Triterpene alcohol and sterol ferulates from rice bran and their anti- inflammatory effects

Article abstract of DOI:10.1021/jf000135o

Six novel feruloyl esters of triterpene alcohols and sterols, viz., two trans-ferulates, cycloeucalenol and 24-methylenecholesterol trans-ferulates, and four cis-ferulates, cycloartenol, 24-methyelenecycloartanol, 24- methylcholesterol, and sitosterol cis-ferulates, besides five known trans- ferulates, cycloartenol (CAR), 24-methylenecycloartanol (24-MCA), 24- methylcholesterol, sitosterol, and stigmastanol trans-ferulates, and one known cis-ferulate, stigmastanol cis-ferulate, were isolated from the methanol extract of edible rice bran. These and eight other synthetic trans- and cis-ferulates of triterpene alcohols and sterols, along with the corresponding free alcohols, were evaluated with respect to their anti- inflammatory activity against 12-O-tetradecanoylphorbol-13-acetate (TPA)induced inflammation (1 μg per ear) in mice. All of the ferulates showed marked inhibitory activity, and their 50% inhibitory dose (ID₅₀) was 0.1-0.8 mg per ear. On the other hand, whereas two free triterpene alcohols, CAR and 24-MCA, showed strong inhibition (ID₅₀ 0.2-0.3 mg/ear), eight free sterols examined showed weaker activity (ID₅₀ 0.7-2.7 mg/ear) than their corresponding ferulates.

Full text of DOI:10.1021/jf000135o

J. Agric. Food Chem. 2000, 48, 2313−2319
2313
Tr iter p en e Alcoh ol a n d Ster ol F er u la tes fr om Rice Br a n a n d Th eir
An ti-in fla m m a tor y Effects
Toshihiro Akihisa,*,† Ken Yasukawa,‡ Miho Yamaura,† Motohiko Ukiya,† Yumiko Kimura,‡
Naoto Shimizu,§ and Koichi Arai
College of Science and Technology, Nihon University, 1-8 Kanda Surugadai, Chiyoda-ku,
Tokyo 101-8308, J apan, College of Pharmacy, Nihon University, 7-7-1 Narashinodai, Funabashi,
Chiba 274-8555, J apan, Application Development Department, Yokogawa Analytical Systems Inc.,
2-11-13 Nakacho, Musashino, Tokyo 101-0006, J apan, and Meikai University School of Dentistry,
1-1 Keyakidai, Sakado, Saitama 350-0283, J apan
Six novel feruloyl esters of triterpene alcohols and sterols, viz., two trans-ferulates, cycloeucalenol
and 24-methylenecholesterol trans-ferulates, and four cis-ferulates, cycloartenol, 24-methyelene-
cycloartanol, 24-methylcholesterol, and sitosterol cis-ferulates, besides five known trans-ferulates,
cycloartenol (CAR), 24-methylenecycloartanol (24-MCA), 24-methylcholesterol, sitosterol, and
stigmastanol trans-ferulates, and one known cis-ferulate, stigmastanol cis-ferulate, were isolated
from the methanol extract of edible rice bran. These and eight other synthetic trans- and cis-ferulates
of triterpene alcohols and sterols, along with the corresponding free alcohols, were evaluated with
respect to their anti-inflammatory activity against 12-O-tetradecanoylphorbol-13-acetate (TPA)-
induced inflammation (1 µg per ear) in mice. All of the ferulates showed marked inhibitory activity,
and their 50% inhibitory dose (ID50) was 0.1-0.8 mg per ear. On the other hand, whereas two free
triterpene alcohols, CAR and 24-MCA, showed strong inhibition (ID50 0.2-0.3 mg/ear), eight free
sterols examined showed weaker activity (ID50 0.7-2.7 mg/ear) than their corresponding ferulates.
Keyw or d s: Rice bran; trans- and cis-feruloyl esters; triterpene alcohol; sterol; antiedema; TPA-
induced ear edema
INTRODUCTION
24-MCA), 24-methylcholesterol (trans-24-MCO), and
sitosterol (trans-SITO) trans-ferulates, isolated from the
methanol extract of rice bran and γ-oryzanol markedly
inhibited the TPA-induced inflammation in mice (Ya-
sukawa et al., 1998). In addition, trans-CAR has been
found to strongly inhibit the tumor-promoting activity
of TPA in DMBA-initiated mice (Yasukawa et al., 1998).
In continuing our study on the feruloyl ester components
of rice bran, we have isolated six novel triterpene alcohol
and sterol ferulates, viz., two trans- and four cis-
ferulates, besides five and one known trans- and cis-
ferulates, respectively. In this paper, we report the
characterization of the novel ferulates as well as the
inhibitory effect against TPA-induced inflammation in
mice of 12 ferulates from rice bran extract and eight
other synthetic ferulates along with the corresponding
10 free triterpene alcohols and sterols.
