Steroids 70 (2005) 886–895

Gram-scale chromatographic purification of�-sitosterolSynthesis and characterization of�-sitosterol oxides

Xin Zhanga, Philippe Geoffroyb, Michel Mieschb, Diane Julien-Davida, Francis Raulc,Dalal Aoude-Wernerd, Eric Marchionia,∗

a Laboratoire de Chimie Analytique et Sciences de l’Aliment (UMR 7512), Faculte de Pharmacie, Universite Louis Pasteur,74 route du Rhin, 67400 Illkirch, France

b Laboratoire de Chimie Organique Synthetique (UMR 7123), Universite Louis Pasteur, 1 rue Blaise Pascal, 67008 Strasbourg, Francec Laboratoire d’Oncologie Nutritionnelle, Inserm UMR S392/IRCAD, 1 place de l’Hopital, 67091 Strasbourg, France

d Aerial, rue Laurent Fries - Parc d’Innovation, 67412 Illkirch, France

Received 28 October 2004; received in revised form 9 June 2005; accepted 9 June 2005Available online 20 July 2005

Abstract

parative,The

tainively,guesntcts,stig-

s ofrs of

milar

are,,

withtos-xide

esul-

An effective purification method for�-sitosterol was developed starting from a commercial source of a phytosterol mixture using preadsorption column chromatography.�-Sitosterol (≥95% purity) was obtained on a gram-scale. Thus, the synthesis of six�-sitosterol oxidesincluding 7�-hydroxy, 7�-hydroxy, 5,6�-epoxy, 5,6�-epoxy, 7-keto, and 5�,6�-dihydroxysitosterol, were successfully carried out.spectral characteristics of all the synthetic intermediates and target compounds (∼95% purity) were well-documented.© 2005 Elsevier Inc. All rights reserved.

Keywords: Phytosterol oxides;�-Sitosterol; Isolation; Column chromatography; Synthesis

1. Introduction

During the last decades, phytosterols have receivedgreat attention due to their biological activities and health-promoting effects[1–3]. Several laboratories have reportedon the ability of phytosterols to lower cholesterol and LDL-cholesterol[4,5].

Like other unsaturated lipids, phytosterols and choles-terols are susceptible to oxidation by heating, exposure toionizing radiation, light, chemical catalysts, or enzymaticprocesses[6–8]. In vitro studies have shown that some choles-terol oxides have cytotoxic, mutagenic, atherogenic, and car-cinogenic activities[9,10]. Due to the structural similaritywith cholesterol and its oxides, phytosterols and their oxideshave received much attention with respect to their biologicalproperties and consequently, their performance in food safetyevaluation[10–12].

∗ Corresponding author. Tel.: +33 3 90244326; fax: +33 3 90244325.E-mail address: eric.marchioni@pharma.u-strasbg.fr (E. Marchioni).

Whereas mammalian and fungal cells generally conone major sterol, cholesterol and ergosterol, respectplants contain complex phytosterols of cholesterol analo[13]. Among phytosterols,�-sitosterol is the most abundaand widely distributed sterol in plants and plant produtogether with other plant sterols such as campesterol,masterol, brassicasterol, dihydrobrassicasterol, et al.[13,14].They are of similar structures only due to the variationthe number and location of double bond, and the numbecarbon atoms composing the side chain. Thus, their siphysical properties lead to the separation very difficult.

Since not all pure individual phytosterols until so faravailable commercially except for�5-stigmasterol (Sigmapurity: 95%) and�-sitosterol (Acros or Sigma, purity: 97%purified from natural plant resource or synthesis, butan excessively high price), individual standards of phyterol oxides are also not available. Thus, phytosterol omixtures are often used in the relevant research[15–17].However, Maguire et al.[18] reported that phytosterol oxidexhibit toxicity similar to that of cholesterol oxides in c

0039-128X/$ – see front matter © 2005 Elsevier Inc. All rights reserved.doi:10.1016/j.steroids.2005.06.003

X. Zhang et al. / Steroids 70 (2005) 886–895 887

tured mammalian cells and further suspected that individualphytosterol oxides might behave differently from an oxidemixture. Therefore, it seems important to obtain individualstandards of phytosterol oxides and, consequently, to researchthe different factors influencing their formation, their concen-tration or possible interconversion in foods, their adsorptionin plasma, and their biological activities.

It has known that the better way to prepare the individualsterol oxides is the synthesis. However, as motioned above,due to the extremely similar molecular structures of sterols,it makes difficulty to obtain enough amounts of individualphytosterols for the chemical synthesis. Hyde and Elliott[19] described a liquid-gel chromatographic separation ofmilligram-scale of sitosterol and campesterol on a SephadexLH-20 column (38- or 88-cm column (×2.5 cm i.d.)) aftersix recycles. Grandgirard et al.[20] isolated milligram-scaleindividual phytosterols, e.g. sitosterol, campesterol and bras-sicasterol, using semi-preparative reversed-phase HPLC after51 injections. But it was still not enough to synthesize theoxides. Considering the great number of oxides to be pre-pared, they used a method adapted from Osada et al.[21]to form several oxides in one step. On the other hand,based on the complex enzymatic synthetic method[2,13] orsolvent crystallization[22,23], the routes to get individualphytosterols were not only incomplete but were also time-consuming.

ctives ,w therc ourk ato-g ela . Six�e l,w ands copicm

2

2

nce),a nifi-a res( as asf s-t nts( bes

usedf radeh d dis-t hic

grade acetonitrile and 2-propanol were purchased fromRiedel-de Haen (Seelze, Germany). An alumina–AgNO3column was prepared as described by Pascal et al.[24].

