Microbial Transformation of Isosteviol and Inhibitory Effects on Epstein-Barr Virus Activation of the Transformation Products

Article abstract of DOI:10.1021/np030393q

Microbial transformation of isosteviol (2), a beyerane-type diterpenoid obtained from stevioside (1) by acid hydrolysis, yielded 7β -hydroxyisosteviol (3), 11β-hydroxyisosteviol (5), and 12β -hydroxyisosteviol (6) by the fungus Aspergillus niger, 17-hydroxyisosteviol (7) by the fungus Glomerella cingulata, and 3 and 7-oxoisosteviol (4) by the fungus Mortierella elongate. The five metabolites, 3-7, along with 1 and 2 were evaluated for their inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation induced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells as a primary screening test for inhibitors of tumor promoters. All the diterpenes tested showed potent inhibitory effects, with the five metabolites 3-7 exhibiting more potent effects.

Full text of DOI:10.1021/np030393q

J . Nat. Prod. 2004, 67, 407-410
407
Micr obia l Tr a n sfor m a tion of Isosteviol a n d In h ibitor y Effects on Ep stein -Ba r r
Vir u s Activa tion of th e Tr a n sfor m a tion P r od u cts
,
†
†
‡
†
§
Toshihiro Akihisa,* Yusuke Hamasaki, Harukuni Tokuda, Motohiko Ukiya, Yumiko Kimura, and
Hoyoku Nishino
‡
College of Science and Technology, Nihon University, 1-8 Kanda Surugdai, Chiyoda-ku, Tokyo 101-8308, J apan,
Department of Biochemistry, Kyoto Prefectural University of Medicine, Kamigyo-ku, Kyoto 602-0841, J apan, and
College of Pharmacy, Nihon University, 7-7-1 Narashinodai, Funabashi-shi, Chiba 274-8555, J apan
Received August 28, 2003
Microbial transformation of isosteviol (2), a beyerane-type diterpenoid obtained from stevioside (1) by
acid hydrolysis, yielded 7â-hydroxyisosteviol (3), 11â-hydroxyisosteviol (5), and 12â-hydroxyisosteviol (6)
by the fungus Aspergillus niger, 17-hydroxyisosteviol (7) by the fungus Glomerella cingulata, and 3 and
7
-oxoisosteviol (4) by the fungus Mortierella elongate. The five metabolites, 3-7, along with 1 and 2 were
evaluated for their inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation induced
by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells as a primary screening
test for inhibitors of tumor promoters. All the diterpenes tested showed potent inhibitory effects, with
the five metabolites 3-7 exhibiting more potent effects.
Stevioside (1) is a sweet-tasting glycoside occurring
abundantly in the leaves of Stevia rebaudiana Bertoni
The molecular formula of 5 was determined as C20
from its HREIMS ([M] m/z 334.2144) as well as from its
H
30
O
4
+
1
3
(
Compositae), which has been popularly used as a sugar
C NMR DEPT. The compound had one secondary hy-
1
-1
substitute in J apan and Brazil for decades. Hydrolysis of
in acid solution produces isosteviol (2; ent-16-keto-
H
droxyl [νmax 3409 cm ; δ 4.16 (1H, ddd, J ) 5.8, 10.4,
67.8 (d)], one ketone [νmax 1731 cm- ; δ
1
1
11.3 Hz); δ
C
C
219.5
beyeran-19-oic acid), a tetracyclic diterpenoid with a bey-
(s)], and one carboxyl [νmax 1691 cm-1; δ
C
180.4 (s)] group,
erane skeleton. The biological activities of 2 include the
in addition to three methyls, eight methylenes, two me-
thines, and four quaternary carbons (see Experimental
Section). These data suggested that one methylene carbon
of 2 was hydroxylated by the fungus. The hydroxylation
2
insect antifeeding action, inhibition of rat liver mitochon-
3
dria functions, decrease of glucose production and inhibi-
4
tion of oxygen uptake in the isolated rat renal tubules,
1
1
and inhibition of D-glucose and D-fructose transport across
the cell membrane in the isolated perfused rat liver.5
Microbial transformations have recently been used to
introduce hydroxyl groups at positions remote from the
had occurred at C-11 by analysis of H- H COSY, HMQC,
13
and HMBC spectra of 5 and comparison of the C NMR
data with those of 2.10 The axial (R) orientation of the
oxymethine proton at C-11 was deduced from the splitting
pattern (H-11; ddd, J ) 5.8, 10.4, 11.3 Hz) of the -CHOH
6
functional group on diterpenoid molecules, and 2 has been
1
reported to produce metabolites hydroxylated at 7R-,
signal in the H NMR spectrum. This was supported from
7
1
â-, 9â-, 12â-, 17-, 1R,7â-, 7â,15â-, 11â,12â-, 12â,15â-, and
the NOESY experiment in which significant NOE correla-
tion was observed between [H-20(10R-Me)-H-11R]. Con-
sequently, the hydroxyl group had an equatorial (â) orien-
tation and metabolite 5 was ent-11R-hydroxy-16-oxobeyeran-
19-oic acid (11â-hydroxyisosteviol).
