Cucurbitane-type triterpenoids from the fruits of Momordica charantia and their cancer chemopreventive effects

Article abstract of DOI:10.1021/np068075p

Thirteen cucurbitane-type triterpene glycosides, including eight new compounds named charantosides I (6), II (7), III (10), IV (11), V (12), VI (13), VII (16), and VIII (17), and five known compounds, 8, 9, 14, 15, and 18, were isolated from a methanol extract of the fruits of Japanese Momordica charantia. The structures of the new compounds were determined on the basis of spectroscopic methods. On evaluation of these triterpene glycosides and five other cucurbitane-type triterpenes, 1-5, also isolated from the extract of M. charantia fruits, for their inhibitory effects on the induction of Epstein-Barr virus early antigen (EBV-EA) by 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells, these compounds showed inhibitory effects on EBV-EA induction with IC₅₀ values of 200-409 mol ratio/32 pmol TPA. In addition, upon evaluation of compounds 1-5 for inhibitory effects against activation of (±)-(E)-methyl-2[(E)-hydroxyimino]-5-nitro-6-methoxy-3-hexemide (NOR 1), a nitrogen oxide (NO) donor, compounds 1-3 showed moderate inhibitory effects. Compounds 1 and 2 exhibited marked inhibitory effects in both 7,12-dimethylbenz[a]anthracene (DMBA)- and peroxynitrite (ONOO^-; PN)-induced mouse skin carcinogenesis tests.

Full text of DOI:10.1021/np068075p

J. Nat. Prod. 2007, 70, 1233-1239
1233
Cucurbitane-Type Triterpenoids from the Fruits of Momordica charantia and Their Cancer
Chemopreventive Effects
Toshihiro Akihisa,*,† Naoki Higo,† Harukuni Tokuda,‡ Motohiko Ukiya,† Hiroyuki Akazawa,† Yuichi Tochigi,†
Yumiko Kimura,§ Takashi Suzuki,§ and Hoyoku Nishino‡
College of Science and Technology, Nihon UniVersity, 1-8 Kanda Surugadai, Chiyoda-ku, Tokyo 101-8308, Japan, Department of Biochemistry
and Molecular Biology, Kyoto Prefectural UniVersity of Medicine, Kamigyo-ku, Kyoto 602-0841, Japan, and College of Pharmacy, Nihon
UniVersity, 7-7-1 Narashinodai, Funabashi-shi, Chiba 274-8555, Japan
ReceiVed December 27, 2006
Thirteen cucurbitane-type triterpene glycosides, including eight new compounds named charantosides I (6), II (7), III
(10), IV (11), V (12), VI (13), VII (16), and VIII (17), and five known compounds, 8, 9, 14, 15, and 18, were isolated
from a methanol extract of the fruits of Japanese Momordica charantia. The structures of the new compounds were
determined on the basis of spectroscopic methods. On evaluation of these triterpene glycosides and five other cucurbitane-
type triterpenes, 1-5, also isolated from the extract of M. charantia fruits, for their inhibitory effects on the induction
of Epstein-Barr virus early antigen (EBV-EA) by 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells, these
compounds showed inhibitory effects on EBV-EA induction with IC50 values of 200-409 mol ratio/32 pmol TPA. In
addition, upon evaluation of compounds 1-5 for inhibitory effects against activation of (()-(E)-methyl-2[(E)-
hydroxyimino]-5-nitro-6-methoxy-3-hexemide (NOR 1), a nitrogen oxide (NO) donor, compounds 1-3 showed moderate
inhibitory effects. Compounds 1 and 2 exhibited marked inhibitory effects in both 7,12-dimethylbenz[a]anthracene
(DMBA)- and peroxynitrite (ONOO-; PN)-induced mouse skin carcinogenesis tests.
The plant Momordica charantia L. (Cucurbitaceae) is com-
monly known as “bitter gourd” or “bitter melon” in English
and is cultivated throughout the world for use as a vegetable as
well as a medicine. M. charantia has been used traditionally
medicinally in developing countries mostly for healing diabetes
and as a carminative and in the treatment of colic.1,2 Pre-
vious investigations have shown that crude extracts of the fruit
of M. charantia possess antidiabetic activity,3,4 and many
cucurbitane-type triterpenoids have been isolated from the
fruits,5-12 seeds,13-15 leaves and vines,16 and stems17 of M.
charantia.
In the course of our search for potential antitumor pro-
moters from natural sources,18,19 we have previously isolated
five cucurbitane-type triterpenoids, 1-5, from the MeOH
extract of the dried fruit of Japanese M. charantia, which is
commonly called “nigauri” or “goya”.20 Our continuing
study on the triterpenoid constituents of the MeOH extract
of M. charantia fruit has now led to the isolation of eight
new (6, 7, 10-13, 16, and 17) and five known (8, 9, 14, 15, and
18) cucurbitane-type triterpene glycosides. This paper de-
scribes the characterization of these compounds and the inhibitory
effects on the activation of Epstein-Barr virus early antigen
(EBV-EA) by 12-O-tetradecanoylphorbol-13-acetate (TPA) in
Raji cells by compounds 1-18 and on the activation of
(()-(E)-methyl-2[(E)-hydroxyimino]-5-nitro-6-methoxy-3-hex-
emide (NOR 1), a nitrogen oxide (NO) donor, by compounds
1-5. In addition, we report the inhibitory effects of (19R,23E)-
5â,19-epoxy-19-methoxycucurbita-6,23,25-trien-3â-ol (1) and
(19R,23E)-5â,19-epoxy-19,25-dimethoxycucurbita-6,23-dien-3â-
ol (2) in 7,12-dimethylbenz[a]anthracene (DMBA)- and peroxyni-
trite (ONOO-; PN)-induced two-stage mouse skin carcinogenesis
tests.
* To whom correspondence should be addressed. Tel: 81-3-
3259-0806. 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.
10.1021/np068075p CCC: $37.00
© 2007 American Chemical Society and American Society of Pharmacognosy
Published on Web 08/09/2007

1234 Journal of Natural Products, 2007, Vol. 70, No. 8
Akihisa et al.
Table 1. 13C NMR Spectroscopic Data (δ values; 150 MHz,
Results and Discussion
C5D5N) for Eight Cucurbitane Glycosides from the Fruits of
Thirteen cucurbitane-type triterpene glycosides, including eight
new compounds, charantosides I (6), II (7), III (10), IV (11), V
(12), VI (13), VII (16), and VIII (17), and five known compounds,
goyaglycoside-c (8),10 goyaglycoside-d (9),10 momordicoside F1
(14),5 momordicoside F2 (15),5 and karaviloside I (18),11 were
isolated from a MeOH extract of the fruits of M. charantia.
Identification of the five known compounds was performed by MS
and 1H NMR spectroscopic comparison of the corresponding
compounds with literature values.
