Inhibitory effects of the constituents of hippophae rhamnoides on 3t3-l1 cell differentiation and nitric oxide production in RAW264.7 Cells

Article abstract of DOI:10.1248/cpb.c12-00835

Three new flavonol glycosides, hippophaeosides A-C (1-3), together with 27 known constituents, were isolated from Hippophae rhamnoides L. leaves. Their structures were determined by spectroscopic analyses. Their inhibitory activities on 3T3-L1 preadipocyte differentiation and triglyceride accumulation in maturing adipocytes, and nitric oxide production in RAW264.7 cells were examined.

Full text of DOI:10.1248/cpb.c12-00835

March 2013
Regular Article
Chem. Pharm. Bull. 61(3) 279–285 (2013)
279
Inhibitory Effects of the Constituents of Hippophae rhamnoides on 3T3-
L1 Cell Differentiation and Nitric Oxide Production in RAW264.7 Cells
a,†
b
b
a
,a
Zhi-Gang Yang, Xiu-Feng Wen, Yong-Hai Li, Keiichi Matsuzaki, and Susumu Kitanaka*
a
b
School of Pharmacy, Nihon University; 7–7–1 Narashinodai, Funabashi, Chiba 274–8555, Japan: and National
Administration Center for Sea Buckthorn Development, Ministry of Water Resources of China; Jia 1 Fuxing Road,
Haidian District, Beijing 100038, P.R. China. Received September 21, 2012; accepted December 1, 2012
Three new flavonol glycosides, hippophaeosides A–C (1–3), together with 27 known constituents, were
isolated from Hippophae rhamnoides L. leaves. Their structures were determined by spectroscopic analyses.
Their inhibitory activities on 3T3-L1 preadipocyte differentiation and triglyceride accumulation in maturing
adipocytes, and nitric oxide production in RAW264.7 cells were examined.
Key words Hippophae rhamnoides; flavonol glycoside; triterpenoid; adipocyte; macrophage
Hippophae rhamnoides L. (Elaeagnaceae), known as “sea spectroscopic data with those in the literature as kaempferol
1
3)
14)
buckthorn,” is a deciduous shrub distributed throughout Asia (4), kaempferol 3-O-rutinoside (5), kaempferol 3-O-β-d-
15)
and Europe. The fruit has been used as a traditional medi- glucopyranosyl-7-O-α-l-rhamnopyranoside (6),
cine for the treatment of cough indigestion and blood stasis 3-O-β-d-glucopyranoside (7), tiliroside (8), quercetin (9),
in China. Investigations of H. rhamnoides have yielded fla- rutin (10),
and tannins. The leaf has been rhamnopyranoside (11), quercetin 3-O-β-d-glucopyranoside
reported to have anti-inflammatory, antioxidant, anti-obesi- (12), quercetin 7-O-α-l-rhamnopyranoside (13), isorham-
netin (14), isorhamnetin 3-O-rutinoside (15), isorhamne-
In our previous study, we investigated anti-obesity and anti- tin 3-O-β-d-glucopyranosyl-7-O-α-l-rhamnopyranoside (16),
inflammatory compounds that not only inhibit triglyceride isorhamnetin 3-O-β-d-glucopyranoside (17),
TG) accumulation by 3T3-L1 adipocytes but also inhibit the (18), methyl gallate (19), 4-hydroxybenzoic acid (20),
secretion of nitric oxide (NO) from RAW264.7 cells. In that protocatechuic acid (21),
(22), ursolic acid (23), ursolic aldehyde (24), 28-nor-urs-
In the present study, the 80% methanol extract of the leaves 12-ene-3β,17β-diol (25), pomolic acid (26), 2α-hydroxy
of H. rhamnoides L. and the chloroform- and ethyl acetate- ursolic acid (27), 23-hydroxy ursolic acid (28), vomifoliol
soluble portions of the extract were observed to inhibit the (29), and β-sitosterol (30) (Fig. 1).
kaempferol
16)
3)
17)
1)
18)
quercetin 3-O-β-d-glucopyranosyl-7-O-α-l-
2
–4)
5,6)
3)
19)
vonols,
triterpenoids,
7
)
4)
20)
21)
8
,9)
4)
22)
23)
ty, and α-glucosidase inhibitory activities.