7
-10
1â,12â,17-positions during microbial transformations.
In continuing our search for potential antitumor promoters
1
(
chemopreventive agents) from natural sources,1 we were
especially interested in microbial transformation of 2 to
obtain various hydroxylated metabolites that might be
more potent than the parent compound.12 This paper
reports the microbial transformation of 2 and evaluation
of the metabolites for their inhibitory effects on Epstein-
Barr virus early antigen (EBV-EA) activation induced by
the tumor promoter 12-O-tetradecanoylphorbol-13-acetate
Biotransformation of 2 by Glomerella cingulata IFO 9767
produced only one metabolite, which was identified as 17-
hydroxyisosteviol (7; ent-17-hydroxy-16-oxobeyeran-19-oic
3,9,14
acid) by spectral comparison with the literature.
Two oxidized metabolites, 7â-hydroxyisosteviol (3)8,13 and
, were obtained from biotransformation of 2 by Mortierella
4
(TPA) in Raji cells as a primary screening test for inhibitors
elongate IFO 8570. The molecular formula of 4 was
of tumor promoters.
+
determined as C20
3
compound had one carboxyl [νmax 1680 cm ; δ
H
28
O
4
from its HREIMS ([M] m/z
1
3
32.1990) as well as from its C NMR DEPT. The
Resu lts a n d Discu ssion
-1
C
179.5] and
-
1
Isosteviol (2) was obtained by hydrolysis of stevioside (1)
two ketone [νmax 1718 and 1742 cm ; δ 212.3 and 217.4]
C
with dilute hydrochloric acid.8
-10,13
groups, in addition to three methyls, eight methylenes, two
methines, and four quaternary carbons (see Experimental
Section). These suggested that one methylene carbon of 2
was oxidized into a keto group by the fungus. The keto
group was shown to be located at C-7 by the analysis of
Incubation of 2 with
Aspergillus niger IFO 4414 yielded three metabolites, 3,
, and 6, of which two were identified as 7â-hydroxyiso-
5
8
,13
steviol (3; ent-7R-hydroxy-16-oxobeyeran-19-oic acid) and
1
1
2â-hydroxyisosteviol (6; ent-12R-hydroxy-16-oxobeyeran-
9-oic acid) by spectral comparison with literature data.
8
1
H- H COSY, HMQC, and HMBC spectra of 4 and
1
1
3
10
comparison of the C NMR data with those of 2, and
metabolite 4 was, therefore, assigned the structure ent-
7,16-dioxobeyeran-19-oic acid (7-oxoisosteviol).
*
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.
Kyoto Prefectural University of Medicine.
College of Pharmacy, Nihon University.
‡
Among the five metabolites, 3-7, obtained from the
biotransformation of 2 by the fungi A. niger, G. cingulata,
§
1
0.1021/np030393q CCC: $27.50
© 2004 American Chemical Society and American Society of Pharmacognosy
Published on Web 02/18/2004

4
08 J ournal of Natural Products, 2004, Vol. 67, No. 3
Akihisa et al.
F igu r e 1. Structures of stevioside (1), isosteviol (2), and metabolites 3-7.
