New ellagitannins from Tiencha and their inhibitory activity against dental plaque

Article abstract of DOI:10.1007/s11418-017-1090-9

Four new galloyl-oxygen-diphenyl (GOD)-type ellagitannins, brambliins A–D (1–4), were isolated from the leaves of Rubus suavissimus. Their structures were elucidated by extensive spectroscopic analyses and the absolute configurations of 1–4 were determined by chemical and phytochemical evidence. These GOD-type ellagitannins inhibited the formation of dental plaque, which is beneficial for oral hygiene.

Full text of DOI:10.1007/s11418-017-1090-9

J Nat Med
DOI 10.1007/s11418-017-1090-9
ORIGINAL PAPER
New ellagitannins from Tiencha and their inhibitory activity
against dental plaque
1
1
1
Fusako Nakano
•
Shozo Miyoshi
•
Toshiyuki Murakami
Received: 5 March 2017 / Accepted: 21 April 2017
Ó The Japanese Society of Pharmacognosy and Springer Japan 2017
Abstract Four new galloyl-oxygen-diphenyl (GOD)-type
ellagitannins, brambliins A–D (1–4), were isolated from
the leaves of Rubus suavissimus. Their structures were
elucidated by extensive spectroscopic analyses and the
absolute configurations of 1–4 were determined by chem-
ical and phytochemical evidence. These GOD-type ellagi-
tannins inhibited the formation of dental plaque, which is
beneficial for oral hygiene.
these oral diseases, and inhibition of dental plaque was
closely involved with a reduction in bad breath [6, 7].
While some research has shown the inhibitory effect of
Tiencha and phenolic compounds such as ellagic acid and
catechins on dental plaque, the effect of ellagitannins has
not yet been researched [8–10]. In this study, we conducted
the isolation and structure elucidations of galloyl-oxygen-
diphenyl (GOD)-type ellagitannins, brambliins A–D (1–4),
and examined the inhibitory effect of R. suavissimus leaf
extract on the formation of dental plaque.
Keywords Rubus suavissimus Á Tiencha Á GOD-type
ellagitannin Á Brambliin Á Anti-dental plaque
Results and discussion
Introduction
Dried leaves of R. suavissimus were extracted with H O
2
The leaves of Rubus suavissimus S. Lee (Rosaceae) have a
long history of use in traditional Chinese herbal medicine
and health-promoting herbal tea (Tiencha). Its beneficial
effects were not well elucidated in the past; however,
recent scientific studies have reported its potent anti-an-
giogenic and anti-allergic activities [1, 2]. In addition to
pharmacological studies, chemical investigations have
found that gallotannins, ellagitannins, flavonoids and
diterpenes are the major constituents of R. suavissimus
under reflux. The extract was concentrated and suspended
in water, then subjected to a Diaion HP-20 column (H O),
2
and eluted with aqueous 20% EtOH as a tannin-rich frac-
tion. The 20% EtOH eluate (8.5 g) was separated by a Diol
silica-gel column (CHCl –MeOH), a Sephadex LH-20
3
column (MeOH–H O ? acetone–H O), and preparative
2
2
HPLC (MeOH–H O) to give four new dimeric ellagitan-
2
nins named brambliins A (1), B (2), C (3), and D (4)
together with known GOD-type ellagitannins, sanguiin H-4
(5) [11], sanguiin H-5 (6) [12], 2,3-O-hexahydroxydiphe-
noyl-4,6-O-sanguisorboyl-(a/b)-glucose (7) [13], rubusa-
viin A (8) [3], ellagic acid (9) [14], brevifolin carboxylic
acid (10) [15], and rubusoside (11) [4] (Fig. 1).
