Brazilian natural medicines. IV. New noroleanane-type triterpene and ecdysterone-type sterol glycosides and melanogenesis inhibitors from the roots of Pfaffia glomerata

Article abstract of DOI:10.1248/cpb.58.690

The ethyl acetate and 1-butanol soluble fractions of the roots of Pfaffia glomerata were found to show inhibitory effects on melanogenesis in theophylline-stimulated B16 melanoma 4A5 cells. From the ethyl acetate and 1-butanol soluble fractions, we isolated a new noroleanane-type triterpene, pfaffianol A, its glycosides, pfaf-fiaglycosides A and B, and ecdysterone-type sterol glycosides, pfaffiaglycosides C, D, and E, together with eight known constituents. The structures of new constituents were determined on the basis of physicochemical and chemical evidence. Among them, pfaffianol A (IC ₅₀=44 μM) and pfaffoside C (IC₅₀=92 μM) substantially inhibited melanogenesis without cytotoxic effects. The inhibitory effects were stronger than that of reference compound, arbutin (IC ₅₀=174 μM).

Full text of DOI:10.1248/cpb.58.690

690
Regular Article
Chem. Pharm. Bull. 58(5) 690—695 (2010)
Vol. 58, No. 5
Brazilian Natural Medicines. IV.1) New Noroleanane-Type Triterpene
and Ecdysterone-Type Sterol Glycosides and Melanogenesis Inhibitors
from the Roots of Pfaffia glomerata
Seikou NAKAMURA,a Gang CHEN,a,b Souichi NAKASHIMA,a Hisashi MATSUDA,a Yuehu PEI,b and
Masayuki YOSHIKAWA*,a
a Kyoto Pharmaceutical University; Misasagi, Yamashina-ku, Kyoto 607–8412, Japan: and b School of Traditional Chinese
Materia Medica, Shenyang Pharmaceutical University; Shenyang 110016, China.
Received January 9, 2010; accepted February 24, 2010; published online March 4, 2010
The ethyl acetate and 1-butanol soluble fractions of the roots of Pfaffia glomerata were found to show
inhibitory effects on melanogenesis in theophylline-stimulated B16 melanoma 4A5 cells. From the ethyl acetate
and 1-butanol soluble fractions, we isolated a new noroleanane-type triterpene, pfaffianol A, its glycosides, pfaf-
fiaglycosides A and B, and ecdysterone-type sterol glycosides, pfaffiaglycosides C, D, and E, together with eight
known constituents. The structures of new constituents were determined on the basis of physicochemical and
chemical evidence. Among them, pfaffianol A (IC50ꢀ44 mM) and pfaffoside C (IC50ꢀ92 mM) substantially inhib-
ited melanogenesis without cytotoxic effects. The inhibitory effects were stronger than that of reference com-
pound, arbutin (IC50ꢀ174 mM).
Key words Pfaffia glomerata; Brazil ginseng; pfaffiaglycoside; noroleanane-type triterpene; ecdysterone-type sterol glycoside;
melanogenesis inhibitor
The Amaranthaceae plant, Pfaffia (P. ) glomerata, which is acetate (EtOAc) and 1-butanol (1-BuOH) soluble fractions of
so-called as Brazil ginseng in Japan, is widely cultivated in the roots of P. glomerata were found to show inhibitory
South American countries such as Brazil, Ecuador, and effects on melanogenesis in theophylline-stimulated B16
Panama. The roots of this plant are used as a Brazilian folk melanoma 4A5 cells. From the EtOAc and 1-BuOH soluble
medicine for a tonic and treatment of diabetes. The extract fractions, we have isolated a new noroleanane-type triter-
from the roots of P. glomerata has been reported to possess pene, pfaffianol A (1), two new noroleanane-type triterpene
gastroprotective,2) anti-oxidant,3) and anti-inflammatory glycosides, pfaffiaglycosides A (2) and B (3), and three
effects.4) On the other hand, ecdysterone has been character- ecdysterone-type sterol glycosides, pfaffiaglycosides C (4), D
ized as the principal constituent from the roots.5,6) However, (5), and E (6), with eight known constituents. Furthermore,
chemical and pharmacological studies on the roots of P. we examined the inhibitory effects of the fractions and prin-
glomerata have not been investigated sufficiently yet. During cipal constituents on melanogenesis in theophylline-stimu-
the course of characterization studies on the bioactive con- lated B16 melanoma 4A5 cells. In this paper, we describe the
stituents of Brazilian natural medicines,1,7—10) the ethyl isolation and structure elucidation of the new constituents
Chart 1
© 2010 Pharmaceutical Society of Japan
∗ To whom correspondence should be addressed. e-mail: myoshika@mb.kyoto-phu.ac.jp

May 2010
691
(1—6) and the effects on melanogenesis of the principal con- (2—6) Pfaffianol A (1) was obtained as a white powder
stituents from the roots of P. glomerata.