Momordica charantia
carbon
6
7
10
11
12
13
16
17a
aglycon
moiety
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
18.7 18.7 18.9 18.9 18.9 18.9 19.7 22.2
27.4 27.4 27.6 27.6 27.6 27.6 26.5 29.3
83.7 83.4 85.4 85.1 85.4 85.1 85.5 86.9
39.2 39.1 39.1 39.0 39.1 39.0 38.4 42.1
85.5 85.5 85.9 85.9 85.8 85.9 84.2 147.5
133.2 133.1 134.7 134.7 134.2 134.2 133.0 120.7
131.6 131.6 129.9 129.9 130.0 130.0 132.5 75.7
42.2 42.2 52.3 52.3 52.3 52.3 44.9 45.8
48.2 48.2 45.3 45.3 45.3 45.3 50.7 50.2
41.7 41.6 40.2 40.1 40.1 40.1 40.8 36.8
23.3 23.4 23.8 23.8 23.9 23.8 21.9 22.6
30.9 31.1 31.0 31.0 31.2 31.2 30.0 29.3
45.4 45.4 45.5 45.5 45.6 45.5 45.4 45.8
48.3 48.4 48.9 48.9 48.9 48.9 47.9 47.9
33.9 33.8 33.4 33.4 33.4 33.4 33.4 35.0
28.3 28.4 28.3 28.3 28.4 28.6 27.8 27.6
50.7 51.4 50.6 50.6 51.3 51.3 50.8 50.2
14.9 14.8 15.0 15.0 15.0 14.9 14.6 15.0
112.4 112.4 80.1 80.2 80.1 79.7 182.0 207.0
36.9 32.9 36.8 36.8 32.8 33.8 36.8 36.3
19.0 19.0 18.9 18.9 19.0 19.9 18.8 19.0
40.1 43.4 40.1 40.1 43.4 43.0 40.1 39.7
129.9 74.8 129.9 129.9 74.9 76.4 129.8 128.4
134.7 127.9 135.2 135.2 127.9 127.4 134.8 137.7
142.6 134.4 142.5 142.5 134.5 135.8 142.5 74.8
114.6 25.8 114.7 114.7 25.8 25.9 114.7 26.1
18.9 18.1 18.9 18.9 18.1 18.5 18.9 26.4
24.4 24.8 25.6 25.6 25.6 25.6 24.0 27.8
21.2 21.3 21.1 21.1 21.1 21.1 20.9 25.8
20.0 20.0 20.2 20.2 20.2 20.2 19.3 18.1
55.9
All of the eight new compounds were suggested to be monogly-
cosides on the basis of the IR absorption bands due to a glycosidic
function (e.g., 6: νmax 3421, 1068, 1036 cm-1 10,11
and an anomeric
)
1
proton signal of the glycosyl moiety observed in their H NMR
spectra. The proposed structures for all of the eight new compounds
as described below were supported by analysis of the HMBC and
NOESY data (Tables S1-S8, Supporting Information), in addition
1
to 13C DEPT, H-1H COSY, and HMQC data.
The molecular formula of compound 6 was determined to be
C37H58O8 on the basis of its HRESIMS (positive-ion mode) ([M +
Na]+, m/z 653.4017). The UV absorptions at λmax 239 (log ꢀ 4.09),
230 (4.28), and 225 (4.26) suggested the presence of a (23E)-∆23,25
-
conjugated diene system in the side chain of the aglycon moiety.20,21
The 13C (Table 1) and 1H NMR spectra (Table 2) of 6 showed the
presence of four tertiary methyls, a secondary methyl, a vinylic
methyl, an O-methyl, an oxymethine, two disubstituted double
bonds, a terminal methylene, and an acetal methine. The 1H NMR
spectrum also showed signals assignable to a â-D-glucopyanosyl
moiety [δH 4.99 (1H, d, J ) 7.9 Hz; H-1′)].10,11 The NMR data of
the aglycon moiety of 6 were in good agreement with those of
compound 1 except that the former exhibited a downfield glyco-
sylation shift for the C-3 signal in the 13C NMR spectrum.12,22
Compound 6 showed a diagnostic NOE correlation between H-1â
and H-19 in the NOESY experiment, which supported the 19R-
stereochemistry.6,23 The 19R-stereochemistry allows very close
spatial orientation of H-19 to H-1â, which is consistent with the
OMe-7
OMe-19
OMe-23
OMe-25
sugar
moiety
1′
2′
3′
4′
5′
57.6 57.6
55.6
55.6 55.3
50.1
1
105.3 102.3 106.7 103.8 106.7 103.7 107.7 107.3
76.2 73.8 75.8 73.1 75.8 73.1 75.3 75.2
78.0 71.7 78.3 72.4 78.3 72.4 78.4 78.8
72.0 69.2 71.9 69.3 71.9 69.3 71.7 71.7
78.7 76.5 78.4 76.2 78.4 76.2 78.8 78.3
63.0 63.3 63.1 63.4 63.1 63.4 63.1 63.1
appearance of the NOE correlation between the H NMR signals
of H-1â and H-19 in the NOESY spectrum. In addition, conjugation
at C-3 of the aglycon moiety with the glucosyl moiety was
supported from the HMBC experiment, which showed cross-
correlations between H-3 (δH 3.75) and the C-1′ (δC 105.3) of the
glucose moiety and between H-1′ of the glucose (δH 4.99) and C-3
of the aglycon (δC 83.7). The above evidence suggested that 6 has
a structure of (19R,23E)-5â,19-epoxy-19-methoxycucurbita-6,23,-
25-trien-3â-ol 3-O-â-D-glucopyranoside (charantoside I).
6′
a Determined at 125 MHz.
from the 13C NMR data of compounds 7 and 13 (Table 1), which
were almost consistent with the ∆δC values [δC(23R) - δC(23S)]
of -0.5 (C-20), -1.0 (C-21), (0 (C-22), -1.0 (C-23), +0.7 (C-
24), -1.8 (C-25), (0 (C-26), and (0 (C-27) calculated from the
13C NMR data of (23R)- and (23S)-cycloart-24-ene-3â,23-diols.25
The above evidence suggested that 7 possesses the structure (19R,-
23R)-5â,19-epoxy-19,23-dimethoxycucurbita-6,24-dien-3â-ol 3-O-
â-D-allopyranoside (charantoside II). As will be mentioned later,
compound 13 (charantoside VI) was deduced to possess a 23S-
stereochemistry.