In the course of our search on potential antitumor-
promoters (cancer chemopreventive agents) from edible
fungi and plants, and from crude herbal drugs, we have
found that various triterpene alcohols and sterols and
their oxygenated derivatives exhibited activity on in vivo
primary screening assay for antitumor-promoters by
inhibiting the inflammatory ear edema induced by 12-
O-tetradecanoylphorbol-13-acetate (TPA) in mice and on
tumor promotion in two-stage carcinogenesis in mouse
skin initiated by 7,12-dimethylbenz[a]anthracene
(DMBA) and promoted by TPA (Akihisa et al., 1997;
Yasukawa et al., 1991, 1997). We were especially
interested to examine rice bran and γ-oryzanol, a
characteristic component of rice bran oil containing
triterpene alcohol and sterol ferulic acid (4-hydroxy-3-
methoxycinnamic acid) esters. γ-Oryzanol has been
reported to possess diverse physiological effects includ-
ing the lowering blood lipids, growth promotion, gona-
dotrophic action, and stimulation of the hypothalamus
(Kahlon et al., 1992, 1994; Seetharamaiah and Chan-
drasekhara, 1988; Shinomiya et al., 1983; Sugano et al.,
1996, 1997, 1999; Yoshino et al., 1989). Our recent study
demonstrated that four trans-feruloyl esters, viz., cyclo-
artenol (trans-CAR), 24-methylenecycloartanol (trans-
MATERIALS AND METHODS
Crystallizations were performed from acetone-MeOH (1:1,
v/v). Melting points measured are uncorrected. Preparative
thin-layer chromatography (TLC) on silica gel (Kieselgel 60G,
Merck; 0.5 mm thick; 20 × 20 cm) was developed using
benzene-CHCl3 (3:1, v/v). Normal-phase high-performance
liquid chromatography (HPLC) was carried out on a silica
column (Silica-1301N column, 4.6 mm i.d. × 30 cm; ERC Co.,
Ltd., Tokyo, J apan) at 25 °C with n-hexanes-ethyl acetate
(EtOAc) (97:3, v/v; 1.3 mL/min) as the mobile phase. Reverse-
phase HPLC was performed on an ODS column (ODS-2152
column, 10 mm i.d. × 25 cm; ERC Co., Ltd.) at 25 °C with
MeOH (4 mL/min) as the mobile phase. Gas-liquid chroma-
tography (GLC) for free triterpene alcohols and sterols was
performed on a Shimadzu GC-14B instrument (Shimadzu Co.,
* Author to whom correspondence should be addressed (fax
+81-3-3293-7572; e-mail akihisa@chem.cst.nihon-u.ac.jp).
† College of Science and Technology, Nihon University.
‡ College of Pharmacy, Nihon University.
§ Yokogawa Analytical Systems Inc.
Meikai University School of Dentistry.
10.1021/jf000135o CCC: $19.00 © 2000 American Chemical Society
Published on Web 05/26/2000

2314 J. Agric. Food Chem., Vol. 48, No. 6, 2000
Akihisa et al.
Kyoto, J apan) using a DB-17 fused-silica capillary column (30
m × 0.3 mm i.d.; column temperature, 275 °C) and nitrogen
as a carrier gas at 60 mL/min (split ratio 60:1). For both HPLC
and GLC, cholesterol (cholest-5-en-3â-ol) was the standard for
the determination of relative retention times (RRt). Ultraviolet
(UV) spectra were recorded in CHCl3. Electron-impact mass
spectra (MS) were recorded on a Hitachi M-80B double-
focusing gas chromatograph-mass spectroscopy (GC-MS) in-
strument (70 eV) using a direct inlet system. 1H Nuclear
magnetic resonance (NMR) spectra were recorded on a J EOL
J NM LA-500 spectrometer at 500 MHz in CDCl3 with tetra-
methylsilane (TMS) as internal standard.
Ch em ica ls a n d Ma ter ia ls. Cycloartenol (CAR), 24-meth-
ylenecycloartanol (24-MCA), cycloeucalenol (CEU), gramisterol
(GRM), citrostadienol (CTR), 24-methylcholesterol (a mixture
of 24R- and 24S-epimers; 24-MCO), 24-methylenecholesterol
(24-MEC), sitosterol (SITO), stigmasterol (STIG), and stig-
mastanol (STAN) (Goad and Akihisa, 1997) were used as the
reference specimens and as the starting materials for the
preparation of their ferulates. Edible rice bran was purchased
at a market in Tokyo (J apan). Four trans-ferulates, trans-CAR,
trans-24-MCA, trans-24-MCO, and trans-SITO also were used
as the reference compounds (Yasukawa et al., 1998). trans-
Ferulic acid was purchased from Tokyo Chemical Industry Co.,
Ltd. (Tokyo, J apan), and 2-chloro-1,3-dimethylimidazolinium
chloride (DMC) was donated by Shiratori Pharmaceutical Co.,
Ltd. (Chiba, J apan). TPA was purchased from ChemSyn
Laboratories (Lenexa, KS). Indomethacin and hydrocortisone
were obtained from Sigma Chemical Co. (St. Louis, MO). The
structures of triterpene alcohols and sterols and their ferulates
described in this paper are shown in Figure 1.
Extr a ction a n d Isola tion . Rice bran (1 kg) was extracted
three times at room temperature for 3 days each with
methanol (MeOH) to give an extract (158 g). A portion (100 g)
of the extract was partitioned between n-hexane-MeOH-H2O
(19:19:2, v/v/v). The n-hexane fraction (42 g) was subjected to
column chromatography on silica gel (800 g) using an n-hex-
anes-EtOAc gradient of 1:0-1:1 (v/v), which yielded a mixture
containing the feruloyl esters of triterpene alcohols and sterols
(11.2 g). Preparative TLC of the mixture yielded purified trans-
ferulate (Rf 0.36; 692 mg) and cis-ferulate (Rf 0.49; 96 mg)
fractions which on reverse-phase HPLC, and when necessarily
on normal-phase HPLC, eventually yielded three trans-feru-
lates, trans-CEU (7.0 mg), trans-24-MEC (5.4 mg), and trans-
STAN (8.1 mg), and five cis-ferulates, cis-CAR (3.8 mg), cis-
24-MCA (16.2 mg), cis-24-MCO (5.4 mg), cis-SITO (5.4 mg),
and cis-STAN (1.9 mg), besides recently reported four trans-
ferulates, trans-CAR (88 mg), trans-24-MCA (177 mg), trans-
24-MCO (88 mg), and trans-SITO (62 mg) (Yasukawa et al.,
1998). All extraction and isolation procedures for ferulates
were carried out in “white” fluorescent light to prevent
isomerization of the ferulates (Ar´ın et al., 1995; Hartley and
J ones, 1975; Van Boven et al., 1996).
