Ellagic acid glycosides with hepatoprotective activity from traditional Tibetan medicine Potentilla anserina

Article abstract of DOI:10.1007/s11418-017-1137-y

Two new gallic acid glycosides, potentillanosides G (1) and H (2), were newly isolated from the methanol extract of the tuberous roots of Potentilla anserina (Rosaceae), together with a known compound, ellagic acid 3-O-α-l-rhamnopyranoside (3). Their structures were elucidated on the basis of chemical and physicochemical evidence. Among the constituents, potentillanoside H (2, IC₅₀?=?99.5?μM) was found to show hepatoprotective activity.

Full text of DOI:10.1007/s11418-017-1137-y

J Nat Med
DOI 10.1007/s11418-017-1137-y
NOTE
Ellagic acid glycosides with hepatoprotective activity
from traditional Tibetan medicine Potentilla anserina
Toshio Morikawa1,2 · Katsuya Imura1 · Yoshinori Akagi1 · Osamu Muraoka1,2
·
Kiyofumi Ninomiya1,2
Received: 5 August 2017 / Accepted: 18 September 2017
© The Japanese Society of Pharmacognosy and Springer Japan KK 2017
Abstract Two new gallic acid glycosides, potentil-
lanosides G (1) and H (2), were newly isolated from the
methanol extract of the tuberous roots of Potentilla anse‑
rina (Rosaceae), together with a known compound, ellagic
acid 3-O-α-l-rhamnopyranoside (3). Their structures were
elucidated on the basis of chemical and physicochemi-
cal evidence. Among the constituents, potentillanoside H
(2, IC50 = 99.5 μM) was found to show hepatoprotective
activity.
the local people [4, 5]. Previous chemical studies on this
plant material revealed the presence of several tannins [6],
favonoids [7], triterpenes [8–10], polysaccharides [2, 3], and
amino acids [4]. In addition, pharmacological activities of
the extracts and/or constituents, such as anti-mutagenic [6],
anti-hepatitis B virus [10], immunomodulatory [3], antitus-
sive [5], and expectorant activities [5], have been reported.
In the course of our studies on bioactive constituents from
the tuberous roots of P. anserina, we have reported the iso-
lation and structure determination of 21 triterpenes as well
as the protective efects of the methanol extract and several
triterpenes against liver injuries induced by d-galactosamine
(d-GalN)/lipopolysaccharide (LPS) in mice [1]. Further
separation of the constituents in the extract allowed us to
isolate two new ellagic acid glycosides, potentillanosides G
(1) and H (2), and a known compound, ellagic acid 3-O-α-
l-rhamnopyranoside (3). Here, we describe the isolation and
structure elucidation of 1 and 2 as well as the protective
efects of the isolates on d-GalN-induced cytotoxicity in
primary cultured mouse hepatocytes.
Keywords Potentillanoside · Ellagic acid glycoside ·
Potentilla anserina · Hepatoprotective activity · Rosaceae
Introduction
Potentilla anserina L. is a Rosaceae plant which is widely
distributed in the western areas of China, particularly in the
Qinghai–Tibetan Plateau. The roots of P. anserina have been
used to treat malnutrition, anemia, diarrhea, hemorrhage,
cough, and sputum in traditional Tibetan medicine [1–5].
For thousands of years, the roots have also been used in
food, such as congee with rice, which is a principal food of
Results and discussion
Isolation
In the present study, we isolated three ellagic acid glyco-
sides, potentillaosides G (1, 0.00098%) and H (2, 0.00033%)
and ellagic acid 3-O-α-l-rhamnopyranoside (3, 0.00048%)
[11], from the ethyl acetate (EtOAc)-soluble and methanol
(MeOH)-eluted fractions using normal-phase silica gel and
reversed-phase ODS CC, and finally preparative HPLC
(Fig. 1).
* Toshio Morikawa
morikawa@kindai.ac.jp
1
Pharmaceutical Research and Technology Institute, Kindai
University, 3-4-1 Kowakae, Higashi-osaka, Osaka 577-8502,
Japan
2
Antiaging Center, Kindai University, 3-4-1 Kowakae,
Higashi-osaka, Osaka 577-8502, Japan
Vol.:(0123456789)
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J Nat Med
Fig. 1 Structures of the isolates (1–8) from the tuberous roots of P. anserina
Structure determination of potentillanosides G (1)
and H (2)
(δC 137.5). In the nuclear Overhauser enhancement spec-
troscopy (NOESY) experiment, the nuclear Overhauser
efect (NOE) correlation was observed between H-1″ and
H-5, as shown in Fig. 2. In addition, methylation of 1 with
iodomethane (CH3I) in the presence of potassium carbon-
ate (K2CO3) gave 1b, which was also obtained by the same
methylation of 5 (Scheme 1). Therefore, the confguration
of the l-arabinofuranosyl linkage in 1 was the same as that
of 5, i.e., α-form. Acid hydrolysis of 1b produced 3,3′,4′-tri-
O-methylellagic acid (1c) [12, 23, 24] as the aglycone, thus
the structure of 1b was elucidated to be 3,3′,4′-tri-O-methyl-
ellagic acid 4-O-α-l-arabinofuranoside. On the basis of the
above-mentioned evidence, the structure of potentillanoside
G was determined to be 3-O-methylellagic acid 4-O-α-l-
arabinofuranoside (1).
Potentillanoside G (1) was obtained as an amorphous pow-
der with negative optical rotation ([α]2D7—21.0 in MeOH).
