Acylated oleanane-type triterpene oligoglycosides from the flower buds of Camellia sinensis var. assamica

Article abstract of DOI:10.1016/j.tet.2014.12.049

Eight new acylated oleanane-type triterpene oligoglycosides, floraassamsaponins I, II, III, IV, V, VI, VII, and VIII, were isolated from the flower buds of Camellia sinensis var. assamica. The chemical structures of new constituents were characterized on

Full text of DOI:10.1016/j.tet.2014.12.049

Accepted Manuscript
Acylated oleanane-type triterpene oligoglycosides from the flower buds of Camellia
sinensis var. assamica
Tomoe Ohta, Seikou Nakamura, Souichi Nakashima, Takahiro Matsumoto, Keiko
Ogawa, Katsuyoshi Fujimoto, Masashi Fukaya, Masayuki Yoshikawa, Hisashi
Matsuda
PII:
S0040-4020(14)01753-0
10.1016/j.tet.2014.12.049
TET 26270
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 24 September 2014
Revised Date: 12 December 2014
Accepted Date: 15 December 2014
Please cite this article as: Ohta T, Nakamura S, Nakashima S, Matsumoto T, Ogawa K, Fujimoto K,
Fukaya M, Yoshikawa M, Matsuda H, Acylated oleanane-type triterpene oligoglycosides from the flower
buds of Camellia sinensis var. assamica, Tetrahedron (2015), doi: 10.1016/j.tet.2014.12.049.
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ACCEPTED MANUSCRIPT
Tetrahedron (Full Paper)
(Text 22, Legends 1, Figure 2, Table 4, Graphical abstract 1)
Acylated oleanane-type triterpene oligoglycosides
from the flower buds of Camellia sinensis var. assamica
Tomoe Ohta, Seikou Nakamura, Souichi Nakashima, Takahiro Matsumoto,
Keiko Ogawa, Katsuyoshi Fujimoto, Masashi Fukaya, Masayuki Yoshikawa,
Hisashi Matsuda*
Kyoto Pharmaceutical University, Misasagi, Yamashina-ku, Kyoto 607-8412, Japan
Abstract− Eight new acylated oleanane-type triterpene oligoglycosides,
floraassamsaponins I, II, III, IV, V, VI, VII and VIII, were isolated from the flower
buds of Camellia sinensis var. assamica. The chemical structures of new constituents
were characterized on the basis of chemical and physicochemical evidence. In the
present study, floraassamsaponins I and II with sugar moiety at 28-position were
obtained from the flower buds of C. sinensis var. assamica. In the past, acylated
triterpene oligoglycosides with sugar moiety at 28-position have not been isolated
from the flower buds of C. sinensis var. sinensis. This finding is interesting from the
perspective of chemotaxonomy of Camellia plants. In addition, acylated oleanane-
type triterpene saponins significantly inhibited the aggregation of Aβ42.
Key words: Camellia sinensis var. assamica; Theaceae; acylated oleanane-type
triterpene; floraassamsaponin
*To whom correspondence should be addressed.
Tel: +81-75-595-4633, Fax: +81-75-595-4768,
E-mail:matsuda@mb.kyoto-phu.ac.jp
1

ACCEPTED MANUSCRIPT
1. Introduction
Tea flowers, which are used as foods or drinks, were divided into two main
species: the flower buds of Camellia sinensis var sinensis and C. sinensis var
assamica. Previously, we have isolated saponins named floratheasaponins A–I and
chakasaponins I–VI with several biological effects such as anti-obesity, anti-
hyperlipidemic, and anti-hyperglycemic effects from the flower buds of C. sinensis
var sinensis cultivated in China and Japan. However, the chemical constituents and
biological activities of the flower buds of C. sinensis var assamica, which were
widely cultivated in south and south-eastern Asia such as India, Sri Lanka, and
Indonesia, have not been characterized. In the course of our studies on the bioactive
constituents of Camellia plants and medicinal flowers,1−8 we examined the
constituents from the flower buds of C. sinensis var assamica cultivated in India. In
this paper, we describe the isolation and structure elucidation of eight new acylated
oleanane-type triterpene oligoglycosides named floraassamsaponins I (1), II (2), III
(3), IV (4), V (5), VI (6), VII (7) and VIII (8) as well as the inhibitory effects of the
isolated compounds on Aβ42 aggregation.
2. Results and Discussion
The methanolic extract (40.8% from the dried flower buds of C. sinensis var.
assamiaca) was partitioned between EtOAc–H2O (1:1) to furnish an EtOAc-soluble
fraction (2.6%) and aqueous layer. The aqueous layer was further extracted with n-
BuOH to give n-BuOH (13.9%) and H2O-soluble fractions (21.9%). The n-BuOH-
soluble fraction was subjected to normal-phase and reversed-phase silica gel column
chromatography (CC) and repeated HPLC to give floraassamsaponins I (1, 0.007%),
II (2, 0.014%), III (3, 0.003%), IV (4, 0.005%), V (5, 0.004%), VI (6, 0.022%), VII (7,
0.018%), and VIII (8, 0.035%) together with floratheasaponins D (9, 0.060%)9, G (10,
0.039%)9 and I (11, 0.042%)9.
2