Another portion (58 g) of the MeOH extract was alkaline
hydrolyzed giving a nonsaponifiable lipid fraction (6.2 g) which
on TLC yielded fractions of triterpene alcohols (Rf 0.27; 396
mg; Fr. A), 4R-methylsterols (Rf 0.22; 12 mg; Fr. B), and sterols
(Rf 0.16; 558 mg; Fr. C). Fr. A contained CAR (45.6% of the
fraction; RRt 1.96 in GLC), 24-MCA (51.2%; RRt 2.14) and
other components (3.2%), Fr. B contained CEU (34.0% of the
fraction; RRt 1.73), GRM (29.8%; RRt 1.73), CTR (15.5%; RRt
2.28), and other components (20.7%), and Fr. C contained 24-
MCO (24.8% of the fraction; RRt 1.28), 24-MEC (1.0%; RRt
1.35), SITO (49.9%; RRt 1.54), STIG (18.5%; RRt 1.38), STAN
(1.4%; RRt 1.54), and other components (4.4%). Reverse-phase
HPLC of the fractions A, B, and C yielded CAR and 24-MCA;
CEU, GRM and CTR; and 24-MCO, 24-MEC, SITO, STIG, and
STAN; respectively. The compositions of individual fractions
were determined based on the GLC and reverse-phase HPLC
data.
F igu r e 1. Structures of cycloartenol (CAR), 24-methylene-
cycloartanol (24-MCA), cycloeucalenol (CEU), gramisterol
(GRM), citrostadienol (CTR), 24-methylcholesterol (24-MCO),
24-methylenecholesterol (24-MEC), sitosterol (SITO), stigma-
sterol (STIG), and stigmastanol (STAN), and their trans- and
cis-ferulates.
yielded 4-propionyl ferulate (1.3 g) (Tamura et al. 1962). Into
the solution of STIG (20 mg), 4-propionyl ferulate (60 mg), and
DMC (40 mg) in dry CH2Cl2 (10 mL) was poured dry pyridine
(30 mL) dropwise under water cooling, and the solution was
stirred for 2 h at room temperature. Water was added to the
reaction mixture, and the CH2Cl2 layer was washed succes-
sively with diluted HCl, saturated NaHCO3 solution, and
water and then dried over Na2SO4. This yielded 4-propionyl
ferulate of STIG. Hydrolysis of a propionyl ester moiety of the
ester by 0.1 M KOH/MeOH solution at 50 °C for 20 min yielded
trans-STIG (11 mg). Two other trans-ferulates, trans-GRM and
P r ep a r a tion of tr a n s-F er u loyl Ester s. To the solution
of trans-ferulic acid (1 g) in dry pyridine (3 mL) in a flask
equipped with a drying tube of CaCl2 was added propionic
anhydride (1 g), and the mixture was stirred for 48 h at room
temperature. Extraction of the reaction mixture with CHCl3

Anti-Inflammatory Effects of Sterol Ferulates from Rice Bran
J. Agric. Food Chem., Vol. 48, No. 6, 2000 2315
Ta ble 1. Rela tive Reten tion Tim es (RRt)a in th e HP LC of Tr iter p en e Alcoh ols, Ster ols, a n d Th eir F er u la tes
free alcohol
trans-ferulate
cis-ferulate
(I) b
(II) c
(I) b
(II) c
(I) b
(II) c
compound (abbreviated and systematic names)
cycloartenol (CAR)
RRt
RRt
RRt
RRt
RRt
RRt
Triterpene Alcohol
0.66
1.09
0.85
1.38
0.24
1.64
(cycloart-24-en-3â-ol)
24-methylenecycloartanol (24-MCA)
0.54
1.20
0.80
1.72
0.25
1.96
[24-methylcycloart-24(241)-en-3â-ol]
4R-Methylsterol
cycloeucalenol (CEU)
0.74
1.07
1.02
1.20
0.86
0.91
0.88
1.37
1.34
1.98
0.29
0.26
0.24
1.65
1.87
2.16
[4R,14R-dimethyl-9â,19-cycloergost-24(241)-en-3â-ol]
gramisterol (GRM)
0.89
0.85
[4R-methylergosta-7,24(241)-dien-3â-ol]
citrostadienol (CTR)
{[24(241)Z]-4R-methylstigmasta-7,24(241)-dien-3â-ol}
Sterol
0.96
24-methylcholesterol (24-MCO)
[(24R,S)-ergost-5-en-3â-ol]
24-methylenecholesterol (24-MEC)
[ergosta-5,24(241)-dien-3â-ol]
sitosterol (SITO)
[(24R)-stigmast-5-en-3â-ol]
stigmasterol (STIG)
[(22E,24S)-stigmasta-5,22-dien-3â-ol]
stigmastanol (STAN)
1.12
0.84
1.28
1.15
1.42
0.79
0.78
0.65
0.68
0.71
1.79
1.38
2.14
1.86
2.58
0.22
0.24
0.19
0.21
0.20
1.92
1.47
2.16
1.89
2.70
1.02
0.92
0.94
0.97
[(24R)-stigmastan-3â-ol]
a
b
Cholesterol: RRt ) 1.00. RRt(I): Silica-1301N column (n-hexane-EtOAc ) 97:3). c RRt(II): ODS-2152 column (MeOH).
trans-CTR, were prepared by the same manner from the
corresponding free alcohols.
MEC, trans-STIG, and trans-STAN) were essentially the same
with that of trans-CEU.