The UV spectrum of 1 exhibited absorption maxima at 254
and 354 nm, while the IR spectrum showed absorption bands
at 3385, 1710, 1611, and 1078 cm−1 ascribable to hydroxy,
carbonyl, and aromatic functionalities. In the positive-ion
FABMS profle, a quasimolecular ion peak was observed
at m/z 471 [M+Na]+, and HRFABMS analysis revealed the
molecular formula to be C20H16O12. The acid hydrolysis of
1 using 1 M hydrochloric acid (HCl)-1,4-dioxane (1:1, v/v)
produced 3-O-methylellagic acid (1a) [12] together with
l-arabinose, which was identifed by HPLC analysis [1,
13–21]. The 1H (Table 1) and 13C NMR (Table 2) spectra
(DMSO-d6) of 1, which were assigned using DEPT, 1H–1H
COSY, HSQC, and HMBC experiments, showed signals
assignable to a methoxy methyl [δ 4.00 (3H, s, 3-OCH3)]
and two aromatic protons [δ 7.42, 7.50 (1H each, both s,
H-5, 5′)] together with an arabinofuranosyl moiety [δ 5.53
(1H, br s, Ara(f)-H-1)]. The 1H and 13C NMR spectroscopic
properties of 1 were quite similar to those of ducheside B (5)
[22]. As shown in Fig. 2, the connectivities of the methoxy
and arabinofuranosyl moieties in 1 were characterized on
the basis of the HMBC experiment. Long-range correlations
were observed between H-5 (δ 7.42) and C-1 (δC 115.4), C-3
(δC 137.5), and C-7 (δC 159.6); H-5′ (δ 7.50) and C-1′ (δC
114.0), C-3′ (δC 139.9), and C-7′ (δC 160.4); H-1″ and C-4
(δC 149.2), and the methoxy methyl proton (δ 4.00) and C-3
Potentillanoside H (2), C25H24O16, was obtained as an
amorphous powder with negative optical rotation ([α]2D8—
52.9 in MeOH). Acid hydrolysis of 2 released ellagic acid
(8) [12, 23, 25, 26] together with l-rhamnose and l-arab-
inose, which were identifed by HPLC analysis. The 1H and
13C NMR (DMSO-d6, Tables 1, 2) spectra of 2 showed sig-
nals ascribable to an ellagic acid moiety [δ 7.60, 7.73 (both
1H, each s, H-5, 5′)] along with a rhamnopyranosyl [δ 5.56
(1H, br s, Rha-H-1), 1.09 (3H, d, J = 6.2 Hz, Rha-H3-6)] and
an arabinofuranosyl moiety [δ 5.65 (1H, br s, Ara(f)-H-1)].
Methyl ether derivatives 2a and 3a were obtained by the
methylation of 2 and 3, respectively, using the above-men-
tioned procedure. The NOE correlations of 2a and 3a in the
NOESY experiment were observed between the following
proton pairs: 4-OCH3 [2a: δ 4.02 (3H, s); 3a δ 4.02 (3H, s)]
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J Nat Med
Table 1 1H NMR spectroscopic data (600 MHz, DMSO-d6) for potentillanosides G (1) and H (2), 1b, 1c, 2a, and 3a
Position
1
1b
1c
2
2a
3a
5
5′
7.42 (s)
7.50 (s)
7.79 (s)
7.66 (s)
7.60 (s)
7.51 (s)
7.60 (s)
7.73 (s)
7.70 (s)
7.80 (s)
7.68 (s)
7.67 (s)
3-OCH3 4.00 (3H, s)
4-OCH3
3′-OCH3
4′-OCH3
4.11 (3H, s)
4.04 (3H, s)
4.02 (3H, s)
4.11 (3H, s)
4.02 (3H, s)
4.07 (3H, s)
4.02 (3H, s)
4.06 (3H, s)
4.02 (3H, s)
4.02 (3H, s)
3.98 (3H, s)
3-O-Rha
1
2
3
4
5
6
5.56 (br s)
4.08 (br s)
3.81 (dd, 3.1, 9.3)
3.34 (dd, 9.3, 9.5)
4.17 (dq, 9.5, 6.2)
1.09 (3H, d, 6.2)
5.48 (d, 1.7)
5.47 (br s)
4.08 (m)
3.78 (m)
3.33 (m)
4.11 (m)
4.06 (dd, 1.7, 3.5)
3.78 (dd, 3.5, 9.5)
3.33 (dd, 9.4, 9.5)
4.10 (m)
1.09 (3H, d, 6.2)
1.09 (3H, 6.2)
4 or 4′-O-Ara(f)
1
2
3
4
5
5.53 (br s)
4.13 (dd, 1.4, 3.8)
3.78 (dd, 3.8, 6.1)
5.67 (d, 1.4)
4.25 (dd, 1.4, 3.9)
3.88 (dd, 3.9, 6.4)
5.65 (br s)
4.34 (br d, ca. 4)
3.88 (dd, 3.8, 5.9)
5.68 (d, 1.6)
4.25 (dd, 1.6, 4.0)
3.88 (dd, 4.0, 6.4)
4.00 (ddd, 3.5, 5.7, 6.1) 3.98 (ddd, 3.4, 5.4, 6.4)
3.99 (ddd, 3.7, 5.5, 5.9) 3.99 (ddd, 3.4, 5.5, 6.4)
3.46 (dd, 5.7, 11.7)
3.52 (dd, 5.4, 12.0)
3.51 (dd, 5.5, 11.9)
3.52 (dd, 5.5, 12.0)
3.52 (dd, 3.5, 11.7)
3.63 (dd, 3.4, 12.0)
3.61 (dd, 3.7, 11.9)
3.62 (dd, 3.4, 12.0)
and H-5 [2a: δ 7.70 (1H, s); 3a: δ 7.68 (1H, s)]; and 4′-OCH3
[3a: δ 4.02 (3H, s)] and H-5′ [3a: δ 7.67 (1H, s)]. In addition,
acid hydrolysis of 2a and 3a gave the corresponding agly-
cones 4,3′-di-O-methylellagic acid (2b) [26] and 4,3′,4′-tri-
O-methylellagic acid (3b) [23, 27], respectively. The proton
and carbon signals due to the 3-O-α-l-rhamnopyranosyl part
in 2a were superimposable on those of 3a, while due to the
4′-O-α-l-arabinofuranosyl part in 2a also resembled those
of 1b. Based on these fndings and comparison of the NMR
data with those of the corresponding derivatives, the struc-
ture of potentillanoside H was determined to be ellagic acid
3-O-α-l-rhamnopyranosyl-4′-O-α-l-arabinofuranoside (2).
