters of plant sterols than in hamsters fed nonesterified plant sterols (P<0.0001). Despite higher (P<0.0001) plant sterol levels in whole erythrocytes of hamsters fed nonesterified plant sterols and fish oil esters of plant sterols compared with hamsters fed no plant sterols, no difference was observed in erythrocyte fragility. The present results show that EPA and DHA esters of plant sterols have a hypocholesterolemic effect in hamsters, and that these new esters of plant sterols exert no detrimental effect on erythrocyte fragility.     

Steryl esters in a cell suspension culture of celery (Apium graveolens)

Arange of analytical techniques was used to investigate the composition of the steryl fatty acyl esters in a cell suspension culture of celery (Apium graveolens). Gas chromatography (GC) and GC-mass spectrometry (GC-MS), using electron ionization (EI) and negative ion chemical ionization (NICI), were employed to characterize the intact steryl esters. Assignments were supported by analysis of the sterol and fatty acid moieties released from the intact molecular species by alkaline hydrolysis. A selectivity for sterol esterification was noted, with the major free sterol, stigmasterol, occurring only in a very small amount in the esterified form. Instead, the precursors to Δ⁵-phytosterols, particularly cycloartenol, predominated in the ester fraction. The pentacyclic triterpene, β-amyrin, was also found as the palmitate and linoleate esters. Changes in composition and abundance of the steryl esters during the different growth phases of a celery cell suspension culture were investigated. The total amount of esterified sterols exceeded that of free sterols throughout the growth cycle. The changes observed during growth highlighted differences between the esters of precursor sterols and those of the 4-desmethyl-sterols, and it is postulated that the various steryl esters perform different functions in cell metabolism.     

Occurrence of 5α-cholesta-7,24-dien-3β-ol and 23-dehydrolophenol in the bean lipids of Vanilla madagascariensis

Eleven 4-desmethylsterols, four 4,4-dimethylsterols, and twelve 4-methylsterols were identified in Vanilla madagascariensis beans. The 4-desmethylsterol fraction was characterized by a high level of 5α-cholesta-7,24(25)-dien-3β-ol (35.3%). The 4-methylsterol fraction was characterized by a high level of 31-norcycloartenol (57.5%) and the presence of 23-dehydrolophenol (9.4%). Cycloartenol (72.9%), cyclosadol (12.7%), parkeol (9.7%), and 24-dehydrotirucallol (4.7%) were identified in the 4,4-dimethylsterol fraction.     

Triterpene alcohols and sterols of vegetable oils

Triterpene alcohols and sterols were separated by thin-layer chromatography and gas-liquid chromatography from the unsaponifiable fractions of the following 18 vegetable oils: linseed, peanut, olive, rice bran, palm kernel, corn, sesame, oiticica, palm, coconut, rapeseed, grape seed, sunflower, poppy seed, castor, tea seed, cocoa butter and soybean. Two triterpene alcohols, cycloartenol and 24-methylene cycloartanol, were found in all of the oils except soybean oil, which contained only cycloartenol. Triterpene alcohols such as α- and β-amyrin, euphorbol, butyrospermol and cyclolaudenol also were encountered occasionally. Three sterols, β-sitosterol, stigmasterol and campesterol were present in all of the oils. In addition a fourth sterol, not yet idenfified, was found in oils of palm, palm kernel and sunflower in varying amounts. This unknown sterol and brassicasterol were found in rapeseed oil in addition to the three sterols that were common to all of the oils studied. Experiment Station for Fats and Oils, National Center for Lipochemistry of National Research Council, Milan, Italy.     

Pollen sterols from three species of sonoran cacti

Cactus bees are important pollinators that contribute to the long-term stability of arid regions in the United States. Since all insects are dependent upon a dietary source of sterol for normal growth, development and reproduction, a study was undertaken to determine neutral sterols available to cactus bees. The total neutral sterol composition of hand-collected pollen was determined for three species of Sonoran cacti by gas-liquid chromatography and mass spectrometry. 24-Methylenecholesterol was the predominant pollen sterol in Engelmann's prickly pear,Opuntia phaeacantha, cholla,O. versicolor, and cardon,Pachycereus pringlei. Two pentacyclic triterpene alcohols, lupeol and moretenol, were also isolated. Since no cholesterol was detected in any of the pollen samples, cactus bees would have to utilize the 24-alkyl sterols unchanged or convert these sterols to cholesterolvia dealkylation.     

