Progress in Lipid Research 41 (2002) 457500 www.elsevier.

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Review

Phytosterols, phytostanols, and their conjugates in foods: structural diversity, quantitative analysis, and health-promoting uses
Robert A. Moreaua,*, Bruce D. Whitakerb, Kevin B. Hicksa
a

Crop Conversion Science and Technology Research Unit, Eastern Regional Research Center, United States Department of Agriculture, Agricultural Research Service, 600 East Mermaid Lane, Wyndmoor, PA 19038, USA b Produce Quality and Safety Laboratory, Beltsville Agricultural Research Center, United States Department of Agriculture, Agricultural Research Service, 10300 Baltimore Avenue, Beltsville, MD 20705, USA Received 1 February 2002; received in revised form 15 March 2002; accepted 22 March 2002

Abstract Phytosterols (plant sterols) are triterpenes that are important structural components of plant membranes, and free phytosterols serve to stabilize phospholipid bilayers in plant cell membranes just as cholesterol does in animal cell membranes. Most phytosterols contain 28 or 29 carbons and one or two carboncarbon double bonds, typically one in the sterol nucleus and sometimes a second in the alkyl side chain. Phytostanols are a fully-saturated subgroup of phytosterols (contain no double bonds). Phytostanols occur in trace levels in many plant species and they occur in high levels in tissues of only in a few cereal species. Phytosterols can be converted to phytostanols by chemical hydrogenation. More than 200 dierent types of phytosterols have been reported in plant species. In addition to the free form, phytosterols occur as four types of conjugates, in which the 3b-OH group is esteried to a fatty acid or a hydroxycinnamic acid, or glycosylated with a hexose (usually glucose) or a 6-fatty-acyl hexose. The most popular methods for phytosterol analysis involve hydrolysis of the esters (and sometimes the glycosides) and capillary GLC of the total phytosterols, either in the free form or as TMS or acetylated derivatives. Several alternative methods have been reported for analysis of free phytosterols and intact phytosteryl conjugates. Phytosterols and phytostanols have received much attention in the last ve years because of their cholesterol-lowering properties. Early phytosterol-enriched products contained free phytosterols and relatively large dosages were required to signicantly lower serum cholesterol. In the last several years two spreads, one containing phytostanyl fatty-acid esters and the other phytosteryl fatty-acid esters, have been commercialized and

Mention of a brand or rm name does not constitute an endorsement by the US Department of Agriculture above others of a similar nature not mentioned. * Corresponding author. E-mail address: rmoreau@arserrc.gov (R.A. Moreau).
0163-7827/02/$ - see front matter Published by Elsevier Science Ltd. PII: S0163-7827(02)00006-1

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were shown to signicantly lower serum cholesterol at dosages of 13 g per day. The popularity of these products has caused the medical and biochemical community to focus much attention on phytosterols and consequently research activity on phytosterols has increased dramatically. Published by Elsevier Science Ltd.

Contents 1. Introduction and a primer on phytosterol nomenclature....................................................................................... 459 2. Structural diversity and phylogenetic distribution of phytosterols ........................................................................ 465 2.1. Occurrence and metabolism of cholesterol in plants ..................................................................................... 465 2.2. Distribution and diversity of major C-24 alkyl phytosterols......................................................................... 466 2.3. Free and conjugated phytosterols in fruits and vegetables............................................................................ 467 2.4. Unique phytosterols and phytosterol conjugates in cereals........................................................................... 470 2.5. Function of 24-alkyl phytosterols and their conjugates ................................................................................ 470 2.6. Steroidal saponins ......................................................................................................................................... 471 2.7. Steroidal glycoalkaloids................................................................................................................................. 472 2.8. Phytoecdysteroids and brassinosteroids ........................................................................................................ 474 3. Methods for the Quantitative Analysis of Phytosterols and Phytostanols............................................................. 476 3.1. Extraction and fractionation of phytosterols ................................................................................................ 476 3.2. Methods for the separation of intact phytosterol classes .............................................................................. 477 3.3. Methods for the separation of molecular species of phytosterol conjugates ................................................. 478 3.4. Methods for hydrolysis of conjugates and separation of free phytosterols ................................................... 479 3.5. Mass spectrometry and NMR of phytosterols .............................................................................................. 481 3.6. Enzymatic assays of phytosterols .................................................................................................................. 482 4. Health-promoting eects of phytosterols, phytostanols, and their esters .............................................................. 483 4.1. Historical perspectives, changing dogma, and critical questions ................................................................... 483 4.2. Active forms and mechanism of action of steryl and stanyl esters................................................................ 485 4.3. Recent clinical studies on phytostanyl esters................................................................................................. 485 4.4. Recent clinical studies on phytosteryl esters.................................................................................................. 486 4.5. Recent clinical studies on free phytosterols and phytostanols....................................................................... 486 4.6. Relative LDL-C lowering ecacy: sterols vs stanols & esters vs free forms ................................................. 487 4.7. Health claims................................................................................................................................................. 488 4.8. Reduction in the risk of coronary heart disease ............................................................................................ 488 4.9. Toxicology, anticancer properties and potential benets .............................................................................. 488 4.10. Eects on absorption of fat soluble vitamins and antioxidants .................................................................... 489 4.11. Dosage levels and frequency.......................................................................................................................... 490 4.12. Phytosterol/phytostanol products: past, present, and in development .......................................................... 491 5. Conclusions ............................................................................................................................................................ 494 Acknowledgements...................................................................................................................................................... 494 References ................................................................................................................................................................... 495

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Nomenclature ASG DGDG FFA FS FSE HDL-C HSE LDL-C Lyso-PC SE PE PI PC SG TAG acylated steryl glycoside digalactosyldiacylglycerol free (non-esteried) fatty acids free sterol ferulate steryl ester high density lipoprotein serum cholesterol hydroxycinnamate steryl ester low density lipoprotein serum cholesterol lysophosphatidylcholine steryl fatty-acid ester phosphatidylethanolamine phosphatidylinositol phosphatidylcholine steryl glycoside triacylglycerol

1. Introduction and a primer on phytosterol nomenclature Phytosterols (plant sterols) are members of the triterpene family of natural products, which includes more than 100 dierent phytosterols and more than 4000 other types of triterpenes [1,2]. Cholesterol is the predominant sterol in animals, wherein free cholesterol serves to stabilize cell membranes and cholesteryl fatty-acid esters are a storage/transport form, usually found associated with triacylglycerols [3]. Plant membranes contain little or no cholesterol and instead contain several types of phytosterols that are similar in structure to cholesterol but include a methyl or ethyl group at C-24. In general, phytosterols are also thought to stabilize plant membranes, with an increase in the sterol/phospholipid ratio leading to membrane rigidication [4]. However, individual phytosterols dier in their eect on membranes stability. Stigmasterol has been reported to have a disordering eect on membranes [5] and the molar ratio of stigmasterol to other phytosterols in the plasma membrane increases during senescence [6]. All triterpenes are synthesized via a pathway that starts with reduction of HMG-CoA (six carbons) to mevalonate (ve carbons). Six mevalonate units are then assembled into two farnesyl diphosphate molecules, which are combined to make squalene (30 carbons or three terpenes). Enzymatic ring closure steps then form cycloartenol (also 30 carbons), and additional enzymatic reactions form common plant triterpenes such as phytosterols, triterpene alcohols, and brassinosteroids (Fig. 1). There have been several excellent reviews on phytosterols, most notably a review by Goad [1], which focused on phytosterol chemistry and analytical methods, and one by Piironen and colleagues [3], which was a very comprehensive review of biological, chemical, and nutrition aspects of phytosterols. The purpose of this review is not to be a comprehensive treatise on all of phytosterol chemistry, structure and function, but to focus on three areas of phytosterol research that may be of interest to scientists largely unacquainted with the eld. The three topics are as follows: (A) the occurrence of various phytosterols in plants and a discussion of the nomenclature, (B)

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Fig. 1. Biosynthesis of phytosterols and other triterpenes.

modern analytical tools used to quantify and identify phytosterols and a comparison of the various analytical methods, and (C) recent advances in the health-promoting and nutraceutical aspects of phytosterols. Phytosterol nomenclature is confusing because international attempts at standardization have been only partially adopted. The two main nomenclatures (Fig. 2) currently utilized follow the IUPAC-IUB recommendations of 1976 and 1989 [1]. Our approach in this review will be to refer to phytosterols by their common (trivial) names and in the latter part of this section we will list the common phytosterols, alternative names, systematic names, molecular masses, and CAS (Chemical Abstracts Service) registry numbers. A convenient way to describe and catalog phytosterols is to divide them into three groups based on the number of methyl groups on carbon-4, two (4-dimethyl), one (4-monomethyl), or none (4-desmethyl). 4-Dimethylsterols and 4a-monomethylsterols are metabolic intermediates in the biosynthetic pathway leading to end-product, 4-desmethyl phytosterols, but they are usually present at low levels in most plant tissues. Cycloartenol and cycloartanol are examples of 4-dimethylsterols, and gramisterol is an example of a 4a-monomethylsterol (Fig. 3).

