Analysis of sterol fractions from twenty vegetable oils

The unsaponifiables separated from 20 vegetable oils were divided into sterol and three other (less polar compound, triterpene alcohol, and 4-methylsterol) fractions by preparative thin layer chromatography. The amounts of the sterol fractions were more than ca. 30% in the unsaponifiables from all of the oils, except tohaku, pumpkin seed, and fagara seed oils. Composition of the sterol fractions were determined by gas liquid chromatography. Individual components of the sterol fractions were identified by gas liquid chromatography and combined gas liquid chromatography-mass spectrometry. β-Sitosterol was found as the most predominant component in the sterol fractions from all oils, except two, i.e. the sterol fraction from pumpkin seed oil contained no detectable amount of β-sitosterol and the sterol fraction from akamegashiwa oil contained Δ⁵-avenasterol as the most abundant component. Campesterol, stigmasterol, Δ⁵-avenasterol, Δ⁷-stigmastenol, and Δ⁷-avenasterol and also trace amounts (at the very least) of cholesterol and brassicasterol were found in most of the oils analyzed. It may be noted that a large amount (ca. 9%) of cholesterol was detected in the sterol fraction from capsicum seed oil. The presence of 24-methylenecholesterol and Δ⁵-avenasterol in the sterol fraction of akamegashiwa oil was demonstrated by isolation of these sterols.     

Determination of sterols by capillary column gas chromatography. Differentiation among different types of olive oil: Virgin,refined, and solvent-extracted

Compositional analysis of the sterol fraction of olive oil can be used to assess the degree of purity of the oil and the absence of admixture with other plant oils. This determination also permits characterization of the type of olive oil in question: virgin, refined, or solvent-extracted. In the present work, 130 samples of olive oil were analyzed, the sterol fractions were separated from the unsaponifiable fraction by silica gel plate chromatography, and later they were analyzed as the trimethylsilyl ether derivatives by capillary column gas chromatography. From the results obtained, it was concluded that this methodology is able to differentiate among virgin, refined, and solvent-extracted olive oils. Stigmasterol, clerosterol, Δ5-avenasterol, Δ7-stigmasterol, and Δ7-avenasterol permit the differentiation of the three types of oil from one another. Campesterol, Δ5, 23-stigmastadienol, β-sitosterol, and Δ5,24-stigmastadienol permit the differentiation of only two oils from each other but confirm the conclusions obtained for other sterols. Correlations between the different sterols of virgin, refined, and solven-extracted olive oil also have been obtained.     

Characterization of Total and Individual Sterols in Canola Sprouts

In this study, the contents of total and individual phytosterols in sprouts made from seeds of seven canola (Brassica napus L.) lines (Acropolis, Banjo, Jetton, KS-7740, KSM3-1-124, Mussette and Virginia), grown at three locations in Virginia (Orange, Petersburg and Suffolk), were determined. Canola sprouts contained, on an average, 36.3 g sterols in 100 g of unsaponifiable matter (UNSAP), 10.7 mg sterols in 1 g of oil and 2.4 mg sterols in 1 g of dry sprouts. The contents of individual phytosterols (μg per g of oil) in canola sprouts were 1,162 brassicasterol, 3,799 campesterol, 34 stigmasterol, 5,359 β-sitosterol, 201 Δ⁵-avenasterol and 97 Δ⁷-stigmastenol. Canola lines had significant effects on the contents of oil, brassicasterol and campesterol. Locations had significant effects on the oil, UNSAP, total sterols, brassicasterol, stigmasterol and β-sitosterol. The oil content in canola sprouts was positively correlated with total sterols and Δ⁵-avenasterol, whereas oil content was negatively correlated with brassicasterol content. In general, the contents of campesterol and β-sitosterol increased with an increase in total sterol content. The concentrations of sterols were in the following decreasing order: β-sitosterol > campesterol > brassicasterol > Δ⁵-avenasterol > Δ⁷-stigmastenol > stigmasterol. These results indicate that canola sprouts may have the potential as a natural source of dietary sterols and might be desirable for human nutrition.     