P r ep a r a tion of cis-F er u loyl Ester s. cis-Ferulates were
prepared from the corresponding trans-ferulates by exposing
to UV light in a similar manner as described in the literature
(Hartley and J ones, 1975; Van Boven et al., 1996). A solution
(5 mg in 100 mL of MeOH) of trans-STIG was irradiated under
N2 with a 100-W mercury-vapor-discharge lamp (UM-102
lamp, Ushio Inc., Tokyo, J apan) having the radiation at 365
nm in an immersion-type silica tube unit for 1 h, and the
suspected cis-STIG and unchanged trans-STIG isomers were
separated by normal-phase HPLC. The same isomerization
reaction yielded four other cis-ferulates, cis-CEU, cis-GRM, cis-
CTR, and cis-24-MEC, from the corresponding trans-ferulates.
Id en tifica tion a n d Ch a r a cter iza tion . Identification of
four trans-ferulates, trans-CAR, trans-24-MCA, trans-24-MCO,
and trans-SITO, and 10 free alcohols, from CAR to STAN
(Table 1), was performed by spectroscopic (UV, MS, and 1H
NMR) and chromatographic (normal- and reverse-phase HPLC)
comparison with reference compounds. Characterization of six
other trans-ferulates, trans-CEU, trans-GRM, trans-CTR,
trans-24-MEC, trans-STIG, and trans-STAN, and 10 cis-
ferulates, from cis-CAR to cis-STAN (Table 1), was undertaken
Citr osta d ien ol tr a n s-F er u la te (tr a n s-CTR). Mp 118-
123 °C. MS m/z (%): 602 (M+, 23), 504 (17), 461 (28), 408 (4),
393 (4), 310 (9), 295 (6), 267 (13), 241 (7), 227 (15), 194 (74),
177 (100). HR-MS: m/z 602.4304 [Calcd for C40H58O4 (M+):
602.4332].
24-Met h ylen ech olest er ol tr a n s-F er u la t e (tr a n s-24-
MEC). Mp 172-173 °C. MS m/z (%): 574 (M+, 16), 447 (4),
380 (100), 365 (10), 296 (24), 253 (15), 194 (74), 177 (71). HR-
MS: m/z 574.4028 [Calcd for C38H54O4 (M+): 574.4019].
Stigm a ster ol tr a n s-F er u la te (tr a n s-STIG). Mp 145-146
°C [lit. mp 147-148 °C (Tamura et al., 1962)]. HR-MS: m/z
588.4242 [Calcd for C39H56O4 (M+): 588.4176]. The mass
fragmentation was essentially the same with that reported for
the same compound (Tanaka and Kato, 1975).
Stigm a sta n ol tr a n s-F er u la te (tr a n s-STAN). Mp 153-
154 °C [lit. mp 156-157 °C (Tamura et al., 1958)]. HR-MS:
m/z 592.4501 [Calcd for C39H60O4 (M+): 592.4488]. The mass
fragmentation was essentially the same with that reported for
the same compound (Evershed et al., 1988).
Cycloa r ten ol cis-F er u la te (cis-CAR). Mp 143-145 °C.
MS m/z (%): 602 (M+, 3), 587 (1), 425 (4), 408 (20), 393 (6),
365 (6), 339 (7), 316 (6), 297 (4), 286 (2), 203 (8), 194 (26),
177 (100). HR-MS: m/z 602.4357 [Calcd for C40H58O4 (M+):
602.4332]. UV λmax nm: 242 (log ꢀ 4.0), 298 (4.2), 318 (4.3).
24-Meth ylen ecycloa r ta n ol cis-F er u la te (cis-24-MCA).
Mp 125-126 °C. MS m/z (%): 616 (M+, 2), 601 (1), 439 (4),
422 (20), 407 (7), 379 (5), 316 (5), 300 (1), 297 (3), 203 (8), 194
(23), 177 (100). HR-MS: m/z 616.4483 [Calcd for C41H60O4
(M+): 616.4488]. The UV spectral data of this compound and
eight cis-ferulates described below were essentially the same
with that of cis-CAR.
1
by spectral (UV, MS, and H NMR) comparison with those of
the above trans-ferulates and with the literature data for the
relevant compounds (Goad and Akihisa, 1997). The melting
points and the mass and UV spectral data of six trans-
ferulates, trans-CEU, trans-GRM, trans-CTR, trans-24-MEC,
trans-STIG, and trans-STAN, and 10 cis-ferulates, from cis-
CAR to cis-STAN (Table 1), are shown below. The cis-ferulates
exhibited the coupling constant between H′-2 and H′-3
(J H′-2,3 ∼ 13 Hz) in the 1H NMR spectra which unambiguously
distinguished them from the trans-diastereoisomers (J H′-2,3
16 Hz) (Kondo et al., 1988; Otsuka et al., 1996).
∼
Cycloeu ca len ol cis-F er u la te (cis-CEU). Amorphous gum.
MS m/z (%): 602 (M+, 14), 587 (2), 425 (10), 408 (100), 393
(43), 353 (7), 325 (6), 311 (6), 283 (12), 194 (27), 177 (62), 108
(35). HR-MS: m/z 602.4335 [Calcd for C40H58O4 (M+): 602.4332].
Gr a m ist er ol cis-F er u la t e (cis-GR M). Mp 128-129 °C.
MS m/z (%): 588 (M+, 45), 573 (2), 504 (2), 461 (18), 394 (11),
379 (9), 310 (4), 269 (9), 267 (12), 241 (6), 227 (16), 194 (92),
177 (100). HR-MS: m/z 588.4163 [Calcd for C39H56O4 (M+):
588.4176].
