Fatty acid synthase inhibitors from Geum japonicum Thunb. var. chinense

Article abstract of DOI:10.1002/cbdv.200700462

Bioassay-guided fractionation of the MeOH extract of Geum japonicum THUNB. var. chinense using the fatty acid synthase inhibition assay led to the isolation of a new dimeric ellagitannin, gemin G (1), together with six known compounds, gemin A (2), casuar

Full text of DOI:10.1002/cbdv.200700462

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CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
Fatty Acid Synthase Inhibitors from Geum japonicum Thunb. var. chinense
by Hongwei Liu*a)b), Jiankuan Lic), Wenhua Zhaob), Li Baod), Xiaohong Songb), Ying Xiab), Xue
Wangb), Chao Zhangb), Xiaozhu Wangb), Xinsheng Yaod), and Ming Lie)
a) Institute of Microbiology, Chinese Academy of Sciences, No. 8 Zhongguancun Beiertiao Road,
Haidian District, Beijing 100071, P. R. China (phone: þ86-10-62566577; fax: þ86-10-62566511;
e-mail: liuhongwei60@yahoo.com.cn)
b) School of Chemical Biology and Pharmaceutical Sciences 62#, Capital University of Medical Sciences,
No. 10 Xitoutiao, You An Men Beijing 100069, P. R. China
c) School of Pharmaceutical Sciences, Shanxi Medical University, Taiyuan 030001, P. R. China
d) Department of Natural Product Chemistry, Shenyang Pharmaceutical University, Wenhua-Road 103,
Shenyang 110016, P. R. China
e) Epithelial Cell Biology Research Centre, The Chinese University of Hong Kong, Hong Kong,
P. R. China
Bioassay-guided fractionation of the MeOH extract of Geum japonicumThunb. var. chinense using
the fatty acid synthase inhibition assay led to the isolation of a new dimeric ellagitannin, gemin G (1),
together with six known compounds, gemin A (2), casuarinin (3), pedunculagin (4), potentillin (5) ,
tellimagrandin II (6), and ellagic acid (7). Their structures were determined on the basis of spectroscopic
analyses. Compounds 1–7 displayed strong inhibitory activities on fatty acid synthase with IC50 values in
the range of 0.21–41.4 mm. Compounds 1–4 exhibited significant antioxidant activities higher than
vitamin C in the ORAC assay. Compounds 1 and 2 also showed weak cytotoxic effects on BGC-823 cell.
Introduction. – Animal fatty acid synthase (E.C.2.3.l.85; FAS) is active as an
homodimer, composed of two identical, 260–270-kDa subunits juxtaposed head-to-tail,
each containing an acyl carrier protein and seven enzymatic active sites, including
acetyl transacylase, malonyl transacylase, b-ketoacyl synthase, b-ketoacyl reductase, b-
hydroxyacyl dehydratase, enoyl reductase, and thioesterase [1]. FAS catalyses the de
novo synthesis of fatty acids from acetyl-CoA and malonyl-CoA in the presence of
NADPH through the reaction which elongates the Ac group by C2 units derived from
malonyl-CoA in a stepwise manner [2][3]. FAS is an important lipogenic enzyme
involved in energy metabolism in vivo and is related to various human diseases such as
cancer and obesity [4]. Human cancer cells express high levels of FAS [5–10]. It has
been reported that FAS inhibitors are selectively cytotoxic to many kinds of human
cancer cells [11–14]. Inhibitors of FAS such as cerulenin, synthetic C75, and
epigallocatechin gallate (EGCG) are known for their effects on some human cancers
[15][16].
The whole plant of Geum japonicum Thunb. var. chinense F. Bolle (Rosaceae) has
been used as diuretic and astringents in traditional Chinese medicine [17]. It is also
used for the treatment of dizziness and headache in some regions of China [18]. In a
previous study of this plant, we isolated two known ellagitannins, casuarinin, and
pedunculagin [19]. The plants of the genus Genum are known to be rich in tannins.
Several hydrolyzable tannins and triterpenoid acids have been isolated from G.
ꢀ 2009 Verlag Helvetica Chimica Acta AG, Zꢁrich

