Improved method for the synthesis of trans-feruloyl-β-sitostanol

Article abstract of DOI:10.1021/jf010703f

Phytosterols and phytostanols are known to lower low-density lipoprotein-cholesterol (LDL-C) levels in humans by up to 15%, and at least two products, Benecol and Take Control, are now on the market as naturally derived fatty acid esters of phytostanols (

Full text of DOI:10.1021/jf010703f

J. Agric. Food Chem. 2001, 49, 4961−4964
4961
Im p r oved Meth od for th e Syn th esis of tr a n s-F er u loyl-â-sitosta n ol
Anthony M. Condo, J r.,† David C. Baker,*,† Robert A. Moreau,‡ and Kevin B. Hicks‡
Department of Chemistry, University of Tennessee, Knoxville, Tennessee 37996-1600, and
Eastern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture,
600 East Mermaid Lane, Wyndmoor, Pennsylvania 19038
Phytosterols and phytostanols are known to lower low-density lipoprotein-cholesterol (LDL-C) levels
in humans by up to 15%, and at least two products, Benecol and Take Control, are now on the
market as naturally derived fatty acid esters of phytostanols (stanol esters) and phytosterols (sterol
esters), respectively. A synthetic process was developed to synthesize gram quantities of trans-
feruloyl-â-sitostanol from ferulic acid and â-sitostanol, with high purity and yields of ∼60%. The
process involves (a) condensation of trans-4-O-acetylferulic acid with the appropriate phytostanol
or phytostanol mixture in the presence of N,N-dicyclohexylcarbodiimide and 4-(dimethylamino)-
pyridine, (b) separation of the trans-4-O-acetylferuloyl products by preparative liquid chromatog-
raphy, (c) selective deacetylation of the feruloyl acetate, and (d) chromatographic purification of
the feruloylated phytostanols. The process was successfully applied to synthesize stanol trans-feruloyl
esters from “Vegetable Stanols”, a mixture of ∼70:30 â-sitostanol and â-campestanol, in comparable
purity and yield.
Keyw or d s: â-Sitostanol; trans-feruloyl-â-sitostanol; phytostanols; stanol esters
INTRODUCTION
also reduce the oxidation of LDL-C, further lowering the
risk of coronary heart disease. To test this hypothesis,
quantities of pure stanol ferulate esters are required.
A method that uses 4-O-acetylferuloyl chloride as the
acylating agent has been published for the synthesis of
these types of esters (7, 8). The present study was
undertaken to develop an improved method for the
synthesis of â-sitostanol ferulate (5a ). Because “soy
stanols” are a more readily available commercial prod-
uct, containing ∼70% â-sitostanol (3a ) and 30% â-cam-
pestanol (3b), the ferulate ester synthesis was also
evaluated with this mixed substrate (3a + 3b).
The published procedures (7, 8) for the chemical
synthesis of trans-feruloyl-â-sitostanol is limited by two
steps: (1) the required preparation of the highly reactive
trans-4-O-acetylferuloyl chloride, which is difficult to
purify and handle, and (2) the deprotection step that
uses sodium borohydride to remove the acetyl protective
group on the trans-feruloylated product. Our goal in the
present work was to develop a more workable synthesis
of the compounds.
Two of the most popular cholesterol-lowering “func-
tional” foods currently on the market are Benecol and
Take Control, with the former containing synthetic fatty
acid esters of phytostanols (stanol esters) derived from
tall oil and the latter containing synthetic fatty acid
esters of phytosterols (sterol esters) derived from soy-
bean oil (1). Phytosterols and phytostanols are known
to lower low-density lipoprotein-cholesterol (LDL-C)
levels in humans by up to 15%, and the FDA has
recently permitted a rare health claim for their use in
low-fat diets (2). Phytosterols and phytostanols are
thought to lower LDL-C by inhibiting the absorption of
cholesterol from the intestine (3). Free phytosterols and
phytostanols are poorly soluble in most foods, and this
has led to problems with formulations and inconsisten-
cies in efficacy. To solve this problem, they are usually
esterified with fatty acids to make them soluble in
margarines and other high-fat food products (4). In the
intestine, fatty acid esters are hydrolyzed by digestive
enzymes, producing the physiologically active free sterol
or stanol (4). We at the USDA laboratories have recently
discovered and patented a natural phytosterol-contain-
ing oil from corn fiber (corn fiber oil) that contains high
levels of natural phytosterol and phytostanol fatty acid
esters and a unique class of stanol esters, the stanol
ferulate esters (also called ferulate phytostanol esters)
(5, 6). Although many clinical studies have been per-
formed on fatty acid esters of stanols and sterols, no
studies have examined the efficacy of stanol ferulate
esters. Because these compounds contain a ferulic acid
moiety, which is a powerful antioxidant, it is proposed
that in addition to reducing LDL-C levels, they might
MATERIALS AND METHODS
Gen er a l P r oced u r es. All reactions were conducted under
a nitrogen atmosphere unless otherwise indicated. Anhydrous
solvents were dried prior to distillation as follows: dichloro-
methane, chloroform, and pyridine were refluxed over calcium
hydride; THF was refluxed over sodium benzophenone ketyl;
and methanol was refluxed with magnesium metal shavings.