Unless otherwise stated, all the chemical reactions inorganic medium were carried out under argon using freshlydistilled anhydrous solvents. After workup, organic phaseswere evaporated under reduced pressure. Trace solvents wereremoved on a vacuum pump. Evolution of chemical reac-tions and column chromatography (Silica gel Si 60, particlesize 40–63�m, Merck, Germany) were monitored by ana-lytical thin-layer chromatography (TLC) with silica gel 60F254 pre-coated aluminium plates (Merck, Germany). Com-ponents on the plates were visualized after spraying with 20%phosphomolybdic acid reagent in ethanol followed by heat-ing at 150◦C.

2.2. GC analysis

Prior to GC analysis, the analytes were transformedto trimethylsilyl (TMS) ethers with 50�L of pyridineand 40�L of N-methyl-N-(trimethylsilyl)trifluoroacetamide(MSTFA) (Sigma, Steinheim, Germany) at ambient temper-ature overnight, then diluted with 410�L of isooctane.

Gas chromatography-mass spectrometry (GC-MS) anal-yses were performed on a Varian Star 3400 GC instrumente rianS oper-a eVa aa werep eres ary:56 tureg11 ws:iw .H ith afl

2

ofp ar-i th a7 rian9 ce).A el,5 thep e/2-p ina was2

The aim of the present study was to develop an effeeparation method for gram-scale purification of�-sitosterolhich is the most representative phytosterol, for the furhemical synthesis of standard phytosterol oxides. Tonowledge, this is the first report of gram-scale chromraphic purification of�-sitosterol by preparative silica gdsorption column and chemical synthesis of its oxides-sitosterol oxides, namely 7�-hydroxy, 7�-hydroxy, 5,6�-poxy, 5,6�-epoxy, 7-keto, and 5�,6�-dihydroxysitosteroere presented in this report. All of these productsynthetic intermediates were characterized by spectrosethods.

. Experimental

.1. Materials and reagents

Generol 95R (Cognis, Saint-Fargeau-Ponthierry, Fracommercial mixture of phytosterols, was an unsapo

ble fraction of soja oil. The composition of the mixtuas percent ratio of the peak areas by GC analysis) wollow: brassicasterol (10%),�5-avenasterol (3%), campeerol (39%) and�-sitosterol (46%), along with small amou2%) impurities. It had a slight yellow color and shouldtored at 4◦C before use.

All organic reagents (analytical grade) and chemicalsor synthesis were commercially available. Technical geptane was purchased from SDS (Peypin, France) an

illed from calcium hydride before use. Chromatograp

quipped with an on-column SPI injector coupled to a Vaaturn 2000 mass sensitive detector (Varian, France)ting in the electron impact (EI) ionization mode at 70nd monitored on the full-scan range (m/z 40–600). Datcquisition and processing, and instrumental controlerformed by Varian Saturn WS software. Analytes weparated with a VF-5ms capillary column (phase station% phenyl–95% dimethylpolysiloxane, thickness of 0.1�m,0 m× 0.25 mm, Varian, France). The column temperaradient was programmed from 105◦C (hold for 2 min) to70◦C at 20◦C/min and then, to 320◦C at 7◦C/min (hold for5 min). The injector operating conditions were as follo

njection volume 1�L; initial injector temperature of 105◦Cas increased to 300◦C at 100◦C/min (hold for 40 min)elium (purity 99.9995%) was used as a carrier gas wow rate of 1 mL/min.

.3. HPLC analysis

Analytical HPLC was used to monitor the processurification of �-sitosterol. The HPLC system was a V

an ProStar 210 pump (Varian, France) equipped wi125-038 sample injector (Rheodyne, USA) and a Va050 Variable Wavelength UV–vis Detector (Varian, FranPolaris C8-A column (250× 4.6 mm i.d. stainless ste

�m particles, Varian, France) was used to separatehytosterols with an isocratic mobile phase (acetonitrilropanol/water = 2:2:1, v/v/v) at a flow rate of 0.5 mL/mt ambient temperature. The UV detector wavelength08 nm, 0.1 AUFS.

888 X. Zhang et al. / Steroids 70 (2005) 886–895

Phytosterols could be dissolved in acetonitrile and injecteddirectly into the HPLC system without any derivatization.

2.4. Other spectrometric analysis

NMR spectra were recorded on a Bruker AC-300 spec-trometer (1H NMR at 300 MHz and13C NMR at 75 MHz)with CDCl3 as the solvent at ambient temperature. Meltingpoints (m.p.) were measured with a BIBBY Stuart Scientific,SMP3 (UK) melting apparatus. Infrared (IR) spectra wererecorded on a Perkin Elmer IR-881 spectrometer. Opticalrotations were measured with a Perkin-Elmer 241 polarime-ter. Microanalyses were performed by Le Service de Micro-analyses de l’Universite Louis Pasteur de Strasbourg.

2.5. Preparative adsorption chromatography forisolation of β-sitosterol (1)

Consideration of large amounts consumption of silica geland organic reagents, a two-column system was designed topurify �-sitosterol. Silica gel was dispersed with heptane andthe columns were slurry packed with slight pressure. Thecolumns were conditioned with the corresponding eluentsbefore use.