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
403
japonicum Thunb., a plant closely related to G. japonicum Thunb. var. chinense [20–
22]. In the course of a continuing search for potential plant anti-FAS agents, the MeOH
extract of the whole plant of G. japonicum Thunb. var. chinense was found to show
significant inhibitory activity against FAS. In this article, we describe the isolation and
structure elucidation of a new dimeric ellagitannin, gemin G (1), along with six known
ellagitannins, gemin A (2), casuarinin (3), pedunculagin (4), potentillin (5), tell-
imagrandin II (6), and ellagic acid (7; Fig. 1), and their inhibitory activity against FAS,
antioxidative activity, and cytotoxicity.
Results and Discussion. – The whole plant of G. japonicum Thunb. var. chinense
was ground and macerated with MeOH at room temperature. The MeOH extract was
suspended in H2O and partitioned with AcOEt and H2O-saturated BuOH, successively.
The BuOH-soluble portion showing strong anti-FAS activity (IC50 2.5 mg/ml) was
subjected to D-101 resin fractionation and further purified by HW-40 gel to yield gemin
G (1), gemin A (2), casuarinin (3), pedunculagin (4), potentillin (5), tellimagrandin II
(6), and ellagic acid (7). The structures of compounds isolated were identified by NMR
and MS analysis, and comparison with literature data. Compounds 1–6 were shown to
be ellagitannins by the color reaction with the NaNO2-AcOH reagent on cellulose TLC
plates [20].
Gemin G (1) was isolated as amorphous powder ([a]D þ71). The HR-TOF-MS (m/z
1569.1626 ([MꢀH]ꢀ )) and NMR analyses revealed the molecular formula as
C68H50O44. The presence of phenolic group(s) in the structure was indicated by its
characteristic color reaction with FeCl3 (violet). UV Absorption lmax (MeOH) at 220
(log e 4.82) and 273 nm (log e 4.73) was indicative of a bibenzyl. The IR absorptions
indicated the presence of OH (3425 cmꢀ1) and C¼O groups (1716 cmꢀ1), and aromatic
rings (1610, 1510, and 1445 cmꢀ1). The 1H- and 13C-NMR spectra of 1 (Tables 1 and 2)
showed close similarity to those of the known ellagitannin gemin A that was also
obtained in our study, indicating that compound 1 was dimeric ellagitannin. The
dimeric nature of compound 1 was further substantiated by the pseudomolecular peak
at m/z 1569 ([MꢀH]ꢀ ) and the corresponding fragment ion peak at m/z 785 in the
1
negative-ion ESI-MS. The H-NMR spectrum showed seven singlets at d 6.64, 6.50,
6.70, 6.83, 7.01 (2 H), 7.04 (2 H), and 7.33, two meta-coupled doublets at d 6.85 (J ¼
2.0), 7.30 (J ¼ 2.0) in the aromatic region, in agreement with the presence of a
dehydrodigalloyl, two hexahydroxydiphenoyl, and two galloyl groups in the molecule.
Acid hydrolysis, followed by GLC analysis of the aldononitrile peracetate derivative,
confirmed the presence of glucose units in the structure. The two anomeric H-atom
signals at d 6.18 (J ¼ 8.0) and 6.31 (J ¼ 4.0) indicated the b- and a-glucosidic linkages
of the two glucose units of 1. The presence of two glucose units in 1 was also indicated
by two sets of the sequentially coupled H-atom signals at d 6.31–3.60, which were fully
1
assigned by H,1H-COSY experiment. The 13C-NMR spectrum of 1 showed twelve
distinctive glucose C-atom signals including those of b- and a-anomeric C-atoms at d
92.9 and 92.2, eight C¼O C-atom signals at d 162.8–167.8 together with a group of
aromatic C-atom signals at d 107.1–147.1.
Based on these data, compound 1 is presumed to be a dimeric ellagitannin
possessing b- and a-glucose units which are linked to each other through the
dehydrodigalloyl group in a way analogous to gemin A. Allocation of each phenolic