Ta ble 1. Percentage of Epstein-Barr Virus Early Antigen
J ASCO IR-300 spectrometer in KBr disks. NMR spectra were
(
EBV-EA) Induciton in the Presence of Compounds 1-7a
recorded with a J EOL LA-400 spectrometer at 400 MHz ( H
1
1
3
NMR) and 100 MHz ( C NMR) in pyridine-d
5
, and chemical
concentration (mol ratio/TPA)
shifts are expressed in δ (ppm) referred to tetramethylsilane
compound
1000
500
100
10
1
(
TMS; H NMR). Electron-impact mass spectra (EIMS) and
1
2
3
4
5
6
7
12.5
12.3
6.9
7.4
4.0
5.7
8.2
8.6
(60)
(60)
(60)
(60)
(60)
(60)
(60)
(70)
46.2
46.7
41.7
35.1
39.8
40.0
42.6
34.2
78.9
80.0
76.3
72.3
74.1
75.1
77.0
82.1
100
98.8
high-resolution EIMS (HREIMS) were recorded on a J EOL
J MS-BU20 spectrometer (70 eV) using a direct inlet system.
FABMS were obtained with a J EOL J MS-BU20 spectrometer
using glycerol as the matrix. Silica gel 60, 220-400 mesh
94.6
94.5
91.0
93.2
95.7
(Merck), was used for open column chromatography. Reversed-
phase preparative HPLC was carried out on a 25 cm × 10 mm
b
i.d. C18 silica column at 25 °C using a Pegasil ODS II column
â-carotene
100
(
Senshu Scientific Co., Ltd., Tokyo, J apan) with MeOH-H O-
2
a
Values represent relative percdentages to the positive control
AcOH (70:30:1, v/v/v) as mobile phase at 2.0 mL/min. A
refractive index detector was used for HPLC. Thin-layer
chromatography (TLC) on 10 × 10 cm silica gel 60G (Merck)
was developed using n-hexanes-EtOAc-AcOH (60:40:1,
v/v/v). Incubations of microorganisms and microbial transfor-
mations were performed on an Incubator Shaker IM41
value. TPA (32 pmol, 20 ng) ) 100%. Values in parentheses are
viability percentages of Raji cells. Reference compound.
b
and M. elongate, metabolites 4 and 5 were new compounds.
-Oxo compound 4 produced along with a 7â-ol (3) from
7
the biotransformation by M. elongate may be a further
oxidative metabolite of the latter. Consistent with this
study, biotransformation of 2 by A. niger has recently been
reported to produce metabolite 3.9
The inhibitory effects of compounds 1-7 on EBV-EA
activation induced by TPA were examined for the primary
screening of antitumor-promoting activities, and the results
are shown in Table 1. Among the compounds tested, the
biotransformation products that underwent hydroxylation
and oxidation, 3-7, exhibited more potent inhibitory
(Yamato Scientific Co., Ltd., Tokyo). Methyl ester derivatives
of 2 and 3 were prepared by treatment with ethereal CH N .
2
2
Ch em ica ls a n d Ma ter ia ls. Stevioside (1) was kindly
donated by Horiuchi Foods, Co., Ltd. (Tokyo, J apan), and its
+
identification was carried out by FABMS ([M - H] m/z 803)
1
13
17,18
and by H and C NMR comparison with the literature.
Potato-dextrose agar, corn steep liquor, and yeast extract from
Nissui Pharmaceutical Co., Ltd. (Tokyo, J apan), glucose, the
EBV cell culture reagents, and n-butyric acid from Nacalai
Tesque, Inc. (Kyoto, J apan), and TPA and â-carotene from
Dojindo Laboratories (Kumamoto, J apan) were purchased.
3
effects, 92-96% inhibition of activation at 1 × 10 mol ratio/
Acid Hyd r olysis of Steviosid e (1). Compound 1 (8.2 g)
was dissolved in H O (140 mL) and then treated with concen-
2
TPA, with preserving high viability (60%) of Raji cells,15
than those of the biotransformation precursor 2 as well as
. The inhibitory effects of the metabolites were almost
trated HCl (2.8 mL). The reaction mixture was refluxed for 2
h. The product was recovered in EtOAc and the solvent
evaporated. The residue (3.8 g) was chromatographed on a
silica gel (180 g) column with a stepwise gradient of n-hex-
anes-EtOAc (95:5 f 0:1; v/v), which yielded isosteviol (2; 2.0
g) from the eluant of n-hexanes-EtOAc (9:1, v/v). Identification
1
equivalent to those of â-carotene, intensively studied in
cancer prevention using animal models.16 Thus, this study
has established that microbial transformation of natural
products (or their derivatives) has a potential value in
developing more potent inhibitors of tumor promotion.