2
4)
15)
gallic acid
2
5)
26)
27)
(
2
8)
methyl brevifolincarboxylate
10–12)
29)
30)
31)
study, several crude drugs were examined.
32)
33)
33)
34)
3
5)
30)
TG accumulation in 3T3-L1 cells, and inhibit NO production
Hippophaeoside A (1) was obtained as a yellow amorphous
in lipopolysaccharide (LPS) and interferon-γ (IFN-γ) activated powder and gave positive Mg–HCl and FeCl tests. The mo-
3
macrophages. Furthermore, an attempt to identify the bioac- lecular formula of 1 was established to be C H O by high
37
44 17
tive compounds present in this plant led to the isolation of resolution (HR)-FAB-MS. The UV spectrum exhibited absorp-
three new flavonol glycosides, Hippophaeosides A (1), B (2), tion maxima at 264nm and 352nm, suggesting the presence of
and C (3), along with 27 known constituents.
aromatic rings in the molecule. The IR spectrum contained
−1
absorption bands for a hydroxyl group (3387cm ), conjugated
−
1
−1
Results and Discussion
The leaves of H. rhamnoides L. were extracted with 80%
carbonyl (1654cm ), and aromatic ring (1597, 1515cm ). The
1
H-NMR spectrum of 1 indicated meta-coupled proton signals
aqueous methanol, followed by evaporation of the solvent at δ 6.45 (1H, d, J=2.0Hz) and 6.73 (1H, d, J=2.0Hz); an
under reduced pressure from the combined extract to give A B -type aromatic proton signal at δ 6.87 (2H, d, J=8.9Hz)
2
2
the 80% methanol extract. The extract [TG and NO in- and 8.07 (2H, d, J=8.9Hz); two sugar anomeric proton signals
hibitory activity: 35% and 63%, respectively (30µg/mL)] at δ 5.31 (1H, d, J=7.6Hz) and 5.61 (1H, d, J=1.4Hz); and one
was dissolved in water and successively partitioned to give methyl proton of 6-deoxyhexose at δ 1.28 (3H, d, J=6.9Hz).
a chloroform layer [82, 67% (30µg/mL)], ethyl acetate layer Kaempferol, d-glucose, and l-rhamnose were identified by
[
45, 60% (30µg/mL)], n-butanol layer [10, 19% (30µg/mL)], the acid hydrolysis of 1 (see Experimental). The anomeric
and aqueous layer [42, 15% (30µg/mL)]. We focused our ef- configuration for glucose was assigned as β from the relatively
forts on investigating the chemical constituents of the chlo- large J values (7.6Hz) of the anomeric proton, and rhamnose
1
roform- and ethyl acetate-soluble fractions. The ethyl acetate was α from the J (173.4Hz) coupling constant between H-1
CH
36,37)
layer was separated by Sephadex LH-20 column chroma- and C-1.
Based on these results, the sugars in 1 were de-
tography to yield four fractions from which 22 compounds termined to be β-d-glucopyranose and α-l-rhamnopyranose.
1
including three new compounds 1–3 were isolated. Eight
The H-NMR data of 1 also showed the presence of one
compounds were isolated from the chloroform layer. Com- monoterpene moiety; an ABX system of three olefinic proton
pounds 4–30 were identified by detailed comparisons of their signals at δ 5.07 (1H, dd, J=10.7, 1.4Hz), 5.24 (1H, dd, J=17.2,
1
.4Hz), and 5.93 (1H, dd, J=17.2, 10.7Hz), an olefinic proton
signal at δ 6.89 (1H, t, J=7.6Hz), two methyl signals at δ 1.29
Present address: RIKEN Plant Science Center; 1–7–22 Suehiro-cho, (3H, s) and 1.88 (3H, s), and two methylene proton signals at
Tsurumi-ku, Yokohama, Kanagawa 230–0045, Japan.
δ 1.65 (2H, m) and 2.28 (2H, m). Furthermore, in conjunction
The authors declare no conflict of interest.