Cycloartanol, also called 9,19-cyclo-5a, 9b-lanostan-3b-ol or cycloartan-3b-ol, C30H52O, MW 428.74, CAS # 4657-58-3. Found in rice bran oil and the rhizomes of Polypodium vulgare. Gramisterol, also called 24-methylenelophenol, C29H49O, MW 412.69, CAS# 1176-52-9.

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Fig. 2. Nomenclature of phytosterols (example: sitosterol=stigmast-5-en-3b-ol=24R-ethylcholest-5-en-3b-ol).

Fig. 3. 4-Monomethyl and 4,4-dimethylphytosterols.

4-Desmethylsterols include the 27-carbon sterol cholesterol (Fig. 4) (ubiquitous and predominant in animals, but also generally present in plants at low levels) and all of the common 28-carbon (Fig. 5) and 29-carbon (Fig. 6) phytosterols, which are typically major membrane structural components in plant cells. Most 4-desmethyl phytosterols have a double bond between carbons 5 and 6 of the ring system and are thus called R 5 phytosterols. However, another group of common desmethylsterols that are abundant in plants of certain families have a double bond

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Fig. 4. C27 4-desmethyl phytosterols.

Fig. 5. C28 4-desmethyl phytosterols. Note that some of these compound are 24a (solid wedge) and some are 24b (dashed wedge). Catalytic hydrogenation (a method currently used in the production of commercial stanyl ester products) of the unsaturated C28 phytosterols can yield two 24-methyl epimers, campestanol (24a=24R) or ergostanol (24b=24S), depending on the phytosterol composition of the original material.

between carbons 7 and 8 instead of 5 and 6, and are hence referred to as R 7 phytosterols. Both R 5 and R 7 desmethylsterols can include a second double bond in the alkyl side chain, most frequently between carbons 22 and 23 or carbons 24 and 28 (carbons 24 and 241 in the 1989 IUPAC nomenclature). The common 29-carbon desmethylsterol stigmasterol (Fig. 6), which includes both C5,6 and (trans) C22,23 double bonds, is, for example, designated as R5,22E. For the C28 and C29 phytosterols the introduction of a methyl or ethyl group at C24 renders this position chiral and thus two epimers are possible. The nomenclature of the conguration of

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Fig. 6. C29 4-desmethyl phytosterols. Note that catalytic hydrogenation (a method currently used in the production of commercial stanyl ester products) of unsaturated C29 phystosterols can yield stigmastanol and/or its 24 b epimer, depending on the phytosterol composition of the original material.

the C24 methyl, C24 ethyl, or C24 ethylidene groups on the C28 and C29 phytosterols requires some explanation. For the seven common C28 phytosterols listed in Fig. 5, three (campesterol, epibrassicasterol, and campestanol) are 24a epimers (with the methyl group indicated as a solid wedge), and the other ve phytosterols are 24b epimers (with the methyl group indicated as a dashed wedge). The 24-methyl epimers are also designated 24R and 24S, which are equivalent to 24a and 24b, respectively, unless there is a double bond at C22,23, in which case the chirality is reversed (24R=24b and 24S=24a). With the C29 phytosterols, the good news is that almost all phytosterols are 24a-ethyl epimers. Unfortunately, three of the common C29 phytosterols have a ) and the resulting ethylidene group can either be cis or trans. The double bond at C24,28 (C24,24R C24 ethylidene in fucosterol is the trans isomer and is designated as a 24E, whereas the C24 ethylidene in R 5-avenasterol and R 5-avenasterol is the cis isomer and is designated as 24Z. Fortunately, the C22,23 double bond in common phytosterols (brassicasterol, epibrassicasterol, stigmasterol, and 7-stigmasterol (spinasterol) only occur as 22E. 27-Carbon 4-desmethylsterols (Fig. 4). Cholesterol, cholest-5-en-3b-ol, C27H46O, mol. wt. 386.65, CAS# 57-88-5. The common sterol in animal tissues. Also occurs in the date palm, Phoenix dactylifera, and in many marine red algae (Rhodophyceae). Desmosterol, also called cholesta-5,24-dien-3b-ol, 24-dehydrocholesterol, C27H44O, MW 384.63, CAS# 313-04-02.

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Lathosterol, also called 5a-cholest-7-en-3b-ol, C27H46O, mol. wt. 386.65. Lathosterol is the C27 precursor to phytoecdysteroids in spinach (Spinacia oleracea). Cholesta-5,7-dien-3A-ol, also called 7-dehydrocholesterol, R 7-cholesterol, and Provitamin D3, C27H44O, MW 384.63, CAS# 434-16-2. 28-Carbon 4-desmethylsterols (Fig. 5) Campestanol (saturated) (24a=24R), also called (24R)-24-methylcholestan-3b-ol or (24R)ergostan-3b-ol, C28H50O, MW 402.70, CAS # 474602. Occurs naturally in corn ber oil, almost exclusively as a ferulate or p-coumarate esters. Generated by catalytic hydrogenation of campesterol or epibrassicasterol. Ergostanol (saturated) (24b=24S), also called (24S)-24-methylcholestan-3b-ol, C28H50O, MW 402.70. Not reported naturally but can be generated by catalytic hydrogenation of brassicasterol, 22-dihydrobrassicasterol, or ergosterol. Campesterol (R 5) (24a=24R), also called (24R)-24-methylcholest-5-en-3b-ol, campest-5-en-3bol, or R 5-24a-methyl-cholesten-3b-ol, (24R)-ergost-5-en-3b-ol, C28H48O, mol. Wt. 400.68, CAS # 474-62-4. Widespread occurrence in plants. 22-Dihydrobrassicasterol (R5) (24b=24S), also called ergost-5-en-3b-ol and 24-epicampesterol, C28H48O, mol. Wt. 400.68, CAS # 4651-51-8. Brassicasterol (R5,22E) (24b=24R), also called (22E)-ergosta-5,22-dien-3b-ol, C28H46O, MW 398.66, CAS # 474-67-9. Found in rapeseed oil from Brassica napus. Epibrassicasterol (R 5,22E) (24a=24S), also called (22E)-(24S)-24-methylcholesta-5,22-dien-3bol or (22E)-campesta-5,22-dien-3b-ol, C28H46O, MW 398.66, CAS # 17472-78-5. Ergosterol (R 5,7,22E) (24b=24R), also called (22E)-ergosta-5,7,22-trien-3b-ol, C28H44O, MW 396.54, CAS # 57-87-4. Occurs in yeasts and many other fungi. 29-Carbon 4-desmethylsterols (Fig. 6) Sitostanol (saturated) (24a=24R), also called stigmastanol, stigmastan-3b-ol, 24a-ethylcholestan-3b-ol, C29H52O, MW 416.40, CAS # 19466-47-8. Occurs in corn ber oil, almost exclusively as a ferulate ester or p-coumarate esters. Sitosterol (R 5) (24a=24R), also called b-sitosterol, stigmast-5-en-3b-ol, 24a-ethylcholest-5-en3b-ol, C29H50O, MW 414.71, CAS # 83-46-5. Widespread occurrence in plants. $ 7-Stigmastenol (7-Stigmastenol) (R 7) (24a=24S), also called, 22-dihydrospinasterol, stigmasta-7-en-3b-ol, C29H50O, MW 414.71, CAS # 521-03-9.