ACCEPTED MANUSCRIPT
OR1
OAc
CH2O
OH
COOH
O
O
HO
HO
OH
O
OH
O
O
OH
H
O
H
H
OH
O
OH
HO
HO
HO
OH
O
O
floraassmsaponin I (1)
H
R1 = Tig
H
OH
OHOH
floraassamsaponin II (2)
R1 = Ang
OR1
OR2
CH2OR3
OH
COOH
O
R4
O
HO
OH
O
O
H
OR5
OH
H
O
HO
O
R1 R2 R3 R4 R5
floraassamsaponin III (3)
floraassamsaponin IV (4)
floraassamsaponin V (5)
floraassamsaponin VI (6)
floraassamsaponin VII (7)
floraassamsaponin VIII (8)
floratheasaponin D (9)
floratheasaponin G (10)
floratheasaponin I (11)
Tig Ac
H
H
OH Glc
OH Gal
OH Gal
H
Tig Ac
Ang Ac
Tig Ac
Ang Ac
OHOH
H
H
H
H
H
H
H
H
Glc
Glc
Glc
Gal
Gal
Gal
H
Tig
H
Ac
H
Ang Ac
Tig Ac
H
Ang
H
Ac
H
H3C
H3C
CH3
H
Glc: -D-glucopyranosyl
Gal: -D-galactopyranosyl
Ang =
Tig =
CH3
O
O
Fig. 1. Structure of saponin constituents isolated from C. sinensis var. assamica
3

ACCEPTED MANUSCRIPT
Floraassamsaponins I (1) and II (2), which were obtained as a white amorphous
21
D
21
D
powder with negative optical rotations (1: [ –18.3, 2: [
α
]
α
]
–12.2 in MeOH),
showed absorption bands due to hydroxy, α,β-unsaturated ester, carboxyl, and ether
components in their IR spectra (1: 3400, 1734, 1717, 1698, and 1078 cm–1, 2: 3400,
1734, 1716, 1697, and 1078 cm–1). FABMS in the positive-ion mode of 1 and 2
revealed a quasimolecular ion peak [M + Na]+ at m/z 1415 from which the common
13
molecular formula C66H104O31 was determined via each HRMS and C NMR data.
Acid hydrolysis of 1 and 2 with 5% aqueous H2SO4-1,4-dioxane yielded
L-arabinose,
D
-galactose, -glucose, -glucuronic acid, and -rhamnose, which were identified by
D
D
L
HPLC analysis of their tolythiocarbamoyl thiazolidine derivatives, respectively.10
The 1H NMR (Table 1) and 13C NMR (Table 3 and 4) spectra (pyridine-d5) of 1 and
2, which were assigned by various NMR experiments,11 showed signals assignable to
an aglycone {seven methyls [1:
s, H3-25, 26, 29, 24, 30, 23, 27), 2:
all s, H3-25, 26, 29, 24, 30, 23, 27)], four methines each bearing an oxygen function
[1: 3.26 (1H, dd-like, H-3), 4.74 (1H, m, H-16)，6.10 (1H, d, J = 10.3 Hz, H–22),
6.58 (1H, d, J = 10.3 Hz, H–21), 2: 3.22 (1H, dd-like, H-3), 4.75 (1H, m, H-16)，
6.03 (1H, d, J = 9.7 Hz, H–22), 6.57 (1H, d, J = 9.7 Hz, H–21)], an olefinic proton
[1: 5.38 (1H, br-s, H-12), 2: 5.33 (1H, br-s, H-12)] and five glycosyl moieties {a
-glucopyranosyl moiety [1: 4.76 (1H, d, J = 5.8 Hz H-1'''''''), 2: 4.77 (1H, d, J
= 5.8 Hz H-1''''''')], a -glucuronopyranosyl moiety [1: 4.97 (1H, d, J = 6.8 Hz, H-
1'), 2: 4.93 (1H, d, J = 6.7 Hz, H-1')], a -galactopyranosyl moiety [1: 5.71 (1H,
d, J = 7.7 Hz H-1''), 2: 5.68 (1H, d, J = 7.6 Hz H-1'')], an -rhamnopyranosyl
6.05 (1H, s, H-1''''), 2: 6.02 (1H, s, H-1'''')], an -arabinopyranosyl
6.14 (1H, d-like, H-1'''), 2:
1.63 (3H, d-like, H3-4'''''), 1.93 (3H, br-s, H3-5'''''), 7.09
(1H, m, H-3''''')], an acetyl moiety [ 1.92 (3H, s, H3-2'''''')], 2: an angeloyl moiety
1.96 (3H, br-s, H3-5'''''), 2.05 (3H, d-like, H3-4'''''), 5.92 (1H, m, H-3''''')], an acetyl
moiety [ 1.91 (3H, s, H3-2'''''')]. The proton and carbon signals due to the aglycone
δ
0.83, 1.02, 1.08, 1.19, 1.27, 1.28, 1.82 (3H each, all
δ
0.80, 0.98, 1.04, 1.12, 1.21, 1.29, 1.80 (3H each,
δ
δ
δ
δ
β
-D
δ
δ
β
-D
δ
δ
β
-D
δ
δ
α
α
-L
moiety [1:
moiety [1:
δ
δ
-L
δ
δ 6.13 (1H, d-like, H-1''')] and two acyl
groups {1: a tigloyl moiety [
δ
δ
[
δ
δ
4

