Chemical structures of constituents from the whole plant of Bacopa monniera

Article abstract of DOI:10.1007/s11418-016-0986-0

Two new dammarane-type triterpene oligoglycosides, bacomosaponins A and B, and three new phenylethanoid glycosides, bacomosides A, B₁, and B₂, were isolated from the whole plant of Bacopa monniera Wettst. The chemical structures of the new constituents were characterized on the basis of chemical and physicochemical evidence. In the present study, bacomosaponins A and B with acyl groups were obtained from the whole plant of B. monniera. This is the first report of acylated dammarane-type triterpene oligoglycosides isolated from B. monniera. In addition, dammarane-type triterpene saponins significantly inhibited the aggregation of 42-mer amyloid β-protein.

Full text of DOI:10.1007/s11418-016-0986-0

J Nat Med
DOI 10.1007/s11418-016-0986-0
ORIGINAL PAPER
Biologically Active Natural Products from Microorganisms
and Plants
Chemical structures of constituents from the whole plant
of Bacopa monniera
1
1
1,2
1,2
•
Tomoe Ohta
Takahiro Matsumoto
•
Seikou Nakamura
1
•
Souichi Nakashima
1
•
Yoshimi Oda
Masayuki Yoshikawa
1
1
•
•
Masashi Fukaya
•
Mamiko Yano
•
1
Hisashi Matsuda
Received: 26 January 2016 / Accepted: 6 March 2016
Ó The Japanese Society of Pharmacognosy and Springer Japan 2016
Abstract Two new dammarane-type triterpene oligogly-
cosides, bacomosaponins A and B, and three new pheny-
lethanoid glycosides, bacomosides A, B , and B , were
potential for the treatment of Alzheimer’s disease (AD) [2–
5]. However, to the best of our knowledge, there is no
report about the potential for the treatment of AD of iso-
lated compounds from B. monniera. In the course of our
studies on bioactive constituents from traditional Asian
medicine [6–12], we examined the constituents from the
whole plant of B. monniera. In this paper, we describe the
isolation and structural elucidation of two acylated dam-
marane-type triterpene oligoglycosides named baco-
mosaponins A (1) and B (2) and three new phenylethanoid
glycosides, bacomosides A (3), B (4), and B (5), and the
1
2
isolated from the whole plant of Bacopa monniera Wettst.
The chemical structures of the new constituents were
characterized on the basis of chemical and physicochemi-
cal evidence. In the present study, bacomosaponins A and
B with acyl groups were obtained from the whole plant of
B. monniera. This is the first report of acylated dammar-
ane-type triterpene oligoglycosides isolated from B. mon-
niera. In addition, dammarane-type triterpene saponins
significantly inhibited the aggregation of 42-mer amyloid
b-protein.
1
2
inhibitory effects of the isolated compounds on Ab42
aggregation.
Keywords Bacopa monniera Á Bacomosaponin Á
Bacomoside Á Ab42 aggregation Á Ayurvedic traditional
medicine
Results and discussion
The MeOH extract (30.4 % from the whole plant of B.
monniera cultivated in India) was partitioned into an
Introduction
EtOAc-H O (1:1, v/v) mixture to furnish an EtOAc-soluble
2
fraction (4.8 %) and an aqueous layer. The aqueous layer
was further extracted with 1-butanol to give 1-butanol-
The Scrophulariaceae plant, Bacopa monniera Wettst, is a
perennial herb and widely cultivated in many countries,
including China, India, and Japan. In the Ayurvedic system
of traditional Indian medicine, B. monniera has been
widely used as a nerve tonic and memory enhancer [1]. In
previous studies, the alcoholic extract was reported to have
(8.8 %) and H O- (16.8 %) soluble fractions. The 1-bu-
2
tanol- and EtOAc-soluble fractions were subjected to nor-
mal- and reversed-phase silica-gel column chromatography
and repeated HPLC. From these fractions, two new triter-
pene oligoglycosides, bacomosaponins A (1, 0.012 %) and
B (2, 0.00078 %), three new phenylethanoid glycosides,
bacomosides A (3, 0.0016 %), B (4, 0.0051 %), and B (5,
1
2
&
Hisashi Matsuda
matsuda@mb.kyoto-phu.ac.jp
0
0
.0046 %), and nine known compounds, bacoside A (6,
3
.097 %) [13], bacopasides VII (7, 0.012 %) [14] and II (8,
1
2
0.062 %) [15], bacopasaponin C (9, 0.0061 %) [16],
monnieraside III (10, 0.0022 %) [17], plantainoside B (11,
0.012 %) [17], cucurbitacins E (12, 0.0011 %) [18] and I
Department of Pharmacognosy, Kyoto Pharmaceutical
University, Misasagi, Yamashina-ku, Kyoto 607-8412, Japan
N.T.H Co., Ltd., 4F Sky-ebisu Bldg., 1-8-11 Ebisu, Shibuya-
ku, Tokyo 150-0013, Japan
123

J Nat Med
(
13, 0.00041 %) [19], and cucurbitacin E 2-O-b-D-glu-
0.55, 0.97, 0.99, 1.25, 1.33, 1.63, 1.75 (3H each, all s, H3-
19, 18, 29, 28, 21, 26, 27), 3.17 (1H, dd-like, H-3), 5.24
(1H, t-like, H-23), 5.47 (1H, d, J = 8.2 Hz, H-24)], four
copyranoside (14, 0.012 %) [20] were isolated (Fig. 1).
