Inhibitory effects of constituents from Euphorbia lunulata on differentiation of 3T3-L1 cells and nitric oxide production in RAW264.7 cells

Article abstract of DOI:10.3390/molecules16108305

A new flavonol galactopyranoside, myricetin 3-O-(2″,3″- digalloyl)-β-D-galactopyranoide (1), and 23 known constituents, including myricetin 3-O-(2″-galloyl)-β-D-galactopyranoide (2), myricitrin (3), myricetin (4), quercetin 3-O-(2″, 3″-digalloyl)-β-D- galactopyranoide (5), quercetin 3-O-(2″-galloyl)-β-D-galactopyranoide (6), hyperin (7), isoquercetrin (8), quercetin (9), kaempferol (10), apigenin (11), luteolin (12), 3-O-methylquercetin (13), 5,7,2',5'-tetrahydroxyflavone (14), 1,3,4,6-tetra-O-galloyl-β-D-glucose (15), 1,2,6-tri-O-galloyl-β- D-glucose (16), 1,3,6-tri-O-galloyl-β-D-glucose (17), gallic acid (18), protocatechuic acid (19), 3,4,5-trimethoxybenzoic acid (20), 2,6-dihydroxyacetophenone (21), 3,3'-di-O-methylellagic acid (22), ellagic acid (23) and esculetin (24) were isolated from Euphorbia lunulata Bge. Their structures were determined by spectroscopic analysis. Isolated hydrolysable tannins, flavonoids, and flavonol galactopyranoside gallates showed significant inhibition of the differentiation of 3T3-L1 preadipocytes and triglyceride accumulation in maturing adipocytes, and nitric oxide production in RAW 264.7 cells.

Full text of DOI:10.3390/molecules16108305

Molecules 2011, 16, 8305-8318; doi:10.3390/molecules16108305
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Inhibitory Effects of Constituents from Euphorbia lunulata on
Differentiation of 3T3-L1 Cells and Nitric Oxide Production in
RAW264.7 Cells
Zhi-Gang Yang 1,†, Liu-Nan Jia 1,†, Yan Shen 2, Atsuko Ohmura 3 and Susumu Kitanaka 1,*
1
School of Pharmacy, Nihon University;, 7-7-1 Narashinodai, Funabashi, Chiba 274-8555, Japan;
E-Mails: yang_zhg@hotmail.com (Z.-G.Y.); deathmetallica82@hotmail.com (L.-N.J.)
2
College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-8510,
Japan; E-Mail: shen.yan@nihon-u.ac.jp (Y.S.)
3
Saitama Prefectural Pharmaceutical Affairs Divison, 3-15-1 Takasago, Saitama, Saitama 330-9301,
Japan; E-Mail: omura.atsuko@pref.saitama.lg.jp (O.A.)
†
These authors contributed equally to this work.
* Author to whom correspondence should be addressed; E-Mail: kitanaka.susumu@nihon-u.ac.jp;
Tel.: +81-47-4655356; Fax: +81-47-4655440.
Received: 9 August 2011; in revised form: 14 September 2011 / Accepted: 27 September 2011 /
Published: 29 September 2011
Abstract: A new flavonol galactopyranoside, myricetin 3-O-(2'',3''-digalloyl)-β-D-
galactopyranoide (1), and 23 known constituents, including myricetin 3-O-(2''-galloyl)-β-
D-galactopyranoide (2), myricitrin (3), myricetin (4), quercetin 3-O-(2'', 3''-digalloyl)-β-D-
galactopyranoide (5), quercetin 3-O-(2''-galloyl)-β-D-galactopyranoide (6), hyperin (7),
isoquercetrin (8), quercetin (9), kaempferol (10), apigenin (11), luteolin (12),
3-O-methylquercetin (13), 5,7,2',5'-tetrahydroxyflavone (14), 1,3,4,6-tetra-O-galloyl-β-D-
glucose (15), 1,2,6-tri-O-galloyl-β-D-glucose (16), 1,3,6-tri-O-galloyl-β-D-glucose (17),
gallic acid (18), protocatechuic acid (19), 3,4,5-trimethoxybenzoic acid (20),
2,6-dihydroxyacetophenone (21), 3,3'-di-O-methylellagic acid (22), ellagic acid (23) and
esculetin (24) were isolated from Euphorbia lunulata Bge. Their structures were
determined by spectroscopic analysis. Isolated hydrolysable tannins, flavonoids, and
flavonol galactopyranoside gallates showed significant inhibition of the differentiation of
3T3-L1 preadipocytes and triglyceride accumulation in maturing adipocytes, and nitric
oxide production in RAW 264.7 cells.

