Mediterr J Nutr Metab (2011) 4:165–172 DOI 10.1007/s12349-010-0049-0

REVIEW

Phytosterols, phytostanols and their esters: from natural to functional foods T. Bacchetti • S. Masciangelo • V. Bicchiega E. Bertoli • Gianna Ferretti

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Received: 14 April 2010 / Accepted: 18 December 2010 / Published online: 20 January 2011 Ó Springer-Verlag 2011

Abstract Phytosterols, phytostanols and their esters are a group of steroid alcohols that occur naturally in plants. As natural constituents of plant structures, phytosterols contribute to the regulation of the fluidity and permeability of cell membranes. They are found mostly in vegetable oils, fruits, nuts, cereals and legumes. The most abundant phytosterols are b-sitosterol, campesterol and stigmasterol. During the last 15 years the market for phytosterols, as dietary supplements, has lead to a rapidly growing worldwide market for functional foods containing phytosterols and stanols. Even though many different clinical trials have clearly demonstrated that phytosterols reduce LDL-cholesterol, it is unclear whether phytosterols have a positive effect on cardiovascular disease. Until now, there are no data related to the effect of phytosterol consumption on the development of cardiovascular diseases. This review focuses on the biochemistry of phytosterols, their metabolism and role in health and in pathological conditions. Keywords

Phytosterols  Phytostanols  Functional foods

Introduction Phytosterols, phytostanols and their esters are a group of steroid alcohols that are natural constituents of plant cell membrane and contribute to the regulation of the fluidity and T. Bacchetti  E. Bertoli  G. Ferretti (&) Dipartimento di Biochimica, Biologia e Genetica, Universita` Politecnica delle Marche, Via Ranieri, 60131 Ancona, Italy e-mail: [email protected] S. Masciangelo  V. Bicchiega Laboratorio Sperimentale di Ricerche Nutrizionali-Istituto Auxologico Italiano (IRCCS), Ospedale S. Giuseppe, Piancavallo (VB), Italy

permeability of cell membranes [1]. They are substrates for the synthesis of numerous secondary plant metabolites and act as biogenic precursors of compounds involved in growth. More than 200 different types of phytosterols have been reported in plant species. The chemical structure of phytosterols differs from that of cholesterol only by minor modifications (Fig. 1). b-Sitosterol, campesterol and stigmasterol (Fig. 1) are the most frequent plant sterols in food and represent about in media 65, 30 and 3% of dietary intake [2]. Plant stanols, on the other hand, are the saturated form of plant sterols, meaning they have no double bond in the sterol ring. Saturation of b-sitosterol, campesterol or stigmasterol gives rise to sitostanol, campestanol or stigmastanol, respectively. In addition to the free form, phytosterols occur as four types of conjugates, in which the 3 -OH group is esterified to a fatty acid or a hydroxycinnamic acid, or glycosylated with a hexose (usually glucose) or a 6-fatty-acyl hexose (Fig. 2) [1]. During the last 15 years the market for phytosterols as dietary supplements, has led to a rapidly growing worldwide market for functional foods containing phytosterols and stanols. Even though many different clinical trials have clearly demonstrated that phytosterols reduce LDLcholesterol, it is unclear whether phytosterols have a positive effect on cardiovascular disease [3]. Until now, there are no data related to the effect of phytosterol consumption on the development of cardiovascular diseases [4]. This review focuses on the biochemistry of phytosterols, their metabolism and role in health and pathological conditions.

Phytosterol and phytostanol content in vegetables and functional foods Phytosterols are abundant in vegetable products, such as vegetable oils, fruits, nuts, grains, and pulses (Table 1).

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Mediterr J Nutr Metab (2011) 4:165–172 Table 1 Content of total phytosterols in oil, fruit, vegetable, cereals and legumes Food source

Total sterol content (mg/100 g)a

Oils Rice bran oil

Fig. 1 Phytosterols more commonly found in vegetable foods

1,055a

Wheat germ oil

919

Corn oil

909

Rape oil

668

Sunflower oil

411

Flax seed oil Cotton seed oil

338a 327a

Soybean oil

320

Oil peanuts

258

Margarine

217

Oil seeds

215

Olive oil

154

Coconut oil

91a

Palm oil

39

Nuts and seeds Sesame

360

Sunflower

300

Pistachios

276

Almond

183

Cashew

158a

Nut Walnut

138 127

Pecan

108a

Peanut

104

Cereals Wheat (germ)