ACCEPTED MANUSCRIPT
1
13
part including glycoside moiety at 28-position in the H and C NMR spectra of 1
and 2 were superimposable on those of foenumoside E,12 while the proton and carbon
signals assignable to the two acyl groups at 21- and 22-positions and oligoglycoside
moiety at 3-position of 1 and 2 are in agreement with those of floratheasaponins G
and D,9 respectively. In addition, the stereostructures of the aglycone parts of 1 and 2
were confirmed by NOESY experiment. Therefore, the aglycone parts of 1 and 2
were found to be barringtogenol C.13 As shown in Fig. 2, the position of the two acyl
groups and the structure of the 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 carbons: H-21 and C-1'''''; H-22 and C-1''''''; H-1'
and C-3; H-1'' and C-2'; H-1''' and C-3'; H-1'''' and C-2'''; H-1''''''' and C-28,
respectively. Consequently, the structure of floraassamsaponins I and II was
determined as 3-O-{[
β
-
D
-galactopyranosyl(1–2)][
-glucuronopyranosyl}-21-O-tigloyl-22-O-
28-O- -glucopyranoside (1) and 3-O-{[
-rhamnopyranosyl(1–2)- -arabinopyranosyl(1–3)]-
28-O-
α
-
L
-rhamnopyranosyl(1–2)-
α
-
L
-
arabinopyranosyl(1–3)]-
β-D
acetylbarringtogenol
C
β
-D
β
-
D
-
galactopyranosyl(1–2)][
α
-
L
α
-L
β
-
D
-
glucuronopyranosyl}-21-O-angeloyl-22-O-acetylbarringtogenol
glucopyranoside (2) , respectively.
C
β
-
D
-
5

ACCEPTED MANUSCRIPT
6

ACCEPTED MANUSCRIPT
Table 1
1H NMR Data of floraassamsaponin I–IV (1–4)
1
2
3
4
position
3.26 (dd-like)
5.38 (br-s)
1.62 (m)
3.22 (dd-like)
5.33 (br-s)
1.60 (m)
3.25 (dd-like)
5.51 (br-s)
4.40 (m)
3.28 (dd-like)
5.51 (br-s)
4.40 (m)
3
12
15
2.05 (m)
2.05 (m)
4.74 (m)
4.75 (m)
4.16 (m)
4.19 (m)
16
21
22
23
24
25
26
27
28
6.58 (d, J = 10.3)
6.10 (d, J = 10.3)
1.28 (s)
1.19 (s)
0.83 (s)
1.02 (s)
1.82 (s)
3.46 (m)
3.98 (m)
6.57 (d, J = 9.7)
6.03 (d, J = 9.7)
1.29 (s)
1.12 (s)
0.80 (s)
0.98 (s)
1.80 (s)
3.44 (m)
3.95 (m)
6.61 (d, J = 10.3)
6.28 (d, J = 10.3)
1.16 (s)
1.08 (s)
0.83 (s)
1.16 (s)
1.86 (s)
3.46 (d-like)
3.72 (d-like)
1.12 (s)
6.60 (d, J = 10.3)
6.23 (d, J = 10.3)
1.26 (s)
1.19 (s)
0.83 (s)
1.01 (s)
1.84 (s)
3.47 (d-like)
3.73 (d-like)
1.12 (s)
1.08 (s)
1.04 (s)
29
1.27 (s)
1.21 (s)
1.34 (s)
1.33 (s)
30
(Tig)
7.09 (m)
(Ang)
5.92 (m)
(Tig)
7.13 (m)
(Tig)
7.13 (m)
21-O-acyl
3'''''
4'''''
1.63 (d-like)
1.93 (br-s)
2.05 (d-like)
1.96 (br-s)
1.66 (d-like)
1.97 (br-s)
1.66 (d-like)
1.97 (br-s)
5'''''
22-O-Ac
2''''''
1.92 (s)
1.91 (s)
1.77 (s)
1.78 (s)
3-O-GlcA
1'
4.97 (d, J = 6.8)
(Gal)
5.71 (d, J = 7.7)
4.93 (d, J = 6.7)
(Gal)
5.68 (d, J = 7.6)
4.92 (d, J = 7.1)
(Glc)
5.87 (d, J = 6.8)
4.96 (d, J = 7.1)
(Gal)
5.71 (d, J = 6.8)
2'-O- glycoside
1''
3'-O-Ara
1'''
6.14 (d-like)
6.05 (s)
6.13 (d-like)
6.02 (s)
6.15 (d-like)
6.06 (s)
6.15 (d-like)
6.06 (s)
2'''-O-Rha
1''''
28-O-Glc
1'''''''
4.76 (d, J = 5.8)
4.77 (d, J = 5.8)
Measured in pyridine-d5 for 1-4 at 600 MHz.
7