[
3, 4]. Previously, we reported that the water extract of the
leaves of R. suavissimus produced a significant reduction in
halitosis [5]. Halitosis is caused by a variety of factors such
as dental decay, diet, periodontal disease, stomatitis, or
internal disease. In particular, dental plaque is related to
Structure determination of brambliins A–D (1–4)
&
Fusako Nakano
f-nakano@maruzenpcy.co.jp
Brambliin A (1) was isolated as a pale brown amorphous
powder. The IR spectrum of 1 showed an absorption band
-
at 1731 cm ascribable to carbonyl and strong absorption
1
1
Maruzen Pharmaceuticals Co.Ltd., 1089-8 Sagata, Shin-ichi,
Fukuyama, Hiroshima 729-3102, Japan
-
bands at 3374 and 1046 cm suggestive of a glycosidic
1
123

J Nat Med
Fig. 1 The chemical structures of compounds from R. suavissimus
structure. The negative-ion ESI-TOF–MS of 1 showed a
trimethylsilyl thiazolidine derivatives [17]. Analysis of
NMR data suggested that 1 contained two glycosyl moi-
eties for the presence of two anomeric protons [d 5.39 (1H,
-
quasimolecular ion peak at m/z 1265.1356 [M–H] and the
molecular formula C H O was determined by high-
54 42 36
1
resolution MS measurement. Acid hydrolysis of 1 with 5%
aqueous sulfuric acid (H SO ) liberated gallic acid, ellagic
d, J = 8.0 Hz), d 6.09 (1H, d, J = 8.5 Hz)] in the H- and
1
3
C-NMR spectra. The aliphatic signals in the range d
3.24–6.10 with large coupling constants (J = 8.3–13.3 Hz)
2
4
acid, sanguisorbic acid dilactone, and D-glucose [16]. This
monosaccharide was identified by HPLC analysis of
indicated that both of the two b-D-glucopyranosyl moieties
1
23

J Nat Med
4
adopted the C conformation. The geminal-coupled glu-
13
HHDP group. Comparison of the C-NMR data for 2 with
1
cose H-6 protons resonated at d 5.23 (1H, dd, J = 13.3,
that of 1 showed acylation shifts of 2 around the 2- and
3-positions of the D-glucopyranosyl moiety. Furthermore,
long-range correlations were observed between glc-2-H [d
5.07 (t-like)] and HHDP-7-C (dc 169.9), and between glc-
3-H [d 4.92 (m)] and HHDP-7 -C (dc 170.1) in the HMBC
experiment. The orientations of the anomer groups were
6
.2 Hz) and 3.70 (1H, d-like), and a large difference in the
chemical shifts of these signals was commonly observed in
the ellagitannins with the hexahydroxydiphenoyl (HHDP)
or the sanguisorboyl groups at the 4,6-positions in glucose
0
[
18]. A pair of characteristic meta-coupled aromatic dou-
0
blets [d 7.25, 7.00 (each d, J = 2.0 Hz)] along with a one-
proton singlet [d 6.59 (s)] suggested the presence of a
sanguisorboyl group [3]. The location of a HHDP group
and a sanguisorboyl group with glucose was assigned from
the HMBC correlations of glc-1-H [d 5.39 (1H, d,
assigned as 1b and 1 b based on the coupling patterns of
0
glc-1-H [d 5.90 (d, J = 8.1 Hz)] and glc-1 -H [d 6.07 (d,
J = 8.3 Hz)]. The CD spectra caused by atropisomerism of
the chiral HHDP and sanguisorboyl groups in 2 also
appeared to have a strong positive Cotton effect at 243 nm
and a negative one at 264 nm, respectively, which was in
agreement with those of (S)-HHDP and (S)-sanguisorboyl
groups [19]. Therefore, the structure of 2 was fully deter-
J = 8.0 Hz)] with galloyl-7-C (d 166.2); glc-4-H [d 4.60
C
(
t-like)] with sang-7-C (dC 167.0); glc-H-6 [d 3.70 (1H,
0
d-like) and d 5.23 (1H, dd, J = 13.3, 6.2 Hz)] with sang-7 -
C (d 167.6); glc-1 -H [d 6.09 (1H, d, J = 8.5 Hz)] with
sang-7 -C (d 170.1); glc-2 -H [d 5.09 (dd, J = 9.5,
8
0
00
mined as 1-O-galloyl-2,3-O-(S)-HHDP-4,6-O-(S)-[7 -O-
0
C
0
0
0
0
[2 ,3 -O-(S)-HHDP-b-D-glucopyranosyl]-sanguisorboyl]-b-
D-glucopyranose.
C
0
.5 Hz)] with HHDP-7-C (d 171.1); and glc-3 -H [d 5.17
C
0
(
dd, J = 9.5 Hz)] with HHDP-7 -C (dC 169.5) (Fig. 2).
On the basis of HR-ESI–MS, 2 and 3 have the same
1
13
molecular formula (C H O ). The H- and C-NMR
68 48 44
Based on the circular dichroism (CD) spectrum, 1 exhibited
a strong positive Cotton effect at 240 nm and a negative
one at 262 nm. Therefore, the atropisomerism of the chiral
HHDP and sanguisorboyl groups was determined to be
S [19]. From the evidence described above, the structure of
spectra of 3 were similar to those of 2, indicating that 3 was
also a GOD-type dimer tannin. The 2D-NMR spectra of 3
was similar to that found in 2, except for the HMBC cor-
0
0
relations of glc-4 -H with HHDP-7-C and glc-6 -H with
0
0
0
0
0
1
was elucidated as 1-O-galloyl-4,6-O-[7 -O-(S)-[2 ,3 -O-
S)-HHDP-b-D-glucopyranosyl]-sanguisorboyl]-b-D-gluco-
pyranose.
HHDP-7 -C observed in 3. The data indicated that one of
0
0
(
the HHDP group was present at glc-4 /glc-6 . These signals
were completely similar to those of sanguiin H-10, which
was isolated from the Rubus species. However, the anomer
configurations of 3 revealed both b-configurations from
their J values (9.3 and 9.0 Hz), while sanguiin H-10
-
A quasimolecular ion peak at m/z 1567.1427 ([M-H] ;
calcd. for 1567.1440) was observed in the negative-ion
HR-ESI–MS of 2, and indicated the molecular formula
1
13
0
C H O . The H- and C-NMR spectra of 2 were
6
appeared as 1-a-D-glucopyranose and 1 -b-D-glucopyranose
moiety [20]. Consequently, the structure of brambliin C (3)
8 48 44
similar to those of 1, and revealed the presence of another
1
1
Fig. 2 H– H COSY and
HMBC correlations of 1
123

J Nat Med
was determined as 1-O-galloyl-2,3-O-(S)-HHDP-4,6-O-
00 0 0
Table 1 Inhibitory effect of dental plaque
[
7 -O-(S)-[4 ,6 -O-(S)-HHDP-b-D-glucopyranosyl]-san-
Compound no.