Cycloeu ca len ol tr a n s-F er u la te (tr a n s-CEU). Mp 155-
156 °C. MS m/z (%): 602 (M+, 4), 408 (34), 393 (18), 302 (1),
300 (2), 283 (5), 194 (50), 177 (80), 55 (100). High-resolution
(HR)-MS: m/z 602.4345 [Calcd for C40H58O4 (M+): 602.4332].
UV λmax nm: 243 (log ꢀ 4.0), 293 (4.2), 319 (4.3).
Gr a m ister ol tr a n s-F er u la te (tr a n s-GRM). Mp 179-180
°C. MS m/z (%): 588 (M+, 44), 573 (2), 504 (2), 461 (16), 394
(10), 379 (7), 310 (4), 269 (11), 267 (12), 241 (7), 227 (15), 194
(91), 177 (100). HR-MS: m/z 588.4197 [Calcd for C39H56O4
(M+): 588.4176]. The UV spectral data of this compound and
four trans-ferulates described below (trans-CTR, trans-24-
Citr osta d ien ol cis-F er u la te (cis-CTR). Mp 92-93 °C. MS
m/z (%): 602 (M+, 20), 504 (16), 461 (35), 408 (5), 393 (6), 310

2316 J. Agric. Food Chem., Vol. 48, No. 6, 2000
Akihisa et al.
Ta ble 2. 1H NMR Da ta (δ/p p m , 500 MHz, CDCl3) of Six tr a n s-F er u loyl Ester s of Ster olsa
proton
trans-CEU
trans-GRM
trans-CTR
trans-24-MEC
trans-STIG
trans-STAN
H-3
H-6
4.65 (dt, 4.9, 10.7)
4.55 (dt, 4.6, 10.9)
4.55 (dt, 4.5, 10.9)
4.74 (tt, 5.5, 11.0)
5.41 (br d, 4.1)
4.75 (m)
5.40 (m)
4.82 (tt, 5.1, 11.4)
H-7
H-18
H-19
5.19 (m)
0.55 (s)
0.87 (s)
5.19 (m)
0.55 (s)
0.86 (s)
0.98 (s)
0.69 (s)
1.05 (s)
0.71 (s)
1.05 (s)
0.66 (s)
0.85 (s)
0.18 (1H, d, 3.9)
0.43 (1H, d, 4.0)
0.90 (d, 6.1)
H-21
H-22
H-23
H-25
H-26
H-27
H-241
0.90 (d, 6.6)
0.90 (d, 6.4)
0.96 (d, 6.8)
1.02 (d, 6.5)
0.91 (d, 6.6)
5.16 (dd, 8.5, 14.9)
5.02 (dd, 8.8, 15.2)
2.24 (sept, 6.7)
1.03 (d, 7.0)
1.03 (d, 7.0)
4.67 (1H, br s)
4.72 (1H, br s)
2.23 (sept, 6.8)
1.03 (d, 6.9)
1.03 (d, 6.9)
4.66 (1H, br s)
4.72 (1H, br s)
2.83 (sept, 6.7)
0.98 (d, 7.0)
0.98 (d, 7.0)
5.11 (q, 6.7)
2.23 (sept, 6.7)
1.03 (d, 7.0)
1.03 (d, 7.0)
0.80 (d, 6.7)
0.85 (d, 6.4)
0.81 (d, 7.0)
0.84 (d, 7.0)
4.66 (1H, br s)
4.72 (1H, br s)
H-242
H-28
H-30
H-2′
H-3′
H-5′
OMe-6′
OH-7′
H-8′
1.59 (d, 6.7)
0.96 (d, 7.0)
0.81 (d, 7.3)
0.85 (d, 7.0)
0.89 (d, 6.1)
0.91 (s)
6.30 (d, 15.9)
7.61 (d, 15.9)
7.04 (d, 1.8)
3.93 (s)
5.82 (s)
6.92 (d, 8.2)
7.08 (dd, 1.8, 8.2)
0.96 (d, 6.3)
6.30 (d, 15.9)
7.61 (d, 15.9)
7.04 (d, 1.9)
3.93 (s)
5.84 (s)
6.92 (d, 8.2)
7.08 (dd, 2.0, 8.8)
6.30 (d, 15.9)
7.61 (d, 15.9)
7.04 (d, 1.9)
3.93 (s)
5.83 (s)
6.92 (d, 8.2)
7.08 (dd, 1.9, 8.4)
6.28 (d, 15.9)
7.60 (d, 15.9)
7.03 (d, 1.8)
3.93 (s)
6.28 (d, 16.0)
7.60 (d, 15.9)
7.03 (d, 1.8)
3.92 (s)
6.27 (d, 15.8)
7.59 (d, 16.1)
7.03 (d, 1.8)
3.92 (s)
5.82 (s)
5.83 (s)
5.83 (s)
6.91 (d, 8.2)
7.07 (dd, 2.1, 8.1)
6.91 (d, 8.2)
7.07 (dd, 2.1, 8.5)
6.91 (d, 8.1)
7.07 (dd, 1.8, 8.1)
H-9′
a
Figures in parentheses denote J values (Hz).
(7), 295 (7), 267 (15), 241 (8), 227 (16), 194 (75), 177 (100).
HR-MS: m/z 602.4312 [Calcd for C40H58O4 (M+): 602.4332].
24-Meth ylch olester ol cis-F er u la te (cis-24-MCO). Mp
103-105 °C. MS m/z (%): 576 (M+, 1), 382 (100), 367 (10),
274 (8), 261 (8), 255 (10), 228 (2), 213 (9), 194 (86), 177 (57).
HR-MS: m/z 576.4217 [Calcd for C38H56O4 (M+): 576.4166].
24-Met h ylen ech olest er ol cis-F er u la t e (cis-24-ME C).
Mp 126-127 °C. MS m/z (%): 574 (M+, 4), 380 (100), 365 (24),
296 (8), 255 (9), 253 (14), 227 (6), 213 (10), 194 (74), 177 (64).