Comparative studies of metabolism of 4-desmethyl, 4-monomethyl and 4,4-dimethyl sterols inManduca sexta

To investigate the metabolism and possible deleterious effects of 4-methyl and 4,4-dimethyl steroids inManduca sexta, the 4,4-dimethyl sterols lanosterol and cycloartenol, the 4-methyl sterol obtusifoliol and the 4,4-dimethyl pentacyclic triterpenoid α-amyrin were fed in an artificial agar-based diet at various concentrations. Utilization and metabolism of these four compounds were compared with sitosterol, stigmasterol, brassicasterol, ergosterol and 24-methylenecholesterol, 24-alkyl sterols that are readily dealkylated and converted to cholesterol inManduca and in most phytophagous insects. None of the 4-methylated compounds significantly inhibited development except at very high dietary concentrations. The Δ²⁴-bonds of lanosterol and cycloartenol were effectively reduced by theManduca Δ²⁴-sterol reductase enzyme, as is the Δ²⁴-bond of desmosterol which, in most phytophagous insects, is an intermediate in the conversion of sitosterol, stigmasterol and other C₂₈ and C₂₉ phytosterols to cholesterol. On the other hand, the 24-methylene substituent of obtusifoliol was not dealkylated. Each of the 4-desmethyl C₂₈ and C₂₉ sterols was readily converted to cholesterol, and a significant amount of 7-dehydro-cholesterol was derived from ergosterol metabolism. The reason for the differences in substrate specificity of these sterols is not clear, but the information may be useful in the development of new, specific, mechanism-based inhibitors of sterol metabolism.     

Free and esterified sterols of the marine dinoflagellateGonyaulax polygramma

Free and esterified sterols of the common marine dinoflagellateGonyaulax polygramma were identified using capillary gas chromatography—mass spectrometry (GC-MS). Fractions containing free 4α-methyl and 4-desmethyl sterols were isolated by column chromatography and shown to consist of at least 20 components. Major sterols included 4α,23,24-trimethyl-5α-cholestan-3β-ol (dinostanol), 4α,23,24-trimethyl-5α-cholest-22E-en-3β-ol (dinosterol), cholest-5-en-3β-ol (cholesterol), 23,24-dimethyl-5α-cholest-22E-en-3β-ol and 23,24-dimethylcholesta-5,22E-dien-3β-ol. Although the same group of sterols was found in the free and esterified sterol fractions, the proportions of individual sterols were quite different. The complexity of the sterol distributions, together with the predominance of dinostanol, distinguishes the sterol composition of this alga from those of other members of theGonyaulacaceae.     

The content and composition of sterols and sterol esters in low erucic acid rapeseed (Brassica napus)

The low temperature crystallization technique for the enrichment of “minor” components, such as sterols and sterol esters, from vegetable oils was applied to low erucic acid rapeseed oils. The recovery of free sterols and sterol esters was estimated by use of¹⁴C-cholesterol and¹⁴C-cholesterol oleate. 80% of the free sterols and 45% of the sterol esters were recovered in the liquid fraction, while in two studies total recoveries were 95% and 99%, respectively. This technique showed some selectivity toward the sterol bound fatty acids when compared to direct preparative thin layer chromatography (TLC) of the crude oil. Gas liquid chromatography (GLC) analysis of the free and esterified sterols as TMS-derivatives showed very little selectivity in the enrichment procedure. The fatty acid patterns of the sterol esters demonstrated, however, a preference in the liquid fraction for those sterol esters which have a high linoleic and linolenic acid content. The content of free sterols was 0.3–0.4% and that of sterol esters 0.7–1.2% of the rapeseed oils in both winter and summer types of low erucic acid rapeseed (Brassica napus) when the lipid classes were isolated by direct preparative TLC of the oils. The free sterols in the seven cultivars or breeding lines analyzed were composed of 44–55% sitosterol, 27–36% campesterol, 17–21% brassicasterol, and a trace of cholesterol. The esterified sterols were 47–57% sitosterol, 36–44% campesterol, 6–9% brassicasterol, and traces of cholesterol and Δ5-avenasterol. The fatty acid patterns of these esters were characterized by ca. 30% oleic acid and ca. 50% linoleic acid, whereas these acids constitute 60% and 20%, respectively, of the total fatty acids in the oil. Little or no variation in sterol and sterol ester patterns with locality within Sweden was observed for the one cultivar of summer rapeseed investigated by the low temperature crystallization technique.     

