Induction of adiponectin by natural and synthetic phenolamides in mouse and human preadipocytes and its enhancement by docosahexaenoic acid

Article abstract of DOI:10.1016/j.lfs.2007.11.016

Adiponectin, the adipose-derived cytokine, plays an important role in preventing metabolic syndromes. To develop new adiponectin inducers, eight species of ferulic esters and amides, and five related compounds were synthesized and tested on the stimulation of adiponectin production in mouse 3T3-L1 and normal human preadipocytes. The ferulamides with an aromatic ring in the N-substituent are very active in inducing adiponectin as compared with the known active compounds, curcumin, [6]-gingerol, and capsaicin, and furthermore the activities of these ferulamides are remarkably stronger than those of the corresponding esters or the straight chain octylamide. The most active compound, N-(2-phenylethyl)ferulamide (7), was found to activate the PPAR (peroxisome proliferator-activated receptor) γ-RXR (retinoid X receptor) α heterodimeric complex in the PPRE (PPAR-responsive element)-driven luciferase reporter assay. The adiponectin production by 7 is synergistically enhanced by coaddition of a PPARγ-specific agonist, pioglitazone (PGZ), or another PPARγ agonist, docosahexaenoic acid (DHA), in cultured preadipocytes. The compound 7 alone did not show a statistically significant effect on the plasma adiponectin level in KK-A^y/Ta mice, while 1% 7 in the diets significantly lowered the blood glucose and triglyceride levels and 0.3% 7 mixed with DHA oil in the diets significantly increased the adiponectin level as compared with the control. These results suggest that the present ferulamides would be useful lead compounds in developing more potent agents for treatment of metabolic syndromes through promoting the endogenous adiponectin production, and that such an activity is possibly enhanced by the coadministration with DHA.