†
*
To whom correspondence should be addressed. e-mail: kitanaka.susumu@nihon-u.ac.jp
© 2013 The Pharmaceutical Society of Japan

2
80
Vol. 61, No. 3
Fig. 1. Structures of Compounds 1–30
Fig. 2. Key HMBC Correlations of Compounds 1, 2, and 3
13
1
1
with the C-NMR, H– H correlation spectroscopy (COSY), acid [(−)-linalool-1-oic acid].
heteronuclear multiple quantum coherence (HMQC), and Furthermore, in the HMBC spectrum, the anomeric proton
heteronuclear multiple bond connectivity (HMBC) spectra signals δ 5.31 (H-1″) and 5.61 (H-1‴) showed long-range cor-
indicated that the monoterpene moiety may be 2,6-dimethyl- relation with the carbon signal at δ 135.7 (C-3) and 163.2 (C-7)
6
-hydroxy-2,7-octadienoic acid. The E form of the trisubstitut- respectively, suggesting the location of the glucose was at C-3,
ed double bond was deduced from the chemical shift of H-3 (δ and rhamnose was at C-7. The sugar proton signal at δ 5.26
.89) because the olefinic proton of the Z-isomer would appear (H-2‴) showed correlations with the carbon signal at δ 169.0
6
38)
at a higher field. Moreover, after alkaline hydrolysis of 1, the (C-1⁗), suggesting the location of the (−)-linalool-1-oic acid
absolute configuration for C-6 was determined to be R by com- was at the C-2 of rhamnose (Fig. 2). Based on the foregoing
parison of its optical rotation with the literature (see Experi- findings, hippophaeoside A (1) was determined to be kaemp-
mental). Therefore, the monoterpene moiety in 1 were deter- ferol
3-O-β-d-glucopyranosyl-7-O-[(6R,2E)-2,6-dimethyl-6-
mined to be (6R,2E)-2,6-dimethyl-6-hydroxy-2,7-octadienoic hydroxy-2,7-octadienoyl-(1→2)]-α-l-rhamnopyranoside.

March 2013
281
Hippophaeoside B (2) was isolated as a yellow powder; the 3)]-α-l-rhamnopyranoside.
molecular formula of 2 was established to be C H O by
HR-FAB-MS. The H-NMR spectral data of 2 were similar to molecular formula of 3 was established to be C H O by
Hippophaeoside C (3) was isolated as a yellow powder; the
37
44 17
1
38
46 18
1
those of 1, except for a sugar proton signal at δ 5.16 (1H, dd, HR-FAB-MS. The H-NMR spectral data of 3 were similar to
J=9.3, 3.4Hz) (Table 1). Acid hydrolysis of 2 yielded kaemp- those of 2, except for a methoxy proton signal at δ 3.93 (3H, s)
ferol, d-glucose, and l-rhamnose (see Experimental). More- and 1,3,5-trisubstituted aromatic ring proton signals at δ 6.89
over, (6R,2E)-2,6-dimethyl-6-hydroxy-2,7-octadienoic acid (1H, d, J=8.3Hz), 7.60 (1H, dd, J=8.3, 1.2Hz), and 7.93 (1H,
was identified by the alkaline hydrolysis of 2 (see Experimen- d, J=1.2Hz) (Table 1). Acid hydrolysis of 3 yielded isorham-
tal). Furthermore, in the HMBC spectrum, the anomeric pro- netin, d-glucose, and l-rhamnose (see Experimental). More-
ton signals δ 5.32 (H-1″) and 5.59 (H-1‴) showed long-range over, (6R,2E)-2,6-dimethyl-6-hydroxy-2,7-octadienoic acid
correlation with the carbon signal at δ 135.7 (C-3) and 163.4 was identified by the alkaline hydrolysis of 3 (see Experimen-
(C-7), respectively, suggesting the location of the glucose to tal). Furthermore, in the HMBC spectrum, the anomeric pro-