+
19
13
8
of 2 was done by EIMS ([M] m/z 318) and C NMR, and
1
13
13
by H and C NMR as its methyl ester derivative.
Micr oor ga n ism s a n d Cu ltu r e Con d ition s. All cultures
were obtained from the Institute of Fermentation (IFO)
Exp er im en ta l Section
Gen er al Exper im en tal P r ocedu r es. Crystallizations were
performed in acetone, and melting points (uncorrected) deter-
mined using a Yanagimoto micromelting point apparatus.
Optical rotations were measured on a J ASCO DIP-380 pola-
rimeter in MeOH at 25 °C. IR spectra were obtained on a
(
Osaka, J apan). Seven microorganisms were used for the
preliminary screening as follows: A. niger IFO 4414, C.
longirostre IFO 9823, G. cingulata IFO 9767, G. fusarioides
IFO 8831, M. elongate IFO 8570, P. funiculosum IFO 31132,
and P. oxacalcium IFO 7000. Stock cultures of the fungi were

Microbial Transformation of Isosteviol
J ournal of Natural Products, 2004, Vol. 67, No. 3 409
stored on potato-dextrose agar medium at 24 °C. Seed cultures
were obtained by transferring fungi from stock cultures to a
yeast extract-corn steep liquor broth medium (YCB; 8 g of
corn steep liquor, 1 g of yeast extract, and 10 g of glucose were
0.55), of which identification was done by spectral comparison
with the literature.9
,14,19
Biotr a n sfor m a tion by M. elon ga te. The procedure was
similar to the one described above for A. niger. Bioconversion
of 2 (500 mg) by M. elongate yielded a crude extract (549 mg),
which was subjected to column chromatography on silica gel
(30 g). Elution of the column with 1.5 L of n-hexanes-EtOAc
(9:1, v/v) yielded fraction A′′ (53 mg), elution with 1.0 L of
n-hexanes-EtOAc (4:1, v/v) gave fraction B′′ (278 mg), which
was identified as unmetabolized 2, and further elution with
2.0 L of n-hexanes-EtOAc (1:1, v/v) afforded fraction C′′ (174
2
suspended in 1 L of H O). Preliminary screenings were
conducted with conical flasks (250 mL) containing 50 mL of
YCB medium. For each fungus two flasks were inoculated with
the seed culture and incubated on a rotary shaker (110 rpm)
for 3 days at 25 °C. Then compound 2 (10 mg) in dimethyl
sulfoxide (DMSO) (0.2 mL) was added to one flask, while the
other one was kept as a control. The fermentation was
continued for 7 more days, after which the mycelium was
filtered off and washed with EtOAc. The combined broth, after
adjusting the acidity at pH 3-4 by diluted HCl, was extracted
three times with EtOAc, and the organic layers were combined
mg). Reversed-phase HPLC of fraction C′′ gave metabolites 3
(75 mg, 15.0%)8 and 4 (30 mg, 6.0%; t
,13
13.0 min, R 0.60).