Inhibition (%)
guisorboyl]-b-D-glucopyranose.
a
Tiencha
61.1 ± 6.0**
Brambliin D (4) was also isolated as a pale brown
amorphous powder, liberating gallic acid, ellagic acid,
sanguisorbic acid dilactone, and D-glucose on acid
hydrolysis with 5% aqueous H SO [16, 17]. The molec-
1
31.1 ± 8.9*
17.4 ± 5.0
17.4 ± 5.0
26.0 ± 8.9
17.8 ± 1.2
32.9 ± 15.9
54.2 ± 7.9
17.9 ± 1.2
28.0 ± 9.4
–
2
3
2
4
ular formula C H O of 4 was clarified from a quasi-
8
4
9 58 57
-
molecular ion peak (m/z 2037.1512 [M-H] ; calcd. for
5
2
037.1562) by high-resolution MS analysis observed in the
6
1
13
negative-ion ESI–MS. The H- and C-NMR spectra of 4
showed 11 carbonyl groups, which were determined to be a
galloyl group, two HHDP groups, and two sanguisorboyl
7
8
9
1
1
groups, respectively. H– H-COSY and HSQC were used
for the assignments of the sugar proton and carbon signals,
and the locations of the acyl groups were determined by the
HMBC correlations between the sugar proton signals and
acyl carbonyl carbons (Fig. 3). On the basis of these
findings, the structure of 4 was determined to be 1-O-gal-
11
Catechin (positive control)
45.8 ± 7.4**
a
Data are mean ± SE from three experiments (n = 3)
p \ 0.05, ** p \ 0.01 vs control
*
00
0
0
loyl-2,3-O-(S)-HHDP-4,6-O-[7 -O-(S)-[2 ,3 -O-(S)-HHDP-
0
0
4
,6 -O-(S)-sanguisorboyl-b-D-glucopyranosyl]-sanguisor-
compared with catechin (positive control). In addition, the
inhibitory effect was not related to molecular size. Previous
research has shown the strong anti-dental plaque activity of
R. suavissimus extract and ellagic acid [8, 9]. However, this
is the first study on anti-dental plaque activity of GOD-type
ellagitannins. These results indicated that although GOD-
type ellagitannins are active principles, the effect each has
on dental plaque is varied. Therefore, further consideration
will be needed to yield any findings about stronger acti-
vators contained in Tiencha.
boyl]-b-D-gluco-pyranose.
Anti-dental plaque activity
The results of the formation of dental plaque assay indi-
cated that four new GOD-type tannins (50 lg/mL) have
strong effects (inhibition: 17.8–31.1%), respectively, while
it is known that Tiencha has a strong effect (61.1%) [8]. As
shown in Table 1, ellagitannins showed strong inhibition
1
1
Fig. 3 H– H COSY and HMBC correlations of 4
1
23

J Nat Med
Conclusion
Plant material
Our investigation into the constituents of the tannin
fraction from R. suavissimus led to the isolation of four
new GOD-type ellagitannins (1–4), named brambliin A–
D. Although several GOD-type ellagitannins have
already been isolated from Rosaceae plants, this appears
to be the first example of the isolation of both b-con-
formation dimer tannins from R. suavissimus. The anti-
dental plaque activity was evaluated for the compounds
The leaves of R. suavissimus were purchased in Qingping
market, China in 2013, and identified by Prof. Xia Nianhe
of South China Botanical Garden.
Extraction and isolation
The leaves of R. suavissimus (220 g) were extracted with
H
2
O under reflux. After filtering the residue, the extract was
concentrated in vacuo to give the H O extract (55.6 g). The
O extract was suspended in water and subjected to Diaion
HP-20 (H O), and eluted with aqueous 20% EtOH and EtOH.
The 20% EtOH eluate was separated by a Diol silica gel
column (CHCl –MeOH) to give 5 fractions (1.1–1.5). Fr. 1.3
(
1–4, 6–9 and 11) and they were found to be effective.
2
We determined that GOD-type ellagitannins are active
principles and that the use of Chinese sweet tea, Tien-
cha, as a complementary functional food can be assumed
to help reduce the occurrence of halitosis caused by
dental plaque.