ACCEPTED MANUSCRIPT
Table 2
13C NMR Data of floraassamsaponin I–IV (1–4)
1
2
3
4
position
-GlcA
1
2
3
4
position
38.8
26.5
89.9
39.7
55.8
18.5
33.1
40.2
46.9
36.8
23.9
38.8
26.5
89.9
39.7
55.8
18.5
33.1
40.2
46.9
36.8
23.9
39.0
26.6
89.7
39.7
55.6
18.8
36.7
41.4
47.1
37.0
24.0
39.0
26.6
89.8
39.6
55.6
18.7
36.6
41.3
47.0
36.9
23.9
1
3-
1'
2'
3'
4'
5'
6'
Ο
105.5 105.6 105.4 105.4
2
79.3
82.8
71.3
77.5
79.3
82.8
71.3
77.5
79.3
82.9
71.4
77.6
79.2
83.0
71.3
77.6
3
4
5
6
172.0 172.2 172.0 171.9
(Gal) (Gal) (Glc) (Gal)
103.8 103.8 103.0 103.7
73.6
75.3
70.0
76.9
62.5
7
8
2'-O-glycoside
9
1''
73.7
75.2
70.0
77.0
62.6
76.5
78.3
72.6
78.2
63.5
73.6
75.3
70.0
76.9
62.5
10
2''
11
3''
124.3 124.3 125.3 125.4
142.3 142.3 143.7 143.7
12
4''
13
5''
41.7
34.6
67.3
46.9
41.0
46.9
36.4
78.8
75.7
28.0
16.9
15.7
16.8
27.5
73.7
29.5
20.3
41.7
34.6
67.2
46.9
41.1
46.9
36.2
78.6
76.0
28.0
16.9
15.8
16.9
27.5
74.0
29.5
20.3
48.3
72.7
67.5
47.7
40.9
46.9
36.4
79.3
74.0
28.1
16.8
15.8
17.5
21.1
63.1
29.5
20.0
(Tig)
48.3
72.6
67.4
47.8
40.8
46.9
36.3
79.2
74.1
28.0
16.9
15.8
17.5
21.1
63.1
29.5
20.0
(Tig)
14
6''
15
3'-O-Ara
1'''
101.2 101.3 101.3 101.2
16
77.0
73.5
69.1
65.6
77.1
73.5
69.2
65.6
77.1
73.8
68.8
65.4
77.0
73.5
68.9
65.7
17
2'''
18
3'''
19
4'''
20
5'''
21
2'''-O-Rha
1''''
102.4 102.5 102.3 102.4
22
72.6
72.7
73.9
70.2
18.3
72.6
72.7
74.0
70.1
18.4
72.5
72.6
74.0
70.2
18.4
72.4
72.6
74.1
70.0
18.3
23
2''''
24
3''''
25
4''''
26
5''''
27
6''''
28
28-O-Glc
1'''''''
2'''''''
3'''''''
4'''''''
5'''''''
6'''''''
104.5 104.5
29
75.2
78.5
71.6
78.1
62.9
75.3
78.4
71.6
78.3
62.9
30
(Tig) (Ang)
21-O-acyl
1'''''
2'''''
3'''''
4'''''
5'''''
22-O-Ac
1''''''
2''''''
168.0 167.8 168.1 168.1
129.4 128.9 129.4 129.5
137.0 137.2 137.0 137.0
14.2
12.4
15.9
21.0
14.2
12.4
14.2
12.4
171.5 171.3 170.8 170.8
21.1 20.7 20.7
21.1
Measured in pyridine-d5 for 1-4 at 150 MHz.
8

ACCEPTED MANUSCRIPT
Floraassamsaponins III (3), IV (4), and V (5), which were obtained as a white
21
D
21
D
amorphous powder with negative optical rotations (3: [
α
]
–11.6, 4: [
α
]
–21.3,
22
D
5: [
α
]
–15.8 in MeOH). Absorption bands due to hydroxy, α,β-unsaturated ester,
carboxyl, and ether components are shown in their IR spectra. FABMS in the
positive-ion mode of 3–5 revealed a quasimolecular ion peak [M+Na]+ at m/z 1269
from which the common molecular formula C60H94O27 was determined via each
13
HRMS and C NMR data. Acid hydrolysis of 3 yielded
L-arabinose,
D
-glucuronic
acid,
D
-glucose, and
-arabinose,
L
-rhamnose. On the other hand, acid hydrolysis of 4 and 5
1
yielded
L
D
-galactose,
D
-glucuronic acid, and
L
-rhamnose. The H NMR
(Table 1 and 2) and 13C NMR (Table 3 and 4) spectra (pyridine-d5) of 3−5, showed
signals assignable to a R1-barrigenol part,14 four glycosyl moieties {a
glucopyranosyl moiety [3: 5.87 (1H, d, J = 6.8 Hz H-1'')] or a -galactopyranosyl
moiety [4: 5.71 (1H, d, J = 6.8 Hz H-1''), 5:
together with -arabinopyranosyl moiety, a
an
1.66 (3H, d-like, H3-4'''''), 1.97 (3H, br-s, H3-5'''''), 7.13 (1H, m, H-3''''')] or an
angeloyl moiety [5: 2.03 (3H, br-s, H3-5'''''), 2.13 (3H, d-like, H3-4'''''), 6.00 (1H, m,
β-D-
δ
β-D
δ
δ
5.71 (1H, d, J = 7.0 Hz H-1'')]
α
-L
β
-D
-glucuronopyranosyl moiety, and
α-L-rhamnopyranosyl moiety}, and two acyl groups {a tigloyl moiety [3 and 4:
δ
δ
H-3''''')] together acetyl moiety}. The proton and carbon signals due to the aglycone
1
part including the acyl moiety in the H and 13C NMR spectra of 4 and 5 were
superimposable on those of chakasaponin III (for 4)15 and saniculasaponin III (for
5).16 The proton and carbon signals assignable to the oligoglycoside moiety at 3-
position are superimposable on those of floratheasaponin E (for 4 and 5).9 On the
other hand, the proton and carbon signals of 3 are in agreement with those of 4,
except for 2'-O-glycoside. Furthermore, the positions of the acyl moieties of 3–5
were confirmed by the DQF COSY and HMBC experiments. In addition, the
structure of the oligoglycoside moiety of 3 determined by the DQF COSY and
1
HMBC experiments and comparison of the H and 13C NMR date with those of
known compounds, yuchasaponins A and C with 3-O-
β
-D
-glucuronopyranosyl and
the structures of
2'-O- -glucopyranosyl Consequently,
β-D
moieties.17
9

