Microbial transformations of two lupane-type triterpenes and anti-tumor-promoting effects of the transformation products

Article abstract of DOI:10.1021/np010424m

Microbial transformation of betulin (1), a lupane-type triterpene obtained from the bark extract of white birch (Betula platyphylla Sukatshev var. japonica), and its chemical oxidation product, betulonic acid (2), by the fungus Chaetomium longirostre yielded 4,28-dihydroxy-3,4-seco-lup-20(29)-en-3-oic acid (3) and 4-hydroxy-3,4-seco-lup-20(29)-ene-3,28-dioic acid (4) from 1, and 4,7β,17-trihydroxy-3,4-seco-28-norlup-20(29)-en-3-oic acid (5) and 7β,15α-dihydoxy-3-oxolup-20(29)-en-28-oic acid (6) from 2. The four metabolites, 3-6, 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 promotion. All of the triterpenes tested showed potent inhibitory effects, with the four metabolites 3-6 exhibiting the more potent effects.

Full text of DOI:10.1021/np010424m

278
J . Nat. Prod. 2002, 65, 278-282
Micr obia l Tr a n sfor m a tion s of Tw o Lu p a n e-Typ e Tr iter p en es a n d
An ti-Tu m or -P r om otin g Effects of th e Tr a n sfor m a tion P r od u cts
Toshihiro Akihisa,*,† Yoshio Takamine,† Kazuo Yoshizumi,† Harukuni Tokuda,‡ Yumiko Kimura,§
Motohiko Ukiya,† Toro Nakahara, Toshihiro Yokochi, Eiichiro Ichiishi,‡ and Hoyoku Nishino‡
College of Science and Technology, Nihon University, 1-8 Kanda Surugadai, Chiyoda-ku, Tokyo 101-8308, J apan,
Department of Biochemistry, Kyoto Prefectural University of Medicine, Kamigyo-ku, Kyoto 602-0841, J apan,
College of Pharmacy, Nihon University, 7-7-1 Narashinodai, Funabashi-shi, Chiba 274-8555, J apan, and
National Institute of Advanced Industrial Science and Technology, Higashi 1-1, Tsukuba, Ibaraki 305-8566, J apan
Received August 27, 2001
Microbial transformation of betulin (1), a lupane-type triterpene obtained from the bark extract of white
birch (Betula platyphylla Sukatshev var. japonica), and its chemical oxidation product, betulonic acid
(2), by the fungus Chaetomium longirostre yielded 4,28-dihydroxy-3,4-seco-lup-20(29)-en-3-oic acid (3)
and 4-hydroxy-3,4-seco-lup-20(29)-ene-3,28-dioic acid (4) from 1, and 4,7â,17-trihydroxy-3,4-seco-28-norlup-
20(29)-en-3-oic acid (5) and 7â,15R-dihydoxy-3-oxolup-20(29)-en-28-oic acid (6) from 2. The four metabolites,
3-6, 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 promotion. All of the triterpenes tested showed
potent inhibitory effects, with the four metabolites 3-6 exhibiting the more potent effects.
Betulin [1; lup-20(29)-ene-3â,28-diol] is a naturally oc-
curring lupane-type triterpene abundantly available from
birch bark.1 Lupane-type triterpenes including 1, betulinic
acid [3â-hydroxylup-20(29)-en-28-oic acid], and their de-
rivatives have been reported to exhibit a variety of biologi-
cal activities, such as anti-inflammatory activity,2-4 and
inhibitory effects on Epstein-Barr virus early antigen
(EBV-EA) activation,5 tumor-promoting activity in two-
stage carcinogenesis in mouse skin promoted by 12-O-
tetradecanoylphorbol-13-acetate (TPA),2,6 human immun-
odeficiency virus (HIV)-1,7 and HIV-1 reverse transcriptase.8
Betulinic acid has also been reported to exhibit anti-tumor
activity against human melanoma.9 We were especially
interested in preparing various structurally modified lu-
pane-type triterpenes from 1 in order to find compounds
with more potent biological activities. Thus, we undertook
the microbial transformation of 1 and its chemically
oxidized product, betulonic acid [2; 3-oxolup-20(29)-en-28-
oic acid], by the filamentous fungus Chaetomium longiros-
tre10 in order to obtain oxygenated derivatives.10-12 This
metabolites were detected in the broth. The metabolites
were not present in the controls undertaken without either
the mycelium or the substrate.
paper reports the microbial transformations of 1 and 2 to
metabolites that exhibited more potent inhibition of tumor-
promoting activities than the parent compounds.