As a slight yellow solid, commercial Generol 95R con-tained white phytosterols component together with minora ofG firstpi min)t per-f centc usedt ctionw d bya rr

L ofc ato-g te( d byg ve.T ndt thea f elu-a umn,t db withs po-r . Thes o tenc y≥ thism d fivet np

1: white solid; m.p. 136–138◦C (literature m.p. value:138–139◦C [19]). 1H NMR (300 MHz, CDCl3) � 5.34 (1H,br s, H-6), 3.55–3.45 (1H, m, H-3), 2.27 (2H, m), 2.26-1.08(28H, m), 1.00 (3H, s, H-19), 0.92 (3H, d,J = 6.5 Hz, H-21), 0.84 (3H, t,J = 7.2 Hz, H-29), 0.83 (3H, d,J = 6.5 Hz,H-26), 0.80 (3H, d,J = 6.6 Hz, H-27), 0.68 (3H, s, H-18).13C NMR (75 MHz, CDCl3) � 140.75, 121.71, 71.79, 56.76,56.05, 50.13, 45.83, 42.30, 39.77, 37.25, 36.50, 36.14, 33.94,31.90, 31.65, 29.14, 28.24, 26.07, 24.30, 23.06, 21.08, 19.82,19.39, 19.03, 18.77, 11.98, 11.85. IR (CCl4), 3622, 3326,2958, 2938, 2901, 2870, 1463, 1432, 1377, 1365, 1052, 1022,1002, 959, 839 cm−1. [�]20

D −34◦ (c 2, CHCl3) (literaturevalue:[�]20

D −38◦ [19]). Anal. calcd for C29H50O: C 83.99,H 12.25. Found C 83.74, H 12.16. GC-MS-EI (as TMS ether),m/z (%): 487 (32), 472 (16), 397 (100), 382 (55), 358 (51),256 (39), 213 (21), 159 (35), 129 (98), 105 (55), 73 (90), 43(63).

2.6. Chemical synthesis

2.6.1. Sitost-5-en-3β-ol acetate (2)Pyridine (289 mg, 3.65 mmol) and a solution of acetyl

chloride (210 mg, 2.67 mmol) in CH2Cl2 (2 mL) were addedto a solution of�-sitosterol1 (1.01 g, 2.44 mmol) in CH2Cl2(20 mL). The reaction mixture was stirred at room temper-ature for 1 h. Water (5 mL) was added to the residue, andto eousH olu-t da e. Theci givec d):mb .03( H,H ,t ,dC .03,4 1.89,3 1.02,12 1243,1f 6.

2lite

( ,8 xedf wedt andw derr silica

mount (2%) of polar or unpolar yellow impurities. 15 generol 95R was dissolved in 80 mL of chloroform andurified on a silica gel chromatographic column 1 (35× 3 cm

.d.) with heptane/ethyl acetate (4:1) as an eluent (5 mL/o remove the impurities quickly. No separation wasormed between phytosterols in column 1, so the peromposition of phytosterols was not changed. TLC waso monitor the separation process. The phytosterol fraas collected and the organic solvents were removerotary evaporator (Rotavapor, Buchi, Switzerland) unde

educed pressure at 40◦C.8 g of the purified phytosterols were dissolved in 30 m

hloroform and subjected to a preparative silica gel chromraphic column 2 (60× 6 cm i.d.) with heptane/ethyl aceta6:1) as the eluent (7 mL/min). This column was operateravity flow and flow rate was controlled by the exit valhe fractions (n × 150 mL) were checked first by TLC a

hen by analytical HPLC. TLC was used only to monitorbsence or presence of phytosterols in the fractions ote. As soon as the sterols were eluted out of this col

he composition of these fractions (n = 15–35) were checkey analytical HPLC as mentioned above. The fractionsimilar mobility of�-sitosterol were pooled together, evaated, and subjected to column chromatography 2 againilica gel could be used repeatedly for 8–10 times. Eight tycles were needed to purify 2 g of�-sitosterol until the purit95% was reached (2–5% campesterol impurity). Withethod, the whole purification processes were repeate

imes. Finally, 10 g (1, 24 mmol) of�-sitosterol were theroduced.

he mixture was extracted with CH2Cl2 (3× 50 mL). Therganic layer was washed sequentially with a 10% aquCl solution (50 mL) and a saturated aqueous NaCl s

ion (50 mL), dried over MgSO4. The mixture was filterend the solvent was evaporated under reduced pressurrude product was purified on a silica gel column (35× 2 cm.d.) with cyclohexane/ethyl acetate (95:5) as an eluent toompound2 as a white solid (1.10 g, 2.40 mmol, 90% yiel.p. 121–122◦C. 1H NMR (300 MHz, CDCl3) � 5.37 (1H,r s, H-6), 4.65-4.51 (1H, m, H-3), 2.31 (2H, m, H-4), 23H, s, CH3COO ), 2.00-1.09 (30H, m, including 1s (3-19) at 1.02 ppm), 0.92 (3H, d,J = 6.5 Hz, H-21), 0.84 (3H

, J = 7.2 Hz, H-29), 0.83 (3H, d,J = 6.5 Hz, H-26), 0.81 (3H, J = 6.6 Hz, H-27), 0.68 (3H, s, H-18).13C NMR (75 MHz,DCl3) � 170.51, 139.64, 122.64, 73.97, 56.68, 56.03, 505.83, 42.30, 39.71, 38.12, 36.99, 36.58, 36.15, 33.93, 31.86, 29.14, 28.24, 27.77, 26.07, 24.28, 23.06, 21.43, 29.81, 19.30, 19.03, 18.77, 11.98, 11.85. IR (CCl4), 2961,947, 2900, 2871, 1733, 1466, 1440, 1375, 1365, 1340,190, 1134, 1032 cm−1. [�]20

D −38◦ (c 1, CHCl3). Anal. calcdor C31H52O2: C 81.52, H 11.48. Found C 81.34, H 11.2

.6.2. 3β-Acetoxysitost-5-en-7-one (3)Pyridinium chlorochromate (20 g, 92.78 mmol) and ce