3-O-α-l-rhamnopyranoside (3). Among them, 2 was investi-
gated as a hepatoprotective constituent. The detailed mecha-
nisms of action as well as the structural requirements of
these ellagic acid derivatives for hepatoprotective activity
should be further examined.
Materials and methods
General
The following instruments were used to obtain spectroscopic
data: specifc rotation, Horiba SEPA-300 digital polarimeter
(l = 5 cm); UV spectra, Shimadzu UV-1600 spectrometer;
Inhibitory efects on d‑GalN‑induced cytotoxicity
1
in primary cultured mouse hepatocytes
IR spectra, Shimadzu FTIR-8100 spectrometer; H NMR
spectra, JNM-ECA600 (600 MHz) and JNM-ECS400
(400 MHz) spectrometers; 13C NMR spectra, JNM-ECA600
(150 MHz) and JNM-ECS400 (100 MHz) spectrometers
with tetramethylsilane as an internal standard; FABMS
and HRFABMS, JEOL JMS-SX 102A mass spectrom-
eter; HPLC detectors, Shimadzu RID-6A refractive index
and SPD-10A UV–VIS detectors and Shodex OR-2 opti-
cal rotation detector; HPLC columns, Cosmosil 5C18-MS-II
(Nacalai Tesque, Inc., Kyoto, Japan, 4.6 mm i.d. × 250 mm
and 20 mm i.d. × 250 mm) for analytical and preparative
purposes, respectively, and Kaseisorb LC NH2-60-5 (Tokyo
Kasei Co., Ltd., Tokyo, Japan, 4.6 mm i.d. × 250 mm) for
identifcation of the sugar part.
As a part of our eforts to characterize hepatoprotective
compounds from natural medicines, we have investigated
several constituents that showed inhibitory efect on d-GalN-
induced cytotoxicity in primary cultured hepatocytes [1, 14,
28–49]. As a continuation of the above study, hepatoprotec-
tive compounds from P. anserina were explored. The results
revealed that potentillanoside H (2, IC50 = 99.5 μM) shows
hepatoprotective activity (Table 3).
In conclusion, two new ellagic acid glycosides, poten-
tillanosides G (1) and H (2), were newly isolated from
the methanol extract of the tuberous roots of P. anse‑
rina together with the known compound, ellagic acid
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J Nat Med
Table 2 13C NMR
Position
1
1b
1ca
2
2a
3a
spectroscopic data (150 MHz,
DMSO-d6) for potentillanosides
G (1) and H (2), 1b, 1c, 2a,
and 3a
1
2
3
4
5
6
7
1′
2′
3′
4′
5′
6′
7′
115.4
142.6
137.5
149.2
114.4
112.9
159.6
114.0
142.6
139.9
152.1
110.6
112.9
160.4
60.9
113.5
142.0
141.2
151.0
112.0
112.2
158.2
112.9
141.1
141.0
154.3
107.7
112.5
158.3
61.4
111.0
141.0
139.9
152.5
111.6
112.3
157.5
112.3
141.5
140.5
153.7
107.5
114.5
157.8
60.9
111.3
142.7
136.8
153.1
111.5
113.9
159.1
114.6
136.3
142.8
146.8
112.4
106.5
158.7
112.8
142.1
137.7
154.8
107.8
112.2
158.2
113.7
141.4
142.2
151.2
112.1
113.6
158.2
113.1
141.2
137.7
154.6
107.9
112.8
158.3
113.4
142.0
141.2
154.3
107.9
112.3
158.2
3-OCH3
4-OCH3
3′-OCH3
4′-OCH3
57.1
61.6
56.9
61.4
57.1
61.3
56.8
61.2
56.8
3-O-Rha
1
2
3
4
5
6
102.6
70.2
70.5
71.6
70.7
17.7
103.3
70.2
70.5
71.5
70.9
17.7
103.3
70.2
70.5
71.5
70.9
17.7
4 or 4′-O-Ara(f)
1
2
3
4
5
107.5
80.7
77.1
87.1
62.0
107.6
82.1
76.6
86.2
61.0
107.9
81.5
76.8
86.3
61.2
107.7
82.2
76.7
86.3
61.2
aMeasured in DMSO-d6 [12]
The following experimental conditions were used for
column chromatography (CC): highly porous synthetic
resin, Diaion HP-20 (Mitsubishi Chemical Co., Tokyo,
Japan); normal-phase silica gel CC, silica gel 60N (Kanto
Chemical Co., Ltd., Tokyo, Japan; 63–210 mesh, spheri-
cal, neutral); reversed-phase ODS CC, Chromatorex ODS
DM1020T (Fuji Silysia Chemical, Ltd., Aichi, Japan;
100–200 mesh); TLC, pre-coated TLC plates with silica
gel 60F254 (Merck, Darmstadt, Germany, 0.25 mm) (nor-
mal-phase) and silica gel RP-18 WF254S (Merck, 0.25 mm)
(reversed-phase); reversed-phase HPTLC, pre-coated TLC
plates with silica gel RP-18 WF254S (Merck, 0.25 mm).