R
f
2
5
7-Oxoisosteviol (4): fine needles, mp 216-219 °C; [R]
-73.8° (c 0.13, MeOH); IR νmax 3416 (OH), 1742 and 1718
D
-
1 1
and dried with anhydrous Na
2
SO
4
. After filtration the solvent
(>CdO), 1680 (COOH) cm ; H NMR (pyridine-d
δ 0.91 (1H, ddd, J ) 3.4, 13.2, 13.2 Hz, H -1), 1.06 (1H, m,
-3), 1.07 (3H, s, H-17), 1.11 (3H, s, H-20), 1.28 (3H, s, H-19),
1.32 (1H, m, H -12), 1.36 (1H, ddt, J ) 12.9, 12.9, 4.6 Hz, H
11), 1.51 (1H, m, H -2), 1.53 (1H, dd, J ) 3.9, 12.2 Hz, H -14),
1.56 (1H, dd, J ) 2.9, 8.8 Hz, H-9), 1.57 (1H, m, H -12), 1.63
(1H, br d, J ) 15.4 Hz, H-5), 1.65 (1H, m, H -1), 1.67 (1H, m,
-11), 2.14 (1H, d, J ) 18.3 Hz, H -15), 2.19 (1H, m, H -2),
2.46 (1H, br d, J ) 13.2 Hz, H -3), 2.64 (1H, dd, J ) 2.8, 12.2
Hz, H -15), 2.75 (1H, dd, J ) 3.9, 18.3 Hz, H -15), 3.05 (1H,
dd, J ) 3.7, 15.4 Hz, H -6), 3.44 (1H, dd, J ) 15.4, 15.4 Hz,
-6); 13C NMR (pyridine-d
, 100 MHz) δ 13.2 (CH , C-20), 19.5
CH , C-2), 20.1 (CH , C-17), 20.2 (CH , C-11), 28.6 (CH , C-19),
6.9 (CH , C-12), 38.2 (C, C-10), 38.3 (CH
C-1), 40.3 (CH , C-6), 43.8 (C, C-4), 46.2 (CH
5
, 400 MHz)
was evaporated in vacuo, producing the crude extract. The
extracts from the experimental flasks were compared to the
controls by TLC. Among the seven fungi examined, three, A.
niger, G. cingulata, and M. elongate, produced metabolites of
a
H
a
a
a
-
a
a
2
reproducibly, and these were used for preparative experi-
b
ments. Preparative experiments were conducted in 500 mL
conical flasks containing 250 mL of YCB liquid medium. The
procedure and conditions were the same as for the preliminary
experiments.
b
H
b
a
b
b
b
b
a
Biotr a n sfor m a tion by A. n iger . Isosteviol (2; 500 mg; R
f
H
b
5
3
value 0.85 in TLC) in 2.0 mL of DMSO was evenly distributed
among 4 flasks containing A. niger cultures, and one was kept
as control. The crude extract (510 mg) obtained was subjected
to column chromatography on silica gel (30 g). The column was
eluted with 0.7 L of n-hexanes-EtOAc (9:1, v/v), 0.8 L of
n-hexanes-EtOAc (4:1, v/v), 0.3 L of n-hexanes-EtOAc (7:3,
v/v), and 0.6 L of EtOAc, which yielded fractions A (53 mg), B
(
2
3
2
3
3
2
2
2
, C-3), 39.7 (CH
, C-15), 46.6 (CH
2
2
,
,
2
C-14), 48.9 (C, C-13), 51.7 (C, C-8), 54.1 (CH, C-5), 55.1 (CH,
C-9), 179.5 (C, C-18), 212.3 (C, C-7), 217.4 (C, C-16); EIMS
+
m/z 332 [M] (33), 319 (4), 314 (8), 286 (11), 276 (100), 258 (4),
2
30 (6), 179 (8), 162 (5), 150 (15), 135 (7), 121 (9), 109 (17);
(
278 mg), C (57 mg), and D (76 mg), respectively. Fraction B
was unmetabolized 2 (retention time (t ) 53.0 min in HPLC,
value 0.85 in TLC) by MS and H NMR analysis. Reversed-
phase HPLC of fraction D yielded metabolites 3 (18 mg, 3.6%;
7.8 min, R 0.58), 5 (23 mg, 4.6%; t 8.3 min, R 0.64), and
(11 mg, 2.2%; t 7.1 min, R 0.59). Identification of 3, as its
HREIMS m/z 332.1984 (calcd for C20 , 332.1990).
28 4
H O
R
1
Meth od of EBV-EA In d u ction Tests. The inhibition of
EBV-EA activation was assayed using Raji cells (virus non-
producer type), the EBV genome-carrying human lymphoblas-
toid cells, which were cultivated in 10% fetal bovine serum-
Roswell Park Memorial Institute (FBS RPMI) 1640 medium
R
f
t
6
R
f
R
f
8
R
f
1
3
8
methyl ester derivative, and 6 was performed by spectral
6
solution. The indicator cells (Raji) (1 × 10 /mL) were incubated
comparison with the literature.
at 37 °C for 48 h in 1 mL of the medium containing n-butyric
acid (4 mM, trigger) and 32 pmol of TPA (20 ng/mL, inducer)
in DMSO, and a known amount of test compound in DMSO.