344

Wheat (bran)

200

Buckwheat(flour)

Fig. 2 Example of phytosterol (a), phytostanol (b) and of phytosterols esterified to fatty acid (c) or glycosylated with a hexose (d)

The levels of phytosterols in some vegetables have recently been quantified. A range between 1.1 and 53.7 mg/100 g of edible portion has been observed in vegetables with the highest concentration found in pea, cauliflower, broccoli, and romaine lettuce. The phytosterol contents in fruits ranged from 1.6 to 32.6 mg/100 g, the highest concentration found in navel orange, tangerine, and mango [5] Phytosterols in nuts range from 95 to 280 mg/100 g. Either in fruits and vegetables, the phytosterol content is modulated by ripening and post-harvest practice [6]. Recently, Marcone et al. [7] have demonstrated that amaranth is a rich dietary source of b-sitosterol and other phytosterols (Table 1).

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99

Wheat (wholemeal bread)

86

Rye (flour)

86

Wheat

69

Wheat (wholemeal flour)

70

Wheat

69

Rye

69

Crackers

67

Muesli

63

Corn (flour) Wheat (bread)

52 44

Rice

30

Wheat (flour)

28

Rice (flour)

23

Corn flakes

22

Puffed rice

20

Fruit Passion fruit

44

Orange

24

Fig

22

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Table 1 continued Food source

Total sterol content (mg/100 g)

Grapefruit

18

Lemon

18

Pineapple

17

Clementine

16

Banana

16a

Peach

15

Apple

13

Pear

12

Cherry

12a

Kiwi

9

Melon

2

Watermelon

1

Vegetables Black olives

a

50

Brussels sprout

43

Cauliflower

40

Broccoli

39

Green olives

35

Mushrooms

18

Celery

17

Carrot

16

Cabbage

11a

Fennel

10

Yam

10a

Onion

8

Leeks

8

Green pepper

7

Tomato Potato (boiler)

5 4

Legumes Pea

135a

Kidney bean

127a

Broad bean

124a

a

Sterol content is presented as the sum of b-sitosterol, campesterol and stigmasterol

In vegetable oils, the technological processes used to extract the oil from fruits or seeds influence the levels of phytosterols. Some authors have demonstrated that phytosterol oxidation occurs in vegetable oils during refining [8, 9]. Phytostanol and their esters are mainly contained in cereals such as corn, wheat, rice and triticale.

Phytosterols and phytostanols in functional foods The first phytostanol ester enriched product, a spread, was launched in Finland in late 1995. Since then, phytostanol and phytosterol enriched products have been launched in

several countries and the market for phytosterols as dietary supplements, has lead to a rapidly growing worldwide market for functional foods in the EU and in the USA [10, 11]. The phytosterols and phytostanols added in functional foods are isolated from vegetable oils or are derived from tall oil (a by-product of wood pulp manufacture). They are purified by distillation, extraction, crystallization and washing resulting in products of high purity. Phytosterol blends derived from either vegetable oils or tall oil may be converted to the corresponding phytostanols by catalytic saturation. Phytosterol and phytostanol esters are a mixture of plant sterol esters obtained by esterification of free plant sterols with fatty acids derived from vegetable oils (olive oil, sunflower oil) or fish oils [1]. A number of different types of dairy applications of phytostanol ester enriched foods have been launched [11]. In addition to margarine and fermented milk drinks, there are salad dressing, spreads, milk, soy, yoghurt, cheesy products, soy and fruit drinks, even sausages and breads, ready-to-eat meals, snack bars and candies. Depending on the manufacturer, the commercial product may be a mixture of the extracted sterols, a mixture of free sterols and stanols, sterol and stanol esters or stanol esters.

Dietary intake Diet of different populations varies widely in the amount of plant foods. In Japan, typical plant sterol intakes are approximately 373 mg/day [12]. It is assumed that the normal western-type diet contains about 200–400 mg cholesterol and 200–400 mg plant sterols. Phytostanols are much less abundant in nature than phytosterols and consequently we typically consume much lower amounts (about 25–50 mg/day). For vegetarians dietary phytosterols have been estimated at almost twice this level. The EPIC Norfolk population study has recently investigated the intake of plant sterols in the British diet, using food frequency questionnaires [13]. Bread and other cereals, vegetables and added fats were the three major food sources of plant sterols. Women showed a higher plant sterol levels in plasma than men (36.4 vs. 32.8 mg/1,000 kJ, p \ 0.001) and higher intakes of plant sterols from vegetables, bread and other cereals, added fats, fruits and mixed dishes, whilst men had higher intakes of plant sterols from cakes, scones and chocolate, potatoes and other foods.