with a positive optical rotation ([a]D26 ꢂ33.5° in MeOH). The
The roots of Brazilian P. glomerata were extracted with IR spectrum of 1 showed absorption bands at 3420, 1710 and
methanol (MeOH). The MeOH extract (20.3% from the 1655 cmꢃ1 ascribable to hydroxy, carboxy and olefin func-
roots) was partitioned into an EtOAc–H2O (1 : 1, v/v) mix- tions. In the electron ionization-mass spectra (EI-MS) of 1, a
ture to furnish an EtOAc-soluble fraction (2.0%) and an molecular ion peak was observed at m/z 456 (Mꢂ), and high-
aqueous phase. The aqueous phase was further extracted resolution EI-MS (HR-EI-MS) analysis revealed the molecu-
1
with 1-BuOH to give 1-BuOH- (4.1%) and H2O- (14.1%) lar formula of 1 to be C29H44O4. The H-NMR (pyridine-d5)
soluble fractions. The EtOAc-soluble fraction [inhibition and 13C-NMR (Table 1) spectra of 1, which were assigned by
(%): 70.4ꢀ1.5 (pꢁ0.01) at 100 mg/ml] and 1-BuOH-soluble various NMR experiments,18) showed signals assignable to
fraction [inhibition (%): 25.0ꢀ3.3 (pꢁ0.01) at 100 mg/ml] five methyls [d 0.87, 1.00, 1.01, 1.24, 1.36 (3H each, all s,
were found to show the inhibitory effects on melanogenesis H3-25, 24, 26, 23, 27)], two methines bearing an oxygen
in theophylline-stimulated B16 melanoma 4A5 cells. The function [d 3.45 (1H, dd, Jꢄ4.0, 11.5 Hz, H-3), 4.66 (1H, dd,
EtOAc-soluble fraction was subjected to normal-phase and Jꢄ4.6, 11.5 Hz, H-16)], three olefinic protons [d 4.77, 4.81
reversed-phase silica gel column chromatography and finally (1H each, both s like, H2-29), 5.52 (1H, dd like, H-12)], and
HPLC to afford a pfaffiaglycoside A (2, 0.00036%) together a carboxy carbon (dC 180.0, C-28). The proton and carbon
with a known compound, 22-oxo-20-hydroxyecdysone (12, signals of 1 in the 1H- and 13C-NMR spectra resembled those
0.0008%).11) The 1-BuOH-soluble fraction was subjected to of akebonoic acid,19) except for the signals around the 16-po-
normal-phase and reversed-phase silica gel column chro- sition. The double quantum filter correlation spectroscopy
matography and finally HPLC to afford pfaffianol A (1, (DQF COSY) experiment on 1 indicated the presence of par-
0.0014%), pfaffiaglycosides B (3, 0.00027%), C (4, 0.00009 tial structures written in bold lines (Fig. 1), and in the het-
%), D (5, 0.00005%), and E (6, 0.00005%), together with eronuclear multiple bond connectivity spectroscopy (HMBC)
seven known compounds, akebonoic acid (7, 0.00008%),12) experiment, long-range correlations were observed between
boussingoside A2 (8, 0.0011%),13) ecdysterone (9, 0.46%),14) the following protons and carbons: H-12 and C-9, 14, 18; H-
taxisterone (10, 0.0023%),15) pterosterone (11, 0.0018%),14) 16 and C-28; H-18 and C-16, 19, 20, 22, 28; H-23 and C-3,
2b,3b,14a,17b-tetrahydroxy-5b-androst-7-en-6-one (13, 4, 5, 24; H-24 and C-3, 4, 5, 23; H-25 and C-1, 5, 9, 10; H-
0.00053%),16) and pfaffoside C (14, 0.0037%).17)
26 and C-7, 8, 9, 14; H-27 and C-8, 15; H-29 and C-20.
Structures of Pfaffianol A (1), Pfaffiaglycosides A—E These results supported that the structure of 1 was noroleana-
Fig. 1. Selected DQF, HMBC and NOE Correlations
Table 1. 13C-NMR (125 MHz) Data for 1—6a)
Carbon
1
2
3
4
5
6
Carbon
1
2
3
4
5
6
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
38.2
28.0
78.0
39.1
55.7
18.7
33.1
39.8
47.2
37.2
23.7
123.5
144.6
44.3
38.9
65.0
50.7
49.6
38.1
28.0
78.0
39.0
55.8
18.7
33.2
39.9
47.2
37.2
23.8
122.9
142.4
44.5
39.4
65.2
51.6
49.7
38.1
26.5
89.1
39.4
55.7
18.3
33.0
39.7
47.0
36.9
23.7
123.5
142.7
44.3
38.5
65.0
50.7
49.6
37.7
68.1
68.0
32.4
51.2
203.7
121.5
166.4
34.2
38.7
20.9
31.8
47.4
84.5
31.5
21.9
53.9
17.9
38.0
68.1
68.0
32.8
51.4
203.7
121.6
166.1
34.4
38.6
21.5
31.8
48.1
84.1
31.9
21.5
49.9
17.8
42.5
69.8
73.4
19
20
21
22
23
24
25
26
27
28
29
41.5
148.3
29.9
32.9
28.7
16.5
15.5
17.2
26.9
41.8
148.2
29.9
31.8
28.7
16.5
15.7
17.4
26.8
41.5
148.3
29.9
32.9
28.1
16.8
15.5
17.2
26.9
24.5
74.3
27.0
45.2
18.8
42.8
77.4
27.1b)
27.2b)
24.7
76.9
21.5
76.0
31.9
47.6
80.9
22.0b)
26.7b)
47.6
14.2
27.4
76.1
20.7
77.6
26.4
39.7
77.2
27.5b)
27.7b)
28.1
132.8
144.4
180.8
127.4
163.6
41.6
24.4
36.8
47.2
142.2
124.2
32.3
55.7
18.5
180.0
107.6
174.8
107.4
180.0
107.6
1ꢅ
2ꢅ
3ꢅ
4ꢅ
5ꢅ
6ꢅ
96.1
73.9
78.3
71.1
79.3
62.3
107.1
75.4
77.8
73.3
78.0
172.4
98.7
75.4
78.7
71.8
78.2
62.9
98.7
75.4
78.8
72.0
78.6
63.4
98.7
75.4
78.8
72.0
78.6
63.4
a) Measured in pyridine-d5; b) reversible.

692
Vol. 58, No. 5
dienoic acid with two hydroxy groups at the 3- and 16-posi- glycoside linkage was determined by a HMBC experiment,
tions. Next, the stereostructure of 1 was characterized by nu- which showed long-range correlations between H-1ꢅ and C-
clear Overhauser enhancement spectroscopy (NOESY) ex- 3. Furthermore, the DQF COSY and HMBC experiments on
periment, which showed NOE correlations between the fol- 3 showed correlations as shown in Fig. 1. This evidence led
lowing proton pairs: H-3a and H3-23; H-16a and H3-27; H- us to formulate the structure of pfaffiaglycoside B (3) as
18 and H3-26; H-19a and H3-27; H3-24 and H3-25; H3-25 shown.
and H3-26 (Fig. 1). On the basis of this evidence, the struc-
ture of pfaffianol A (1) was characterized as shown.