Detection was carried out by spraying 1% Ce(SO4)2—10%
aqueous H2SO4, followed by heating.
Plant material
This item has been described in a previous report [1].
Extraction and isolation
Potentillanosides G (1) and H (2) and ellagic acid 3-O-α-
l-rhamnopyranoside (3) were isolated from previously
reported fractions: fraction 10–3 (195.8 mg), originally
obtained from the EtOAc-soluble fraction (0.58%) of the
methanol extract (23.0%) from the dried tuberous roots of P.
anserina and fractions 4–5 (334.9 mg) and 4–6 (128.6 mg)
obtained from the MeOH-eluted fraction (0.73%) [1]. Frac-
tion 10–3 (195.8 mg), obtained from the EtOAc-soluble
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J Nat Med
fraction, was subjected to HPLC [MeOH—1% aqueous
acetic acid (AcOH) (35:65, v/v)] to give potentillanoside
G (1, 73.4 mg, 0.00098%) together with ellagic acid (8,
21.3 mg). Fraction 4 (4.05 g), obtained from the MeOH-
eluted fraction, was subjected to reversed-phase ODS CC
[125 g, MeOH–H2O (20:80 → 30:70 → 40:60 → 60:40 →
95:5, v/v) → MeOH → acetone] to give seven fractions [Fr.
4–1 (238.5 mg), Fr. 4–2 (334.3 mg), Fr. 4–3 [= l-tryptophan
(1.17 g, 0.0155%)], Fr. 4–4 (295.6 mg), Fr. 4–5 (334.9 mg),
Fr. 4–6 (128.6 mg), and Fr. 4–7 (1.30 g)], as described previ-
ously [1]. Fraction 4–5 (334.9 mg) was subjected to HPLC
[MeOH–1% aqueous AcOH (30:70, v/v)] to give potentil-
lanoside H (2, 24.7 mg, 0.00033%) together with quercetin
3-O-sambubioside (11.0 mg). Fraction 4–6 (128.6 mg) was
subjected to HPLC [MeOH—1% aqueous AcOH (30:70,
v/v)] to give ellagic acid 3-O-α-l-rhamnopyranoside (3,
35.5 mg, 0.00048%) [11] along with potentillanoside F
(18.3 mg) and ellagic acid 4-O-α-l-arabinofuranoside (4,
22.6 mg).
Fig. 2 1H–1H COSY, HMBC, and NOE correlations of 1 and 2
Scheme 1 Conversion of ellagic acid derivatives. Reagents and
conditions: i) 1 M HCl-1,4-dioxane (1:1, v/v), 80 °C, 1 h, 1a (78%
from 1, 85% from 5), 1c (88% from 1b), 8 (77% from 2, 86% form
3), 2b (88% from 2a), 3b (73% from 3a); ii) CH3I, K2CO3/DMSO,
r.t., 45 min, 1b (94% from 1, 94% from 5), 2a (99% from 2), 3a (91%
from 3)
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J Nat Med
Table 3 Inhibitory efect of the constituents of ellagic acid derivatives (1–8) on d-GalN-induced cytotoxicity in primary cultured mouse hepato-
cytes
Treatment
Inhibition (%)a
0 μM
IC50 (μM)
3 μM
− 1.4 1.2
10 μM
− 1.6 1.1
30 μM
100 μM
Potentillanoside G (1)
Potentillanoside H (2)
0.0 2.2
0.0 0.6
0.0 0.5
0.0 1.1
0.0 0.7
0.0 1.2
0.0 0.5
0.0 0.5
0.0 0.3
− 0.7 0.6
2.4 0.5
3.1 0.5
2.4 0.9
5.2 1.4
3.7 0.6
10.8 1.8
5.8 0.5
6.0 0.7
4.8 1.1
6.6 2.0
4.9 0.5
6.9 0.2
0.0 2.2
14.2 2.8b
14.2 1.3c
7.8 0.4
7.7 0.7
11.2 2.8c 50.7 3.6c
11.1 1.0c 26.3 2.1c
99.5
Ellagic acid 3-O-α-l-rhamnopyranoside (3)
Ellagic acid 4-O-α-l-arabinofuranoside (4) [1]
Ducheside B (5)
8.4 1.2
2.3 1.4
16.2 3.3c
3.7 0.9
Gallic acid (6) [1]
26.0 5.6c 35.9 7.5c
33.1 1.7c 65.8 4.1c
Gallic acid methyl ester (7) [1]
Ellagic acid (8) [1]
53.7
38.8
6.0 1.1
45.2 8.8c 77.0 5.5c
14.7 1.4c
Silybind [1]
aEach value represent the mean SEM, (n = 4)
Signifcantly diferent from the control group, bp < 0.05, cp < 0.01
dCommercial silybin was purchased from Funakoshi Co., Ltd. (Tokyo, Japan)
Potentillanoside G (1)
column, Kaseisorb LC NH2-60-5, 4.6 mm i.d. × 250 mm
(Tokyo Kasei Co., Ltd.); detection, optical rotation [Sho-
dex OR-2 (Showa Denko Co., Ltd., Tokyo, Japan); mobile
phase, CH3CN–H2O (85:15, v/v); fow rate 0.5 mL/min].