Molecules 2011, 16
8306
Keywords: Euphorbia lunulata Bge.; flavonol galactopyranoside gallate; phenolic
constituents; adipocyte; macrophage
1. Introduction
Euphorbia lunulata Bge. (Euphorbiaceae) is a perennial herbaceous plant that has long been used in
China as a traditional crude drug for the treatment of chronic bronchitis. Previous investigations of
E. lunulata Bge. have yielded some flavonoids and coumarins that have been reported to have
anticancer, antioxidant, insulin and interleukin-10 (IL-10) mimicking activities [1-5].
It is well known that obesity is often accompanied by hyperglycemia, hypertension, and
hyperlipidemia, which are together known as metabolic syndrome. Obesity is the result of an increase
in the number and size of adipocytes, in which adipocytes accumulate a large amount of lipids and
become enlarged. Cultured 3T3-L1 adipocytes show many properties similar to those of normal
adipocytes, lipids are accumulated during the differentiation and maturing process, the cytosolic
enzyme glycerol-3-phosphate dehydrogenase (GPDH) appears to have an important role in the
conversion of glycerol into triglyceride (TG), and the level of activity of GPDH increases during the
conversion of 3T3 cells. Recent studies indicate that obesity is associated with low-grade chronic
inflammation of adipose tissues, and the obese adipose tissue is characterized by increased infiltration
of macrophages. In a coculture system of 3T3-L1 adipocytes and RAW264 macrophages, marked
increases in secretion levels of inflammatory mediators such as TNF-α, MCP-1, and nitric oxide (NO)
were observed [6-8]. NO mediates diverse functions by acting on various cells through interactions
with different molecular targets, and excessive NO production is involved in various types of
inflammation [9]. Therefore, as a part of our continuing investigation of anti-adipogenesis and
anti-inflammatory agents from natural sources [10], the chemical composition of the whole plant of
E. lunulata Bge. has been examined.
In the present study, the 70% acetone extract of the whole plant of E. lunulata Bge. and the ethyl
acetate soluble portion of the extract were observed to inhibit the accumulation of TG in 3T3-L1 cells,
and inhibit NO production in lipopolysaccharide (LPS) and interferon-γ (IFN-γ) activated
macrophages. Furthermore, an attempt to identify the bioactive compounds present in this plant led to
the isolation of a new flavonol galactopyranoside gallate, myricetin 3-O-(2'',3''-digalloyl)-β-D-
galactopyranoide (1), and 23 known constituents, including myricetin 3-O-(2''-galloyl)-β-D-
galactopyranoide (2) [11], myricitrin (3) [12], myricetin (4) [13], quercetin 3-O-(2'',3''-digalloyl)-β-D-
galactopyranoide (5) [2], quercetin 3-O-(2''-galloyl)-β-D-galactopyranoide (6) [14], hyperin (7) [15],
isoquercetrin (8) [16], quercetin (9) [17], kaempferol (10) [18], apigenin (11) [19], luteolin (12) [19],
3-O-methylquercetin (13) [20], 5,7,2',5'-tetrahydroxyflavone (14) [21], 1,3,4,6-tetra-O-galloyl-β-D-
glucose (15) [22], 1,2,6-tri-O-galloyl-β-D-glucose (16) [23], 1,3,6-tri-O-galloyl-β-D-glucose (17) [24],
gallic acid (18) [25], protocatechuic acid (19) [26], 3,4,5-trimethoxybenzoic acid (20) [27],
2,6-dihydroxyacetophenone(21) [28], 3,3'-di-O-methylellagic acid (22) [29], ellagic acid (23) [30] and
esculetin (24) [31] (Figure 1). Among these compounds, 1–4, 8, 11–17, and 19–22 were isolated from
E. lunulata Bge. for the first time. We also describe their inhibitory activity towards the differentiation

Molecules 2011, 16
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of 3T3-L1 preadipocytes and TG accumulation in maturing adipocytes, and their inhibition of NO
production in RAW 264.7 cells in this contribution.
