Isolation of 9-hydroxy-10E,12Z-octadecadienoic acid, an inhibitor of fat accumulation from Valeriana fauriei

Article abstract of DOI:10.1271/bbb.110994

An EtOH extract of Valeriana fauriei was found to exhibit potent inhibition of fat accumulation against 3T3-L1 murine adipocytes. After performing several chromatographic steps, we successfully isolated the conjugated linoleic acid derivative, 9-hydroxy-10E,12Z-octadecadienoic acid (9-HODE). Synthesized 9-HODE and its analogs showed inhibitory activity against fat accumulation.

Full text of DOI:10.1271/bbb.110994

Biosci. Biotechnol. Biochem., 76 (6), 1233–1235, 2012
Note
Isolation of 9-Hydroxy-10E,12Z-octadecadienoic Acid, an Inhibitor
of Fat Accumulation from Valeriana fauriei
y
Keiji YUKI,1 Mariko IKEDA,1 Kenji MIYAMOTO,1; Osamu OHNO,2
y
3;
Kaoru YAMADA,3 and Daisuke UEMURA
1Department of Biosciences and Informatics, Keio University, 3-14-1 Hiyoshi, Yokohama 223-8522, Japan
2Department of Chemistry, Keio University, 3-14-1 Hiyoshi, Yokohama 223-8522, Japan
3Department of Chemistry, Kanagawa University, 2946 Tsuchiya, Hiratsuka 256-1293, Japan
Received January 6, 2012; Accepted February 21, 2012; Online Publication, June 7, 2012
[doi:10.1271/bbb.110994]
An EtOH extract of Valeriana fauriei was found to
exhibit potent inhibition of fat accumulation against
3T3-L1 murine adipocytes. After performing several
chromatographic steps, we successfully isolated the
conjugated linoleic acid derivative, 9-hydroxy-
10E,12Z-octadecadienoic acid (9-HODE). Synthesized
9-HODE and its analogs showed inhibitory activity
against fat accumulation.
Pure Chemical Industries) was used to determine the
amount of triglyceride in the 3T3-L1 cells, and Cell
Counting Kit-8 (Dojindo Laboratories) was used to
determine the cell viability. Both the fat accumulation
(FA) and cell viability (CV) rates were determined by
dividing the absorbance value of a sample by the
absorbance of the control which had been exclusively
treated with a vehicle. An FA rate of 50% and CV rate
of 50% are respectively presented as the EC50 and IC50
values.
Key words: Valeriana fauriei; 3T3-L1 murine adipo-
cytes; inhibitor; fat accumulation
The rhizomes and roots of V. fauriei (2 kg) purchased
from Tochimoto Tenkaido Co. (Japan) were extracted
with 80% aqueous ethanol over 2 weeks. The concen-
trated extract (125 g) was partitioned between ethyl
acetate and water, and the ethyl acetate layer was
partitioned between 90% aqueous methanol and hexane.
Since the 90% aqueous methanol layer showed inhib-
itory effects on fat accumulation (66% FA rate and 93%
CV rate at a 100 mg/mL conc.), the methanol fraction
was separated by ODS column chromatography with
stepwise elution by 70% aqueous MeOH to MeOH. The
80% aqueous MeOH fraction (73% FA rate and 93% CV
rate at a 50 mg/mL conc.) was separated by silica-gel
column chromatography with stepwise elution by