Sterol composition of 19 vegetable oils

The unsaponifiables from 19 vegetable oils were divided into a sterol and three other fractions by thin-layer chromatography. All except olive and palm kernel oils gave the sterol fraction in a large quantity. Compositions of the sterol fractions were determined by gas liquid chromatography. Identification of each sterol was carried out by gas liquid chromatography and combined gas chromatograph-mass spectrometry. Campesterol, stigmasterol and β-sitosterol were present in all oils, and a minor amount of cholesterol in majority of the oils. Brassicasterol occurrence was widespread but its content was extremely small in oils other than rapeseed oil. Other sterols, presumably δ⁷-stigmastenol and δ⁵- and δ⁷-avenasterol were detected in most of the oils.     

Phytosterols,triterpene alcohols,and phospholipids in seed oil from white lupin

This study was conducted to determine effects of genotypes and growing environment on phytosterols, triterpene alcohols, and phospholipids (PL) in lupin (Lupinus albus L.) oil from seven genotypes grown in Maine and Virginia. The unsaponifiable lipid (UNSAP) and phospholipid (PL) fractions ranged from 2.1 to 2.8% and from 2.6 to 2.8% of oil, respectively. UNSAP in lupin oil contained 19.9 to 28.7% sterols and 17.3 to 22.0% triterpene alcohols. Growing location significantly affected contents of total PL, PS, phosphatidylglycerol, β-sitosterol, campesterol, and β-amyrin. Genotypic effects were significant for stigmasterol. PC (32.6 to 46.3% of PL), PE (21.6 to 32% of PL), and PS (11.2 to 17.9% of PL) were the major PL in lupin oil. The concentration of PL classes in lupin oil were in the following descending order: PC>PE>PS>PI>phosphatidic acid > lysophosphatidylcholine > phosphatidylglycerol > diphosphatidylglycerol. In descending order of abundance, the sterols present in lupin oil were: β-sitosterol > campesterol > stigmasterol > Δ⁵-avenasterol > Δ⁷-stigmastenol Lupeol was the most prominent triterpene alcohol in lupin seed oil. In general, growing environment had a much greater influence on lupin oil characteristics than the genotypes.     

Effect of extraction system, stage of ripeness, and kneading temperature on the sterol composition of virgin olive oils

Comparative extraction trials were carried out among a classical pressing, a dual-, and a three-phase centrifugation system using olive crops of Koroneiki variety. Two different kneading temperatures, 30 and 45°C, were tested at three stages of ripeness for two consecutive years of harvest, 1995–1996 and 1996–1997. Composition of the sterol fraction was determined in the resulting olive oil samples (n=72). Stigmasterol was found to be affected by the extraction system; it was obtained in the highest amount in the pressing system. The ratio campesterol/stigmasterol was significantly higher in oils extracted by dual- and three-phase centrifugation. Sterols were significantly affected by the ripening stage of the fruit. During December, the ratio campesterol/stigmasterol reached the maximal and β-sitosterol the minimal values; this appears to be the optimal period for harvesting the olives. Comparison of the different kneading temperatures showed that at 30°C, Δ⁵-avenasterol and campesterol/stigmasterol ratio reached higher values than at 45°C.     

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.     

