Plant Sterols and Tocols Profile of Vegetable Oils Consumed in Egypt

Abstract

The aim of this study was to quantify specific phytosterols/-stanols (campesterol, β-sitosterol, stigmasterol, β-sitostanol, and campestanol) and tocols (tocopherol and tocotrienol) in the vegetable oils (corn oil, sunflower oil, blended oil, and palm oil) available in the Egyptian market. Gas-liquid chromatography and high performance liquid chromatography procedures were applied to 12 vegetable oils. The best source of phytosterols was the corn oil (4814 μg/g oil) samples while palm oil samples had the lowest phytosterols level (660 μg/g). The mean value of total phytosterols was 2872 and 3443 μg/g for sunflower and blended oil samples, respectively. β-Sitosterol was the dominant phytosterol (ca. 70% of the total phytosterols), followed by campesterol (ca. 16%) and stigmasterol (ca. 9.4%), while β-sitostanol and campestanol were detected in some oils in small amounts or traces. The levels of mean total tocols in corn, sunflower, blended, and palm oil samples were 891.4, 716.1, 707.5, and 311.8 μg/g, respectively. In sunflower and blended oil samples, α-tocopherol was the main tocol, which accounted for more than 80% of total tocols, while α-tocopherol was found in lower levels in corn and palm oils accounting for ca. 40% of total tocols.

Keywords:

• Egyptian market
• Vegetable oils
• Phytosterols
• Phytostanols
• Tocols
• Tocopherols
• Tocotrienols
• Gas liquid chromatography
• High performance liquid chromatography
• Egyptian vegetable oils database
• Vegetable oils and their derived products

INTRODUCTION

Vegetable oils and derived products are a major source of phytosterols (ST) and tocols (TO) in the diet. They contribute to 20 and 41% of the total α-tocopherol equivalent intake in the US and in Finland, respectively,[1, 2] and to 26, 39, and 50% of the total ST intake in Holland, Finland, and Spain, respectively.[3–5 5 Phytosterols are isoprenoid compounds with a sterol nucleus and an alkyl chain. Most plant sterols have a double bond in position C–5 in the nucleus, while others are totally saturated and called stanols.[6] In this work, the term “phytosterols, ST” is used for both sterols and stanols. Together with cholesterol, ST are chemically classified as 4-desmethyl sterols, wherein more than 250 types of ST have been reported.[7] The most common ST are sitosterol (24α-ethylcholest-5en-3β-ol), campesterol (24α-methyl-5-cholesten-3β-ol), and stigmasterol (5,22-cholestadien-24α-ethyl-3βol). Phytosterols exist in four different forms in foods: free, esterified to fatty acids, sugar moieties, or phenolic acid.[7]

Data on the ST content of many edible oils have been published;[8, 9] however, more information about the ST profile in commercial vegetable oils is still needed. Some of the available data on the ST composition of edible oils are for crude oilseed extracts.[10] These data may not apply to commercially available refined oils due to the effects of processing on ST content and composition. For example, refining has been reported to decrease the ST levels of crude oils. On the other hand, in many old reports ST values were from colorimetric, not chromatographic, analysis. Differences in analytical methods might also confound inter-study comparison of values for different oils. In addition, many older studies involved thin-layer chromatographic separation and/or colorimetric quantification of sterols and lacked validation of quantitative recovery.[11] Recently, gas chromatography with flame ionization detection is most frequently applied in ST analysis.[12–16 16

Several reviews on the health effects of ST were recently published.[17, 18] Most of them focus on the clinical trials of the cholesterol-lowering properties with ST and the preventive impacts this may have on coronary heart disease. A preventive impact on the development of colon cancer has been suggested through findings of animal and in vitro studies. Moreover, ST has been widely studied for their hypocholesterolemic, anticarcinogenic, and other health impacts.[19] Unlike sterols that compete with cholesterol for absorption and are themselves present in serum, stanols are virtually unabsorbable. Still, they are capable of decreasing total and low density lipoprotein (LDL) cholesterol as well as ST serum levels.[7, 20] To study the relation between ST intake and health outcome, validated databases are needed. Earlier studies have made an attempt to quantify ST intake; they show that ST are usually consumed in the range of 100–400 mg/day.[21, 22] It is, however, likely that these estimates would change with the choice of database.