Figure 1. Structures of compounds 1–24.
2. Results and Discussion
Whole plants of Euphorbia lunulata Bge. were extracted with 70% aqueous acetone, followed by
evaporation of the solvent under reduced pressure from the combined extract to give the 70% acetone
extract. The extract [TG and NO inhibitory activity: 50%, 89%, respectively (100 μg/mL)] was
dissolved in water and successively partitioned to give a chloroform layer [68%, 71% (30 μg/mL)],
ethyl acetate layer [70%, 76% (30 μg/mL)], n-butanol layer [41%, 23% (30 μg/mL)], and aqueous
layer [32%, 8% (30 μg/mL)]. We have focused our efforts on investigating the chemical constituents
of the ethyl acetate soluble fraction, which was separated by Diaion HP-20 column chromatography to
yield eight fractions from which twenty three compounds were isolated. One compound was isolated
from the chloroform layer.

Molecules 2011, 16
8308
Compound 1 was obtained as a yellow amorphous powder and gave positive Mg-HCl and FeCl3
tests. The molecular formula of 1 was established as C35H28O21 by HR-FAB-MS. The UV spectrum
exhibited absorption maxima at 268 and 355 nm, suggesting the presence of aromatic rings in the
molecule. The IR spectrum contained absorption bands for a hydroxyl group (3,396 cm−1), ester
carbonyl (1,703 cm−1) and conjugated carbonyl (1,656 cm−1), and aromatic ring (1,608, 1,497 cm−1).
1
The H-NMR spectrum of 1 indicated meta-coupled proton signals at δ 6.31 (1H, d, J = 2.1 Hz) and
6.14 (1H, d, J = 2.1 Hz), and an A2-type aromatic proton signal at δ 7.27 (2H, s). These data for the
aromatic region matched those of myricetin 3-O-(2''-galloyl)-β-D-galactopyranoide (2) and myricetin (4),
indicating the aglycone was most likely a myricetin (Table 1). Except for the aglycone signals, the 1H-
13
and C-NMR spectra of 1 showed the presence of two galloyl groups [δH 7.03, 7.02, (each 2H, s);
δC 166.4 (C=O), 166.2 (C=O), 145.1 (×4), 138.8, 138.7, 119.1, 118.7, 109.4 (×2), 109.3 (×2)], and a
4
sugar of the anomeric proton signal at δ 5.94 (1H, d, J = 8.1 Hz). The sugar was found to be C1
1
galactopyranose with the help of H-1H COSY. These data coupled with the FAB-MS data indicated
that 1 may be a digallate of myricetin galactopyranoside. Myricetin, gallic acid, and D-galactose were
13
identified by hydrolysis of 1 (see Experimental). Furthermore, comparative analysis of the C-NMR
data of the aglycone carbons in 1 with those of 4 showed an upfield shift of C-3 (Δδ 1.6 ppm), a
downfield shift of C-2 (Δδ 9.7 ppm) and C-4 (Δδ1.8 ppm), suggesting the location of the sugar was at
C-3 [32]. This was further supported by HMBC correlations, the anomeric proton signal δ 5.94 (H-1'')
showed long-range correlation with the carbon signal at δ 134.0 (C-3) (Figure 2). The locations of the
two galloyl groups in 1 were confirmed to be at C-2'' and C-3'' of the galactosyl moiety by the
remarkable downfield shifts of H-2'' (δ 5.81, dd, J = 10.2, 8.1 Hz), and H-3'' (δ 5.18, dd, J = 10.2, 3.0 Hz)
signals, a finding which was further supported by HMBC correlations between the sugar protons at
δ5.81 and δ5.18 with the carbon signals (δ 166.4, 166.2) of the two galloyl carbonyls (Figure 2). Based
on the foregoing findings, 1 was determined to be myricetin 3-O-(2'',3''-digalloyl)-β-D-galactopyranoide.