Genotype and growing location effects on phytosterols in canola oil

There is little information available about phytosterols in canola (Brassica napa L.) oil and the effects of genotype and growing locations from Virginia and the mid-Atlantic region of the United States, a potential area for the establishment of domestic production to provide edible oil. Our objectives were to characterize the phytosterols, phospholipids, unsaponifiable matter, and FA in oil from Virginia-grown canola. Among 11 canola genotypes grown at two locations during 1995–1996 significant variations existed for oil content and FA profiles, but not for contents of phospholipids, unsaponifiable matter, total phytosterols, campesterol, stigmasterol, and β-sitosterol, Total phytosterol content in the oil of Virginia-grown canola varied from 0.7 to 0.9% with a mean of 0.8%. This concentration compared favorably with oil from Canadian canola, which typically contains 0.5 to 1.1% total phytosterols. The mean contents of brassicasterol, campesterol, stigmasterol, β-sitosterol, Δ⁵-avenasterol, and Δ⁷-stigmatenol as percentages of total phytosterols in Virginia-grown canola were: 9.7, 32.0, 0.6, 49.3, 4.99, and 3.5%, respectively. Growing location did not affect phytosterols in Virginia-grown canola oil but had significant effects on contents of phospholipids, and saturated (myristic, stearic, and arachidic) and unsaturated (palmitoleic, linoleic, linolenic, eicosenoic, and erucic) FA.     

Technological Factors Affecting Sterols in Australian Olive Oils

Sterols are important lipids related to the quality of olive oil and broadly used for checking its genuineness. Recent analyses have identified that some Australian olive oils would not meet international standards for total content of sterols or for certain individual components. Several research works indicate that there are some significant correlations between cultural and processing practices and sterols content and composition. In this work the horticultural and processing practices that may have an impact on the sterol content and profile of the most important Australian varieties were analysed. The information generated with this study aims to solve a legislation problem as well as maximising the nutritional and health benefits of Australian olive oils. The evaluation was undertaken using three different varieties and the processing practices evaluated were: irrigation, fruit size, maturity, malaxing time, malaxing temperature and delays between harvest and process. The total content of sterols and their composition in olive oil is strongly influenced by genetic factors and year. Processing practices particularly affect triterpene dialcohols and stigmasterol while horticultural practices and fruit characteristics tend to affect more significantly other sterols such as β-sitosterol, sitostanol, Δ5-avenasterol and Δ7-avenasterol.     

Unsaponifiable matter of green and blue-green algal lipids as a factor of biochemical differentiation of their biomasses: II. Terpenic alcohol and sterol fractions

The composition of the terpenic alcohol and sterol fractions of the unsaponifiables of ten algal species, fiveMyxophyceae and fiveChlorophyceae, is discussed. The major component of the terpenic fraction is phytol, a diterpenic alcohol. Minor amounts of straight chain and triterpenic alcohols are also present. Practically all the species examined contain ten components in the sterol fraction: cholesterol, brassicasterol, Δ⁵-ergostenol, poriferasterol, Δ⁷-ergostenol, clionasterol, chondrillasterol, Δ⁵-avenasterol, Δ⁷-chondrillastenol, and an unidentified component. Identification of the sterols was made by gas chromatography-mass spectrometry, and a 24 S configuration was assumed. The prokaryotic blue-green algae are characterized by a higher content in cholesterol (3.5–14%) than the eucaryotic green algae (0–2.5). Also, brassicasterol, poriferasterol, clionasterol, and Δ⁵-avenasterol are more abundant in blue-green algae. Δ⁷-Ergostenol, chondrillasterol, and Δ⁷-chondrillastenol predominate, on the contrary, in green algae.     

Improvement of Canola Oil Frying Stability by Bene Kernel Oil’s Unsaponifiable Matter

The anti-rancidity effect of the unsaponifiable matter fraction of bene kernel (UFB) oil on canola oil (CAO) during frying was compared to that of tert-butyl hydroquinone (TBHQ). The UFB was separated into hydrocarbons (12.9%), carotenes (9.6%), tocopherols and tocotrienols (65.8%, mainly γ-tocopherol), linear and triterpenic alcohols (3.8%), methyl sterols (2.8%), sterols (3.0%, mainly β-sitosterol, stigmasterol, Δ⁵-avenasterol, and Δ⁷-avenasterol, respectively), and triterpenic dialcohols (2.2%). The results obtained from the measurements of the total polar compounds, the conjugated diene value, the carbonyl value, and total tocopherols showed that the stability of CAO improves similarly in the presence of UFB or TBHQ, and even more in the presence of UFB in some cases (especially inhibition of oxidized triglyceride monomers and triglyceride dimers). The analysis of polar components showed that the antioxidative additives were more effective to resist the formation of thermo-oxidative than hydrolytic products during the frying of CAO.     