Effect of phytosterols on intestinal absorption of cholesterol and fat-soluble vitamins Although cholesterol and phytosterols are structurally similar, their metabolism and intestinal absorption are very

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Fig. 3 Absorption, transport and metabolism of dietary phytosterols. Dietary phytosterols and cholesterol are assembled in micelles. Their uptake by intestinal epithelial cells involves NPC1L1 (Niemann–Pick C1 Like 1) protein. In the enterocytes phytosterols, are esterified by ACAT-2 (acetyl-coenzyme A acetyltransferase 2), packed into

chylomicrons (CM), and drained into the lymph system via the basolateral membrane. Non-esterified cholesterol and phytosterols are pumped back into the intestinal lumen via the ATP-binding cassette transporters (ABCG5 and ABCG8). TG triglycerides

different. Phytosterols and phystanols are very poorly absorbed in human gut and the small amount that is absorbed is actively re-excreted in bile. It has been suggested that the low absorption of plant sterols with respect to cholesterol may represent a defence response. Despite the fact that molecular mechanisms involved in intestinal absorption are not completely elucidated, it has been demonstrated that phytosterols competitively inhibit intestinal absorption of cholesterol [14]. As shown in Fig. 3, both cholesterol and phytosterols require the Niemann–Pick C1 Like 1 (NCP1L1), a protein localized in the brush border membrane to enter therein enterocytes [15]. In enterocytes, cholesterol is transported in the endoplasmic reticulum, where it is esterified via the enzyme acetyl-coenzyme A acetyltransferase 2 (ACAT2), packed into chylomicrons, and drained into the lymph system via the basolateral membrane. Non-esterified cholesterol and phytosterols are pumped back into the intestinal lumen via the ATP-binding cassette transporters (ABC). ABCG5 and ABCG8 are present in the apical membrane of enterocytes and are also expressed in the liver. The complex mechanisms of the transport of sterols are responsible for the assimilation of about 50% of cholesterol. As far as concerned the intestinal absorption of plant sterols range from 0.4 to 5% for phytosterols, and from 0.02 to 0.3% for their saturated counterparts. Consequently, plant stanol concentrations in serum are much lower than plant sterol concentrations. The major part of assimilated phytosterols is directly eliminated via the liver, where they are probably not converted to bile acids and thus excreted much faster into bile compared with

cholesterol [16] (Fig. 3). The biliary excretion rate of phytosterols is opposite to their absorption rate. Contrasting results have been reported about the effect of phytosterol and phytostanols on absorption of fat-soluble vitamins. Fahy et al. [16] using cultured Caco-2 cells, have demonstrated that phytosterols interfere with alphatocopherol or beta-carotene uptake. Studies in vivo [17, 18] have shown that in addition to reducing cholesterol absorption in normocholesterolemic men, plant sterol esters reduced the bioavailability of beta-carotene and alpha-tocopherol more than did plant free phytosterols [18]. However, other authors have reported no significant effects on fat-soluble vitamin levels even at high intake of plant stanols (9 g/day) [19]. No effect of cholesterolstandardized beta-carotene, alpha-tocopherol and lutein concentrations were observed by other authors [20]. Therefore, a structure–function relationship has been suggested that deserves future studies.

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Plasma levels in normal and pathological conditions In circulation, phytosterols are associated with lipoproteins and are mainly localized in low-density lipoproteins (LDL). A wide range of serum-free phytosterol levels (1–17 mg/L; 2.5–41 lmol/L; \1–10% of total sterol) has been demonstrated [21]. Plasma levels of campesterol, b-sitosterol and stigmasterol are, respectively, 14.2 lM (0.58 mg/dL), 7.9 lM (0.327 mg/dL) and 0.26 lM (0.011 mg/dL) [23]. Dietary supplements are reported to increase serum phytosterols levels by 200% [22]. Patients

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affected by phytosterolemia, have a mutation in one of their sterolin genes that allows phytosterols (including stanols) to be more easily absorbed [23]. Therefore, phytosterol levels are much higher (about 1 mM), 10–25 times more phytosterols in their circulation than a normal individual. The fact that patients with this disease present an aggressive vascular disease process despite nearly normal cholesterol levels, brought up the question whether phytosterols are endowed with atherogenous potential [24]. Higher levels of phytosterols (about 60 lmol/L) have been also observed in plasma of subjects on parenteral nutrition (PN) [25]. Phytosterols present in PN-lipid emulsions have been linked to phytosterolemia and cholestatic liver disease. A striking association between phytosterolemia and cholestatic liver disease has been demonstrated by Clayton et al. [26] in pediatric patients after long-term parenteral nutrition. Jansen et al. [27] have demonstrated that dietary plant sterols pass the blood–brain barrier and accumulate in the brain where they may exert brain cell type specific effects.