Compound 7 was assigned a molecular formula of C38H62O9, as
determined from its [M + Na]+ ion at m/z 685.4290 in the
HRESIMS. The 13C (Table 1) and 1H NMR spectra (Table 2) of 7
showed the presence of four tertiary methyls, a secondary methyl,
two vinylic methyls, two O-methyls, both a di- and a trisubustituted
double bond, an oxymethine, and an acetal methine, in addition to
a â-D-allopyranosyl function.10-12 The 13C and 1H NMR spectra of
7 were very similar to those of compound 9,10 except for the signals
due to a side chain of the aglycon moiety. Compound 7 exhibited
1H NMR signals for the side-chain protons at δH 1.08 (3H, d, J )
6.2 Hz; a secondary methyl), 1.71 and 1.74 (each 3H and br s; two
vinylic methyls), 3.28 (3H, s; an O-methyl), 4.13 (1H, dt, J ) 9.0,
3.1 Hz; an allylic oxymethine), and 5.22 (1H, d, J ) 9.0 Hz; an
olefinic methine). This permitted the positioning of the double bond
at C-24 and the methoxyl group at C-23 in the side chain and was
used to formulate a (23ê)-23-methoxy-∆24 side-chain structure.24
The stereochemistry at C-23 of 7 was deduced to be R by
comparison of its side-chain 13C NMR signals with those of
compound 13 and (23R)- and (23S)-cycloart-24-ene-3â,23-diols.24
Thus, the ∆δC values [δC(7) - δC(13)] for the side-chain signals
were calculated as -0.9 (C-20), -0.9 (C-21), +0.4 (C-22), -1.6
(C-23), +0.5 (C-24), -1.4 (C-25), -0.1 (C-26), and -0.4 (C-27)
Compound 10 exhibited a [M + Na]+ ion at m/z 623.3914 in
the HRESIMS, compatible with the molecular formula, C36H56O7.
1
The 13C (Table 1) and H NMR spectra (Table 2) of 10 showed
the presence of four tertiary methyls, a secondary methyl, a vinylic
methyl, an oxymethylene, a terminal methylene, two disubstituted
double bonds, and a â-D-glucopyranosyl functionality. The UV
absorptions at λmax 238, 229, and 224 suggested that the aglycon
moiety possesses a (23E)-∆23,25 side chain.20,21 The above evidence,
coupled with the spectroscopic comparison with compounds 6 and
14, led to the formulation of the structure of 10 as (23E)-5â,19-
epoxycucurbita-6,23,25-trien-3â-ol 3-O-â-D-glucopyranoside (cha-
rantoside III).
Compound 11 was assigned the molecular formula C36H56O7
(HRESIMS m/z 623.3919 [M + Na]+) and exhibited 13C (Table 1)

Table 2. 1H NMR Spectroscopic Data (δ values; 600 MHz, C5D5N) for Eight Cucurbitane Glycosides from the Fruits of Momordica charantiaa
proton(s)
6
7
10
11
12
13
16
17b
aglycon moiety
1R
1.44
1.90
1.78
1.44
1.90
1.74
1.34
1.77
1.78
1.34
1.78
1.80
2.33
1.35
1.77
1.78
2.37
1.35
1.77
1.78
2.34
1.47
1.70
1.77
1.57
1.82
1.92
1â
2R
2â
2.18 (dq, 13.5, 3.5)
3.75 (br s)
6.18 (dd, 2.4, 9.6)
5.63 (dd, 3.5, 9.6)
3.13 (br s)
2.47 (dd, 5.9, 12.7)
1.74
1.63
1.61
1.52
1.31 (2H)
2.13 (dq, 13.1, 3.7) 2.33
3.70 (br s)
6.15 (dd, 2.4, 9.6)
5.61 (dd, 3.8, 9.6)
3.13 (br s)
2.47 (dd, 5.8, 12.7) 2.28
1.76
1.67
1.66
2.46 (dd, 3.5, 13.0)
3.68 (br s)
6.33 (dd, 2.0, 9.7)
5.63 (dd, 3.1, 9.7)
2.57 (t, 2.0)
2.67 (dd, 5.5, 12.4)
1.71
2.41 (dt, 5.5, 15.2)
1.51
1.58
1.28
1.20
1.91
1.30
1.49
0.87 (s)
2.44 (dq, 13.2, 2.6)
3.74 (br s)
6.07 (d, 4.9)
3.48 (br d, 5.4)
2.19 (s)
2.57
1.44
2.59
1.58
1.52
1.34
1.14
1.94
1.33
1.55
0.94 (s)
10.21 (s)
3R
3.71 (br s)
3.66 (br s)
3.70 (br s)
3.65 (br s)
6.19 (d, 9.6)
5.56 (dd, 3.8, 9.6)
2.31 (br s)
2.29
1.64
1.34
1.61
1.51
1.27
1.20
1.98
1.35
6
7
6.21 (dd, 2.0, 9.6)
5.58 (dd, 3.8, 9.6)
2.32
6.20 (dd, 2.1, 9.6)
5.58 (dd, 3.5, 9.6)
2.34
2.31
1.62
1.33
1.54
1.46
1.30
1.22
1.95
1.32
1.47
0.78 (s)
3.61 (d, 8.3)
3.77 (d, 8.3)
1.51
6.20 (dd, 2.0, 9.6)
5.56 (dd, 3.8, 9.6)
2.31
2.29
1.62
1.34
1.61
1.52
1.26
1.19
1.95
1.43
1.45
0.79 (s)
3.59 (d, 7.9)
3.73 (d, 7.9)
1.92
8â
10R
11R
11â
12R
12â
15R
15â
16R
16â
17R
18
1.63
1.33
1.56
1.45
1.28
1.21
1.94
1.32
1.61
1.30 (2H)
1.94
1.31
1.51
0.90 (s)
4.86 (s)
1.96
1.43
1.48
0.95 (s)
4.88 (s)
1.47
1.53
0.78 (s)
3.61 (d, 8.0)
3.74 (d, 8.0)
1.49
0.78 (s)
3.58 (d, 8.3)
3.75 (d, 8.3)
1.55
19a
19b
20
1.50
1.94
1.48
1.53
21
0.95 (d, 5.8)
1.84
2.30 (br dd, 5.8, 13.4)
5.77 (ddd, 5.8, 8.2, 15.1) 4.13 (dt, 9.0, 3.1)
6.31 (d, 15.1)
4.98 (s)
5.03 (s)
1.92 (s)
0.86 (s)
1.50 (s)
1.08 (d, 6.2)
1.07
1.86
0.93 (d, 5.8)
1.84
2.31
0.94 (d, 6.2)
1.84
2.30
1.05 (d, 6.5)
1.05
1.85
1.04 (d, 5.5)
1.57
1.72
4.11 (dt, 8.9, 5.1)
5.16 (br d, 8.9)
1.75 (s)
0.92 (d, 5.8)
1.81
0.98 (d, 5.4)
1.85
2.24 (br d, 5.4, 13.5)
5.65
5.57 (d, 15.7)
1.72 (s)
22a
22b
23
2.28 (br dd, 6.2, 13.4)
5.76 (ddd, 6.2, 8.6, 15.1)
6.30 (d, 15.1)
4.98 (br s)
5.03 (br s)
1.91 (s)
0.94 (s)
1.60 (s)
0.82 (s)