ACCEPTED MANUSCRIPT
floraassamsaponins III (3), IV (4), and V (5) were characterized as shown.
Floraassamsaponins VI (6), VII (7) and VIII (8), isolated as a white amorphous
22
powder with negative specific rotation (6: [
α
]
22 –13.2, 7: [
α
]
22 –22.7, 8: [
α]
D
–
D
D
35.9 in MeOH). The common molecular formula C60H94O26 of 6–8 was determined
individually from the quasimolecular ion peak [m/z 1253 (M+Na)+] in positive-ion
FABMS and HRMS measurement. Acid hydrolysis of 6–8 yielded
L-arabinose, D-
1
13
glucose,
D
-glucuronic acid, and
L
-rhamnose. The H NMR (Table 2) and C NMR
(Table 3 and 4) spectra (pyridine-d5) of 6−8, showed signals assignable to a
barringtogenol C part,13 four glycosyl moieties, and two acyl groups {a tigloyl moiety
for 6 and 8 or an angeloyl moiety for 7 together acetyl moiety}. The proton and
1
13
carbon signals due to the aglycone part including the acyl moiety in the H and C
NMR spectra of 6 and 7 were superimposable on those of floratheasaponins G and
D,9 respectively, while the proton and carbon signals assignable to the oligoglycoside
moiety at 3-position of 6 and 7 are in agreement with those of 3. In addition, the
proton and carbon signals in the 1H and 13C NMR spectra of 8 were superimposable
on those of 6 except for the signals around the 22- and 28-postions of 8. The position
of the two acyl groups of 8 were determined by HMBC experiments, which showed
long-range correlations between the following proton and carbons: H-21 and C-1''''';
H2-28 and C-1''''''. On the basis of those findings and the detail DQF COSY and
HMBC experiments, the structures of floraassamsaponins VI (6), VII (7) and VIII (8)
were characterized as shown.
10

ACCEPTED MANUSCRIPT
Table 3
1H NMR Data of floraassamsaponin V–VIII (5–8)
5
6
7
8
position
3.23 (dd-like)
5.50 (br. s)
4.40 (m)
3.25 (dd-like)
5.42 (br. s)
1.63 (m)
3.27 (dd-like)
5.41 (br. s)
1.66 (m)
3.23 (dd-like)
5.46 (br. s)
1.69 (m)
3
12
15
1.90 (m)
1.88 (m)
1.91 (m)
4.19 (m)
4.47 (m)
4.48 (m)
4.42 (m)
16
21
22
23
24
25
26
27
28
6.62 (d, J = 10.3)
6.23 (d, J = 10.3)
1.26 (s)
1.19 (s)
0.82 (s)
1.00 (s)
1.85 (s)
3.46 (d-like)
3.72 (d-like)
1.11 (s)
6.61 (d, J = 10.3)
6.28 (d, J = 10.3)
1.18 (s)
1.10 (s)
0.80 (s)
0.85 (s)
1.87 (s)
3.41 (d-like)
3.65 (d-like)
1.11 (s)
6.64 (d, J = 10.3)
6.24 (d, J = 10.3)
1.18 (s)
1.09 (s)
0.80 (s)
0.85 (s)
1.85 (s)
3.41 (d-like)
3.64 (d-like)
1.10 (s)
6.49 (d, J = 9.6)
4.54 (d, J = 9.6)
1.17 (s)
1.08 (s)
0.82 (s)
0.98 (s)
1.84 (s)
3.25 (d-like)
4.21 (d-like)
1.12 (s)
29
1.31 (s)
1.34 (s)
1.32 (s)
1.30 (s)
30
(Ang)
6.00 (m)
(Tig)
7.11 (m)
(Ang)
6.00 (m)
(Tig)
7.04 (m)
21-O-acyl
3'''''
4'''''
2.13 (d-like)
2.03 (br-s)
1.67 (d-like)
1.97 (br-s)
2.13 (d-like)
2.03 (br-s)
1.70 (d-like)
1.96 (br-s)
5'''''
22-O-Ac
2''''''
1.78 (s)
1.92 (s)
1.94 (s)
28-O-Ac
2''''''
1.93 (s)
3-O-GlcA
1'
4.96 (d, J = 7.1)
(Gal)
5.71 (d, J = 7.0)
4.91 (d, J = 7.1)
(Glc)
5.86 (d, J = 6.2)
4.91 (d-like)
(Glc)
5.86 (d, J = 6.0)
4.92 (d-like)
(Glc)
5.84 (d, J = 6.0)
2'-O-glycoside
1''
3'-O-Ara
1'''
6.14 (d-like)
6.06 (s)
6.15 (d-like)
6.08 (s)
6.17 (d-like)
6.09 (s)
6.16 (d-like)
6.05 (s)
2'''-O-Rha
1''''
Measured in pyridine-d5 for 5-8 at 600 MHz.
11