H
2
2
3
was subjected to the Sephadex LH-20 column (MeOH–H2-
O ? acetone–H O), then a 50% acetone eluted fraction was
2
followed by Develosil RPAQUEOUS-AR-5 (MeOH–
H O = 18:82) to yield sanguiin H-4 (5, 8.7 mg, 0.004%),
2
Experimental
General
sanguiiin H-5 (6, 33 mg, 0.015%), and brevifolin carboxylic
acid (10, 47 mg, 0.021%). Fr. 1.3 (1.6 g) was chro-
matographed on the Sephadex LH-20 column (MeOH–H2-
O ? acetone–H O), and a 50% acetone eluted fraction was
followed by Develosil RPAQUEOUS-AR-5 (MeOH–
2
The following instruments were used to obtain physical
data: IR spectra, JASCO FT/IR-460 spectrometer; CD
spectra, JASCO J-1500 spectrometer; MS and high-reso-
H O = 20:80) to afford the new dimeric ellagitannin named
2
brambliin A (1, 23 mg, 0.010%) together with known GOD-
type ellagitannins, 2,3-O-hexahydroxydiphenoyl-4,6-O-san-
guisorboyl-(a/b)-glucose (7, 94 mg, 0.042%) and rubusaviin
A (8, 20 mg, 0.009%). Fr. 1.5 was applied to the Sephadex
LH-20 column (MeOH–H O ? acetone–H O), then a 50%
1
lution MS, Waters Xevo G2-Tof MS; H-NMR spectra,
1
3
JEOL ECS-400 (400 MHz) spectrometer; C-NMR spec-
tra, JEOL ECS-400 (100 MHz) spectrometer; analytical
HPLC, Agilent 1200 Series [Column: Develosil RPAQU-
EOUS-AR-5 (150 9 4.6 mm i.d., Nomura Chemical Co.,
Ltd.), Wakosil II 5C18HG (250 9 4.6 mm i.d., Wako Pure
Chemical Industries, Ltd.); analytical UHPLC, Waters
ACQUITY UPLC [Column: ACQUITY UPLC BEH C18
2
2
acetone eluted fraction was purified by HPLC on Develosil
RPAQUEOUS-AR-5 (MeOH–H O = 25:75), yielding
brambliin B (2, 44 mg, 0.020%), brambliin C (3, 24 mg,
.011%), and brambliin D (4, 16 mg, 0.007%). The MeOH
2
0
(
50 9 1.8 mm i.d., Waters)]; preparative HPLC, YMC LC-
forte/R [Column: Develosil RPAQUAOUS-AR-5
250 9 20 mm i.d., Nomura Chemical Co., Ltd.), YMC
eluate from the Diaion HP-20 column was separated by the
ODS column (MeOH-H O), and further applied to a silica gel
2
(
column (CHCl –MeOH), then recrystallized in MeOH to give
3
triart C18 (250 9 20 mm i.d., YMC Co., Ltd)]. The fol-
lowing materials were used for chromatography: synthetic
absorbent resin column chromatography, Diaion HP-20
rubusoside (11, 2.4 g, 1.1%).
21
Brambliin A (1): pale brown amorphous powder, [a]D
-
58.8° (c = 0.06, MeOH). High-resolution negative-ion
(
Mitsubishi Chemical Co., Ltd.); normal-phase DIOL silica
-
ESI-TOF–MS: calcd for C H O [M–H] : 1265.1378;
5
4 41 36
gel column chromatography (Fuji Silysia, Ltd); Sephadex
LH-20 column chromatography (GE Healthcare Life Sci-
ence Co., Ltd.); Toyo Pearl HW-40C column chromatog-
raphy (Tosoh Bioscience Co., Ltd.); normal-phase HPTLC,
silica gel 60 F254 (Merck); reversed-phase HPTLC, silica
gel RP-18WF254s (Merck). Detection on HPTLC was
achieved by spraying with 10% aqueous H SO solution
found: 1265.1356. IR (KBr): 3375, 1732, 1617, 1515,
-1
1
446, 1213, 1046 cm
;
CD (MeOH) [h] (nm):
8.2 9 10 (240), –4.3 9 10 (262), ?3.2 9 10 (283),
5
5
4
?
-
5
1.5 9 10 (310); H-NMR (methanol-d ): 3.24 (1H, t,
1
4
J = 8.3 Hz, glc-3-H), 3.39 (1H, dd, J = 10.1, 6.2 Hz, glc-
0
5
3
-H), 3.48 (1H, t-like, glc-2-H), 3.61 (1H, m, glc-5 -H),
0
2
4
.70 (1H, d-like, glc-6-H), 3.76 (1H, m, glc-6 -H), 3.81
0
followed by heating. Gallic acid was purchased from Wako
Pure Chemical Industries, Ltd., brain heart infusion (BHI)
broth was purchased from Eiken Chemical Co., Ltd., and
all other chemicals used in this experiment were of ana-
lytical and reagent grade.