Bacomosaponins A (1) and B (2) were obtained as a
white amorphous powder with negative specific rotations
glycosyl moieties {a b-D-glucopyranosyl moiety [1: d 5.00
1
9
19
0
(
1H, d, J = 6.8 Hz, H-1 )] or a a-L-arabinopyranosyl
(
1: ½aŠ -2.9; 2: ½aŠD –3.8, both in MeOH). The IR spectra
D
0
moiety [2: d 4.70 (1H, d, J = 6.8 Hz, H-1 )] together with
a-L-arabinofuranosyl moiety [1: d 6.24 (1H, d, J = 2.7 Hz,
H-1 ), 2: d 6.04 (1H, d, J = 2.8 Hz, H-1 )], a b-D-glu-
copyranosyl moiety [1: d 5.22 (1H, d, J = 7.6 Hz, H-1 ),
: d 5.08 (1H, d, J = 7.6 Hz, H-1 )], and an a a-L-ara-
of 1 and 2 showed absorption bands assignable to hydroxyl,
acetyl, and olefin groups [1: 3400, 1743, 1635, and
-
083 cm , 2: 3392, 1745, 1643, and 1076 cm ]. Their
1
-1
00
00
1
0
00
molecular formulas (1: C H O , 2: C H O ) were
5
6
88 24
55 86 23
0
00
2
determined from the quasimolecular ion peaks (1: m/z 1167
[
1
FABMS and by HRMS measurement. The H-NMR
?
M ? Na] , 2: m/z 1137 [M ? Na] ) in the positive-ion
?
binopyranosyl moiety [1: d 4.99 (1H, d, J = 6.9 Hz,
0
000
0000
H-1 ), 2: d 4.93 (1H, d, J = 6.9 Hz, H-1 )]}, and two
1
3
000000
acetyl moieties [1: d 1.99, 2.08 (3H each, both s, H-2
,
(
Table 1) and C-NMR (Table 2) spectra (pyridine-d ) of
5
0
0000
000000 00000
2
), 2: d 1.93, 1.97 (3H each, both s, H-2 , 2 )].
1
and 2, which were assigned by various NMR experi-
Alkaline hydrolysis of 1 and 2 with 5 % aqueous potassium
carbonate furnished bacopasaponin F (1a) [21] and baco-
pasaponin E (2a) [21]. As shown in Fig. 2, the position of
the two acetyl groups and the structure of the
ments, showed signals assignable to triterpene part [1: d
0
.58, 1.03, 1.04, 1.26, 1.38, 1.69, 1.81 (3H each, all s, H3-
9, 18, 29, 28, 21, 26, 27), 3.25 (1H, dd-like, H-3), 5.30
1
(
1H, t-like, H-23), 5.50 (1H, d, J = 8.2 Hz, H-24), 2: d
Fig. 1 Structures of the compounds isolated from Bacopa monniera
1
23

J Nat Med
1
Table 1 H NMR (600 MHz) data of compounds 1 and 2 (pyridine-
13
Table 2 C NMR (150 MHz) data of compounds 1 and 2 (pyridine-
d
5
)
5
d )
Position
1
2
Position
1
2
3
3.25 (dd-like)
2.90 (m)
1.03 (s)
3.17 (dd-like)
2.87 (m)
1
38.6
26.7
89.1
39.7
56.0
18.2
35.9
37.4
52.8
37.0
21.6
28.3
36.1
53.7
37.4
38.7
26.7
88.6
39.8
56.1
18.2
35.9
37.1
52.9
37.3
21.6
28.3
36.1
53.7
37.4
110.2
55.0
18.8
16.3
76.3
24.6
41.9
68.7
127.3
133.7
25.6
18.2
27.7
16.5
65.8
1
1
1
2
2
2
2
2
2
2
3
3
8
9
1
3
4
6
7
8
9
0
2
0.97 (s)
3
0.58 (s)
0.55 (s)
4
1.38 (s)
1.33 (s)
5
5.30 (t-like)
5.50 (d, J = 8.2)
1.69 (s)
5.24 (t-like)
5.47 (d, J = 8.2)
1.63 (s)
6
7
8
1.81 (s)
1.75 (s)
9
1.26 (s)
1.25 (s)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
1.04 (s)
0.99 (s)
4.03 (m)
4.05 (m)
4
.20 (m)
4.18 (m)
3
2
3
2
3
4
-O-glycoside
0
Glc
Ara (p)
1
5.00 (d, J = 6.8)
4.70 (d, J = 6.8)
0
-O-Ara (f)
0
110.1
54.9
18.8
16.2
76.3
24.5
41.9
68.8
127.2
133.7
25.6
18.2
27.7
16.5
65.9
Glc
0
1
6.24 (d, J = 2.7)
5.22 (d, J = 7.6)
4.99 (d, J = 6.9)
2.08 (s)
6.04 (d, J = 2.8)
5.08 (d, J = 7.6)
4.93 (d, J = 6.9)
1.97 (s)
0
-O-Glc
0
00
1
0-O-Ara (p)
000
0
1
0
000
0
-O-Ac
0000
2
0
000
-O-Ac
00000
0
2
1.99 (s)
1.93 (s)
2
2
2
6
7
8
oligoglycoside moiety of 1 and 2 were also confirmed by
DQF COSY and HMBC experiments, which showed long-
range correlations between the following proton and car-
29
30
3-O-glycoside
0
Ara (p)
105.6
0
00
0
000
0
0000
bons: H-1 and C-3; H-1 and C-2 ; H-1 and C-3 ; H-1
1
105.0
79.1
88.5
70.2
77.8
62.6
0
000
00000
0000
000000
0
and C-20; H-3 and C-1 ; H-4 and C-1 , respec-
tively. Consequently, the structure of bacomosaponins A
and B was determined as 3-O-{b-D-glucopyranosyl
2
77.1
83.6
68.6
65.8
0
3
0
4
0
(
1 ? 3)[a-L-arabinofuranosyl(1 ? 2)]-b-D-glucopyranosyl}
20-O-(3,4-diacetyl-a-L-arabinopyranosyl) jujubogenin (1)
5
0
-
6
0
and 3-O-{b-D-glucopyranosyl(1 ? 3)[a-L-arabinofuranosyl
1 ? 2)]-a-L-arabinopyranosyl}-20-O-(3,4-diacetyl-a-L-ara-
binopyranosyl) jujubogenin (2), respectively.