40 g) were added to a solution of compound2 (3.842 g.41 mmol) in benzene (300 mL). The mixture was reflu

or 15 h using a Dean-Stark apparatus and then, alloo cool to room temperature. The residue was filteredashed with Et2O, and the solvent was evaporated un

educed pressure. The crude product was purified on a

X. Zhang et al. / Steroids 70 (2005) 886–895 889

gel column (35× 2 cm i.d.) with cyclohexane/ethyl acetate(95:5) as an eluent to give compound3 as a white solid(3.96 g, 8.41 mmol, 63% yield): m.p. 163–164◦C. 1H NMR(300 MHz, CDCl3) � 5.69 (1H, br s, H-6), 4.75-4.65 (1H, m,H-3), 2.60-2.35 (3H, m, H-4), 2.25 (1H, br t,J = 9.0 Hz), 2.05-1.85 (6H, m, including 1s (3H, CH3COO ) at 2.04 ppm),1.75-1.45 (6H, m), 1.40-0.95 (17H, m, including 1s (3H, H-19) at 1.20 ppm), 0.92 (3H, d,J = 6.5 Hz, H-21), 0.84 (3H, t,J = 7.4 Hz, H-29), 0.82 (3H, d,J = 6.7 Hz, H-26), 0.80 (3H, d,J = 6.3 Hz, H-27), 0.67 (3H, s, H-18).13C NMR (75 MHz,CDCl3) � 201.93, 170.26, 163.82, 126.69, 72.20, 54.67,49.94, 49.80, 45.80, 45.41, 43.10, 38.30, 37.73, 36.07, 35.99,33.92, 29.11, 28.54, 27.34, 26.30, 26.07, 23.03, 21.24, 21.15,19.78, 19.03, 18.91, 17.24, 11.96. IR (CCl4), 2958, 2873,1737, 1678, 1465, 1373, 1239, 1181, 1035 cm−1. [�]20

D −91◦(c 1, CHCl3). Anal. calcd for C31H50O3: C 79.10, H 10.71.Found: C 78.99, H 10.57.

2.6.3. 3β-Hydroxysitost-5-en-7-one (7-ketositosterol)(4)

Sodium carbonate (390 mg, 3.67 mmol) was added toa solution of 3 (348 mg, 0.74 mmol) in a mixture ofMeOH/H2O (9:1, 35 mL). After being stirred at room tem-perature for 15 h, the solvent was removed under reducedpressure, and then, water (20 mL) was added. The aque-ous layer was extracted with CHCl (3× 50 mL) and EtO(a e. Theci givec ld):mb .20( ),1 m),02H2 5.80,4 1.17,2 8.91,1 75,1 m[ ,H er),m 0),1

2,

0 ,0 ew H( s-s rentm ture

was allowed to reach room temperature. Water (5 mL) wasadded, the mixture was extracted with CH2Cl2 (2× 10 mL)and saturated NaCl solution, and then, dried over MgSO4.The resulting precipitate was filtered, washed with waterand dried under reduced pressure. The crude product waspurified on a silica gel column (35× 2 cm i.d.) with cyclo-hexane/ethyl acetate (9:1) as an eluent to give5 as a whitesolid (186 mg, 0.39 mmol, 78% yield): m.p. 132.5–134◦C.1H NMR (300 MHz, CDCl3) � 5.63 (1H, d,J = 5.1 Hz, H-6), 4.75-4.55 (1H, m, H-3), 3.84 (1H, br s, H-7), 2.40-2.35(2H, m), 2.03-1.80 (6H, m, including 1s (3H, CH3COO )at 2.03 ppm), 1.80-1.05 (23H, m), 1.00 (3H, s, H-19), 0.93(3H, d,J = 6.5 Hz, H-21), 0.84 (3H, t,J = 7.2 Hz, H-29), 0.83(3H, d, J = 6.6 Hz, H-26), 0.81 (3H, d,J = 6.7 Hz, H-27),0.68 (3H, s, H-18).13C NMR (75 MHz, CDCl3) � 170.42,145.18, 124.74, 73.37, 65.24, 55.68, 49.38, 45.81, 42.19,42.10, 39.11, 37.90, 37.47, 36.74, 36.11, 33.89, 30.16, 29.11,28.28, 27.49, 26.89, 25.89, 24.26, 23.04, 21.36, 20.65, 19.79,19.01, 18.79, 18.17, 11.98, 11.62. IR (CCl4), 2959, 2867,1734, 1465, 1435, 1374, 1369, 1243, 1030, 1010 cm−1. [�]20

D−62◦ (c 1, CHCl3). Anal. calcd C31H52O3: C 78.76, H 11.09.Found C 78.39, H 11.06.

2.6.5. Sitost-5-ene-3β,7α-diol (7α-hydroxysitosterol) (6)The same procedure as for compound4 was used: com-

pound 5 (163 mg, 0.35 mmol), MeOH/HO (9:1, 20 mL),N ona ol,6� 50( 80-1HH -1 3,6 7.51,3 4.30,2 3. IR(( .F( (6),1

2ix-

t ,1 ure.N ns.T in,h andtE Oa e. Theci give

2 2 220 mL). The organic layer was dried over MgSO4, filtered,nd the solvents were removed under reduced pressurrude product was purified on a silica gel column (35× 2 cm

.d.) with cyclohexane/ethyl acetate (4:1) as an eluent toompound4 as a white solid (251 mg, 0.58 mmol, 80% yie.p. 121–124◦C. 1H NMR (300 MHz, CDCl3) � 5.68 (1H,r s, H-6), 3.70-3.60 (1H, m, H-3), 2.55-2.30 (3H, m), 21H, br t,J = 9.0 Hz), 2.10-1.75 (6H, m), 1.75-1.40 (6H, m.40-1.00 (15H, m, including 1s (3H, H-19) at 1.19 pp.92 (3H, d,J = 6.5 Hz, H-21), 0.84 (3H, t,J = 7.4 Hz, H-9), 0.83 (3H, d,J = 6.6 Hz, H-26), 0.81 (3H, d,J = 6.5 Hz,-27), 0.68 (3H, s, H-18).13C NMR (75 MHz, CDCl3) �02.37, 165.19, 126.08, 70.48, 54.69, 49.95, 49.92, 45.41, 43.09, 41.81, 38.69, 38.27, 36.34, 36.07, 33.93, 39.11, 28.54, 26.32, 26.07, 23.04, 21.21, 19.79, 19.03, 17.30, 11.96. IR (CCl4), 3620, 3412, 2958, 2943, 2873, 16625, 1464, 1370, 1385, 1379, 1296, 1180, 1058, 947 c−1.�]20

D −98◦ (c 1, CHCl3). Anal. calcd for C29H48O2: C 81.2511.29. Found C 81.31, H 11.32. GC-MS-EI (as TMS eth/z (%): 501 (80), 486 (41), 411 (26), 396 (100), 270 (187 (20), 174 (28), 161 (30), 129 (41), 73 (76), 43 (45).