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Stigmasterol (R 5,22E) (24a=24S), also called (22E)-stigmasta-5,22-dien-3b-ol or 24a-ethylcholesta-5,22E-dien-3b-ol, C29H48O, MW 412.69, CAS # 83-48-7. Widespread occurrence in plants. Fucosterol (R5,24E), also called [24(28)E]-stigmasta-5,24(28)-dien-3b-ol, [24(24R )E]-stigmasta)-dien-3b-ol, or 24E-ethylidenecholesta-5,24(28)-dien-3b-ol, C29H48O, MW 412.69, 5,24(24R CAS # 17605-67-3. Found in coconut pollen, Cocos nucifera, and in many brown algae, e.g. Fucus vesiculosus. $ 5-Avenasterol (5-Avenasterol) (R 5,24Z), also called isofucosterol, 28-isofucosterol, 29-isofucosterol, 24Z-ethylidenecholesta-5,24(28)-dien-3b-ol, [24(28)Z]-stigmasta-5,24(28)-dien-3b)Z]-stigmasta-5,24(28R )-dien-3b-ol, C29H48O, MW 412.69, CAS # 18472-36-1. ol, or [24(24R Found as a major phytosterol in oats, and in signicant levels in other plant materials. $ 7-Stigmasterol (7-Stigmasterol) (R 7,22E) (24a=24S), also called spinasterol, (22E)-stigmasta7,22-dien-3b-ol, C29H48O, MW 412.69, CAS # 481-18-4. $ 7-Avenasterol (7-Avenasterol)(R 7,24Z), also called avenasterol, (24Z)-24-ethylidenecholesta7,24(28)-dien-3b-ol, C29H48O, MW 412.69, CAS # 23290-26-8. In all plant tissues, phytosterols occur in ve common forms (Fig. 7): as the free alcohol (FS), as fatty-acid esters (SE), as steryl glycosides (SG), and as acylated steryl glycosides (ASG). The last three forms (SE, SG, and ASG) are generically called phytosterol conjugates. In free phytosterols (FS), the 3b-OH group on the A-ring of the sterol nucleus is underivatized, whereas in the three conjugates the OH is covalently bound with another constituent. The OH group is ester-linked with a fatty acid in SE and linked by a 1-O-b-glycosidic bond with a hexose (most commonly glucose) in SG (rst reported by Power and Salway in 1919 [7]). The third group of phytosterol conjugates, ASG, dier from SG by the addition of a fatty acid esteried to the 6-OH of the hexose moiety (rst reported by Lepage in 1964) [8]. Seeds of corn and rice and other grains contain a fourth type of phytosterol conjugate, phytosteryl hydroxycinnamic-acid esters (HSE), in which the sterol 3b-OH group is esteried to ferulic or p-coumaric acid (Fig. 7).

2. Structural diversity and phylogenetic distribution of phytosterols 2.1. Occurrence and metabolism of cholesterol in plants There is a widespread misconception, perhaps fostered by the nutritional information printed on packages and containers of various foods of plant origin, that plant tissues are devoid of cholesterol. The fact is that this C27 sterol, which is predominant in animals and a contributing factor in human cardiovascular disease, often accounts for 12% of the total plant sterols, and can compose 5% or more in select plant families, species, organs, or tissues. Despite the fact that the edible portion of some crop plants can include cholesterol as a signicant portion of the total phytosterols, it should be noted that this is inconsequential in the human diet relative to the amount of cholesterol in meat and dairy products. Many species of the Solanaceae (Nightshade

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Fig. 7. Structures of phytosterol conjugates. The sites of cleavage via alkaline hydrolysis (saponication) and acid hydrolysis are indicated with arrows.

family) include relatively high levels of cholesterol [9]. In total sterols from pericarp tissue of mature-green tomato (Lycopersicon esculentum) fruit, cholesterol constituted e 6%, and in the SE fraction it ranged from 15 to 20% [10,11]. A study of the sterol composition of seed oil from 13 species of Solanum showed that in six species the combined FS plus SE fractions contained R 5% cholesterol, and the level in one species, S. pseudocapsicum, ranged from 10 to 22% [12]. A recent molecular-genetic study demonstrated that the activity of sterol methyltransferase 1 (SMT1) governs the level of cholesterol in plants [13]. SMT1 catalyzes the rst step in the production of C28 and C29 phytosterols, methylation of cycloartenol to 24-methylene cycloartenol. In mature Arabidopsis plants bearing an smt1 null mutation, cholesterol was the major sterol and composed 26% of the total phytosterols, compared with 6% in wild-type plants. There is ample evidence that in plants cholesterol serves as a precursor in the synthesis of steroidal saponins and alkaloids, as well as ecdysteroids (insect molting hormones) and other pregnane- and androstane-type steroids (see Sections 2.6, 2.7, and 2.8) [1417]. In medicinal herbs and food plants, steroidal saponins and alkaloids are of interest because of their potential pharmacological activity and/or toxicity in animals. 2.2. Distribution and diversity of major C-24 alkyl phytosterols The most commonly encountered phytosterols in higher plants are the 24a-methyl (24R) sterol campesterol (Fig. 5), the 24a-ethyl (24R) sterol sitosterol (Fig. 6), and the 24a-ethyl (24S, due to

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the 22E double bond) sterol stigmasterol (Fig. 6). Each of these possesses one double bond at C5,6 in the B-ring of the sterol nucleus (R5). Typically, campesterol occurs in approximately a 2:1 ratio with its 24b-methyl epimer ergost-5-en-3b-ol (22-dihydrobrassicasterol, Fig. 5) [18], which is not readily apparent because the two isomers are not resolved by GLC or HPLC [19]. The stereochemistry at C-24 is determined by the particular sterol methyltransferase which converts the initial phytosterol precursor cycloartenol (Fig. 3) to 24-methylene cycloartenol [20]. Another 24b-methyl R 5 sterol found predominantly in species of the Brassicaceae (also known as the Cruciferae or Cruciferaceae, Mustard family) is brassicasterol (ergosta-5,22E-dien-3b-ol, Fig. 5), which generally accounts for less than 10% of the total phytosterols in crops such as cabbage, broccoli, and canola. A second major group of desmethyl sterols found in relatively few plant families includes 22dihydrospinasterol (7-stigmastenol, stigmast-7-en-3b-ol) and spinasterol (7-stigmasterol, stigmasta-7,22E-dien-3b-ol) as predominant constituents (Fig. 6). These are the R7 equivalents of the 24a-ethyl R 5 sterols sitosterol and stigmasterol, respectively, and apparently fulll the same function as membrane structural components. Crops from the Cucurbitaceae (Cucumber family), including melon, squash, cucumber, and pumpkin, all contain close to 100% R 7 phytosterols, whereas outside of this family the only crop plant with such a high percentage appears to be spinach, unless one includes tea as well (Camellia sinensis, a member of the Theaceae) [21]. Studies early in the last decade revealed that R7 sterols are more widely distributed than previously thought, and are abundant in many species from ve families within the order Caryophyllales, including the Amarathaceae, Caryophyllaceae, Chenopodiaceae, Phytolaccaceae, and Portulacaceae [21,22]. Often these species include a blend of R 7 and R5 phytosterols, as exemplied by the crop plants Beta vulgaris (table beet), with a R 7 to R 5 ratio of about 7:3, and Chenopodium quinoa (Inca wheat), with a R 7 to R 5 ratio of about 1:3 [23]. An interesting quirk of cucurbit crops is that the seeds and seedlings generally contain high levels of 24b-ethylcholesta7,25-dienol and 24b-ethylcholesta-7,22,25-trienol, as well as small amounts of R 5 phytosterols, which largely disappear as the plants grow to maturity [19,24,25]. 2.3. Free and conjugated phytosterols in fruits and vegetables The sterol lipid composition of mature-green tomato fruit is much like that of tomato leaves, and is quite unusual relative to most plant tissues and organs in that ASG accounts for more than half, and ASG plus SG compose 8590%, of the total sterols [10,26]. With tomato fruit ripening, a substantial increase in sterol synthesis was accompanied by marked changes in sterol composition and conjugation [10,27]. An increase in the ratio of stigmasterol to sitosterol, the two major phytosterols, was evident in all four sterol lipid classes but was most pronounced in FS [10,27]. Although stigmasterol diers from sitosterol only by the 22E-double bond in the alkyl side chain, it has been elegantly demonstrated that these two sterols have markedly dierent eects on the permeability, ordering, and uidity of plant phospholipid vesicles [28,29]. In addition to the large increase in stigmasterol with ripening, there was a reapportioning of sterols among the four steryl lipid classes. Both FS and SG increased at the expense of ASG, and the level of SE was 10-fold higher in red-ripe compared with mature-green fruit [10]. A less dramatic increase in SE has been reported to occur with senescence of both tomato leaves and potato tubers [26]. In a sterol-overproducing tobacco mutant cell line that was selected for resistance to a triazole sterol biosynthesis