Tocopherols and tocotrienols, together abbreviated as tocols (TO), are a group of fat soluble antioxidants with a chromanol ring and a hydrophobic side chain (phytyl in the case of tocopherols, isoprenyl in the case of tocotrienols). Tocopherols (α-, β-, γ-, and δ-tocopherol) and the corresponding tocotrienols differ by the number and positions of methyl substituents on the phenolic part of the chromanol ring. Despite similar absorption from the gastrointestinal tract, α-tocopherol is preferentially resecreted by incorporation into lipoproteins by the hepatic α-tocopherol transfer protein, resulting in the highest plasma and tissue levels. Therefore, the Institute of Medicine defined natural α-tocopherol as the only TO contributing toward meeting the vitamin E requirement.[23] Analysis of TO is usually performed by normal phase high performance liquid chromatography (NP-HPLC). Vegetable oils contain TO mainly in unconjugated form and can be directly injected into the NP-HPLC system after dilution.[14, 15] Many industrial fats, however, contain water, emulsifiers, and possibly added α-tocopherol esters. Thus, their analysis requires saponification in order to avoid degradation of TO in alkaline solution.[24]

The market situation of vegetable oils in Egypt has changed dramatically. Cottonseed oil and hydrogenated vegetable oils have disappeared almost completely from the market, and items like sunflower oil, corn oil, blended oils, and palm oil have found their way into the shelves of retail stores. The aim of this work was to provide up-to-date and validated data on the levels of the individual TO and ST in the main vegetable oils currently available on the Egyptian market in order to enable an update of the Egyptian food composition database. To the best of knowledge, this is the first database for ST and TO in the vegetable oils consumed in Egypt. Valid data would represent a welcome addition since they could be used to generate data on the health impacts of bioactive ST and TO.

MATERIALS AND METHODS

Vegetable Oil Samples and Chemicals

Commercially available vegetable oil samples (corn, sunflower, blended oil [mixture of soybean, sunflower, and palm oils], and palm) from different producers readily available in the Egyptian market (Arma, Savola, and Mazola) were bought in different retail outlets in Zagazig (Egypt) in October 2008. Since cottonseed oil is hardly found in the Egyptian market, these four edible oils represent the main oils found in the market and were advertised as suitable for different kinds of edible application (cooking, frying, etc.). Multiple samples (three samples from the same oil type) were assayed to evaluate potential variability in the ST and TO distribution. Standards used for ST characterization were purchased from Supelco (Bellefonte, PA, USA). Standards used for TO were purchased from Merck (Darmstadt, Germany). Reagents and chemicals used were of the highest purity available.

Gas Liquid Chromatography (GLC) Analysis of Fatty Acids

Fatty acids were transesterified into methyl esters (FAME) using N-trimethylsulfoniumhydroxide (Macherey-Nagel, Düren, Germany) according to the procedure reported by Ramadan and Mörsel.[15] FAME were identified on a Shimadzu GC-14A equipped with flame ionization detector (FID) and C-R4AX chromatopac integrator (Kyoto, Japan). The flow rate of the carrier gas helium was 0.6 mL/min and the split value with a ratio of 1:40. A sample of 1 μL was injected on a 30 m × 0.25 mm × 0.2 μm film thickness Supelco SP™-2380 (Bellefonte, PA, USA) capillary column. The injector and FID temperature was set at 250°C. The initial column temperature was 100°C programmed by 5°C/min until 175°C and kept 10 min at 175°C, then 8°C/min until 220°C and kept 10 min at 220°C. A comparison between the retention times of the samples with those of authentic standard mixture (Sigma, St. Louis, MO, USA; 99% purity specific for GLC), run on the same column under the same conditions, was made to facilitate identification.