Profiling of Phytosterols, Tocopherols and Tocotrienols in Selected Seed Oils from Botswana by GC–MS and HPLC

The phytosterol, tocopherol, and tocotrienol profiles for mkukubuyo, Sterculia africana, manketti, Ricinodendron rautanenni, mokolwane, Hyphaene petersiana, morama, Tylosema esculentum, and moretologa-kgomo, Ximenia caffra, seed oils from Botswana have been determined. Normal-phase HPLC analysis of the unsaponifiable matter showed that among the selected oils, the most abundant tocopherol and tocotrienol were γ-tocopherol (2232.99 μg/g) and γ-tocotrienol (246.19 μg/g), detected in manketti and mkukubuyo, respectively. Mokolwane oil, however, contained the largest total tocotrienol (258.47 μg/g). Total tocol contents found in manketti, mokolwane, mkukubuyo, morama, and moretologa-kgomo oils were 2238.60, 262.40, 246.20, 199.10, and 128.0 μg/g, respectively. GC–MS determination of the relative percentage composition of phytosterols showed 4-desmethylsterols as the most abundant phytosterols in the oils, by occurring up to 90% in moretologa-kgomo, mkukubuyo, and manketti seed oils, with β-sitosterol being the most abundant. Mokolwane seed oil contained the largest percentage composition of 4,4-dimethylsterols (45.93%). Besides 4-desmethylsterols (75%), morama oil also contained significant amounts of 4,4-dimethylsterols and 4-monomethylsterols (15.72% total). GC–MS determination of the absolute amounts of 4-desmethylsterols, after SPE fractionation of the unsaponifiable matter, confirmed that β-sitosterol was the most abundant phytosterol in the test seed oils, with manketti seed oil being the richest source (1326.74 μg/g). The analysis showed total 4-desmethylsterols content as 1617.41, 1291.88, 861.47, 149.15, and 109.11 μg/g for manketti, mokolwane, mkukubuyo, morama, and moretologa-kgomo seed oils, respectively.     

The content and composition of sterols and sterol esters in sunflower and poppy seed oils

The composition and proportion of free sterols and sterol esters in crude sunflower and poppy seed oils were determined, using preparative thin layer chromatography followed by gas chromatography with cholesterol as an internal standard. Free sterols and sterol esters were also isolated in a liquid fraction obtained by low temperature crystallization (−80 C) of the oils and enriched with minor lipid classes. This enrichment procedure provided a liquid fraction suitable for studies of minor components in the oils. However, selectivity towards sterol esters was observed since sterols esterified to very long chain fatty acids (C₂₀–C₂₄) were preferentially retained in the precipitate. The proportions of free and esterified sterols were found to be 0.34 and 0.28%, respectively, in the sunflower oil, whereas the corresponding figures for poppy seed oil were 0.33% and 0.05%. Sunflower oil was characterized by a relatively high percentage of Δ7-sterols, preferentially obtained in the esterified fraction, and by very long chain saturated fatty acids of sterol esters. The sterols in poppy seed oil were composed almost entirely of campesterol, stigmasterol, sitosterol and Δ5-avenasterol, although their percentage distributions were remarkably different in the free and esterified fraction.     

Distribution of free and conjugated sterols in orange and tangor juice sacs

Comparative studies of the sterol composition of four sterol fractions, vis., free sterols, sterol esters, sterol glucosides and esterified sterol glucosides, were conducted on the juice sacs of six varieties of oranges and two tangor varieties. The sterols quantified in each fraction were β-sitosterol, campesterol, stigmasterol, cholesterol, 24-ethylidene cholesterol, brassicasterol and 24-methylene cholesterol. Each variety showed its own intrinsic composition for these sterols in the four sterol fractions. So. Market. Nutr. Res. Div., ARS, USDA.     