5.77 (ddd, 6.2, 6.8, 14.8) 5.78 (ddd, 6.2, 8.1, 15.1) 4.13 (dt, 9.9, 3.1)
6.31 (d, 14.8)
4.98 (s)
5.03 (s)
1.92 (s)
0.93 (s)
1.54 (s)
0.87 (s)
24
5.22 (d, 9.0)
1.74 (br s)
6.31 (d, 15.1)
4.98 (s)
5.04 (s)
1.92 (s)
0.90 (s)
5.22 (dt, 8.6, 1.4)
1.75 (d, 1.1)
26a
26b
27
28
29
1.71 (br s)
0.82 (s)
1.44 (s)
0.93 (s)
1.71 (d, 1.0)
0.93 (s)
1.53 (s)
1.73 (s)
0.89 (s)
1.47 (s)
0.86 (s)
1.34 (s)
1.14 (s)
1.34 (s)
0.78 (s)
3.20 (s)
1.48 (s)
0.87 (s)
30
0.90 (s)
0.89 (s)
OMe-7
OMe-19
OMe-23
OMe-25
sugar moiety
1′
2′
3′
4′
5′
3.45 (s)
3.48 (s)
3.28 (s)
3.30 (s)
3.30 (s)
3.23 (s)
4.99 (d, 7.9)
4.00 (t, 7.9)
4.26 (t, 8.9)
4.22 (t, 8.9)
3.99
5.46 (d, 7.6)
3.88 (dd, 2.0, 7.6)
4.71 (t, 2.0)
4.18 (dd, 2.0, 9.7)
4.45
4.92 (d, 7.9)
4.00 (t, 7.9)
4.22 (t, 8.9)
4.20 (t, 8.9)
3.98
5.40 (d, 7.9)
3.94 (dd, 2.4, 7.9)
4.69 (t, 2.4)
4.18 (br d, 9.6)
4.46
4.91 (d, 7.9)
3.99 (t, 7.9)
4.23 (t, 9.0)
4.19 (t, 9.0)
5.38 (d, 7.9)
3.92 (dd, 2.7, 7.9)
4.68 (t, 2.7)
4.86 (d, 7.9)
3.97 (dd 7.9, 8.9)
4.20 (t, 8.9)
4.86 (d, 8.3)
3.87 (t, 8.3)
4.20 (t, 8.9)
4.15 (t, 8.9)
3.94
4.17 (br dd, 2.4, 9.2) 4.13 (t, 8.9)
3.97 (ddd, 3.1, 5.5, 8.2) 4.45
3.95
6′a
6′b
4.42 (dd, 4.4, 11.3)
4.59 (br d, 11.3)
4.37 (dd, 4.8, 11.3) 4.41 (dd, 5.5, 11.7)
4.52 (dd, 2.0, 11.3) 4.57 (br d, 11.7)
4.38 (dd, 5.1, 11.7)
4.53 (br d, 11.7)
4.40 (dd, 5.5, 11.7)
4.57 (dd, 2.4, 11.7)
4.37 (dd, 5.1, 11.7)
4.52 (dd, 2.4, 11.7)
4.38 (dd, 5.5, 12.1)
4.56 (dd, 2.4, 12.1)
4.36 (dd, 5.1, 11.7)
4.54 (dd, 2.4, 11.7)
b
a Figures in parentheses denote J values (Hz). Determined at 500 MHz.

1236 Journal of Natural Products, 2007, Vol. 70, No. 8
Akihisa et al.
Table 3. Inhibitory Effects on the Induction of Epstein-Barr Virus Early Antigen and Inhibitory Ratio (I.R.) on NOR 1 Action of
Compounds Isolated from Momordica charantia and Reference Compounds
concentration (mol ratio/TPA)a
1000
500
100
10
IC50b (mol ratio/
32 pmol TPA)
IR (%) for
NOR1 action
compound
% to control (% viability)
1
2
3
4
5
(19R,23E)-5â,19-epoxy-19-methoxy-
cucurbita-6,23,25-trien-3â-ol
(19R,23E)-5â,19-epoxy-19,25-dimethoxy-
cucurbita-6,23-dien-3â-ol
(23E)-5â,19-epoxy-19-methoxycucurbita-
6,23-diene-3â,25-diol
(23E)-3â-hydroxy-7â-methoxycucurbita-
5,23,25-trien-19-al
(23E)-3â-hydroxy-7â,25-dimethoxy-
cucurbita-5,23-dien-19-al
charantoside I
charantoside II
goyaglycoside-c
goyaglycoside-d
charantoside III
charantoside IV
charantoside V
charantoside VI
momordicoside F1
momordicoside F2
charantoside VII
0 (70)
0 (70)
0 (70)
0 (70)
0 (70)
18.5
19.0
27.6
22.5
33.7
60.2
42.4
72.5
75.4
77.5
82.7
83.9
94.5
95.3
97.3
203
200
315
291
328
2.0
1.9
1.9
1.6
1.5
6
7
8
12.6 (70)
12.3 (70)
13.5 (70)
11.2 (70)
12.9 (70)
12.1 (70)
10.5 (70)
12.7 (70)
11.3 (70)
9.1 (70)
35.4
35.0
33.7
34.2
36.0
35.0
30.6
35.9
31.5
29.7
28.1
38.5
39.2
81.1
82.1
80.0
80.3
82.1
81.1
77.0
82.0
78.1
76.9
75.1
82.5
83.7
100
372
373
398
358
384
371
376
380
385
362
361
401
409
100
100
100
100
100
100
100
100
100
100
100
100
9
10
11
12
13
14
15
16
17
18
9.9 (70)
15.3 (70)
16.1 (70)
charantoside VIII
karaviloside I
reference compounds
â-carotene
8.6 (70)
0 (70)
27.4 (>80)
34.2
22.8
63.3
82.1
81.8
83.3
100
100
100
400
341
572
curcumin
glycyrrhizin
carboxy-PTIO
2.2
8.0
a Values represent percentages relative to the positive control value. TPA (32 pmol, 20 ng) ) 100%. Values in parentheses are viability percentages
b
of Raji cells. IC50 represents the mol ratio to TPA that inhibits 50% of positive control (100%) activated with 32 pmol of TPA.
and 1H NMR signals (Table 2) almost indistinguishable from those
of 10 except for resonances due to a glycosyl moiety. Compound
11 exhibited NMR signals for a â-D-allopyranosyl function as the
glycosyl moiety, and consequently, this was characterized as (23E)-
5â,19-epoxycucurbita-6,23,25-trien-3â-ol 3-O-â-D-allopyranoside
(charantoside IV).
and a â-D-glucopyranosyl unit. The NMR spectra of 16 were quite
similar to those of 6 except that the former showed a carbonyl signal
(δC 182.0) instead of the O-methyl and an acetal methine signal
observed for the latter. This suggested that C-19 of 16 is part of a
5â,19-γ-lactone ring, which was supported by observation of 13C-
1H long-range couplings from H-8, H-10, and H-11â to C-19 in
the HMBC spectrum (Table S7, Supporting Information). Thus,
the structure of 16 was assigned as (23E)-3â-hydroxycucurbita-6,-
23,25-trien-5â,19-olide 3-O-â-D-glucopyranoside (charantoside VII).
Compound 12 exhibited a [M + Na]+ ion at m/z 655.4194 in
the HRESIMS, corresponding to a molecular formula of C37H60O8.