1
Table 1. H- and 13C-NMR Spectral Data of compounds 1, 2 and 4 [(300/75 MHz, in
methanol-d4, TMS, δ (ppm) (J = Hz)].
1
2
4
Position
δH
δC
δH
δC
δH
δC
2
3
156.4
134.0
177.6
104.6
161.7
98.5
156.6
134.0
177.4
104.7
161.7
98.5
146.7
135.6
175.8
103.3
161.1
98.1
4
4a
5
6
6.14 (d 2.1)
6.31 (d 2.1)
6.13 (d 2.1)
6.30 (d 2.1)
6.17 (d 2.1)
6.36 (d 2.1)
7
164.5
93.3
164.4
93.3
164.2
93.2
8
8a
1'
2'
3'
156.6
120.6
108.6
144.9
156.8
120.8
108.6
144.9
156.8
121.8
107.3
145.4
7.27 (s)
7.25 (s)
7.32 (s)

Molecules 2011, 16
8309
Table 1. Cont.
1
2
4
Position
δH
δC
δH
δC
δH
δC
4'
5'
6'
136.6
144.9
136.6
144.9
108.6
100.2
73.5
136.1
145.4
107.3
7.27 (s)
108.6
7.25 (s)
7.32 (s)
1''
2''
5.94 (d 8.1)
100.1
5.77 (d 7.8)
5.43 (dd 9.9, 7.8)
3.84 (dd 9.9, 3.6)
3.95 (d 3.6)
5.81 (dd 10.2, 8.1)
5.18 (dd 10.2, 3.0)
4.28 (d 3.0)
74.8
3''
70.7
72.4
4''
66.9
69.5
5''
3.75 (m)
76.3
3.61 (m)
76.4
6''
3.71, 3.69 (m)
60.8
3.71, 3.69 (m)
61.0
1'''
119.1, 118.7
109.4, 109.3
145.1
120.4
109.5
145.0
138.5
166.7
2''',6'''
3''',5'''
4'''
7.03, 7.02 (s)
7.12 (s)
138.8, 138.7
166.4, 166.2
C=O
Figure 2. Key HMBC correlations of 1.
Compounds 2–24 were identified as known compounds by detailed comparisons of their spectroscopic
data with that in the literature.
Compounds 1–24 were next evaluated for their inhibitory effects on triglyceride accumulation and
GPDH activity in 3T3-L1 cells at the concentration of 30 μM (Figure 3). The major component,
quercetin (9, 0.038% yield), which has been reported to have inhibitory effects on triglyceride
accumulation, was used as a positive control [10,33]. As shown in Figure 3, the hydrolysable tannins,
15–17, 22 and 23 showed significant inhibition of adipogenesis in 3T3-L1 adipocytes, with TG inhibition
values of 75.7%, 72.2%, 56.6%, 64.2% and 72.8%, respectively. The flavonol galactopyranoside
gallates, 1, 2, 5 and 6 showed moderate TG inhibitory activities (43.7%, 30.5%, 55.5% and 32.5%
inhibition, respectively). Comparing 1 with 2 and 5 with 6, compounds 1, 5 showed stronger TG and
GPDH activity inhibition than 2 and 6, respectively; these results suggest that the galloyl group may

Molecules 2011, 16
8310
enhance their activities. The flavonols, 7, 8, 9, 10 and 13 showed moderate TG inhibitory activities
(35.8%, 27.7%, 42.4%, 40.8% and 25.9% inhibition, respectively), which were stronger than the
flavones, 11, 12 and 14 with TG inhibitions 20.3%, 13.7%, and 15.9%, respectively. The
phenylpropanoids 18–21 and coumarin 24 showed weak inhibitory effects on TG and GPDH activity.