2 -O-Ara (f)
00
(
1
109.8
83.8
77.6
84.8
62.5
110.1
83.8
77.8
84.8
62.1
00
2
Bacomoside A (3), obtained as a white amorphous
00
3
27
0
0
0
0
powder with negative specific rotation (½aŠ -3.9 in
4
5
0
D
MeOH), showed absorption bands due to hydroxyl, ester
carbonyls, and aromatic rings in the IR spectrum (3415,
3 -O-Glc
-
1
705, 1609 cm ). Positive-ion FABMS revealed a
0
0
00
00
1
1
2
3
4
104.6
75.5
78.3
71.5
104.9
75.2
78.1
71.4
?
quasimolecular ion [M ? Na] at m/z 473 from which the
molecular formula C H O was deduced via HRMS and
2
000
000
1 22 11
1
3
1 13
C NMR data. The H-NMR (CD OD) and C-NMR
3
123

J Nat Med
Table 2 continued
DQF COSY and HMBC experiments (Fig. 2). Namely,
long-range correlations were observed between the fol-
Position
1
2
0
0
lowing proton and carbon pairs: H-1 and C-8; H-2 and
00
0
0
00
00
5
6
78.5
62.3
78.4
62.5
C-7 . Finally, acid hydrolysis of 3 with 5 % aqueous
H SO -1,4-dioxane liberated 4-hydroxybenzoic acid,
2
4
2
0-O-Ara (p)
which was identified by HPLC analysis, together with a D-
glucose, which was identified by HPLC of its tolylthio-
carbamoyl thiazolidine derivatives [22]. Consequently, this
evidence led us to formulate the structures of bacomoside
A (3) as shown.
0
0
0
0
0
000
000
000
000
000
1
2
3
4
5
98.6
70.0
74.5
69.0
63.5
98.6
70.0
74.5
69.0
63.5
Bacomosides B (4) and B (5), isolated as a white
1
2
0
000
0
27
D
3
-O-Ac
0000
amorphous powder with positive specific rotation (4: ½aŠ
27
1
2
170.5
20.8
170.5
20.8
?
5.1; 5: ½aŠ ?22.0, both in MeOH), showed absorption
D
0
0000
bands due to hydroxyl, ester carbonyls and aromatic rings
in the IR spectrum. The common molecular formula
C H O of 4 and 5 was determined individually from the
0
000
0
4
-O-Ac
00000
1
2
170.5
20.7
170.5
20.7
2
4 28 12
0
00000
?
quasimolecular ion peak [m/z 531 (M ? Na) ] in positive-
ion FABMS and HRMS measurement. Acid hydrolysis of 4
and 5 yielded trans-caffeic acid and D-glucose, respec-
(
Table 3) spectra of 3 showed signals assignable to an
aglycone part [d 4.88, 4.94 (1H each, both d, J = 15.8 Hz,
H -8), 6.66 (1H, d, J = 8.9 Hz, H-5), 7.31 (1H, d,
1
13
tively. The H-NMR (CD
spectra of 4 and 5 showed signals assignable to an aglycone
parts {4: d 3.13 (3H, s, 7-OCH ), [3.52 (1H, m), 3.93 (1H,
dd, J = 11.0, 8.2 Hz), H -8], 4.15 (1H, dd-like, H-7), 6.56
OD) and C-NMR (Table 3)
3
2
J = 1.8 Hz, H-2), 7.33 (1H, dd, J = 8.9, 1.8 Hz, H-6)], D-
0
3
glucopyranosyl moiety [d 4.75 (1H, d, J = 8.2 Hz, H-1 )],
2
and 4-hydroxybenzoyl moiety [d 6.81 (1H, d, J = 8.9 Hz,
(1H, dd, J = 8.2, 1.8 Hz, H-6), 6.68 (1H, d, J = 1.8 Hz,
0
0
00
00 00
1
H-3 , 5 ), 7.89 (1H, d, J = 8.9 Hz, H-2 , 6 )]. The H- and
H-2), 6.77 (1H, d, J = 8.2 Hz, H-5), 5: d 3.14 (3H, s,
1
3
C-NMR spectra of 3 were superimposable on those of
monnieraside III, except for the signals around the 7-po-
7-OCH
-8], 4.17 (1H, dd-like, H-7), 6.58 (1H, dd, J = 8.2,
1.8 Hz, H-6), 6.68 (1H, m, H-2), 6.72 (1H, m, H-5)}, a
3
), [3.66 (1H, m), 3.77 (1H, dd, J = 11.6, 3.4 Hz),
H
2
sition. The structure of 3 was characterized by means of
Fig. 2 Important 2D NMR correlations of 1–5
1
23

J Nat Med
1 13
Table 3 H NMR (600 MHz) and C NMR (150 MHz) data of compounds 3–5 (CD
3
OD)
Position
3
4
5
d
H
d
C
d
H
d
C
d
H
d
C
1
2
3
4
5
