Analysis of Phytosterols in Foods M.J
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View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Repositori d'Objectes Digitals per a l'Ensenyament la Recerca i la Cultura Journal of Pharmaceutical and Biomedical Analysis 41 (2006) 1486–1496 Review Analysis of phytosterols in foods M.J. Lagarda, G. Garc´ıa-Llatas, R. Farre´ ∗ Nutrition and Food Chemistry, Faculty of Pharmacy, University of Valencia, Burjassot, Spain Received 11 January 2006; received in revised form 21 February 2006; accepted 22 February 2006 Available online 18 April 2006 Abstract Phytosterols are bioactive compounds, one of their most studied and outstanding properties being their cholesterol-lowering activity. This explains the growing interest in the phytosterol contents of foods as either intrinsic or added components. The different steps (extraction, saponification, clean up, chromatographic determination) of plant sterol determination are reviewed, and emphasis is placed on the methods used to assay different phytosterols in food. © 2006 Elsevier B.V. All rights reserved. Keywords: Phytosterols; Food; Diet; Gas chromatography; Liquid chromatography Contents 1. Introduction ........................................................................................................... 1486 2. Physiological functions of phytosterols ................................................................................... 1488 3. Phytosterol contents in foods ............................................................................................ 1488 4. Estimated average intakes of phytosterols................................................................................. 1488 5. Analytical methods..................................................................................................... 1489 5.1. Sample storage conditions ........................................................................................ 1489 5.2. Sample preparation .............................................................................................. 1489 5.2.1. Solvent extraction........................................................................................ 1489 5.2.2. Saponification ........................................................................................... 1490 5.2.3. Purification/enrichment techniques ........................................................................ 1490 5.3. Determination ................................................................................................... 1490 5.3.1. Gas chromatography ..................................................................................... 1490 5.3.2. Liquid chromatography................................................................................... 1491 6. Conclusions ........................................................................................................... 1495 Acknowledgment ...................................................................................................... 1495 References ............................................................................................................ 1495 1. Introduction structure (steroid nucleus, 3-hydroxyl group, 5, 6 double bond). Phytosterols contain an extra methyl or ethyl group, or dou- Plant sterols (phytosterols) are bioactive components of all ble bond; most phytosterol side chains contain 9–10 carbon vegetable foods. They are 28- or 29-carbon alcohols and resem- atoms, instead of 8 as found in cholesterol. Phytosterols have ble cholesterol in vertebrates in terms of both function (stabi- been classified as 4-demethylsterols of the cholestane series, lization of phospholipid bilayers in plant cell membranes) and all of which have double bonds at the C-5 position of the ring [1–3]. In plants, more than 200 different types of phytosterols ∗ Corresponding author. Tel.: +34 963544950; fax: +34 963544954. have been reported the most abundant being -sitosterol (24- E-mail address: [email protected] (R. Farre).´ ␣-ethylcholesterol), campesterol (24-␣-methylcholesterol) and 0731-7085/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jpba.2006.02.052 M.J. Lagarda et al. / Journal of Pharmaceutical and Biomedical Analysis 41 (2006) 1486–1496 1487 Fig. 1. Structures of the most commonly phytosterols found in food. stigmasterol (22, 24-␣-ethylcholesterol). Ergosterol 7,22, 24- In 1999, a panel of independent scientists in the United States ␣-methylcholesterol is the principal sterol of yeast and is found concluded that plant sterol esters can be Generally Recognized as in corn, cotton seed, peanut and linseed oils [3]. Structures of Safe (GRAS) for use as an ingredient in vegetable oil spreads at the most commonly phytosterols found in food are included