Gas Liquid Chromatography (GLC-FID) Analysis of ST

Separation of ST was performed after saponification of the oil samples without derivatization according to Ramadan, Zayed, and El-Shamy.[16] Oils (250 mg) were refluxed with 5 mL of ethanolic potassium hydroxide solution (6%, w/v) and a few anti-bumping granules for 60 min. The unsaponifiables were first extracted 3 times with 10 mL of petroleum ether, the extracts were then combined and washed 3 times with 10 mL of neutral ethanol/water (1:1, v/v), and then dried overnight with anhydrous sodium sulphate. The extract was evaporated in a rotary evaporator at 25°C under reduced pressure and then ether was completely evaporated under nitrogen. GLC analyses of unsaponifiable residues were carried out using a Mega Series (HRGC 5160, Carlo Erba Strumentazione; Milan, Italy) equipped with FID. The following parameters were performed: DB 5 column (J&W Scientific, Falsom, CA, USA) packed with 5% phenylmethylpolysiloxan, 30 m length, 0.25 mm i.d., 1.0 μm film thickness; carrier gas (helium) flow 38 mL/min (split-splitless injection was used). Detector and injector were set at 280°C. The oven temperature was kept constant at 310°C and the injected volume was 2 μL. The repeatability of the analytical procedure was tested and the relative standard deviation of three repeated analyses of a single sample was <5%. Quantitative analyses were performed with a Shimadzu (C-R6A Chromatopac; Kyoto, Japan) integrator.

Normal Phase High Performance Liquid Chromatography (NP-HPLC) Analysis of TO

NP-HPLC was selected to avoid extra sample treatment (e.g., saponification) according to Ramadan and Mörsel.[14] Analysis was performed with a solvent delivery LC-9A HPLC (Shimadzu, Kyoto, Japan). The chromatographic system included a model 87.00 variable wavelength detector and a 250 × 4 mm i.d. LiChrospher-Si 60, 5 μm column (Knauer, Berlin, Germany). Separation of TO was based on isocratic elution when the solvent flow rate was maintained at 1 mL/min at a column back-pressure of about 65–70 bar. The solvent system selected for elution was isooctane/ethyl acetate (96:4, v/v) with detection at 295 nm. Twenty μL of the diluted solution of oil in the mobile phase were directly injected into the HPLC column. Tocols were identified by comparing their retention times with those of authentic standards. All work was carried out under subdued light conditions. All the experiments were repeated at least three times when the variation on any one was routinely less than 5%. All experimental procedures were performed in duplicate and their mean values (± standard deviation) were given.

RESULTS AND DISCUSSION

Fatty Acid Profile of Vegetable Oils

Vegetable oil samples in this work were characterized for their fatty acid composition. In corn, sunflower, and blended oils, the major omega-6 fatty acid is linoleic acid, representing more than 59% of the total fatty acids, followed by oleic acid, palmitic acid, stearic acid, and linolenic acid, respectively. In palm oil, palmitic acid (45%) followed by oleic acid (37%) were the main fatty acids. As shown in Fig. 1, blended oil recorded the highest level of polyunsaturated fatty acids (PUFA, 64%), followed by sunflower oil (61%) and corn oil (60%), respectively. Saturated fatty acids accounted for more than 50% of total fatty acids in palm oil followed by monounsaturated fatty acids (38.4%), while PUFA recorded the lowest amount (9.7%) in this oil.

Figure 1 Fatty acid composition (%) of vegetable oils found in the Egyptian market.