1
Assignments of all of the 13C and H NMR spectral data
Resu lts a n d Discu ssion
for 3-6 (Table 1) described in this paper have been done
1
with the aid of DEPT, H-1H COSY, HMQC, HMBC, and
Since C. longirostre has been reported to efficiently
transform oleanonic acid (3-oxo-olean-12-en-28-oic acid), a
pentacyclic triterpene, into ring-A cleaved and hydroxy-
lated compounds,10 we opted to use this fungus for trans-
formation of 1 and 2.
To evaluate the ability of C. longirostre to metabolize
compounds 1 and 2, preliminary experiments were con-
ducted in 500 mL flasks containing a four-day-old cultures
of the fungus. After addition of the substrates, the fermen-
tation was continued for two more weeks, after which
phase-sensitive NOESY experiments.
Incubation of 1 with C. longirostre on a preparative scale
resulted in formation of 3 in 3% yield based on weight
relative to starting material, along with two other minor
uncharacterized metabolites and unmetabolized 1. The
molecular formula of 3 was determined as C30H50O4 from
its HREIMS ([M - H2O]+, m/z 456.3603), positive ESIMS
([M + Na]+, m/z 497), and negative ESIMS ([M - H]-, m/z
473) as well as from its DEPT. The compound had a
carboxyl group [νmax 1710, 1640 cm-1; δC 177.4 (s)] and a
1-hydroxy-1-methylethyl group [νmax 3410 cm-1; δC 28.0 (q),
34.0 (q), and 74.8 (s); δH 1.45 and 1.50 (each 3H and s)]
but showed no evidence for the secondary hydroxyl group
at C-3 and the geminal dimethyl group at C-4 present in
1.13 These data, in combination with the diagnostic EIMS
* 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.
National Institute of Advanced Industrial Science and Technology.
10.1021/np010424m CCC: $22.00
© 2002 American Chemical Society and American Society of Pharmacognosy
Published on Web 02/08/2002

Microbial Transformations of Triterpenes
J ournal of Natural Products, 2002, Vol. 65, No. 3 279
Ta ble 1. 13C (125 MHz) and 1H (500 MHz) NMR Spectral Data CDCl3) for Four Lupane-type Triterpenoids (3-6)
3
4
5
6
C
no.
δC
δH
HMBC (HfC)b
δC
δH
δC
δH
δC
δH
a
a
a
a
1
2
3
4
5
6
7
8
9
35.3 t 2.12, 3.10
30.1 t 2.62, 3.01
177.4 s
35.3 t 2.13, 3.10 br t (10.0) 35.5 t 2.16, 3.18 br t (10.0)
39.8 t 1.32,1.80
34.4 t 2.50 (2H)
216.1 s
30.1 t 2.62, 3.02
177.4 s
30.2 t 2.59, 2.96
177.4 s
74.8 s
74.8 s
74.4 s
47.0 s
52.1 d 1.70
11.8 t 1.59, 1.69
33.9 t 1.34, 1.67
41.1 s
4, 6, 10, 23
7
5, 6
52.1 d 1.70
22.8 t 1.60 (2H)
34.1 t 1.40, 1.68
41.0 s
50.1 d 1.95
34.1 t 2.02, 2.07
74.3 d 4.07 dd (3.9, 10.7)
46.4 s
52.1 d 1.54 dd (2.4, 12.4)
29.8 t 1.82, 1.92
72.2 d 4.17 dd (4.6, 10.7)
49.6 s
50.3 d 1.39
37.3 s
42. 4 d 1.80
10, 11, 14, 26
42.5 d 1.86
41.7 s
44.2 d 1.75
41.9 s
10 41. 6 s
11 21.1 t 1.21, 1.68
12 26.0 t 1.29, 1.83
13 37.8 d 1.84
14 43.4 s
21.3 t 1.26, 1.71
26.3 t 1.31, 1.99 br d (11.5) 27.8 t 1.03, 1.89
38.8 d 2.82 br t (11.5)
43.3 s
21.8 t 1.33, 1.80
21.7 t 1.42 (2H)
26.2 t 1.38, 1.99
38.3 d 2.71
14, 18
47.6 d 1.35