be at C-3, and rhamnose to be at C-7. The HMBC correlations ton signals δ 5.46 (H-1″) and 5.59 (H-1‴) showed long-range
between the δ 5.16 (H-3‴) and δ 169.4 (H-1⁗), indicated that correlation with the carbon signal at δ 135.5 (C-3) and 163.3
the (−)-linalool-1-oic acid was located at the C-3 of rham- (C-7), respectively, suggesting the location of the glucose to
nose (Fig. 2). Thus, the structure of hippophaeoside B (2) be at C-3, and rhamnose to be at C-7. The HMBC correlations
was determined to be kaempferol 3-O-β-d-glucopyranosyl-7- between the δ 5.16 (H-3‴) and δ 169.4 (H-1⁗), indicated that
O-[(6R,2E)-2,6-dimethyl-6-hydroxy-2,7-octadienoyl-(1→ the (−)-linalool-1-oic acid was located at the C-3 of rhamnose
1
13
Table 1. H- and C-NMR Spectral Data of Compounds 1, 2 and 3 [(600/150MHz, in Methanol-d , TMS, δ (ppm) (J=Hz)]
4
1
2
3
Position
δH
δC
δH
δC
δH
δC
2
3
4
159.6
135.7
179.7
107.6
162.9
100.6
163.2
95.7
158.0
122.6
132.4
116.2
161.8
116.2
132.4
159.6
135.7
179.7
107.5
162.9
100.6
163.4
95.7
158.0
122.6
132.4
116.2
161.8
116.2
132.4
159.1
135.5
179.5
107.5
162.8
100.6
163.3
95.6
157.9
122.7
114.3
148.4
151.1
116.0
124.0
56.8
4
5
6
7
8
8
1
2
3
4
5
6
a
6.45 (d 2.0)
6.73 (d 2.0)
6.49 (d 2.0)
6.77 (d 2.0)
6.48 (d 1.7)
6.74 (d 1.7)
a
′
′
′
′
′
′
8.07 (d 8.9)
6.87 (d 8.9)
8.07 (d 8.9)
6.88 (d 8.9)
7.93 (d 1.2)
6.87 (d 8.9)
8.07 (d 8.9)
6.88 (d 8.9)
8.07 (d 8.9)
6.89 (d 8.3)
7.60 (dd 8.3, 1.2)
3.93 (s)
3
1
2
3
4
5
6
6
1
2
3
4
5
6
1
2
3
4
5
6
7
8
8
9
′-OCH3
″
″
″
″
5.31 (d 7.6)
3.44 (m)
3.42 (m)
3.30 (m)
3.22 (m)
3.70 (dd 12.0, 2.4)
3.53 (dd 12.0, 5.5)
5.61 (d 1.4)
5.26 (m)
4.07 (dd 9.2, 3.1)
3.50 (m)
103.8
75.8
78.0
71.4
78.5
62.7
5.32 (d 7.3)
3.44 (m)
3.42 (m)
3.30 (m)
3.21 (m)
103.7
75.8
78.0
71.4
78.5
62.7
5.46 (d 7.2)
3.47 (m)
3.45 (m)
3.30 (m)
3.25 (m)
3.74 (m)
3.55 (dd 12.1, 5.5)
5.59 (brs)
4.24 (m)
5.16 (dd 9.6, 3.4)
3.74 (m)
103.3
75.9
78.0
71.4
78.7
″
″a
″b
‴
‴
‴
‴
‴
‴
⁗
⁗
⁗
⁗
⁗
⁗
⁗
⁗a
⁗b
⁗
3.70 (m)
62.5
3.55 (dd 12.0, 5.5)
5.59 (d 1.7)
4.24 (m)
5.16 (dd 9.3, 3.4)
3.74 (m)
97.1
73.4
70.4
74.1
71.4
99.8
69.5
75.2
71.0
71.5
99.7
69.4
75.2
71.0
71.5
3.67 (m)
1.28 (d 6.9)
3.70 (m)
1.28 (d 5.8)
3.70 (m)
1.28 (d 6.2)
18.2
18.1
18.1
169.0
128.3
145.2
24.7
41.7
73.7
169.4
128.6
144.6
24.7
41.7
73.7
169.4
128.6
144.6
24.6
41.7
73.6
6.89 (t 7.6)
2.28 (m)
1.65 (m)
6.95 (t 7.5)
2.28 (m)
1.65 (m)
6.95 (t 7.5)
2.28 (m)
1.65 (m)
5.93 (dd 17.2, 10.7)
5.24 (dd 17.2, 1.4)
5.07 (dd 10.7, 1.4)
1.88 (s)
146.0
112.5
5.93 (dd 17.5, 10.6)
5.24 (dd 17.5, 1.4)
5.07 (dd 10.6, 1.4)
1.89 (s)
146.0
112.5
5.93 (dd 17.5, 10.6)
5.25 (dd 17.5, 1.4)
5.07 (dd 10.6, 1.4)
1.89 (s)
145.9
112.5
12.5
27.9
12.5
27.9
12.5
27.8
1
0⁗
1.29 (s)
1.29 (s)
1.29 (s)

2
82
Vol. 61, No. 3
Fig. 3. Inhibition of TG Content and GPDH Activity by the Isolated Compounds 1–29 in 3T3-L1 Adipocytes
3T3-L1 adipocytes were harvested after 8d of treatment with compounds (1–22, 29: at the concentration of 30µm; 23–28: at the concentration of 10µm) or vehicle, and
assayed for total triglyceride (TG) content and glycerol-3-phosphate dehydrogenase (GPDH) activity. The reported values are the mean± S.D. (n=3).