6
7
128.4
115.8
152.5
146.5
115.7
123.2
196.1
131.3
115.0
146.4
146.3
116.2
120.0
83.5
131.2
116.2
146.4
146.2
114.8
119.7
84.6
7.31 (d, J = 1.8)
6.68 (d, J = 1.8)
6.68 (m)
6.66 (d, J = 8.9)
6.77 (d, J = 8.2)
6.56 (dd, J = 8.2, 1.8)
4.15 (dd-like)
6.72 (m)
7.33 (dd, J = 8.9, 1.8)
6.58 (dd, J = 8.2, 1.8)
4.17 (dd-like)
3.14 (s)
7
-OCH
3
3.13 (s)
56.9
57.2
8
4.88 (d, J = 15.8)
71.3
3.93 (dd, J = 11.0, 8.2)
3.52 (m)
74.3
3.77 (dd, J = 11.6, 3.4)
3.66 (m)
74.9
4
.94 (d, J = 15.8)
8
-O-Glc
0
1
4.75 (d, J = 8.2)
5.00 (dd, J = 9.6, 8.2)
3.66 (m)
101.7
75.1
76.3
71.7
78.3
62.7
4.50 (d, J = 7.6)
4.78 (m)
102.1
75.1
76.2
71.7
78.1
62.6
4.63 (d, J = 7.6)
4.82 (m)
103.0
75.2
76.2
71.7
78.1
62.6
0
2
0
3
3.58 (m)
3.51 (m)
0
4
3.44 (m)
3.37 (m)
3.39 (m)
0
5
3.38 (m)
3.28 (m)
3.33 (m)
0
6
3.71 (dd, J = 12.4, 6.2)
3.68 (dd, J = 12.4, 6.2)
3.87 (dd, J = 12.4, 2.0)
3.68 (m)
3
.91 (dd, J = 12.4, 2.0)
3.88 (dd, J = 12.4, 2.0)
0
2
-O-acyl
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
2
3
4
5
6
7
8
9
122.4
133.1
116.0
163.4
116.0
133.1
167.5
127.8
115.2
146.8
149.5
116.5
123.0
147.0
115.4
168.3
127.8
115.1
146.8
149.5
116.5
122.9
147.0
115.4
168.5
7.89 (d, J = 8.9)
6.81 (d, J = 8.9)
7.05 (d, J = 1.8)
7.06 (d, J = 1.8)
6.81 (d, J = 8.9)
7.89 (d, J = 8.9)
6.78 (d, J = 8.2)
6.79 (d, J = 8.2)
6.94 (dd, J = 8.2, 1.8)
7.57 (d, J = 15.8)
6.29 (d, J = 15.8)
6.97 (dd, J = 8.2, 1.8)
7.61 (d, J = 15.8)
6.34 (d, J = 15.8)
D-glucopyranosyl moiety [4: d 4.50 (1H, d, J = 7.6 Hz,
AD is a neurodegenerative disorder characterized by the
0
0
H-1 ), 5: d 4.63 (1H, d, J = 7.6 Hz, H-1 )], and trans-
aggregation of 42-mer amyloid b-protein (Ab ) [23].
4
2
0
0
caffeoyl moiety [4: d 6.29 (1H, d, J = 15.8 Hz, H-8 ), 6.78
Previously, the alcoholic extract of B. monniera was
reported to be available for AD [2–5]. In the present study,
the 1-butanol- and EtOAc-soluble fractions were found to
have inhibitory effects on Ab42 aggregation [inhibition
(%): 22.3 ± 1.5 (P \ 0.01), 29.6 ± 0.6 (P \ 0.01),
respectively at 100 lM] by the Thioflavin T (Th-T)
method. Furthermore, inhibitory effects of the constituents
(1–11, 1a, and 2a) were shown on Ab42 aggregation. As a
result, compound 7 [inhibition (%): 25.6 ± 4.5 (P \ 0.05)
at 10 lM, 39.5 ± 11.6 (P \ 0.05) at 100 lM] significantly
inhibited the aggregation of Ab42 in a concentration-de-
pendent manner, but compounds 1, 8, and 9 inhibited it
only at a higher concentration [inhibition (%) 1:
44.0 ± 16.9 (P \ 0.05), 8: 67.7 ± 14.7 (P \ 0.01), 9:
00
(
1H, d, J = 8.2 Hz, H-5 ), 6.94 (1H, dd, J = 8.2, 1.8 Hz,
0
0
00
H-6 ), 7.05 (1H, d, J = 1.8 Hz, H-2 ), 7.57 (1H, d,
0
0
00
J = 15.8 Hz, H-7 ), 5: d 6.34 (1H, d, J = 15.8 Hz, H-8 ),
00
6
.79 (1H, d, J = 8.2 Hz, H-5 ), 6.97 (1H, dd, J = 8.2,
0
0
00
1
.8 Hz, H-6 ), 7.06 (1H, d, J = 1.8 Hz, H-2 ), 7.61 (1H, d,
0
0
1
13
J = 15.8 Hz, H-7 )]. The H- and C-NMR spectra of 4
and 5 were similar to those of plantainoside B, except for
the signals due to the 7-methoxy group. The connectivities
of the trans-caffeoyl group and the D-glucopyranosyl
moiety in 4 and 5 were elucidated on the basis of HMBC
experiments as shown in Fig. 2. On the basis of those
findings, the structures of bacomoside B (4) and B (5)
1
2
were characterized as shown.