Identifcation of l-rhamnose [tR: 12.0 min (negative opti-
cal rotation)] from 2 and l-arabinose [tR: 16.2 min (pos-
itive optical rotation)] from 1 and 2 in the H2O-eluted
fraction was carried out by comparing the retention time
and optical rotation with those of authentic samples [1,
13–21]. Through a similar procedure, 1a [3.0 mg, 85%
from 5 (5.0 mg)], 3,3′-di-O-methylellagic acid [1c, 1.9 mg,
88% from 1b (3.0 mg)] [12, 23], 4,3′-di-O-methylellagic
acid [2b, 1.0 mg, 88% from 2a (2.0 mg)] [26], 4,3′,4′-tri-
O-methylellagic acid [3b, 1.0 mg, 73% from 3a (2.0 mg)]
[23, 27], and 8 [2.9 mg, 86% from 3 (5.0 mg)] were puri-
fed from each EtOAc-soluble fraction by normal-phase
silica gel CC.
An amorphous powder, [α]2D7—21.0 (c 0.10, MeOH); UV
[MeOH, nm (log ε)]: 254 (4.53), 354 (4.03); IR (KBr) vmax
:
3385, 1710, 1611, 1078 cm−1; 1H (600 MHz, DMSO-d6) and
13C NMR (150 MHz, DMSO-d6): shown in Tables 1 and 2;
Positive-ion FABMS m/z: 471 [M+Na]+; HRFABMS m/z:
471.0547 [M+Na]+ (calcd for C20H16O12Na, 471.0539).
Potentillanoside H (2)
An amorphous powder, [α]2D8—52.9 (c 0.10, MeOH); UV
[MeOH, nm (log ε)]: 247 (4.98), 352 (4.17); IR (KBr) vmax
:
3385, 1744, 1613, 1078 cm−1; 1H (600 MHz, DMSO-d6) and
13C NMR (150 MHz, DMSO-d6): shown in Tables 1 and 2;
positive-ion FABMS m/z: 603 [M+Na]+; HRFABMS m/z:
603.0967 [M+Na]+ (calcd for C25H24O16Na, 603.0962).
Acid hydrolysis of 1–3, 5, 1b, 2a, and 3a
Methylation of 1–3 and 5
A solution of 1 or 2 (each 5.0 mg) in 1 M HCl–1,4-di-
oxiane (1:1, v/v, 1.0 mL) was stirred at 80 °C for 1 h.
After cooling, the reaction mixture was neutralized with
Amberlite IRA-400 (OH− form) and the resin was removed
by fltration. After removal of the solvent from the fltrate
under reduced pressure, the residue was partitioned in
EtOAc–H2O (1:1, v/v), and the solvents removed in vacuo
from the EtOAc-soluble fraction and an aqueous phase,
respectively. The EtOAc-soluble fraction was purifed
by normal-phase silica gel CC [500 mg, hexane–EtOAc
(3:1, v/v)] to furnish 3-O-methylellagic acid (1a, 2.8 mg,
78%, from 1) [12] or ellagic acid (8, 2.0 mg, 77% from 2)
[12, 23, 25, 26]. In turn, the aqueous layer was subjected
to HPLC analysis under the following conditions: HPLC
A solution of 1 (5.0 mg) in DMSO (1.0 mL) was treated
with iodomethane (CH3I, 0.1 mL) in the presence of
K2CO3 (5.0 mg), and the mixture was stirred at room tem-
perature for 45 min. The reaction mixture was poured into
ice water and was extracted with EtOAc. The EtOAc layer
was successively washed with 5% aqueous HCl, saturated
aqueous NaHCO3, and brine, then dried over MgSO4 pow-
der, and fltrated under reduced pressure. The residue was
purifed by HPLC [MeOH–1% aqueous AcOH (60:40,
v/v)] to give 1b (5.0 mg, 94%). Through a similar proce-
dure, 1b [5.0 mg, 94% from 5 (5.0 mg)], 2a [3.1 mg, 99%
from 2 (3.0 mg)] and 3a [3.0 mg, 91% from 3 (3.0 mg)]
were obtained.
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J Nat Med
Compound 1b
added to 100 μL of the fresh medium containing d-GalN
(2 mM), with or without the test sample, and the hepato-
cytes were cultured for 44 h. The medium was exchanged
with 100 μL of the fresh medium, and 10 μL of MTT (5 mg/
mL in phosphate-bufered saline) solution was added to the
medium. After 4 h of cultivation, the medium was removed,
and 100 μL of isopropanol containing 0.04 M HCl was then
added to dissolve the formazan produced in the cells. The
optical density (O.D.) of the formazan solution was meas-
ured by a microplate reader at 570 nm (reference: 655 nm).
Inhibition (%) was obtained using the following formula:
An amorphous powder, [α]2D7—9.2 (c 0.12, MeOH); UV
[MeOH, nm (log ε)]: 248 (4.70), 365 (4.11); IR (KBr) vmax
:
3380, 1744, 1609, 1098 cm−1; 1H (600 MHz, DMSO-d6) and
13C NMR (150 MHz, DMSO-d6): shown in Tables 1 and 2;
Positive-ion FABMS m/z: 499 [M+Na]+; HRFABMS m/z:
499.0886 [M+Na]+ (calcd for C22H20O12Na, 499.0852).
Compound 2a
An amorphous powder, [α]2D8—49.9 (c 0.11, MeOH); UV
[
Inhibition (%) = (O.D. (sample) − O.D. (control))
[MeOH, nm (log ε)]: 251 (4.69), 363 (4.05); IR (KBr) vmax
:
]
3385, 1734, 1618, 1072 cm−1; 1H (600 MHz, DMSO-d6) and
13C NMR (150 MHz, DMSO-d6): shown in Tables 1 and 2;
positive-ion FABMS m/z: 631 [M+Na]+; HRFABMS m/z:
631.1267 [M+Na]+ (calcd for C27H28O16Na, 631.1275).