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CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
Fig. 1. Structures of compounds 1–7 isolated from Geum japonicum Thunb. var. chinense

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
405
Table 1. 1H-NMR Data for Gemin G (1) (500 MHz, (D6)acetone; d in ppm, J in Hz)
d(H)
d(H)
b-Glucose
HꢀC(1’)
HꢀC(2’)
HꢀC(3’)
HꢀC(4’)
HꢀC(5’)
CH2(6’)
a-Glucose
6.18 (d, J¼8.0)
5.57 (t, J¼9.0)
5.84 (t, J¼9.0)
5.18–5.21 (m)
4.51–4.53 (m)
5.35–5.37 (m),
3.87–3.89 (m)
6.31 (d, J¼4.0)
3.88 (m)
3.98 (t, J¼9.0)
4.84 (t, J¼10.0)
4.19 (m)
HꢀC(2)
HꢀC(3)
HꢀC(4)
HꢀC(5)
CH2(6)
5.05 (dd, J¼6.5, 13.0),
3.60 (d, J¼13.0)
ArꢀH
ArꢀH
HꢀC(1’c)
HꢀC(1’g)
HꢀC(2’c,2’g)
HꢀC(3’c,3’g)
HꢀC(4’c)
HꢀC(6’c)
7.30 (d, J¼2.0)
6.85 (d, J¼2.0)
7.04 (s)
7.01 (s)
6.50 (s)
HꢀC(1c)
7.33 (s)
HꢀC(4c)
HꢀC(6c)
6.64 (s)
6.83 (s)
6.70 (s)
Table 2. 13C-NMR Data for Gemin G (1) (125 MHz, (D6)acetone; d in ppm)
d(C)
d(C)
b-Glucose
C(1’)
C(2’)
C(3’)
C(4’)
a-Glucose
92.9
70.8
72.3
69.8
72.3
63.0
C(1)
C(2)
C(3)
C(4)
C(5)
C(6)
92.2
72.1
72.5
71.9
70.1
62.3
C(5’)
C(6’)
Carbonyl C-atoms
C(1’a)
C(2’a)
C(3’a)
C(4’a)
C(6’a)
164.1
164.7
165.5
166.8
167.3
C(1a)
162.8
C(4a)
C(6a)
167.6
167.8
ester groups on the glucopyranose ring was confirmed by HMBC correlations (Fig. 2).
The H- and C-atom signals for two glucose moieties, and the phenolic ester C¼O
1
signals were assigned by H,1H-COSY, HSQC, and HMBC experiments.
The configuration of the hexahydroxybiphenyl groups in 1 was determined as (S)
on the basis of CD-spectral measurements and the specific-rotation value. The strong
positive Cotton effect at 237 nm (13.4ꢁ104) for 1 indicated that the hexahydroxybi-
phenyl groups in 1 have the (S)-configuration which is identical with that established
for these groups in gemin A (2; 237 nm, 19.3ꢁ104; Fig. 3) [20]. This conclusion is also
in accordance with the empirical rule in which the sign and amplitude of the Cotton
effect around 235 nm reflect the configuration and the number of the hexahydroxy-
biphenyl groups in the molecules of ellagitannins [23]. Based on the above chemical
and spectral interpretation, the structure for compound 1 was established. Compound 1
was designated as gemin G.