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inhibitor, the excess sterol was mainly metabolic intermediates that were esteried to fatty acids (SE) and sequestered in cytoplasmic lipid droplets. It was concluded that SE are involved in removing improper sterols from the FS pool, thereby assuring proper sterol composition in cell membranes [3032]. SE from tomato fruit were also enriched in sterol intermediates and even late in ripening there was preferential esterication of sitosterol over stigmasterol [10]. Storage of mature-green tomato fruit at 2 v C for 1121 days (chilling) resulted in about a twofold increase in the level of FS. Tomatoes do not ripen at 2 v C and the increase in stigmasterol noted with ripening of the same lot of fruit at 15 v C was greatly attenuated [11,33,34]. In microsomal membranes from pericarp of Pik-Red fruit stored at 2 v C, the doubling of FS was oset by a decrease in ASG, suggesting that sterol glycosylation and esterication are inhibited at low temperature. This increase in the FS:ASG ratio may be a means of acclimation to low temperature. The esteried fatty acids in tomato ASG are about 7580% saturated [33,34], so a drop in the proportion of ASG might serve to increase the uidity of cell membranes. In accord with a possible role of ASG in thermal acclimation, a 3-day heat treatment of mature-green tomatoes at 38 v C elicited a marked increase in ASG at the expense of SG and FS [35]. Four days after green tomatoes chilled for 21 days at 2 v C were transferred to 20 v C, the distribution of sterols in ASG, SG, FS, and SE had essentially returned to the pre-storage levels (63, 19, 16, and 3 mol%, respectively), but the percentage of stigmasterol had risen dramatically [34]. These fruit subsequently showed symptoms of chilling injury, delayed and uneven ripening and extensive decay. Changes in sterol lipid content and composition in pericarp tissue during ripening of bell pepper fruit are much less dramatic than in tomato fruit [36]. In contrast to tomato, sitosterol and campesterol, in a ratio of about 3:1, are the major sterols in all four sterol lipid classes in bell pepper. Only small amounts of stigmasterol are present. FS is the most abundant of the sterol lipid classes, ranging from e 55 to 75% of the total sterols in pericarp from fruit of dierent cultivars and separate harvests [36,37]. The level of FS changed little with ripening and SE rose only slightly, from e 5 to 7% of the total sterols. The only signicant change among the sterol lipid classes, which occurred both with ripening of fruit on the plant and during a 2-week storage of maturegreen fruit at 2 v C, was a 50100% increase in SG, largely balanced by a decline in ASG [36,37]. The sterol lipid distribution in microsomal membranes isolated from bell pepper pericarp tissue was shown to change much more with ripening in the eld in summer than with ripening in the greenhouse in spring [38]. Overall, SG and ASG composed greater proportions of total sterol lipids in microsomes than in whole pericarp, and a pronounced increase in microsomal SG with ripening was compensated by declines of both ASG and FS. In microsomes from red-ripe eldgrown fruit, SG, FS, and ASG comprised 50, 28, and 22 mol% of the total sterols, respectively. A very informative early study by Hartmann and Benveniste [39] found that a burst of respiration and metabolic activity following slicing of potato tuber tissue is accompanied by a sharp increase in de novo sterol synthesis (that utilized radiolabeled acetate). The rst sterol product, cycloartenol, was converted to desmethyl sterols only after several hours of aging. Initially, 28-isofucosterol composed 30% of the FS fraction and pulse-chase labeling indicated that it was the precursor to sitosterol and stigmasterol. In retrospect, this work provided the rst indication that regulation of carbon ow into end product (24a methyl or ethyl, 4,14-desmethyl) sterols occurs at a step beyond squalene cyclization and formation of cycloartenol [3,40]. As stated in Section 2.2, phytosterols of the cucurbit crops are predominantly R 7, 24a-ethyl, but also include an interesting array of unusual minor constituents. The peel and outer pericarp

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tissues of zucchini squash (Cucurbita pepo) were shown to have 7-stigmasterol (spinasterol) and 7-stigmastenol (22-dihydrospinasterol) as the dominant FS, each at close to 40% of the total [41]. Minor sterols included 7-isoavenasterol (24E-ethylidene) at about 8%, and 7-campestenol, 8,22stigmastadienol, sitosterol, stigmastanol, 8(9)-stigmastenol, 7,25(27)-stigmastadienol, and 7-avenasterol (24Z-ethylidene), all in the range of e 1 to 3%. FS and SE were present in about a 12:1 ratio; ASG and SG were not analyzed. In a postharvest study of muskmelons (Cucumis melo), the sterol lipid composition was analyzed in plasma membrane (PM) isolated from hypodermal mesocarp tissue of mature fruit kept for either 1 night at 4 v C (pre-storage condition) or for 7 days at 4 v C plus 3 days at 21 v C (post-storage condition) [42]. In PM from pre-storage melons, FS, ASG, and SG comprised 43, 37, and 20 mol% of the total sterol lipids, respectively, whereas in PM from post-storage fruit, the corresponding percentages were 57, 32, and 11. The ratio of 7-stigmasterol to 7-stigmastenol, the two predominant sterols, was about 1.1 in FS and about 0.5 in both ASG and SG. The PM sterol composition did not change signicantly during storage in any of the sterol lipid classes. Pre-storage gamma irradiation of whole fruit at 1.0 kGy caused a transient decline in PM H+ATPase activity, which was associated with both an increase in the proportion of FS and decrease in the 7-stigmasterol:7-stigmastenol ratio in all three sterol lipid classes. By the end of storage, however, the sterol lipid content and composition was similar in PM from control and irradiated melons, and H+-ATPase activity was higher in PM from the irradiated fruit. Apple (Malus domestica) fruit are the antithesis of tomato with respect to sterol conjugates, and are surely more representative of the majority of plant tissues. At harvest, the concentrations of FS, SG, and ASG in outer cortical tissue of Golden Delicious apple were 145, 97, and 3 nmol/g fresh weight, respectively [43]. The mole ratio of FS:SG:ASG changed from 59:40:1 at harvest to 65:33:2 after 15 weeks of storage at 0 v C plus 1 week at 20 v C, mainly due to a decline in SG from 97 to 73 nmol/g fresh weight. It is interesting that a 4-day heat treatment at 38 v C just after harvest also induced a specic reduction in SG to 77 nmol/g fresh weight. The level of ASG was increased after a longer duration of cold storage (6 months at 0 v C), but the concentrations of FS and SG remained about the same [44]. The sterol composition of both FS and SG included 90 95% sitosterol and changed little with storage. Minor sterols in the FS fraction were identied as campesterol, stigmasterol, 5,7-stigmastadienol, and 5,25(27)-stigmastadienol [43]. Carrot (Daucus carota) storage root tissue appears to respond to wounding in much the same way that potato tuber tissue does, i.e. there is an induction of de novo phytosterol synthesis associated with cell membrane proliferation and repair [45]. However, tissues from the two storage organs do dier markedly with respect to enzymatic hydrolysis of glycerolipids directly after wounding, which is extensive in potato but minimal in carrot [46]. In a pair of studies, freshly shredded carrots were stored at 10 v C and 95% relative humidity for up to 10 days, and samples were taken periodically for analysis of membrane sterol lipid and glycerolipid contents [45,47]. After 10 days of storage, total sterol lipids (FS+ASG+SG) in the shredded tissue had increased by 2428%. In the rst study, the mole ratio of FS:ASG:SG in the carrot tissues changed from 76:17:7 immediately after shredding to 64:30:6 after 10 days, whereas in the second study the change was from 56:29:15 to 60:33:7. The tissue concentrations of ASG and FS increased in both storage experiments, whereas SG remained the same or declined. In the FS fraction, sitosterol composed 5668%, stigmasterol 1628%, and campesterol 1012%. The only signicant change in FS composition with storage was an increase in the stigmasterol:sitosterol ratio from 0.30 to 0.45 during the last 5 days at 10 v C.