trans-Ferulic acid and acetic anhydride were purchased from
Aldrich Chemical Co. and used without further purification.
â-Sitostanol (3a ) was purchased from Research Plus, Inc., and
used without further purification. To maximize the yield of
the coupled products 4a and 4b, the starting material was
dried for 18 h to remove residual ethanol (78 °C, <1 Torr).
“Vegetable Stanols” were obtained from AC Humko, Inc.
(Memphis, TN) and used without further purification. Thin-
layer chromatography (TLC) was carried out on E. Merck
Silica Gel 60 aluminum-backed plates. Visualization was
* Corresponding author [telephone (865) 974-1066; fax (865)
974-1536; e-mail dcbaker@utk.edu].
† University of Tennessee.
‡ U.S. Department of Agriculture.
10.1021/jf010703f CCC: $20.00 © 2001 American Chemical Society
Published on Web 09/05/2001

4962 J. Agric. Food Chem., Vol. 49, No. 10, 2001
Condo et al.
Sch em e 1. P r ep a r a tion of tr a n s-4-O-Acetylfer u lic
Acid
150 mL). The organic extracts were combined and dried over
anhydrous MgSO4 and evaporated to yield a white solid. The
solid was dissolved in a minimal amount of THF and chilled
at 0 °C overnight to precipitate any residual DCU. The solution
was filtered, the solvent was evaporated, and the residual solid
was chromatographed using PLC with 2:4:1 chloroform/hex-
ane/EtOAc as the eluent to yield 4a as a white solid (29.0 g,
43%). Two additional products were recovered and identified
as ethyl 4-O-acetylferulate (5.8 g, 22%) and unreacted â-sito-
stanol (3a , 11.9 g, 29%). 1H and 13C NMR as well as EIMS
were used to characterize the minor products. Characterization
data for the major product 4a are as follows: TLC Rf (5:1
petroleum ether/EtOAc) 0.24; mp 162 °C [lit. (7) 156 °C];
[R]2D0 +20.6° (c 0.7, CHCl3); IR (cm-1) 2938, 2867, 1760, 1711,
1639, 1600, 1512, 1155, 1073; 1H NMR δ 0.6-2.0 (â-sitostanol
moiety, ∼50H), 2.33 (s, 3H), 3.86 (s, 3H), 4.83 (m, 1H), 6.37
(d, 1H, J ) 16.2 Hz), 7.10 (m, 3H), 7.62 (d, 1H, J ) 15.9 Hz);
13C NMR δ 12.06, 12.15, 12.34, 18.80, 19.09, 19.91, 20.76,
21.27, 23.08, 24.28, 26.04, 27.63, 28.33, 28.67, 29.12, 32.04,
33.92, 34.15, 35.51, 36.21, 36.80, 39.98, 42.60, 44.68, 45.79,
54.19, 55.86, 56.12, 56.39, 73.96, 110.95, 118.88, 121.08,
123.08, 133.36, 141.06, 143.41, 151.11, 166.15, 168.64; EIMS
accomplished by a combination of UV exposure (254 nm) and
treatment with a phosphomolybdic acid dip. Solvents were
evaporated at aspirator vacuum at 25 °C unless otherwise
indicated. Preparative liquid chromatography (PLC) was car-
ried out on a Waters Prep LC/System 500A with a flow rate
of 250 mL/min of the indicated solvent system and with
refractive index detection. Melting points were taken on a
Thomas-Hoover capillary melting point apparatus and are
uncorrected. Optical rotations were measured at the sodium
D line at 20 °C using a Perkin-Elmer 241 polarimeter (1-dm
1
cell). H and 13C NMR spectra were recorded at 300.087 and
(70 eV), m/z 634.5 [M+], 592.4 [M+ - CH3CO], 398.4 [M+
-
75.464 MHz, respectively, on a Varian Mercury series spec-
trometer as solutions in CDCl3 with an internal standard of
C12H12O5]. Anal. Calcd for C41H62O5: C, 77.56; H, 9.84.
