Progress in Lipid Research 41 (2002) 457–500 www.elsevier.



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

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 carbon–carbon 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 different 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 esterified 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 five years because of their cholesterol-lowering properties. Early phytosterol-enriched products contained free phytosterols and relatively large dosages were required to significantly 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 firm 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: (R.A. Moreau).
0163-7827/02/$ - see front matter Published by Elsevier Science Ltd. PII: S0163-7827(02)00006-1



R.A. Moreau et al. / Progress in Lipid Research 41 (2002) 457–500

were shown to significantly lower serum cholesterol at dosages of 1–3 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 effects 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 efficacy: 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 benefits .............................................................................. 488 4.10. Effects 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-esterified) 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 different 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 rigidification [4]. However, individual phytosterols differ in their effect on membranes stability. Stigmasterol has been reported to have a disordering effect 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 (five 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 field. The three topics are as follows: (A) the occurrence of various phytosterols in plants and a discussion of the nomenclature, (B)


R.A. Moreau et al. / Progress in Lipid Research 41 (2002) 457–500

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.

Nomenclature of phytosterols (example: sitosterol=stigmast-5-en-3b-ol=24R-ethylcholest-5-en-3b-ol). 6) phytosterols. Most 4-desmethyl phytosterols have a double bond between carbons 5 and 6 of the ring system and are thus called R 5 phytosterols.R. 5) and 29-carbon (Fig. 2. 4-Monomethyl and 4. However. 4-Desmethylsterols include the 27-carbon sterol cholesterol (Fig. but also generally present in plants at low levels) and all of the common 28-carbon (Fig. Moreau et al. Fig. which are typically major membrane structural components in plant cells. 3. another group of common desmethylsterols that are abundant in plants of certain families have a double bond .A. 4) (ubiquitous and predominant in animals.4-dimethylphytosterols. / Progress in Lipid Research 41 (2002) 457–500 461 Fig.

The nomenclature of the configuration of . 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. Moreau et al. 4. for example.462 R. designated as R5. C27 4-desmethyl phytosterols.22E. which includes both C5. / Progress in Lipid Research 41 (2002) 457–500 Fig.A. and are hence referred to as R 7 phytosterols. is. campestanol (24a=24R) or ergostanol (24b=24S). Fig. The common 29-carbon desmethylsterol stigmasterol (Fig. depending on the phytosterol composition of the original material. C28 4-desmethyl phytosterols. 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. Both R 5 and R 7 desmethylsterols can include a second double bond in the alkyl side chain. 6).23 double bonds.6 and (trans) C22. most frequently between carbons 22 and 23 or carbons 24 and 28 (carbons 24 and 241 in the 1989 IUPAC nomenclature). 5. Note that some of these compound are 24a (solid wedge) and some are 24b (dashed wedge). between carbons 7 and 8 instead of 5 and 6.

R. stigmasterol. and the other five phytosterols are 24b epimers (with the methyl group indicated as a ‘‘dashed wedge’’). The common sterol in animal tissues. 27-Carbon 4-desmethylsterols (Fig. unless there is a double bond at C22. depending on the phytosterol composition of the original material. the C22.23. which are equivalent to 24a and 24b. epibrassicasterol. epibrassicasterol. wt. The double bond at C24. or C24 ethylidene groups on the C28 and C29 phytosterols requires some explanation. in which case the chirality is reversed (24R=24b and 24S=24a). Phoenix dactylifera. For the seven common C28 phytosterols listed in Fig. also called cholesta-5. Unfortunately. mol. Cholesterol. C29 4-desmethyl phytosterols.23 double bond in common phytosterols (brassicasterol. C27H46O. 386.28 (C24.A.65. . CAS# 57-88-5. The 24-methyl epimers are also designated 24R and 24S. CAS# 313-04-02. Also occurs in the date palm. the C24 methyl. and 7-stigmasterol (spinasterol) only occur as 22E. cholest-5-en-3b-ol. Desmosterol. C24 ethyl. whereas the C24 ethylidene in R 5-avenasterol and R 5-avenasterol is the cis isomer and is designated as 24Z. Fortunately.24-dien-3b-ol. the good news is that almost all phytosterols are 24a-ethyl epimers.24R C24 ethylidene in fucosterol is the trans isomer and is designated as a 24E. / Progress in Lipid Research 41 (2002) 457–500 463 Fig. respectively. 24-dehydrocholesterol. three (campesterol. 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. MW 384.63. Moreau et al. 6. C27H44O. three of the common C29 phytosterols have a ) and the resulting ethylidene group can either be cis or trans. 5. 4). and in many marine red algae (Rhodophyceae). With the C29 phytosterols. and campestanol) are 24a epimers (with the methyl group indicated as a ‘‘solid wedge’’).

also called 7-dehydrocholesterol. CAS# 434-16-2. 400. C29H50O. also called. MW 416. .22-trien-3b-ol. or R 5-24a-methyl-cholesten-3b-ol. C29H52O. C28H50O. also called (22E)-ergosta-5. Campesterol (R 5) (24a=24R). also called stigmastanol. also called ergost-5-en-3b-ol and 24-epicampesterol. mol. CAS # 4651-51-8. almost exclusively as a ferulate ester or p-coumarate esters. also called (22E)-(24S)-24-methylcholesta-5. mol. also called 5a-cholest-7-en-3b-ol.7. CAS # 474–60–2.7-dien-3A-ol. CAS # 474-67-9. / Progress in Lipid Research 41 (2002) 457–500 Lathosterol. Wt. MW 396. C28H46O.63.22-dien-3b-ol. 386. CAS # 57-87-4. 28-Carbon 4-desmethylsterols (Fig. (24R)-ergost-5-en-3b-ol.54.7.65. also called (22E)-ergosta-5. R 7-cholesterol. Brassicasterol (R5. 29-Carbon 4-desmethylsterols (Fig. also called (24R)-24-methylcholestan-3b-ol or (24R)ergostan-3b-ol. C27H46O. campest-5-en-3bol. and Provitamin D3. Found in rapeseed oil from Brassica napus. stigmast-5-en-3b-ol.22E) (24b=24R). Sitosterol (R 5) (24a=24R). 24a-ethylcholest-5-en3b-ol.464 R. MW 384. 22-Dihydrobrassicasterol (R5) (24b=24S). also called (24R)-24-methylcholest-5-en-3b-ol. C29H50O. 22-dihydrospinasterol. Occurs naturally in corn fiber oil.22E) (24b=24R).22E) (24a=24S). Lathosterol is the C27 precursor to phytoecdysteroids in spinach (Spinacia oleracea).66. MW 414. 400. CAS # 521-03-9. almost exclusively as a ferulate or p-coumarate esters. Widespread occurrence in plants. MW 398. Not reported naturally but can be generated by catalytic hydrogenation of brassicasterol. Occurs in corn fiber oil. Cholesta-5.68. CAS # 474-62-4. or ergosterol. also called (24S)-24-methylcholestan-3b-ol. CAS # 19466-47-8. C28H48O. Widespread occurrence in plants. Wt. MW 414.68. $ 7-Stigmastenol (7-Stigmastenol) (R 7) (24a=24S).70. Ergostanol (saturated) (24b=24S). Moreau et al.A.22-dien-3b-ol. stigmasta-7-en-3b-ol. Generated by catalytic hydrogenation of campesterol or epibrassicasterol. 24a-ethylcholestan-3b-ol. 22-dihydrobrassicasterol. MW 402.71.71.70. C27H44O. 5) Campestanol (saturated) (24a=24R). also called b-sitosterol. 6) Sitostanol (saturated) (24a=24R).22-dien-3bol or (22E)-campesta-5. MW 402. CAS # 83-46-5. wt.66. Ergosterol (R 5. C28H44O. CAS # 17472-78-5.40. C28H48O. C28H46O. MW 398. Epibrassicasterol (R 5. mol. stigmastan-3b-ol. Occurs in yeasts and many other fungi. C28H50O.