Changes in Δ5- and Δ7-sterols during germination and seedling development ofCucurbita maximaduring germination and seedling development ofCucurbita maxima

While seeds ofCucurbita maxima contain both Δ⁵- and Δ⁷-sterols, the former, which have been described earlier, now have been found to disappear during germination. This suggests that a function exists for the Δ⁵-compounds only in the early part of the life cycle ofC. maxima, unlike most of the other higher plants studied. In contrast to the Δ⁵-sterols, the level of Δ⁷-sterols increased during germination as well as during seedling development and maturation. The period of transition between germination and seedling development appeared to be of special importance in terms of sterol changes. This period represented a surge of sterol biosynthesis with an ontogenetic shift in sterol composition from approximately equal amounts of 24α- and 24β-ethyl stereochemistry to a predominance of the former. The sterol composition of the mature plants included only about 5% of the 24β-ethylsterols. The configurational relationships were demonstrated by high resolution¹H-NMR. The sterols of the mature plants were: 25(27)-dehydrochondrillasterol, 24β-ethyl-25(27)-dehydrolathosterol, avenasterol, spinasterol, 22-dihydrospinasterol and 24ξ-methyllathosterol. Based on the changes which occurred in the relative amounts of the Δ⁷-sterols, it did not appear that the Δ⁵-components were being converted to their Δ⁷-analogs. A portion of this work was presented at the meeting of the American Oil Chemists' Society in May, 1985 in Philadelphia.     

Variation in lipid composition of niger seed (Guizotia abyssinica Cass.) Samples collected from different regions in ethiopia

Niger seed samples were collected from different regions in Ethiopia for determination of oil content, and of fatty acid, tocopherol and sterol composition in the seed oil by gas-liquid chromatography and high-performance liquid chromatography methods. There was a large variation in oil content, ranging from 29 to 39%. More than 70% of the fatty acids was linoleic acid (18∶2) in all samples analyzed. The other predominant fatty acids were palmitic (16∶0), stearic (18∶0) and oleic (19∶1) at a range of 6 to 11% each. Total polar lipids recovered after preparative thin-layer chromatography comprised a small fraction of the total lipids. They had higher 16∶0 and lower 18∶2 contents than the triacylglycerols.α-Tocopherol was the predominant tocopherol in all samples, 94–96% of the total amounting to 630–800 μg/g oil. More than 40% of the total sterols wasβ-sitosterol,ca. 2000μg/g oil. The other major sterols were campesterol and stigmasterol, ranging from 11 to 14%. The Δ5- and Δ7-avenasterols were in the range of 4 to 7%. From the samples studied, no conclusion could be drawn regarding the influence of altitude or location on oil content, tocopherol and/or sterol contents. The results of the present study on niger seed oil are discussed in comparison with known data for common oils from Compositae,viz, safflower and sunflower.     

A method for the separation of seed oil steryl esters and free sterols: Application to peanut and corn oils

A method for separating and quantitating seed oil steryl esters and free sterols was developed using a combination of preparative column, thin layer (TLC), and gas liquid chromatography (GLC). Cholesteryl heneicosanoate and cholesterol served as internal standards. The method was applied to corn-oil samples (Mazola, Kroger) obtained from the local market and peanut-oil samples prepared in the laboratory from commercial varieties of peanuts (Florunner, Starr). Concentration (mg/100 g oil; mean ± SD) of steryl esters and free sterols in the 4 oils were: Mazola, 1420±40 and 370±8; Kroger, 950±40 and 320±4; Florunner, 74±0.5 and 150±3; and Starr, 51±0.5 and 130±2. Sitosterol was the major sterol in both the free sterol and steryl ester fractions of all oils and together with campesterol, stigmasterol and Δ⁵-avenasterol made up 90–95% of all sterols. Steryl esters of peanut oil contained higher proportions of linoleic acid and long-chain acids (C₂₀–C₂₄) than did whole oil. Corn-oil steryl esters also contained a higher proportion of linoleic acid than did whole oil. Squalene was the major hydrocarbon of all oils with the remaining hydrocarbon fraction consisting of a mixture of compounds. Presented at the AOCS meeting, Toronto, May 1982.     