.6.4. 3β-Acetoxysitost-5-ene-7α-ol (5)A solution of l-selectride (1 M in THF, 0.65 mL

.65 mmol) was added dropwise to a solution of3 (235 mg

.5 mmol) in THF (6 mL) at−78◦C. The resulting mixturas stirred at−78◦C for 1.5 h. Water (0.75 mL), 6 M NaO

0.75 mL), and 30% H2O2 (0.75 mL) were added succeively to the reaction mixture to give a clean and transpaixture. The dry ice was removed, and the reaction mix

2a2CO3 (55 mg, 0.53 mmol). After workup, the reactifforded compound6 as a white solid (96 mg, 0.22 mm5% yield): m.p. 219–220◦C. 1H NMR (300 MHz, CDCl3)5.60 (1H, d,J = 5.1 Hz, H-6), 3.85 (1H, br s, H-7), 3.65-3.

1H, m, H-3), 2.40-2.20 (2H, m), 2.05-1.80 (4H, m), 1..00 (23H, m), 0.99 (3H, s, H-19), 0.93 (3H, d,J = 6.5 Hz,-21), 0.84 (3H, t,J = 7.3 Hz, H-29), 0.83 (3H, d,J = 6.6 Hz,-26), 0.81 (3H, d,J = 6.6 Hz, H-27), 0.68 (3H, s, H8). 13C NMR (75 MHz, CDCl3) � 146.24, 123.85, 71.35.36, 55.70, 49.41, 45.82, 42.25, 42.13, 42.01, 39.16, 37.39, 37.00, 36.10, 33.90, 31.36, 29.11, 28.28, 25.89, 23.05, 20.70, 19.80, 19.02, 18.80, 18.24, 11.99, 11.6CHCl3), 2960, 2934, 2863, 1458, 1375 cm−1. [�]20

D −77◦c 1, CHCl3). Anal. calcd for C29H50O2: C 80.87, H 11.70ound C 80.78, H 11.79. GC-MS-EI (as di-TMS ether),m/z%): 575 (1), 485 (100), 470 (4), 396 (1), 253 (2), 12905 (4), 73 (22), 43 (9).

.6.6. 3β-Acetoxysitost-5-ene-7β-ol (7)CeCl3·7H2O (1.04 g, 2.78 mmol) was dissolved in a m

ure THF/MeOH (2:1, 15 mL), and compound3 (625 mg.33 mmol) in THF (15 mL) was added at room temperataBH4 (105 mg, 2.78 mmol) was added in small portiohe mixture was stirred at room temperature for 15 mydrolyzed with a 10% aqueous HCl solution (10 mL),

hen, extracted successively with CH2Cl2 (3× 50 mL) andt2O (50 mL). The organic layer was dried over MgS4,nd the solvents were removed under reduced pressurrude product was purified on a silica gel column (35× 2 cm.d.) with cyclohexane/ethyl acetate (94:6) as an eluent to

890 X. Zhang et al. / Steroids 70 (2005) 886–895

compound7 as a white solid (560 mg, 1.18 mmol, 89% yield):m.p. 67–68◦C. 1H NMR (300 MHz, CDCl3) � 5.30 (1H, brs, H-6), 4.70-4.50 (1H, m, H-3), 3.84 (1H, d,J = 7.8 Hz,H-7), 2.40-2.25 (2H, m), 2.05-1.95 (4H, m, including 1s(3H, CH3COO ) at 2.03 ppm), 1.95-1.75 (4H, m), 1.75-1.00(24H, m, including 1s (3H, H-19) at 1.05 ppm), 0.92 (3H, d,J = 6.5 Hz, H-21), 0.84 (3H, t,J = 7.2 Hz, H-29), 0.83 (3H, d,J = 6.6 Hz, H-26), 0.81 (3H, d,J = 6.7 Hz, H-27), 0.68 (3H, s,H-18).13C NMR (75 MHz, CDCl3) � 170.47, 142.34, 126.30,73.44, 73.20, 55.89, 55.34, 48.17, 45.83, 42.91, 40.77, 39.50,37.59, 36.65, 36.50, 36.09, 33.96, 29.13, 28.54, 27.70, 26.40,26.09, 23.05, 21.37, 21.02, 19.80, 19.07, 19.02, 18.82, 11.97,11.81. IR (CCl4), 3606, 2958, 2872, 1734, 1466, 1374, 1243,1135, 1085, 1033, 951, 903 cm−1. [�]20

D −6◦ (c 1, CHCl3).Anal. calcd for C31H52O3: C 78.76, H 11.09. Found C 78.54,H 11.18.

2.6.7. Sitost-5-ene-3β,7β-diol (7β-hydroxysitosterol) (8)The same procedure as for compound4 was used: com-

pound 7 (560 mg, 1.18 mmol), MeOH/H2O (9:1, 50 mL),Na2CO3 (741 mg, 6.99 mmol). After workup, the reactionafforded compound8 as a white solid (332 mg, 0.77 mmol,76% yield): m.p. 156–158◦C.1H NMR (300 MHz, CDCl3) �5.28 (1H, br s, H-6), 3.83 (1H, dd,J = 7.5 Hz, H-7), 3.60-3.45(1H, m, H-3), 2.40-2.15 (2H, m), 2.05-1.95 (1H, m), 1.90-0.95 (29H, m, including 1s (3H, H-19) at 1.04 ppm), 0.92( 3( 9( ,7 0.89,3 6.38,2 1.82.I 209,1f 58.G ),4 ).