in which the sterol 3b-OH group is esterified to ferulic or p-coumaric acid (Fig. MW 412. MW 412. The fact is that this C27 sterol.1. [24(24R )E]-stigmasta)-dien-3b-ol. and ASG) are generically called ‘‘phytosterol conjugates. also called isofucosterol.69. MW 412.22-dien-3b-ol.R. C29H48O.24(28)-dien-3b-ol. phytosterols occur in five common forms (Fig. $ 7-Avenasterol (7-Avenasterol)(R 7. differ from SG by the addition of a fatty acid esterified to the 6-OH of the hexose moiety (first reported by Lepage in 1964) [8].69. or 24E-ethylidenecholesta-5.24E). MW 412. and in many brown algae. and as acylated steryl glycosides (ASG). ol. and in significant levels in other plant materials. CAS # 18472-36-1. CAS # 23290-26-8.22E-dien-3b-ol. it should be noted that this is inconsequential in the human diet relative to the amount of cholesterol in meat and dairy products. CAS # 83-48-7.22E) (24a=24S). Widespread occurrence in plants. as steryl glycosides (SG). or tissues. often accounts for 1–2% of the total plant sterols. which is predominant in animals and a contributing factor in human cardiovascular disease.22E) (24a=24S).A. 29-isofucosterol.69.24(24R CAS # 17605-67-3. whereas in the three conjugates the OH is covalently bound with another constituent. Cocos nucifera. Structural diversity and phylogenetic distribution of phytosterols 2.24(28)-dien-3b)Z]-stigmasta-5. the 3b-OH group on the A-ring of the sterol nucleus is underivatized. and can compose 5% or more in select plant families. Seeds of corn and rice and other grains contain a fourth type of phytosterol conjugate. also called avenasterol. e. CAS # 481-18-4.g. Found in coconut pollen.24(28)-dien-3b-ol.22-dien-3b-ol or 24a-ethylcholesta-5. also called [24(28)E]-stigmasta-5. Many species of the Solanaceae (Nightshade .24(28R )-dien-3b-ol. 2. C29H48O. Occurrence and metabolism of cholesterol in plants There is a widespread misconception.24(28)-dien-3b-ol. 7): as the free alcohol (FS). C29H48O.24Z). perhaps fostered by the nutritional information printed on packages and containers of various foods of plant origin. 7). 24Z-ethylidenecholesta-5. [24(28)Z]-stigmasta-5. Fucus vesiculosus. $ 7-Stigmasterol (7-Stigmasterol) (R 7. (22E)-stigmasta7. The last three forms (SE.’’ In free phytosterols (FS). 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 (first reported by Power and Salway in 1919 [7]). Moreau et al. ASG. also called (22E)-stigmasta-5.69. C29H48O.24(28)-dien-3b-ol. or [24(24R Found as a major phytosterol in oats. that plant tissues are devoid of cholesterol. The third group of phytosterol conjugates. $ 5-Avenasterol (5-Avenasterol) (R 5. In all plant tissues. / Progress in Lipid Research 41 (2002) 457–500 465 Stigmasterol (R 5. (24Z)-24-ethylidenecholesta7. also called spinasterol. C29H48O.24Z). phytosteryl hydroxycinnamic-acid esters (HSE). 5. 28-isofucosterol. SG. species. organs. as fatty-acid esters (SE). Despite the fact that the edible portion of some crop plants can include cholesterol as a significant portion of the total phytosterols. Fucosterol (R5.69. MW 412.

S. and the level in one species. There is ample evidence that in plants cholesterol serves as a precursor in the synthesis of steroidal saponins and alkaloids. Structures of phytosterol conjugates.8) [14–17]. / Progress in Lipid Research 41 (2002) 457–500 Fig. family) include relatively high levels of cholesterol [9].and androstane-type steroids (see Sections 2. cholesterol constituted e 6%. cholesterol was the major sterol and composed 26% of the total phytosterols. and the 24a-ethyl (24S. the 24a-ethyl (24R) sterol sitosterol (Fig. Moreau et al.2. SMT1 catalyzes the first step in the production of C28 and C29 phytosterols. In mature Arabidopsis plants bearing an smt1 null mutation.7. In medicinal herbs and food plants. pseudocapsicum.6. 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).A. In total sterols from pericarp tissue of mature-green tomato (Lycopersicon esculentum) fruit. The sites of cleavage via alkaline hydrolysis (saponification) and acid hydrolysis are indicated with arrows. methylation of cycloartenol to 24-methylene cycloartenol. and in the SE fraction it ranged from 15 to 20% [10. and 2. steroidal saponins and alkaloids are of interest because of their potential pharmacological activity and/or toxicity in animals. due to . as well as ecdysteroids (insect molting hormones) and other pregnane. 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. ranged from 10 to 22% [12]. 2.11]. 7. compared with 6% in wild-type plants. A recent molecular-genetic study demonstrated that the activity of sterol methyltransferase 1 (SMT1) governs the level of cholesterol in plants [13]. 6).466 R.