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Table 4
13C NMR Data of floraassamsaponin V–VIII (5–8)
5
6
7
8
position
-GlcA
5
6
7
8
position
39.0
26.6
89.5
39.7
55.6
18.8
36.7
41.4
47.1
37.0
24.0
38.9
26.5
89.9
39.8
55.7
18.4
33.1
40.1
46.9
36.7
23.8
38.8
26.5
89.9
39.7
55.7
18.4
33.1
40.1
46.9
36.7
23.8
38.8
26.5
89.7
39.6
55.7
18.4
33.1
40.0
46.9
36.7
23.8
1
3-
1'
2'
3'
4'
5'
6'
Ο
105.5 105.4 105.6 105.5
2
79.3
82.7
71.4
77.6
79.1
83.0
71.3
77.6
79.4
82.8
71.3
77.4
79.0
82.8
71.2
77.5
3
4
5
6
171.9 172.0 172.1 172.0
(Gal) (Glc) (Glc) (Glc)
103.7 102.9 103.0 103.0
73.6
75.2
70.1
76.9
62.6
7
8
2'-O-glycoside
9
1''
76.4
78.3
72.5
78.2
63.6
76.4
78.3
72.7
78.3
63.6
76.4
78.4
72.6
78.2
63.5
10
2''
11
3''
125.4 124.0 124.0 123.7
143.7 142.7 142.8 142.7
12
4''
13
5''
48.3
72.6
67.5
47.8
40.9
46.8
36.2
78.8
74.0
28.0
16.9
15.8
17.5
21.2
63.2
29.4
20.1
41.7
34.7
68.1
48.0
40.1
47.2
36.5
79.4
74.3
27.8
16.7
15.6
16.9
27.5
63.7
29.6
20.2
41.7
34.6
68.1
48.0
40.0
47.2
36.3
79.1
74.4
27.8
16.8
15.6
16.7
27.5
63.8
29.5
20.3
41.8
34.6
67.6
47.0
40.5
47.2
36.3
81.6
71.4
27.4
17.0
15.7
17.0
27.4
66.5
29.8
20.1
(Tig)
14
6''
15
3'-O-Ara
1'''
101.2 101.1 101.3 101.2
16
76.9
73.5
69.0
65.7
77.0
73.5
69.1
65.8
77.0
73.4
68.9
65.4
76.6
73.6
69.0
65.4
17
2'''
18
3'''
19
4'''
20
5'''
21
2'''-O-Rha
1''''
102.4 102.2 102.3 102.2
22
72.6
72.7
74.0
70.0
18.3
72.5
72.7
74.0
70.0
18.3
72.5
72.7
74.0
70.2
18.3
72.5
72.6
74.0
70.0
18.3
23
2''''
24
3''''
25
4''''
26
5''''
27
6''''
28
29
30
(Ang) (Tig) (Ang)
21-O-acyl
1'''''
2'''''
3'''''
4'''''
5'''''
22-O-Ac
1''''''
2''''''
28-O-Ac
1''''''
2''''''
167.9 168.0 167.9 168.5
128.9 129.5 129.0 129.8
137.1 137.0 137.2 136.8
15.9
20.7
14.3
12.4
15.9
21.0
14.1
12.4
170.7 171.1 171.0
21.0 20.9 20.9
170.7
20.8
Measured in pyridine-d5 for 5-8 at 150 MHz.
12

ACCEPTED MANUSCRIPT
Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by
aggregation of 42-mer amyloid β-protein (Aβ42).18 Previously, various constituents
such as flavonoids and tannins inhibited Aβ42 aggregation.19 However, to the best of
our knowledge, there is no report about the inhibitory effects of acylated triterpene
glycosides. In the present study, the n-BuOH soluble fraction showed the inhibitory
effects of Aβ42 aggregation. Therefore, inhibitory effects of acylated triterpene
glycosides, floraassamsaponins 2, 3, 4, and 7, on Aβ42 aggregation were examined.
As the results, compounds 3, 4 and 7 significantly inhibited the aggregation of Aβ42.
[inhibition (%) 3: 73.4±8.0 (p<0.01), 4: 68.8±8.4 (p<0.01) and 7: 56.9±13.2 (p<0.01)
at 100 µM, respectively]. Their effects were almost equivalent that of positive control,
morin [inhibition (%): 54.0±11.2 (p<0.01) at 100 µM]. Compound 2 had weak
effects [inhibition (%): 18.3±5.4 (p<0.01) at 100 µM]. The detailed experiments
including in vivo study should be studied further.
In conclusion, eight new acylated oleanane-type triterpenoid saponins,
floraassamsaponins I (1), II (2), III (3), IV (4), V (5), VI (6), VII (7) and VIII (8) were
isolated from the flower buds of C. sinensis var. assamica cultivated in India. In the
present study, floraassamsaponins I and II with a glucose at 28-position were
obtained as major compounds from the flower buds of C. sinensis var. assamica. In
the past, acylated triterpene oligoglycosides with sugars at 28-position have not been
isolated from Camellia plants including the flower buds of C. sinensis var. sinensis.
This finding is interesting from the perspective of chemotaxonomy of Camellia
plants. In addition, further study for the development of acylated oleanane-type
triterpenoid saponins as the AD therapy are expected.
3. Experimental
The following instruments were used to obtain physical data: specific rotations,
Horiba SEPA-300 digital polarimeter (l = 5 cm); IR spectra, Shimadzu FTIR-8100
13