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2.4. Unique phytosterols and phytosterol conjugates in cereals Several unique phytosterols and phytosterol conjugates have been reported in cereal grains. Seitz [48] reported trans-hydroxycinnamate esters of phytosterols (HSE, including steryl ferulate and p-coumarate esters) of phytosterols in corn, wheat, rye, rice, and triticale. Norton [49,50] extended these studies and separated several additional molecular species of hydroxycinnamate esters from rice bran and corn bran. We reported that the levels of hydroxycinnamate esters in corn ber were higher than in corn bran or any other grain [51]. In a recent paper, we compared the levels of SE, FS, and HSE in 66 accessions of Zea, teoscinte and Jobs tears, and identied several corn accessions with very high levels of HSE and total phytosterols [52]. Both Seitz and Norton noted that sitostanol was the predominant phytosterol in corn HSE [48,50], whereas cycloartanol and 24-methylene cycloartenol were the predominant phytosterols in rice bran HSE (called oryzanol). Piironen and colleagues [53] recently compared the composition of total sterols (free+bound) in rye, oats, barley, wheat, corn and other grains. They found that all grains contained signicant levels of phytostanols (sitostanol and campestanol) in the total phytosterol fractions. Recent studies from our lab indicate that most of the phytostanols in corn are esteried in either SE or HSE, and all of the HSE is localized in the aleurone cells which form a single layer in corn, and fractionates into the corn ber fraction during wet milling [54]. Since commercial corn oil is obtained by extracting corn germ, the levels of HSE and phytostanols in corn germ oil are very low [55,56]. 2.5. Function of 24-alkyl phytosterols and their conjugates In plant as in animal cells, the plasma membrane is greatly enriched in sterols relative to other cell membranes [57,58]. The profound eects of sterols on the physical properties of membranes are well documented [59]. Through interaction with phospholipids in a one to two stoichiometry, sterols condense the bilayer, reduce bulk uidity and permeability, and broaden or eliminate phospholipid phase transitions [60,61]. Sterolphospholipid interactions inuence membrane functions such as simple diusion, carrier-mediated diusion, and active transport, and also modulate the activities of membrane-bound enzymes or receptors [62]. Although a wide variation in sterol structure can be accommodated to fulll the bulk membrane structural requirement [59], there appear to be other, more subtle functions or specic situations, such as salt stress, for which sterol structure becomes more critical [63,64]. Evidence from sterol biosynthesis inhibitor studies indicate that phytosterols also play an essential role in plant cell division [6567]. Compared with work on membrane phospholipids, the role of sterol lipids in pre- and postharvest plant physiology has received little attention [3,68,69]. A sharp increase in the sterol:phospholipid ratio in microsomal membranes during plant senescence is associated with loss of membrane function [70,71]. Changes in sterol composition likely to aect membrane function [28,29,57] can occur with greening, shading, maturation, aging, or ripening of plant tissues [10, 7274] . There is evidence that free sterols are tightly bound to the plasma membrane H+-ATPase and may be essential for activity of this critical enzyme [75]. Further work has shown that H+ATPase activity is dependent upon the kind and amount of sterol present in a reconstituted system [76,77]. Finally, Zelazny and colleagues [78] showed that free sterols in the plasma membrane of the marine alga Dunaliella are absolutely required for sensing osmotic changes.

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Sterol conjugation, the conversion of free sterols (FS) to steryl esters (SE), steryl glycosides (SG), or acylated steryl glycosides (ASG), is another potentially important aspect of membrane lipid metabolism. Like FS, SG and ASG are membrane structural components, whereas SE appear to be largely excluded from membranes [31,79], possibly because of their relatively low solubility in a phospholipid bilayer [80]. Metabolic studies have shown that interconversion of sterols and sterol conjugates is quite rapid, suggesting a regulatory function [81]. Based on reports that sterol interconversions are controlled by phytohormone levels and environmental factors such as light, temperature, and water stress, it has been postulated that they are involved in the regulation of membrane properties in response to changing conditions [62]. Kesselmeier and colleagues [82] reported an increase in the levels of SG and ASG during the enzymatic preparation of protoplasts from oat leaves. In the course of our work, we have found that the extent of sterol glycosylation and esterication can be dramatically altered in response to ozone stress in snapbean leaves [83], growth conditions in bell pepper fruit [38], freezing stress in potato leaf plasma membrane [84], chilling stress in tomato fruit [34], and challenge by fungal elicitors, cellulase, xylanase, or copper ions in tobacco cells [85]. In particular, it appears that dierent types of stress can promote conversion of FS to ASG [82,83,85]. Recently, Peng and colleagues [86] reported that sitosterol-b-glucoside serves as a primer for cellulose synthase in plants. Since it is thought that most of the ASG and SG is localized in the plasma membrane [84,85], the involvement of SG in a biosynthetic process that occurs adjacent to the plasma membrane is a reasonable hypothesis. 2.6. Steroidal saponins Steroidal saponins consist of a furostanol- or spirostanol-based aglycone (Fig. 8) and an oligosaccharide of typically 25 hexose or pentose moieties attached to the 3b-OH of the steroid nucleus. These saponins are abundant in a number of monocot species, including the crops onion, garlic, and leek (Allium spp.), Asparagus, oats (Avena sativa), and yam (Dioscorea spp.), and have also been described in several solanaceous crops, including eggplant (Solanum melongena), bell pepper (Capsicum annuum), and tomato, and the leguminous herb fenugreek (Trigonella foenumgraecum) [16, 8790]. Glucosylation of yamogenin (25S-spirost-5-en-3b-ol) at the 3b-OH by a steroid-specic UDP-glucose-dependent glucosyl transferase, the rst step in generation of a steroidal saponin from the sapogenin aglycone, was demonstrated in a preparation from Asparagus plumosus [89]. Also, the furostanol saponins are usually present as 26-O-glucosides that are cleaved by specic 26-O-b-glucosidases. In oats, one such enzyme converts avenacosides A and B to antifungal 26-desglucoavenacosides [87], whereas another 26-O-b-glucosidase cloned from Costus speciosus (wild ginger) transforms furostanol saponins lacking the F-ring to the corresponding spirostanol saponins (Fig. 8) [91]. Variations in sapogenin spirostan and furostan structures (e.g. addition of hydroxy groups at C-2 and/or C-22, and saturation versus unsaturation at C5,6), and the myriad possible arrangements of oligosaccharides glycosylated to the 3-b-OH, allow for many dierent steroidal saponins. Dozens have been isolated from the monocot and dicot crops cited above, including at least 17 from Asparagus spp., 10 from leek, and eight from fenugreek [87,88,90]. Among these, a number are of pharmacological interest, such as porrigenin C, which showed antiproliferative activity on four tumor cell lines [90], and a spirostanol glycoside from fruit of Asparagus ocinalis with marked spermatocidal activity [88]. Yams, wild alliums, and fenugreek have drawn much attention as rich

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Fig. 8. Steroidal sapogenins (aglycones) and saponins.

sources of diosgenin and yamogenin (Fig. 8), which are used in production of various steroids. Finally, preparations of steroidal saponins from Dioscorea, fenugreek, and particularly the Indian puncture plant, Tribulus terrestris, are currently widely marketed on the Internet as herbal substitutes for anabolic steroids and ViagraTM because of their reported stimulation of testosterone production. 2.7. Steroidal glycoalkaloids Steroidal glycoalkaloids appear to be ubiquitous in members of the Solanaceae and among the solanaceous crops they are most abundant and diverse in wild and cultivated potato (Solanum spp.) [15,87,92]. The steroidal alkaloid aglycones have solanidane- or spirosolane-based struc-