Found: C, 77.58; H, 9.88. 1H and 13C NMR data are consistent
with published literature values (7).
1
TMS. H chemical shifts are reported as δ (parts per million)
downfield from the TMS standard. 13C chemical shifts are
reported as δ (parts per million) relative to CDCl3 (77.00 ppm)
as standard. Infrared spectra were obtained on a Bomem MB
series spectrometer as KBr pellets. Elemental analyses were
furnished by Atlantic Microlab, Inc., of Norcross, GA, and are
within (0.4% of the theoretical values. Electron-impact mass
spectra (EIMS) were obtained on a VG-ZAB-EQ mass spec-
trometer (Manchester, U.K.) using an ionization potential of
70 eV.
In a similar experiment, using a sample (633 mg, 1.52 mmol)
of 3a that had been dried for 18 h (78 °C, <1 Torr), 489 mg
(61%) of coupled product 4a was obtained, and no ethyl 4-O-
acetylferulate was detected.
3-O-(tr a n s-4-F er u loyl)-â-sitosta n ol (5a ). Compound 4a
(28.5 g, 44.9 mmol) was dissolved in a 2:1 CHCl3-MeOH solvent
mixture (450 mL). K2CO3 (1.24 g, 8.98 mmol, 0.20 equiv) was
added, and the mixture was refluxed for 6 h. TLC analysis
revealed the consumption of the starting material and the
appearance of a new spot with a decreased Rf value. The
reaction was quenched by the addition of satd aq NH4Cl (100
mL). The layers were separated, and the organic layer was
washed with H2O (2 × 100 mL) and dried over MgSO4. PLC
using 2:4:1 CHCl3/hexane/EtOAc as the eluent yielded a white
solid (23.5 g, 88%) that was recrystallized (CHCl3-MeOH) to
give the final product 5a as fine white crystals (19.0 g, 71%):
TLC: Rf (5:1 petroleum ether/EtOAc) 0.14; HPLC (normal
phase): tR 26.2 min; mp 156-157 °C (lit. (7) 156 °C); [R]D20
+22.3° (c 1.33, CHCl3); IR (cm-1): 3528, 3408, 2938, 2867,
HP LC Con d ition s. Ester products [â-sitostanol ferulate
(5a ) and â-campestanol ferulate (5b), with retention times of
35.4 and 33.9 min, respectively] were separated and quantified
via a reversed-phase HPLC system consisting of a Hewlett-
Packard (Avondale, PA) model 1100 pump and autosampler,
with the effluent entering first a Hewlett-Packard model 1100
UV detector at 320 nm and then an Alltech-Varex model MKIII
evaporative light-scattering detector (Deerfield, IL). The col-
umn (100 × 3 mm) was a Chromsep glass cartridge packed
with LiChrosorb7RP18 (7 µm) by Varian-Chrompack (Bridge-
water, NJ ). The 60-min solvent gradient program (0.5 mL/min)
was for a ternary solvent system of solvents A/B/C: at 0 min,
10:10:80; at 10 min, 10:10:80; at 40 min, 10:0:90; at 50 min,
10:0:90; at 52 min, 10:10:90; and at 60 min, ) 10:10:90, A/B/C
(v/v/v), where A ) 2-PrOH, B ) water, and C ) MeOH.
Normal-phase HPLC was used to separate various stanol
esters from the free-OH stanols. Normal-phase HPLC was
carried out using the same HPLC system and flow rate as
above with a LiChrosorb Diol column (100 × 3 mm) and a 60-
min solvent program consisting of solvents A/B: at 0 min, 100:
0; at 8 min, 100:0; at 10 min, 75:25; at 40 min, 75:25; at 41
min, 100:0; and at 60 min, 100:0, where A ) 1000:1 hexane/
HOAc and B ) 100:1 hexane/2-PrOH.