42.7 s
48.6 s
15 27.7 t 1.12, 1.92
16 30.0 t 1.33, 2.45
17 48.6 s
14, 16, 27
14, 17, 28
30.4 t 1.32, 1.88
32.8 t 1.62, 2.63
56.7 s
32.3 t 1.81, 2.84 dt (13.6, 3.9) 69.1 d 4.69 br d (9.2)
34.5 t 2.05, 2.22
79.0 s
42.3 t 1.94, 3.18 br d (10.7)
55.3 s
18 49.1 d 1.73
19 48.3 d 2.63 ddd
13, 16, 17, 19, 28
13, 18, 20, 21, 29, 30 47.7 d 3.54 br t (12.4)
49.7 d 1.80
53.1 d 1.88
55.4 d 2.59
49.6 d 1.85
47.7 d 3.55
(5.6, 11.0, 11.2)
20 151.2 s
151.2 s
151.5 s
151.1 s
21 30.5 t 1.50, 2.17
22 34.8 t 1.16, 2.41
23 28.5 q 1.45 s
24 34.0 q 1.49 s
25 21.0 q 1.17 s
26 16.4 q 1.09 s
27 14.9 q 1.13 s
28 59. 5 t 3.69 d (10.7),
4.12 d (10.7)
17
31.2 t 1.53, 2.26
37.5 t 1.57, 2.28
28.4 a 1.43 s
34.0 q 1.48 s
21.0 q 1.14 s
16.6 q 1.14 s
14.8 q 1.14 s
178.9 s
30.5 t 1.92, 2.23
38.6 t 1.69, 2.04
28.6 q 1.48 s
33.7 q 1.48 s
21.3 q 1.26 s
10.1 q 1.37 s
14.4 q 1.28 s
31.9 t 1.59, 2.36 br t (9.1)
37.7 t 1.83, 2.25 br t (9.3)
26.5 q 1.14 s
21.2 q 1.02 s
15.6 q 0.88 s
11.3 q 1.36 s
9.1 q 1.38 s
178.7 s
17, 18, 21, 28
4, 5, 24
4, 5, 6, 23
1, 5, 9, 10
7, 8, 9, 14
8, 13, 14, 15
16, 22
29 110.0 t 4.75 s, 4.91 d (1.2) 19, 30
110.0 t 4.76 s, 4.95 d (1.2)
19.5 q 1.77 s
109.6 t 4.87 t (1.5), 5.14 d (2.4) 110.1 t 4.82 s, 4.98 d (1.2)
20.8 q 2.04 s 19.7 q 1.83 s
30 19.3 q 1.74 s
19, 20, 29
a
b
Figures in parentheses denote J values (hertz). J values not included were not determined. HMBC spectral data.
fragmentations observed at m/z 398 [M - C3H6O]+, formed
from cleavage of the C-4-C-5 bond with 1H transfer, and
367 (m/z 398-CH2OH), and m/z 383 [M - CH2CH2COOH
- H2O]+, corresponding to the loss of C-1-C-3 and H2O,
suggested a 4-hydroxy-3,4-seco-3-oic acid partial structure
negative ESIMS ([M - H]-, m/z 487) as well as from its
DEPT. Compared to 2,14 metabolite 4 had new carboxyl
[δC 177.4 (s)] and 1-hydroxy-1-methylethyl [νmax 3420 cm-1
;
δC 28.4 (q), 34.0 (q), and 74.8 (s); δH 1.43 and 1.58 (each
3H and s)] groups with the disappearance of the oxo group
at C-3. The diagnostic EIMS fragmentations were observed
for 4 at m/z 430 [M - C3H6O]+ and m/z 415 [M - CH2CH2-
COOH]+, which, in combination with the data mentioned
above, suggested a partial structure of 4-hydroxy-3,4-seco-
3-oic acid for 4. Furthermore, comparison of the 13C and
1H NMR data (Table 1) of 4 with those of 214 and 3 allowed
characterization of 4 as 4-hydroxy-3,4-seco-lup-20(29)-ene-
3,28-dioic acid. The NOE correlations observed for 4 (Figure
1) were consistent with the proposed structure.
1
for 3. This was further supported from the 13C and H NMR
data (Table 1) and from spectral comparison with 1.13 Thus,
metabolite 3 was characterized as 4,28-dihydroxy-3,4-seco-
lup-20(29)-en-3-oic acid. The proposed stereochemistry of
3 was supported from NOE correlations observed between
[H-24-H-25 (10â-Me)-H-26 (8â-Me)-H-13â-H-19â and
H-28 (17â-CH2OH)] on the â-face and [H-5R-H-9R-H-27
(14R-Me)-H-18R] on the R-face of the molecule (Figure 1).