Phytosterols and cardiovascular risk Many controlled clinical studies have demonstrated the ability of phytosterol to reduce blood cholesterol levels in hyper- and normo-cholesterolaemic subjects [3, 28]. Meta-analyses of more than 40 clinical studies indicate that phytosterols taken as dietary supplements (phytosterols incorporated into fat spreads as well as other food matrices) can induce a reduction of LDL-cholesterol up to 15%. The hypocholesterolemic effect has been related to the ability of phytosterols to interfere with the uptake of both dietary and biliary cholesterol in the intestinal tract [29]. The Mediterranean diet is rich in plant foods containing phytosterol and other bioactive compounds. The PREDIMED study planned to investigate the effect of the increase in phytosterol intake from natural foods has shown that small amounts of phytosterols in natural foods appear to be bioactive in lowering cholesterol in subjects with high cardiovascular risk [30]. In addition to an effect on LDL-cholesterol levels, studies in vitro have demonstrated that plant sterols prevent hyperproliferation of vascular smooth muscle cell that play a role in atherosclerosis development [31].

Interactions between phytosterols and lipid-lowering drugs The mechanisms of action of plant sterols are different from those of lipid-lowering drugs such as 3-hydroxy-3-methylglutaryl coenzyme A inhibitors (statins). Clinical trials have

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been planned to analyze the effect of plant sterols/stanols in combination with statins or other lipid-lowering drugs on normocholesterolemic subjects and hypercholesterolemic patients [32–36]. Till now the results have demonstrated that plant sterols/stanols, when administered in addition to statins, favorably affect total and LDL-cholesterol [32, 33]. Future randomized trials examining the impact of plant sterols/stanols in combination with statins are needed. In fact it has been demonstrated that statins modulate plasma levels of phytosterols. In particular, long-term treatment with large doses of statins increase serum plant sterol concentrations and frequently their ratios to cholesterol [34]. Enhanced intestinal absorption or decreased biliary secretion could explain these findings. Other authors have investigated the effect of association between fibrates and plant sterol enriched spreads. It has been suggested that phytosterol-enriched spread may represent a useful adjunctive therapy for hypercholesterolemic patients [35]. However, the effect of fibrate on plant sterol absorption has not been investigated till now. Other studies carried out in animal models have investigated the effect of the combination of phytosterols and omega-3 fatty acids [36]. The consumption of omega-3 fatty acids is associated with a significant reduction in plasma cholesterol (TC) and triglyceride (TG) concentrations, proaggregatory factors, arrhythmic eicosanoid and thromboxane A2 levels that represent cardiovascular risk factors. Therefore, it has been suggested that combination of phytosterols and n-3 may further reduce cardiovascular risk factors [36]. Human studies are needed to confirm these effects and to investigate its long-term effects.

Effect of phytosterol against lipid peroxidation: in vivo and in vitro studies Oxidative damage of plasma low-density lipoprotein (LDL) and high-density lipoproteins (HDL) plays a key role in the molecular mechanism of atherosclerosis [37, 38]. Oxidized LDL show functional alterations and abnormal interaction with endothelial and circulating cells. Recent studies have demonstrated that plant sterol could exert a modulator effect against oxidative damage [39]. In vivo a decrease of plasma lipid peroxidation has been demonstrated in subjects consuming spread containing plant sterols. The effect was observed at plasma concentration of 14.5 ± 7.3 lmol/L of b-sitosterol and 12.5 ± 5.0 lmol/L of campesterol [40]. Homma et al. [40] have demonstrated that the significant decrease in the levels of plasma LDL-C, and ApoB, in subjects whose diet was supplemented with 2 g/day plant sterols, was associated with a decrease of oxidized–LDL. Other authors have confirmed that lipoprotein changes after diet supplementation with phytosterol are associated with a slight decrease in plasma isoprostanes, a marker of free-radical