Full text of DOI:10.1016/j.lfs.2007.11.016

Available online at www.sciencedirect.com
Life Sciences 82 (2008) 290–300
www.elsevier.com/locate/lifescie
Induction of adiponectin by natural and synthetic phenolamides in mouse and
human preadipocytes and its enhancement by docosahexaenoic acid
⁎
Yoshimitsu Yamazaki , Yasuhiro Kawano, Masami Uebayasi
Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi,
Tsukuba, Ibaraki 305-8566, Japan
Received 7 July 2007; accepted 28 November 2007
Abstract
Adiponectin, the adipose-derived cytokine, plays an important role in preventing metabolic syndromes. To develop new adiponectin inducers, eight
species of ferulic esters and amides, and five related compounds were synthesized and tested on the stimulation of adiponectin production in mouse 3T3-
L1 and normal human preadipocytes. The ferulamides with an aromatic ring in the N-substituent are very active in inducing adiponectin as compared
with the known active compounds, curcumin, [6]-gingerol, and capsaicin, and furthermore the activities of these ferulamides are remarkably stronger
than those of the corresponding esters or the straight chain octylamide. The most active compound, N-(2-phenylethyl)ferulamide (7), was found to
activate the PPAR (peroxisome proliferator-activated receptor) γ-RXR (retinoid X receptor) α heterodimeric complex in the PPRE (PPAR-responsive
element)-driven luciferase reporter assay. The adiponectin production by 7 is synergistically enhanced by coaddition of a PPARγ-specific agonist,
pioglitazone (PGZ), or another PPARγ agonist, docosahexaenoic acid (DHA), in cultured preadipocytes. The compound 7 alone did not show a
statistically significant effect on the plasma adiponectin level in KK-Ay/Ta mice, while 1% 7 in the diets significantly lowered the blood glucose and
triglyceride levels and 0.3% 7 mixed with DHA oil in the diets significantly increased the adiponectin level as compared with the control. These results
suggest that the present ferulamides would be useful lead compounds in developing more potent agents for treatment of metabolic syndromes through
promoting the endogenous adiponectin production, and that such an activity is possibly enhanced by the coadministration with DHA.
© 2007 Elsevier Inc. All rights reserved.
Keywords: Adiponectin; Phenolic compounds; Ferulic acid; Amide; Docosahexaenoic acid; Thiazolidinedione; 3T3-L1; Preadipocyte; Diabetes
Introduction
et al., 2004), xanthohumol (Nozawa, 2005), polymethoxylated
flavones (Li et al., 2006), catechins (Cho et al., 2007), and sulfatide
(Bruun et al., 2007) have been reported to enhance adiponectin
production as safer agents for treating or preventing the metabolic
syndromes. This variety of compounds may reflect the complex
signaling cascade in the adipogenesis and the regulation of
adiponectin gene expression involving many transcription factors
(Rosen et al., 2000; Seo et al., 2004; Fox et al., 2006). Recently, we
found that the curry pigment, curcumin (1), and the pungent
components in ginger and red pepper, [6]-gingerol (2) and
capsaicin (3), stimulated adiponectin production in cultured mouse
and human preadipocytes (Yamazaki and Kawano, 2005;
Yamazaki et al., 2006a,b). The up-regulation of adiponectin
expression by capsaicin was also reported by other investigators
(Hsu and Yen, 2007). These phenolic compounds have the com-
mon structural feature of a guaiacol unit being bound to a hydro-
phobic moiety through a hydrophilic linker (Fig. 1). Following
Metabolic syndromes such as type 2 diabetes and athero-
sclerosis are closely related to decrease in the plasma concentration
of adiponectin, an adipocyte-derived cytokine (Fang and Sweeney,
2006). The pathophysiologically low adiponectin level in the
blood of diabetes patients is improved by thiazolidinedione anti-
diabetics such as pioglitazone (PGZ) and rosiglitazone (Eguchi
et al., 2007), but these drugs have some side effects (Nesto et al.,
2004). Several new synthetic drugs (e.g., telmisartan (Moriuchi
et al., 2007), bezafibrate (Hiuge et al., 2007), and rimonabant
(Hollander, 2007)) have been found to function as adiponectin
inducers, while a few natural products such as anthocyanin (Tsuda
⁎
Corresponding author. Tel./fax: +81 29 861 6063.
E-mail address: y.yamazaki@aist.go.jp (Y. Yamazaki).
0024-3205/$ - see front matter © 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.lfs.2007.11.016