25

wasa( -t hen,d eds( usNv e thec solep -a tion( col-u yla )a

9: m.p. 117–122◦C. 1H NMR (300 MHz, CDCl3) � 5.00-4.90 (1H, m, H-3), 2.89 (1H, d,J = 4.3 Hz, H-6), 2.13 (1H,dd, J = 11.8 Hz, H-4�), 2.05-1.75 (7H, m, including 1s (3H,CH3COO ) at 2.01 ppm), 1.75-0.90 (27H, m, including 1s(3H, H-19) at 1.07 ppm), 0.89 (3H, d,J = 6.5 Hz, H-21),0.84 (3H, t,J = 7.2 Hz, H-29), 0.82 (3H, d,J = 6.4 Hz, H-26), 0.80 (3H, d,J = 6.5 Hz, H-27), 0.60 (3H, s, H-18).13CNMR (75 MHz, CDCl3) � 170.22, 71.40, 65.18, 59.17, 56.77,55.76, 45.81, 42.43, 42.32, 39.35, 36.13, 34.98, 33.88, 32.12,29.85, 29.13, 28.75, 28.09, 27.21, 26.09, 24.05, 23.04, 21.34,20.57, 19.81, 19.02, 18.69, 15.85, 11.96, 11.84.[�]20

D −22◦(c 1, CHCl3). Anal. calcd for C31H52O3: C 78.76, H 11.09.Found C 78.73, H 11.23.

10: m.p. 110–111◦C;1H NMR (300 MHz, CDCl3) � 4.85-4.70 (1H, m, H-3), 3.07 (1H, br s, H-6), 2.20-1.75 (9H,m, including 1s (3H, CH3COO ) at 2.03 ppm), 1.75-0.95(26H, m, including 1s (3H, H-19) at 0.99 ppm), 0.89 (3H, d,J = 6.4 Hz, H-21), 0.84 (3H, t,J = 7.2 Hz, H-29), 0.83 (3H,d, J = 6.7 Hz, H-26), 0.80 (3H, d,J = 6.5 Hz, H-27), 0.63(3H, s, H-18).13C NMR (75 MHz, CDCl3) � 170.55, 71.34,63.58, 62.51, 56.16, 56.06, 50.96, 45.80, 42.27, 39.76, 38.00,36.66, 36.08, 35.01, 33.87, 32.46, 29.72, 29.12, 28.16, 27.20,26.02, 24.18, 23.04, 21.91, 21.32, 19.80, 18.73, 17.03, 11.97,11.75. IR (CCl4), 2959, 2872, 1737, 1467, 1435, 1418, 1386,1377, 1363, 1243, 1125, 1037, 952, 935 cm−1. Anal. calcdf 1.

2(

-p ,N ona ol,8�( m,iHH ).1 5,5 4.85,3 6.89,2 1.96,1 89,1C C-M ),4 27),1

2(

-p ,N on

3H, d,J = 6.5 Hz, H-21), 0.84 (3H, t,J = 7.2 Hz, H-29), 0.83H, d,J = 6.6 Hz, H-26), 0.81 (3H, d,J = 6.7 Hz, H-27), 0.63H, s, H-18).13C NMR (75 MHz, CDCl3) � 143.46, 125.453.34, 71.40, 55.96, 55.37, 48.26, 45.83, 42.92, 41.72, 49.55, 36.94, 36.43, 36.09, 33.97, 31.55, 29.13, 28.54, 26.09, 23.05, 21.07, 19.81, 19.15, 19.02, 18.83, 11.97, 1

R (CCl4), 3604, 3449, 2957, 2878, 1464, 1384, 1217, 1044, 1007, 947 cm−1. [�]20

D +6◦ (c 1, CHCl3). Anal. calcdor C29H50O2: C 80.87, H 11.70. Found C 80.38, H 11.C-MS-EI (as di-TMS ether),m/z (%): 575 (1), 485 (10070 (3), 396 (1), 253 (1), 129 (5), 105 (4), 73 (22), 43 (8

.6.8. 5,6α-Epoxy-5α-sitostan-3β-ol acetate (9) and,6β-epoxy-5β-sitostan-3β-ol acetate (10)

m-CPBA (70% aqueous solution, 80 mg, 0.33 mmol)dded to a solution of2 (150 mg, 0.33 mmol) in CH2Cl210 mL) containing NaHCO3 (50 mg, 0.59 mmol). The mixure was stirred at room temperature for 24 h and tiluted with CH2Cl2 (20 mL). The organic layer was washuccessively with a saturated aqueous NaHCO3 solution5× 10 mL), water (2× 20 mL), and a saturated aqueoaCl solution (2× 20 mL) and dried over MgSO4. The sol-ents were evaporated under reduced pressure to givrude product (170 mg). NMR analysis indicated theresence of the two epoxides9 and10 (9:10 = 3:1; 67% overll yield). In order to separate the two compounds, a frac100 mg) of the crude product was purified on an aluminamn treated with AgNO3 (75× 1 cm i.d.) with hexane/ethcetate (97:3) as an eluent to give9 (fractions 55–78: 68 mgnd10 (fractions 118-148: 23 mg) as white solids.

or C31H52O3: C 78.76, H 11.09. Found C 78.77, H 11.2

.6.9. 5,6α-Epoxy-5α-sitostan-3β-ol5,6α-epoxysitosterol) (11)