Pfaffiaglycoside C (4) was obtained as a white powder
with negative optical rotation ([a]D23 ꢃ16.1° in MeOH). The
Pfaffiaglycoside A (2) was obtained as a white powder IR spectrum of 4 showed absorption bands at 3430, 1670,
with a positive optical rotation ([a]D23 ꢂ27.9° in MeOH). The and 1074 cmꢃ1 assignable to hydroxy, unsaturated carbonyl,
IR spectrum of 2 showed strong absorption bands at 3430 and ether functions. In the positive-ion FAB-MS of 4, a qua-
and 1072 cmꢃ1 suggestive of the glycosidic structure together simolecular ion peak was observed at m/z 649 (MꢂNa)ꢂ.
with absorption bands at 1718 and 1655 cmꢃ1 due to carboxy The HR-FAB-MS analysis revealed the molecular formula
and olefin functions. In the positive-ion fast atom bombard- of 4 to be C33H54O11. The acid hydrolysis of 4 liberated D-
1
ment (FAB)-MS of 2, a quasimolecular ion peak was ob- glucose, which was identified by HPLC analysis.20) The H-
served at m/z 641 (MꢂNa)ꢂ. The HR-FAB-MS analysis re- NMR (pyridine-d5) and 13C-NMR (Table 1) spectra18) of 4
vealed the molecular formula of 2 to be C35H54O9. Acid hy- showed signals due to five methyls [d 1.05, 1.12, 1.34, 1.37,
drolysis of 2 liberated the aglycone, pfaffianol A (1), together 1.50 (3H each, all s, H3-19, 18, 26, 27, 21)], two methines
with D-glucose, which was identified by HPLC analysis using bearing an oxygen function [d 4.15 (1H, m, H-2), 4.21 (1H,
an optical rotation detector.20) The 1H-NMR (pyridine-d5) and m, H-3)], an olefinic proton [d 6.19 (1H, d, Jꢄ2.0 Hz, H-7)],
13C-NMR (Table 1) spectra18) of 2 showed signals assignable a carbonyl carbon (dC 203.7, C-6), and a b-D-glucopyranosyl
to an aglycon moiety [d 0.91, 0.98, 1.06, 1.19, 1.28 (3H moiety [d 4.96 (1H, d, Jꢄ8.0 Hz, H-1ꢅ)]. The proton and car-
1
each, all s, H3-25, 24, 26, 23, 27), 3.40 (1H, dd, Jꢄ4.1, bon signals of the aglycon part in the H- and 13C-NMR
11.5 Hz, H-3), 4.60 (1H, dd, Jꢄ4.0, 11.5 Hz, H-16), 4.73, spectra of 4 were superimposable on those of taxisterone
4.77 (1H each, both s like, H2-29), 5.43 (1H, dd like, H-12)], (10),15) except for the signals due to the terminal side chain
together with a b-D-glucopyranosyl moiety [d 6.10 (1H, d, part (C-25—C-27), while the proton and carbon signals due
Jꢄ8.0 Hz, H-1ꢅ)]. The position of the glycoside linkage was to the glycoside moiety including terminal side chain part
determined by a HMBC experiment, which showed long- (C-25—C-27) were very similar to those of ecdysterone
range correlations between H-1ꢅ and C-28. Furthermore, the 25-O-b-D-glucopyranoside.24) As shown in Fig. 2, the DQF
DQF COSY and HMBC experiments on 2 showed correla- COSY experiment on 4 indicated the presence of partial
tions as shown in Fig. 1. This evidence led us to formulate structures written in bold lines, and in the HMBC experi-
the structure of pfaffiaglycoside A (2) as shown.
ment, long-range correlations were observed between the fol-
Pfaffiaglycoside B (3), obtained as a white powder with a lowing protons and carbons: H-2 and C-4; H-3 and C-1; H-5
positive optical rotation ([a]D23 ꢂ35.3° in MeOH), showed and C-3, 4, 6, 10; H-7 and C-5, 6, 8, 9; H-17 and C-13, 15,
strong absorption bands at 3430 and 1070 cmꢃ1 suggestive of 16, 20; H-18 and C-12, 13, 14, 17; H-19 and C-1, 5, 9, 10;
the glycosidic structure together with absorption bands at H-21 and C-20, 22; H-26, 27 and C-24, 25; H-1ꢅ and C-25.
1710 and 1655 cmꢃ1 due to carboxy and olefin functions On the basis of this evidence, the structure of pfaffiaglyco-
in the IR spectrum. The molecular formula C35H52O10 was side C (4) was characterized as shown.
determined from the positive-ion FAB-MS at m/z 655
Pfaffiaglycoside D (5), obtained as a white powder with
(MꢂNa)ꢂ and by HR-FAB-MS measurement. Acid hydroly- positive optical rotation ([a]D23 ꢂ23.8° in MeOH), showed
sis of 3 liberated the aglycone, pfaffianol A (1), together with absorption bands at 3430, 1670, and 1072 cmꢃ1 assignable to
D-glucuronic acid, which was identified by GLC analysis of hydroxy, unsaturated carbonyl, and ether functions in the IR
1
the trimethylsilyl thiazolidine derivative.21—23) The H-NMR spectrum. The positive-ion FAB-MS of 5 exhibited a quasi-
(pyridine-d5) and 13C-NMR (Table 1) spectra18) of 3 showed molecular ion peak at m/z 693 (MꢂNa)ꢂ. The molecular for-
signals assignable to an aglycon moiety [d 0.87, 1.00, 1.01, mula C35H58O12 of 5 was determined from the quasimolec-
1.24, 1.36 (3H each, all s, H3-25, 24, 26, 23, 27), 3.45 (1H, ular ion peak and by HR-FAB-MS measurement. The acid
dd, Jꢄ4.0, 11.5 Hz, H-3), 4.66 (1H, dd, Jꢄ4.6, 11.5 Hz, H- hydrolysis of 5 liberated D-glucose, which was identified by
16), 4.78, 4.82 (1H each, both s like, H2-29), 5.52 (1H, dd HPLC analysis.20) The H-NMR (pyridine-d5) and 13C-NMR
1
like, H-12)], together with a b-D-glucopyranosiduronic acid (Table 1) spectra18) of 5 showed signals assignable to an agly-
moiety [d 4.98 (1H, d, Jꢄ8.0 Hz, H-1ꢅ)]. The position of the con part [d 1.03 (3H, t, Jꢄ7.4 Hz, H3-29), 1.05, 1.21, 1.31,
Fig. 2. Selected DQF and HMBC Correlations

May 2010
693
Table 2. Inhibitory Effects of Constituents on Melanogenesis in B16 Melanoma 4A5 Cells
Inhibition (%) for melanogenesis
Compound No.