(
Fig. 2). Thus, the structure of hippophaeoside C (3) was Table 2. Inhibition by Isolated Compounds of NO Production Stimulated
determined to be isorhamnetin 3-O-β-d-glucopyranosyl-7-O- by LPS and IFN-γ in RAW264.7 Cells
[
(6R,2E)-2,6-dimethyl-6-hydroxy-2,7-octadienoyl-(1→3)]-α-l-
Compound
IC50 (µm)
Compound
IC50 (µm)
rhamnopyranoside.
Compounds 1–30 were next evaluated for their inhibitory
effects on triglyceride accumulation and glycerol-3-phosphate
dehydrogenase (GPDH) activity in 3T3-L1 cells (Fig. 3).
Quercetin (9) was used as a positive control (it has been re-
1
2
3
4
5
6
7
8
9
68.8
67.0
52.3
16
17
18
19
20
21
22
23
24
25
26
27
28
29
>100
>100
>100
>100
>100
>100
>100
17.8
>100
>100
15.0
23.6
12.5
>100
17.5
18.2
10,12,39)
>100
>100
>100
26.9
ported to have inhibitory effects on TG accumulation).
In general, the flavonol in free form showed stronger activ-
ity than the glycosides at 30µm (Fig. 3). The flavonols, 4, 9,
and 14 showed significant TG inhibitory activities in 3T3-L1
adipocytes, with TG inhibition values of 45.6%, 42.2%, and
20.6
1
1
0
1
>100
>100
>100
>100
25.1
4
4.3%, respectively. The new flavonol glycosides, 1, 2, and 3,
showed moderate TG inhibitory activities (28.9, 25.5, 30.1%
inhibition, respectively). The other flavonol glycosides 5–8,
12
13
14
1
0–13, and 15–17 showed moderate inhibitory effects on TG
and GPDH activity. The major flavonol glycoside, tiliroside (8,
.05% yield), showed 25.1% TG inhibitory activity, and has
1
5
>100
Aminoguanidine
0
4
0)
been reported to have anti-obesity effects in mice. Compar-
ing 2 with 6, 3 with 16, and 8 with 7, compounds 2, 3, and 8 18.2, 20.6, and 16.9µm, respectively (Table 2). Most of the fla-
showed stronger inhibition of TG and GPDH activity than 6, vonol glycosides, 5–7, 10–13, and 15–17 did not show inhibi-
16, and 7, respectively. These results suggest that the linaloyl tory effects on NO production. However, the new compounds
or coumaroly group at the sugar moiety of flavonol glycosides 1, 2, and 3, and the major flavonol glycoside 8 showed stron-
may enhance their activities. In addition, some of flavonoids ger activities than glycosides 6, 7, and 16, with IC50 values of
have been reported to inhibit triglyceride accumulation in 68.8, 67.0, 52.3, and 26.9µm, respectively, suggesting that the
1
1,12,41–43)
3
T3-L1 adipocytes.
showed weak inhibitory effects on TG and GPDH activity at tant for their activities. Many flavonoids are also reported to
0µm. The triterpenoids 23, 26, 27, and 28 showed significant have inhibitory effects on NO production in activated macro-
The phenylpropanoids 18–22 linaloyl or coumaroly groups at the sugar moiety are impor-
3
11,45–47)
TG inhibitory activities at 10µm, with TG inhibition values phages.