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tures, the latter being closely related to the spirostanol sapogenins [92]. The common solanidane aglycones include solanidine (solanid-5-en-3b-ol) and demissidine (5a-solanidan-3b-ol), and common spirosolane aglycones include solasodine (22R,25R-spirosol-5-en-3b-ol), soladulcidine (22R,25R,5a-spirosolan-3b-ol), tomatidenol (22S,25S-spirosol-5-en-3b-ol), and tomatidine (22S,25S,5a-spirosolan-3b-ol) (Fig. 9). Two trisaccharides of solanidine, a-solanine (3b-O-galactose-rhamnose1glucose2) and a-chaconine (3b-O-glucose-rhamnose1rhamnose2), are the main steroidal glycoalkaloids in S. tuberosum. Two analogous trisaccharides of tomatidenol, a- and b-solamarine, respectively, as well as two tetrasaccharides of demissidine, have been introduced into cultivated potato by crossbreeding with wild species [87,92]. In tomato, tomatidine is the principal aglycone and its tetrasaccharide a-tomatine (3b-O-galactose-glucose-xylose1galactose2) is the major glycoalkaloid, whereas the major constituents in eggplant are the aglycone solasodine and its trisaccharide solasonine (3b-O-galactose-rhamnose1glucose2) [93]. Accumulation of solanine and chaconine in potato tubers is closely associated with light-induced greening (chlorophyll synthesis), although metabolically these appear to be independent events [94,95]. Similarly, in tomato fruit a-tomatine accumulates in immature green fruit but is essentially absent in fully ripe fruit. Radiolabeling experiments showed that young tomato fruit synthesized a-tomatine from cholesterol and that the decline in a-tomatine concentration with ripening could be attributed to loss of biosynthetic capacity combined with an increased rate of degradation [96]. The steroidal glycoalkaloids are toxic to humans and other mammals. Fortuitously, they are poorly absorbed by the gastrointestinal tract, where they are partially hydrolyzed to the less toxic aglycones, which in turn are rapidly excreted [97]. At moderately high concentrations (e 20 mg/ 100 g fresh weight), these alkaloids have a bitter taste and create a burning sensation in the mouth and throat. They are intense irritants of the gastrointestinal tract due to disruption of cell membranes, and also act as cholinesterase inhibitors, which can severely depress the central nervous system [94,95,97,98]. A toxicological study in which four glycoalkaloids or their respective aglycones were fed to mice indicated that a-chaconine is more damaging to the liver than a-solanine, solasonine, or a-tomatine, and although solanidine and solasodine (but not tomatidine) caused

Fig. 9. Steroidal aglycones commonly found in glycoalkaloids of the Solanaceae.

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signicant liver enlargement, this was reversible upon removal of the alkaloids from the diet [93]. A recent extension of this work determined that among the aglycones, tomatidine, with a saturated steroid nucleus, is essentially non-toxic relative to the 5-unsaturated solanidine and solasodine [99]. On the other side of the ledger, there have been a number of reports indicating that some steroidal alkaloids have potential pharmacological or therapeutic applications [98]. For example, solasodine has hypocholesterolaemic and antiatherosclerotic eects [100], whereas its glycosides (solasonine and solamargine) show selective toxicity against cancer cells [101]. 2.8. Phytoecdysteroids and brassinosteroids Phytoecdysteroids, analogues of insect-molting hormones (ecdysteroids) that play an essential role in insect development and maturation, are found in higher plant species representing over 100 families [23,102]. More than 150 dierent phytoecdysteroid structures have been reported [103], the most commonly encountered being 20-hydroxyecdysone (Fig. 10), which is identical to the principal molting hormone isolated from insects [23]. Although denitive proof is still lacking, it is widely accepted that production of phytoecdysteroids in plants deters predation by non-adapted insects (as well as other invertebrates such as nematodes), either by functioning as antifeedants or via disruption of development [102]. The occurrence, biosynthesis, and distribution of these plant steroids have been studied most extensively in species of the Chenopodiaceae (Goosefoot family). Accumulation of phytoecdysteroids produced in young, developing tissues generally occurs in apical leaves and owers and ultimately in seeds [104,105]. Appreciable levels were detected in seeds of about 35% of the species tested [102]. Among chenopod crop species, leaves and seeds of spinach (Spinacia olereacea) are particularly rich in phytoecdysteroids and levels are also high in Chenopodium quinoa (the Inca mother grain currently being developed as a dryland crop in the western USA), whereas in table beet (Beta vulgaris) these steroids are scarcely detectable [23,102]. The biosynthetic origin of phytoecdysteroids is an interesting and as yet incomplete story. A number of studies have shown incorporation of radiolabeled cholesterol into these plant steroids, leading to the conclusion that cholesterol serves as the sterol precursor [9,15]. However, in spinach, which produces exclusively R 7 rather than R5 sterols (see Section 2.2), it has been shown

Fig. 10. Phytoecdysteroida plant steroid that serves as an insect molting hormone. 20-Hydroxyecdysone is synthesized from lathosterol in spinach (Spinacia oleracea) and is identical with the naturally-occurring hormone in insects.

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that lathosterol (cholest-7-en-3b-ol) is the direct precursor of 20-hydroxyecdysone [106]. Whether there are separate biosynthetic routes to phytoecdysteroids utilizing cholesterol and lathosterol, and whether cholesterol is converted to lathosterol in some plants by consecutive hydrogenation and dehydrogenation steps (Fig. 4), are presently open questions [23]. Aside from this issue, it is also known that many plants produce ecdysteroids that are alkylated at C-24 and thus are likely derived from C-24 methyl and ethyl phytosterols [107]. In the last decade, brassinosteroids have come to be recognized as an important new class of potent steroid hormones in plants, operative in the nanomolar range or lower as are their steroid counterparts in animal cells [108,109]. Over 40 of these plant steroids have been fully characterized, the most active compound being brassinolide (Fig. 11), which was also the rst to be isolated [110]. Mutants that are decient in the synthesis of or responsiveness to brassinosteroids are typically characterized by a dwarf phenotype when grown in the light and deetiolation when grown in the dark, and are also often impaired in reproduction [108110]. The complete, dual biosynthetic pathways of brassinolide in Catharanthus roseus and Arabidopsis have recently been elucidated through detailed metabolic studies in which deuteriumlabeled intermediates were supplied to cultured cells of C. roseus and various Arabidopsis mutants [109111]. Campesterol is the mainstream desmethyl phytosterol from which brassinolide and its immediate precursor castasterone (Fig. 11), another active brassinosteroid, are derived. It was recently determined that the brassinolide-decient Arabidopsis mutant dim (also called dwf1) lacks the ability to convert 24-methylenecholesterol to campesterol via a 24-methyldesmosterol intermediate [109]. An interesting observation from this study, unrelated to brassinolide synthesis, was that dim mutant plants accumulate very high levels of isofucosterol, indicating that the DIM gene product also performs the reduction of isofucosterol to sitosterol via the stigmasta-5,24(25)-dienol intermediate. The initial steps in the dual pathways from campesterol to castasterone and brassinolide involve the conversion of campesterol to campestanol. This is actually a three-step process that includes 24-methylcholest-4-en-3-one and 24-methyl-5a-cholestan-3-one as intermediates. A recent study of the brassinolide-decient Arabidopsis mutant det2 showed that the DET2 gene product saturates the 4-double bond in the A-ring of 24-methylcholest-4-en-3-one to

Fig. 11. Brassinosteroids: a recently discovered class of plant hormones derived from campesterol and other phytosterols.

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yield 24-methyl-5a-cholestan-3-one [111]. In the subsequent metabolism of campestanol to castasterone, there are two alternate routes dubbed the early and late C6-oxidation pathways, which refers to whether the 6-keto group present on the B-ring of castasterone is introduced during the rst step (campestanol to 6-oxocampestanol) or during the last step (6-deoxocastasterone to castasterone) [110]. It is interesting that the products of two genes in Arabidopsis, DWF4 and CPD, are cytochrome P450-like enzymes that perform the sequential hydroxylations at C22 and C23 of the alkyl side chain in both the early and late C6-oxidation pathways of brassinolide biosynthesis [109,110].