Conc. (mM)
0
1
3
10
30
100
Pfaffianol A (1)
Pfaffiaglycoside B (3)
Boussingoside A2 (8)
Ecdysterone (9)
Taxisterone (10)
Pterosterone (11)
12
0.0ꢀ3.1
0.0ꢀ2.0
0.0ꢀ3.4
0.0ꢀ2.9
0.0ꢀ2.2
0.0ꢀ3.3
0.0ꢀ2.9
0.0ꢀ6.4
0.0ꢀ1.4
ꢃ1.0ꢀ3.4
ꢃ4.0ꢀ2.3
ꢃ3.3ꢀ4.6
ꢃ6.6ꢀ2.2
ꢃ2.5ꢀ2.3
2.2ꢀ4.9
7.9ꢀ5.0
ꢃ1.2ꢀ1.6
4.2ꢀ2.9
ꢃ6.6ꢀ3.1
ꢃ1.1ꢀ6.6
ꢃ3.5ꢀ2.7
0.6ꢀ3.2
34.7ꢀ3.7**
ꢃ17.0ꢀ4.2
2.5ꢀ8.6
ꢃ27.2ꢀ18.3
1.9ꢀ3.5
ꢃ9.1ꢀ3.2
7.4ꢀ2.2
1.3ꢀ5.8
80.0ꢀ2.5**
ꢃ12.6ꢀ4.3
22.1ꢀ2.4*
ꢃ6.1ꢀ7.6
ꢃ10.1ꢀ6.4
ꢃ3.3ꢀ3.0
ꢃ0.5ꢀ2.9
2.3ꢀ4.2
0.9ꢀ3.4
ꢃ4.4ꢀ1.8
0.6ꢀ2.5
1.8ꢀ3.0
4.5ꢀ2.3
ꢃ4.2ꢀ3.1
ꢃ8.9ꢀ5.1
ꢃ5.4ꢀ3.5
ꢃ2.6ꢀ4.0
2.2ꢀ2.1
13
ꢃ1.3ꢀ5.2
10.8ꢀ1.9**
Pfaffoside C (14)
28.3ꢀ2.2**
51.4ꢀ1.2**
Inhibition (%) for melanogenesis
30
Conc. (mM)
0
10
10.6ꢀ0.6**
100
300
1000
Arbutin
0.0ꢀ1.4
20.4ꢀ0.5**
38.1ꢀ0.9**
61.5ꢀ0.6**
83.7ꢀ0.5**
Each value represents the meanꢀS.E.M. (nꢄ4). Significantly different from the control ∗ pꢁ0.05, ∗∗ pꢁ0.01. The cell viabilities of compounds, 1, 3, 8—14, and arbutin at
100 mM are more than 86%.
1.47, 1.59 (3H each, all s, H3-19, 18, 26, 27, 21), 3.98 (1H, C-17, 20, 22; H-22 and C-23, 24; H-26, 27 and C-24, 25; H-
m, H-22), 4.15 (1H, m, H-2), 4.20 (1H, m, H-3), 6.29 (1H, d, 1ꢅ and C-25. On the basis of this evidence, pfaffiaglycoside E
Jꢄ2.0 Hz, H-7)] and a b-D-glucopyranosyl moiety [d 5.09 (6) was characterized as shown.
(1H, d, Jꢄ8.0 Hz, H-1ꢅ). The proton and carbon signals of
Inhibitory Effects of Constituents on Melanogenesis in
1
the aglycon part in the H-NMR spectra of 5 were similar to B16 Melanoma 4A5 Cells Melanin production is princi-
those of makisterone C,25) except for the signals due to the pally responsible for skin color, and melanin pigmentation is
terminal side chain part (C-25—C-27), while the proton and a major defense mechanism against ultraviolet rays from the
carbon signals due to the glycoside moiety were superimpos- sun. On the other hand, the excess melanin formation after
able on those of ecdysterone 25-O-b-D-glucopyranoside.24) In sunlight exposure for long periods of time causes some
the HMBC experiment, long-range correlations were ob- dermatological disorders such as melasma, freckles, post-
served between the following protons and carbons: H-2 and inflammatory melanoderma, and solar lentigines. To develop
C-4; H-3 and C-1; H-5 and C-3, 4, 6, 10; H-7 and C-5, 6, 8, melanogenesis inhibitors, we have reported the inhibitory
9; H-17 and C-13, 15, 16, 20; H-18 and C-12, 13, 14, 17; H- effects of several diaryheptanoids and flavonoids in theo-
19 and C-1, 5, 9, 10; H-21 and C-17, 20, 22; H-26, 27 and C- phylline-stimulated B16 melanoma 4A5 cells recently.26,27)
24, 25; H-29 and C-24; H-1ꢅ and C-25. On the basis of this As a continuing of these studies, the inhibitory effects of the
evidence, the structure of pfaffiaglycoside D (5) was charac- constituents from the roots of P. glomerata on melanogenesis
terized as shown.
were examined. Among the principal constituents, pfaffianol
Pfaffiaglycoside E (6), obtained as a white powder with A (1, IC50ꢄ44 mM) and pfaffoside C (14, IC50ꢄ92 mM) sub-
positive optical rotation ([a]D23 ꢂ24.6° in MeOH), showed stantially inhibited melanogenesis without cytotoxic effects.
absorption bands due to hydroxy, unsaturated carbonyl, and The effects of 1 and 14 were stronger than that of reference
ether functions in the IR spectrum. The molecular formula compound, arbutin (IC50ꢄ174 mM). On the other hand, ecdys-
C33H50O12 of 6 was determined from the positive-ion FAB- terone (9) and its derivatives 10—13 did not show such
MS [m/z 661 (MꢂNa)ꢂ] and by HR-FAB-MS measurement. effects (Table 2).
The acid hydrolysis of 6 liberated D-glucose, which was iden-
1
tified by HPLC analysis.20) The H-NMR (pyridine-d5) and
Experimental
13C-NMR (Table 1) spectra18) of 6 showed signals assignable
to an aglycon part [d 1.42, 1.43, 1.47, 1.58, 1.74 (3H each,
all s, H3-18, 26, 27, 21, 19), 3.84 (1H, m, H-22), 4.08 (1H,
General Experimental Procedures The following instruments were
used to obtain physical data: specific rotations, Horiba SEPA-300 digital
polarimeter (lꢄ5 cm); IR spectra, Shimadzu FTIR-8100 spectrometer; EI-
MS and HR-EI-MS, JEOL JMS-GCMATE mass spectrometer; FAB-MS and
HR-FAB-MS, JEOL JMS-SX 102A mass spectrometer; 1H-NMR spectra,
JEOL EX-270 (270 MHz), JNM-LA500 (500 MHz), and JEOL ECA-600K
(600 MHz) spectrometers; 13C-NMR spectra, JEOL EX-270 (68 MHz) JNM-
LA500 (125 MHz), and JEOL ECA-600K (150 MHz) spectrometers with
tetramethylsilane as an internal standard; HPLC detector, Shimadzu RID-6A
refractive index detector; and HPLC column, COSMOSIL 5C18-MS-II
(250ꢆ4.6 mm i.d.) and (250ꢆ20 mm i.d.) columns were used for analytical
and preparative purposes, respectively.