Our results are in agreement with these earlier
of 27.8%, 36.4%, 27.7%, and 24.0%, respectively, which were studies. The phenylpropanoids 18–22 did not show inhibition
stronger than those of 24 and 25. These results showed that of NO production. The triterpenoids 23, 26, 27, and 28 showed
the carboxyl group at the C-28 of triterpenoids may enhance stronger NO production inhibitory effects than 24 and 25,
their activities.
with IC50 values of 17.8, 15.0, 23.6, and 12.5µm, respectively.
Compounds 1–29 were also examined with respect to their These results showed that the carboxyl group at C-28 may
inhibition of NO production stimulated by LPS and IFN-γ enhance their activities. Our results suggest that the extracts
in RAW264.7 cells (Table 2). In the assay, aminoguanidine of the leaves of H. rhamnoides L. and the isolated compounds
(which has been reported to have inhibitory effects on NO might be sources of anti-obesity and anti-inflammatory agents
production in LPS-activated RAW264.7 macrophages by to improve the symptoms of the metabolic syndrome.
down-regulation of the expression of inducible nitric oxide
10,44)
synthase (iNOS)) was used as a positive control.
flavonol in free form 4, 9, and 14 showed stronger NO pro-
duction inhibitory effects than glycosides, with IC50 values of obtained in MeOH on a Shimadzu UV-160 spectrophotometer,
The Experimental
General Experimental Procedure The UV spectrum was

March 2013
283
and the IR spectrum was recorded on a JASCO FT/IR 4200/ fractions (Frs. 2-1–4). Fraction 2-3 was purified using ODS
type A spectrophotometer. Optical rotations were measured C.C. [CH OH–H O (0:100→100:0 v/v)] to give 23 (3000mg),
3
2
in MeOH on a JASCO P-1020 polarimeter. The NMR spectra 26 (5mg), 27 (60mg), 28 (4mg) and 29 (18mg). Fraction 2-2
were recorded on a JEOL ECA 600MHz spectrometer, with was purified using ODS C.C. [CH OH–H O (50:50→100:0
3
2
tetramethylsilane (TMS) as an internal standard. The MS v/v)] to give 24 (8mg), 25 (11mg) and 30 (670mg).
2
5
were obtained on a JEOL GCmate mass spectrometer. Col-
umn chromatography was carried out with Sephadex LH-20 (c=0.1, MeOH); negative FAB-MS (m/z): 759 [M−H] , 597,
(
Hippophaeoside A (1): Yellow powder; [α]D −112.8°
−
−
Pharmacia), Chromatorex ODS (Fuji Silysia Chemical Ltd.) 447, 285, 151; HR-FAB-MS m/z: Found 759.2499 [M−H]
and Silica gel 60N (Kanto Chemical Co., Inc.). Thin-layer (Calcd 759.2499 for C H O ); UV (MeOH) λ (logε): 206
37
43 17
max
−
1
chromatography (TLC) was performed on Merck TLC plates (4.68), 264 (4.34), 352 (4.27); IR (KBr) νmax cm 3387 (OH),
0.25mm thickness), with compounds visualized by spray- 2927, 1702, 1654 (C=O), 1597, 1515 (aromatic C=C), 1491,
(
1
13
ing with 5% (v/v) H SO in ethanol solution and then heating 1460,1346, 1282, 1209, 1066, 922; H- and C-NMR data: see
2
4
on a hot plate. HPLC was performed on a JASCO PU-2089 Table 1.
apparatus equipped with JASCO UV-2075. Cosmosil 5C18 Hippophaeoside B (2): Yellow powder; [α]D −119.0° (c=0.1,
2
5
−
MSII (10×150mm i.d.), Cosmosil 5C18 PAQ (10×150mm MeOH); negative FAB-MS (m/z): 759 [M−H] , 596, 447,
−
i.d.), Cosmosil π-NAP (10×150mm i.d.) and YMC Pack-Ph 285, 151; HR-FAB-MS m/z: Found 759.2501 [M−H] (Calcd
(
10×150mm i.d.) column was used for preparative purposes. 759.2499 for C H O ); UV (MeOH) λ
(logε): 206 (4.73),
max
−1
37
43 17
A Cosmosil Sugar-D (4.6×250mm i.d.) column was used for 265 (4.39), 352 (4.31); IR (KBr) νmax cm 3421, 2929, 1692,
the detection of d-glucose and l-rhamnose. 1653 (C=O), 1597, 1515 (aromatic C=C), 1491, 1460,1348,
Plant Material Whole plants of H. rhamnoides L. were 1283, 1208, 1068, 922; H- and C-NMR data: see Table 1.