123

J Nat Med
9
8.2 ± 14.5 (P \ 0.01) at 100 lM, respectively]. Their
effects were almost equivalent to that of the positive con-
mixture to furnish an EtOAc-soluble fraction (31.8 g,
4.8 %) and an aqueous phase. The aqueous phase was
further extracted with n-BuOH to give an n-BuOH-soluble
trol, morin [inhibition (%): 34.0 ± 7.8 (P \ 0.01) at
1
00 lM]. Compounds 1a, 4, 5, 10, and 11 had weak effects
fraction (57.9 g, 8.8 %) and an H O-soluble fraction
2
[inhibition (%) 1a: 13.3 ± 0.7 (P \ 0.01), 4: 16.5 ± 1.5
(P \ 0.01), 5: 11.9 ± 1.5 (P \ 0.01), 10: 16.9 ± 1.5
(P \ 0.01), and 11: 9.7 ± 2.2 (P \ 0.01) at 100 lM,
(110.3 g, 16.8 %). A part of the n-BuOH-soluble fraction
(30.0 g) was subjected to normal-phase silica gel column
chromatography [900 g, CHCl ? CHCl :MeOH (100:1
3
3
respectively]. On the other hand, compounds 2, 2a, 3, 6, 12,
1
? 20:1 ? 10:1 ? 8:1 ? 6:1 ? 4:1,
v/v) ? CHCl3–
3, and 14 did not show such the effects.
MeOH–H O (8:2:0.03 ? 10:3:1 ? 7:3:1 ? 6:4:1, v/v/v,
2
lower layer) ? MeOH] to give seven fractions [Fr. B1
(
219.5 mg), Fr. B2 (696.3 mg), Fr. B3 (1.51 g), Fr. B4
(8.70 g), Fr. B5 (4.28 g), Fr. B6 (11.01 g), Fr. B7
328.5 mg)]. Fraction B4 (8.70 g) was subjected to
Experimental
General
(
reversed-phase silica gel column chromatography [425 g,
MeOH:H O (30:70 ? 40:60 ? 50:50 ? 60:40 ? 70:30,
2
The following instruments were used to obtain physical
data: specific rotations, a Horiba SEPA-300 digital
polarimeter (l = 5 cm); IR spectra, a Thermo Electron
Nexus 470; EIMS and HREIMS, JEOL JMS-GCMATE
mass spectrometer; FABMS and HRFABMS, JEOL JMS-
v/v) ? MeOH] to give five fractions [Fr. B4–1 (3.55 g),
Fr. B4–2 (312.3 mg), Fr. B4–3 (396.6 mg), Fr. B4–4
(3.65 g), Fr. B4–5 (1.09 g)]. Fraction B4–4 (3.65 g) was
subjected to reversed-phase silica gel column chromatog-
raphy
[180 g,
MeCN:H O
2
(20:80 ? 30:70 ?
1
SX 102A mass spectrometer; H-NMR spectra, JEOL
40:60 ? 50:50, v/v) ? MeOH] to give ten fractions [Fr.
B4–4–1 (39.7 mg), Fr. B4–4–2 (168.8 mg), Fr. B4–4–3
(320.9 mg), Fr. B4–4–4 (103.5 mg), Fr. B4–4–5 (1.09 g),
Fr. B4–4–6 (913.6 mg), Fr. B4–4–7 (178.3 mg), Fr. B4–4–
8 (4.9 mg), Fr. B4–4–9 (5.9 mg), Fr. B4–4–10 (18.4 mg)].
Fr. 4–4–5 (1.09 g) was purified by HPLC [MeCN:H2-
1
3
JNM-ECA 600 (600 MHz) spectrometer; C-NMR spec-
tra, JEOL JNM-ECA 600 (150 MHz) spectrometers;
HPLC, SPD-10Avp UV–VIS detectors. COSMOSIL 5C18-
MS-II columns (250 9 4.6 mm i.d., 250 9 10 mm i.d.,
and 250 9 20 mm i.d.) were used for analytical and
preparative purposes. The following materials were used
for chromatography: normal-phase silica gel column
chromatography, Silica gel BW-200 (Fuji Silysia Chemi-
cal, 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, 0.25 mm) (or-
dinary phase) and Silica gel RP-18 F254S (Merck, 0.25 mm)
O:CH COOH (31:69:0.3), COSMOSIL 5C18-MS-II] to
3
give 6 (145.7 mg), 8 (88.0 mg), and 1 (18.5 mg). Fr. 4–4–7
(178.3 mg) was purified by HPLC [MeCN:H O:CH
2
3-
COOH (33:67:0.3), COSMOSIL 5C18-MS-II] to give 8
(8.8 mg), 7 (40.0 mg), 9 (19.6 mg), and 2 (25.1 mg). The
EtOAc-soluble fraction (31.8 g) was subjected to normal-
phase silica gel column chromatography [1.25 kg, n-hexane:
CHCl (10:1 ? 1:1 ? 1:10, v/v) ? CHCl :MeOH (100:
3
3
(
reversed phase); reversed-phase HPTLC, precoated TLC
1 ? 50:1 ? 30:1 ? 15:1 ? 7:1 ? 5:1, v/v) ? MeOH]
to give 18 fractions [Fr. E1 (149.8 mg), Fr. E2 (11.0 mg),
Fr. E3 (5.4 mg), Fr. E4 (15.4 mg), Fr. E5 (8.9 mg), Fr. E6
(476.8 mg), Fr. E7 (92.7 mg), Fr. E8 (60.3 mg), Fr. E9
plates with Silica gel RP-18 WF254S (Merck, 0.25 mm).