Furthermore, effects of isolated compounds on cell viability of 3T3-L1 cells were measured by MTT
assay. As shown in Figure 3, the isolated compounds of 15–17, 22 and 23 had effects on viability of
3T3-L1 at concentration of 30 μM. However, these compounds at low concentrations (10, 3, 1 μM,
respectively) suppressed the differentiation of 3T3-L1 preadipocytes and TG accumulation in maturing
adipocytes without exerting cytotoxicity (Figure 4). The hydrolysable tannin, 15 and 22 showed
significant inhibitions of TG content and GPDH activity at the concentration of 10 μM, 15 with values
of 45.6% and 44.2%, 22 with values of 42.0% and 34.7%, respectively.
Figure 3. (A) Inhibition of TG Content and GPDH Activity by the isolated compounds in
3T3-L1 Adipocytes; (B) Cell viability of 3T3-L1 cells by MTT assay.
Notes: (A) 3T3-L1 adipocytes were harvested after 8 days of treatment with compound (30 μM) or
vehicle, and assayed for total triglyceride (TG) content and glycerol-3-phosphate dehydrogenase
(GPDH) activity; (B) Cell viability was determined by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-
diphenyl-2H-tetrazolium bromide (MTT) assay after 8 days of treatment with compound (30 μM)
or vehicle. Data are expressed as a percentage of the control (vehicle) levels. The reported values
are the mean ± SD (n = 3), * p < 0.05, ** p < 0.01 vs. the control using Student’s t-test.

Molecules 2011, 16
Figure 4. (A) Inhibition of TG Content and GPDH Activity by compounds 9, 15-17, 22
8311
and 23 at low concentrations in 3T3-L1 Adipocytes; (B) Cell viability of 3T3-L1 cells by
MTT assay.
Notes: (A) 3T3-L1 adipocytes were harvested after 8 days of treatment with compounds 9, 15–17,
22 and 23 (30, 10, 3, 1 μM, respectively) or vehicle, and assayed for total triglyceride (TG)
content and glycerol-3-phosphate dehydrogenase (GPDH) activity; (B) Cell viability was
determined by MTT assay after 8 days of treatment with compounds or vehicle. Data are expressed
as a percentage of the control (vehicle) levels. The reported values are the mean ± SD (n = 3),
* p < 0.05, ** p < 0.01 vs. the control using Student’s t-test.
Compounds 1–24 also examined with respect to their inhibition of NO production stimulated by
LPS and IFN-γ in RAW 264.7 cells (Table 2). In the assay, aminoguanidine, which has been reported
to have inhibitory effects on NO production in LPS activated RAW 264.7 macrophages by down-
regulation of inducible nitric oxide synthase (iNOS), was used as a positive control (IC50 17.5 μM) [34].
As shown in Table 2, in general, all compounds except 3, 7, 8, 18, 20, 21 and 24 showed NO
production inhibitory activities with IC50 values lower than 100 μM. Furthermore, the hydrolysable
tannins, 15 and 17, showed slightly stronger NO production inhibitory effects than aminoguanidine,
with IC50 values of 14.2 and 16.9 μM, respectively; compounds 16, 22 and 23 showed moderate NO
inhibitory activity, with IC50 values of 21.7, 18.5 and 26.1 μM, respectively. These results showed that
the hydrolysable tannins were the significantly active compounds in E. lunulata Bge. In addition, the
free flavonoids, 4, 9, 10, 11, 12, 13 and 14, showed strong or moderate NO inhibitory activity, with
IC50 values of 37.2, 20.6, 21.5, 17.9, 23.8, 42.6 and 18.5μM, respectively. The flavonol glycosides, 3, 7
and 8, showed no inhibition. However, the flavonol galactopyranoside gallates, 1, 2, 5 and 6, showed

Molecules 2011, 16
8312
little or moderate NO inhibitory activity, with IC50 values of 62.3, 48.6, 56.8 and 43.2 μM, respectively.