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CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
Fig. 2. 1H,1H-COSY (—) and HMBC Correlations (H!C) of compound 1
Fig. 3. CD Spectra of compounds 1 and 2 in MeOH
The structures for compounds 2–7 were determined by spectral analysis and
comparison with published data [20–22].
Compounds 1–7 showed reversible fast-binding inhibition against FAS activity with
the IC50 values of 0.79ꢂ0.04, 0.21ꢂ0.02, 8.01ꢂ0.11, 3.19ꢂ0.07, 2.51ꢂ0.08, 2.40ꢂ0.06,
and 41.40ꢂ0.45 mm, respectively. The epigallocatechin gallate (EGCG), which has
been reported to be an inhibitor of FAS, exhibited an IC50 value of 52.03ꢂ3.11 mm in
our assay. The number of galloyl or hexahydroxybiphenyldicarbonyl moieties contrib-
utes most to the inhibition of fatty acid synthase. It has been shown that the galloyl
moiety of green tea catechins is the critical structural feature to inhibit FAS [24]. The
inhibitory activity of compound 3 is lower than that of compounds 4–6, indicating that
the intactness of the sugar ring also influences the activity. Since FAS inhibitors are

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
407
reported to be selectively cytotoxic to many kinds of human cancer cells, we tested the
cytotoxicity of compounds 1–4 in vitro. Compounds 1 and 2 showed weak cytotoxic
activity towards BGC-823 cells with IC50 values of 82.5ꢂ2.2 and 75.6ꢂ3.5 mg/ml,
respectively.
The antioxidant activity of compounds 1–4 was evaluated by the Oxygen Radical
Absorbance Capacity (ORAC) assay (Table 3). Vitamin C was used as positive control.
Compounds 1–4 displayed potent peroxyl radical scavenging activity higher than that
of vitamin C, which was in accordance with the strong antioxidant properties reported
for galloyl groups. [25][26].
Table 3. Antioxidative Values of Compounds 1–4a)
Concentration [mm]
ORAC [mm trolox equiv.]
1
2
3
4
Vitamin C
100
50
25
12.5
6.25
3.125
99.2ꢂ1.3
95.6ꢂ1.8
40.8ꢂ0.4
24.7ꢂ2.6
14.3ꢂ1.0
6.4ꢂ1.2
98.0ꢂ1.3
84.3ꢂ6.0
31.3ꢂ6.2
20.1ꢂ2.6
11.8 ꢂ2.2
7.5ꢂ1.3
96.8ꢂ1.1
73.1ꢂ2.0
38.3ꢂ0.5
22.9ꢂ1.1
16.5ꢂ1.3
11.8 ꢂ0.5
96.0ꢂ0.8
58.3ꢂ9.7
31.6ꢂ3.6
15.6ꢂ1.4
10.4ꢂ0.7
6.2ꢂ2.8
62.8ꢂ0.2
31.9ꢂ0.1
15.8ꢂ0.2
7.2ꢂ0.5
3.8ꢂ0.7
1.7ꢂ0.4
a) ORAC Values are expressed as relative trolox equivalents. Data were expressed as meanꢂSD.
Vitamin C was used as positive control.
The chemistry and biology of ellagitannins have achieved great progress recently.
Some dimeric ellagitannins promoted tumor necrosis in various mouse xenograft
models at concentrations well below toxic levels. Biological mechanism-of-action
studies led to a proposal that the ellagitannins were operating indirectly through
endogenous effectors such as the cytokines interleukin 1b (IL-1b) and cytokine tumor
necrosis factor alpha (TNFa) [27–29]. Our study is the first report of FAS inhibitory
activity of ellagitannins, which may open new fields of application for these compounds,
in particular in the treatment of obesity.
The authors thank Dr. Hao Gao and Xue Zhang for ESI-MS and NMR measurements. The work was
supported by the National Natural Science Foundation of China (NSFC, 20502016) and the Scientific
Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry, China.
Experimental Part
General. 2,2-Azobis (2-amidinopropane) dihydrochloride (AAPH), sodium fluorescein (FL), and 6-
hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) were purchased from Wako Pure
Chemical Industries, Ltd. (Osaka, Japan). Acetyl-CoA and malonyl-CoA were purchased from Sigma,
NADPH was from Fluka. All other reagents were local products of anal. grade. Column chromatography
(CC): D101 resin (Anhui Sanxing Scientific Corporation, China) and HW-40F (Tosoh Corporation,
Japan) as packing materials. Anal. TLC: cellulose (funakoshi), with BuOH/AcOH/H2O 4 :1:5 (upper
layer). GC: HP-5890 SERIES II spectrometer, with a SE30 cap. column (12 m, 0.22 mm i.d.), hydrogen
flame ionization detector (FID, 2708), the column temp. was 170–2508 with the rate of 58/min, and the
carrier gas was N2 (30 ml/min). Optical rotations: Jasco P-1020 polarimeter. CD Spectra: Jasco-810
spectropolarimeter. UV Spectra: Shimadzu UV2401PC UV/VIS spectrophotometer in MeOH. IR