∕(O.D. (normal) − O.D. (control)) × 100.
Statistical analysis
Values are expressed as mean SEM. One-way analysis of
variance (ANOVA), followed by Dunnett’s test, was used
for statistical analysis. Probability (p) values <0.05 were
considered signifcant.
Compound 3a
An amorphous powder, [α]2D7—18.0 (c 0.11, MeOH); UV
[MeOH, nm (log ε)]: 247 (4.74), 370 (4.17); IR (KBr) vmax
:
Acknowledgements This work was supported by the MEXT-Sup-
ported Program for the Strategic Research Foundation at Private Uni-
versities, 2014–2018, Japan (S1411037, TM) as well as JSPS KAK-
ENHI, Japan, Grant numbers 15K08008 (TM), 15K08009 (KN), and
16K08313 (OM). Financial support by the Kobayashi International
Scholarship Foundation, Japan, is also acknowledged (TM).
3380, 1739, 1609, 1063 cm−1; 1H (600 MHz, DMSO-d6) and
13C NMR (150 MHz, DMSO-d6): shown in Tables 1 and 2;
positive-ion FABMS m/z: 513 [M+Na]+; HRFABMS m/z:
513.1003 [M+Na]+ (calcd for C23H22O12Na, 513.1009).
Bioassay
References
Reagents
1. Morikawa T, Ninomiya K, Imura K, Yamaguchi T, Akagi Y,
Yoshikawa M, Hayakawa T, Muraoka O (2014) Hepatoprotective
triterpenes from traditional Tibetan medicine Potentilla anserina.
Phytochemistry 102:169–181
LPS (from Salmonella enteritidis), minimum essential
medium, and Williams’ E medium were purchased from
Sigma-Aldrich Chemical (St. Louis, MO, USA). Fetal calf
serum (FCS) was obtained from Life Technologies (Rock-
ville, MD, USA). Other chemicals were procured from Wako
Pure Chemical Industries, Co., Ltd. (Osaka, Japan), and
96-well microplates were purchased from Sumitomo Bake-
lite Co., Ltd. (Tokyo, Japan).
2. Wang J, Zhang J, Zhao B, Wang X, Wu Y, Yao J (2010) A com-
parison study on microwave-assisted extraction of Potentilla
anserina L. polysaccharides with conventional method: molecule
weight and antioxidant activities evaluation. Carbohydrate Polym
80:84–93
3. Chen J-R, Yang Z-Q, Hu T-J, Yan Z-T, Niu T-X, Wang L, Cui
D-A, Wang M (2010) Immunomodulatory activity in vitro and
in vivo of polysaccharide from Potentilla anserina. Fitoterapia
81:1117–1124
Efects on d‑GalN‑induced cytotoxicity in primary cultured
mouse hepatocytes
4. Xia L, You J (2011) The determination of amino acids composi-
tion of the traditional food Potentilla anserina L. root by high-
performance liquid chromatography via fuorescent determination
and mass spectrometry. Int J Food Sci Technol 46:1164–1170
5. Guo T, Wei JQ, Ma JP (2016) Antitussive and expectorant activi-
ties of Potentilla anserina. Pharm Biol 54:807–811
The hepatoprotective efects of the isolated constituents were
determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide (MTT) colorimetric assay using
primary cultured mouse hepatocytes [1, 48, 49]. Hepato-
cytes were isolated from male ddY mice (30–35 g) by the
collagenase perfusion method. A cell suspension at 4 × 104
cells in 100 μL Williams’ E medium containing FCS (10%),
penicillin G (100 units/mL), and streptomycin (100 μg/mL)
was inoculated in a 96-well microplate and precultured for
4 h at 37 °C under a 5% CO2 atmosphere. The medium was
6. Schimmer O, Lindenbaum M (1995) Tannins with antimutagenic
properties in the herb of Alchemilla species and Potentilla anse‑
rina. Planta Med 61:141–145
7. Kombal R, Glasl H (1995) Flavan-3-ols and favonoids from
Potentilla anserina. Planta Med 61:484–485
8. Li Q, Hui J, Shang D, Wu L, Ma X (2003) Investigation of the
chemical constituents of the roots of Potentilla anserina L. in
Tibet. Chin Pharm J 55:179–184
1 3

J Nat Med
25. Nawwar MAM, Hussein SAM, Merfort I (1994) NMR spectral
analysis of polyphenols from Punica granatum. Phytochemistry
36:793–798
9. Chu L, Wang L, Zhang Z, Gao H, Huang J, Sun B, Wu L (2008)
Studies on the chemical constituents of Potentilla anserine L.