HR-MS: m/z 574.4060 [Calcd for C38H54O4 (M+): 574.4019].
Sitoster ol cis-F er u la te (cis-SITO). Mp 102-103 °C. MS
m/z (%): 590 (M+, 4), 396 (58), 381 (7), 288 (5), 275 (5), 255
(7), 213 (5), 194 (100), 177 (46). HR-MS: m/z 590.4347 [Calcd
for C39H58O4 (M+): 590.4332].
Stigm a ster ol cis-F er u la te (cis-STIG). Mp 150-151 °C.
MS m/z (%): 588 (M+, 1), 394 (86), 379 (4), 351 (7), 282 (5),
255 (23), 213 (8), 194 (64), 177 (31), 55 (100). HR-MS: m/z
588.4177 [Calcd for C39H56O4 (M+): 588.4176].
Stigm a sta n ol cis-F er u la te (cis-STAN). Mp 120-121 °C.
MS m/z (%): 592 (M+, 32), 398 (2), 383 (4), 257 (4), 215 (7),
194 (100), 177 (14). HR-MS: m/z 592.4382 [Calcd for C39H60O4
(M+): 592.4488].
An im a ls. Female ICR mice were obtained from J apan SLC
(Shizuoka, J apan). The animals were housed in an air-
conditioned specific pathogen free room (22-23 °C) lit from
08:00 to 20:00. Food and water were available ad libitum.
Assa y of TP A-In d u ced In fla m m a tion Ea r Ed em a . TPA
(1 µg) dissolved in acetone (20 µL) was applied to the right
ear of ICR mice by means of a micropipet. A volume of 10 µL
was delivered to both the inner and outer surfaces of the ear.
The samples or their vehicles, MeOH-CHCl3-H2O (2:1:1, v/v/
v; 20 µL), as control, were applied topically about 30 min before
TPA treatment. For ear thickness determinations, a pocket
thickness gauge with a range of 0-9 mm, graduated at 0.01
mm intervals and modified so that the contact surface area
was increased to reduce the tension, was applied to the tip of
the ear. The ear thickness was measured before treatment (a),
and 6 h after TPA treatment (b ) TPA alone; b′ ) TPA plus
sample). The following values were then calculated:
Each value was the mean of individual determinations from
five mice. The 50% inhibitory dose (ID50) values were deter-
mined by the method of probit-graphic interpolation for four
dose levels.
Sta tistica l An a lysis. Statistical analysis was carried out
by Student’s t-test.
RESULTS AND DISCUSSION
We have isolated and characterized six novel feruloyl
esters of triterpene alcohols and sterols, viz., two trans-
ferulates, trans-CEU and trans-24-MEC, and four cis-
ferulates, cis-CAR, cis-24-MCA, cis-24-MCO (The ratio
of the 24R:24S stereoisomers was estimated to be 2:3
based on the 1H NMR data), and cis-SITO, as minor
feruloyl ester constituents from the MeOH extract of
edible rice bran in this study along with five known
trans-ferulates, trans-CAR, trans-24-MCA, trans-24-
MCO, trans-SITO, and trans-STAN (Diack and Saska,
1994; Evershed et al., 1988; Kondo et al., 1988; Norton,
1995; Rogers et al., 1993; Seitz, 1989; Tamura et al.,
1958, 1962; Tanaka, 1971; Yasukawa et al., 1998), and
one known cis-ferulate, cis-STAN (Kondo et al., 1988).
In addition, 10 triterpene alcohols and sterols, from CAR
to STAN (Table 1), were isolated from the nonsaponi-
fiable lipid fraction of the MeOH extract.
This is the first instance for the characterization of
naturally occurring ferulate of 4R-methylsterol, viz.,
trans-CEU. Among the five cis-ferulates characterized
from rice bran extract in this study, cis-STAN has
previously been isolated accompanied with its 24-methyl
homologue and their trans-ferulate isomers (Kondo et
al., 1988). Daylight and long-wavelength UV radiation
induce cis-trans isomerization of feruloyl esters (Ar´ın
et al., 1995; Hartley and J ones, 1975; Van Boven et al.,
1996). Even though all extraction and isolation proce-
dures were undertaken with devoid of daylight, there
is a high possibility as artifacts (Van Boven et al., 1996)
for the five cis-ferulates characterized from rice bran
extract in this study which remains to be clarified in
the future. Natural occurrence of several cis-cinnamic
acid derivative esters, viz., the cis-ferulate of loganic
acid, an iridoid glucoside acyl ester, in Alangium pla-
Edema A is induced by TPA alone (b - a)
Edema B is induced by TPA plus sample (b′ - a)
Inhibitory ratio (%) )
[(Edema A - Edema B)/Edema A] × 100

Anti-Inflammatory Effects of Sterol Ferulates from Rice Bran
J. Agric. Food Chem., Vol. 48, No. 6, 2000 2317

2318 J. Agric. Food Chem., Vol. 48, No. 6, 2000
Akihisa et al.