The following experimental materials were used for chromatography:
normal-phase silica gel column chromatography, Silica gel BW-200 (Fuji
Silysia Chemical, Ltd., 150—350 mesh); reversed-phase silica gel column
chromatography, Chromatorex ODS DM1020T (Fuji Silysia Chemical, Ltd.,
100—200 mesh); TLC, precoated TLC plates with Silica gel 60F254 (Merck,
m, H-3), 4.46 (1H, m, H-2), 7.47 (1H, dd like, H-15)] and a
b-D-glucopyranosyl moiety [d 5.06 (1H, d, Jꢄ8.0 Hz, H-1ꢅ)].
The proton and carbon signals of the aglycon part in the
1H- and 13C-NMR spectra of 6 were similar to those of
podecdysone B 25-O-b-D-glucopyranoside,24) except for the
signals around the B ring part (C-5—C-10). As shown in Fig.
2, the DQF COSY experiment on 6 indicated the presence of
partial structures written in bold lines, and in the HMBC ex-
periment, long-range correlations were observed between the
following protons and carbons: H-2 and C-1, 3, 4; H-3 and
C-2, 4, 5; H-4 and C-3, 5, 6, 10; H-15 and C-8, 13, 16, 17;
H-18 and C-12, 13, 14, 17; H-19 and C-1, 5, 9, 10; H-21 and 0.25 mm) (ordinary phase) and Silica gel RP-18 F254S (Merck, 0.25 mm)

694
Vol. 58, No. 5
(reversed phase); reversed-phase HPTLC, precoated TLC plates with Silica 600 MHz) d: 0.85 (1H, m, H-5), 0.91, 0.98, 1.06, 1.19, 1.28 (3H each, all s,
gel RP-18 WF254S (Merck, 0.25 mm); and detection was achieved by spray- H3-25, 24, 26, 23, 27), 2.25 (1H, dd like, H-19b), 2.69 (1H, dd, Jꢄ12.5,
ing with 1% Ce(SO4)2–10% aqueous H2SO4 followed by heating.
Plant Material The roots of Pfaffia glomerata, which were cultivated in 11.5 Hz, H-3), 4.60 (1H, dd, Jꢄ4.0, 11.5 Hz, H-16), 4.73, 4.77 (1H each,
Brazil, were purchased from Tamura Pharmaceutical Co., Ltd., in 2007. A both s like, H2-29), 5.43 (1H, dd like, H-12), 6.10 (1H, d, Jꢄ8.0 Hz, H-1ꢅ);
12.5 Hz, H-19a), 3.16 (1H, dd, Jꢄ4.1, 12.5 Hz, H-18), 3.40 (1H, dd, Jꢄ4.1,
voucher of the plant is on file in our laboratory (2007. Brazil-01).
13C-NMR data see Table 1; positive-ion FAB-MS m/z: 641 [MꢂNa]ꢂ; HR-
Extraction and Isolation The roots of P. glomerata (4.75 kg) were cut FAB-MS m/z: 641.3672 (Calcd for C35H54O9Na [MꢂNa]ꢂ, 641.3666).
and extracted four times with MeOH under reflux. Evaporation of the sol-
Pfaffiaglycoside B (3): A white powder; [a]D23 ꢂ35.3° (cꢄ0.30, MeOH);
vent under reduced pressure provided the MeOH extract (963 g, 20.3%), IR (KBr) nmax 3430, 2945, 1710, 1655, 1070 cmꢃ1; H-NMR (pyridine-d5,
1
which was partitioned into an EtOAc–H2O (1 : 1, v/v) mixture to furnish an
600 MHz) d: 0.85 (1H, m, H-5), 0.87, 1.00, 1.01, 1.24, 1.36 (3H each, all s,
EtOAc-soluble fraction (97 g, 2.0%) and aqueous layer. The aqueous layer H3-25, 24, 26, 23, 27), 2.25 (1H, dd like, H-19b), 2.69 (1H, dd, Jꢄ12.5,
was extracted with 1-BuOH to give 1-BuOH- (195 g, 4.1%) and H2O- 12.5 Hz, H-19a), 3.35 (1H, dd, Jꢄ4.1, 12.5 Hz, H-18), 3.45 (1H, dd, Jꢄ4.0,
(671 g, 14.1%) soluble fractions. The EtOAc-soluble fraction (97 g) was sub- 11.5 Hz, H-3), 4.66 (1H, dd, Jꢄ4.6, 11.5 Hz, H-16), 4.78, 4.82 (1H each,
jected to normal-phase silica gel column chromatography [2.0 kg, CHCl3– both s like, H2-29), 5.52 (1H, dd like, H-12), 4.98 (1H, d, Jꢄ8.0 Hz, H-1ꢅ);
MeOH (50 : 1→20 : 1, v/v)→CHCl3–MeOH–H2O (10 : 3 : 1→7 : 3 : 1→6 : 4 : 13C-NMR data see Table 1; positive-ion FAB-MS m/z: 655 [MꢂNa]ꢂ; HR-
1, v/v)→MeOH] to give 4 fractions [Fr. 1 (3.0 g), Fr. 2 (25.5 g,), Fr. 3 FAB-MS m/z: 655.3463 (Calcd for C35H52O10Na [MꢂNa]ꢂ, 655.3458).
(30.5 g), Fr. 4 (23.0 g)]. Fraction 3 (30.5 g) was separated by reversed-phase
silica gel column chromatography [750 g, MeOH–H2O (50 : 50→60 : 40→
Pfaffiaglycoside C (4): A white powder; [a]D23 ꢃ16.1° (cꢄ0.13, MeOH);
IR (KBr) nmax 3430, 2926, 1670, 1074 cmꢃ1 1H-NMR (pyridine-d5, 600
;
70 : 30→80 : 20, v/v)→MeOH] to give 11 fractions [Fr. 3-1, Fr. 3-2, Fr. 3-3, MHz) d: 1.05, 1.12, 1.34, 1.37, 1.50 (3H each, all s, H3-19, 18, 26, 27, 21),
Fr. 3-4 (101 mg), Fr. 3-5, Fr. 3-6, Fr. 3-7, Fr. 3-8 (3.21 g), Fr. 3-9, Fr. 3-10, 2.55 (1H, ddd, Jꢄ4.1, 13.1, 13.1 Hz, H-12), 2.81 (1H, dd, Jꢄ8.9, 8.9 Hz, H-
Fr. 3-11]. Fraction 3-4 (101 mg) was purified by HPLC [MeOH–H2O 17), 2.98 (1H, dd, Jꢄ4.0, 14.0 Hz, H-5), 3.56 (1H, m, H-9), 4.15 (1H, m, H-
(80 : 20, v/v)] to give 22-oxo-20-hydroxyecdysone (12, 41 mg). Fraction 3-8 2), 4.21 (1H, m, H-3), 4.96 (1H, d, Jꢄ8.0 Hz, H-1ꢅ), 6.19 (1H, d, Jꢄ2.0 Hz,
(3.21 g) was purified by reversed-phase silica gel column chromatography H-7); 13C-NMR data see Table 1; positive-ion FAB-MS m/z: 649 [MꢂNa]ꢂ;
[60 g, MeOH–H2O (60 : 40→70 : 30→80 : 20, v/v)] and HPLC [MeOH–H2O HR-FAB-MS m/z: 649.3571 (Calcd for C33H54O11Na [MꢂNa]ꢂ, 649.3564).