Incubation of 2 afforded three metabolites, 4-6, in 6%,
12%, and 4% yield, respectively, based on weight relative
to starting material. The molecular formula of 4 was
determined as C30H48O5 from its HREIMS ([M - H2O]+,
m/z 470.3396), positive ESIMS ([M + Na]+, m/z 511), and
Metabolite 5 was determined to have the molecular
formula of C29H48O5, which is one carbon less than that of
2, from its HREIMS ([M]+, m/z 476.3504), positive ESIMS
([M + Na]+, m/z 499), negative ESIMS ([M - H]-, m/z 475),
and DEPT. The presence of a 4-hydroxy-3,4-seco-3-oic acid
structural moiety in the molecule, similar to metabolites
3 and 4, was indicated from the 13C NMR signals at δC
177.4 (s; carboxyl carbon at C-3) and δC 28.6 (q; C-23), 33.7
(q; C-24), and 74.4 (s; C-4), arising from the 1-hydroxy-1-
methylethyl group at C-5, and from EIMS fragmentations
observed at m/z 418 [M - C3H6O]+ and m/z 400 [M - CH2-
CH2COOH - H]+. The two other oxygen atoms were shown
to be present as a secondary hydroxyl [δC 74.3 (d); δH 4.07
(dd; J ) 3.9, 10.7 Hz, axially oriented methine)] and a
tertiary â-hydroxyl at C-17 [δC 79.0 (s)], which replaced
the carboxyl group at C-17â, accounting for the loss of a
carbon unit. The axially oriented hydroxymethine (>CHOH;
δH 4.07; dd) was linked with H-5R (δH 1.95) via a methylene
group (δH 2.02 and 2.07), as revealed from the 1H-1H
COSY spectrum, suggesting that the secondary hydroxyl
group was located at C-7 with a â-orientation. From these
1
data, in combination with the 13C and H NMR data (Table

280 J ournal of Natural Products, 2002, Vol. 65, No. 3
Akihisa et al.
F igu r e 1. Major NOE correlations (r f) for 3-6. Drawings correspond to energy-minimized conformations of triterpenes. Calculations were
performed using MacroModel Ver. 6.0 with extended MM3 parameters. The conformation with minimum steric energy was obtained through a
Metropolis Monte Carlo procedure.20
1), metabolite 5 was characterized as 4,7â,17-trihydroxy-
3,4-seco-28-norlup-20(29)-en-3-oic acid. This was supported
from significant NOE correlations observed between [H-24-
H-25 (10â-Me)-H-26 (8â-Me)-H-13â-H-19â] on the â-face
and [H-5R-H-7R and H-9R-H-27 (14R-Me)-H-18R] on the
R-face of the molecule (Figure 1).
cleavage of bonds C-13-C18 and C-14-C-15 with concomi-
tant loss of H2O and 1H transfer.
In summary, oxidative ring cleavage, hydroxylation, and
decarboxylation were observed in this study of the trans-
formation of 1 and 2 with the fungus C. longirostre IFO
9873. The microbial oxidative cleavage of ring-A of a
triterpene to give 4-hydroxy-3,4-seco-3-oic acid has previ-
ously been observed in the transformation of eburicoic acid
[3â-hydroxy-24-methyllanosta-8,24(241)-dien-21-oic acid] by
the fungus Glomerella fusarioides ATCC 955215 and ole-
anonic acid by C. longirostre RF-109510 and has been
suggested to arise by a Baeyer-Villiger type oxidation of
the 3-ketone followed by hydrolysis of the resulting seven-
membered ring lactone.10,15 Formation of metabolite 5
requires decarboxylation followed by hydroxylation at C-17
of substrate 2. Although dealkylation-hydroxylation of the
sterol cycloartenol (9â,19-cycloart-24-en-3â-ol) to afford
17â-hydroxyandrosta-4,8(14)-dien-3-one by Mycobacterium
sp. (NRRL B-3805) has been previously reported,16 this
appears to be the first report of such a biotransformation
of a tritepene. 7â,15R-Dihydroxylation has recently been
reported also in the transformation of betulinic acid by
Bacillus megaterium ATCC 13368.12
Metabolite 6, determined to have the molecular formula
C
30H46O5 from its HREIMS ([M - H2O]+, m/z 468.3237),
positive ESIMS ([M + Na]+, m/z 509), and negative ESIMS
([M - H]-, m/z 485) and from its DEPT, possesses two
oxygen atoms more than substrate 2. The two oxygen
atoms were shown to be present as secondary hydroxyl
groups at C-7 and C-15. One of the hydroxyls at C-7 [δC
72.2 (d)] with a â (equatorial)-orientation was located from