0
(
1H, m, glc-4 -H), 3.89 (1H, m, glc-6 -H), 4.60 (1H, t-like,
0
glc-4-H), 5.09 (1H, dd, J = 9.5, 8.5 Hz, glc-2 -H), 5.17
0
(
1H, t-like, glc-3 -H), 5.23 (1H, dd, J = 13.3, 6.2 Hz, glc-
6
-H), 5.39 (1H, d, J = 8.3 Hz, glc-1-H), 6.10 (1H, d,
0
0
J = 8.5 Hz, glc-1 -H), 6.43 (1H, s, HHDP-3 -H), 6.59 (1H,
123

J Nat Med
0
0
s, sang-3-H), 6.65 (1H, s, HHDP-3-H), 7.00 (1H, d,
4)-7-C], 169.2 [sang (glc-6)-7 -C], 169.5 [HHDP (glc-2 )-
0
00
J = 2.0 Hz, sang-2 -H), 7.09 (2H, s, galloyl-2,6-H), 7.25
7-C], 169.9 [HHDP (glc-2)-7-C], 170.1 [HHDP (glc-3)-7 -
0 0
0
0
13
1H, d, J = 2.0 Hz, sang-6 -H). C-NMR (methanol-d4):
(
C], 171.1 [HHDP (glc-3 )-7 -C].
0
0
23
Brambliin C (3): pale brown amorphous powder, [a]D
-80.1° (c = 0.1, MeOH). High-resolution negative-ion
6
5
1.89 (glc-6 -C), 64.0 (glc-6-C), 67.9 (glc-4 -C), 73.4 (glc-
0
-C), 73.6 (glc-4-C), 74.5 (glc-2-C), 76.1 (glc-2 -C), 76.3
0
0
0
-
ESI-TOF–MS: calcd for C H O [M–H] : 1567.1440;
(
glc-3-C), 79.4 (glc-5 -C), 81.0 (glc-3 -C), 92.9 (glc-1 -C),
6
8 48 44
0
9
1
6.1 (glc-1-C), 107.7 (HHDP-3-C), 107.9 (HHDP-3 -C),
08.5 (sang-3-C), 110.4 (galloyl-2, 6-C), 110.4, 110.6
found: 1567.1436. IR (KBr): 3385, 1732, 1617, 1515,
-
1
1446, 1197, 1048 cm
5
;
CD (MeOH) [h] (nm):
?6.6 9 10 (239), -2.0 9 10 (264), -1.9 9 10 (284),
0
0
5
3
(
sang-2 -C), 112.1, 115.4, 115.4, 116.3, 116.5 (galloyl-1-
00
5
1
C), 120.0, 120.3 (sang-1 -C), 121.0, 126.0, 126.2, 126.8,
-9.2 9 10 (308); H-NMR (methanol-d ): 3.70 (1H,
4
0
0
0
1
1
1
35.6 (HHDP-5-C), 137.3 (HHDP-5 -C), 137.4 (sang-5-C),
0
t-like, glc-3 -H), 3.85 (1H, t-like, glc-2 -H), 3.93 (1H, dd,
0
0
37.5, 138.9, 139.7 (galloyl-4-C), 140.5 (sang-4 -C),
0
J = 9.3, 6.8 Hz, glc-5-H), 4.01 (2H, d-like, glc-6, 6 -H),
0
41.0, 142.6, 144.7, 145.2 (HHDP-4-C), 145.7 (HHDP-4 -
0
4.04 (1H, dd, J = 9.0, 6.3 Hz, glc-5 -H), 4.62 (1H, t-like,
0
C), 145.9, 146.0, 146.5 (galloyl-3, 5-C), 146.8 (sang-5 -C),
glc-3-H), 4.84 (1H, m, glc-4-H), 5.02 (1H, t-like, glc-4-H),
0
0
0
00
1
1
1
49.2 (sang-3 -C), 166.2 (galloyl-7-C), 167.0 (sang-7 -C),
0
5.09 (1H, t-like, glc-2-H), 5.15 (1H, dd, J = 13.4, 6.3 Hz,
0
67.6 (sang-7-C), 169.5 (HHDP-7 -C), 170.1 (sang-7 -C),
71.1 (HHDP-7-C).
glc-6 -H), 5.74 (1H, dd, J = 13.4, 6.8 Hz, glc-6-H), 5.75
(1H, d, J = 9.3 Hz, glc-1-H), 5.79 (1H, d, J = 9.0 Hz, glc-
0 0
2
4
Brambliin B (2): pale brown amorphous powder, [a]D
1 -H), 6.32 [1H, s, HHDP (glc-3)-3 -H], 6.40 [1H, s, HHDP
0
0
0
-
72.7° (c = 0.1, MeOH). High-resolution negative-ion
(glc-4 )-3-H], 6.45 [1H, s, HHDP (glc-6 )-3 -H], 6.68 [1H,
0
-
ESI-TOF–MS: calcd for C H O [M–H] : 1567.1440;
s, HHDP (glc-2)-3-H], 6.73 [1H, s, sang (glc-6)-3 -H], 6.98
0 00
6
8 48 44
found: 1567.1427. IR (KBr): 3384, 1731, 1617, 1514,
-
(2H, s, galloyl-2, 6-H), 7.23 [1H, s, sang (glc-1 )-2 -H],
1
0 00
13
7.27 [1H, s, sang (glc-1 )-6 -H]. C-NMR (methanol-d4):
1
446, 1196, 1049 cm
;
CD (MeOH) [h] (nm):
3.5 9 10 (243), -1.8 9 10 (264), ?1.8 9 10 (285),
5
5
4
0 0
62.5 (glc-6-C), 63.2 (glc-6 -C), 68.3 (glc-4-C), 72.0 (glc-4 -
?