collected in Neimenggu, China, in October 2008 and were
identified by Professor Naili Wang (Shenyang Pharmaceutical MeOH); negative FAB-MS (m/z): 789 [M−H] , 626, 477,
1
13
2
5
Hippophaeoside C (3): Yellow powder; [α]D −105.0° (c=0.1,
−
−
University, China). Voucher specimens have been deposited at 327, 314, 151; HR-FAB-MS m/z: Found 789.2613 [M−H]
the Laboratory of Pharmacognosy, School of Pharmacy, Nihon (Calcd 789.2605 for C H O ); UV (MeOH) λ (logε): 207
3
8
45 18
max
−
1
University.
(4.77), 253 (4.44), 355 (4.31); IR (KBr) νmax cm 3412 (OH),
Extraction and Isolation The leaves of H. rhamnoides 2931, 1693, 1655 (C=O), 1597, 1515 (aromatic C=C), 1491,
1
13
L. (6.0kg) were extracted three times with 80% methanol 1460,1345, 1287, 1206, 1067, 922; H- and C-NMR data: see
and concentrated to give the extract. This extract (1.4kg) was Table 1.
dissolved and suspended in water (6L) and partitioned into a
chloroform layer (150g), ethyl acetate layer (60g), n-butanol Samples (8mg) of 1, 2, and 3 were separately dissolved in 2m
layer (237g) and water layer (933g).
HCl and heated at 90°C for 2h. After cooling, the reaction
Acid Hydrolysis of 1, 2, 3 and Identification of Sugar
The ethyl acetate layer was subjected to Sephadex LH-20 mixture was neutralized and then partitioned between ethyl
column chromatography (C.C.) and eluted with CH OH–H O acetate and water. Kaempferol was isolated from the ethyl
3
2
(0:100→100:0 v/v) to afford four fractions (Frs. 1–4). Frac- acetate layers of 1 and 2; isorhamnetin was isolated from
tion 2 was purified using ODS C.C. [CH OH–H O (0:100→ the ethyl acetate layer of 3, which were identified by TLC
3
2
100:0 v/v)] to yield four fractions (Frs. 2-1–4). 18 (1200mg) comparison with authentic samples. The water layers were
was crystallized from Fr. 2-1 using MeOH–H O. Fr. 2-2 was then separately analyzed by HPLC under the following condi-
2
purified using Silica gel C.C. [CHCl –MeOH (100:0→0:100 tions: column, Cosmosil Sugar-D (4.6×250mm i.d.); solvent,
3
v/v)] and reverse-phase HPLC eluted with CH OH–H O to CH CN–H O=75:25; flow rate 0.8mL/min; detector, Shodex
3
2
3
2
give 19 (570mg), 20 (14mg) and 21 (5mg). Fraction 2-3 was OR-2. d-Glucose and l-rhamnose present in the water layer
purified using Silica gel C.C. [CHCl –MeOH (100:0→0:100 were identified by comparing its retention time (t ) and polar-
3
R
v/v)] and reverse-phase HPLC eluted with CH OH–H O to ity with those of an authentic sample; d-glucose (t : 8.5min,
3
2
R
give 5 (6mg), 6 (12mg), 10 (11mg), 11 (17mg), 15 (31mg), 16 positive polarity), l-rhamnose (t : 6.0min, negative polarity).
