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.     

Influence of the vegetable oil refining process on free and esterified sterols

The influence of the refining process on the distribution of free and esterified phytosterols in corn, palm, and soybean oil was studied. Water degumming did not affect the phytosterol content or its composition. A slight increase in the content of free sterols was observed during acid degumming and bleaching due to acid-catalyzed hydrolysis of steryl esters. A significant reduction in the content of total sterols during neutralization was observed, which was attributed to a reduction in the free sterol fraction. Free sterols probably form micelles with soaps and are transferred into the soapstock. The steryl ester content remained constant during all neutralization experiments, indicating that hydrolysis of steryl esters did not take place during neutralization. During deodorization, free sterols are distilled from the oil, resulting in a gradual reduction in the total sterol content as a function of the deodorization temperature (220–260°C). A considerable increase in the steryl ester fraction was found during physical refining, probably owing to a heat-promoted esterification reaction between free sterols and FA.     

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.     

The Content and Composition of Total,Free, and Esterified Sterols of Lotus Plumule Oil by GC–MS/FID

This study investigated the content and composition of total, free, and esterified sterols of three varieties of lotus plumule oil (Hunan lotus, Jiangxi lotus, and Fujian lotus) using GC–MS/FID. The fatty acid composition of sterol fatty acid esters (SFAE) was also analyzed and compared with that of triglycerides. Results showed that total sterol of lotus plumule oil (12.10–14.21 g/100 g) was higher than that of other plant oils (corn germ oil, 1.11 g/100 g; rapeseed oil, 0.78 g/100 g). No significant difference was found among the total sterol contents of the three types of lotus plumule oils (p > 0.05). Most sterol existed in ester forms (81.8–89.1%) rather than in free forms (8.4–10.1%). β‐Sitosterol (71.4–73.4%), and campesterol (6.2–7.5%) were the predominant fractions of free sterols. β‐Sitosterol (41.3–53.7%) and ?5‐avenasterol (27.1–31.1%) were the predominant fractions of esterified sterols, followed by campesterol (12.1–13.0%) and ?7‐avenasterol (3.4–3.7%). Linoleic acid (63.6–65.8%), oleic acid (8.3–10.4%), and behenic acid (9.0–9.9%) were the main fatty acids of SFAE, which were different from those of triglycerides. The results from this study suggest that lotus plumule oil may be a good resource of SFAE and can be used as a supplemental ingredient in functional foods.     

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.     

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.     

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.     

Phytosterols and steryl esters in diverse Cucurbita,Cucumis and Citrullus seed oils

Δ⁷‐Phytosterols present in pumpkin seed oil are significant for the prevention of prostate disorders. Herbal medicines are increasingly offered as dried kernels or concentrated ethanolic extracts of Cucurbita pepo seeds. Until now, the pumpkin seeds of C. pepo have almost exclusively been used for this purpose. Only a few data concerning the sterol content of other Cucurbitoideae seeds are available. Therefore, we isolated, identified, and quantified the free and esterified phytosterols of 12 Cucurbita, 3 Cucumis, and 3 Citrullus seed oils. The total sterol content of these seeds ranged from 297 mg per 100 g oil in Cucurbita maxima ‘Turk's Turban’ to 814 mg per 100 g oil in Citrullus lanatus ‘Sugar Baby Watermelon’, equivalent to 64 to 193 mg per 100 g seeds respectively. These were mainly Δ⁷‐sterols (˜82%) with the steryl esters acounting for ˜32% of the total sterol content.     

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.     

Freie und gebundene Sterine in rohen und raffinierten Palmölen,Teil I: Gehalt und Zusammensetzung der freien Sterine,Sterinester, freien und acylierten Steringlucoside

Free and Bound Sterols in Raw and Refined Palm Oil, Part I: Content and Composition of Sterols, Sterol Esters, Free and Acylated Sterol Glucosides A method for separation and quantitative determination of free sterols, sterol esters, free and acylated sterol glucosides in fats was developed and applied to the investigation of raw and refined palm oil. During refining the content and the composition of all 4 sterol fractions are varying characteristically. Some samples showed an evident increase of the cholesterol level during certain refining steps. Since none of the 4 investigated sterol fractions has such a high cholesterol level, sterols must be present in bound form, which can be liberated during refining and can be determined afterwards by conventional test methods.     