1
The 13C (Table 1) and H NMR spectra (Table 2) of 12 exhibited
the presence of four tertiary methyls, a secondary methyl, an
O-methyl, two vinylic methyls, an oxymethylene, both a di- and a
trisubstituted double bond, and a â-D-glucopyranosyl moiety. The
NMR data for the ring system of the aglycon and the glycosyl
moieties of 12 were superimposable on those of 10, whereas the
NMR data for the side-chain moiety of 12 were almost indistin-
guishable from those of 7. Therefore, the structure of compound
12 was assigned as (23R)-5â,19-epoxy-23-methoxycucurbita-6,24-
dien-3â-ol 3-O-â-D-glucopyranoside (charantoside V).
Compound 13 gave a [M + Na]+ ion in the HRESIMS at m/z
655.4167, consistent with a molecular formula of C37H60O8. The
13C (Table 1) and 1H NMR spectra (Table 2) of 13 suggested it to
be a stereoisomer of compound 12. The NMR data showed that 13
possesses a â-D-allopyranosyl group as a glycosyl moiety. In
addition, as mentioned above, comparison of the side-chain 13C
NMR data with those of compound 7 and (23R)- and (23S)-cycloart-
24-ene-3â,23-diols24,25 made it possible to assign the stereochem-
istry at C-23 of 13 as S. Consequently, the structure of 13 was
characterized as (23S)-5â,19-epoxy-23-methoxycucurbita-6,24-dien-
3â-ol 3-O-â-D-allopyranoside (charantoside VI).
The molecular formula of compound 17 was determined as
C38H62O9 from its HRESIMS ([M + Na]+ m/z 685.4294). The 13
C
(Table 1) and 1H NMR spectra (Table 2) of 17 showed the presence
of six tertiary methyls, a secondary methyl, two O-methyls, both a
di- and a trisubstituted double bond, an aldehyde, and a â-D-
glucopyranosyl unit. The NMR data for the aglycon moiety of 17
were very similar to those of compound 520 except that the former
exhibited a downfield glycosylation shift for the C-3 signal in the
13C NMR spectrum.12,22 The above evidence suggested that 17 has
the structure (23E)-3â-hydroxy-7â,25-dimethoxycucurbita-5,23-
dien-19-al 3-O-â-D-glucopyranoside (charantoside VIII).
Acid hydrolysis of 6, 10, 12, 16, and 17 gave D-glucose as a
sugar identified by HPLC, whereas 7, 11, and 13 yielded D-allose,
which supported the proposed structures of the eight new glycosides.
The inhibitory effects on the induction of EBV-EA induced by
TPA were examined as a preliminary evaluation of antitumor-
promoting activity. Table 3 shows the inhibitory effects of 13
cucurbitane-type triterpene glycosides, 6-18, isolated in this study,
and five cucurbitane-type triterpenes, 1-5, isolated recently from
an n-hexane-soluble fraction of the MeOH extract of M. charantia
fruits,20 against TPA (32 pmol)-induced EBV-EA activation in Raji
cells. All of the compounds tested caused high viability (70%) of
Raji cells even at 32 nmol (mol ratio of compound to TPA ) 1000:
1), indicating their very low cytotoxicity at this high concentration.
Each compound tested showed an inhibitory effect, with an IC50
Compound 16 possesses the molecular formula C36H54O8 as
determined from the HRESIMS ([M + Na]+ m/z 637.3694). The
13C (Table 1) and 1H NMR spectra (Table 2) exhibited the presence
of four tertiary methyls, a secondary methyl, a vinylic methyl, a
terminal methylene, two disubstituted double bonds, a carbonyl,

Cucurbitane-Type Triterpenoids from Momordica charantia
Journal of Natural Products, 2007, Vol. 70, No. 8 1237
Figure 1. Inhibitory effect of (19R,23E)-5â,19-epoxy-19-methoxycucurbita-6,23,25-trien-3â-ol (1) and (19R,23E)-5â,19-epoxy-19-
dimethoxycucurbita-6,23-dien-3â-ol (2) on mouse skin carcinogenesis induced by DMBA and TPA. (A) Percentage of mice bearing papillomas.
(B) Average number of papillomas per mouse. b, positive control, TPA (1.7 nmol) alone (group I); O, TPA (1.7 nmol) + 85 nmol of 1
(group II); 4, TPA (1.7 nmol) + 85 nmol of 2 (group III). At 20 weeks of promotion, the number of papillomas per mouse differed
significantly (p < 0.01, using the Student’s t-test) between the groups treated with compounds 1 and 2 and the control group. The number
(standard deviations are shown in parentheses) of papillomas per mouse for each group was 9.3 (1.6), 4.0 (1.4), and 3.9 (1.3) for groups
I, II, and III, respectively.
Figure 2. Inhibitory effect of (19R,23E)-5â,19-epoxy-19-methoxycucurbita-6,23,25-trien-3â-ol (1) and (19R,23E)-5â,19-epoxy-19-
dimethoxycucurbita-6,23-dien-3â-ol (2) on mouse skin carcinogenesis induced by PN and TPA. (A) Percentage of mice bearing papillomas.
(B) Average number of papillomas per mouse. b, positive control, PN (390 nmol) + TPA (1.7 nmol) alone (group I); O, PN (390 nmol)
+ 0.0025% of 1 (2 weeks) + TPA (1.7 nmol) (group II); 4, PN (390 nmol) + 0.0025% of 2 (2 weeks) + TPA (1.7 nmol) (group III). At
20 weeks of promotion, the number of papillomas per mouse differed significantly between the groups treated with compounds 1 and 2 and
the control group (p < 0.01, using the Student's t-test). The number (standard deviations are shown in parentheses) of papillomas per mouse
for each group was 7.0 (1.9), 4.7 (1.8), and 4.5 (1.3) for groups I, II, and III, respectively.
value (concentration of 50% inhibition with respect to positive
control) of 200-409 mol ratio/32 pmol TPA. As such, these
compounds were comparable with or more potent than the reference
compound, â-carotene (IC50 ) 397 mol ratio/32 pmol TPA), a
vitamin A precursor studied widely in cancer chemoprevention
animal models. Of the compounds tested, five triterpenes without
glycosyl moieties, 1-5, exhibited more potent inhibitory effects
(IC50 values 200-328 mol ratio/32 pmol TPA) than the triterpenes
with glycosyl units, 6-18 (IC50 values 358-409 mol ratio/32 pmol
TPA). Since inhibitory effects against EBV-EA induction have been
demonstrated to correlate with those against tumor promotion in
vivo,26 compounds 1-5 are potential antitumor promoters.