The sterol composition of freshly harvested compared to stored seeds of rape,sunflower and poppy

The composition of the free sterols and the sterol esters of freshly harvested seeds of rape, sunflower and poppy was compared to that of stored seeds. The sterol composition of rapeseed was not changed during storage, whereas in sunflower seed the free sterols had less of Δ5-avenasterol and Δ7-stigmastenol in ten-month-old seeds compared to fresh seeds. The greatest relative changes were observed for esterified sterols in poppy seed, with a drop in the percentage of Δ5-avenasterol from 25.3% in freshly harvested to 16.9% in seeds stored for 10 months.     

Differential uptake of cholesterol and plant sterols by rat erythrocytes in vitro

The in vitro uptake of radioactively labeled cholesterol and the plant sterol β-sitosterol has been examined in rat erythrocytes. From mixed micellar solutions containing egg yolk phospholipid and sodium taurocholate, the erythrocytes showed a nonlinear uptake of the two sterols. The uptake leveled off after about 45 min with the attainment of a 1∶1 total sterol-to-phospholipid ratio within the cell membrane, as determined on a mass basis. From soltuions containing egg yolk phospholipid, or purified egg yolk phosphatidylcholine, a preference for cholesterol over the plant sterol was observed, increasing with time from a cholesterol/β-sitosterol uptake ratio of unity (the media ratio) to a maximum of 2 after a 60-min incubation. Correction of the data for nonspecifically bound sterol increased the ratio to a maximum of 5 at the 30-min time point. The increase in the cholesterol/β-sitosterol uptake ratio with time, following an initial nonspecific association, showed that penetration of the plasma membrane by the sterol was required for the selectivity to be expressed. The presence of lysophosphatidylcholine or bovine serum albumin did not exert any noticeable influence over the extent of selectivity of absorption. Replacement of the egg yolk phospholipid with synthetic dipalmitoyl-phosphatidylcholine led to a loss of the sterol selectivity. No evidence was found to support a selective extraction of sterol from the erythrocyte membrane to account for the observed effects, nor was there any sign of a mass accumulation of phospholipid during the incubation. It is suggested that the media phospholipid influences the membrane permeability toward cholesterol and β-sitosterol. Presented in part at the 72nd annual meeting of the American Oil Chemists' Society, New Orleans, LA, May 1981.     

Acid-catalyzed isomerization of fucosterol and Δ5-avenasterol

This work shows that fucosterol, Δ⁵-avenasterol, and similar ethylidene-side chain sterols can undergo acid-catalyzed isomerization to give a mixture of five isomers. Four isomers formed from fucosterol were analyzed, using gas chromatography-mass spectrometry, and were characterized as Δ⁵-avenasterol two Δ^5,23-stigmastadienols, and Δ^5,24(250)-stigmastadienol. When the unsaponifiables fraction from oat oil was subjected to acid hydrolysis, the two Δ^5,23-stigmastadienol isomers and Δ^5,24(25)-stigmastadienol were detected while fucosterol coeluted with sitosterol. Interisomerization of ethylidene-side chain sterols represents a limitation to the use of the acid hydrolysis method in the determination of sterols in food and other plant materials rich in these sterols, e.g., oat lipids.     