as well as small amounts of R 5 phytosterols. Studies early in the last decade revealed that R7 sterols are more widely distributed than previously thought. and ASG plus SG compose 85–90%. The stereochemistry at C-24 is determined by the particular sterol methyltransferase which converts the initial phytosterol precursor cycloartenol (Fig. broccoli. with a R 7 to R 5 ratio of about 7:3. Chenopodiaceae. Fig. and apparently fulfill the same function as membrane structural components.24. 6).6 in the B-ring of the sterol nucleus (R5).22.22E-dien-3b-ol. 3) to 24-methylene cycloartenol [20]. Mustard family) is brassicasterol (ergosta-5. squash. and pumpkin. An increase in the ratio of stigmasterol to sitosterol. of the total sterols [10. the two major phytosterols. and the level of SE was 10-fold higher in red-ripe compared with mature-green fruit [10]. In addition to the large increase in stigmasterol with ripening. including melon. Caryophyllaceae. A less dramatic increase in SE has been reported to occur with senescence of both tomato leaves and potato tubers [26]. and fluidity of plant phospholipid vesicles [28. all contain close to 100% R 7 phytosterols. which is not readily apparent because the two isomers are not resolved by GLC or HPLC [19]. respectively. stigmast-7-en-3b-ol) and spinasterol (7-stigmasterol. 5) [18].25-dienol and 24b-ethylcholesta-7.25-trienol.27]. whereas outside of this family the only crop plant with such a high percentage appears to be spinach. Fig. it has been elegantly demonstrated that these two sterols have markedly different effects on the permeability.3. Another 24b-methyl R 5 sterol found predominantly in species of the Brassicaceae (also known as the Cruciferae or Cruciferaceae.29]. Phytolaccaceae. 2. was evident in all four sterol lipid classes but was most pronounced in FS [10.22E-dien-3b-ol) as predominant constituents (Fig. including the Amarathaceae. and Chenopodium quinoa (‘‘Inca wheat’’). there was a reapportioning of sterols among the four steryl lipid classes. / Progress in Lipid Research 41 (2002) 457–500 467 the 22E double bond) sterol stigmasterol (Fig. with a R 7 to R 5 ratio of about 1:3 [23]. 6).R. Often these species include a blend of R 7 and R5 phytosterols.25]. and are abundant in many species from five families within the order Caryophyllales.26]. and canola. Crops from the Cucurbitaceae (Cucumber family). A second major group of desmethyl sterols found in relatively few plant families includes 22dihydrospinasterol (7-stigmastenol. which generally accounts for less than 10% of the total phytosterols in crops such as cabbage. Moreau et al. ordering.27]. Both FS and SG increased at the expense of ASG. as exemplified by the crop plants Beta vulgaris (table beet). and is quite unusual relative to most plant tissues and organs in that ASG accounts for more than half. stigmasta-7. An interesting quirk of cucurbit crops is that the seeds and seedlings generally contain high levels of 24b-ethylcholesta7. Free and conjugated phytosterols in fruits and vegetables The sterol lipid composition of mature-green tomato fruit is much like that of tomato leaves. With tomato fruit ripening. Although stigmasterol differs from sitosterol only by the 22E-double bond in the alkyl side chain. Each of these possesses one double bond at C5. cucumber. and Portulacaceae [21. a substantial increase in sterol synthesis was accompanied by marked changes in sterol composition and conjugation [10. These are the R7 equivalents of the 24a-ethyl R 5 sterols sitosterol and stigmasterol.A. a member of the Theaceae) [21]. 5).22]. In a sterol-overproducing tobacco mutant cell line that was selected for resistance to a triazole sterol biosynthesis . which largely disappear as the plants grow to maturity [19. unless one includes tea as well (Camellia sinensis. campesterol occurs in approximately a 2:1 ratio with its 24b-methyl epimer ergost-5-en-3b-ol (22-dihydrobrassicasterol. Typically.

As stated in Section 2. Storage of mature-green tomato fruit at 2 v C for 11–21 days (chilling) resulted in about a twofold increase in the level of FS. and a pronounced increase in microsomal SG with ripening was compensated by declines of both ASG and FS. 4. SG.37].34]. 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. The level of FS changed little with ripening and SE rose only slightly.37]. but the percentage of stigmasterol had risen dramatically [34]. In microsomes from red-ripe fieldgrown fruit. 16. It was concluded that SE are involved in removing ‘‘improper’’ sterols from the FS pool. The esterified fatty acids in tomato ASG are about 75–80% saturated [33. SG. These fruit subsequently showed symptoms of chilling injury. In accord with a possible role of ASG in thermal acclimation. in a ratio of about 3:1. phytosterols of the cucurbit crops are predominantly R 7. In retrospect.40]. and SE had essentially returned to the pre-storage levels (63. / Progress in Lipid Research 41 (2002) 457–500 inhibitor. and ASG comprised 50. thereby assuring ‘‘proper’’ sterol composition in cell membranes [30–32]. The only significant change among the sterol lipid classes. respectively. are the major sterols in all four sterol lipid classes in bell pepper. the distribution of sterols in ASG. sitosterol and campesterol. FS. so a drop in the proportion of ASG might serve to increase the fluidity of cell membranes.468 R. FS is the most abundant of the sterol lipid classes. SG and ASG composed greater proportions of total sterol lipids in microsomes than in whole pericarp. but also include an interesting array of unusual minor constituents. and 3 mol%. was a 50–100% increase in SG. Four days after green tomatoes chilled for 21 days at 2 v C were transferred to 20 v C. largely balanced by a decline in ASG [36. The first sterol product. Overall. The peel and outer pericarp . was converted to desmethyl sterols only after several hours of aging. respectively). which occurred both with ripening of fruit on the plant and during a 2-week storage of maturegreen fruit at 2 v C. the doubling of FS was offset by a decrease in ASG. This increase in the FS:ASG ratio may be a means of acclimation to low temperature. 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]. delayed and uneven ripening and extensive decay. Initially.2. FS. The sterol lipid distribution in microsomal membranes isolated from bell pepper pericarp tissue was shown to change much more with ripening in the field in summer than with ripening in the greenhouse in spring [38].34]. 28-isofucosterol composed 30% of the FS fraction and pulse-chase labeling indicated that it was the precursor to sitosterol and stigmasterol. Moreau et al.14-desmethyl) sterols occurs at a step beyond squalene cyclization and formation of cycloartenol [3. this work provided the first indication that regulation of carbon flow into end product (24a methyl or ethyl. 24a-ethyl. 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].A. SE from tomato fruit were also enriched in sterol intermediates and even late in ripening there was preferential esterification of sitosterol over stigmasterol [10]. In contrast to tomato. 28. the ‘‘excess’’ sterol was mainly metabolic intermediates that were esterified to fatty acids (SE) and sequestered in cytoplasmic lipid droplets. In microsomal membranes from pericarp of ‘Pik-Red’ fruit stored at 2 v C. from e 5 to 7% of the total sterols. and 22 mol% of the total sterols. 19. cycloartenol. ranging from e 55 to 75% of the total sterols in pericarp from fruit of different cultivars and separate harvests [36. Only small amounts of stigmasterol are present. 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). suggesting that sterol glycosylation and esterification are inhibited at low temperature.