Ta ble 4. In h ibitor y Effect of Tr iter p en e Alcoh ols a n d
Ster ols, Th eir F er u la tes, a n d Refer en ce Com p ou n d s on
TP A-In d u ced In fla m m a tion in Micea
cation with both trans- and cis-ferulic acids enhanced
the activity as for the former two sterol groups (0.1-
0.5 mg/ear) of which activity was almost comparable
with that of indomethacin (0.3 mg/ear). As for the
triterpene alcohols, CAR and 24-MCA, esterification
with trans-ferulic acid exerted almost no influence (0.2
mg/ear) on the activity while with cis-ferulic acid
reduced the activity (0.5-0.8 mg/ear).
trans-CAR has been proved in our recent study to
possess marked antitumor-promoting activity in two-
stage carcinogenesis in mouse skin (Yasukawa et al.,
1998). Taking this into consideration, it is highly
probable that the ferulates of 4R-methylsterols and
sterols described in this paper also are effective anti-
tumor-promoters. Rice bran which contains various
triterpene alcohols and sterols as the feruloyl esters is
suggested to have potential for the chemoprevention of
cancer.
free alcohol
ID50 I.R.
trans-ferulate
ID50 I.R.
cis-ferulate
ID50 I.R.
compound
CAR
(mg/ear) (%) (mg/ear) (%) (mg/ear) (%)
Triterpene Alcohol
0.3
0.2
92b
85
0.2c
0.2c
79c
88c
0.8
0.5
47
62
24-MCA
4a-Methylsterol
CEU
GRM
CTR
1.9
0.7
0.7
38
66
60
0.2
0.5
0.5
86
72
64
0.4
0.5
0.5
68
55
72
Sterol
24-MCO
24-MEC
SITO
STIG
STAN
1.7
2.7
1.8
1.9
1.0
44
28
43
44
53
0.3c
0.4
85c
77
0.3
0.5
0.3
0.3
0.1
84
66
74
87
94
0.2c
0.2
93c
93
0.1
94
Reference Compound
ACKNOWLEDGMENT
quercetin
indomethacin
1.6
0.3
40
96
We thank Shiratori Pharmaceutical Co., Ltd. (Chiba,
J apan) for the generous gift of DMC (2-chloro-1,3-
dimethylimidazolinium chloride).
a
ID50
: 50% inhibitory dose. I.R.: inhibitory ratio. Unless
otherwise stated, the I.R. was at 1 mg/ear, and p < 0.01 by
b
Student’s t-test as compared to control group. I.R. at 0.5 mg/ear.
c Values taken from the literature (Yasukawa et al., 1998).
LITERATURE CITED
tanifolium (Alangiacae) (Otsuka et al., 1996), the cis-
ferulates of C22-C29 ω-hydroxy fatty acids in the bark
of Virola species (Myristicaceae) (Kawanishi and Hash-
imoto, 1982), and taraxerol cis-p-coumarate in Rhizo-
phora apiculata (Rhizophoraceae) (Kokpol and Chava-
siri, 1990), has previously been reported.
Akihisa, T.; Yasukawa, K.; Kasahara, Y. Triterpenoids from
the flowers of Compositae and their anti-inflammatory
effects. Curr. Topics Phytochem. 1997, 1, 137-144.
Ar´ın, M. J .; D´ıez, M. T.; Resines, J . A. High-performance liquid
chromatographic determination of phenolic acids isolated
from plants. J . Liq. Chromatogr. 1995, 18, 4183-4192.
Diack, M.; Saska, M. Separation of vitamin E and γ-oryzanols
from rice bran by normal-phase chromatography. J . Am. Oil
Chem. Soc. 1994, 71, 1211-1217.
Endo, T.; Ueno, K.; Inaba, Y. Studies on the ferulates contained
in rice bran oil. I. Analysis of the ferulates by means of GLC
and TLC. J . J pn. Oil Chem. Soc. 1968, 17, 344-348.
Evershed, R. P.; Spooner, N.; Prescott, M. C.; Goad, L. J .
Isolation and characterization of intact steryl ferulates from
seeds. J . Chromatogr. 1988, 440, 23-35.
Goad, L. J .; Akihisa, T. Analysis of Sterols; Blackie Academic
and Professional: London, 1997.
Hartley, R. D.; J ones, E. C. Effect of ultraviolet light on
substituted cinnamic acids and the estimation of their cis
and trans isomers by gas chromatography. J . Chromatogr.
1975, 107, 213-218.
Kahlon, T. S.; Saunders: R. M.; Sayre, R. N.; Chow, F. I.; Chiu,
M. M.; Betschart, A. A. Cholesterol-lowering effects of rice
bran and rice bran oil fractions in hypercholesterolemic
hamsters. Cereal Chem. 1992, 69, 485-489.
Kahlon, T. S.; Chow, F. I.; Sayre, R. N. Cholesterol-lowering
properties of rice bran. Cereal Foods World 1994, 39, 99-
103.
To compare the inhibitory effects on TPA-induced
inflammation in mice of free triterpene alcohols (CAR
and 24-MCA) and sterols (from CEU to STAN; Table 1)
with their trans- and cis-ferulates, we have prepared
three trans-ferulates, trans-GRM, trans-CTR, and trans-
STIG, and five cis-ferulates, cis-CEU, cis-GRM, cis-CTR,
cis-24-MEC, and cis-STIG, from the corresponding free
alcohols for their evaluation of the anti-inflammatory
effects, among which trans-STIG is a known ferulate
in rice bran oil (Endo et al., 1968). Table 1 summarizes
the retention data on normal- and reverse-phase col-
umns in the HPLC of 10 free alcohols (from CAR to
STAN) and their trans- and cis-ferulates. Table 2 shows
the 1H NMR spectral data for two novel trans-ferulates,
trans-CEU and trans-24-MEC, and, in addition, four
known trans-ferulates, trans-GRM, trans-CTR, trans-
1
STIG, and trans-STAN, since the full H NMR data for
1
the latter four are unavailable in the literature. The H
NMR spectral data for 10 cis-ferulates, from cis-CAR
to cis-STAN (Table 1), are shown in Table 3.