3. Methods for the quantitative analysis of phytosterols and phytostanols 3.1. Extraction and fractionation of phytosterols Most common methods for the extraction of lipids also extract phytosterols. Nonpolar solvents such as hexane (commonly used to extract most types of vegetable oils), quantitatively extract free phytosterols (FS) and phytosteryl fatty-acid esters (SE) [1,3]. The extraction of FS, SE, and ferulate phytosteryl esters from corn ber was compared with four dierent solvents (hexane, methylene chloride, ethanol, and isopropanol) and each solvent extracted R95% of these three sterol lipid classes [51]. Steryl glycosides (SG) and fatty-acylated steryl glycosides (ASG) are only partially extracted with hexane, and increasing the polarity of the solvent gave a higher percentage of extraction [1]. We routinely use the Bligh and Dyer chloroformmethanol extraction method to extract all sterol lipid classes [112]. Even after chloroform-methanol extraction, additional phytosterols are sometimes liberated by subsequent acid or alkaline hydrolysis, suggesting that there may be pools of bound or evasive phytosterols in some plant tissues (personal communication, V. Piironen). Additional research is necessary to provide a better understanding of the optimal extraction methods that are required to assure complete phytosterol extraction. Once a lipid extract has been prepared it is often necessary to separate (fractionate) one or more of the sterol lipid classes. Traditionally, open column LC (liquid chromatography), with either silicic acid or Floricil as the solid phase column packing, was used to fractionate the various lipid classes. In the last decade, open column LC has generally been replaced by similar methods using pre-packed SPE (solid phase extraction) cartridge columns. We recently reported a silica SPE method to purify hydroxycinnamate steryl esters (HSE) in corn ber oil [113]. In a later section we will present several SPE methods for the fractionation of free phytosterols before GC analysis. Preparative thin-layer chromatography can be used to separate and fractionate sterol lipid classes. After spotting the sample(s) on a TLC plate, the plate is developed with an appropriate solvent mixture, and each spot containing a specic sterol lipid class is scraped into a tube and eluted with solvent. Finally, silver ion (argentation) chromatography (either silver-impregnated TLC or HPLC) can be employed as a fractionation technique that separates phytosterols based on their total number of carboncarbon double bonds [1]. Before the development of micro-scale GLC and HPLC analytical methods, precipitation of cholesterol with digitonin (a type of steroidal saponin) was a technique commonly used to remove other lipids and obtain a cholesterol-enriched fraction. Digitonin precipitation also has been used in studies of both fungal sterols [114] and phytosterols [115] to isolate the FS fraction (binding of

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digitonin to a sterol, and consequent precipitation of the complex, requires a free 3b-OH group). However, there is evidence that certain types of phytosterols are not quantitatively precipitated by digitonin, and this technique is now infrequently used to isolate free phytosterols. 3.2. Methods for the separation of intact phytosterol classes Thin-layer chromatography was traditionally used for qualitative separation of phytosterol lipid classes. Grunwald and Huang [116] compared ve dierent TLC methods for separation of the four common sterol lipid classes in plant tissues (FS, SE, SG, and ASG). Numerous other TLC methods for separation of phytosterol lipid classes have been reported [1,3]. Numerous high performance liquid chromatography (HPLC) methods have been developed to both qualitatively separate and quantitatively analyze phytosterol lipid classes (Table 1). In general, polar or normal phase HPLC columns (e.g. silica, DIOL, Amino, CN) are used to separate phytosterol lipid classes (Table 1) [117123] and reversed phase HPLC columns (e.g. C18=ODS, C8, or phenyl) are used to separate molecular species (individual compounds) that comprise a lipid class (see next section and Table 2). Although methods have been reported for the simultaneous analysis of both nonpolar (FS, SE, and sometimes HSE) and polar (SG and ASG) sterol lipid classes [119,121], we nd that the most accurate way to quantify these lipids is to analyze the polar and nonpolar classes separately (Figs. 12 and 13) [85,120]. In our methodology, the ltered total lipid extract is injected in two dierent HPLC systems, both equipped with a Diol column but programmed for dierent solvent gradients. The three nonpolar sterol lipid classes are quantitatively analyzed with a hexane-based gradient that starts at 0% isopropanol and increases to 0.25% during 40 min (Fig. 12), resulting in retention times of 2, 21, and 28 min for SE, FS, and HSE [51]. The two polar sterol lipid classes are quantitatively analyzed with a gradient that starts at 90/10, hexane/isopropanol, and increases to 45/50/5, hexane/isopropanol/water, resulting in retention times of 6 and 11 min for ASG and SG, respectively, with the other glycolipids and phospholipids eluting over the range of 1550 min (Fig. 13).
Table 1 Methods for the TLC and HPLC analysis of intact phytosterol classes Sample Chromatography Gradient SE HSE FS ASG SG Date Y Y Y Y Y Y Y Y Y Y Y Y 1989 2001 1984 1984 1990 1994 1993 1996 1999 Reference [116] [117] [118] [118] [119] [120] [121] [51] [122]

Standards and various plants TLC-Silica Fruit and vegetables TLC-Silica Peanut and corn oils TLC-Silica Peanut and corn oils Low pressure LC-Silica Spinach leaves HPLC-Silica Tobacco cells HPLC-Cyano Wheat our HPLC-Silica Corn ber HPLC-DIOL Rice bran oil HPLC-Prep Nova-Pak HR Silica Zooplankton lipids HPLC-Alumina Soy lecithin HPLC-DIOL

Steps Y Y Y

Y Y

Y Y Y Y Y

Y Y Y

Y Y Y

Y Y

Y Y Y

1999 [123] Fig. 9 Unpublished

SE, fatty acid ester; HSE, hydroxycinnamic-acid esters; FS, free alcohol; ASG, acylated steryl glycoside; SG, steryl glycoside.

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Table 2 Methods for the HPLC analysis of molecular species of free phytosterols and intact phytosterol conjugates Sample Synthetic standards Oat leaves and seeds Fruits and vegetables Grains Corn and rice Rice bran oil Column Zorbax ODS RP-hexyl Luna C18 HPLC Zorbax C18 Deltabond C18 Microsorb-MV C18 SE Y Y Y Y Y Y Y Y FS HSE ASG SG Date 1983 1985 2001 1989 1995 1999 Reference [124] [125] [117] [48] [50] [122]

SE, fatty acid ester; HSE, hydroxycinnamic-acid esters; FS, free alcohol; ASG, acylated steryl glycoside; SG, steryl glycoside.

Fig. 12. HPLC Chromatogram of nonpolar lipids including SE, FS, and HSE, using a DIOL column and detection via an evaporative light scattering detector [51].

3.3. Methods for the separation of molecular species of phytosterol conjugates When the ve phytosterol lipid classes (FS, SE, HSE, SG, and ASG) have been separated by TLC or HPLC, it is sometimes useful to study the individual compounds (molecular species) within each class. Billheimer and colleagues [124] reported the rst reversed phase HPLC method to separate molecular species of SE (stigmasteryl oleate and campesteryl palmitate are two examples of SE molecular species). Kesselmeier and colleagues [125] reported a method to separate molecular species of FS and SG, and also stated that the method could be used to analyze SG molecular species obtained by partial hydrolysis of ASG (selective cleavage of the fatty acid

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Fig. 13. HPLC Chromatogram of polar lipids including ASG and SG, using a DIOL column and detection via an evaporative light-scattering detector (R. Moreau, unpublished results).

from the 6-OH of the hexose moiety). A recent report described an HPLC method to separate intact molecular species of ASG and SG, as well as the three other common plant glycolipid classes [117]. Several methods have been reported for separating molecular species of HSE from corn ber oil, rice bran oil, and lipids of other grains (Table 2) [124,125]. The most abundant molecular species of HSE in corn ber oil and rice bran oil are sitostanyl ferulate and cycloartanyl ferulate, respectively [50]. 3.4. Methods for hydrolysis of conjugates and separation of free phytosterols Total phytosterols (including free, esteried and glycosylated) can be quantied by hydrolysis and subsequent GC analysis of the combined FS. This method of phytosterol analysis has been the most widely used. SE, HSE, and the fatty acid-hexose ester linkage in ASG can be hydrolyzed via saponication (alkaline hydrolysis with 12 N KOH or NaOH), whereas the glycosydic linkages in SG and ASG require acid hydrolysis (46 N HCl). The points of hydrolytic cleavage of the four phytosterol conjugates are indicated in Fig. 7. Since vegetable oil samples usually contain primarily SE and little or no SG or ASG, alkaline hydrolysis alone is sucient to cleave all of the conjugated phytosterols [55]. Toivo and colleagues [126,127] developed an elaborate, routine method for hydrolysis of phytosterol conjugates that includes both acidic and alkaline steps. However, these authors noted that additional studies would be necessary to optimize the conditions for acid hydrolysis of phytosterol conjugates. One artifact observed with this method is that if plant tissues contained R 5-avenasterol, acid hydrolysis caused its isomerization to fucosterol and several 5,23- and 5,24(25)-stigmastadienols [128]. Similarly, we found that during acid hydrolysis of SG, R5-avenasterol (isofucosterol) is lost and lathosterol (cholest-7-en-3b-ol), included as an internal standard, was isomerized to cholesterol (B.D. Whitaker, unpublished observation). Kamal-Eldin et al. [128] suggested that replacing acid plus alkaline hydrolysis protocols with enzymatic hydrolysis would solve this problem. Kesselmeier and colleagues [125] reported an enzymatic method (using a commercial b-glucosidase) to hydrolyze SG, but this