The same procedure as for compound4 was used: comound 9 (75 mg, 0.16 mmol), MeOH/H2O (9:1, 20 mL)a2CO3 (34 mg, 0.32 mmol). After workup, the reactifforded compound11 as a white solid (60 mg, 0.14 mm8% yield): m.p. 109–110◦C. 1H NMR (300 MHz, CDCl3)4.00-3.80 (1H, m, H-3), 2.89 (1H, d,J = 4.4 Hz, H-6), 2.06

1H, dd,J = 12.7 Hz), 2.00-1.75 (4H, m), 1.70-0.90 (28H,ncluding 1s (3H, H-19) at 1.05 ppm), 0.89 (3H, d,J = 6.6 Hz,-21), 0.84 (3H, t,J = 7.2 Hz, H-29), 0.82 (3H, d,J = 6.4 Hz,-26), 0.80 (3H, d,J = 6.6 Hz, H-27), 0.61 (3H, s, H-18

3C NMR (75 MHz, CDCl3) � 68.74, 65.67, 59.29, 56.85.78, 45.81, 43.45, 42.55, 42.33, 39.87, 39.40, 36.13, 33.89, 32.40, 31.10, 30.16, 29.89, 29.13, 28.82, 28.09, 26.08, 24.05, 23.04, 20.64, 19.82, 19.02, 18.69, 15.92, 11.85. IR (CCl4), 3620, 3438, 2940, 2870, 1466, 1377, 10037, 966 cm−1. [�]20

D −40◦ (c 1, CHCl3). Anal. calcd for29H50O2: C 80.87, H 11.70. Found C 80.71, H 11.82. GS-EI (as TMS ether),m/z (%): 503 (15), 488 (7), 485 (613 (46), 398 (20), 395 (60), 253 (25), 211 (18), 159 (35 (35), 129 (12), 105 (70), 73 (100), 43 (25).

.6.10. 5,6β-Epoxy-5β-sitostan-3β-ol5,6β-epoxysitosterol) (12)

The same procedure as for compound4 was used: comound 10 (38 mg, 0.08 mmol), MeOH/H2O (9:1, 10 mL)a2CO3 (17 mg, 0.16 mmol). After workup, the reacti

X. Zhang et al. / Steroids 70 (2005) 886–895 891

afforded compound12 as a white solid (30 mg, 0.07 mmol,87% yield): m.p. 145–147◦C. 1H NMR (300 MHz, CDCl3)� 3.75-3.60 (1H, m, H-3), 3.05 (1H, d,J = 2.3 Hz, H-6),2.10-1.90 (4H, m), 1.90-1.70 (2H, m), 1.70-0.90 (27H, m,including 1s (3H, H-19) at 0.99 ppm), 0.89 (3H, d,J = 6.5 Hz,H-21), 0.84 (3H, t,J = 7.2 Hz, H-29), 0.83 (3H, d,J = 6.5 Hz,H-26), 0.80 (3H, d,J = 6.7 Hz, H-27), 0.64 (3H, s, H-18).13CNMR (75 MHz, CDCl3) � 69.44, 63.71, 62.91, 56.22, 56.11,51.33, 45.80, 42.28, 42.24, 39.83, 37.23, 36.08, 34.85, 33.88,32.61, 31.05, 29.77, 29.12, 28.16, 26.04, 24.19, 23.05, 21.99,19.81, 19.01, 18.73, 17.04, 11.97, 11.75. IR (CCl4), 3620,2936, 2870, 1466, 1383, 1054, 954 cm−1. [�]20

D +12◦ (c 1,CHCl3). Anal. calcd for C29H50O2: C 80.87, H 11.70. FoundC 79.03, H 11.60. GC-MS-EI (as TMS ether),m/z (%): 503(24), 488 (12), 485 (11), 413 (52), 398 (38), 395 (38), 253(32), 211 (36), 197 (45), 135 (67), 129 (45), 105 (90), 73(100), 43 (91).

2.6.11. Sitost-5-en-3β,5α,6β-triol(5α,6β-dihydroxysitosterol) (13)

�-Sitosterol1 (180 mg, 0.446 mmol) was suspended in95% formic acid (15 mL) at 75◦C to form a homogeneousmedium. The mixture was allowed to reach room tem-perature, and 30% hydrogen peroxide (3 mL) was added.

The mixture was stirred for 15 h at room temperature. Hotwater (300–400 mL) was added, and the resulting white pre-cipitate was filtered, dissolved in methanol (30 mL), andstirred for 30 min in the presence NaOH (60 mg). The sol-vent was removed under reduced pressure to give a whiteresidue (260 mg), which was purified on a silica gel col-umn (35× 2 cm i.d.) with cyclohexane/ethyl acetate (1:1)as an eluent to give the compound13 as a white solid(53 mg, 0.12 mmol, 25% yield): m.p. 244–245◦C. 1H NMR(300 MHz, CDCl3 + CD3OD): 4.15-4.00 (1H, m, H-3), 3.54(1H, s, H-6), 2.15-1.95 (2H, m), 1.90-1.00 (33H, m, includ-ing 1s (3H, H-19) at 1.18 ppm), 0.90 (3H, d,J = 6.5 Hz,H-21), 0.85 (3H, t,J = 7.2 Hz, H-29), 0.83 (3H, d,J = 6.5 Hz,H-26), 0.80 (3H, d,J = 6.7 Hz, H-27), 0.67 (3H, s, H-18).13C NMR (75 MHz, CDCl3 + CD3OD): 75.46, 75.10, 66.90,55.86, 55.71, 45.52, 45.13, 42.40, 39.71, 37.79, 35.86, 33.71,33.59, 31.94, 30.00, 28.80, 27.92, 25.76, 23.78, 22.69, 20.82,19.34, 18.55, 18.30, 16.19, 11.67, 11.48. IR (CHCl3): 3696,3424, 2933, 2861, 1602, 1463, 1373, 1039 cm−1. [�]20

D +8◦(c 1, MeOH). Anal. calcd for C29H52O3, C 77.62, H 11.68.Found C 76.60, H 11.40. GC-MS-EI (as tris-TMS ether),m/z(%): 575 (13), 560 (34), 545 (22), 485 (84), 470 (28), 432(100), 395 (11), 380 (4), 351 (12), 253 (2), 129 (17), 105(18), 73 (66), 43 (30).