1
1707, 1634, 1594, 1514, 1173, 1074; H NMR: δ 0.6-2.0 (â-
sitostanol moiety, ∼50H), 3.91 (s, 3H), 4.83 (m, 1H), 5.96 (s,
1H), 6.2 (d, J ) 15.9 Hz, 1H), 6.91 (d, J ) 7.8 Hz, 1H), 7.05
(m, 2H), 7.60 (d, J ) 16.2 Hz, 1H); 13C NMR: δ 12.06, 12.14,
12.33, 18.79, 19.09, 19.91, 21.26, 23.08, 24.27, 26.04, 27.67,
28.33, 28.67, 29.12, 32.03, 33.91, 34.19, 35.50, 36.20, 36.81,
39.97, 42.58, 44.66, 45.78, 54.19, 55.86, 56.12, 56.38, 73.68,
109.07, 114.57, 115.99, 122.88, 126.93, 144.25, 146.54, 147.63,
166.63; EIMS (70 eV) m/z: 592.4 [M+], 398.4 [M+ - C10H10O4].
Anal. Calcd for C39H60O4: C, 79.01; H, 10.20. Found: C, 78.73;
H, 10.21.
3-O-(tr a n s-4-O-Acetylfer u loyl)-â-sitostan ol (4a) an d 3-O-
(tr a n s-4-O-acetylfer u loyl)-â-cam pestan ol (4b). By the same
procedure used for the preparation of 4a , trans-4-O-acetylferu-
lic acid (2, 8.86 g, 37.5 mmol) and Vegetable Stanols (69.0%
3a and 30.4% 3b, 17.2 g, 41.3 mmol, 1.1 equiv) were dissolved
in dry CH2Cl2 (75 mL) and reacted with DCC (8.52 g; 41.3
mmol; 1.1 equiv) and DMAP (504 mg; 4.13 mmol; 0.10 equiv).
Workup as for 4a gave a residual solid that upon PLC (5:1
petroleum ether/EtOAc) gave 4a and 4b as a white solid (9.25
g, 39%) that was an inseparable mixture by chromatographic
techniques. The characterization data for the mixture are as
follows: TLC Rf (5:1 petroleum ether/EtOAc) 0.24; mp 160-
161 °C; [R]2D0 +22.2° (c 0.18, CHCl3); IR (cm-1) 2946, 2862,
1764, 1710, 1639, 1596, 1514, 1155, 1078; 1H NMR δ 0.6-2.0
(â-sitostanol/â-campestanol moieties, ∼50H), 2.32 (s, 3H), 3.85
(s, 3H), 4.83 (m, 1H), 6.36 (d, J ) 16.2 Hz, 1H), 7.10 (m, 3H),
7.61 (d, J ) 15.9 Hz, 1H); 13C NMR δ 12.05, 12.14, 12.33,
15.44*, 15.51*, 17.65*, 18.32*, 18.72*, 18.80*, 18.91*, 19.10,
tr a n s-4-O-Acetylfer u lic Acid (2). trans-Ferulic acid (1,
25.0 g, 128.7 mmol) was dissolved in distilled pyridine (100
mL) in a 250-mL round-bottom flask (Scheme 1). Acetic
anhydride (65 mL) was added, and the mixture was stirred
overnight in the dark. The solution was diluted with toluene
(50 mL) and evaporated to yield a yellow syrup. Subsequent
addition and evaporation of toluene (3 × 50 mL) yielded an
off-white solid that was dried for 18 h (78 °C, <1 Torr) prior
1
to use. H and 13C NMR data were consistent with published
literature values (9).
3-O-(tr a n s-4-O-Acetylfer u loyl)-â-sitosta n ol (4a ). trans-
4-O-Acetylferulic acid (2, 23.2 g; 98.2 mmol) and â-sitostanol
(3a , 49.0 g; 108 mmol; 1.1 equiv) were dissolved in dry CH2-
Cl2 (750 mL). DCC (1.0 M in CH2Cl2, 108.0 mL; 108 mmol; 1.1
equiv) and DMAP (1.20 g; 9.82 mmol; 0.10 equiv) were added,
and the mixture was stirred for 18 h. The DCU that formed
was filtered off, and the solution was extracted with H2O (2 ×
150 mL), 10% HOAc (2 × 150 mL), and again with H2O (2 ×

Synthesis of trans-Feruloyl-â-sitostanol
J. Agric. Food Chem., Vol. 49, No. 10, 2001 4963
Sch em e 2. P r ep a r a tion of
3-O-(tr a n s-F er u loyl)-â-sitosta n ol a n d -â-ca m p esta n ol
O4 (4b): C, 78.96; H, 10.17. Found: C, 78.98; H, 10.24. Note:
Resonances in the 13C spectrum attributable to the minor
component 5b are denoted by an (*).