the 1H-1H COSY spectrum in which the diagnostic one-
bond cross-correlations were observed between the 1H
signal of the axially oriented hydroxymethine [>CHOH;
δH 4.17 (dd; J ) 4.6, 10.7 Hz); H-7R] and H-5R (δH 1.54)
via a methylene group (δH 1.82 and 1.92; H-6). The other
hydroxyl group was located at C-15 with an R (equatorial)-
orientation based on the HMBC spectrum (see Experimen-
tal Section) in which a three-bond correlation was observed
between H-27 [14R-Me; δH 1.38 (s)] and the 13C signal of
the hydroxyl carbon [δC 69.1 (d)] possessing an axially
oriented hydroxymethine [>CHOH; δH 4.69 (br d; J ) 9.2
Hz); H-15â]. On the basis of these observations, in combi-
The inhibitory effects of the substrates 1 and 2, their
microbial metabolites 3-6, and the reference substance
â-carotene on EBV-EA activation induced by TPA were
examined as a preliminary evaluation of their potential to
inhibit tumor-promotion, and the results are shown in
Table 2. All compounds evaluated showed inhibitory effects
on EBV-EA activation at 1 × 102 mol ratio concentration
(compound/TPA) and exhibited significant activity at a high
concentration (90-100% inhibition at 1 × 103 mol ratio
concentration) while not adversely affecting the viability
of Raji cells. The inhibitory effects of all compounds tested
were equivalent to or more potent than those of â-carotene,
a vitamin A precursor that has been intensively studied
in cancer prevention using animal models.17 Among the
compounds tested, the biotransformation products that
underwent ring-opening and hydroxylation exhibited more
potent activity than their corresponding precursors. Since
1
nation with the 13C and H NMR data (Table 1), metabolite
6 was characterized as 7â,15R-dihydoxy-3-oxolup-20(29)-
en-28-oic acid. This structure was supported by NOE
correlations observed between [H-24 (4â-Me)-H-25 (10â-
Me)-H-26 (8â-Me)-H-13â-H-15â and H-19â] on the â-face
and [H-23 (4R-Me)-H-5R-H-7R (and H-9R)-H-27 (14R-
Me)-H-18R] on the R-face of the molecule (Figure 1). The
13C NMR data of 6 (Table 1) were in accord with those
reported for 3â,7â,15R-trihydroxylup-20(29)-en-28-oic acid,12
except for the expected differences in the 13C signals of C-3
and adjacent carbons, further supporting the proposed
structure of 6. A fragmentation ion at m/z 271 (C19H27O)
observed as a base peak in the EIMS of 6 could be
accounted for by the loss of D- and E-rings resulting from

Microbial Transformations of Triterpenes
J ournal of Natural Products, 2002, Vol. 65, No. 3 281
Ta ble 2. Percentage of Epstein-Barr Virus Early Antigen
Induction in the Presence of Compounds 1-6 with Respect to a
Positive Control (100%)a
The organic solvent was removed in vacuo, and the aqueous
residue was extracted with EtOAc (2 × 40 mL). The EtOAc
layer was washed with water, dried over Na2SO4, and con-
centrated under reduced pressure. Preparative TLC (Si gel G;
developing solvent: n-hexanes-EtOAc-HOAc ) 85:15:1,
v/v/v; 2 developments) of the residue gave betulonic acid (2;
316 mg; RtR 1.40 in HPLC). The physical and spectral data of
2 were in agreement with the reported data for betulonic
acid.14,19
F u n gu s a n d Cu ltu r e Con d ition s. Stock culture of the
fungus Chaetomium longirostre IFO 9873 obtained from the
Institute for Fermentation (IFO), Osaka, was stored on potato-
dextrose agar medium (PDA, Nissui Pharmaceutical Co., Ltd.,
Tokyo) at 24 °C. A seed culture was grown in a 500 mL flask
containing 300 mL of potato-dextrose broth medium (PDB; 39.5
g of Nissui PDA powder was suspended in 1 L of H2O and the
agar was removed by filtration). After incubation at room
temperature under stirring with a magnetic stirrer for 4 days,
the whole culture was transferred into a 5 L culture flask
containing 2.3 L of PDB and incubated for 2 days under
aeration by bubbling and stirring. The cells were harvested
and washed with H2O by filtration, obtaining 70 g fresh weight
of mycelium.