(80 : 20, v/v)] to give pfaffiaglycoside A (2, 15 mg). A part of the 1-BuOH-
Pfaffiaglycoside D (5): A white powder; [a]D23 ꢂ23.8° (cꢄ0.10, MeOH);
soluble fraction (179 g) was subjected to normal-phase silica gel column IR (KBr) nmax 3430, 2934, 1670, 1072 cmꢃ1
;
1H-NMR (pyridine-d5, 600
chromatography [3 kg, CHCl3 : MeOH : H2O (50 : 10 : 1→40 : 10 : 1→30 : MHz) d: 1.03 (3H, t, Jꢄ7.4 Hz, H3-29), 1.05, 1.21, 1.31, 1.47, 1.59 (3H
10 : 1→7 : 3 : 1→6 : 4 : 1)→MeOH] to give 15 fractions [Fr. 1, Fr. 2, Fr. 3, Fr. each, all s, H3-19, 18, 26, 27, 21), 2.55 (1H, m, H-12), 2.98 (1H, dd, Jꢄ9.0,
4 (3.13 g), Fr. 5, Fr. 6, Fr. 7 (4.00 g), Fr. 8, Fr-9, Fr. 10, Fr. 11, Fr. 12, Fr. 13, 9.0 Hz, H-17), 3.04 (1H, dd, Jꢄ4.0, 12.4 Hz, H-5), 3.60 (1H, m, H-9), 3.98
Fr. 14 (27.86 g), Fr. 15 (100.67 g)]. Fraction 4 (3.13 g) was separated by re- (1H, m, H-22), 4.15 (1H, m, H-2), 4.20 (1H, m, H-3), 5.09 (1H, d, Jꢄ8.0
versed-phase silica gel column chromatography [60 g, MeOH : H2O (50 :
Hz, H-1ꢅ), 6.29 (1H, d, Jꢄ2.0 Hz, H-7); 13C-NMR data see Table 1; positive-
50→60 : 40→70 : 30→80 : 20→MeOH) to give 4 fractions [Fr. 4-1, Fr. 4-2 ion FAB-MS m/z: 693 [MꢂNa]ꢂ; HR-FAB-MS m/z: 693.3829 (Calcd for
(205 mg), Fr. 4-3, Fr. 4-4]. Fraction 4-2 (205 mg) was purified by HPLC C35H58O12Na [MꢂNa]ꢂ, 693.3826).
[MeOH–H2O (70 : 30, v/v)] to give akebonoic acid (7, 22 mg). Fraction 7
(4.00 g) was separated by reversed-phase silica gel column chromatography IR (KBr) nmax 3430, 2926, 1675, 1075 cmꢃ1
Pfaffiaglycoside E (6): A white powder; [a]D23 ꢂ24.6° (cꢄ0.10, MeOH);
;
1H-NMR (pyridine-d5, 600
[80 g, MeOH : H2O (50 : 50→60 : 40→70 : 30→80 : 20)→MeOH] to give 14 MHz) d: 1.42, 1.43, 1.47, 1.58, 1.74 (3H each, all s, H3-18, 26, 27, 21, 19),
fractions [Fr. 7-1, Fr. 7-2, Fr. 7-3, Fr. 7-4, Fr. 7-5, Fr. 7-6, Fr. 7-7, Fr. 7-8, Fr. 2.25 (1H, dd, Jꢄ9.0, 9.0 Hz, H-17), 3.11 (1H, dd, Jꢄ11.0, 12.0 Hz, H-4a),
7-9, Fr. 7-10, Fr. 7-11, Fr. 7-12, Fr. 7-13 (300 mg), Fr. 7-14]. Fraction 7-13 3.84 (1H, m, H-22), 4.08 (1H, m, H-3), 4.46 (1H, m, H-2), 7.47 (1H, dd like,
(300 mg) was purified by HPLC [MeOH–H2O (70 : 30, v/v)] to give pfaffi- H-15), 5.06 (1H, d, Jꢄ8.0 Hz, H-1ꢅ); 13C-NMR data see Table 1; positive-
anol A (1, 59 mg). Fraction 14 (27.86 g) was separated by reversed-phase sil- ion FAB-MS m/z: 661 [MꢂNa]ꢂ; HR-FAB-MS m/z: 661.3196 (Calcd for
ica gel column chromatography [80 g, MeOH : H2O (20 : 80→30 : 70→40 : C33H50O12Na [MꢂNa]ꢂ, 661.3200).
60→50 : 50→60 : 40→80 : 20)→MeOH] to give 13 fractions [Fr. 14-1, Fr.
14-2, Fr. 14-3, Fr. 14-4 (230 mg), Fr. 14-5 [ꢄecdysterone (9, 20.6 g)], Fr. 14-
Acid Hydrolyses of Pfaffiaglycosides A—E (2—6) A solution of 2—6
(4.0 mg each for 2 and 3, 1.0 mg each for 4—6) in 1.0 M aqueous HCl (1 ml)
6 (422 mg), Fr. 14-7, Fr. 14-8 (325 mg), Fr. 14-9, Fr. 14-10, Fr. 14-11, Fr. 14- and 1,4-dioxane (1 ml) was heated under reflux for 3 h, respectively. After
12, Fr. 14-13]. Fraction 14-4 (230 mg) was purified by HPLC [MeOH–H2O cooling, the reaction mixture was poured into ice-water and neutralized with
(40 : 60, v/v)] to give 2b,3b,14a,17b-tetrahydroxy-5b-androst-7-en-6-one Amberlite IRA-400 (OHꢃ form), and the resin was removed by filtration. On
(13, 23 mg). Fraction 14-6 (422 mg) was purified by HPLC [MeOH–H2O removal of the solvent from the filtrate, the residue was passed through a
(45 : 55, v/v)] to give taxisterone (10, 100 mg) and pterosterone (11, 80 mg).