FS. Moreau et al. there is an induction of de novo phytosterol synthesis associated with cell membrane proliferation and repair [45]. tissues from the two storage organs do differ markedly with respect to enzymatic hydrolysis of glycerolipids directly after wounding. and H+-ATPase activity was higher in PM from the irradiated fruit. Minor sterols in the FS fraction were identified as campesterol. mainly due to a decline in SG from 97 to 73 nmol/g fresh weight.e. In a pair of studies. respectively [43]. In a postharvest study of muskmelons (Cucumis melo). Pre-storage gamma irradiation of whole fruit at 1. 37. and 3 nmol/g fresh weight.45 during the last 5 days at 10 v C. 32. stigmastanol.R. 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. FS and SE were present in about a 12:1 ratio. respectively. Minor sterols included 7-isoavenasterol (24E-ethylidene) at about 8%. whereas SG remained the same or declined. stigmasterol. and 5. and 11. ASG. SG. / Progress in Lipid Research 41 (2002) 457–500 469 tissues of zucchini squash (Cucurbita pepo) were shown to have 7-stigmasterol (spinasterol) and 7-stigmastenol (22-dihydrospinasterol) as the dominant FS.47].25(27)-stigmastadienol.0 kGy caused a transient decline in PM H+ATPase activity.7-stigmastadienol. 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]. Apple (Malus domestica) fruit are the antithesis of tomato with respect to sterol conjugates. In the first study.A. but the concentrations of FS and SG remained about the same [44]. In the FS fraction. stigmasterol 16–28%. whereas in the second study the change was from 56:29:15 to 60:33:7. .30 to 0. The sterol composition of both FS and SG included 90– 95% sitosterol and changed little with storage. and campesterol 10–12%. By the end of storage. In PM from pre-storage melons. was about 1. however. 7. The PM sterol composition did not change significantly during storage in any of the sterol lipid classes. 8(9)-stigmastenol. However. total sterol lipids (FS+ASG+SG) in the shredded tissue had increased by 24–28%. Carrot (Daucus carota) storage root tissue appears to respond to wounding in much the same way that potato tuber tissue does. and 7-campestenol. the corresponding percentages were 57.25(27)-stigmastadienol [43]. freshly shredded carrots were stored at 10 v C and 95% relative humidity for up to 10 days. and SG comprised 43.5 in both ASG and SG. ASG and SG were not analyzed. each at close to 40% of the total [41]. and are surely more representative of the majority of plant tissues. 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. i. and ASG in outer cortical tissue of ‘Golden Delicious’ apple were 145. and 7-avenasterol (24Z-ethylidene). The tissue concentrations of ASG and FS increased in both storage experiments. The level of ASG was increased after a longer duration of cold storage (6 months at 0 v C). whereas in PM from post-storage fruit. After 10 days of storage. the concentrations of FS. which is extensive in potato but minimal in carrot [46]. 97. the two predominant sterols. It is interesting that a 4-day heat treatment at 38 v C just after harvest also induced a specific reduction in SG to 77 nmol/g fresh weight.1 in FS and about 0. The ratio of 7-stigmasterol to 7-stigmastenol. and samples were taken periodically for analysis of membrane sterol lipid and glycerolipid contents [45. 8. all in the range of e 1 to 3%. the sterol lipid content and composition was similar in PM from control and irradiated melons. 5. At harvest. and 20 mol% of the total sterol lipids.22stigmastadienol. The only significant change in FS composition with storage was an increase in the stigmasterol:sitosterol ratio from 0. sitosterol. sitosterol composed 56–68%. 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.

The profound effects of sterols on the physical properties of membranes are well documented [59]. corn and other grains. Both Seitz and Norton noted that sitostanol was the predominant phytosterol in corn HSE [48.29.58]. Since commercial corn oil is obtained by extracting corn germ.50].50] extended these studies and separated several additional molecular species of hydroxycinnamate esters from rice bran and corn bran. Recent studies from our lab indicate that most of the phytostanols in corn are esterified in either SE or HSE. Changes in sterol composition likely to affect membrane function [28. whereas cycloartanol and 24-methylene cycloartenol were the predominant phytosterols in rice bran HSE (called ‘‘oryzanol’’).56]. such as salt stress. wheat. teoscinte and Job’s tears. wheat.71]. Although a wide variation in sterol structure can be accommodated to fulfill the bulk membrane structural requirement [59]. Unique phytosterols and phytosterol conjugates in cereals Several unique phytosterols and phytosterol conjugates have been reported in cereal grains. and triticale. barley.470 R. maturation. rye. rice. 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].5. and fractionates into the corn fiber fraction during wet milling [54].and postharvest plant physiology has received little attention [3. oats. aging. Piironen and colleagues [53] recently compared the composition of total sterols (free+bound) in rye. Zelazny and colleagues [78] showed that free sterols in the plasma membrane of the marine alga Dunaliella are absolutely required for sensing osmotic changes. Sterol–phospholipid interactions influence membrane functions such as simple diffusion.A. Seitz [48] reported trans-hydroxycinnamate esters of phytosterols (HSE. and all of the HSE is localized in the aleurone cells which form a single layer in corn. Further work has shown that H+ATPase activity is dependent upon the kind and amount of sterol present in a reconstituted system [76. shading.69]. carrier-mediated diffusion. 72–74] . They found that all grains contained significant levels of phytostanols (sitostanol and campestanol) in the total phytosterol fractions. for which sterol structure becomes more critical [63. the levels of HSE and phytostanols in corn germ oil are very low [55. FS. Moreau et al. 2. We reported that the levels of hydroxycinnamate esters in corn fiber were higher than in corn bran or any other grain [51].68.77]. Norton [49. and broaden or eliminate phospholipid phase transitions [60. Through interaction with phospholipids in a one to two stoichiometry. more subtle functions or specific situations. reduce bulk fluidity and permeability.61].57] can occur with greening. Evidence from sterol biosynthesis inhibitor studies indicate that phytosterols also play an essential role in plant cell division [65–67]. the role of sterol lipids in pre. sterols condense the bilayer. Function of 24-alkyl phytosterols and their conjugates In plant as in animal cells. or ripening of plant tissues [10. the plasma membrane is greatly enriched in sterols relative to other cell membranes [57.64]. A sharp increase in the sterol:phospholipid ratio in microsomal membranes during plant senescence is associated with loss of membrane function [70. and HSE in 66 accessions of Zea. we compared the levels of SE. / Progress in Lipid Research 41 (2002) 457–500 2. Finally. Compared with work on membrane phospholipids. and active transport. including steryl ferulate and p-coumarate esters) of phytosterols in corn. there appear to be other. In a recent paper. . and also modulate the activities of membrane-bound enzymes or receptors [62]. and identified several corn accessions with very high levels of HSE and total phytosterols [52].4.