Levels and Profile of Phytosterols/-stanols in the Vegetable Oils

Vegetable oils are generally regarded as important sources for ST since, in general, they contain relatively higher ST amounts than fruits, vegetables, and fruits.[20, 25] Phytosterols/-stanols (ST) concentrations for all oils are listed in Table 1. In total, 12 oil items were analyzed. Corn oil samples had the highest levels of total ST (4814 μg/g) and palm oil samples had the lowest ST content (660 μg/g). The mean values of total ST content were 2872 and 3443 μg/g for sunflower and palm oil samples, respectively. The total ST contents determined in this work were generally in line with those listed in the literature.[11] Recently, comparable levels of total ST contents were found in sunflower oil consumed in Spain and the Netherlands (492 and 436 mg/100 g).[5] Corn oil proved to be the best source of ST, followed by blended oil and sunflower oil as also reported by others.[11]

Vegetable oils are complex media because they contain several different phytosterols/-stanols. β-Sitosterol is among the most representative ST along with campesterol, stigmasterol, and other various types of minor ST.[26] As expected, the predominant ST in all vegetable oil samples were β-sitosterol, stigmasterol, and campesterol. Low levels of sitostanol and campestanol were present in all of the samples but a relatively higher concentration of sitostanol was found in corn (130 μg/g). In corn oil, the percentages of individual PS were β-sitosterol (54.9%), campesterol (13.3%), and stigmasterol (6.2%) being the most abundant ST. These data are similar to those reported by Cercaci et al.[27] Generally, in the samples under investigation, it could be noted that β-sitosterol was the dominant ST (ca. 70% of the total concentration of all five ST), followed by campesterol (ca. 16%) and stigmasterol (ca. 9.4%), while β-sitostanol (ca. 1.4%) and campestanol (ca. 2.1%) were detected in some oils in small amounts (Fig. 2).

Table 1  Levels of phytosterols/-stanols (μg/g) in vegetable oils consumed in Egypt

	Corn oil			Sunflower oil			Blended oil			Palm oil
	Sample 1	Sample 2	Sample 3	Sample 1	Sample 2	Sample 3	Sample 1	Sample 2	Sample 3	Sample 1	Sample 2	Sample 3
Campesterol	810 ± 0.1	818 ± 0.4	819 ± 0.4	295 ± 0.2	292 ± 0.3	296 ± 0.2	382 ± 0.3	381 ± 0.3	381 ± 0.2	175 ± 0.2	170 ± 0.2	177 ± 0.3
Campestanol	55 ± 0.1	57 ± 0.6	59 ± 0.3	44 ± 0.2	48 ± 0.1	41 ± 0.2	54 ± 0.1	56 ± 0.3	54 ± 0.1	34 ± 0.1	27 ± 0.2	29 ± 0.1
Stigmasterol	382 ± 0.3	384 ± 0.5	384 ± 0.5	266 ± 0.5	269 ± 0.5	270 ± 0.4	315 ± 0.5	318 ± 0.5	319 ± 0.7	71 ± 0.2	75 ± 0.1	79 ± 0.2
β-Sitosterol	3418 ± 0.4	3415 ± 0.5	3411 ± 0. 8	2214 ± 0.6	2209 ± 0.6	2213 ± 0.9	2607 ± 0.7	2604 ± 0.6	2612 ± 0.5	382 ± 0.5	388 ± 0.6	371 ± 0.4
Sitostanol	130 ± 0.4	130 ± 0.2	133 ± 0.3	42 ± 0.3	40 ± 0.2	45 ± 0.2	54 ± 0.2	59 ± 0.1	66 ± 0.1	ND ^a	ND	ND
Total ST	4795	4804	4806	2861	2858	2865	3412	3418	3432	662	660	656
Results are given as the average of triplicate determinations ± standard deviation.
^aNot detected.

In the comparison of the analyzed ST levels in vegetable oils in the Egyptian market to that of recent ST databases for vegetable oils found in the western countries,[6, 11] it could be observed that the ST levels in corn oil from the Egyptian market is significantly lower than the amounts of sterols in the corn oil in the western market (Table 2). On the other hand, the ST concentrations in palm oil from the Egyptian market are higher than the amounts of ST in the palm oil in the western market. Concerning sunflower oil, the ST levels resemble that of Normén et al.[11] and Schwartz et al.[6] Several factors may affect the ST content, including plant variety, growing season, shipping, storage, and preparation.