Zhongguo Xiandai Zhongyao 10:10–12
26. Sato T (1991) Comparative spectroscopic characterization of
synthesized isomers of di-O-methylated ellagic acids. Phyto-
chem Anal 2:271–273
10. Zhao Y-L, Cai G-M, Hong X, Shan L-M, Xiao X-H (2008) Anti-
hepatitis B virus activities of triterpenoid saponin compound from
Potentilla anserina L. Phytomedicine 15:253–258
27. Kosuge T, Ishida H, Yokota M, Yoshida M (1984) Studies on
antihemorrhagic substances in herbs classifed as hemostatics
in Chinese medicine. III. On the antihemorrhagic principle in
Sanguisorba ofcinallis L. Chem Pharm Bull 32:4478–4481
28. Matsuda H, Ninomiya K, Morikawa T, Yoshikawa M (1998)
Inhibitory efect and action mechanism of sesquiterpenes from
11. Li L, Zhao Y, Liu W, Feng F, Xie N (2013) HPLC with quadrupole
TOF-MS and chemometrics analysis for the characterization of
Folium Turpiniae from diferent regions. J Sep Sci 36:2552–2561
12. Bai N, He K, Roller M, Zheng B, Chen X, Shao Z, Peng T, Zheng
Q (2008) Active compounds from Lagerstroemia speciosa,
insulin-like glucose uptake-stimulatory/inhibitory and adipocyte
diferentiation-inhibitory activities in 3T3-L1 cells. J Agric Food
Chem 56:11668–11674
Zedoariae Rhizoma on
d-galactosamine/lipopolysaccharide-
induced liver injury. Bioorg Med Chem Lett 8:339–344
29. Matsuda H, Morikawa T, Ninomiya K, Yoshikawa M (2001)
Hepatoprotective constituents from Zedoariae Rhizoma: abso-
lute stereostructures of three new carabrane-type sesquiter-
penes, curcumenolactonea A, B, and C. Bioorg Med Chem
9:909–916
13. Morikawa T, Imura K, Miyake S, Ninomiya K, Matsuda H,
Yamashita C, Muraoka O, Hayakawa T, Yoshikawa M (2012)
Promoting the efect of chemical constituents from the fowers
of Poacynum hendersonii on adipogenesis in 3T3-L1 cells. J Nat
Med 66:39–48
14. Chaipech S, Morikawa T, Ninomiya K, Yoshikawa M, Pongpiri-
yadacha Y, Hayakawa T, Muraoka O (2012) Structures of two new
phenolic glycosides, kaempferiaosides A and B, and hepatopro-
tective constituents from the rhizomes of Kaempferia parvifora.
Chem Pharm Bull 60:62–69
30. Morikawa T, Matsuda H, Ninomiya K, Yoshikawa M (2002)
Medicinal foodstufs. XXIX. potent protective efects of sesquit-
erpenes and curcumin from Zedoariae Rhizoma on liver injury
induced by d-galactosamine/lipopolysaccharide or tumor necrosis
factor-α. Biol Pharm Bull 25:627–631
15. Morikawa T, Chaipech S, Matsuda H, Hamao M, Umeda Y, Sato
H, Tamura H, Kon’i H, Ninomiya K, Yoshikawa M, Pongpiriya-
dacha Y, Hayakawa T, Muraoka O (2012) Antidiabetogenic oli-
gostilbenoids and 3-ethyl-4-phenyl-3,4-dihydroisocoumarins from
the bark of Shorea roxburghii. Bioorg Med Chem 20:832–840
16. Morikawa T, Ninomiya K, Zhang Y, Yamada T, Nakamura S, Mat-
suda H, Muraoka O, Hayakawa T, Yoshikawa M (2012) Flavonol
glycosides with lipid accumulation inhibitory activity from Sedum
sarmentosum. Phytochemistry Lett 5:53–58
31. Yoshikawa M, Xu F, Morikawa T, Ninomiya K, Matsuda H (2003)
Anastatins A and B, new skeletal favonoids with hepatoprotective
activities from the desert plant Anastatica hierochuntica. Bioorg
Med Chem Lett 13:1045–1049
32. Yoshikawa M, Morikawa T, Kashima Y, Ninomiya K, Matsuda
H (2003) Structures of new dammarane-type triterpene saponins
from the fower buds of Panax notoginseng and hepatoprotective
efects of principal ginseng saponins. J Nat Prod 66:922–927
33. Xu F, Morikawa T, Matsuda H, Ninomiya K, Yoshikawa M (2004)
Structures of sesquiterpenes and hepatoprotective constituents
from the Egyptian herbal medicine Cyperus longus. J Nat Prod
67:569–576
17. Chaipech S, Morikawa T, Ninomiya K, Yoshikawa M, Pongpori-
yadacha Y, Hayakawa T, Muraoka O (2012) New fav-3-en-3-ol
glycosides, kaempferiaosides C and D, and acetophenone glyco-
sides, kaempferiaosides E and F, from the rhizomes of Keampferia
parvifora. J Nat Med 66:486–492
34. Matsuda H, Morikawa T, Xu F, Ninomiya K, Yoshikawa M (2004)
New isofavones and pterocarpanes with hepatoprotective activity
from the stems of Erycibe expansa. Planta Med 70:1201–1209
35. Yoshikawa M, Nishida N, Ninomiya K, Ohgushi T, Kubo M,
Morikawa T, Matsuda H (2006) Inhibitory efects of coumarin
and acetylene constituents from the roots of Angellica furcijuga on
d-galactosamine/lipopolysaccharide-induced liver injury in mice
and on nitric oxide production in lipopolysaccharide-activated
mouse peritoneal macrophages. Bioorg Med Chem 14:456–463
36. Morikawa T (2007) Search for bioactive constituents from several
medicinal food: hepatoprotective, antidiabetic, and antiallergic
activities. J Nat Med 61:112–126
18. Morikawa T, Chaipech S, Matsuda H, Hamao M, Umeda Y, Sato
H, Tamura H, Ninomiya K, Yoshikawa M, Pongpiriyadacha Y,