Biot r a n sfor m a t ion of Bet u lin (1) a n d Isola t ion of
Meta bolites. Betulin (1; 200 mg/4 mL DMSO) was dispersed
to the mycelium (70 g fr. wt), suspended in 3 L of H2O in a 5
L culture flask, and incubated for 2 weeks at room temperature
under aeration by bubbling and stirring. After incubation, the
mycelium was filtered off and washed with EtOAc. The broth,
after adjusting the acidity at pH 3-4 by diluted HCl, was
extracted three times with EtOAc, and the organic layers were
combined. The extract (130 mg) was then subjected to column
chromatography on Si gel (10 g). Elution with 500 mL of
n-hexanes-EtOAc (9:1, v/v) and 600 mL of EtOAc gave fraction
A (63 mg), from which unmetabolized betulin (1) was recovered
(38 mg) after rechromatography. Subsequent elution with
MeOH (1 L) gave fraction B (50 mg), from which compound 3
(6 mg; RtR 0.52 in HPLC) was isolated along with two minor
uncharacterized compounds by reversed-phase HPLC.
4,28-Dih yd r oxy-3,4-seco-lu p -20(29)-en -3-oic a cid (3):
mol ratio concentration (compound/TPA)
compound
1000
500
100
10
1
2
3
4
5
6
4.7 (70)
9.9 (70)
0 (60)
0 (60)
0 (60)
3.5 (60)
15.6 (>80)
33.3
45.7
24.7
24.7
20.0
42.6
54.3
74.2
79.3
71.6
71.2
66.4
76.5
96.8
100
94.7
94.1
89.1
96.9
glycyrrhetic acidb
100
100
a
Values represent relative percentages to the positive control
value. Lower values reflect higher potency. TPA (32 pmol, 20 ng)
) 100%. Values in parentheses are viability percentages of Raji
b
cells. Reference compound.
the inhibitory effects against EBV-EA activation have been
demonstrated to closely parallel inhibition of tumor promo-
tion in vivo,18 the lupane-type triterpenes, and especially
the biotransformation products obtained in this study, may
be valuable inhibitors of promotion (potential cancer
chemopreventive agents). Thus, this study has established
that microbial transformation of natural products has a
potential value in developing more potent inhibitors of
tumor promotion.
Exp er im en ta l Section
Gen er al Exper im en tal P r ocedu r es. Crystallizations were
performed in MeOH, and melting points reported are uncor-
rected. IR spectra were recorded in KBr disks. NMR spectra
were recorded at 500 MHz (1H NMR) and 125 MHz (13C NMR)
in C5D5N with tetramethylsilane (TMS) as internal standard.
EIMS and HREIMS were recorded at 70 eV. Positive and
negative ESIMS were recorded on an Agilent Technologies
1100 LC/MSD SL instrument. Column chromatography was
carried out on silica gel (Si gel 60, 220-400 mesh; Merck).
Reversed-phase HPLC was carried out on an a reversed-phase
(C18) column (Pegasil ODS II column, 25 cm × 10 mm i.d.;
Senshu Scientific Co., Ltd., Tokyo, J apan) at 25 °C with a
refractive index detector and MeOH-H2O-AcOH (80:20:1, v/v/
v; 3.0 mL/min) as a mobile phase. Betulin (1; retention time
52 min) was the standard for the determination of relative
retention time (RtR) in HPLC of the triterpenes described in
this paper.
Ma ter ia ls. A tree of Betula platyphylla Sukatchev var.
japonica (Miq.) Hara was collected in September 1998, at
Bifuka-cho, Hokkaido (J apan), and kindly donated by Mr. Ryoji
Hamatani, The Bifuka-cho Forest Owner’s Cooperative (Bi-
fuka-cho, Hokkaido, J apan). Identification of the plant mate-
rial was done by Mr. R. Hamatani. A voucher specimen has
been deposited at College of Science and Technology, Nihon
University. Compound 1 used as the authentic specimen was
described previously.2
Isola tion of Betu lin (1). The outer bark (124 g) separated
from the birch tree (3.6 kg) was powdered and oven-dried (98
g) and was then extracted by soaking in MeOH at room
temperature for 3 days each for three times to give an extract
(54.7 g). The extract was crystallized from acetone-MeOH,
which gave a crystallized portion (13.9 g), consisting most
exclusively of compound 1, which upon column chromatogra-
phy on a Si gel (700 g) column with a stepwise gradient of
n-hexane-EtOAc [9:1 (2.4 L), 6:1 (2.1 L), 4:1 (3.0 L), 1:1 (5.1
L); v/v] as eluant yielded purified compound 1 (12.2 g; RtR 1.00
in HPLC). The physical and spectral data of 1 were in
agreement with the reported data for betulin.13
P r ep a r a tion of Betu lon ic Acid (2) fr om Betu lin (1). To
a solution of compound 1 (1.0 g) in acetone (50 mL) was added
dropwise freshly prepared J ones’ reagent at 0 °C.19 The
solution was stirred for 1.5 h at 0 °C, quenched with MeOH
(25 mL), and stirred for 5 min, and H2O (40 mL) was added.