The highly inhibitory compounds 1-5 were then evaluated for
their scavenging activity against NO generation by NOR 1 in a
cultured cell system using an in vitro screening model for NO
scavenging.27 Table 3 shows the inhibitory ratios (I.R.) of these
compounds and two reference compounds, the natural product
glycyrrhizin and the synthetic NO scavenger carboxy-PTIO, on
NOR 1 activity. Among the isolates tested, three compounds, 1-3,
exhibited moderate inhibitory effects (I.R. 1.9-2.0) that were almost
equivalent to the value for glyzyrrhizin (I.R. 2.2). NO is known to
be involved in several potential toxic mechanisms and is a mutagen
and can cause mutations in both microorganisms and mammalian
cells.28,29
On the basis of the results of the in vitro assays described above,
we evaluated subsequently the inhibitory effects of compounds 1
and 2 in two tumor models in mouse skin. The incidence (%) of
papilloma-bearing mice and the average numbers of papillomas per
mouse in a two-stage carcinogenesis test in mouse skin using
DMBA as an inhibitor and TPA as a promoter are presented in
Figures 1A and 1B, respectively. The incidence of the papilloma-
bearing mice was high and 100% at 10 weeks promotion in group
I (positive control). Further, more than five and nine papillomas
were formed per mouse at 10 and 20 weeks of promotion,
respectively. The formation of papillomas in mouse skin was
delayed and the mean numbers of papillomas per mouse were
reduced by treatment with 1 and 2. Thus, in groups II (treated with
1) and III (treated with 2), the percentage ratios of papilloma-bearing
mice were only 33% (II and III) at 10 weeks and 80% (II and III)
at 20 weeks, and the mean papillomas per mouse were 1.7 (II) and
2.1 (III) at 10 weeks and 4.0 (II) and 3.9 (III) at 20 weeks.
Compounds 1 and 2 were then evaluated on their inhibitory
effects against the initiation of PN, and the results are shown in
Figure 2. In the positive control group (group I), the first papilloma
appeared after 7 weeks and the incidence of the papilloma-bearing
mice was 100% after 12 weeks (Figure 2A). Oral administration
of 1 and 2 exhibited a significant inhibitory effect in the PN-induced
and TPA-promoted experiment in mice. When 1 (group II) and 2

1238 Journal of Natural Products, 2007, Vol. 70, No. 8
Akihisa et al.
to further chromatography on ODS (185 g), which was eluted
successively with solvents of decreasing polarity [MeOH-H2O, 7:3
f 1:0], and yielded 48 fractions, H1-H48, each of which was
recovered from 100 mL of eluting solution. Isolation of the following
13 compounds was performed by preparative HPLC: compound 13
[3.5 mg; retention time (tR) 32.8 min] from fraction H19 (95 mg) by
HPLC system IV; compound 17 (1.7 mg; tR 20.4 min) from fraction
H20 (75 mg) by HPLC system III; compound 7 (2.2 mg; tR 22.0 min)
from fraction H21 (260 mg) by HPLC system II; compounds 8 (8.4
mg; tR 27.6 min), 12 (10.0 mg; tR 24.4 min), and 16 (15.9 mg; tR 20.0
min) from fraction H22 (487 mg) by HPLC system III; compounds 9
(18.5 mg; tR 45.6 min), 14 (30.3 mg; tR 34.0 min), and 15 (18.7 mg; tR
40.8 min) from fraction H23 (306 mg) by HPLC system IV; compounds
6 (7.1 mg; tR 25.2 min) and 18 (3.4 mg; tR 38.0 min) from fraction
H25 (283 mg) by HPLC system I; and compounds 10 (7.7 mg; tR 24.8
min) and 11 (7.2 mg; tR 28.4 min) from fraction H27 (154 mg) by
HPLC system I.
(group III) (0.0025% each) were administrated in drinking water,
from 1 week before to 1 week after the initiation treatment, whereas
the first papilloma appeared after 8 weeks, the incidence of the
papilloma-bearing mice was 80% even after 20 weeks of promotion
in both groups II and III. In the average number of papillomas per
mouse (Figure 2B), 1 and 2 reduced the number of papillomas
compared to the control group. Whereas approximately 7 papillomas
per mouse were observed after 20 weeks of promotion in group I,
only 4.7 (group II) and 4.5 (group III) papillomas were observed
after the same period of promotion. From these results, 1 and 2
appear to be effective for the inhibition of PN-initiated carcino-
genesis on mouse skin. PN (ONOO-), which is produced by the
reaction of NO with superoxide, is a potent tumor-initiating agent27
as well as an oxidant and nitrating and hydroxylating agent.
Compounds 1 and 2 may be suggested as being able to intercept
and neutralize potent chemical carcinogens, such as reactive oxygen
species (ROS; superoxide, and peroxy and hydroxy radicals) and
NO donors.
(19R,23E)-5â,19-Epoxy-19-methoxycucurbita-6,23,25-trien-3â-
ol 3-O-â-D-glucopyranoside (6; charantoside I): amorphous solid;
[R]25 -116.9 (c 0.47, MeOH); UV (EtOH) λmax (log ꢀ) 239 (4.09),
D
230 (4.28), 225 (4.26) nm; IR νmax 3421, 1647, 1068, 1036 cm-1
;
13C
From the results of in vitro EBV-EA induction, in vitro NOR 1
inhibition, and in vivo two-stage carcinogenesis tests, the cucur-
bitane-type triterpenes from the MeOH extract of M. charantia
fruits, especially, compounds 1 and 2, may be useful as agents that
inhibit chemical carcinogenesis.
and 1H NMR, see Tables 1 and 2, respectively; HRESIMS m/z 653.4017
(calcd for C37H58O8Na [M + Na]+, 653.4029).
(19R,23R)-5â,19-Epoxy-19,23-dimethoxycucurbita-6,24-dien-3â-
ol 3-O-â-D-allopyranoside (7; charantoside II): amorphous solid;
[R]25 -80.6 (c 0.32, MeOH); IR νmax 3431, 1080, 1036 cm-1 13C
;
D
and 1H NMR, see Tables 1 and 2, respectively; HRESIMS m/z 685.4290
Experimental Section
(calcd for C38H62O9Na [M + Na]+, 685.4291).
General Experimental Procedures. Crystallizations were performed
in MeOH, and melting points were determined on a Yanagimoto micro
melting point apparatus and are uncorrected. Optical rotations were
measured on a JASCO P-1020 polarimeter in MeOH at 25 °C. UV
spectra on a Shimadzu UV-2200 spectrometer and IR spectra on a
JASCO FTIR-300E spectrometer were recorded in EtOH and KBr disks,
respectively. NMR spectra were recorded with a JEOL ECA-600 (1H,
600 MHz; 13C, 150 MHz) or with a JEOL LA-500 (1H, 500 MHz; 13C,
125 MHz) spectrometer in C5D5N with tetramethylsilane as an internal
standard. HRESIMS were recorded on an Agilent 1100 LC/MSD TOF
(time-of-flight) system [ionization mode: positive; nebulizing gas (N2)
pressure: 35 psi; drying-gas (N2): flow, 12 L/min, temp, 325 °C;
capillary voltage: 3000 V; fragmentor voltage: 225 V]. Silica gel
(Kieselgel 60, 230-400 mesh, Merck) and octadecyl silica gel
(Chromatorex-ODS, 100-200 mesh; Fuji Silysia Chemical, Ltd., Aichi,
Japan) were used for open column chromatography. Reversed-phase
preparative HPLC was carried out on an octadecyl silica column
(Pegasil ODS II column, 25 cm × 10 mm i.d.; Senshu Scientific Co.,
Ltd., Tokyo, Japan) at 25 °C at a flow rate of 2.0 mL/min with the
eluent MeCN-H2O [9:1 (HPLC system I), 17:3 (HPLC system II),
4:1 (HPLC system III), and 3:1 (HPLC system IV)], and normal-phase
analytical HPLC on an aminopropyl silica column (Senshu PAK NH2-
1251-N, 25 cm × 4.6 mm i.d.; Senshu Scientific Co., Ltd.) at 25 °C at
a flow rate of 1.0 mL/min with the eluent MeCN-H2O [9:1 (HPLC
system V)].