RESULTS AND DISCUSSION
The process described herein proceeds directly from
trans-4-O-acetylferulic acid (2), which is easily prepared
by acetylation of ferulic acid (1) in acetic anhydride/
pyridine, to give a product identical with that reported
by Ebenezer (9). Thus, activation of 2 with N,N-di-
cyclohexylcarbodiimide (10) and condensation with â-
sitostanol (3a ) in the presence of 4-(dimethylamino)-
pyridine proceeded smoothly to give the coupled product,
trans-4-O-acetylferuloyl-â-sitostanol (4a ). The coupled
product was purified by crystallization and filtration of
the byproduct, N,N-dicyclohexylurea, and the crude
product was subjected to preparative liquid chromatog-
raphy (normal phase, silica gel), which served to sepa-
rate the unreacted starting material and another byprod-
uct, ethyl trans-4-O-acetylferulate. The origin of the
latter was traced to ethanol, which was present in the
â-sitostanol starting material (33 mol % ethanol; Re-
search Plus, Inc.). Removal of the ethanol by thorough
drying of the â-sitostanol raised the yield from 43 to 61%
of pure 4a and eliminated the ethyl ester byproduct.
Removal of the acetate protecting group required a
selective hydrolytic procedure that favored acetate
hydrolysis over hydrolysis of the trans-ferulate ester
linkage. The published procedure (7, 8) used sodium
borohydride, a hydride reducing agent, which is not a
common reagent to accomplish ester reductions, owing
to the resistance of nonactivated esters to the action of
borohydride. We found that a simple transesterification
process that makes use of potassium carbonate in
refluxing chloroform/methanol was ideal for the task
(11). Thus, refluxing the acetate-protected 4a with
potassium carbonate in a 2:1 mixture of chloroform/
anhydrous methanol proceeded to give the product 5a
as an analytically pure material in 71% yield after
recrystallization. The product was characterized by
NMR spectroscopy, by mass spectrometry, and by
elemental analysis. Normal-phase (silica gel) HPLC and
TLC (silica gel) of the product showed a single sub-
stance.
The acylation process was adapted to the processing
of a mixture of stanols, termed Vegetable Stanols (AC
Humko, Inc.), that was made up of a 7:3 mixture of
â-sitostanol (3a ) and â-campestanol (3b) (reversed-
phase HPLC analysis showed 69.0% 3a and 30.4% 3b).
Soy oil phytosterols are typically comprised of about 51%
â-sitosterol, 16% stigmasterol, and 22% â-campesterol
(12), and upon hydrogenation to reduce sterols to
stanols, the composition of vegetable (soy) stanols is
about 67% â-sitostanol and 22% â-campestanol. Pro-
cessing the mixture as for pure â-sitostanol was carried
out to give a ∼7:3 mixture of the protected coupled
products 4a and 4b, as well as the free-hydroxy trans-
feruloyl esters 5a and 5b, in comparable yield. At no
stage in the synthesis were the products separable by
either crystallization or normal-phase chromatography
(silica gel); an analytical-scale separation was achieved
by reversed-phase HPLC using a ternary solvent gradi-
ent. The products, either 4a and 4b or 5a and 5b, could
be discerned most readily from one another by inspec-
tion of their 13C NMR spectra, which gave separate
signals for a number of the resonances.
19.89, 20.28*, 20.59*, 20.70, 21.27, 23.10, 24.27, 26.09, 27.63,
28.32, 28.67, 29.16, 30.32*, 30.61*, 31.47*, 32.03, 32.44*,
33.71*, 33.93, 34.16, 35.51, 35.93*, 36.20, 36.81, 38.83*, 39.07*,
39.99, 42.60, 44.68, 45.81, 54.21, 55.84, 56.14, 56.19*, 56.40,
79.92, 110.99, 118.89, 121.04, 123.05, 133.35, 141.10, 143.37,
151.13, 166.10, 168.55; EIMS (70 eV), m/z (4a ) 634.4 [M+],
592.4 [M+ - CH3CO], 398.4 [M+ - C12H12O5], (4b) 620.4 [M+],
578.4 [M+ - CH3CO], 384.4 [M+ - C12H12O5]. Anal. Calcd for
70% C41H62O5 (4a ) and 30% C40H60O5 (4b): C, 77.51; H, 9.81.