83. 8) [91]. is another potentially important aspect of membrane lipid metabolism. wild alliums. Also. These saponins are abundant in a number of monocot species. one such enzyme converts avenacosides A and B to antifungal 26-desglucoavenacosides [87]. / Progress in Lipid Research 41 (2002) 457–500 471 Sterol conjugation. 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.g. the conversion of free sterols (FS) to steryl esters (SE).90].or spirostanol-based aglycone (Fig.A. including the crops onion. Moreau et al.6). and water stress. including eggplant (Solanum melongena). freezing stress in potato leaf plasma membrane [84]. cellulase. Asparagus. 8) and an oligosaccharide of typically 2–5 hexose or pentose moieties attached to the 3b-OH of the steroid nucleus. allow for many different steroidal saponins. Glucosylation of yamogenin (25S-spirost-5-en-3b-ol) at the 3b-OH by a steroid-specific UDP-glucose-dependent glucosyl transferase. growth conditions in bell pepper fruit [38].. Variations in sapogenin spirostan and furostan structures (e. which showed antiproliferative activity on four tumor cell lines [90]. Among these. the furostanol saponins are usually present as 26-O-glucosides that are cleaved by specific 26-O-b-glucosidases. 2. and tomato. 87–90]. steryl glycosides (SG). Recently. SG and ASG are membrane structural components. and a spirostanol glycoside from fruit of Asparagus officinalis with marked spermatocidal activity [88].85]. was demonstrated in a preparation from Asparagus plumosus [89]. chilling stress in tomato fruit [34]. Steroidal saponins Steroidal saponins consist of a furostanol. it has been postulated that they are involved in the regulation of membrane properties in response to changing conditions [62]. bell pepper (Capsicum annuum). temperature. the involvement of SG in a biosynthetic process that occurs adjacent to the plasma membrane is a reasonable hypothesis. Kesselmeier and colleagues [82] reported an increase in the levels of SG and ASG during the enzymatic preparation of protoplasts from oat leaves. and fenugreek have drawn much attention as rich . a number are of pharmacological interest.85]. 10 from leek. and yam (Dioscorea spp. In oats.). suggesting a regulatory function [81]. In particular. it appears that different types of stress can promote conversion of FS to ASG [82.88. Dozens have been isolated from the monocot and dicot crops cited above. In the course of our work. and challenge by fungal elicitors. possibly because of their relatively low solubility in a phospholipid bilayer [80]. such as porrigenin C. xylanase. Since it is thought that most of the ASG and SG is localized in the plasma membrane [84. the first step in generation of a steroidal saponin from the sapogenin aglycone.79]. and the myriad possible arrangements of oligosaccharides glycosylated to the 3-b-OH. or copper ions in tobacco cells [85]. including at least 17 from Asparagus spp. garlic. Yams. and the leguminous herb fenugreek (Trigonella foenumgraecum) [16. or acylated steryl glycosides (ASG).). Like FS. and saturation versus unsaturation at C5. oats (Avena sativa). and have also been described in several solanaceous crops. and leek (Allium spp. Based on reports that sterol interconversions are controlled by phytohormone levels and environmental factors such as light. we have found that the extent of sterol glycosylation and esterification can be dramatically altered in response to ozone stress in snapbean leaves [83]. Peng and colleagues [86] reported that sitosterol-b-glucoside serves as a primer for cellulose synthase in plants. and eight from fenugreek [87.R. Metabolic studies have shown that interconversion of sterols and sterol conjugates is quite rapid. addition of hydroxy groups at C-2 and/or C-22. whereas SE appear to be largely excluded from membranes [31.6.

92]. Steroidal sapogenins (aglycones) and saponins. 2. Moreau et al. / Progress in Lipid Research 41 (2002) 457–500 Fig. are currently widely marketed on the Internet as herbal substitutes for anabolic steroids and ViagraTM because of their reported stimulation of testosterone production. Tribulus terrestris. fenugreek.7. 8. Finally.A.) [15.87. and particularly the Indian puncture plant. which are used in production of various steroids. sources of diosgenin and yamogenin (Fig.or spirosolane-based struc- . 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. preparations of steroidal saponins from Dioscorea. 8). The steroidal alkaloid aglycones have solanidane.472 R.

5a-spirosolan-3b-ol) (Fig. have been introduced into cultivated potato by crossbreeding with wild species [87. At moderately high concentrations (e 20 mg/ 100 g fresh weight). The common solanidane aglycones include solanidine (solanid-5-en-3b-ol) and demissidine (5a-solanidan-3b-ol). which can severely depress the central nervous system [94. soladulcidine (22R. 9. and although solanidine and solasodine (but not tomatidine) caused Fig. are the main steroidal glycoalkaloids in S. as well as two tetrasaccharides of demissidine. 9).5a-spirosolan-3b-ol).25S-spirosol-5-en-3b-ol). Two analogous trisaccharides of tomatidenol.97. where they are partially hydrolyzed to the less toxic aglycones.and b-solamarine. a-solanine (3b-O-galactose-rhamnose1glucose2) and a-chaconine (3b-O-glucose-rhamnose1rhamnose2).25S. respectively. tomatidine is the principal aglycone and its tetrasaccharide a-tomatine (3b-O-galactose-glucose-xylose1galactose2) is the major glycoalkaloid.A. and also act as cholinesterase inhibitors.R. the latter being closely related to the spirostanol sapogenins [92]. and common spirosolane aglycones include solasodine (22R. and tomatidine (22S. Two trisaccharides of solanidine. .95]. Accumulation of solanine and chaconine in potato tubers is closely associated with light-induced greening (chlorophyll synthesis). in tomato fruit a-tomatine accumulates in immature green fruit but is essentially absent in fully ripe fruit.25R. Steroidal aglycones commonly found in glycoalkaloids of the Solanaceae. tuberosum. solasonine. / Progress in Lipid Research 41 (2002) 457–500 473 tures. In tomato. The steroidal glycoalkaloids are toxic to humans and other mammals. a. They are intense irritants of the gastrointestinal tract due to disruption of cell membranes. 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].95. whereas the major constituents in eggplant are the aglycone solasodine and its trisaccharide solasonine (3b-O-galactose-rhamnose1glucose2) [93]. although metabolically these appear to be independent events [94.98].25R-spirosol-5-en-3b-ol). tomatidenol (22S. 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. they are poorly absorbed by the gastrointestinal tract. Fortuitously. Similarly. Moreau et al. or a-tomatine. these alkaloids have a bitter taste and create a burning sensation in the mouth and throat. which in turn are rapidly excreted [97].92].