Associations between ST intake and cancer or coronary heart disease (CHD) risk to date were difficult to assess because of the lack of data covering ST levels in foods. New databases[6] have been recently published. The aim was to create databases that could be used in epidemiological studies. Due to the high contribution of ST from different vegetable oils, databases seem, therefore, to be of the highest importance. In addition, due to the high bioavailability of ST in fatty food items, it is thus recommended that these should be included in sufficient numbers in ST databases.[11]

Levels and Profile of Tocols in the Vegetable Oils

Tocol levels in the vegetable oil samples are given in Table 3. α-Tocopherol and γ-tocopherol proved to be the main TO in vegetable oils and fats.[28] The main function of α-tocopherol is that of a radical-chain breaking antioxidant in membranes and lipoproteins, as well as in foods. Despite lower plasma concentrations, other TO are still capable of exerting antioxidant and biological activities. γ-Tocopherol has been reported to be more potent than α-tocopherol in decreasing platelet aggregation, LDL oxidation, and delaying intra-arterial thrombus formation.[29] Likewise, tocotrienols are discussed in the context of reducing the risk of breast cancer and have been shown to inhibit cholesterol biosynthesis.[30] Hence, concurrent administration of various TO may result in increased antioxidant, antitumor, and hypocholesterolemic potential. In order to take into account the biological activities of other TO, some nutrient databases still include them in the form of α-tocopherol equivalents.[6, 31]

Table 2  Average levels of ST (μg/g) in vegetable oils consumed in Egypt compared with ST levels in vegetable oils consumed in some western countries

	Corn oil				Sunflower oil				Palm oil
	Recent study	Normén et al. (2007)	Phillips et al. (2002)	Schwarz et al. (2008)	Recent study	Normén et al. (2007)	Phillips et al. (2002)	Schwarz et al. (2008)	Recent study	Normén et al. (2007)	Phillips et al. (2002)
Campesterol	815	2150	1241	1710	294	520	270	340	174	90	148
Campestanol	57	140	88	150	44	0	12	68	30	0	0
Stigmasterol	383	660	532	480	268	470	176	280	75	60	87
β-Sitosterol	3414	5860	4630	5150	2212	3090	1941	2060	380	240	395
Sitostanol	131	280	138	290	42	30	29	51	ND ^a	ND	10
Total ST	4800	9090	6629	7780	2860	4110	2428	2799	659	390	640
^aNot detected.

Table 3  Levels of tocols (μg/g) in vegetable oils consumed in Egypt

	Corn oil			Sunflower oil			Blended oil			Palm oil
	Sample 1	Sample 2	Sample 3	Sample 1	Sample 2	Sample 3	Sample 1	Sample 2	Sample 3	Sample 1	Sample 2	Sample 3
α-Tocopherol	415 ± 1.1	417.6 ± 1.5	419 ± 1.6	665 ± 2.8	663 ± 2.8	664 ± 2.7	655 ± 2.3	652 ± 2.2	658 ± 2.6	125 ± 0.7	124 ± 0.6	127 ± 0.8
α-Tocotrienol	13 ± 0.5	14.8 ± 0.6	16 ± 0.7	ND	ND	ND	ND	ND	ND	142 ± 1.2	140 ± 1.1	145 ± 1.0
β-Tocopherol	20 ± 0.7	19.7 ± 0.5	19.5 ± 0.5	29 ± 0.4	27 ± 0.7	25.1 ± 0.8	25.1 ± 0.7	27.2 ± 0.8	27.7 ± 0.8	ND	ND	ND
γ-Tocopherol	417 ± 1.6	418.6 ± 2.1	419 ± 2.3	26 ± 0.3	24 ± 0.2	25.3 ± 0.2	26.4 ± 0.5	24.8 ± 0.4	26.3 ± 0.4	ND	ND	ND
β-Tocotrienol	ND ^a	ND	ND	ND	ND	ND	ND	ND	ND	28 ± 0.6	29.5 ± 0.6	26.9 ± 0.5
δ-Tocopherol	20 ± 0.6	22 ± 0.5	23 ± 0.6	ND	ND	ND	ND	ND	ND	ND	ND	ND
γ-Tocotrienol	ND	ND	ND	ND	ND	ND	ND	ND	ND	16.2 ± 0.3	14.9 ± 0.3	17 ± 0.2
Total tocols	885	892.7	896.5	720	714	714.4	706.5	704	712	311.2	308.4	315.9
Results are given as the average of triplicate determinations ± standard deviation.
^aNot detected.