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initiated lipid peroxidation [41]. Contrasting results have been reported in vitro. An antioxidant effect exerted by phytosterols has been demonstrated on liposomal membranes oxidized by AMVN (2,20 -azobis-2,40 -dimethylvaleronitrile). Using platelets oxidized in the presence of iron, van Rensburg et al. [42] have shown that 20 lM b-sitosterol decreased the levels of TBARS (thiobarbituric acid reactive substances). However at higher concentrations (40-100 lM), b-sitosterol increased lipid peroxidation [42]. Mora-Ranjeva MP et al. have demonstrated that b-sitosterol induces a significant decrease of TBARS on human keratinocytes oxidized with UVA treatment, whilst stigmasterol increases lipid peroxidation. The effect has been observed when phytosterols replace 50% of cholesterol in keratinocytes [43]. Using macrophages activated by PMA, Moreno et al. have demonstrated a lower production of ROS after treatment with b-sitosterol (50-250 lM) [44]. In our study we have demonstrated that b-sitosterol, stigmasterol and campesterol (5–50 lM) exert a protective effect against Cu??-induced lipid peroxidation of LDL. Moreover, using the analysis of fluorescence emission spectra of tryptophan and Laurdan we demonstrated that phytosterols prevent the alterations of apoprotein structure and physico-chemical properties associated with coppertriggered lipid peroxidation of LDL [45].

Conclusions Even though many different clinical trials have clearly demonstrated that phytosterols reduce LDL-cholesterol, it is unclear whether phytosterols have a positive effect on cardiovascular disease [20]. Some studies have recently demonstrated that there is evidence that elevated levels of plant sterols are associated with an increased cardiovascular risk [46]. In a clinical study it has been demonstrated that patients, who were consuming plant sterol ester enriched margarine, had increased concentrations of plant sterols in vascular tissue [47]. Further mechanistic data suggest that vascular deposits of sterols, when compared with cholesterol, result in increased oxidation and release of oxygen radicals [48]. This hypothesis is supported by the study of Rajaratnam et al. [49] who found that in postmenopausal women, plant sterols were independently associated with coronary heart disease in a multivariate analysis. Larger epidemiological studies reported similar data. Results of the PROCAMstudy showed that patients affected by myocardial infarction or sudden cardiac death had increased plant sterol concentrations [50]. Upper normal levels of plant sterols were associated with a three-fold increase of risk for coronary events among men in the highest tertile of coronary risk according to the

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PROCAM-algorithm. Similar data are available for the plant sterol campesterol from the MONICA/KORA-study. In this prospective study, campesterol correlated directly with the incidence of acute myocardial infarction [51]. However not all such studies confirm an association between phytosterols and coronary artery disease (CAD). The EPIC Norfolk population trial, a prospective study, failed to show a relationship between b-sitosterol and CAD [52]. Contrasting results have been reported about the effect of phytosterols and phytostanols on absorption of fat-soluble vitamins. A lower beta-carotene and alphatocopherol bioavailability described by some authors [17], has not been confirmed in other studies [19, 20] therefore it has been hypothesized that the effect could be related to phytosterol structures. Consumers, in particular children, pregnant and breastfeeding women, should be informed about the possible negative effect on fat-soluble vitamins absorption. Dietary advice to consume an additional daily serving of a high-carotenoid vegetable or fruit should be inserted in food labels of all products containing sterol or stanols [53]. Drug–plant sterol interactions have also been demonstrated [32–36]. The studies on the combined effect of phytosterols in association with lipid-lowering drugs (statins, fibrates) have confirmed a significant decrease of LDL-cholesterol levels. However, plant sterol levels are also increased during drug treatment despite the reduced LDL-cholesterol concentrations [34]. Future studies are necessary to better investigate the role of statins and others lipid-lowering drugs on plant sterol absorption and longterm effects. Recently released guidelines are more critical regarding food supplementation with phytosterols and draw attention to significant safety issues [54, 55]. In conclusion, there are no data available that functional foods supplemented with plant sterol esters reduce cardiovascular disease [56, 57]. Patients affected by sitosterolemia whose levels of phytosterol are 20–25 higher than normal subjects, have an increased risk for cardiovascular disease [2]. The subjects having hereditary sitosterolemia are highly advised to refrain from consuming foods supplemented with phytosterols. Conflict of interest

None.

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