CHCl3/MeOH (19/1, 9/1, and 2/1). The 9/1 CHCl3/
MeOH fraction (68% FA rate and 94% CV rate at a
50 mg/mL conc.) showed four spots on the TLC plate
[Rf ¼ 0:63, 0.70, 0.73, and 0.80 (CHCl3/
MeOH ¼ 9 : 1)]. These spots were separated by prepa-
rative TLC (CHCl3/MeOH ¼ 9 : 1) to afford an active
compound (0.6 mg; 50% FA rate and 81% CV rate at a
20 mg/mL conc.) with an Rf value of 0.70. To determine
the structure, this active compound was analyzed by 1H-
NMR (300 MHz, CDCl3), using high-resolution electro-
spray ionization mass spectrometry (HR-ESIMS) with a
Waters LCT Premier XE and ESIMS with a Brucker
Esquire 3000 Plus in the negative ESI mode. Character-
Such lifestyle-related diseases as cancer, cardiovas-
cular disease, hypertension, hyperlipidemia, and diabe-
tes are rapidly growing epidemics in developed coun-
tries, and obesity is one of the causes.1) The Ministry of
Health, Labor and Welfare in Japan has reported
increasing obesity rate in the population. Although such
approaches for anti-obesity as low-calorie foods have
been proposed, we have focused on inhibitors of fat
accumulation, because they could be applied to the
development of anti-obesity drugs.
Our previous study screened the fat accumulation
inhibitors from various sources. We found that the
mushroom, Coriolus versicolor, and plant, Valeriana
fauriei, potently inhibited fat accumulation against 3T3-
L1 murine adipocytes. The highly N-methylated cyclic
heptapeptide, (ꢀ)-ternatin, was successfully isolated
from C. versicolor as a novel inhibitor of fat accumu-
lation.2–4) V. fauriei is a Chinese herbal medicine and is
used to treat hysteroepilepsy and cardiac palpitations.
These effects have been mediated by such terpenoid
glycosides as kessoglycol diacetate and kessoglycol ꢀ-
monoacetate from V. fauriei.5,6) A novel iridoid glyco-
side and a sesquiterpenoid have been isolated from
V. fauriei and showed NGF-potentiating activity.7)
However, there are no reports on the inhibition of fat
accumulation. We report in this study the isolation and
determination of fat accumulation inhibitors from
V. fauriei.
1
istic H-NMR peaks of the active compound are shown
in the experimental section. Its molecular formula was
determined to be C18H32O3 by HR-ESIMS (m=z
295.2262 ½M ꢀ Hꢁꢀ, as calculated for C18H31O3,
295.2273). The ESI mass spectrum showed major peaks
at m=z 295 (½M ꢀ Hꢁꢀ), and an MS/MS analysis gave
To isolate the fat accumulation inhibitors, we used
the assay system with 3T3-L1 murine adipocytes as
described previously.2) LabAssayꢀ Triglyceride (Wako
y
To whom correspondence should be addressed. Kenji MIYAMOTO, Fax: +81-45-566-1783; E-mail: kmiyamoto@bio.keio.ac.jp; Daisuke UEMURA,
Fax: +81-463-58-9684; E-mail: uemura@kanagawa-u.ac.jp
Abbreviations: NGF, nerve growth factor; PPARꢁ, peroxisome proliferator-activated receptor ꢁ; GPCR, G protein-coupled receptor; FA, fat
accumulation; CV, cell viability