Sterols and Oxidized Sterols in Feed Ingredients Obtained from Chemical and Physical Refining Processes of Fats and Oils

The by-products obtained from conventional chemical and physical refining processes for edible fats and oils are important sources of valuable fatty components such as sterols, tocopherols, fatty acids, etc., and are also used as ingredients in animal feed formulations. Reports on sterol composition and content are limited, and the levels of oxidized sterols in these valuable by-products are unknown. This study analyzed by-product fractions from European refineries intended for use as ingredients in animal feeds for their content and composition of sterols and sterol oxidation products. The complex mixtures of sterol oxidation products were separated and quantified by multidimensional capillary columns, a medium polar DB-17MS and an apolar DB-5MS, in GC and GC–MS. Sterol content ranged from 0.l to 3.4 and 0.03 to 5.0 g/100 g in the by-product fractions collected from chemical and physical refining processes, respectively, while the corresponding ranges for sterol oxidation products were 0.02–17 and 0.02–1.5 mg/100 g.     

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.     

Minor Components in Vegetable Oil Methyl Esters I: Sterols in Rape Seed Oil Methyl Ester

Rape seed oil methyl ester (RME), a diesel fuel substitute and a technical product of increasing importance, contains a number of minor components, which may influence its technical properties in various ways, Sterols, the main constituents of the unsaponifiable matter in rape seed oil, represent an important group of such minor components, which are then found in rape seed oil methyl ester. The concentration and composition of the total sterols in the fraction of unsaponifiables were determined in several RME samples of different origin by the usual procedure of saponification and isolation of the sterol fraction, followed by gas chromatographic analysis. The concentration and composition of the free sterols present in RME and the content of sterol esters were also determined by on-line LC-GC. The total sterol content in the RME samples varied between 0.70% and 0.81%. The sterol fraction of RME composed as follows: 0.4% cholesterol, 9.0% brassicasterol, 37.7% campesterol, 0.4% stigmasterol, 48.0% β-sitosterol, 2.8% Δ⁵-avenasterol, and 0.5% Δ⁷-stigmasterol. By on-line LC-GC 0.24-0.34% free sterols and 0.55-0.71% sterol esters have been found in the RME samples.     

Determination of free and esterified sterols in potential new oil seed crops by coupled on-line liquid chromatography-gas-chromatography

In vegetable oil, free and esterified sterols are characteristic constituents of the unsaponifiable matter and provide rich information about the oil quality. The sterol pattern of plants discussed as potential new oil seed crops have been determined by using coupled on-line normal phase liquid chromatography-gaschromatography (LC-GC). The content and composition of free sterols as well as the concentration of sterol esters have been analyzed in seed oils of different varieties of crambe (Crambe abyssinica), meadowfoam (Limnanthes alba, L. douglasii(, cape marigold (Dimorphotheca sinuata);, lesquerella (Lesquerella fendleri);, iron weed (Vernonia galamensis, V. petitiana);, spurge (Euphorbia lagascae, E. lathyris);, and various species of cuphea (Cuphea lanceolata, C. lutea, C. paucipetala, C. viscosissima, C. wrightii);.     

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.     

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.     

Effects of processing on the quality and stability of three unconventional Sudanese oils

In a refining experiment, on a laboratory scale, crude oils from Sclerocarya birrea (SCO), sorghum bugs (SBO), water‐extracted melon bugs (MBO H₂O) and solvent‐extracted melon bugs (MBO SOL) were processed by alkali refining. Quality changes were characterized by the determination of free fatty acids (FFA), peroxide value, tocopherols, sterols, phosphatides and stability against oxidation (Rancimat test). In addition, the fatty acid composition was determined. It is clear that the contents of phosphatides, peroxides, tocopherols, sterols as well as oxidative stability were reduced during processing, while FFA were nearly totally removed. The content of phosphorus was reduced in SCO, SBO, MBO H₂O and MBO SOL by 26, 19, 12, and 78%, respectively, while complete oil processing removed 95, 99, 96 and 99% of the FFA in crude oils, respectively. The level of total tocopherols decreased during processing by 38.7, 83.8, 100, and 33.3%, respectively. The color decreased through the processing steps up to bleaching; then, in the deodorization step, it darkened sharply in all samples. No change in the fatty acid composition was observed. The order of oxidation stability was crude > degummed > deodorized > neutralized > bleached, in SCO; and crude > degummed > neutralized > bleached = deodorized, in MBO H₂O; and crude > degummed > deodorized > neutralized > bleached in MBO SOL; while in SBO, the order of oxidative stability was deodorized > crude > degummed > neutralized = bleached. Total sterols decreased by 42–92% in the processed oils, compared with crude oils.     