Chemicals and Materials. Sliced and dried fresh whole fruits of
“nigauri” (M. charantia), cultivated in Okinawa prefecture, Japan, in
the summer of 2002, used in this study was purchased from Taiyo Co.,
Ltd. (Osaka, Japan).20 The following chemicals were purchased: TPA
from ChemSyn Laboratories (Lenexa, KS), DMBA and glyzyrrhizin
from Sigma Chemical Co. (St. Louis, MO), the EBV cell culture
reagents and n-butanoic acid from Nacalai Tesque, Inc. (Kyoto, Japan),
peroxynitrite (PN) solution, NOR 1, and carboxy-PTIO from Dojindo
Laboratories (Kumamoto, Japan).
Extraction and Isolation. Sliced and dried fruit material of M.
charantia (3.6 kg) was extracted with MeOH and yielded a MeOH
extract (261 g) after evaporation of the solvent in vacuo. The extract
was partitioned between H2O and EtOAc, giving an EtOAc-soluble
fraction (44 g). The EtOAc fraction was further partitioned between
n-hexane-MeOH-H2O (19:19:1), which yielded n-hexane (13.0 g) and
MeOH-H2O (27.4 g) soluble fractions. Column chromatography on
silica gel (516 g) of the MeOH-H2O fraction, eluted successively with
solvents of increasing polarity [n-hexane-EtOAc, 1:1 (2.9 L), 1:4 (1.8
L), 0:1 (2.7 L); EtOAc-MeOH, 4:1 (2.1 L), 1:1 (3.6 L), 3:7 (4.5 L)],
afforded 13 fractions, A-M, listed in increasing order of polarity.
Fraction H (16.0 g), eluted with EtOAc-MeOH (4:1), was subjected
(23E)-5â,19-Epoxycucurbita-6,23,25-trien-3â-ol 3-O-â-D-glucopy-
ranoside (10; charantoside III): amorphous solid; [R]25 -85.2 (c
D
0.19, MeOH); UV (EtOH) λmax (log ꢀ) 238 (4.13), 230 (4.35), 224 (4.27)
nm; IR νmax 3406, 1645, 1080, 1036 cm-1 13C and 1H NMR, see Tables
;
1 and 2, respectively; HRESIMS m/z 623.3914 (calcd for C36H56O7Na
[M + Na]+, 623.3923).
(23E)-5â,19-Epoxycucurbita-6,23,25-trien-3â-ol 3-O-â-D-allopy-
ranoside (11; charantoside IV): colorless needles; mp 256-260 °C;
[R]25 -152.8 (c 0.16, MeOH); UV (EtOH) λmax (log ꢀ) 238 (4.15),
D
229 (4.32), 225 (4.27) nm; IR νmax 3421, 1645, 1086, 1034 cm-1
;
13C
and 1H NMR, see Tables 1 and 2, respectively; HRESIMS m/z 623.3919
(calcd for C36H56O7Na [M + Na]+, 623.3923).
(23R)-5â,19-Epoxy-23-methoxycucurbita-6,24-dien-3â-ol 3-O-â-
D-glucopyranoside (12; charantoside V): colorless needles; mp 235-
240 °C; [R]25D -68.5 (c 0.76, MeOH); IR νmax 3423, 1084, 1032 cm-1
;
1
13C and H NMR, see Tables 1 and 2, respectively; HRESIMS m/z
655.4194 (calcd for C37H60O8Na [M + Na]+, 655.4185).
(23S)-5â,19-Epoxy-23-methoxycucurbita-6,24-dien-3â-ol 3-O-â-
D-allopyranoside (13; charantoside VI): amorphous solid; [R]25
D
-73.5 (c 0.48, MeOH); IR νmax 3392, 1084, 1033 cm-1
;
13C and H
1
NMR, see Tables 1 and 2, respectively; HRESIMS m/z 655.4167 (calcd
for C37H60O8Na [M + Na]+, 655.4185).
(23E)-3â-Hydroxycucurbita-6,23,25-trien-5â,19-olide 3-O-â-D-
glucopyranoside (16; charantoside VII): colorless needles; mp 258-
262 °C; [R]25 -120.4 (c 0.79, MeOH); UV (EtOH) λmax (log ꢀ) 238
D
(4.07), 229 (4.34), 224 (4.26) nm; IR νmax 3423, 1745, 1643, 1080,
1038 cm-1 13C and 1H NMR, see Tables 1 and 2, respectively;
;
HRESIMS m/z 637.3694 (calcd for C36H54O8Na [M + Na]+, 637.3716).
(23E)-3â-Hydroxy-7â,25-dimethoxycucurbita-5,23-dien-19-al 3-O-
â-D-glucopyranoside (17; charantoside VIII): amorphous solid; [R]25
D
+12.4 (c 0.20, MeOH); IR νmax 3427, 1070, 1036 cm-1
;
13C and H
1
NMR, see Tables 1 and 2, respectively; HRESIMS m/z 685.4294 (calcd
for C38H62O9Na [M + Na]+, 685.4291).
Acid Hydrolysis of Compounds 6, 7, 10-13, 16, and 17. A solution
of triterpene glycoside (2 mg each) in 1 M HCl-MeOH (5 mL) was
heated under reflux for 1 h. The reaction mixture was partitioned
between EOAc and H2O, and the H2O layer was neutralized by passing
through Amberlite MB-3. The H2O eluate was concentrated, and the
residue was subjected directly to HPLC system V for the identification
of the sugar moiety of the glycosides. The sugar from 6, 10, 12, 16,
and 17 was identified as D-glucose (tR 22.1 min), whereas that from 7,
11, and 13 as D-allose (tR 17.2 min).
In Vitro EBV-EA Activation Experiment. For the protocol for
this in vitro assay, refer to two previous articles.26,30
In Vitro NOR 1 Inhibition Experiment. For the protocol for this
in vitro assay, refer to two previous articles.27,30

Cucurbitane-Type Triterpenoids from Momordica charantia
Journal of Natural Products, 2007, Vol. 70, No. 8 1239
(8) El-Gengaihi, S.; Karawya, M. S.; Selim, M. A.; Motawe, H. M.;
Ibrahim, N.; Faddah, L. M. Pharmazie 1995, 50, 361-362.