1234
K. YUKI et al.
ion fragments at m=z 277 (½ðM ꢀ H2OÞ ꢀ Hꢁꢀ) and 171
([(M ꢀ CH3(CH2)4CHCHCHCH2) ꢀ H]ꢀ). Based on
these spectral data, we identified the active compound
as the known but unusual fatty acid, 9-hydroxy-
10E,12Z-octadecadienoic acid (9-HODE, 3) (Fig. 1).
activity of the (S)-enantiomer than that of the (R)-acid.
Interestingly, 13-HODE showed slightly greater activity
(EC50 ¼ 17 mg/mL, IC50 ¼ 41 mg/mL) than that of 9-
HODE (Fig. 2B). The methyl esters (4 and 6) were also
subjected to these assays, but indicated no inhibitory
activity (data not shown). These results show that the
structure of a conjugated diene with a hydroxyl group at
the allylic position and a carboxyl group may be
important for the biological activities of 9-HODE.
Although the inhibitory activity of 9-HODE and 13-
HODE against fat accumulation has not been previously
reported, it has been reported that conjugated linoleic
acid (CLA, 2) had such biological activities such as
anti-tumor and hypocholesterolemic effects.11) Interest-
ingly, we found that CLA (2) had weak inhibitory
activity against fat accumulation (EC50 ¼ 316 mg/mL,
IC50 ¼ 375 mg/mL) when compared with 9-HODE and
13-HODE.
1
The H-NMR and MS/MS peaks assigned to the active
compound were in good agreement with those reported
previously for 9-HODE.8,9) The enantiomeric excess of
isolated 9-HODE was determined by an HPLC analysis
after converting to the corresponding methyl ester.10)
The results indicate that the absolute configuration of
isolated 9-HODE was the (R)-enantiomer (>95% e.e.).
We next synthesized 9-HODE and its analogs by
using a reported method.9) Methyl linoleate (1) was
added to a mixture of SeO2 and dichloromethane in an
argon atmosphere. The reaction mixture was stirred for
24 h at room temperature, before a 10% NaCl solution
was added and the mixture extracted with dichloro-
methane. The extract contained the 9-HODE methyl
ester (4) and 13-hydroxy-9Z,11E-octadecadienoic acid
(13-HODE) methyl ester (6). These compounds were
purified by preparative HPLC and subsequent hydrolysis
to afford racemic 9-HODE (3) and racemic 13-HODE
(5), and their inhibitory activity against fat accumulation
was evaluated. Figure 2A shows that synthesized
9-HODE significantly inhibited fat accumulation in
3T3-L1 cells (EC50 ¼ 20 mg/mL, IC50 ¼ 29 mg/mL).
These results are relatively higher than those for (R)-9-
HODE isolated from V. fauriei (EC50 ¼ 40 mg/mL,
IC50 ¼> 40 mg/mL). The results indicate the stronger
It has been reported that 9-HODE and 13-HODE
showed antitumorial activities.9) The methyl esters (4
and 6) had very weak activity compared with the
corresponding acids. Intriguingly, 9-HODE and 13-
HODE showed more potent inhibition of fat accumu-
lation and antitumor activity than CLA, although these
activities were lost by methyl esterification.
It is crucial for future applications to elucidate the
mechanisms for the inhibitory activities of 9-HODE
against fat accumulation. 9-HODE has been reported to
act as a ligand of both PPARꢁ12) and G protein-coupled
receptor G2A.13,14) PPARꢁ is a transcriptional factor
belonging to the nuclear receptor super-family and is
known to promote the differentiation of adipocytes.
Experiments with the heterozygous mouse,15) inhibitors
of PPARꢁ,16) and Pro12Ala SNP in humans17) have
shown a suppression of obesity by incremental serum
leptin levels.18) However, it has been reported that
PPARꢁ activated by troglitazone, a potent agonist,
promoted the differentiation of small adipocytes and the
apoptosis of large adipocytes.19) Troglitazone thus led to
the miniaturization of adipocytes. It is likely that 9-
HODE may activate PPARꢁ and miniaturize adipocytes
in the same way as troglitazone, leading to decreased fat
accumulation. G2A is a stress-inducible G protein-
coupled receptor (GPCR) which is known to have such
biological functions as those involving inflammatory
response, proliferation, and differentiation. It has been
reported that 9-HODE acted as a ligand of G2A and
released a variety of cytokines.13,14) However, intra-
CO2Me
Linoleic acid methyl ester (1)
CO2H
10E,12Z-Octadecadienoic acid (CLA, 2)
OH
12
10
CO2R
CO2R
18
18
16
14
8
6
4
2
9
9-Hydroxy-10E,12Z-octadecadienoic acid (9-HODE, R=H, 3)
methyl ester (R=Me, 4)
OH
12
10
9
16
14
8
6
4
2
13-Hydroxy-9Z,11E-octadecadienoic acid (13-HODE, R=H, 5)
methyl ester (R=Me, 6)
Fig. 1. Structures of Linoleic Acid Methyl Ester 1, Conjugated
Linoleic Acid 2, Allylic Hydroxylated Derivatives 3 and 5, and
Corresponding Methyl Esters 4 and 6.
100
100
A
B
80
60
40
20
0
80
60
40
20
0
0
20
40
60
80
100
0
20
40
60
80
100
9-HODE (µg/mL)
13-HODE (
µ
g/mL)
Fig. 2. Inhibitory Effects of Fat Accumulation on 3T3-L1 Murine Adipocytes.
3T3-L1 cells were treated with 9-HODE (A), 13-HODE (B), and the vehicle (control) for 1 week during differentiation. The fat accumulation
(filled circles) and cell viability (unfilled circles) were then determined. Data are presented as the mean and standard deviation (SD) of four tests
and are shown as a percentage of the control value.

Fat Accumulation Inhibitor from V. fauriei
1235
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