ACCEPTED MANUSCRIPT
spectrometer; FABMS and HRFABMS, a JEOL JMS-SX 102A mass spectrometer;
1H NMR spectra, JEOL ECA-600K (600 MHz) spectrometers with tetramethylsilane
as an internal standard; 13C NMR spectra, and JEOL ECA-600K (150 MHz)
spectrometers with tetramethylsilane as an internal standard; HPLC detector,
Shimadzu RID-6A refractive index detector and SPD-10Avp UV-VIS detectors; and
HPLC column, Cosmosil 5C18-MS-II (Nacalai Tesque Inc., 250 × 4.6 mm i.d.) and
(250 × 20 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, 0.25 mm) (ordinary phase) and Silica gel RP-18
F254S (Merck, 0.25 mm) (reversed phase); reversed-phase HPTLC, precoated TLC
plates with Silica gel RP-18 WF254S (Merck, 0.25 mm); and detection was achieved
by spraying with 1% Ce(SO4)2–10% aqueous H2SO4 followed by heating.
3.1. Plant Material
Flower buds of C. sinensis var. assamica were cultivated in India, in 2009. The plant
was identified by one of the authors (M. Y.). A voucher of the plant is on file in our
laboratory [FCSA-1].
3.2. Extraction and Isolation
The dried flower buds of C. sinensis var. assamica (687.4 g) were extracted three
times with MeOH for three hours under reflux. Evaporation of the solvent under
reduced pressure provided a MeOH extract (280.4 g, 40.8%). A part of the MeOH
extract (270.4 g) from the extract was partitioned into an EtOAc–H2O (1:1, v/v)
mixture to furnish an EtOAc-soluble fraction (17.3 g, 2.6%) and an aqueous phase.
The aqueous phase was further extracted with n-BuOH to give an n-BuOH-soluble
14

ACCEPTED MANUSCRIPT
fraction (92.1 g, 13.9%) and an H2O-soluble fraction (144.8 g, 21.9%). The n-BuOH
fraction (78.9 g) was subjected to ordinary-phase silica gel column chromatography
(CC) [2.4 kg, → CHCl → CHCl –MeOH–H O (7:3:1 → 65:35:10) → MeOH] to
3
3
2
give 5 fractions {Fr. 1 (10.3 g), Fr. 2 (2.9 g), Fr. 3 (31.6 g), Fr. 4 (22.9 g), Fr. 5 (5.1
g)}. Fraction 3 (30.3 g) was subjected to reversed-phase silica gel CC [600 g,
MeOH:H2O (20:80 →30:70 →40:60 →50:50 →60:40 →70:30 →80:20
→90:10→ MeOH)] to give 17 fractions {Fr. 3-1 (20.7 mg), Fr. 3-2 (18.6 g), Fr. 3-3
(1.3 g), Fr. 3-4 (419.7 mg), Fr. 3-5 (1.3 g), Fr. 3-6 (238.2 mg), Fr. 3-7 (568.2 g), Fr.
3-8 (1.0 g), Fr. 3-9 (5.7 g), Fr. 3-10 (282.9 mg), Fr. 3-11 (172.3 mg), Fr. 3-12 (392.2
mg), Fr. 3-13 (899.4 mg), Fr. 3-14 (471.2 mg), Fr. 3-15 (148.3 mg), Fr. 3-16 (80.5
mg), Fr. 3-17 (30.7 mg)}. Fraction 3-8 (1.0 g) was separated by HPLC [MeOH–H2O
in 1% AcOH (70:30)] to give 1 (35.4 mg), 2 (77.8 mg), 3 (18.0 mg), 4 (29.6 mg), 5
(19.9 mg), 6 (23.2 mg), 9 (58.6 mg) and 10 (62.5 mg). Fraction 3-9 (1.0 g) was
separated by HPLC [MeOH–H2O in 1% AcOH (70:30)] to give 6 (17.1 mg), 7 (18.1
mg), 8 (34.7 mg), 9 (49.3 mg), 10 (17.1 mg) and 11 (41.7 mg). The known
compounds were identified by comparison of their physical data ([
NMR, MS) with reported values.
α
]D, 1H NMR, 13C
21
D
3.2.1. Floraassamsaponin I (1). White amorphous powder; [
α
]
–18.3º (c 0.8,
1
MeOH); IR (KBr) vmax 3400, 1734, 1717, 1698, 1078 cm-1; H NMR (pyridine-d5,
13
600 MHz)
δ
given in Table 1; C NMR (pyridine-d5, 150 MHz)
δ
given in Table 3
and 4; positive-ion FABMS m/z 1415 [M + Na]+; HRFABMS m/z 1415.6458 (calcd
for C66H104O31Na [M + Na]+, 1415.6459).
3.2.2. Floraassamsaponin II (2). White amorphous powder; [α]
21 –12.2º (c 0.8,
D
1
MeOH); IR (KBr) vmax 3400, 1734, 1716, 1697, 1078 cm-1; H NMR (pyridine-d5,
13
600 MHz)
δ
given in Table 1; C NMR (pyridine-d5, 150 MHz)
δ
given in Table 3
and 4; positive-ion FABMS m/z 1415 [M + Na]+; HRFABMS m/z 1415.6462 (calcd
for C66H104O31Na [M + Na]+, 1415.6459).
15