In contrast, the descending orders of the NO inhibitory activities were: 4 > 2 > 1 > 3 (myricetin
derivatives), and 9 > 6 > 5 > 7, 8 (quercetin derivatives). These results showed that the free flavonoids
exerted stronger activities than the corresponding glycosides, and the conjugation of gallic acid may
have enhanced the activities of the flavonoid glycosides, even though gallic acid (18) itself showed no
inhibition. Furthermore, effects of isolated compounds on cell viability of RAW264.7 cells were
measured by MTT assay. As shown in Figure 5, the isolated compounds had no effects on viability of
RAW264.7 at concentration of IC50 values.
Table 2. Inhibition by isolated compounds of NO production stimulated by LPS and IFN-γ
in RAW 264.7 cells.
Compound
IC50 (μM)
Compound
IC50 (μM)
1
2
3
4
5
6
7
8
9
10
11
12
62.3
48.6
>100
37.2
56.8
43.2
>100
>100
20.6
21.5
17.9
23.8
17.5
13
14
15
16
17
18
19
20
21
22
23
24
42.6
18.5
14.2
21.7
16.9
>100
19.8
>100
>100
18.5
26.1
>100
Aminoguanidine
Figure 5. Effects of isolated compounds on cell viability of RAW264.7 cells by MTT assay.
Notes: RAW264.7 cells were treated with isolated compounds at indicated concentrations (100, 30,
10 μM, respectively), cell viability was measured by the MTT assay. Data are expressed as a
percentage of the control levels; A: aminoguanidine.
3. Experimental
3.1. General
The UV spectrum was obtained in MeOH on a Shimadzu UV-160 spectrophotometer, and the IR
spectrum was recorded on a JASCO FT/IR 300E spectrophotometer. Optical rotations were measured

Molecules 2011, 16
8313
in MeOH on a JASCO P-1020 polarimeter. The NMR spectra were recorded on a Varian Mercury 300
spectrometer, with TMS as an internal standard. The MS were obtained on a JEOL GCmate mass
spectrometer. Column chromatography was carried out with Diaion HP-20 (Mitsubishi Chemical Co.),
Sephadex LH-20 (Pharmacia), MCI Gel CHP20P (Mitsubishi Chemical Co.) and Chromatorex ODS
(Fuji Silysia Chemical Ltd.). Thin-layer chromatography (TLC) was performed on Merck TLC plates
(0.25 mm thickness), with compounds visualized by spraying with 5% (v/v) H2SO4 in ethanol solution
and then heating on a hot plate. HPLC was performed on a JASCO PU-2089 apparatus equipped with
JASCO UV-2075 and Shodex OR-2. Senshu Pak Silica 60-5(10 × 150 mm i.d.), YMC-Pack Pro C18
(10 × 150 mm i.d.), YMC-Pack ODS Al (10 × 150 mm i.d.) and Cosmosil 5C18 PAQ (10 × 150 mm
i.d.) was used for preparative purposes. A CAPCELL Pak NH2 column (4.6 × 250 mm i.d.) was used
for the detection of D-galactose.
3.2. Plant Material
Whole plants of E. lunulata Bge. were collected in Hebei Province, China, in October 2006 and were
identified by Professor Naili Wang (Shenyang Pharmaceutical University, China). Voucher specimens
have been deposited at the Laboratory of Pharmacognosy, School of Pharmacy, Nihon University.
3.3. Extraction and Isolation
The whole plants of E. lunulata Bge. (4.75 kg) were extracted twice with 70% acetone and
concentrated to give the extract. This extract (721 g) was dissolved and suspended in water (3 L) and
partitioned into a chloroform layer (81 g), ethyl acetate layer (50 g), n-butanol layer (81 g) and water
layer (490 g). The ethyl acetate layer was subjected to Diaion HP-20 column chromatography and
eluted with CH3OH-H2O to afford eight fractions. Fractions 1 and 2 were purified using Sephadex LH-20
column chromatography and eluted with CH3OH-H2O to give 3 (856 mg) and 16 (88 mg), respectively.