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). Although definitive proof is still lacking. it has been shown Fig. biosynthesis. whereas its glycosides (solasonine and solamargine) show selective toxicity against cancer cells [101]. developing tissues generally occurs in apical leaves and flowers and ultimately in seeds [104. leading to the conclusion that cholesterol serves as the sterol precursor [9. in spinach. whereas in table beet (Beta vulgaris) these steroids are scarcely detectable [23. The occurrence. Appreciable levels were detected in seeds of about 35% of the species tested [102]. Accumulation of phytoecdysteroids produced in young.A. solasodine has hypocholesterolaemic and antiatherosclerotic effects [100]. Among chenopod crop species.8. 20-Hydroxyecdysone is synthesized from lathosterol in spinach (Spinacia oleracea) and is identical with the naturally-occurring hormone in insects.102]. 2. 10). this was reversible upon removal of the alkaloids from the diet [93]. Moreau et al.474 R. . Phytoecdysteroid—a plant steroid that serves as an insect molting hormone. there have been a number of reports indicating that some steroidal alkaloids have potential pharmacological or therapeutic applications [98]. is essentially non-toxic relative to the 5-unsaturated solanidine and solasodine [99].105]. which is identical to the principal molting hormone isolated from insects [23]. However. For example. and distribution of these plant steroids have been studied most extensively in species of the Chenopodiaceae (Goosefoot family). A recent extension of this work determined that among the aglycones. The biosynthetic origin of phytoecdysteroids is an interesting and as yet incomplete story.102]. are found in higher plant species representing over 100 families [23. the most commonly encountered being 20-hydroxyecdysone (Fig. 10. which produces exclusively R 7 rather than R5 sterols (see Section 2. either by functioning as antifeedants or via disruption of development [102].2).15]. Phytoecdysteroids and brassinosteroids Phytoecdysteroids. tomatidine. / Progress in Lipid Research 41 (2002) 457–500 significant liver enlargement. On the other side of the ledger. analogues of insect-molting hormones (ecdysteroids) that play an essential role in insect development and maturation. A number of studies have shown incorporation of radiolabeled cholesterol into these plant steroids. with a saturated steroid nucleus. More than 150 different phytoecdysteroid structures have been reported [103]. it is widely accepted that production of phytoecdysteroids in plants deters predation by non-adapted insects (as well as other invertebrates such as nematodes).

was that dim mutant plants accumulate very high levels of isofucosterol. Moreau et al. Brassinosteroids: a recently discovered class of plant hormones derived from campesterol and other phytosterols. This is actually a three-step process that includes 24-methylcholest-4-en-3-one and 24-methyl-5a-cholestan-3-one as intermediates. / Progress in Lipid Research 41 (2002) 457–500 475 that lathosterol (cholest-7-en-3b-ol) is the direct precursor of 20-hydroxyecdysone [106]. 11. A recent study of the brassinolide-deficient 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. Campesterol is the mainstream desmethyl phytosterol from which brassinolide and its immediate precursor castasterone (Fig.109]. 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. are presently open questions [23]. and are also often impaired in reproduction [108–110].R. the most active compound being brassinolide (Fig. another active brassinosteroid. roseus and various Arabidopsis mutants [109–111]. 11). brassinosteroids have come to be recognized as an important new class of potent steroid hormones in plants. Over 40 of these plant steroids have been fully characterized. unrelated to brassinolide synthesis. which was also the first to be isolated [110].24(25)-dienol intermediate. Mutants that are deficient 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. 11). The initial steps in the dual pathways from campesterol to castasterone and brassinolide involve the conversion of campesterol to campestanol. 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. An interesting observation from this study. Whether there are separate biosynthetic routes to phytoecdysteroids utilizing cholesterol and lathosterol. The complete. indicating that the DIM gene product also performs the reduction of isofucosterol to sitosterol via the stigmasta-5. are derived. operative in the nanomolar range or lower as are their steroid counterparts in animal cells [108.A. . It was recently determined that the brassinolide-deficient Arabidopsis mutant dim (also called dwf1) lacks the ability to convert 24-methylenecholesterol to campesterol via a 24-methyldesmosterol intermediate [109]. 4). and whether cholesterol is converted to lathosterol in some plants by consecutive hydrogenation and dehydrogenation steps (Fig.

3. Traditionally. Digitonin precipitation also has been used in studies of both fungal sterols [114] and phytosterols [115] to isolate the FS fraction (binding of . The extraction of FS. 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 carbon–carbon double bonds [1]. V. there are two alternate routes dubbed the ‘‘early’’ and ‘‘late’’ C6-oxidation pathways. DWF4 and CPD. In a later section we will present several SPE methods for the fractionation of free phytosterols before GC analysis. We recently reported a silica SPE method to purify hydroxycinnamate steryl esters (HSE) in corn fiber oil [113]. Piironen). the plate is developed with an appropriate solvent mixture. SE. It is interesting that the products of two genes in Arabidopsis. Once a lipid extract has been prepared it is often necessary to separate (fractionate) one or more of the sterol lipid classes.3]. Finally. methylene chloride. Extraction and fractionation of phytosterols Most common methods for the extraction of lipids also extract phytosterols. Before the development of micro-scale GLC and HPLC analytical methods. 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. Moreau et al. and each ‘‘spot’’ containing a specific sterol lipid class is scraped into a tube and eluted with solvent. Even after chloroform-methanol extraction.1. quantitatively extract free phytosterols (FS) and phytosteryl fatty-acid esters (SE) [1. 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. additional phytosterols are sometimes liberated by subsequent acid or alkaline hydrolysis. 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. In the last decade. was used to fractionate the various lipid classes. Additional research is necessary to provide a better understanding of the optimal extraction methods that are required to assure complete phytosterol extraction. Preparative thin-layer chromatography can be used to separate and fractionate sterol lipid classes. and increasing the polarity of the solvent gave a higher percentage of extraction [1]. ethanol. After spotting the sample(s) on a TLC plate.110]. / Progress in Lipid Research 41 (2002) 457–500 yield 24-methyl-5a-cholestan-3-one [111]. In the subsequent metabolism of campestanol to castasterone.476 R. ‘‘open column’’ LC (liquid chromatography). Methods for the quantitative analysis of phytosterols and phytostanols 3. We routinely use the Bligh and Dyer chloroform–methanol extraction method to extract all sterol lipid classes [112].A. suggesting that there may be pools of ‘‘bound’’ or ‘‘evasive’’ phytosterols in some plant tissues (personal communication. which refers to whether the 6-keto group present on the B-ring of castasterone is introduced during the first step (campestanol to 6-oxocampestanol) or during the last step (6-deoxocastasterone to castasterone) [110]. with either silicic acid or Floricil as the solid phase column packing. Nonpolar solvents such as hexane (commonly used to extract most types of vegetable oils). open column LC has generally been replaced by similar methods using pre-packed SPE (solid phase extraction) cartridge columns. and ferulate phytosteryl esters from corn fiber was compared with four different solvents (hexane.