Smears were made from the cell suspension. The activated
cells were stained by high-titer EBV-EA-positive sera from
nasopharyngeal carcinoma patients and were detected by a
conventional indirect immunofluorescence technique. In each
assay, at least 500 cells were counted, and the experiments
were repeated twice. The average extent of EA induction was
determined and compared with that on positive control experi-
ments in which the cells were treated with n-butyric acid plus
TPA where the extent of EA induction was ordinarily more
than around 40%. The viability of treated Raji cells was
1
1â-Hyd r oxyisosteviol (5): fine needles, mp 210-213 °C;
5
2
[
R]
O), 1691 (COOH) cm ; H NMR (pyridine-d
3H, s, H-17), 1.18 (1H, m, H -3), 1.31 (1H, m, H-5), 1.32 (3H,
s, H-20), 1.38 (1H, m, H -1), 1.40 (3H, s, H-19), 1.45 (1H, dd,
J ) 2.8, 11.6 Hz, H -14), 1.51 (1H, d, J ) 10.4 Hz, H-9), 1.53
1H, m, H -7), 1.54 (1H, m, H -2), 1.59 (1H, dd, J ) 3.7, 11.6
Hz, H -14), 1.62 (1H, m, H -7), 1.78 (1H, dd, J ) 11.3, 11.3
Hz, H -12), 1.83 (1H, d, J ) 18.6 Hz, H -15), 2.11 (2H, m, H-6),
.13 (1H, ddd, J ) 2.8, 5.8, 11.3 Hz, H -12), 2.32 (1H, br d, J
14.0 Hz, H -2), 2.49 (1H, br d, J ) 12.8 Hz, H -3), 2.87 (1H,
b
dd, J ) 3.7, 18.6 Hz, H -15), 3.25 (1H, br d, J ) 13.4 Hz, H -
D
-64.6° (c 0.13, MeOH); IR νmax 3409 (OH), 1731 (>Cd
-1 1
5
, 400 MHz) δ 1.08
(
a
a
a
(
a
a
b
b
a
a
2
)
b
b
b
b
1
3
1
(
2
), 4.16 (1H, ddd, J ) 5.8, 10.4, 11.3 Hz, H-11); C NMR
pyridine-d , 100 MHz) δ 14.3 (CH , C-20), 19.7 (CH , C-17),
, C-2), 22.4 (CH , C-6), 29.9 (CH , C-19), 38.8 (CH
, C-1), 43.2 (CH
, C-12), 49.2 (CH
, C-14), 57.6 (CH, C-5), 60.3 (CH, C-9), 67.8
20
assayed by the Trypan Blue staining method.
5
3
3
0.1 (CH
2
2
3
2
2
,
,
Ack n ow led gm en t. We thank Horiuchi Foods, Co., Ltd.
(Tokyo, J apan) for the generous gift of stevioside used in this
study. This study was supported in part by Grants-in-Aid from
the Ministry of Education, Science and Culture, and the
Ministry of Health and Welfare of J apan, and this study was
also supported in part by a grant from the National Cancer
Institute (CA 177625).
C-3), 40.3 (C, C-10), 40.5 (C, C-8), 42.7 (CH
C-7), 44.4 (C, C-4), 49.1 (CH
2
2
2
, C-15), 49.3
(
(
[
(
C, C-13), 54.0 (CH
2
CH, C-11), 180.4 (C, C-18), 219.5 (C, C-16); EIMS m/z 334
+
M] (47), 316 (100), 301 (10), 298 (15), 289 (21), 271 (41), 257
16), 243 (10), 227 (16), 215 (12), 149 (34), 135 (49), 121 (96),
09 (99); HREIMS m/z 334.2144 (calcd for C20
1
30 4
H O , 334.2144).
Biotr a n sfor m a tion by G. cin gu la ta . The procedure was
Refer en ces a n d Notes
similar to the one described for A. niger. From 500 mg of 2, a
crude extract (526 mg) was obtained, which was then fraction-
ated by column chromatography on silica gel (30 g). Elution
with 0.5 L of n-hexanes-EtOAc (9:1, v/v) yielded 37 mg of
fraction A′, elution with 1.0 L of n-hexanes-EtOAc (9:1, v/v)
followed by 0.5 L of n-hexanes-EtOAc (4:1, v/v) gave fraction
B′ (249 mg), identified as unmetabolized 2, and further elution
with 0.5 L of n-hexanes-EtOAc (4:1, v/v) and 0.6 L of EtOAc
afforded fraction C′ (192 mg). Reversed-phase HPLC of fraction
(
1) Soejarto, D. D.; Kinghorn, A. D.; Farnsworth, N. R. J . Nat. Prod. 1982,
45, 590-599.