Detection was achieved by spraying with 1 % Ce(SO ) –
4
2
1
0 % aqueous H SO , followed by heating.
2 4
(
4.02 g), Fr. E10 (796.3 mg), Fr. E11 (1.40 g), Fr. E12
(638.7 mg), Fr. E13 (1.35 g), Fr. E14 (2.40 g), Fr. E15
4.25 g), Fr. E16 (4.61 g), Fr. E17 (4.70 g), Fr. E18
Plant material
(
The dried whole plants of B. monniera (2 kg) cultivated in
India were purchased from NTH India Pvt. Ltd. (2013). A
voucher of the plant is on file in our laboratory (BM-NTH-
(513.9 mg)]. Fraction E9 (4.02 g) was subjected to normal-
phase silica gel column chromatography [200 g, n-hexane–
EtOAc (50:1 ? 40:1 ? 30:1 ? 20:1 ? 10:1 ? 5:1 ?
3:1 ? 2:1 ? 1:1 ? 1:2, v/v) ? MeOH] to give 24 frac-
tions [Fr. E9–1 (71.5 mg), Fr. E9–2 (40.7 mg), Fr. E9–3
(249.6 mg), Fr. E9–4 (303.1 mg), Fr. E9–5 (250.1 mg), Fr.
E9–6 (163.8 mg), Fr. E9–7 (253.3 mg), Fr. E9–8
(209.6 mg), Fr. E9–9 (183.2 mg), Fr. E9–10 (83.1 mg), Fr.
E9–11 (107.3 mg), Fr. E9–12 (222.4 mg), Fr. E9–13
(208.8 mg), Fr. E9–14 (226.6 mg), Fr. E9–15 (142.6 mg),
Fr. E9–16 (228.4 mg), Fr. E9–17 (137.1 mg), Fr. E9–18
(131.3 mg), Fr. E9–19 (111.0 mg), Fr. E9–20 (114.1 mg),
1
).
Extraction and isolation
The whole plant of B. monniera (2 kg) were extracted three
times with methanol under reflux for 3 h. Evaporation of
the solvent under reduced pressure provided a methanol
extract (608.8 g, 30.4 %). A part of the MeOH extract
(
237.1 g) was partitioned into an EtOAc–H O (1:1, v/v)
2
1
23

J Nat Med
-
1
1
Fr. E9–21 (49.2 mg), Fr. E9–22 (510.0 mg), Fr. E9–23
1076 cm ; H-NMR (pyridine-d , 600 MHz): given in
5
1
3
(
(
270.3 mg), and Fr. E9–24 (181.2 mg)]. Fr. E9–19
Table 1;
C-NMR (pyridine-d , 150 MHz): given in
5
?
111.0 mg) was purified by HPLC [MeCN:H O (50:50),
2
Table 2; positive-ion FAB-MS: m/z 1137 [M ? Na] ; HR-
FAB-MS: m/z 1137.5461 (Calcd. for C H O Na
COSMOSIL 5C18-MS-II] to give 12 (5.5 mg). Fraction
E11 (1.2 g) was subjected to normal-phase silica gel col-
umn chromatography [60 g, CHCl ? CHCl –MeOH
5
5 86 23
?
[M ? Na] : m/z 1137.5458).
Bacomoside A (3): A white amorphous powder; ½aŠ
27
3
3
D
(200:1 ? 100:1 ? 50:1 ? 30:1 ? 20:1 ? 10:1, v/v) ?
MeOH] to give 11 fractions [Fr. E11–1 (16.4 mg), Fr.
-
3.9 (c = 0.7, MeOH); IR (KBr): m
609 cm ; H-NMR (CD OD, 600 MHz) and C-NMR
3415, 1705,
max
-
1
1
13
1
3
E11–2 (42.1 mg), Fr. E11–3 (38.6 mg), Fr. E11–4
(
CD OD, 150 MHz): given in Table 3; positive-ion FAB-
3
?
MS: m/z 473 [M ? Na] ; HR-FAB-MS: m/z 473.1064
(
43.1 mg), Fr. E11–5 (345.7 mg), Fr. E11–6 (293.8 mg),
Fr. E11–7 (88.0 mg), Fr. E11–8 (151.3 mg), Fr. E11–9
58.6 mg), Fr. E11–10 (66.3 mg), Fr. E11–11 (25.2 mg)].
Fr. E11–6 (293.8 mg) was purified by HPLC [MeCN:H O
?
Calcd. for C H O Na [M ? Na] : m/z 473.1060).
(
21 22 11
(
27
D
Bacomoside B (4): A white amorphous powder; ½aŠ
1
2
?
5.1 (c = 0.7, MeOH); IR (KBr): m 3450, 1705,
max
605 cm ; H-NMR (CD OD, 600 MHz) and C-NMR
(30:70), COSMOSIL 5C18-MS-II] to give 13 (1.8 mg).
Fraction E13 (1.35 g) was subjected to reversed-phase
-1
1
13
1
3
(
CD OD, 150 MHz): given in Table 3; positive-ion FAB-
3
silica gel column chromatography [60 g, MeOH:H O
2
?