ACCEPTED MANUSCRIPT
3.2.3. Floraassamsaponin III (3). White amorphous powder; [α]
21 –11.6º (c 0.5,
D
1
MeOH); IR (KBr) vmax 3400, 1734, 1718, 1700, 1078 cm-1; H NMR (pyridine-d5,
13
600 MHz)
δ
given in Table 1; C NMR (pyridine-d5, 150 MHz)
δ
given in Table 3
and 4; positive-ion FABMS m/z 1269 [M + Na]+; HRFABMS m/z 1269.5885 (calcd
for C60H94O27Na [M + Na]+, 1269.5880).
3.2.4. Floraassamsaponin IV (4). White amorphous powder; [α]
21 –21.3º (c 0.8,
D
1
MeOH); IR (KBr) vmax 3400, 1734, 1716, 1698, 1078 cm-1; H NMR (pyridine-d5,
13
600 MHz)
δ
given in Table 1; C NMR (pyridine-d5, 150 MHz)
δ
given in Table 3
and 4; positive-ion FABMS m/z 1269 [M + Na]+; HRFABMS m/z 1269.5876 (calcd
for C60H94O27Na [M + Na]+, 1269.5880).
22
D
3.2.5. Floraassamsaponin V (5). White amorphous powder; [
α
]
–15.8º (c 1.1,
1
MeOH); IR (KBr) vmax 3400, 1734, 1717, 1700, 1078 cm-1; H NMR (pyridine-d5,
13
600 MHz)
δ
given in Table 2; C NMR (pyridine-d5, 150 MHz)
δ
given in Table 3
and 4; positive-ion FABMS m/z 1269 [M + Na]+; HRFABMS m/z 1269.5890 (calcd
for C60H94O27Na [M + Na]+, 1269.5880).
3.2.6. Floraassamsaponin VI (6). White amorphous powder; [α]
22 –13.2º (c 0.8,
D
1
MeOH); IR (KBr) vmax 3400, 1734, 1719, 1700, 1076 cm-1; H NMR (pyridine-d5,
13
600 MHz)
δ
given in Table 2; C NMR (pyridine-d5, 150 MHz)
δ
given in Table 3
and 4; positive-ion FABMS m/z 1253 [M + Na]+; HRFABMS m/z 1253.5941 (calcd
for C60H94O26Na [M + Na]+, 1253.5931).
3.2.7. Floraassamsaponin VII (7). White amorphous powder; [α]
22 –22.7º (c 0.8,
D
1
MeOH); IR (KBr) vmax 3400, 1732, 1717, 1700, 1078 cm-1; H NMR (pyridine-d5,
13
600 MHz)
δ
given in Table 2; C NMR (pyridine-d5, 150 MHz)
δ
given in Table 3
16

ACCEPTED MANUSCRIPT
and 4; positive-ion FABMS m/z 1253 [M + Na]+; HRFABMS m/z 1253.5917 (calcd
for C60H94O26Na [M + Na]+, 1253.5931).
3.2.8. Floraassamsaponin VIII (8). White amorphous powder; [α]
22 –35.9º (c 0.2,
D
1
MeOH); IR (KBr) vmax 3400, 1734, 1716, 1698, 1078 cm-1; H NMR (pyridine-d5,
13
600 MHz)
δ
given in Table 2; C NMR (pyridine-d5, 150 MHz)
δ
given in Table 3
and 4; positive-ion FABMS m/z 1253 [M + Na]+; HRFABMS m/z 1253.5935 (calcd
for C60H94O26Na [M + Na]+, 1253.5931).
3.2.9. Acid Hydrolysis and Monosaccharide Composition of Floraassamsaponin
I –VIII. To determine the absolute configurations of constituent monosaccharides of
1–8, the reported method10 was used with slight modifications as follows.
Compounds 1–8 (1.0–2.0 mg) were dissolved in 5% aqueous H2SO4–1,4-dioxane
(1:1, v/v, 2.0 mL), and each solution was heated at 90 °C for 3 h. After extraction
three times with EtOAc, the aqueous layer was neutralized with Amberlite IRA-400
(OH– form). After drying in vacuo, the residue was dissolved in pyridine (0.1 mL)
containing L-cysteine methyl ester hydrochloride (0.5 mg) and heated at 60 °C for 1 h.
A solution of o-tolylisothiocyanate (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 5C18-AR-II (Nacalai Tesque), 250 × 4.6 mm i.d.
(5
flow rate: 0.8 mL/min; column temperature: 35 °C] to identify the derivatives of
constituent monosaccharides ( -galactose, -glucose, -glucuronic acid, -arabinose
and -rhamnose) in 1–8 by comparison of their retention times with those of
authentic samples (tR: -galactose; 44.1 min, -galactose; 46.9 min, -glucose; 51.6
min, -glucose; 50.0 min, -glucuronic acid; 52.7 min, -glucuronic acid; 44.0 min,
µm); mobile phase: MeCN–0.05M H3PO4 (18:82, v/v); detection: UV (250 nm);
D
D
D
L
L
D
L
D
L
D
L
17

ACCEPTED MANUSCRIPT
D
-arabinose; 63.4 min,
L-arabinose; 58.4 min, D-rhamnose; 88.4 min, L-rhamnose;
92.7 min).
3.2.10. Aβ42 aggregation assay
The inhibitory effects of test compounds against Aβ42 aggregation were evaluated
with Thioflavin T (Th-T) method using SensoLyte Thiofavin T β-Amyloid
Aggregation Kit (ANA SPEC) with a slight modification according to the
manufacturer's instructions. Briefly, 2 µL of Th-T (2 mM) were added to each wells
of 384 black plate (Nunc). Next, 1 µL of test compounds and 17 µL of Aβ42 peptide
solutions were added. After 30 min incubation at 37 °C, the fluorescence intensity
(ex: 440 nm and em: 480 nm) was measured with a fluorescence plate reader
(FLUOstar OPTIMA, BMG Labtechnologies).
Acknowledgments
This work was partly supported in part by a Ministry of Education, Culture, Sports,
Science and Technology (MEXT)-Supported Program for the Strategic Research
Foundation at Private Universities and by JSPS KAKENHI Grant Number 26460135.
References and Notes
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19 Ono, K.; Hamaguchi, T.; Naiki, H.; Yamada, M. Biochim. Biophys. Acta 2006,
1762, 575–586.
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Legends
Figure 1. Structure of saponin constituents isolated from C. sinensis var. assamica
Figure 2. Important 2D-NMR Correlation of 1
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Graphical Abstract
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