Found: C, 77.51; H, 9.94. Note: Resonances in the 13C
spectrum attributable to the minor component 4b are denoted
by an asterisk (*).
3-O-(tr a n s-4-F er u loyl)-â-sitosta n ol (5a ) a n d 3-O-(tr a n s-
4-F er u loyl)-â-ca m p esta n ol (5b). Compounds 4a and 4b
(9.20 g, 14.6 mmol) were deacylated [1:1 CHCl3/MeOH (100
mL), K2CO3 (202 mg, 1.46 mmol)] according to the procedure
used for 4a to give the final products 5a and 5b (Scheme 2) as
fine white crystals (7.30 g, 84%) after PLC (5:1 petroleum
ether/EtOAc) and recrystallization (CHCl3/MeOH). The char-
acterization data for the mixture of isomers are as follows:
TLC Rf (5:1 petroleum ether/ethyl acetate) 0.14; HPLC (re-
versed-phase) tR 33.9 min (5b) and tR 35.4 min (5a ); mp 157
°C; [R]2D0 +19.8° (c 0.23, CHCl3); IR (cm-1) 3531, 3408, 2936,
2867, 1705, 1633, 1594, 1513, 1172, 1074; 1H NMR δ 0.6-2.0
(â-sitostanol/â-campestanol moieties, ∼50H), 3.88 (s, 3H), 4.82
(m, 1H), 5.86 (s, 1H), 6.27 (d, J ) 15.9 Hz, 1H), 6.91 (d, J )
7.8 Hz, 1H), 7.05 (m, 2H), 7.59 (d, J ) 15.9 Hz, 1H); 13C NMR
δ 12.08, 12.18, 12.37, 15.47*, 15.54*, 17.67*, 18.34*, 18.74*,
18.82, 18.93*, 19.11, 19.92, 20.31*, 20.62*, 21.29, 23.12, 24.30,
26.11, 27.70, 28.35, 28.71, 29.18, 30.34*, 30.64*, 31.51*, 32.07,
32.48*, 33.74*, 33.96, 34.24, 35.55, 35.96*, 36.23, 36.85, 38.87*,
39.10*, 40.02, 42.63, 44.72, 45.85, 54.25, 55.92, 56.10*, 56.17,
56.22*, 56.43, 73.70, 109.10, 114.57, 116.10, 122.91, 127.01,
144.23, 146.56, 147.63, 166.63; EIMS (70 eV), m/z (5a ) 592.4
[M+], 398.4 [M+ - C10H10O4], (5b) 578.4 [M+], 384.4 [M+
10H10O4]. Anal. Calcd for 70% C39H60O4 (4a ) and 30% C38H58
-
-
The process described herein offers a straightforward
process to trans-feruloylated â-sitostanol, as well as to
C

4964 J. Agric. Food Chem., Vol. 49, No. 10, 2001
Condo et al.
during consumption of stanol ester margarine. Am. J .
Clin. Nutr. 2000, 71, 1095-1102.
a mixture of â-sitostanol and â-campestanol esters. The
process should be amenable to modest-sized synthesis
of related products. Compounds 5a and 5b are undergo-
ing evaluation as cholesterol-lowering agents. Details
of those studies will be reported elsewhere.
(5) Moreau, R. A.; Powell, M. J .; Hicks, K. B. Extraction
and quantitative analysis of oil from commercial corn
fiber. J . Agric. Food Chem. 1996, 44, 2149-2154.
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ABBREVIATIONS AND NOMENCLATURE USED
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Nomenclature: â-Sitostanol (also known as stigmas-
tanol) is (3â,5R)-stigmastan-3-ol [CAS Registry No. 83-
45-4]; â-campestanol is (3â,5R,24R)-ergostan-3-ol [CAS
Registry No. 474-60-2]; trans-ferulic acid is 4-hydroxy-
3-methoxy-trans-cinnamic acid [CAS Registry No. 537-
98-4]. (CAS Registry No. were provided by the author.)
Abbreviations: DCC, N,N-dicyclohexylcarbodiimide;
DCU, N,N-dicyclohexylurea; DMAP, 4-(dimethylamino)-
pyridine; LDL-C, low-density lipoprotein-cholesterol.
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Received for review May 31, 2001. Revised manuscript received
J uly 24, 2001. Accepted J uly 25, 2001.
J F010703F