fine needles, mp 171-173 °C; IR νmax 3410 (OH), 1710, 1640
1
(COOH), 880 (CdCH2) cm-1
;
13C and H NMR data, see Table
1; EIMS m/z 456 [M - H2O]+ (7), 441 [M - Me - H2O]+ (1),
425 [M - CH2OH - H2O]+ (12), 398 [M - C3H6O - H2O]+
(14), 385 (11), 383 [M - CH2CH2COOH - H2O]+ (11), 367 [M
- C3H6O - CH2OH]+ (39), 357 (13), 345 (9), 327 (3), 315 (4),
271 (6), 257 (6), 245 (10), 234 [M - C14H22O2 - H2O]+ (15),
221 (13), 215 (13), 203 (m/z 234 - CH2OH) (46), 189 [M -
C
15H24O2 - H2O]+ (55), 95 (100); HREIMS m/z 456.3603 (calcd
for C30H48O3, 456.3602); positive ESIMS m/z 497 [M + Na]+;
negative ESIMS m/z 473 [M - H]-.
Biotr a n sfor m a tion of Betu lon ic Acid (2) a n d Isola tion
of Meta bolites. The procedure was similar to that described
above for the transformation of 1. From 150 mg of transformed
2 we obtained an EtOAc extract (120 mg), which was fraction-
ated by column chromatography on Si gel. Elution with
n-hexanes-EtOAc (9:1, v/v; 850 mL) followed by EtOAc (500
mL) gave fraction A′ (48 mg), from which unmetabolized 2 (16
mg) was recovered after rechromatography. Further elution
with MeOH (1 L) yielded fraction B′ (53 mg). Reversed-phase
HPLC of the fraction yielded metabolites 4 (6 mg; RtR 0.44 in
HPLC), 5 (18 mg; RtR 0.18), and 6 (9 mg; RtR 0.28).
4-Hyd r oxy-3,4-seco-lu p -20(29)-en e-3,28-d ioic a cid (4):
fine needles, mp 197-199 °C; IR νmax 3420 (OH), 1698, 1642
1
(COOH), 881 (CdCH2) cm-1
;
13C and H NMR data, see Table
1; HMBC: H-1fC-2, C-5, C-9, C-10; H-2fC-1; H-5fC-4, C-6,
C-10, C-23, C-24, C-25; H-6fC-5; H-9fC-8, C-10, C-11, C-26;
H-13fC-14, C-18, C-27; H-16fC-14, C-18, C-28; H-18fC-28;
H-19fC-18, C-21, C-29, C-30; H-21fC-18, C-19, C-22; H-22fC-
17, C-20, C-21, C-28; H-23fC-5, C-24; H-24fC-5, C-23;
H-25fC-1, C-5, C-9, C-10; H-26fC-7, C-9; H-27fC-13, C-15;
H-29fC-19, C-30; H-30fC-19, C-20, C-29; EIMS m/z 470 [M
- H2O]+ (13), 455 [M - Me - H2O]+ (4), 430 [M - C3H6O]+
(42), 424 (6), 415 [M - CH2CH2COOH]+ (12), 412 (m/z 430-
H2O) (48), 397 (m/z 415 - H2O) (36), 384 [M - C3H6O -

282 J ournal of Natural Products, 2002, Vol. 65, No. 3
Akihisa et al.
COOH]+ (49), 369 (17), 366 (20), 357 (24), 343 (42), 325 (13),
312 (13), 259 (20), 235 (42), 234 (40), 219 (36), 203 (61), 189
(100); HREIMS m/z 470.3396 (calcd for C30H46O4, 470.3393);
positive ESIMS m/z 511 [M + Na]+; negative ESIMS m/z 487
[M - H]-.
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 EA induction was compared
with that of positive control experiments using n-butyric acid
plus TPA, in which EA induction was ordinarily around 30%.
4,7â,17-Tr ih yd r oxy-3,4-seco-28-n or lu p -20(29)-en -3-oic
a cid (5): fine needles, mp 145-146 °C; IR νmax 3445 (OH),
Ack n ow led gm en t. We thank Mr. Ryoji Hamatani, The
Bifuka-cho Forest Owner’s Cooperative (Hokkaido, J apan), and
Dr. Naoto Shimizu, Yokogawa Analytical Systems, Co., Ltd.
(Tokyo, J apan), for the bark material of the birch tree and
ESIMS analysis, respectively. This work was supported in part
by a grant “Research and Development of Nanoscience” from
the Ministry of Education, Science, Sports and Culture to
promote multidisciplinary research projects and also supported
in part by Grants-in-an Aid from the Ministry of Education,
Science, Sports and Culture, and the Ministry of Health and
Welfare, J apan.