-
4
1
7.3 9 10 (310); H-NMR (methanol-d ): 3.62 (1H, ddd,
0
0
C), 72.5 (glc-5 -C), 72.7 (glc-2 -C), 72.9 (glc-5-C), 74.5
0
4
0
J = 9.4, 4.9, 2.0 Hz,glc-5 -H), 3.77 (1H, dd, J = 12.7,
0
(glc-3 -C), 74.5 (glc-2-C), 76.1 (glc-3-C), 91.3 (glc-1-C),
0 0
0
4
.9 Hz, glc-6 -H), 3.78 (1H, t-like, glc-4 -H), 3.83 (1H, m,
95.0 (glc-1 -C), 106.2 [HHDP (glc-3)-3 -C], 106.8 [HHDP
0
0
0
glc-5-H), 3.86 (1H, d-like, glc-6-H), 3.93 (1H, dd,
0
(glc-6 )-3 -C], 107.0 [HHDP (glc-4 )-3-C], 107.3 [HHDP
0
J = 12.7, 2.0 Hz, glc-6 -H), 4.92 (1H, m, glc-3-H), 4.95
0
(glc-2)-3-C], 107.6 [sang (glc-6)-3 -C], 109.1 (galloyl-2,
0 00 0 00
(
1H, m, glc-4-H), 5.07 (2H, t-like, glc-2-H, glc-2 -H), 5.12
0
6-C), 110.4 [sang (glc-1 )-2 -C], 111.7 [sang (glc-1 )-6 -
0 0
C], 113.9 [HHDP (glc-4 )-1-C], 114.1 [HHDP (glc-3)-1 -C,
(
1H, t-like, glc-3 -H), 5.44 (1H, dd, J = 13.3, 6.2 Hz, glc-
0
0
6
-H), 5.90 (1H, d, J = 8.1 Hz, glc-1-H), 6.07 (1H, d,
0
HHDP (glc-4 )-1-C], 114.4, 114.5 [sang (glc-6)-1 -C],
0
0
0
0
J = 8.3 Hz, glc-1 -H), 6.20 [1H, s, HHDP (glc-2 )-3 -H],
115.0 [HHDP (glc-6 )-1 -C], 115.6 [HHDP (glc-2)-1-C],
0
6
3
.33 [1H, s, HHDP (glc-2)-3-H], 6.42 [1H, s, HHDP (glc-
0
117.9 [sang (glc-6)-2 -C], 118.3 (galloyl-1-C), 120.0 [sang
0 00
0
0
)-3 -H], 6.65 [1H, s, HHDP (glc-3 )-3 -H], 6.67 [1H, s,
0
(glc-1 )-1 -C], 124.4, 124.6, 124.8, 124.9, 125.7, 134.9,
0 0
135.8 [HHDP (glc-6 )-5 -C], 136.0, 136.1 [HHDP (glc-3)-
0
sang (glc-6)-3 -H], 6.97 [1H, d, J = 1.8 Hz, sang (glc-1 )-
0
0
0
0
0
2
-H], 7.07 (2H, s, galloyl-2, 6-H), 7.11 [1H, d-like, sang
5 -C, HHDP (glc-4 )-5-C], 136.2 [sang (glc-6)-5 -C], 136.4
[HHDP (glc-2)-5-C], 137.6, 137.8, 139.3, 140.9 [sang (glc-
0
00
13
glc-1 )-6 -H]. C-NMR (methanol-d ): 61.9 (glc-6 -C),
0
(
4
0
0
00
6
3.6 (glc-6-C), 67.9 (glc-4 -C), 69.9 (glc-4-C), 74.5 (glc-5-
0
1 )-4 -C], 141.0 [galloyl-4-C], 143.1, 143.4, 144.1, 144.3,
C), 76.0 (glc-2 -C), 76.1 (glc-2-C), 77.2 (glc-3-C), 79.4
0
144.4, 144.4, 144.6, 144.6, 144.8, 145.2 (galloyl-3, 5-C),
0
0
0
0
00
00
(
glc-5 -C), 81.0 (glc-3 -C), 92.7 (glc-1 -C), 92.9 (glc-1-C),
145.5 [sang (glc-1 )-5 -C], 147.0 [sang (glc-1 )-3 -C],
164.5 [galloyl-7-C], 165.5 [sang (glc-4)-7-C], 165.6 [sang
0
1
1
1
07.5 [HHDP (glc-2)-1-C], 107.7 [HHDP (glc-3)-1 -C],
0
0
0
00
0
07.9 [HHDP (glc-3 )-1 -C], 108.5 [HHDP (glc-2)-1-C],
0
(glc-1 )-7 -C], 167.8 [sang (glc-6)-7 -C], 168.0 [HHDP
0
00
0
08.6 [sang (glc-6)-1 -C], 110.0 [sang (glc-1 )-2 -C], 110.5
0
(glc-4 )-7-C], 168.3 [HHDP (glc-2)-7-C], 168.6 [HHDP
0 0 0
00
(
galloyl-2, 6-C), 112.6 [sang (glc-1 )-6 -C], 115.2, 115.4,
0
(glc-3)-7 -C], 168.7 [HHDP (glc-6 )-7 -C].