Fatty acid composition of individual plasma steryl esters in phytosterolemia and xanthomatosis

The bulk of the plasma plant sterol in phytosterolemia occurs in the esterified form and is carried mostly in the low and high density lipoproteins. We have determined the fatty acid composition of the individual plasma steryl esters from a newly discovered subject with phytosterolemia and xanthomatosis. For this purpose the intact steryl esters were subject to high temperature gas liquid chromatography (GLC) on a polar capillary column, which separated the major esters on the basis of molecular weight and degree of unsaturation of the fatty acids. The saturated and unsaturated sterols esterified to saturated, monoenoic, dienoic and tetraenoic fatty acids were identified by GLC analysis of the sterol moieties of the corresponding AgNO₃-TLC fractions of the steryl esters. The GLC results were confirmed by reversed phase high performance liquid chromatography combined with mass spectrometry via direct liquid inlet interface. It was found that, in general, each fatty acid was esterified to the same complement of sterols, and that the esterified sterols possessed a composition comparable to that of the free plasma sterols, which was comprised of about 75% cholesterol, 6% campesterol, 4% 22,23-dihydrobrassicasterol and 15% β-sitosterol. The fatty acid composition of the steryl esters differed from that of the 2-position of the plasma phosphatidylcholines, which contained significantly less palmitic and oleic and more linoleic acid. On the basis of these results and a review of the literature it is suggested that the plasma cholesteryl and plant steryl esters in phytosterolemia originate from both synthesis in plasma via the lecithin-cholesterol acyltransferase and synthesis in tissues via the acylCoA-cholesterol acyltransferase.     

Freie und gebundene Sterine in rohen und raffinierten Palmölen,Teil III: Verhalten der sterinhaltigen Lipoproteine bei der Raffination

Free and Bound Sterols in Raw and Refined Palm Oils, Part III: Behaviour of Sterol Containing Lipoproteins during Refining Studies have been carried out about the behaviour of sterol containing lipoproteins during refining. By neutralization and bleaching they are partly separated together with the free fatty acids. One part, however, is splitted mainly during deodorization. Thus content and composition of lipoproteins are changed during refining. The sterols released from lipoproteins are determined together with free sterols. Because of their high cholesterol content increased cholesterol values can be found in sterol determination of refined palm oils.     

Investigation of lipids profiles of Nigella, lupin and artichoke seed oils to be used as healthy oils

Nigella sativa, lupin and artichoke seed oils have been investigated. The oils were subjected to detailed studies using gas chromatographic analysis (GLC) for fatty acids (FA, as methyl esters) and whole sterols (as silyl derivatives). Whereas, high pressure liquid chromatography (HPLC) was employed for determination of molecular species of triacylglycerols (TAG), four sterol lipids (free and acylated sterols, FS and AS, and free and acylated sterylglycosides, FSG and ASG, as their anthroylnitrile derivatives) as well as tocopherol patterns (T). The results showed that the three seed oils are rich in oleic and linoleic acids whereas, lupin had high linolenic acid content. It was found that the TAGs of the three oils showed some similarity with sunflower oil. Lupin oil had higher sterol content and it was very rich in campe- and β-sitosterol. Nigella sativa oil had a high content of isofucosterol, whereas artichoke oil was unique in having a high content of 5-stigma-, 7-stigma-, and avena- sterol. Concerning the FS and AS, Nigella sativa oil had the highest content, whereas artichoke oil had the highest content of FSG and ASG. Nigella sativa and lupin oils contained over 90 % γ-T while, artichoke oil comprised about 100 % α-T. It is recommended to use the three oils as healthy oils and folk medicine.