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CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
Spectra: Shimadzu FTIR 8400 spectrophotometer as KBr discs. NMR Spectra: Bruker AVANCE 500
NMR spectrometer (500 MHz for 1H, 125 MHz for 13C) with Me4Si as internal standard. ESI-MS: Bruker
Esquire 2000 mass spectrometer. HR-TOF-MS: Waters Maldi Micro MX instrument.
Plant Material. The whole plants of Geum Japonicum Thunb. var. chinense were collected in
Guizhou Province in September 2005 and identified by M. L. A voucher specimen (YZXDN-2004) was
deposited with the School of Chemical Biology and Pharmaceutical Science of Capital University of
Medical Science, China.
Extraction and Isolation. The air-dried whole plants of Geum JaponicumThunb. var. chinense (5 kg)
were ground and macerated with 20 l of MeOH at r.t. for a week (3 times). After evaporation of solvent
in vacuo, the residue (530 g; IC50 ¼7.5 mg/ml) was suspended in H2O (1000 ml), and partitioned with
AcOEt (1000 ml) and BuOH (1000 ml) successively. The BuOH-soluble fraction exhibited significant
FAS inhibitory activity (IC50 ¼2.5 mg/ml). The active BuOH-soluble fraction (90 g) was first subjected to
CC on D101 resin (˘4ꢁ40 cm, 2000 ml) eluted with MeOH/H2O (100 :0!0 :100) to afford nine
fractions. Fr. 4 was found to be active in FAS assay (IC50 ¼1.5 mg/ml). Fr. 4 (13 g) was further
chromatographed on HW-40 gel by gradient elution with MeOH/H2O (30, 40, 50, 60, 70, 80, and 100%)
and MeOH/H2O/acetone (7:2 :1 and 6 :2 :2), and the eluents were combined by cellulose TLC analysis
to yield compounds 1 (MeOH/H2O/acetone 6 :2 :2; 20 mg), 2 (MeOH/H2O/acetone 7:2 :1; 100 mg), 3
(70% MeOH/H2O; 8 mg), 4 (60% MeOH/H2O; 10 mg), 5 (80% MeOH/H2O; 12 mg), 6 (80% MeOH/
H2O; 15 mg), 7 (50% MeOH/H2O; 10 mg).
Gemin G (1): Amorphous powder. [a]D27 ¼ þ71 (c¼1.0, MeOH). UV (MeOH): lmax (log e) 220
(4.82), 273 (4.73) nm. CD (MeOH): [q]237 þ13.4ꢁ104, [q]263 ꢀ2.4ꢁ104, [q]285 þ3.4ꢁ104. IR (KBr): 3425,
1716, 1610, 1510, 1445, 1050. 1H- and 13C-NMR ((D6)Acetone): see Tables 1 and 2. ESI-MS (neg.): 1569
([MꢀH]ꢀ ), 785 ([Mꢀ785]ꢀ ). HR-TOF-MS: 1569.1626 ([MꢀH]ꢀ , C68H49O4ꢀ4 ; calc. 1569.1597).
Acid hydrolysis of 1 and 2. A soln. of 1 and 2 (each 10 mg) in aq. 2n CF3COOH was heated at 1008
for 2 h in a sealed vial. The mixture was evaporated in vacuo. The obtained residue was dissolved in H2O
and extracted twice with AcOEt. The AcOEt extracts was purified by CC (silica gel; CHCl3/MeOH 10 :1,
9 :1, 7:1, 5 :1) to give gallic acid (1.5 mg) and ellagic acid (2.0 mg). Glucose was identified in the aq. layer
by GLC as aldononitrile peracetate derivative, using standard aldononitrile peracetate of d-glucose as a
reference sample [30].
Preparation and Activity Assay of FAS. The FAS from duck liver was purified according to an
improved method described previously by Tian et al. [31]. The assay for FAS activity, which was
described in [16], was performed with a Shimadzu UV2550 double-wavelength/double-beam spectro-