24
Brambliin D (4): pale brown amorphous powder, [a]D
-32.2° (c = 0.1, MeOH). High-resolution negative-ion
1
15.4, 115.5, 116.2. 116.4 [sang (glc-6)-1 -C], 119.5 (gal-
loyl-1-C), 119.6, 120.9, 126.0, 126.1, 126.3, 126.8, 137.3,
0
0
-
ESI-TOF–MS: calcd for C H O [M–H] : 2037.1562;
1
37.3 [HHDP (glc-3)-5 -C], 137.4 [sang (glc-6)-5 -C,
0
8
9 58 57
0
HHDP (glc-3 )-5 -C, HHDP (glc-2)-5-C], 137.6, 139.1,
0
found: 2037.1503. IR (KBr): 3392, 1739, 1617, 1516, 1446,
-1
00
5
1
39.3, 140.9 (galloyl-4-C), 141.5 [sang (glc-1 )-4 -C],
42.6, 144.5, 144.7, 145.4, 145.7, 145.8 [HHDP (glc-2)-4-
1196, 1048 cm ; CD (MeOH) [h] (nm): ?4.4 9 10
(243), -2.6 9 10 (267), -9.2 9 10 (286), -1.4 9 10
5
3
5
1
0
0
1
(307); H-NMR (methanol-d ): 3.79 (1H, m, glc-5-H), 3.86
C], 145.9 [HHDP (glc-3)-4 -C], 146.7 [sang (glc-6)-4 -C],
0
4
00
0
1
46.7 [galloyl-3, 5-C], 148.7 [sang (glc-1 )-3 -C], 166.2
0
(1H, m, glc-5 -H), 3.93 (2H, dd, J = 13.0, 6.0 Hz, glc-6-H,
00
0
0
[
sang (glc-1 )-7 -C], 166.3 (galloyl-7-C), 167.0 [sang (glc-
glc-6 -H), 4.89 (3H, m, glc-3-H, glc-3 -H, glc-4-H), 4.96
1
23

J Nat Med
0
(
1H, m, glc-4 -H), 5.07 (1H, t-like, glc-2-H), 5.13 (1H,
0
[column: ACQUITY UPLC BEH C18, 50 9 2.1 mm i.d,;
mobile phase: MeCN-0.1% aqueous formic acid (10:
90 ? 40: 60), detection UV (254 nm); flow rate: 0.4 mL/
min; column temperature: 40 °C] to determine the presence
of gallic acid (t : 0.85 min), sanguisorbic acid dilactone (t :
t-like, glc-2 -H), 5.39 (1H, dd, J = 13.0, 6.0 Hz, glc-6-H),
0
5
.55 (1H, dd, J = 13.0, 6.0 Hz, glc-6 -H), 5.85 (1H, d,
0
J = 8.7 Hz, glc-1-H), 5.95 (1H, d, J = 8.4 Hz, glc-1 -H),
0
6
3
.15 [1H, s, HHDP (glc-3)-3 -H], 6.29 [1H, s, HHDP (glc-
0
R
R
0
)-3 -H], 6.32 [1H, s, HHDP (glc-2)-3-H], 6.35 [1H, s,
0
1.65 min), and ellagic acid (9, t : 2.19 min). The aqueous
R
-
layer was neutralized with Amberlite IRA-400 (OH form)
0
HHDP (glc-2 )-3-H], 6.63 [1H, s, sang (glc-6)-3 -H], 6.71
0
0
[
1H, s, sang (glc-6 )-3 -H], 6.92 [1H, d, J = 1.7 Hz, sang
00
and the resin was removed by filtration. After removal of the
solvent in vacuo, the residue was dissolved in pyridine
(0.5 mL) containing L-cysteine methyl ester hydrochloride
(0.5 mg) and heated at 60 °C for 1 h. A solution of o-tolyl
isothiocyanate (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 HPLC [column: Wakosil II 5C18HG,
250 9 4.6 mm i.d.; mobile phase: MeCN-0.1% aqueous TFA
(18: 82); detection UV (250 nm); flow rate: 0.8 mL/min;
column temperature: 35 °C] to identify the derivative of D-
glucose in brambliins A–D (1-4) by comparisons of retention
times with those of authentic samples (D-glucose; 13.27 min.
L-glucose; 14.46 min).