Free and esterified sterols of cotton buds and anthers

Capillary gas liquid chromatography analyses were conducted on free and esterified sterol fractions of cotton (Gossypium hirsutum cv. Stoneville 213) floral buds and anthers. The free sterols of both cotton buds and anthers consist mainly of the common plant sterols sitosterol, stigmasterol and 24ζ-methylcholest-5-en-3β-ol. The composition of esterified sterols of cotton buds and anthers were similar, and consisted of pollinastanol, 31-norcycloartanol, cycloartenol, 31-norcycloartenol, 24-dehydropolinastanol and sitosterol. Desmosterol was also present in both the free and esterified sterols of anthers. The identities of the sterols were confirmed by gas chromatography-mass spectrometry analyses. Esterified sterols accounted for 46.7 and 88.7% of total sterols of cotton bud and anthers, respectively. The ratio of esterified sterol to free sterol per gram of tissue is about 8∶1 in anthers. The Δ⁵-sterols of the esterified sterols of cotton buds and anthers account for only 17 and 9.2% of the total sterols, respectively.     

Analysis of free and esterified sterols in vegetable oils

In vegetable oils, phytosterols occur as free sterols or as steryl esters. Few analytical methods report the quantification of esterified and free sterols in vegetable oils. In this study, esterified and free sterols were separated by silica gel column chromatography upon elution with n-hexane/ethyl acetate (90∶10 vol/vol) followed by n-hexane/diethyl ether/ethanol (25∶25∶50 by vol). Both fractions were saponified separately and the phytosterol content was quantified by GC. The analytical method for the analysis of esterified and free sterols had a relative standard deviation of 1.16% and an accuracy of 93.6–94.1%, which was comparable to the reference method for the total sterol analysis. A large variation in the content and distribution of the sterol fraction between different vegetable oils can be observed. Corn and rapeseed oils were very rich in phytosterols, which mainly occurred as steryl esters (56–60%), whereas the majority of the other vegetable oils (soybean, sunflower, palm oil, etc.) contained a much lower esterified sterol content (25–40%). No difference in the relative proportion of the individual sterols among crude and refined vegetable oils was observed.     

Composition of unsaponifiable lipid from seed oils ofPanax ginseng andP. quiquefolium

The composition of the unsaponifiable lipid, which consisted of squalene, squalene-2,3-oxide, triterpene alcohols, 4α-methyl-sterols, and sterols, of the seed oils ofPanax ginseng andP. quiquefolium, was determined. Squalene was the most abundant component (54%) of the unsaponifiable lipid fromP. ginseng, whereas sterols constituted the principal components of unsaponifiable lipid fromP. quiquefolium. Both ginseng seed oils showed specific features in sterol composition, i.e., 28-isofucosterol (40%) and 24-ethyl-22E-dehydrocholesterol (66%) constituted the most predominant components of the sterol fractions fromP. ginseng andP. quiquefolium seed oils, respectively.     

4-demethyl-,4-monomethyl-and 4,4-dimethylsterols in some vegetable oils

The content of 4-demethyl-, 4-monomethyl- and 4,4-dimethylsterols in 13 vegetable oils was found to vary between 0.10–1.4%, 0.01–0.08% and 0.02–0.29%, respectively. The largest amount of demethylsterols was found in maize and wheat germ oils, whereas the largest amounts of the dimethylsterols were found in olive and linseed oils. The predominating demethylsterols were sitosterol, campesterol, stigmasterol and Δ⁵-avenasterol. Among the 4-monomethylsterols, obtusifoliol, gramisterol, cycloeucalenol and citrostadienol predominated, but usually more than 10 components were found in this fraction. The composition of the 4,4-dimethylsterol fraction was also rather complex, with the 9,19-cyclopropanesterols together with α- and β-amyrin predominating. In most of the oils, characteristically high or low percentages of some sterols were found, and a few specific sterols were also noted. A scheme useful for characterization is presented.