(2) Nanayakkara, N. P. D.; Kloke, J . A.; Compadre, C. M.; Hussain, P.
A.; Pezzuto, J . M.; Kinghorn, A. D. J . Nat. Prod. 1987, 50, 434-441.
(3) Bracht, A. K.; Alvarez, M.; Bracht, A. Biochem. Pharmacol. 1985,
3
4, 873-882.
(4) Yamamoto, N. S.; Kelmer Bracht, A. M.; Ishii, E. L.; Kemmelmeier,
F. S.; Alvarez, M.; Bracht, A. Experientia 1985, 41, 55-57.
5) Ishii, E. L.; Schwab, A. J .; Bracht, A. Biochem. Pharmacol. 1987, 36,
(
1
417-1433.
(6) Atta-ur-Rahman; Farooq, A.; Anjum, S.; Choudhary, M. I. Curr. Med.
Chem. 1999, 3, 309-326.
R f
C′ gave 17-hydroxyisosteviol (7; 40 mg, 8.0%; t 12.2 min, R

4
10 J ournal of Natural Products, 2004, Vol. 67, No. 3
Akihisa et al.
(
(
(
7) Bearder, J . R.; Frydman, V. M.; Gaskin, P.; MacMillan, J .; Wels, C.
M.; Phinney, B. O. J . Chem. Soc., Perkin Trans. 1 1976, 173-183.
8) de Oliveira, B. H.; Strapasson, R. A. Phytochemistry 1996, 43, 393-
(15) The viability percentages of Raji cells are around 80% even if the
incubation (at 37 °C for 48 h) was undertaken without test compound.
(
(
(
16) Murakami, A.; Ohigashi, H.; Koshimizu, K. Biosci. Biotech. Biochem.
999, 60, 1-8.
17) Van Calsteren, M.-R.; Bussi e` re, Y.; Bissonnette, M. C. Spectroscopy
993, 11, 143-156.
18) Ishikawa, H.; Kitahara, S.; Ohtani, K.; Ikuhara, C.; Tanaka, O. Agric.
Biol. Chem. 1990, 54, 3137-3143.
3
95.
1
9) de Oliveira, B. H.; dos Santos, M. C.; Leal, P. C. Phytochemistry 1999,
5
1, 737-741.
1
(
10) Hsu, F.-L.; Hou, C.-C.; Yahng, L.-M.; Cheng, J .-T.; Chi, T.-Ch.; Liu,
P.-C.; Lin, S.-J . J . Nat. Prod. 2002, 65, 273-277.
(
11) Akihisa, T.; Yasukawa, K. In Studies in Natural Products Chemistry,
Vol. 25, Bioactive Natural Products (Part F); Atta-ur-Rahman, Ed.;
Elsevier Science B.V.: Amsterdam, 2001; pp 43-87.
(19) Compadre, C. M.; Hussain, R. A.; Nanayakkara, N. P. D.; Pezzuto,
J . M.; Kinghorn, A. D. Biomed. Environ. Mass Spectrom. 1988, 15,
211-222.
(
12) Akihisa, T.; Takamine, Y.; Yoshizumi, K.; Tokuda, H.; Kimura, Y.;
Ukiya, M.; Nakahara, T.; Yokochi, T.; Ichiishi, E.; Nishino, H. J . Nat.
Prod. 2002, 65, 278-282.
(20) Takaishi, Y.; Ujita, K.; Tokuda, H.; Nishino, H.; Iwashima, A.; Fujita,
T. Cancer Lett. 1992, 65, 19-26.
(
13) Avent, A. G.; Hanson, J . R.; de Oliveira, B. H. Phytochemistry 1990,
2
9, 2712-2715.
(14) Avent, A. G.; Hanson, J . R.; Hitchcock, P. B.; de Oliveira, B. H. J .
Chem. Soc., Perkin Trans. 1 1990, 2661-2665.
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