Sep-Pack C18 cartridge by elution with H2O and then MeOH. The H2O elu-
Fraction 14-8 (325 mg) was purified by HPLC [MeOH–H2O (45 : 55, v/v)] to
ate obtained from 2, 4—6 was concentrated and the residue was subjected to
give pfaffiaglycoside B (3, 11.6 mg). Fraction 15 (100.67 g) was separated HPLC analysis to identify the D-glucose under the following conditions:
by reversed-phase silica gel column chromatography [80 g, MeOH : H2O HPLC column, Kaseisorb LC NH2-60-5, 4.6 mm i.d.ꢆ250 mm; detection,
(20 : 80→30 : 70→40 : 60→50 : 50→60 : 40→80 : 20, v/v)→MeOH] to give optical rotation [Shodex OR-2 (Showa Denko Co., Ltd., Tokyo, Japan)]; mo-
16 fractions [Fr. 15-1, Fr. 15-2, Fr. 15-3, Fr. 15-4, Fr. 15-5, Fr. 15-6, Fr. 15-7, bile phase, MeCN–H2O (75 : 25, v/v); flow rate, 0.80 ml/min; column tem-
Fr. 15-8, Fr. 15-9 (1.90 g), Fr. 15-10, Fr. 15-11, Fr. 15-12 (6.10 g), Fr. 15-13]. perature, room temperature. Identification of D-glucose present in the H2O
Fraction 15-9 (1.90 g) was furthermore purified by reversed-phase silica gel eluate was carried out by comparison of its retention time and optical rota-
column chromatography [50 g, MeOH–H2O (20 : 80→40 : 60→60 : 40→
80 : 20, v/v)] and HPLC [MeOH–H2O (40 : 60, v/v)] to give pfaffiaglycoside The H2O eluate obtained from 3 was concentrated and the residue was
C (4, 3.8 mg), pfaffiaglycoside D (5, 2.0 mg), and pfaffiaglycoside E (6,
treated with L-cysteine methyl ester hydrochloride (3 mg) in pyridine (0.5
tion with that of an authentic sample [tR: 9.8 min (positive optical rotation)].
2.0 mg). Fraction 15-12 (6.10 g) was furthermore purified by reversed-phase ml) at 60 °C for 2 h. After reaction, the solution was treated with N,O-
silica gel column chromatography [50 g, MeOH–H2O (20 : 80→40 : 60→60 : bis(trimethylsilyl)trifluoroacetamide (0.1 ml) at 60 °C for 2 h. The super-
40→80 : 20, v/v)] and HPLC [MeOH–H2O (45 : 55, v/v)] to give boussingo-
side A2 (8, 46 mg) and pfaffoside C (14, 160 mg). The known compounds
natant was then subjected to GLC analysis to identify the derivatives of
D-glucuronic acid under the following conditions: GLC column, Supeluco
STBTM-1, 0.25 mm i.d.ꢆ30 m; column temperature, 230 °C; detector tem-
perature, 230 °C; injector temperature, 230 °C; carrier gas, N2. Identification
of D-glucuronic acid present in the H2O eluate obtained from 3 was carried
out by comparison of its retention time with that of an authentic sample [tR:
23.9 min]. The MeOH eluate obtained from 2 and 3 was concentrated and
1
were identified by comparison of their physical data ([a]D, H-NMR, 13C-
NMR, and MS) with reported values.
Pfaffianol A (1): A white powder; [a]D26 ꢂ33.5° (cꢄ0.30, MeOH); IR
(KBr) nmax 3420, 2943, 1710, 1655 cmꢃ1; 1H-NMR (pyridine-d5, 600 MHz)
d: 0.85 (1H, dd like, H-5), 0.87, 1.00, 1.01, 1.24, 1.36 (3H each, all s, H3-25,
24, 26, 23, 27), 2.25 (1H, dd like, H-19b), 2.69 (1H, dd, Jꢄ12.5, 12.5 Hz, the residue was purified by reversed-phase silica gel column chromatogra-
H-19a), 3.35 (1H, dd, Jꢄ4.1, 12.5 Hz, H-18), 3.45 (1H, dd, Jꢄ4.0, 11.5 Hz, phy [MeOH : H2O (50 : 50→70 : 30) to give pfaffianol A (1, 1.9 mg from 2,
H-3), 4.66 (1H, dd, Jꢄ4.6, 11.5 Hz, H-16), 4.77, 4.81 (1H each, both s like,
1.8 mg from 3), was identified by comparison of their physical data ([a]D,
H2-29), 5.52 (1H, dd like, H-12); 13C-NMR data see Table 1; EI-MS m/z: 1H-NMR, 13C-NMR, and MS) with those of isolated pfaffianol A (1).
456 [M]ꢂ; HR-EI-MS m/z: 456.3241 (Calcd for C29H44O4 [M]ꢂ, 456.3239).
Pfaffiaglycoside A (2): A white powder; [a]D23 ꢂ27.9° (cꢄ0.50, MeOH);
Reagents for Bioassay Methods Dulbecco’s modified Eagle’s medium
(DMEM, 4500 mg/l glucose) was purchased from Sigma-Aldrich (St. Louis,
MO, U.S.A.); fetal bovine serum (FBS), penicillin, and streptomycin were
1
IR (KBr) nmax 3430, 2936, 1718, 1655, 1072 cmꢃ1; H-NMR (pyridine-d5,

May 2010
695
purchased from Gibco (Invitrogen, Carlsbad, CA, U.S.A.); the Cell Counting
Kit-8TM was from Dojindo Lab. (Kumamoto, Japan); and the other chemi-
cals were purchased from Wako Pure Chemical Co., Ltd. (Osaka, Japan).
Cell Culture Murine B16 melanoma 4A5 cells (RCB0557) were
obtained from Riken Cell Bank (Tsukuba, Japan), and the cells were grown
in DMEM supplemented with 10% FBS, penicillin (100 units/ml), and
streptomycin (100 mg/ml) at 37 °C in 5% CO2/air. The cells were harvested
by incubation in phosphate-buffered saline (PBS) containing 1 mM ethylene-
diaminetetraacetic acid (EDTA) and 0.25% trypsin for ca. 5 min at 37 °C
and were used for the subsequent bioassays.