3]. In our methodology. hexane/isopropanol/water. steryl glycoside. Moreau et al. Although methods have been reported for the simultaneous analysis of both nonpolar (FS. and this technique is now infrequently used to isolate free phytosterols. 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 flour HPLC-Silica Corn fiber 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. fatty acid ester. Amino. resulting in retention times of 2. SG. The three nonpolar sterol lipid classes are quantitatively analyzed with a hexane-based gradient that starts at 0% isopropanol and increases to 0. and sometimes HSE) and polar (SG and ASG) sterol lipid classes [119. polar or ‘‘normal phase’’ HPLC columns (e. and HSE [51]. resulting in retention times of 6 and 11 min for ASG and SG.R. In general. acylated steryl glycoside. / Progress in Lipid Research 41 (2002) 457–500 477 digitonin to a sterol. . and 28 min for SE. 9 Unpublished SE. The two polar sterol lipid classes are quantitatively analyzed with a gradient that starts at 90/10. respectively. hydroxycinnamic-acid esters. free alcohol. and consequent precipitation of the complex. and ASG).2. both equipped with a Diol column but programmed for different solvent gradients. SE. Numerous high performance liquid chromatography (HPLC) methods have been developed to both qualitatively separate and quantitatively analyze phytosterol lipid classes (Table 1). SE. or phenyl) are used to separate molecular species (individual compounds) that comprise a lipid class (see next section and Table 2). Grunwald and Huang [116] compared five different TLC methods for separation of the four common sterol lipid classes in plant tissues (FS. with the other glycolipids and phospholipids eluting over the range of 15–50 min (Fig. Numerous other TLC methods for separation of phytosterol lipid classes have been reported [1. HSE.121]. hexane/isopropanol. 13). CN) are used to separate phytosterol lipid classes (Table 1) [117–123] and ‘‘reversed phase’’ HPLC columns (e.25% during 40 min (Fig. 12). DIOL. C18=ODS.g. requires a free 3b-OH group).g. we find that the most accurate way to quantify these lipids is to analyze the polar and nonpolar classes separately (Figs. C8. 3. However. there is evidence that certain types of phytosterols are not quantitatively precipitated by digitonin. 21. FS. the filtered total lipid extract is injected in two different HPLC systems. ASG. 12 and 13) [85. silica. FS. SG. and increases to 45/50/5.A. Methods for the separation of intact phytosterol classes Thin-layer chromatography was traditionally used for qualitative separation of phytosterol lipid classes.120].

3. acylated steryl glycoside. 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 . Methods for the separation of molecular species of phytosterol conjugates When the five phytosterol lipid classes (FS. SE. HSE. / Progress in Lipid Research 41 (2002) 457–500 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. HPLC Chromatogram of nonpolar lipids including SE. 3. steryl glycoside. HSE. and HSE. ASG. SG. Fig. FS. Billheimer and colleagues [124] reported the first reversed phase HPLC method to separate molecular species of SE (stigmasteryl oleate and campesteryl palmitate are two examples of SE molecular species). free alcohol.A. 12. and ASG) have been separated by TLC or HPLC. using a DIOL column and detection via an evaporative light scattering detector [51]. it is sometimes useful to study the individual compounds (molecular species) within each class. Kesselmeier and colleagues [125] reported a method to separate molecular species of FS and SG. SG. FS. hydroxycinnamic-acid esters. Moreau et al.478 R.

respectively [50]. unpublished results). these authors noted that additional studies would be necessary to optimize the conditions for acid hydrolysis of phytosterol conjugates.R. This method of phytosterol analysis has been the most widely used.D. HSE. 13. 7. as well as the three other common plant glycolipid classes [117]. R5-avenasterol (isofucosterol) is ‘‘lost’’ and lathosterol (cholest-7-en-3b-ol).23. acid hydrolysis caused its isomerization to fucosterol and several 5. from the 6-OH of the hexose moiety). Similarly. [128] suggested that replacing acid plus alkaline hydrolysis protocols with enzymatic hydrolysis would solve this problem. Moreau. but this . Several methods have been reported for separating molecular species of HSE from corn fiber oil. Whitaker. A recent report described an HPLC method to separate intact molecular species of ASG and SG. alkaline hydrolysis alone is sufficient to cleave all of the conjugated phytosterols [55]. The points of hydrolytic cleavage of the four phytosterol conjugates are indicated in Fig.24(25)-stigmastadienols [128]. Kamal-Eldin et al. However. HPLC Chromatogram of polar lipids including ASG and SG.125]. was isomerized to cholesterol (B. and lipids of other grains (Table 2) [124. SE. routine method for hydrolysis of phytosterol conjugates that includes both acidic and alkaline steps. using a DIOL column and detection via an evaporative light-scattering detector (R. The most abundant molecular species of HSE in corn fiber oil and rice bran oil are sitostanyl ferulate and cycloartanyl ferulate. Since vegetable oil samples usually contain primarily SE and little or no SG or ASG. included as an internal standard. Methods for hydrolysis of conjugates and separation of free phytosterols Total phytosterols (including free. whereas the glycosydic linkages in SG and ASG require acid hydrolysis (4–6 N HCl).A. esterified and glycosylated) can be quantified by hydrolysis and subsequent GC analysis of the combined FS. 3. rice bran oil. and the fatty acid-hexose ester linkage in ASG can be hydrolyzed via saponification (alkaline hydrolysis with 1–2 N KOH or NaOH). Moreau et al. Toivo and colleagues [126.4. we found that during acid hydrolysis of SG. unpublished observation). Kesselmeier and colleagues [125] reported an enzymatic method (using a commercial b-glucosidase) to hydrolyze SG. One artifact observed with this method is that if plant tissues contained R 5-avenasterol.127] developed an elaborate.and 5. / Progress in Lipid Research 41 (2002) 457–500 479 Fig.