Fraction 3 was purified using Sephadex LH-20 and ODS column chromatography, and reverse-phase
HPLC eluted with CH3OH-H2O to give 1 (9 mg), 2 (13 mg), 5 (15 mg), 6 (607 mg), 7 (200 mg), 9 (68 mg),
15 (39 mg), 16 (5 mg) and 17 (7 mg). Fraction 4 was purified using CHP20P column chromatography
and reverse-phase HPLC eluted with CH3OH-H2O to give 3 (10 mg), 4 (8 mg), 8 (11 mg) and 20 (26 mg).
Fraction 5 was purified using CHP20P column chromatography and reverse-phase HPLC eluted with
CH3OH-H2O to give 9 (1,800 mg), 12 (18 mg) and 14 (6 mg). Fraction 6 was purified using CHP20P
column chromatography and reverse-phase HPLC eluted with CH3OH-H2O to give 10 (9 mg), 11 (10 mg),
13 (6 mg), 21 (5 mg), 22 (125 mg) and 23 (68 mg).The chloroform layer was subjected to silica gel
column chromatography and reverse-phase HPLC to give 24 (25 mg).
Myricetin 3-O-(2'',3''-digalloyl)-β-D-galactopyranoide (1). Yellow amorphous powder; Negative
25
HR-FAB-MS m/z: 783.1040 (Calcd for C35H27O21, 783.1043); [α]D −18.9° (c = 0.10, MeOH). UV
(MeOH) λmax nm (log ε): 210 (4.62), 268 (4.27), 355 (3.96); IR (KBr) νmax cm−1: 3,396, 1,703, 1,656,
1
13
1,608, 1,497, 1,451, 1,352, 1,300, 1,272, 1,231, 1,075, 1,036, 942, 876, 760; H- and C-NMR data,
see Table 1.

Molecules 2011, 16
8314
3.4. Acid Hydrolysis of 1 and Identification of Sugar
Compound 1 (5 mg) was dissolved in 10% H2SO4 and heated at 85 °C for 3 h. After cooling, the
reaction mixture was neutralized by passage though an Amberlite IRA-93ZU (Organo) column and
then partitioned between ethyl acetate and water. The ethyl acetate layer was concentrated and the
concentrate was passed through an ODS column, and then successively eluted with 20% MeOH
and 50% MeOH. Gallic acid was recovered from the 20% MeOH fraction, and myricetin from the
50% fraction by direct comparison with authentic samples. Silica gel TLC of the water layer in
BuOH:Me2CO:H2O (4:5:1) showed the sugar was most likely a galactose (Rf 0.22) by direct
comparison with authentic sample. The water layer was then analyzed by HPLC under the following
conditions: column, CAPCELL Pak NH2 (4.6 × 250 mm i.d.); solvent, CH3CN:H2O = 75:25; flow rate
1.0 mL/min; detector, Shodex OR-2. D-galactose present in the water layer was identified by
comparing its retention time and polarity with those of an authentic sample; tR (min):16.8 min
(D-galactose, positive polarity).
3.5. Triglyceride (TG) Content and Glycerol-3-phosphate Dehydrogenase (GPDH) Activity in 3T3-L1
Cells [10,35]
3T3-L1 preadipocytes (American Type Culture Collection, Manassas, VA, USA) were subcultured
in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% newborn calf serum (Gibco) at
37 °C under a humidified 5% CO2 atmosphere. Briefly, 2 days after reaching confluence (day 0), the
3T3-L1 preadipocytes were induced by switching the differentiation medium to DMEM containing
10% fetal bovine serum (FBS) (SAFC Biosciences), 500 μM 3-isobutyl-1-methylxanthine (Sigma),
1 μM dexamethasone (Sigma), and 10 μg/mL insulin (Sigma) for three days. The medium was then
replaced with DMEM containing 10% FBS and 5 μg/mL insulin, and was changed every 2 or 3 days.