R
(8.3mg) and 22 (24mg). Fraction 3 was purified using Silica
Alkaline Hydrolysis of 1, 2, and 3 Samples (8mg) of 1,
gel C.C. [CHCl –MeOH (100:0→0:100 v/v)] to yield six frac- 2, and 3 were separately dissolved in 1% aqueous NaHCO3
3
tions (Frs. 3-1–6). Fraction 3-3 was purified using ODS C.C. and heated at 90°C for 2h. After cooling, the reaction mixture
[
CH OH–H O (0:100→100:0 v/v)] to afford five fractions was neutralized and then partitioned between chloroform and
3
2
(
Frs. 3-3-1–5). Fraction 3-3-3 was purified using Silica gel water. The monoterpene was isolated from the chloroform
C.C. and reverse-phase HPLC eluted with CH OH–H O to layer, which was identified as (6R,2E)-2,6-dimethyl-6-hy-
3
2
give 1 (21mg), 2 (30mg) and 3 (35mg). Fraction 3-3-2 was droxy-2,7-octadienoic acid [(−)-linalool-1-oic acid] from the
1
13
purified using reverse-phase HPLC eluted with CH OH–H O
H- and C-NMR data and by comparison of its optical rota-
3
2
2
5
48)
to give 7 (30mg) and 17 (30mg). Fraction 3-4 was purified tion value [α]D −15.0° (c=0.1, CHCl ) with literature.
3
using ODS C.C. [CH OH–H O (0:100→100:0 v/v)] to give
Triglyceride (TG) Content and Glycerol-3-phosphate De-
3
2
10,12)
1
2 (130mg). Fraction 4 was purified using Silica gel C.C. hydrogenase (GPDH) Activity in 3T3-L1 Cells
3T3-L1
[
CHCl –MeOH (100:0→0:100 v/v)] to yield four fractions preadipocytes (American Type Culture Collection, Manassas,
3
(
Frs. 4-1–4). Fraction 4-4 was purified using ODS C.C. to give VA, U.S.A.) were subcultured in Dulbecco’s modified Eagle’s
4
(5mg), 8 (1400mg), 9 (5mg), 13 (15mg) and 14 (6mg).
medium (DMEM) containing 10% newborn calf serum
The chloroform layer was subjected to Silica gel C.C. and (Gibco) at 37°C under a humidified 5% CO2 atmosphere.
eluted with CHCl –MeOH (100:0→0:100 v/v) to yield four Briefly, 2d after reaching confluence (day 0), the 3T3-L1
3
fractions (Fr. 1–4). Fraction 2 was purified using Silica gel preadipocytes were induced by switching the differentiation
C.C. [n-hexane–EtOAc (100:0→0:100 v/v)] to yield four medium to DMEM containing 10% fetal bovine serum (FBS)

2
84
Vol. 61, No. 3
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SAFC Biosciences), 500µm 3-isobutyl-1-methylxanthine
(Sigma), 1µm dexamethasone (Sigma), and 10µg/mL insu-
(2007).
lin (Sigma) for three days. The medium was then replaced
with DMEM containing 10% FBS and 5µg/mL insulin, and
was changed every 2 or 3d. The 3T3-L1 preadipocytes were
Ganju L., Padwad Y., Singh R., Karan D., Chanda S., Chopra M. K.,
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seeded at 1.0×10 cells/mL onto 24-well plates (Sumitomo
8)
Nishi S., Saito Y., Koaze H., Hironaka K., Kojima M., Nippon
Shokuhin Kagaku Kogaku Kaishi, 54, 477–481 (2007).
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 8d of incubation.
After removing the medium, the cells were washed twice with
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1
5
00µL of phosphate buffered saline (PBS). The cells were
1
collected in 500µL of cold sonication buffer (pH 7.5–25mm
Tris buffer containing 1mm ethylenediaminetetraacetic acid
(
tion, the cell lysate was used to measure the TG content with
LabAssay™ 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
1
1
1
EDTA)) and sonicated in ice-cold water. After centrifuga-
1
1
1
Cell Co., Ltd.), according to the manufacturer's protocol. In- 15) Özden S., Dürüst N., Toki K., Saito N., Honda T., Phytochemistry,
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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
dimethyl sulfoxide (DMSO) (control) were divided by the
amount of DNA for each well). Cell viability was confirmed
by microscopic observation, and was measured using the
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1
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1
0)
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were cultured in Ham’s F12 medium with 10% FBS (SAFC
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6-well plates (Sumitomo Bakelite, MS-8096R, Tokyo) and
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to the culture simultaneously with both Escherichia coli LPS
2
2
6
9
2
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2
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2
3
Acknowledgments This study was financially supported
by a Grant-in-Aid for Scientific Research (No. 24590028)
from the Ministry of Education, Culture, Sports, Science, and
Technology (MEXT) of Japan and by the Nihon University
Multidsciplinary Research Grant (No. 12-021).
3
3
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