MS: m/z 531 [M ? Na] ; HR-FAB-MS: m/z 531.1473
(
20:80 ? 30:70 ? 40:60 ? 50:50 ? 60:40 ? 70:30 ?
0:20 ? 90:10, v/v) ? MeOH] to give 20 fractions [Fr.
E13–1 (53.7 mg), Fr. E13–2 (22.1 mg), Fr. E13–3
38.2 mg), Fr. E13–4 (26.0 mg), Fr. E13–5 (132.6 mg), Fr.
E13–6 (70.2 mg), Fr. E13–7 (186.8 mg), Fr. E13–8
62.5 mg), Fr. E13–9 (38.8 mg), Fr. E13–10 (21.3 mg), Fr.
E13–11 (33.5 mg), Fr. E13–12 (17.6 mg), Fr. E13–13
41.5 mg), Fr. E13–14 (28.7 mg), Fr. E13–15 (107.9 mg),
Fr. E13–16 (48.6 mg), Fr. E13–17 (218.6 mg), Fr. E13–18
32.2 mg), Fr. E13–19 (15.6 mg), Fr. E13–20 (6.1 mg)].
Fr. E13–7 (186.8 mg) was purified by HPLC [MeOH: H O
?
Calcd. for C H O Na [M ? Na] : m/z 531.1478).
(
24 28 12
8
2
D
7
Bacomoside B (5): A white amorphous powder; ½aŠ
2
?
22.0 (c = 0.2, MeOH); IR (KBr): m 3455, 1690,
max
614 cm ; H-NMR (CD OD, 600 MHz) and C-NMR
(
-
1
1
13
1
3
(
CD OD, 150 MHz): given in Table 3; positive-ion FAB-
3
(
?
MS: m/z 531 [M ? Na] ; HR-FAB-MS: m/z 531.1483
?
Calcd. for C H O Na [M ? Na] : m/z 531.1478).
(
24 28 12
(
Alkaline hydrolyses
(
2
A solution of 1 and 2 (each 1.0 mg) in 5 % aqueous K CO
2
3
(
30:70), COSMOSIL 5C18-MS-II] to give 14 (62.0 mg).
Fraction E16 (4.6 g) was subjected to reversed-phase silica
gel column chromatography [280 g, MeOH:H O (10:90
(0.2 mL) was stirred at room temperature for 1 h. The
reaction solution was neutralized with Dowex HCR W 9 2
?
H form) and the resin was removed by filtration. Eva-
2
(
? 20:80 ? 30:70 ? 40:60 ? 60:40 ? 80:20, v/v) ?
MeOH] to give 20 fractions [Fr. E16–1 (195.7 mg), Fr.
poration of the solvent under reduced pressure from the
filtrate furnished 1a (0.8 mg, from 1) and 2a (0.8 mg, from
2
E16–2 (39.2 mg), Fr. E16–3 (38.2 mg), Fr. E16–4
), which were identified by comparison of their physical
1
(
355.8 mg), Fr. E16–5 (535.2 mg), Fr. E16–6 (479.3 mg),
Fr. E16–7 (231.4 mg), Fr. E16–8 (623.1 mg), Fr. E16–9
364.1 mg), Fr. E16–10 (227.4 mg), Fr. E16–11 (63.3 mg),
data ([a] , H-NMR and MS) with reported values [21].
D
(
Acid hydrolyses
Fr. E16–12 (254.4 mg), Fr. E16–13 (154.5 mg), Fr.
E16–14 (142.8 mg), Fr. E16–15 (85.9 mg), Fr. E16–16
Compounds 3, 4, and 5 (each 3.0 mg) were dissolved in
5
(
366.0 mg)]. Fr. E16–4 (355.8 mg) was purified by HPLC
% aqueous H SO –1,4-dioxane (1:1, v/v, 1.0 mL), and
2 4
{
(1) [MeCN:H O:CH COOH (12:88:0.3), COSMOSIL
2 3
each solution was heated at 90 °C for 3 h. After being
cooled, the reaction mixture was neutralized with Amber-
-
lite IRA-400 (OH form) and the resin was removed by
5
C18-MS-II], (2) [MeCN:H O:CH COOH (10:90:0.3),
2
3
COSMOSIL 5C18-MS-II]} to give 11 (62.0 mg), 3
(
7.7 mg), 10 (10.5 mg), 4 (24.5 mg), and 5 (22.3 mg).
Bacomosaponin A (1): A white amorphous powder;
filtration. After removal of the solvent from the filtrate
under reduced pressure, the residue was partitioned in
1
D
9
½
aŠ -2.9 (c = 0.1, MeOH); IR (KBr): m
3400, 1743,
max
EtOAc–H O (1:1, v/v), and the solvents removed in vacuo
2
-
1 1
1
635, 1083 cm ; H-NMR (pyridine-d , 600 MHz): given
5
from the EtOAc-soluble fraction and aqueous phase,
respectively. The EtOAc-soluble fraction was analyzed by
reversed-phase HPLC [column: COSMOSIL 5C18-MS-II
1
3
in Table 1; C-NMR (pyridine-d , 150 MHz): given in
5
?
Table 2; positive-ion FAB-MS: m/z 1167 [M ? Na] ; HR-
FAB-MS: m/z 1167.5560 (Calcd. for C H O Na
5
6 88 24
(Nacalai Tesque), 250 9 4.6 mm i.d. (5 lm); mobile
?
M ? Na] : m/z 1167.5563).