photometer at 378 by following the decrease of NADPH at 340 nm. The mixture contained 100 mm
potassium phosphate buffer, pH 7.0, 1 mm EDTA, 3 mm acetyl-CoA, 10 mm malonyl-CoA, 35 mm
NADPH, and 10 mg duck liver FAS in a total volume of 2.0 ml. Fast-binding inhibition was determined
by adding the tested compounds to the reaction system before the reaction was initiated by the addition
of FAS. After incubation at 378 for 5 min, the absorbance at 340 nm was measured. The remaining
activity of FAS was assayed as Ai, and the control activity with solvent instead of test compounds was
assayed as A0. The fast-binding inhibition was calculated by the formula: I¼(1ꢀAi/A0)ꢁ100%.
Inhibition of FAS was measured for tested compounds at different concentrations in triplicate, and IC50
values were obtained from the dose-response curves of inhibitors by software Graph 2.0. EGCG was used
as positive control in tests.
Cytotoxicity Bioassay. The effects of compounds 1–4 on cell growth were investigated using gastric
cancer cell line (BGC-823) purchased from Institute of Medicament Research of Chinese Academy of
Medical Sciences and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100
units/ml of penicillin, and 100 mg/ml of streptomycin. Cells were maintained at 378 in a humidified
atmosphere of 5% CO2 in air. The effect on the cell viability was determined by MTT (¼ 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium hydrobromide) method in triplicate [32]. Fluorour-
acil was used as positive control with an IC50 value of 5.5ꢂ0.3 mg/ml.
Oxygen Radical Absorbance Capacity (ORAC) Assay. The ORAC assay is based on free radical
damage to a fluorescent probe. Evaluation of peroxyl-radical scavenging activity was based on the
protocol described in [33–35]. In our assay, sodium fluorescein was used as a fluorescent probe, AAPH
as a free radical initiator, and Trolox as a standard. The 180-ml test soln. contained 20 ml of sample soln.,

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
409
20 ml of phosphate buffer (7.5 mm), and 140 ml of AAPH soln. (12.8 mm). The reaction was started by the
addition of 20 ml of fluorescein (63 nm) to the test soln. The fluorescence intensity was measured with
Genios multi-detection microplate reader (Tecan; ex. 485 nm, em. 538 nm) every 2 min until the
fluorescence was close to zero. The well-known antioxidant vitamin C and the solvent DMSO were used
as the positive control and the blank, resp. The ORAC value is calculated by dividing the area under the
sample curve by the area under the Trolox curve, with both areas being corrected by subtracting the area
under the blank curve. One ORAC unit is defined as the net area of protection provided by Trolox at a
final concentration 1 mmol lꢀ1. The area under the curve for the sample is compared to the area under the
curve for Trolox, and the antioxidative value is expressed in mmol of Trolox equiv. per l. The experiments
were carried out in triplicate and repeated for three times.
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Received December 18, 2007