Varietal and crop year effects on lipid composition of walnut (Juglans regia) genotypes

The FA, unsaponifiable, and volatile constituents of oil from three walnut varieties from two consecutive crop years were studied. The walnut oils (WO) were rich in PUFA and low in saturated FA. The tocopherol fraction consisted mainly of γ-tocopherol. High contents of β-sitosterol were found, together with campesterol and Δ⁵-avenasterol in similar amounts. Methylsterols present in WO were identified as cycloartenol, cyclolaudenol, cycloeucalenol, and 24-methylenecycloartanol. The hydrocarbon fraction was characterized by the predominance of C₁₄–C₂₀ n-alkanes. The major volatiles were aldehydes produced through the linoleic acid oxidative pathway. FA, methylsterols, and some hydrocarbons presented statistically significant differences among varieties. Most of this variation was due to the genotype. The Franquette variety was noteworthy by its higher oil and oleic acid contents. In contrast, tocopherols and volatile compounds showed minor differences among varieties; they were strongly influenced by the crop year. Chemical data were subjected to principal component analysis. The parameters that gave the greatest discrimination between the walnut varieties were oleic and linolenic acids, tetradecane, eicosane, tetracosane, cycloartenol, and 24-methylenecycloartanol. These components presented the major varietal influences and could be useful to determine the identity of walnut genotypes.     

Oil composition of pistachio nuts (Pistacia vera L.) from Turkey

The oil content, fatty acid and sterol compositions and other parameters of pistachio nut (Pistacia vera L.) samples corresponding to five different varieties, all cultivated in Turkey were determined. Mean values were 59.69 ± 1.80% for fat content, 0.9143 ± 0.006 for specific gravity of the oil, 1.4693 ± 0.004 for refractive index, 94.23 ± 1.510 for iodine value, and 188.2 ± 3.80 for saponification value. Fatty acids identified in the oil samples were palmitic, palmitoleic, stearic, oleic, and linoleic acids with oleic acid as the dominant fatty acid (68.78 ± 2.05%). Halebi variety had the highest level of oleic acid among the varieties studied. The sterols isolated from the unsaponifiable fraction were campesterol, stigmasterol, β-sitosterol, and Δ⁵-avenasterol with β-sitosterol as the major constituent (84.95 ± 2.80%). Higher levels of β-sitosterol were found in Kιrmιzι variety. The high level of oil, oleic acid and β-sitosterol content was found in all the varieties studied. Fat content, iodine value, palmitic acid and oleic acid content significantly differed between varieties.     

The lipids of the common house cricket,Acheta domesticus L. III. Sterols

Sterols constitute 1.95% of the total extractable lipids ofAcheta domesticus L., of which 18% are esterified. The free sterols consist of cholestane-3β-ol (0.5%), Δ⁵-cholestene-3β-ol (83.5%), Δ⁷-cholestene-3β-ol (2.3%) Δ^5,7-cholestadiene-3β-ol (3%), Δ^5,22-cholestadiene-3β-ol (4%), Δ^5,7,22-cholestatriene-3β-ol (0.2%), campestane-3β-ol (0.03%), Δ⁵-campestene-3β-ol (1.0%), Δ⁷-campestene-3β-ol (trace), Δ^5,7-campestadiene-3β-ol (0.2%), stigmastane-3β-ol (0.09%), Δ⁵-stigmastene-3β-ol (2.1%), Δ⁷-stigmastene-3β-ol (0.04%), Δ^5,7-stigmastadiene-3β-ol (0.4%), Δ^5,22-stigmastadiene-3βol (0.1%). The same sterols are present in the esterified sterol fraction. Δ⁷-Sterols and Δ^5,7-sterols are present in significantly larger amounts in the esterified fraction than in the free sterol fraction. By a comparison with the sterols of the cricket food, it is clear thatA. domesticus is capable of removing methyl and ethyl groups from C-24 of sterols of the campestane and stigmastane type. The ability to introduce a Δ⁷ double bond into saturated and Δ⁵-sterols is indicated, and it is suggested that Δ⁷-sterols of the C₂₇, C₂₈, and C₂₉ sterol series may be intermediates in the conversion of Δ⁵-sterols to Δ^5,7-sterols. Associate Professor, Department of Chemistry, University of Michigan, Ann Arbor, Mich.; Alfred P. Sloan Foundation Fellow, 1968–68. Public Health Service Predoctoral Fellow, 1968–67.