Ftad

ig. 1. (a) GC chromatogram of Generol 95R as TMS ether derivatives on a Vhickness of 0.1�m, 60 m× 0.25 mm). (b) Analytical HPLC chromatogram of Gcetonitrile:2-propanol:water (2:1:1, v/v/v) as mobile phase at a flow rate ofescribed in Section2. Peaks are identified as: (1) brassicasterol; (2) campesterol

F-5ms capillary column (phase stationary: 5% phenyl–95% dimethylpolysiloxane,enerol 95R on a Polaris C8-A column (250× 4.6 mm i.d., 5�m particles) with0.5 mL/min. UV detection was at 208 nm, 0.1 AUFS. Analytical conditionsare

; (3) �-sitosterol; (4) �5-avenasterol.

892 X. Zhang et al. / Steroids 70 (2005) 886–895

3. Results and discussion

3.1. Gram-scale separation of β-sitosterol bypreparative silica gel column chromatography

The GC-MS analysis of Generol 95R as TMS ethers(Fig. 1(a)) and HPLC analysis (Fig. 1(b)) indicated that fourmain phytosterols, including brassicasterol,�5-avenasterol,campesterol, and�-sitosterol, are present in Generol95R.

Due to their extremely similar molecular structures, these�5-sterols remained in a single spot on a TLC plate, thenno satisfactory separation could be achieved. However, as

mentioned above, Hyde and Elliott[19] described a columnchromatographic separation of 25 mg mixture of sitosteroland campesterol on a Sephadex LH-20 column (hydropho-bic long-chain alky esters). Recycling six times on a 38- or88-cm column (×2.5 cm i.d.) provided these two pure indi-vidual sterols (milligram-scale) as measured by gas–liquidchromatography. This showed that chromatographic separa-tion of mg amounts of closely related sterols, differing onlyby one methyl group, was possible. The small differences insterol retention on the chromatographic column allowed theirfinal separation.

Based on this principle, in the present work, the scaled-upseparation between different phytosterols was performed by

Scheme 1. Synthesis of 7-ketositosterol (4), 7�-hydro

xysitosterol (6), and 7�-hydroxysitosterol (8).

X. Zhang et al. / Steroids 70 (2005) 886–895 893

preparative silica gel adsorption chromatography with suffi-cient length and larger diameter.

Commercial Generol 95R was first purified on a silica gelcolumn 1 (35× 3 cm i.d.). With heptane/ethyl acetate (4:1) asan eluent (5 mL/min), the unpolar impurities were eluted outof the column before sterols fractions, the polar impuritieswere retained and discarded in the column. The composition

of four phytosterols was not changed in this process. Phy-tosterol fractions were then collected to isolate�-sitosterolon silica gel column 2 (60× 6 cm i.d.). With heptane/ethylacetate (6:1) as the eluent,�-sitosterol was eluted slightlyprior than other phytosterols. So the separation between themcould be performed in the silica gel column step by step. Eightto ten cycles were necessary to purify 2 g of�-sitosterol until

Scheme 2. Synthesis of 5,6�-epoxysitoster

ol (11) and 5,6�-epoxysitosterol (12).

894 X. Zhang et al. / Steroids 70 (2005) 886–895

Scheme 3. Synthesis of 5�,6�-dihydroxysitostrerol (13).

the desired purity (≥95%, as measured by HPLC and GC)was reached. Analytical HPLC was used to check the com-position of phytosterols in these fractions.

The elution order of phytosterols on the silica gel col-umn (�-sitosterol→ campesterol→ brassicasterol and�5-avenasterol) was exactly in contrast to the order obtained byRP-HPLC on a Polaris C8-A column (brassicasterol and�5-avenasterol→ campesterol→ �-sitosterol.

3.2. Chemical synthesis

The synthesis of several oxides starting from�-sitosterolwas achieved according to the method developed for the syn-thesis of cholesterol oxides[25,26]. Attention was focusedon the synthesis of phytosterol oxides resulting from the oxi-dation of the C5–C6 double bond. The allylic oxidation ofthe double bond gave rise to 7-keto, 7�-hydroxy, and 7�-hydroxysitosterol. This double bond was also subjected to anepoxidation reaction, affording the two epoxy stereoisomersand, finally, to a hydroxylation reaction, which stereospecif-ically gave 5�,6�-dihydroxysitosterol. The stereochemistryof the different oxidized compounds was determined by com-parison NMR data with those given for cholesterol analogs[26].

3.2.1. Allylic oxidation-

w iumc ,a y to7 tod oro-h the�d fw thed(

3e

C fca( eg-

nated with silver nitrate, the two epoxide isomers were readilyseparated[24,25]. Finally, a deprotection reaction yielded5,6�-epoxysitosterol11 and 5,6�-epoxysitosterol12 as purecompounds, respectively (Scheme 2).

3.2.3. Hydroxylation reaction5�,6�-Dihydroxysitosterol13 was directly obtained from

�-sitosterol1 via a hydroxylation reaction of the C5–C6 dou-ble bond, which was achieved in the presence of formic acidand hydrogen peroxide (Scheme 3). The yield was modest(25%), and we were unable to increase it in spite of manymodifications of the experimental conditions.

Acknowledgments

The authors thank Dr. Jennifer Wytko for her help in thepreparation of the manuscript. This work was supported bya grant from the Ministere de la Jeunesse, de l’EducationNationale et de la Recherche, France (RARE 015 No. 02 P0640).

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sm,

adsthe

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ntra-Nutr

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ides:oods.

tos-

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