Hayakawa T, Muraoka O (2012) Anti-hyperlipidemic constitunets
from the bark of Shorea roxburghii. J Nat Med 66:516–524
19. Morikawa T, Ninomiya K, Miyake S, Miki Y, Okamoto M,
Yoshikawa M, Muraoka O (2013) Flavonol glycosides with lipid
accumulation inhibitory activity and simultaneous quantitative
analysis of 15 polyphenols and cafeine in the fower buds of
Camellia sinensis from diferent regions by LCMS. Food Chem
140:353–360
20. Morikawa T, Ninomiya K, Kuramoto H, Kamei I, Yoshikawa M,
Muraoka O (2016) Phenylethanoid and phenylpropanoid glyco-
sides with melanogenesis inhibitory activity from the fowers of
Narcissus tazetta var. chinensis. J Nat Med 70:89–101
21. Ninomiya K, Motai C, Nishida E, Kitagawa N, Yoshihara K, Hay-
akawa T, Muraoka O, Li X, Nakamura S, Yoshikawa M, Matsuda
H, Morikawa T (2016) Acylated oleanane-type triterpene saponins
from the fowers of Bellis perennis show anti-proliferative activi-
ties against human digestive tract carcinoma cell lines. J Nat Med
70:435–451
37. Li N, Morikawa T, Matsuda H, Ninomiya K, Li X, Yoshikawa M
(2007) New favanone oligoglycosides, theafavanosides I, II, III,
and IV, with hepatoprotective activity from the seeds of tea plant
(Camellia sinensis). Heterocycles 71:1193–1201
38. Ninomiya K, Morikawa T, Zhang Y, Nakamura S, Matsuda H,
Muraoka O, Yoshikawa M (2007) Bioactive constituents from
Chinese natural medicines. XXIII. Absolute structures of new
megastigmane glycosides, sedumosides A4, A5, A6, H, and I, and
hepatoprotective megastigmanes from Sedum sarmentosum. Chem
Pharm Bull 55:1185–1191
22. Ye L, Yang JS (1996) New ellagic glycosides and known triterpe-
noids from Duchesnea indica Focke. Acta Pharm Sin 31:844–848
23. Khac DD, Tran-Van S, Campos AM, Lallemand J-Y, Fetizon M
(1990) Ellagic compounds from Diplopanax stachyanthus. Phy-
tochemistry 29:251–256
39. Zhang Y, Morikawa T, Nakamura S, Ninomiya K, Matsuda H,
Muraoka O, Yoshikawa M (2007) Bioactive constituents from
Chinese natural medicines. XXV. New favonol bisdesmosides,
sarmenosides I, II, III, and IV, with hepatoprotective activity from
Sedum sarmentosum. Heterocycles 71:1565–1576
24. Zhang T, Zhang C-F, Wang Z-T, Xu L-S (2005) Studies on chemi-
cal constituents of Dendrobium trigonopus Rchb. f. Chin J Nat
Med 3:30–33
40. Nakamura S, Li X, Matsuda H, Ninomiya K, Morikawa T,
Yamaguti K, Yoshikawa M (2007) Bioactive constituents from
Chinese natural medicines. XXVI. chemical structures and
1 3

J Nat Med
hepatoprotective efects of constituents from roots of Rhodiola
45. Morikawa T (2010) Search for TNF-α sensitivety degradation
principles from medicinal foods—hepatoprotective amide con-
stituents from Thai natural medicine Piper chaba. Yakugaku
Zasshi 130:785–791
sachalinensis. Chem Pharm Bull 55:1505–1511
41. Matsuda H, Ninomiya K, Morikawa T, Yasuda D, Yamaguchi
I, Yoshikawa M (2008) Protective efects of amide constitu-
ents from the fruit of Piper chaba on d-galactosamine/TNF-α-
induced cell death in mouse hepatocytes. Bioorg Med Chem Lett
18:2038–2042
46. Morikawa T, Pan Y, Ninomiya K, Imura K, Matsuda H, Yoshi-
kawa M, Yuan D, Muraoka O (2010) Acylated phenylethanoid
oligoglycosides with hepatoprotective activity from the desert
plant Cistanche tubulosa. Bioorg Med Chem 18:1882–1890
47. Nakamura S, Xu F, Ninomiya K, Nakashima S, Oda Y, Mori-
kawa T, Muraoka O, Yoshikawa M, Matsuda H (2014) Chemical
structures and hepatoprotective efects of constituents from Cassia
auriculata leaves. Chem Pharm Bull 62:1026–1031
42. Ninomiya K, Morikawa T, Xie H, Matsuda H, Yoshikawa M
(2008) Bioactive constituents from Chinese natural medicines.
XXXI. Hepatoprotective principles from Sinocrassula indica:
structres of sinocrassosides A8, A9, A10, A11, and A12. Heterocy-
cles 75:1983–1995
43. Nakamura S, Okazaki Y, Ninomiya K, Morikawa T, Matsuda H,
Yoshikawa M (2008) Medicinal fowers. XXIV. Chemical struc-
tures and hepatoprotective efects of constituents from fowers of
Hedychium coronarium. Chem Pharm Bull 56:1704–1709
44. Matsuda H, Ninomiya K, Morikawa T, Yasuda D, Yamaguchi I,
Yoshikawa M (2009) Hepatoprotective amide constituents from
the fruit of Piper chaba: structural requirements, mode of action,
and new amides. Bioorg Med Chem 17:7313–7323
48. Ninomiya K, Miyazawa S, Ozeki K, Matsuo N, Muraoka O,
Kikuchi T, Yamada T, Tanaka R, Morikawa T (2016) Hepatopro-
tective limonoids from andiroba (Carapa guianensis). Int J Mol
Sci 17:591
49. Ninomiya K, Chaipech S, Kunikata Y, Yagi R, Pongpiriyadacha
Y, Muraoka O, Morikawa T (2017) Quantitative determination
of stilbenoids and dihydroisocoumarins in Shorea roxburghii and
evaluation of their hepatoprotective activity. Int J Mol Sci 18:451
1 3