(
glc-1)-2 -H], 7.20 (2H, s, galloyl-2, 6-H), 7.07 [1H, s, sang
0 00 00
(
glc-1 )-2 -H], 7.09 [1H, d, J = 1.7 Hz, sang (glc-1)-6 -H],
0 00
13
.15 [1H, s, sang (glc-1 )-6 -H]. C-NMR (methanol-d4):
7
6
5
0
0
2.3 (glc-6-C, glc-6 -C), 68.6 (glc-4-C, glc-4 -C), 73.1 (glc-
0
0
-C), 73.3 (glc-5-C), 74.7 (glc-2-C), 74.8 (glc-2 -C), 76.0
0
0
(
[
[
glc-3-C, glc-3 -C), 91.3 (glc-1-C), 91.5 (glc-1 -C), 106.2
0
HHDP (glc-2)-3-C), 106.5 [HHDP (glc-2 )-3-C], 107.2
0
0
0
0
HHDP (glc-3)-3 -C, HHDP (glc-3 )-3 -C, sang (glc-6)-3 -
0
0
00
C], 107.5 [sang (glc-6 )-3 -C], 108.3 (sang-2 -C), 109.1
0
0
0
0
0
[
sang (glc-1 )-2 -C, galloyl-2, 6-C], 111.2 (sang-6 -C),
0 00
11.5 [sang (glc-1 )-6 -C], 113.8 [HHDP (glc-2)-1-C],
1
1
0
0
0
14.2 [HHDP (glc-3 )-1 -C], 114.3 [HHDP (glc-3)-1 -C,
0
HHDP (glc-2 )-1-C], 114.7, 115.3, 115.3, 117.6, 118.3
0
00
00
(
galloyl-1-C), 119.8 [sang (glc-1 )-1 -C], 120.4 (sang-1 -
Effect on formation of dental plaque
C), 124.5, 124.6, 124.7, 124.8, 134.0, 134.6, 136.0, 136.2,
0
0
0
0
1
1
1
1
37.6 [HHDP (glc-3 )-5 -C], 137.8 [sang (glc-6 )-5 -C],
Test tubes were weighed prior to the start of the experi-
ment, then a BHI broth (5.35 mL) was added and auto-
claved. After autoclaving for 15 min, the test sample
solution (0.15 mL) and a culture solution of Streptococcus
mutans 6715 (0.5 mL) was added to the BHI test tubes
(5.35 mL), incubated overnight at 37 °C and cultivated for
40 h at 37 °C. The final concentration of Tiencha in the
incubation mixture was 500 lg/mL, and of the isolated
compounds (1–4, 6–9, and 11) was 50 lg/mL. After the
supernant of the cultivation solution was removed, the test
tubes were washed with distilled water 3 times, and dried at
105 °C for 5 h. The test tubes were weighed [22].
38.1, 138.9, 139.4, 140.8 (galloyl-4-C), 141.1 [sang (glc-
0
00
)-4 -C], 141.4, 143.0, 143.1, 143.3, 143.4, 144.0, 144.1,
00
44.2, 144.3, 144.7, 144.8, 145.1 (sang-5 -C), 145.3 (gal-
0
00
00
loyl-3, 5-C), 145.5 [sang (glc-1 )-5 -C], 147.1 (sang-3 -C),
0 00
1
1
47.2 [sang (glc-1 )-3 -C], 164.7 [galloyl-7-C, sang (glc-
0
00
0
0
0
)-7 -C], 165.7 [sang (glc-3)-7 -C, sang (glc-3 )-7 -C, sang
0
0
0
(
glc-4)-7-C, sang (glc-4 )-7-C], 167.8 [sang (glc-6 )-7 -C],
0 0
67.9 [sang (glc-6)-7 -C], 168.2 [HHDP (glc-2 )-7-C],
1
1
00
68.6 [HHDP (glc-2)-7-C], 169.3 (sang-7 -C).
Preparation of sanguisorbic acid dilactone
The percent inhibition was calculated as
A solution of 7 (40 mg) in 5% aq. H SO (3 mL) was
2
4
%
Inhibition ¼ ð1 À ðCÀDÞ=ðAÀBÞÞ Â 100:
where A = weight of the test tube with H O after cultiva-
stirred under reflux for 2 h. After cooling, the reaction
mixture was extracted with ethyl acetate and the ethyl
acetate extract was washed with saturated aqueous
NaHCO and NaCl and dried over MgSO . Evaporation of
2
tion, B = weight of the test tube with H O before cultiva-
2
tion, C = weight of the test tube with test sample solution
after cultivation, and D = weight of the test tube with test
sample solution before cultivation.
3
4
the solvent from the filtrate subjected to HPLC [column:
YMC triart C18, 250 9 20 mm i.d.; mobile phase: MeCN-
0
.1% aqueous TFA (15: 85); flow rate: 9.99 mL/min] gave
Acknowledgements We are grateful to Prof. Yoshiki Kashiwada and
Mr. Kanji Niwa of Tokushima University for the measurement of CD
spectra for structure determination. We also thank Prof. Xia Nianhe of
South China Botanical Garden for plant determination.
sanguisorbic acid dilactone (4 mg). The structure was
identified by a comparison with reported values [21].
Acid hydrolysis of brambliins A–D (1–4)
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