3) de Souza Daniel J. F., Alves K. Z., da Silva Jacques D., da Silva e
Souza, P. V., de Carvalho M. G., Freire R. B., Ferreira D. T., Freire M.
F. I., Indian J. Pharmacol., 37, 174—178 (2005).
4) Neto A. G., Costa J. M. L. C., Belati C. C., Vinholis A. H. C., Posse-
bom L. S., Da Silva Filho A. A., Cunha W. R., Carvalho J. C. T., Bas-
tos J. K., e Silva M. L. A., J. Ethnopharmacol., 96, 87—91 (2005).
5) Zimmer A. R., Bruxel F., Bassani V. L., Gosmann G., J. Pharm. Bio-
med. Anal., 40, 450—453 (2006).
6) Shiobara Y., Inoue S., Kato K., Nishiguchi Y., Oishi Y., Nishimoto N.,
de Oliveira F., Akisue G., Akisue, M. K., Hashimoto G., Phytochem-
istry, 32, 1527—1530 (1993).
7) Nakamura S., Hongo M., Sugimoto S., Matsuda H., Yoshikawa M.,
Phytochemistry, 69, 1565—1572 (2008).
Melanogenesis The melanoma cells (2.0ꢆ104 cells/400 ml/well) were
seeded into 24-well multiplates. After 24 h of culture, a test compound and
theophylline 1 mM were added and incubated for 72 h. The cells were har-
vested by incubating with PBS containing 1 mM EDTA and 0.25% trypsin,
and then the cells were washed with PBS. The cells were treated with NaOH
1 M (120 ml/tube, 80 °C, 30 min) to yield a lysate, an aliquot (100 ml) of the
lysate was transferred to a 96-well microplate, and the optical density of
8) Yoshikawa M., Nakamura S., Ozaki K., Kumahara A., Morikawa T.,
Matsuda H., J. Nat. Prod., 70, 210—214 (2007).
9) Matsuda H., Nishida N., Yoshikawa M., Chem. Pharm. Bull., 50,
429—431 (2002).
each well was measured with a microplate reader (Model 550, Bio-Rad Lab- 10) Yoshikawa M., Shimada H., Nishida N., Li Y., Toguchida I., Matsuda
oratories) at 405 nm (reference: 655 nm). The test compound was dissolved H., Chem. Pharm. Bull., 46, 113—119 (1998).
in dimethylsulfoxide (DMSO), and the final concentration of DMSO in the 11) Rudel D., Bathori M., Gharbi J., Girault J. P., Racz I., Melis K., Szen-
medium was 0.1%. The production of melanin was corrected based on cell drei K., Lafont R., Planta Med., 58, 358—364 (1992).
viability. Inhibition (%) was calculated using the following formula, and 12) Ikuta A., Itokawa H., Phytochemistry, 25, 1625—1628 (1986).
IC50 values were determined graphically.
13) Espada A., Rodriguez J., Villaverde M. C., Riguera R., Can. J. Chem.,
68, 2039—2044 (1990).
14) Nishimoto N., Shiobara Y., Fujino M., Inoue S., Takemoto T., De
Oliveira, F., Akisue G., Akisue M. K., Hashimoto G., Phytochemistry,
26, 2505—2507 (1987).
inhibition (%)ꢄ[(AꢃB)/A]/(C/100)ꢆ100
where A and B indicate the optical density of vehicle- and test compound-
treated groups, respectively, and C indicates cell viability (%).
15) Nakano K., Nohara T., Tomimatsu T., Nishikawa M., Phytochemistry,
21, 2749—2751 (1982).
Cell Viability The melanoma cells (5.0ꢆ103 cells/100 ml/well) were
seeded into 96-well microplates and incubated for 24 h. After 70 h incuba-
tion with theophylline 1 mM and a test compound, 10 ml of WST-8 solution
(Cell Counting Kit-8TM) was added to each well. After a further 2 h in cul-
ture, the optical density of the water-soluble formazan produced by the cells
was measured with a microplate reader (Model 550, Bio-Rad Laboratories,
Hercules, CA, U.S.A.) at 450 nm (reference: 655 nm). The test compound
was dissolved in DMSO, and the final concentration of DMSO in the
medium was 0.1%. Cell viability (%) was calculated using the following for-
mula.
16) Cochrane J. S., Hanson J. R., J. Chem. Soc., 1971, 3730—3732 (1971).
17) Nishimoto N., Nakai S., Takagi N., Hayashi S., Takemoto T.,
Odashima S., Kizu H., Wada Y., Phytochemistry, 23, 139—142
(1984).
1
18) The H- and 13C-NMR spectra of 1—6 were assigned with the aid of
distortionless enhancement by polarization transfer (DEPT), double
quantum filter correlation spectroscopy (DQF COSY), heteronuclear
multiple quantum coherence spectroscopy (HMQC), and heteronuclear
multiple bond connectivity spectroscopy (HMBC) experiments.
19) Wu S. H., Yang S. M., Wu D. G., Cheng Y. W., Peng Q., Helv. Chim.
Acta, 88, 259—265 (2005).
cell viability (%)ꢄA/Bꢆ100
where A and B indicate the optical density of vehicle- and test compound-
treated groups, respectively.
Statistical Analyses Values are expressed as meanꢀS.E.M. One-way
analysis of variance followed by Dunnett’s test was used for statistical analy-
ses.
20) Nakamura S., Li X., Matsuda H., Ninomiya K., Morikawa T., Yama-
guti K., Yoshikawa M., Chem. Pharm. Bull., 55, 1505—1511 (2007).
21) Hara S., Okabe H., Mihashi K., Chem. Pharm. Bull., 34, 1843—1845
(1986).
22) Matsuda H., Nakamura S., Murakami T., Yoshikawa M., Heterocycles,
71, 331—341 (2007).
Acknowledgments This research was supported by the 21st COE Pro-
gram, Academic Frontier Project, and a Grant-in Aid for Scientific Research
from the Ministry of Education, Culture, Sports, Science and Technology of
Japan. The authors thank Mr. Nobuyuki Izumi for financial support.
23) Yoshikawa M., Nakamura S., Kato Y., Matsuhira K., Matsuda H.,
Chem. Pharm. Bull., 55, 598—605 (2007).
24) Nishimoto N., Shiobara Y., Inoue S., Fujino M., Takemoto T., Yeoh C.
L., De Oliveira F., Akisue G., Akisue M. K., Hashimoto G., Phyto-
chemistry, 27, 1665—1668 (1988).
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