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). Although cholesterol esterase has been used to hydrolyze carotenoid esters [129]. which may result in peak tailing and poor resolution [1].3. and enzymatic methods may prove to be superior to acidic and/or alkaline hydrolyses.6).480 R. but some methods show good separation and quantification of underivatized phytosterols (Fig. Clearly. / Progress in Lipid Research 41 (2002) 457–500 method has been used by only a few researchers. 14). the quantitative GC analysis of FS can be achieved using one of the numerous published methods (Table 3) [130–136]. and could perhaps also be used to hydrolyze phytosteryl fatty-acid esters. 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 fiber 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. After hydrolysis of conjugates.A. Moreau et al. . Several reversed phase HPLC procedures for the separation of FS have also been reported [1. solid phase extraction. The enzyme ‘‘cholesterol ester hydrolase’’ is used to hydrolyze cholesteryl fatty-acid esters during the routine enzymatic assay of cholesterol (see Section 3.83– 85]. more research is required to perfect hydrolysis methods for phytosterols.125] but the separation and sensitivity are generally lower than with GC. Although quantitative GC analysis of underivatized phytosterols appears to be accurate and reliable. some experts prefer derivatizing phytosterols to prevent dehydration and decomposition. we are unaware of studies employing it to hydrolyze phytosteryl fatty-acid esters. Table 3 Methods for the hydrolysis and GC analysis of free phytosterols (unless otherwise noted. which also enabled us to identify ASG fatty acids by GC analysis of their methyl esters) [35. We used a modified version of the enzymatic method [125] to analyze FS derived from SG (and ASG by including mild alkaline methanolysis.

15) can be used to predict fragments in phytosterols with other structures. Moreau et al. accurate structural identification 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. or as TMS ethers (M++72). Moreau. Mass spectrometry and NMR of phytosterols Mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (NMR) are valuable tools for phytosterol identification. whereas m/e 296 is much more abundant in the . Because the fragmentation pattern of phytosterols is different when they are analyzed in each of these three forms.R. One complicating factor when employing GC–MS for identification of phytosterols is the fact that published spectra include data for free phytosterols (Fig. or TMS). Several common free phytosterols are included in two ‘‘on-line’’ databases: http://webbook.u-tokyo. 14.e. GC–MS chromatogram of sterols and stanols in saponified corn fiber oil (R. brassicasterol and epibrassicasterol) or isomers (e. 3. employing GC–MS of phytosteryl acetates. showed that R 5-avenasterol (24Z) always has a longer GC retention time than fucosterol (24E). Since many of the common phytosterols occur as epimers (e. GC separation and/or identification of these structurally similar compounds can be challenging. unpublished results).g.m.5. and that these two isomers can be distinguished by the mass spectra of their Electron impact (EI) GC–MS has been extensively employed to identify free phytosterols. Another study [138].ac. as acetate esters (M++42).nist. We will present a broad overview of these topics but would urge the reader to consult previous reviews [1] for more details. R 5-avenasterol and fucosterol). acetate. free. Goad’s 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.A. In a recent study reporting the acid-induced isomerization of R 5-avenasterol and fucosterol [128]. The several common points of EI–MS fragmentation ( M-60=m/e 394 is much more abundant in the fucosterol EI spectrum.g. small differences in the GC-MS fragmentation of TMS derivatives were used to document this isomerization. 15) and for their TMS-ether and acetate-ester chemistry and http://lipid. / Progress in Lipid Research 41 (2002) 457–500 481 Fig. Rahier and Benveniste [137] described the mass spectra of six common phytosterols which were each analyzed by GC–MS as the free form (M+).

482 R. Enzymatic assays of phytosterols Enzymatic methods [141] and specific test kits have been developed and marketed to measure cholesterol in blood and other samples.g. 13 C nuclear magnetic resonance (NMR) has been reported to be a valuable tool for the chemical identification of phytosterols since each of the 28 or 29 carbons in the molecule can potentially be examined individually [140].compared with 24Z-ethylidene phytosteryl acetates were also reported elsewhere [139]. 3.6. Other emerging forms of ‘‘soft’’ ionization mass spectrometry (e. specifically atmospheric pressure chemical ionozation mass spectroscopy (APCI–MS). Moreau et al. Another study employed HPLC–MS.A. -avenasterol spectrum. 15. Most of these kits include cholesterol ester hydrolase to hydrolyze cholesteryl fatty-acid esters and cholesterol oxidase (from animal or microbial sources) 5 . electrospray and MALDI-TOF) could also potentially be used to analyze phytosterols and their intact conjugates.19]. 1H and 13C NMR have proven to be essential in the structural elucidation and quantification of 24R. Electron impact mass spectrum of sitosterol (MW 414) and its major fragment ions (spectrum was kindly provided by A. Nunez). Unique EI–MS fragments of 24E. to provide structural information about SG and ASG in red bell pepper fruit [117]. / Progress in Lipid Research 41 (2002) 457–500 Fig.and 24S-epimers of C-24 methyl and ethyl phytosterols [1.

we are not aware that this claim has been validated. Recent strategies for lowering serum Fig. / Progress in Lipid Research 41 (2002) 457–500 483 to oxidize cholesterol. there is a potential to develop enzymatic methods for the rapid quantification of phytosterols in foods.1. 16. Note that oxidation of both sterols shifts the R 5 double bond to R 4. and critical questions Phytosterols are natural components of human diets. Cholesterol oxidase reaction with cholesterol and stigmasterol. and consequently we typically consume much lower amounts ( e 25 mg/day) in our diets [145. Historical perspectives. The amount of H2O2 produced can then be measured by one of several spectrophotometric or fluorometric assays and used to calculate the amount of cholesterol in the sample. 16) [142]. dietary phytosterols have been estimated at almost twice this level [146]. the development of convenient enzymatic assays using microtiter plates is very attractive. phytostanols. and for each molecule of cholesterol that is oxidized. In the West. Moreau et al. cereals. Health-promoting effects of phytosterols. and their esters 4. 16). and found that the values varied considerably. . Common dietary sources of phytostanols are corn. Sterol chemists and biochemists focused their efforts on cholesterol because elevated serum cholesterol levels were shown to be a prominent risk factor for cardiovascular disease (CVD). For vegetarians. changing dogma. rye. Enzyme kinetic data were reported for cholesterol oxidase from Nocardia erythropolis [143]. This is roughly equivalent to the amount of cholesterol ( e 300 mg/day) consumed. Obviously. largely derived from vegetable oils. the existence and dietary effects of these minor sterols were largely ignored and poorly understood. 4. Smith and Brooks [143] compared the Km and Vmax values for the oxidation of several phytosterols by cholesterol oxidase from Nocardia erythropolis (Fig.146].R.145]. When one considers the high cost of GC and HPLC instruments that are currently required to analyze phytosterols. Phytostanols are much less abundant in nature than phytosterols. and rice. wheat. one molecule of H2O2 is produced (Fig. fruits and vegetables [144. we consume an average of e 250 mg per day of phytosterols. with the current interest in phytosterols.A. For many years. Although the manufacturers of some of these kits indicate that the kits can also be used to measure phytosterols.

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