Ten each of free alcohols and their trans- and cis-
ferulates were evaluated with respect to their anti-
inflammatory activity against TPA-induced inflamma-
tion in mice, and the inhibitory activities were compared
with those of quercetin, a known antitumor-promoter,
and indomethacin, a commercially available anti-
inflammatory drug, as shown in Table 4. Whereas one
free 4R-methylsterol, CEU (ID50 1.9 mg/ear), and four
free sterols, 24-MCO, 24-MEC, SITO, and STIG (1.7-
2.7 mg/ear), exhibited inhibitory activities weaker than
or almost equivalent with that of quercetin (1.6 mg/ear),
the others inhibited markedly the TPA-induced inflam-
mation with 0.1-1.0 mg/ear of the 50% inhibitory dose.
While the activities of free 4R-methylsterols (0.7-1.9
mg/ear) and sterols (1.0-2.7 mg/ear) were weaker than
those of triterpene alcohols (0.2-0.3 mg/ear), esterifi-
Kawanishi, K.; Hashimoto, Y. Long chain esters of Virola
species. Phytochemistry 1982, 26, 749-752.
Kokpol, U.; Chavasiri, W. Taraxeryl cis-p-hydroxycinnamate,
a novel taraxeryl from Rhizophora apiculata. J . Nat. Prod.
1990, 53, 953-955.
Kondo, Y.; Nakajima, K.; Nozoe, S.; Suzuki, S. Isolation of
ovulatory-active substances from crops of J ob’s Tears (Coix
lacryma-jobi L. var. ma-yuen Stapf.). Chem. Pharm. Bull.
1988, 36, 3147-3152.
Norton, R. A. Quantitation of steryl ferulate and p-coumarate
esters from corn and rice. Lipids 1995, 30, 269-274.
Otsuka, H.; Kashima, N.; Nakamoto, K. A neolignan glycoside
and acylated iridoid glucosides from stem bark of Alangium
platanifolium. Phytochemistry 1996, 42, 1435-1438.
Rogers E. J .; Rice, S. M.; Nicolosi, R. J .; Carpenter, D. R.;
McClelland, C. A.; Romanczyk, L. J ., J r. Identification and
quantitation of γ-oryzanol components and simultaneous

Anti-Inflammatory Effects of Sterol Ferulates from Rice Bran
J. Agric. Food Chem., Vol. 48, No. 6, 2000 2319
assessment of tocols in rice bran oil. J . Am. Oil Chem. Soc.
1993, 70, 301-307.
Seetharamaiah, G. S.; Chandrasekhara, N. Effect of oryzanol
on fructose induced hypertriglyceridaemia in rats. Indian
J . Med. Res. 1988, 88, 278-281.
Seitz, L. M. Stanol and sterol esters of ferulic and p-coumaric
acids in wheat, corn, rye, and triticale. J . Agric. Food Chem.
1989, 37, 662-667.
Shinomiya, M.; Morisaki, N.; Matsuoka, N.; Izumi, S.; Saito,
Y.; Kumagai, A.; Mitani, K.; Morita, S. Effects of γ-oryzanol
on lipid metabolism in rats fed high-cholesterol diet. Tohoku
J . Exp. Med. 1983, 141, 191-197.
Sugano, M.; Tsuji, E. Rice bran oil and human health. Biomed.
Environm. Sci. 1996, 9, 242-246.
Sugano, M.; Tsuji, E. Rice bran oil and cholesterol metabolism.
Am. Soc. Nutr. Sci. 1997, 17, 521S-524S.
Sugano, M.; Koba, K.; Tsuji, E. Health benefits of rice bran
oil. Anticancer Res. 1999, 19, 3651-3658.
Tamura, T.; Sakaedani, N.; Matsumoto, T. Isolation of dihydro-
â-sitosteryl ferulate from corngerm oil. Nippon Kagaku
Zasshi 1958, 79, 1011-1014.
Tamura, T.; Takeda, M.; Murakami, K.; Yoshida, A.; Matsu-
moto, T. On some ferulates. J . J pn. Oil Chem. Soc. 1962,
11, 454-458.
Tanaka, A. Separation and quantitative analysis of ferulates
in rice bran oil by thin-layer chromatography. J . J pn. Oil
Chem. Soc. 1971, 20, 792-797.
Tanaka, A.; Kato, A. Mass spectra of several ferulates. J . J pn.
Oil Chem. Soc. 1975, 24, 570-574.
Van Boven, M.; Daenens, P.; Tytgat, J .; Cokelaere, M. Deter-
mination of simmondsins and simmondsin ferulates in
jojoba meal and feed by high-performance liquid chroma-
tography. J . Agric. Food Chem. 1996, 44, 2239-2243.
Yasukawa, K.; Takido, M.; Matsumoto, T.; Takeuchi, M.,
Nakagawa, S. Sterol and triterpene derivatives from plants
inhibit the effects of a tumor promoter, and sitosterol and
betulinic acid inhibit tumor formation in mouse skin two-
stage carcinogenesis. Oncology 1991, 48, 72-76.
Yasukawa, K.; Akihisa, T. Anti-inflammatory and antitumor-
promoting activities of sterols, triterpene alcohols and their
derivatives. Recent Res. Devel. Oil Chem. 1997, 1, 115-125.
Yasukawa, K.; Akihisa, T.; Kimura, Y.; Tamura, T.; Takido,
M. Inhibitory effect of cycloartenol ferulate, a component
of rice bran, on tumor promotion in two-stage carcinogenesis
in mouse skin. Biol. Pharm. Bull. 1998, 21, 1072-1076.
Yoshino, G.; Kazumi, T.; Amano, M.; Tateiwa, M.; Yamasaki,
T.; Takashima, S.; Iwai, M.; Hatanaka, H.; Baba, S. Effects
on gamma-oryzanol on hyperlipidemic subjects. Curr.
Therap. Res. 1989, 45, 543-552.
Received for review J anuary 31, 2000. Revised manuscript
received April 4, 2000. Accepted April 5, 2000.
J F000135O