[
phase: MeCN–H O in 0.3 % AcOH (15:85, v/v); detection:
2
1
D
9
Bacomosaponin B (2): A white amorphous powder; ½aŠ
UV (254 nm); flow rate: 1.0 mL/min; column temperature:
-
3.8 (c = 0.1, MeOH); IR (KBr): m
3392, 1745, 1643,
max
35 °C] to give 4-hydroxybenzoic acid (t 8.3 min from 3)
R
123

J Nat Med
and trans-caffeic acid (tR 9.2 min from 4 and 5). The
aqueous layer was dissolved in pyridine (0.5 mL) con-
taining L-cysteine methyl ester hydrochloride (0.5 mg) and
heated at 60 °C for 1 h. A solution of o-tolylisothiocyanate
Acylated oleanane-type triterpene oligoglycosides from the
flower buds of Camellia sinensis var. assamica. Tetrahedron
7
1:846–851
8
9
. Matsumoto T, Nakamura S, Ohta T, Fujimoto K, Yoshikawa M,
Ogawa K, Matsuda H (2014) A rare glutamine derivative from
the flower buds of daylily. Org Lett 16:3076–3078
. Matsumoto T, Nakamura S, Nakashima S, Fujimoto K, Yoshi-
kawa M, Ohta T, Ogawa K, Matsuda H (2014) Lignan dicar-
boxylates and terpenoids from the flower buds of Cananga
odorata and their inhibitory effects on melanogenesis. J Nat Prod
77:990–999
0. Nakamura S, Fujimoto K, Matsumoto T, Nakashima S, Ohta T,
Ogawa K, Matsuda H, Yoshikawa M (2013) Acylated sucroses
and acylated quinic acids analogs from the flower buds of Prunus
mume and their inhibitory effect on melanogenesis. Phytochem-
istry 92:128–136
1. Nakamura S, Nakashima S, Oda Y, Yokota N, Fujimoto K,
Matsumoto T, Ohta T, Ogawa K, Maeda S, Nishida S, Matsuda
H, Yoshikawa M (2013) Alkaloids from Sri Lankan curry-leaf
(Murraya koenigii) display melanogenesis inhibitory activity:
structures of karapinchamines A and B. Bioorg Med Chem
(
0.5 mg) in pyridine (0.1 mL) was added to the mixture
and heated at 60 °C for 1 h. The reaction mixture was
analyzed by reversed-phase HPLC [column: COSMOSIL
5
C18-AR-II (Nacalai Tesque), 250 9 4.6 mm i.d. (5 lm);
mobile phase: MeCN–H O in 1 % AcOH (18:82, v/v);
2
1
detection: UV (250 nm); flow rate: 0.8 mL/min; column
temperature: 35 °C] to identify the derivatives of con-
stituent monosaccharides (D-glucose) in 3, 4, and 5 by
comparison of their retention times with those of authentic
1
samples (t : D-glucose; 48.9 min, L-glucose; 41.2 min).
R
Ab42 aggregation assay
2
1:1043–1049
The inhibitory effects of test compounds against Ab42
aggregation were evaluated by the Th-T method using the
SensoLyte Thiofavin T b-Amyloid Aggregation Kit (ANA
SPEC), with a slight modification according to the manu-
facturer’s instructions. Briefly, 2 lL of Th-T (2 mM) were
added to each well of a 384 black plate (Nunc). Next, 1 lL
of test compounds and 17 lL of Ab42 peptide solutions
were added. After 30 min of incubation at 37 °C, the flu-
orescence intensity (ex: 440 nm and em: 480 nm) was
measured with a fluorescence plate reader (FLUOstar
OPTIMA, BMG Labtech).
1
1
2. Matsumoto T, Nakamura S, Nakashima S, Yoshikawa M, Fuji-
moto K, Ohta T, Morita A, Yasui R, Kashiwazaki E, Matsuda H
(2013) Diarylheptanoids with inhibitory effects on melanogenesis
from the rhizomes of Curcuma comosa in B16 melanoma cells.
Bioorg Med Chem Lett 23:5178–5181
3
3. Rastogi S, Pal R, Kulshreshtha DK (1994) Bacoside A –A
triterpenoid saponin from Bacopa monniera. Phytochemistry
36:133–137
14. Zhou Y, Shen YH, Zhang C, Su J, Liu RH, Zhang WD (2007)
Triterpene saponins from Bacopa monnieri and their antide-
pressant effects in two mice models. J Nat Prod 70:652–655
5. Chakravarty AK, Sarkar T, Masuda K, Shiojima K, Nakane T,
Kawahara N (2001) Bacopaside I and II: two pseudojujubogenin
glycosides from Bacopa monniera. Phytochemistry 58:553–556
6. Garai S, Mahato SB, Ohtani K, Yamasaki K (1996) Dammarane-
type triterpenoid saponins from Bacopa monniera. Phytochem-
istry 42:815–820
7. Chakaravarty AK, Sarkar T, Takahisa N, Kawahara N, Masuda K
(2002) New phenylethanoid glycosides from Bacopa monnieri.
Chem Pharm Bull 50:1616–1618
18. Yoshikawa M, Morikawa T, Kobayashi H, Nakamura A, Mat-
suhira K, Nakamura S, Matsuda H (2007) Bioactive saponins and
glycosides. XXVII. Structures of new cucurbitane-type triterpene
glycosides and antiallergic constituents from Citrullus colocyn-
this. Chem Pharm Bull (Tokyo) 55:428–434
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1
1
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