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method has been used by only a few researchers. We used a modied version of the enzymatic method [125] to analyze FS derived from SG (and ASG by including mild alkaline methanolysis, which also enabled us to identify ASG fatty acids by GC analysis of their methyl esters) [35,83 85]. The enzyme cholesterol ester hydrolase is used to hydrolyze cholesteryl fatty-acid esters during the routine enzymatic assay of cholesterol (see Section 3.6), and could perhaps also be used to hydrolyze phytosteryl fatty-acid esters. Although cholesterol esterase has been used to hydrolyze carotenoid esters [129], we are unaware of studies employing it to hydrolyze phytosteryl fatty-acid esters. Clearly, more research is required to perfect hydrolysis methods for phytosterols, and enzymatic methods may prove to be superior to acidic and/or alkaline hydrolyses. After hydrolysis of conjugates, the quantitative GC analysis of FS can be achieved using one of the numerous published methods (Table 3) [130136]. Most GC methods for phytosterol analysis include derivatization to form either trimethylsilyl (TMS) ethers (using BSTFA or a related silylating reagent) or phytosteryl acetates (via acetylation with pyridine and acetic anhydride), but some methods show good separation and quantication of underivatized phytosterols (Fig. 14). Although quantitative GC analysis of underivatized phytosterols appears to be accurate and reliable, some experts prefer derivatizing phytosterols to prevent dehydration and decomposition, which may result in peak tailing and poor resolution [1]. Several reversed phase HPLC procedures for the separation of FS have also been reported [1,3,125] but the separation and sensitivity are generally lower than with GC.

Table 3 Methods for the hydrolysis and GC analysis of free phytosterols (unless otherwise noted, all columns were capillary columns) Sample Oat leaf and seeds Cucumber Tomato fruit Bell pepper Yeast Tobacco cells Vegetable oils Oat seeds Fruit juices Vegetable oils Vegetable oils Oat seeds Vegetable oils Grains Corn ber oil Grains
a

Alkaline hydrolysis Y

Acid hydrolysis Y

Enzymatic hydrolysis Y

Derivatization None Acetylation None None Silylation None Silylation Silylation None Silylation Silylation Silylation None Silylation None Silylation

SPEa

Column OV-1 OV-17 SP2100, packed column SPB-1 SPB-1 SPB-1 OV-1 DB-5ms DB-5ms NB-17 DB 17 HT DB-5ms SAC-5 NB-17 SAC-5 RTX-5w/ INTEGRA

Date 1985 1987 1988 1989 1989 1994 1996 1998 1998 1998 1999 1999 2000 2000 2000 2002

Reference [125] [19] [10] [130] [131] [85] [132] [128] [133] [134] [135] [136] [55] [126] [54] [53]

Y Y Y Y Y Y Y Y Y Y Y Y

Y Y

Y Y Y Y Y

Y Y Y

SPE, solid phase extraction.

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Fig. 14. GCMS chromatogram of sterols and stanols in saponied corn ber oil (R. Moreau, unpublished results).

3.5. Mass spectrometry and NMR of phytosterols Mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (NMR) are valuable tools for phytosterol identication. Goads review [1] contains an excellent comprehensive discussion of these topics and it also contains a listing of MS electron impact ion fragments and NMR resonances for all of the common phytosterols. We will present a broad overview of these topics but would urge the reader to consult previous reviews [1] for more details. Electron impact (EI) GCMS has been extensively employed to identify free phytosterols. Several common free phytosterols are included in two on-line databases: http://webbook.nist.gov/ chemistry and http://lipid.bio.m.u-tokyo.ac.jp. One complicating factor when employing GCMS for identication of phytosterols is the fact that published spectra include data for free phytosterols (Fig. 15) and for their TMS-ether and acetate-ester derivatives. The several common points of EIMS fragmentation (Fig. 15) can be used to predict fragments in phytosterols with other structures. Rahier and Benveniste [137] described the mass spectra of six common phytosterols which were each analyzed by GCMS as the free form (M+), as acetate esters (M++42), or as TMS ethers (M++72). Because the fragmentation pattern of phytosterols is dierent when they are analyzed in each of these three forms, accurate structural identication is possible only if the spectrum of the unknown compound is matched with a library spectrum of a known compound in the same form (i.e. free, acetate, or TMS). Since many of the common phytosterols occur as epimers (e.g. brassicasterol and epibrassicasterol) or isomers (e.g. R 5-avenasterol and fucosterol), GC separation and/or identication of these structurally similar compounds can be challenging. In a recent study reporting the acid-induced isomerization of R 5-avenasterol and fucosterol [128], small dierences in the GC-MS fragmentation of TMS derivatives were used to document this isomerization. Another study [138], employing GCMS of phytosteryl acetates, showed that R 5-avenasterol (24Z) always has a longer GC retention time than fucosterol (24E), and that these two isomers can be distinguished by the mass spectra of their acetates. M-60=m/e 394 is much more abundant in the fucosterol EI spectrum, whereas m/e 296 is much more abundant in the

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Fig. 15. Electron impact mass spectrum of sitosterol (MW 414) and its major fragment ions (spectrum was kindly provided by A. Nunez).

-avenasterol spectrum. Unique EIMS fragments of 24E- compared with 24Z-ethylidene phytosteryl acetates were also reported elsewhere [139]. Another study employed HPLCMS, specically atmospheric pressure chemical ionozation mass spectroscopy (APCIMS), to provide structural information about SG and ASG in red bell pepper fruit [117]. Other emerging forms of soft ionization mass spectrometry (e.g. electrospray and MALDI-TOF) could also potentially be used to analyze phytosterols and their intact conjugates. 13 C nuclear magnetic resonance (NMR) has been reported to be a valuable tool for the chemical identication of phytosterols since each of the 28 or 29 carbons in the molecule can potentially be examined individually [140]. 1H and 13C NMR have proven to be essential in the structural elucidation and quantication of 24R- and 24S-epimers of C-24 methyl and ethyl phytosterols [1,19]. 3.6. Enzymatic assays of phytosterols Enzymatic methods [141] and specic test kits have been developed and marketed to measure cholesterol in blood and other samples. Most of these kits include cholesterol ester hydrolase to hydrolyze cholesteryl fatty-acid esters and cholesterol oxidase (from animal or microbial sources)

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to oxidize cholesterol, and for each molecule of cholesterol that is oxidized, one molecule of H2O2 is produced (Fig. 16) [142]. The amount of H2O2 produced can then be measured by one of several spectrophotometric or uorometric assays and used to calculate the amount of cholesterol in the sample. Although the manufacturers of some of these kits indicate that the kits can also be used to measure phytosterols, we are not aware that this claim has been validated. Smith and Brooks [143] compared the Km and Vmax values for the oxidation of several phytosterols by cholesterol oxidase from Nocardia erythropolis (Fig. 16), and found that the values varied considerably. Obviously, with the current interest in phytosterols, there is a potential to develop enzymatic methods for the rapid quantication of phytosterols in foods. When one considers the high cost of GC and HPLC instruments that are currently required to analyze phytosterols, the development of convenient enzymatic assays using microtiter plates is very attractive.

4. Health-promoting eects of phytosterols, phytostanols, and their esters 4.1. Historical perspectives, changing dogma, and critical questions Phytosterols are natural components of human diets. In the West, we consume an average of e 250 mg per day of phytosterols, largely derived from vegetable oils, cereals, fruits and vegetables [144,145]. This is roughly equivalent to the amount of cholesterol ( e 300 mg/day) consumed. For vegetarians, dietary phytosterols have been estimated at almost twice this level [146]. Phytostanols are much less abundant in nature than phytosterols, and consequently we typically consume much lower amounts ( e 25 mg/day) in our diets [145,146]. Common dietary sources of phytostanols are corn, wheat, rye, and rice. For many years, the existence and dietary eects of these minor sterols were largely ignored and poorly understood. Sterol chemists and biochemists focused their eorts on cholesterol because elevated serum cholesterol levels were shown to be a prominent risk factor for cardiovascular disease (CVD). Recent strategies for lowering serum

Fig. 16. Cholesterol oxidase reaction with cholesterol and stigmasterol. Note that oxidation of both sterols shifts the R 5 double bond to R 4. Enzyme kinetic data were reported for cholesterol oxidase from Nocardia erythropolis [143].