The 3T3-L1 preadipocytes were seeded at 1.0 × 105 cells/mL onto 24-well plates (Sumitomo
Bakelite, MS-80240, Tokyo) and incubated at 37 °C. A test sample was added to the medium on day 0,
and added at the time of every medium change during the 8 days of incubation. After removing the
medium, the cells were washed twice with 500 μL of PBS. The cells were collected in 500 μL of cold
sonication buffer (pH 7.5 25 mM Tris buffer containing 1 mM EDTA) and sonicated in ice-cold water.
After centrifugation, the cell lysate was used to measure the TG content with LabAssayTM Triglyceride
(WAKO Pure Chemical Industries Ltd.), GPDH activity with a GPDH Assay Kit (TaKaRa Bio Inc.),
and DNA quantity with a DNA Quantity Kit (Primary Cell Co., Ltd.), according to the manufacturer's
protocol. Inhibition of TG and GPDH activity was calculated using the following formula: Inhibition
(%) = [(Cn − S)/Cn] × 100, where S is TG amount or GPDH activity when cells incubated with sample
and divided by the amount of DNA for each well; Cn is TG amount or GPDH activity when cells
incubated with DMSO (control) were divided by the amount of DNA for each well). Cell viability was
confirmed by microscopic observation, and was measured using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-
diphenyl-2H-tetrazolium bromide (MTT) assay method.

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3.6. NO Production in Activated Macrophage-Like Cell Line, RAW 264.7 [10,36]
The macrophage-like cell line, RAW 264.7, was obtained from American Type Culture Collection.
The cells were cultured in Ham’s F12 medium with 10% FBS (SAFC Biosciences) at 37 °C under a
humidified 5% CO2 atmosphere. The RAW264.7 cells were seeded at 1.2 × 106 cells/mL onto 96-well
plates (Sumitomo Bakelite, MS-8096R, Tokyo) and then incubated at 37 °C for 2 h. A test sample was
then added to the culture simultaneously with both Escherichia coli LPS (100 ng/mL) and recombinant
mouse IFN-γ (0.33 ng/mL), and the cells were incubated at 37 °C, usually for 16 h. The amount of
nitrite in culture supernatants was measured using the Griess assay. Cytotoxicity was measured using
the MTT assay method.
3.7. Statistical Analysis
The values represent mean ± S.D. of at least three experiments. Statistical significant differences
were evaluated by the one-way ANOVA followed by the Student’s t-test for paired populations, and a
P value of 0.05 or less among the means were considered significant.
4. Conclusions
Obesity is associated with low-grade chronic inflammation of adipose tissues, and obese adipose
tissue is characterized by increased infiltration of macrophages. The whole plant extract of E. lunulata
Bge. showed inhibitory effects on the differentiation of 3T3-L1 cells and NO production in RAW264.7
cells. Bioassay-directed investigation of active constituents, led to the identification of a new flavonol
galactopyranoside gallate, myricetin 3-O-(2'',3''-digalloyl)-β-D-galactopyranoide (1) and 23 known
phenolic constituents. The isolated compounds, especially hydrolysable tannins, flavonoids, and
flavonol galactopyranoside gallates, showed significant inhibition towards the differentiation of
3T3-L1 adipocytes and nitric oxide production in RAW 264.7 cells. These studies suggest that the
extract of E. lunulata Bge. and the isolated compounds might be a source of anti-obesity and anti-
inflammatory agents to improve the symptoms of metabolic syndrome.
Acknowledgments
This study was financially supported by a Grant-in-Aid for Scientific Research (20590016) and by
the ‘‘High-Tech Research Center (2007-2011)’’ Project for Private Universities, with a matching fund
subsidy from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan.
Conflict of Interest
The authors declare no conflict of interest.
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