(9) Begum, S.; Ahmed, M.; Siddiqui, B. S.; Khan, A.; Saify, Z. S.; Arif,
M. Phytochemistry 1997, 44, 1313-1320.
In Vivo Two-stage Mouse Skin Carcinogenesis Assay Initiated
by DMBA. For the protocol for this in vivo assay, refer to a previous
article.30
In Vivo Two-stage Mouse Skin Carcinogenesis Assay Initiated
by PN. Tumors were induced with PN and promoted with TPA. The
animals were divided into two experimental groups (15 mice each).
The back of each mouse was shaved with surgical clippers, and the
mice were topically treated with PN (390 nmol) in NaOH (1 mM, 0.1
mL) as an initiation treatment. For group I (positive control group), 1
week after the initiation, papilloma formation was promoted by the
twice weekly application of TPA (1 µg, 1.7 nmol) in acetone (0.1 mL)
on the skin (no papilloma formation was seen with topical application
of the acetone solvent alone). For groups II and III (test groups),
0.0025% of test compounds (1 and 2; 2.5 mg/100 mL each in drinking
water) were administered orally for 2 weeks before the promotion
treatment (1 week both before and after the initiation) and subsequently
promoted by the twice a week application with TPA (1.7 nmol) in
acetone (0.1 mL). The incidence of papilloma bearers and numbers of
papillomas per mouse were detected weekly for 20 weeks. Skin
papillomas were first counted when they reached approximately 1 mm
× 1 mm in size. The Student’s t-test was used for statistical analyses
of the numbers of papillomas per mouse. The animal weights were not
statistically different between any of the groups during the in vivo assay
period.
(10) Murakami, T.; Emoto, A.; Matsuda, H.; Yoshikawa, M. Chem.
Pharm. Bull. 2001, 49, 54-63.
(11) Nakamura, S.; Murakami, T.; Nakamura, J.; Kobayashi, H.; Matsuda,
H.; Yoshikawa, M. Chem. Pharm. Bull. 2006, 54, 1545-1550.
(12) Harinantenaina, L.; Tanaka, M.; Takaoka, S.; Oda, M.; Mogami, O.;
Uchida, M.; Asakawa, Y. Chem. Pharm. Bull. 2006, 54, 1017-1021.
(13) Okabe, H.; Miyahara, Y.; Yamauchi, T.; Miyahara, K.; Kawasaki,
T. Chem. Pharm. Bull. 1980, 28, 2753-2762.
(14) Miyahara, Y.; Okabe, H.; Yamauchi, T. Chem. Pharm. Bull. 1981,
29, 1561-1566.
(15) Dutta, P. K.; Chakravarty, A. K.; Chowdhury, U. S.; Pakrashi, S. C.
Indian J. Chem., Sect. B 1981, 20, 669-671.
(16) Yasuda, M.; Iwamoto, M.; Okabe, H.; Yamauchi, T. Chem. Pharm.
Bull. 1984, 32, 2044-2047.
(17) Chang, C.-I.; Chen, C.-R.; Liao, Y.-W.; Cheng, H.-L.; Chen, Y.-C.;
Chou, C.-H. J. Nat. Prod. 2006, 69, 1168-1171.
(18) 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) Akihisa, T.; Yasukawa, K.; Tokuda, H. In Studies in Natural Products
Chemistry, Vol. 29. BioactiVe Natural Products (Part J); Atta-ur-
Rahman, Ed.; Elsevier Science B.V.: Amsterdam, 2003; pp 73-
126.
(20) Kimura, Y.; Akihisa, T.; Yuasa, N.; Ukiya, M.; Suzuki, T.; Toriyama,
M.; Motohashi, S.; Tokuda, H. J. Nat. Prod. 2005, 68, 807-809.
(21) Fujimoto, Y.; Ikekawa, N. Chem. Pharm. Bull. 1976, 24, 825-828.
(22) Matsumoto, K.; Kasai, R.; Ohtani, K.; Tanaka, O. Chem. Pharm.
Bull. 1990, 38, 2030-2032.
Acknowledgment. The authors thank Prof. K.-H. Lee (University
of North Carolina) for his comments and advice. This work was
supported, in part, by a grant “Academic Frontier” Project for Private
Universities: Matching Fund Subsidy from MEXT (Ministry of
Education, Culture, Sports, Science and Technology of Japan) 2002-
2006.
(23) Mulholland, D. A.; Sewram, V.; Osborne, R.; Pegel, K. H.; Connolly,
J. D. Phytochemistry 1997, 45, 391-395.
(24) Lago, J. H. G.; Brochini, C. B.; Roque, N. F. Phytochemistry 2002,
60, 333-338.
1
Supporting Information Available: 13C and H NMR, HMBC,
(25) The side-chain 13C NMR data (125 MHz, CDCl3) of cycloart-24-
ene-3â,23-diols were reported as follows:24 23R: δC 18.1 (C-21),
18.3 (C-27), 25.4 (C-26), 33.0 (C-20), 44.5 (C-22), 66.1 (C-23), 129.1
(C-24); 23S: δC 18.3 (C-27), 19.1 (C-21), 25.4 (C-26), 33.5 (C-20),
44.5 (C-22), 67.3 (C-23), 128.4 (C-24).
and NOESY NMR data for compounds 6-18. This information is
available free of charge via the Internet at http://pubs.acs.org.
References and Notes
(1) Raman, A.; Lau, C. Phytomedicine 1996, 2, 349-362.
(2) Grover, J. K.; Yadav, S. P. J. Ethnopharmacol. 2004, 93, 123-132.
(3) Guevara, A. P.; Lim-Sylianco, C. Y.; Dayrit, F. M.; Finch, P.
Phytochemistry 1989, 28, 1721-1724.
(26) Takaishi, Y.; Ujita, K.; Tokuda, H.; Nishino, H.; Iwashima, A.; Fujita,
T. Cancer Lett. 1992, 65, 19-26.
(27) Tokuda, H.; Okuda, Y.; Mukainaka, T.; Okuda, M.; Ichiishi, E.;
Nishino, H.; Takasaki, M.; Konoshima, T. In Portland Press
Proceedings 16, Biology of Nitrogen Oxide, Part 7; Portland Press:
London, 2000; p 201.
(4) Rathi, S. S.; Grover, J. K.; Vats, V. Phytother. Res. 2002, 16, 236-
243.
(5) Okabe, H.; Miyahara, Y.; Yamauchi, T. Chem. Pharm. Bull. 1982,
30, 3977-3986.
(28) Moncada, S.; A. Higgs, A. N. Engl. J. Med. 1993, 329, 2002-2012.
(29) Nathan, C. FASEB J. 1992, 6, 3051-3064.
(6) Okabe, H.; Miyahara, Y.; Yamauchi, T. Chem. Pharm. Bull. 1982,
30, 4334-4340.
(30) Akihisa, T.; Tokuda, H.; Hasegawa, D.; Ukiya, M.; Kimura, Y.; Enjo,
F.; Suzuki, T.; Nishino, H. J. Nat. Prod. 2006, 69, 38-42.
(7) Fatope, M. O.; Takeda, Y.; Yamashita, H.; Okabe, H.; Yamauchi,
T. J. Nat. Prod. 1990, 53, 1491-1497.
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