1
1709, 1641 (COOH), 884 (CdCH2) cm-1
;
13C and H NMR data,
see Table 1; HMBC: H-1fC-3; H-5fC-4, C-6, C-7, C-10, C-23,
C-24, C-25; H-6fC-5, C-7, C-8; H-7fC-5, C-6, C-8, C-14, C-26,
C-27; H-9fC-1, C-5, C-8, C-10, C-11, C-25, C-26; H-11fC-9;
H-12fC-9, C-13; H-13fC-12, C-18, C-19, C-27; H-15fC-13,
C-14, C-16, C-17, C-27; H-16fC-14, C-15, C-17; H-18fC-12,
C-17, C-19, C-22; H-19fC-13, C-18, C-20, C-21, C-29, C-30;
H-21fC-17, C-18, C-19, C-20, C-22; H-22fC-17, C-18, C-19,
C-21; H-23fC-4, C-5, C-24; H-24fC-4, C-5, C-23; H-25fC-1,
C-5, C-9, C-10; H-26fC-7, C-8, C-9, C-14; H-27fC-8, C-14,
C-15, C-16, C-26; H-29fC-19, C-20, C-30; H-30fC-19, C-20,
C-29; EIMS m/z 476 [M]+ (2), 458 [M - H2O]+ (25), 440 [M -
2H2O]+ (37), 429 (6), 418 [M - C3H6O]+ (7), 400 [M - CH2-
CH2COOH - H]+ (26), 382 (m/z 400 - H2O) (26), 376 (19),
367 (10), 339 (10), 205 (22), 201 (34), 189 (52), 175 (54), 161
(51), 121 (86), 81 (100); HREIMS m/z 476.3504 (calcd for
Refer en ces a n d Notes
(1) Hayek, E. W. H.; J ordis, U.; Moche, W.; Sauter, F. Phytochemistry
1989, 28, 2229-2242.
(2) Yasukawa, K.; Yu, S. Y.; Yamanouchi, S.; Takido, M.; Akihisa, T.;
Tamura, T. Phytomedicine 1995, 1, 309-313.
C
29H48O5, 476.3502); positive ESIMS m/z 499 [M + Na]+;
(3) Manez, S.; Recio, M. C.; Giner, R. M.; Rios, J . L. Eur. J . Pharmacol.
1997, 334, 103-105.
negative ESIMS m/z 475 [M - H]-.
(4) Safayhi, H.; Sailer, E.-R. Planta Med. 1997, 63, 487-493.
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7â,15r-Dih yd oxy-3-oxolu p -20(29)-en -28-oic a cid (6): fine
needles, mp 264-266 °C; IR νmax 3454 (OH), 1706, 1641
(COOH), 1692 (CdO), 881 (CdCH2) cm-1
;
13C and 1H NMR
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data, see Table 1; HMBC H-1fC-2, C-3, C-10, C-25; H-2fC-
1, C-3, C-10; H-5fC-1, C-4, C-6, C-9, C-10, C-23, C-24, C-25;
H-6fC-5, C-7, C-10; H-7fC-6, C-8, C-26; H-9fC-11, C-26;
H-11fC-9, C-13; H-13fC-12; H-15fC-16; H-16fC-14, C-28;
H-18fC-19, C-21, C-22; H-19fC-30; H-23fC-3, C-5, C-24;
H-24fC-3, C-5, C-23; H-25fC-1, C-5, C-10; H-26fC-7, C-8,
C-9, C-14; H-27fC-8, C-13, C-14, C-15; H-29fC-19, C-30;
H-30fC-19, C-20, C-29; EIMS m/z 468 [M - H2O]+ (66), 453
(13), 450 [M - 2H2O]+ (18), 440 (6), 435 (12), 422 (6), 407 (5),
389 (11), 343 (3), 271 (100), 262 (12), 243 (11), 234 (8), 218
(12), 203 (18); HREIMS m/z 468.3239 (calcd for C30H44O4,
468.3237), 271.2062 (calcd for C19H27O, 271.2061); positive
ESIMS m/z 509 [M + Na]+; negative ESIMS m/z 485 [M -
H]-.
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
solution (Nacalai Tesque, Inc., Kyoto, J apan). The indicator
cells (Raji) (1 × 106/mL) were incubated at 37 °C for 48 h in 1
mL of the medium containing n-butyric acid (4 mM, trigger),
32 pmol of TPA (20 ng/mL, inducer) in dimethyl sulfoxide
(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
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NP010424M