Vegetable oils contain TO mainly in unconjugated form and can be directly injected into the normal phase HPLC system after dilution.[6, 14, 32] Rapeseed, corn, and sunflower oils contained high levels of tocopherols, whereas palm oil is the best source of tocotrienols. Wheat germ oil was found to be by far the best source of α-tocopherol, followed by sunflower and olive oils. With the exception of wheat germ oil in which β-tocopherol turned out to be the second most abundant tocopherol, both β- and δ-tocopherol proved to be minor constituents. γ-Tocopherol occurred in the highest levels in camelina, linseed, cold-pressed rapeseed, and corn oils.[6] In this investigation, the levels of mean total TO in corn, sunflower, blended, and palm oil samples were 891.4, 716.1, 707.5, and 311.8 mg/kg, respectively. It was clear that corn, sunflower, and blended oil samples contained high levels of TO, while palm oil samples contained the lowest level. The levels of individual TO in the investigated vegetable oils are given in Table 3. In sunflower and blended oil samples, α-tocopherol was the main TO, which accounted for more than 80% of total TO, while α-tocopherol found in lower levels in corn and palm oils accounted for more than 40% of the total TO (Fig. 3). γ-Tocopherol was the major TO in corn oil (417.2 mg/kg), wherein β- and δ-tocopherol were present in minor amounts or traces. In the recent TO database for vegetable oils and industrial fats purchased in Finland,[6] total tocopherols in sunflower oil was 630 μg/g, while in corn oil the total tocopherols amount was 660 μg/g and total tocotrienols level was 25. Palm oil samples were characterized by its high levels of tocotrienols, wherein α-tocotrienol was the major tocotrienol followed by β-tocotrienol and γ-tocotrienol, respectively.

Figure 3 Percentages of tocols in the vegetable oils found in the Egyptian market.

CONCLUSIONS

In Egypt, vegetable oil production is centralized with few manufacturing companies and a few small ones processing cold-pressed healthier oils. Therefore, although the selection of oil brands in supermarkets may be different, the overall market situation in Zagazig is representative for that in all of Egypt. Epidemiological studies on ST and TO consumption and health impact are difficult to find, however, because few databases are found. The aim of the present study was to survey for the first time the ST and TO composition of a variety of the main commercially available edible oils in the Egyptian market. The TO and ST levels of oil samples were determined using validated NP-HPLC and GLC-FID methods. There is a wide spread of concentrations of PS and TO in the edible oils consumed in Egypt. However, no significant differences were observed among the samples from the same oil type. This may be due to using the same plant source as it is known that about 90% of vegetable oils found in the Egyptian market is imported. Corn oil, one of the most commonly used oils in Egypt, proved to be the best source of both TO and ST. Since this survey database is meant to be used in other prospective cohort studies, a slight overestimation of ST and TO levels may be less important since the database only needs to rank the ST and TO intake of people in order to study associations with morbidity and mortality.

ACKNOWLEDGMENTS

The author would like to thank the Science and Technology Development Fund (STDF, Project ID 21) and the Egyptian Ministry of Higher Education for the financial support.