in a level up to 13.3% by weight. The United States FDA acknowl- Fig. 1. edged the GRAS notification for the intended conditions of use In nature, sterols can be found as free sterols or as four types [6]. In Europe, the Scientific Committee on Food approved the of conjugates in which the 3-hydroxyl group is esterified to use of phytosterol esters in yellow spreadable fats at a maximum a fatty acid or a hydroxycinnamic acid, or glycosylated with a level of 8% of free sterols in 2000, after a thorough review of hexose (usually glucose) or a 6-fatty acyl hexose. Glycosides the safety of phytosterols, phytostanols and their esters [7]. More are the most common form found in cereals [2]. recently, the European Union has issued a commission regula- Phytostanols, a fully saturated subgroup of phytosterols, are tion, EC 608/2004 of 31 March 2004, concerning the labeling of intrinsic constituents of cereals (corn, wheat, rye and rice), fruits foods and food ingredients with added phytosterols, phytosteryl and vegetables, but their concentrations are generally lower than esters, phytostanols and/or phytostanyl ester [8]. those of unsaturated plant sterols [4,5]. At present, several functional food product types such as Phytosterol potency in decreasing serum low-density lipopro- spreadable fats, yoghurts and milk, with free phytosterols or phy- tein (LDL) cholesterol levels and thus in protecting against tosteryl fatty acid esters or phytostanyl fatty acid esters added cardiovascular diseases, has led to the development of functional at high levels, are available in the market—especially in several foods enriched with plant sterols. European countries [5]. 1488 M.J. Lagarda et al. / Journal of Pharmaceutical and Biomedical Analysis 41 (2006) 1486–1496 2. Physiological functions of phytosterols In foods, plant sterols occur as free sterols, fatty acyl esters, glycosides and fatty acyl glycosides, with substitutions occur- Initially, it was shown that soybean sterols could inhibit ring at C3. Accordingly, the total sterol content is defined by the increases in plasma and liver cholesterol normally seen the sum of all of them, whereas cholesterol occurs either as free in cholesterol-fed chicks, and also reduce the severity of alcoholic sterol or as cholesteryl esters [30–32]. atherosclerotic lesions [9–11]. Contrary to popular belief, the Cereal products are a significant natural plant sterol source use of phytosterols for lowering serum cholesterol levels is not (free, esterified and steryl glycosides). The total sterol contents new. For instance, -sitosterol has been used since the 1950s of rye (95.5 mg/100 g, wb), wheat (69.0), barley (76.1) and oat as a supplement and as a drug for lowering serum cholesterol (44.7) have been reported in Finland [28]. A median total phy- levels in hypercholesterolemic individuals [12]. Since then, sev- tosterol concentration of 49 mg/100 g (range 4.1–344) edible eral clinical studies have been made on the effect upon blood portion has been reported for some cereal foods commonly con- lipids of 4-demethyl plant sterols or stanols, either in their free sumed in Sweden and in the Netherlands, -sitosterol generally or esterified form. These compounds reduce total cholesterol being the dominant form (62%), followed by campesterol (21%) and LDL-cholesterol levels through a reduction in cholesterol and, in smaller concentrations, stigmasterol (4%), -sitostanol absorption. Properly solubilized free sterols and esterified sterols (4%) and campestanol (2%) [33]. Free and esterified sterol con- possess similar cholesterol-lowering activity [13–16]. tents of 52.7 and 52.8 mg/100 g, respectively, have been reported Phytosterols and phytostanols inhibit the uptake of dietary in spelt (Triticum spelta) and winter wheat (Triticum aestivum), and endogenously produced cholesterol from the gut. The exact which also contain 123.8 and 112.6 mg/100 g, respectively, of mechanism by which this occurs is not fully understood, though glycosylated sterols [34]. several theories have been proposed. One theory suggests that Phytosterols have also been quantified in nuts and seeds in the intestine, cholesterol already marginally soluble is pre- commonly consumed in the United States—the highest and cipitated into a non-absorbable state by the presence of added lowest phytosterol contents corresponding to sesame seed and phytosterols and stanols. A second theory is based on the fact wheat germ (400–413 mg/100 g), and the lowest to Brazil nuts that cholesterol must enter bile salt and phospholipids containing (95 mg/100 g). Beta-sitosterol, 5-avenasterol and campesterol “mixed micelles” to be absorbed into the bloodstream. Choles- were found to be the predominant forms. It should be noted that terol is only marginally soluble in these