Y. Yamazaki et al. / Life Sciences 82 (2008) 290–300
291
Fig. 1. Structures of the compounds.
this molecular architecture, we tried to synthesize a stronger adip-
onectin inducer.
ferulamide. This product was dissolved in 500 ml ethanol, and then
mixed with 12 ml hydrazine hydrate. After standing at room
temperature for 1 h, the solution was added with 12 ml acetic acid.
The solvent was removed by a rotary evaporator and the residue
was mixed with 500 ml ethyl acetate and 100 ml water in a
separatory funnel. The organic layer was washed twice with
200 ml saturated brine, dried over sodium sulfate, and then eva-
porated to give an oil, which was crystallized from benzene. One
half amount of the crude product was recrystallized from ethanol to
give 10.6 g 7 as needles (81% yield), mp 153–154 °C, MS m/z
297.1355 (M+) (calcd. C18H19NO3 =297.1364), 1H NMR
(CDCl3) δ: 2.889 (t, 2, J=7 Hz, CH2), 3.664 (dt, 2, J=7 and
6 Hz, CH2), 3.903 (s, 3, OCH3), 5.597 (t, 1, J=6 Hz, NH), 5.896 (s,
1, OH), 6.173 (d, 1, J=15.5 Hz, CH_CH), 6.894 (d, 1, J=8 Hz,
H-5), 6.968 (d, 1, J=2 Hz, H-2), 7.041 (dd, 1, J=8 and 2 Hz, H-6),
7.2–7.4 (m, 5, phenyl), and 7.539 (d, 1, J=15.5 Hz, CH_CH).
The other ferulic derivatives, except for 11 were synthesized by
the same method as described above with the corresponding
alcohol or amine, and isolated by crystallization or preparative
TLC to show the following properties: 1-octyl ferulate (4) — oil,
MS m/z 306.1833 (M+) (calcd. C18H26O4=306.1830), 1H NMR
(CDCl3) δ: 0.886 (t, 3, J=7 Hz, CH3), 1.2–1.5 (10, (CH2)5), 1.699
(qt, 2, J=7 Hz, CCH2C), 3.928 (s, 3, OCH3), 4.189 (t, 2, J=7 Hz,
CH2O), 5.874 (s, 1, OH), 6.294 (d, 1, J=16 Hz, CH_CH), 6.916
(d, 1, J=8 Hz, H-5), 7.035 (d, 1, J=2 Hz, H-2), 7.076 (dd, 1, J=8
and 2 Hz, H-6), and 7.691 (d, 1, J=16 Hz, CH_CH); N-(1-octyl)
ferulamide (5) — oil, MS m/z 305.2029 (M+) (calcd. C18H27
NO3=305.1989), 1H NMR (CDCl3) δ: 0.887 (t, 3, J=7 Hz, CH3),
1.2–1.5 (10, (CH2)5), 1.580 (qt, 2, J=7 Hz, CCH2C), 3.393 (t, 2,
Materials and methods
Chemicals
PGZ and cis-4,7,10,13,16,19-docosahexaenoic acid (DHA)
were purchased from Alexis Biochemicals (San Diego, CA) and
Cayman Chemical Company (Ann Arbor, MI), respectively.
Curcumin was obtained from Nagara Science Ltd. (Gifu, Japan)
and [6]-gingerol and capsaicin were from Wako Pure Chemical
Industries Ltd. (Osaka, Japan).
Synthesis
N-(2-Phenylethyl)ferulamide (7) was synthesized as follows.
To an ice-cold solution of 25 ml β-phenethylamine and 50 ml
pyridine in 100 ml toluene was added dropwise a 350 ml toluene
solution containing 30 g O-acetylferuloyl chloride prepared from
ferulic acid by the previous method (Hatfield et al., 1991). The
reaction mixture was stirred at room temperature overnight, and
divided into two portions. Each portion was mixed with 500 ml
ethyl acetate and washed successively with 150 and 100 ml water,
150 and 100 ml 10% citric acid, 150 and 100 ml 10% sodium
bicarbonate, and 100 and 50 ml saturated sodium chloride so-
lutions. The organic layers were combined, dried over sodium
sulfate, and then evaporated to give an oil, which was crystallized
from ethyl acetate to afford 32.6 g O-acetyl-N-(2-phenylethyl)

292
Y. Yamazaki et al. / Life Sciences 82 (2008) 290–300
J=7 Hz, CH2N), 3.932 (s, 3, OCH3), 5.589 (br. s, 1, NH), 5.871
(br. s, 1, OH), 6.255 (d, 1, J=15 Hz, CH_CH), 6.918 (d, 1,
J=8 Hz, H-5), 7.007 (d, 1, J=2 Hz, H-2), 7.073 (dd, 1, J=8 and
2 Hz, H-6), and 7.555 (d, 1, J=15 Hz, CH_CH); β-phenethyl
ferulate (6) — oil, MS m/z 298.1194 (M+) (calcd. C18H18O4=
298.1204), 1H NMR (CDCl3) δ: 3.018 (t, 2, J=7 Hz, CH2), 3.924
(s, 3, OCH3), 4.419 (dt, 2, J=7 and 6 Hz, CH2), 5.896 (br. s,
1, OH), 6.270 (d, 1, J=15.9 Hz, CH_CH), 6.912 (d, 1, J=8 Hz,
H-5), 7.017 (d, 1, J=2 Hz, H-2), 7.062 (dd, 1, J=8 and 2 Hz, H-6),
7.2–7.4 (m, 5, phenyl), and 7.598 (d, 1, J=15.9 Hz, CH_CH); N-
(3-phenylpropyl)ferulamide (8) — fine needles from benzene/
hexane/acetone, mp 75–78 °C, MS m/z 311.1510 (M+) (calcd. C19
(CH2)2), 2.652 (t, 2, J=7 Hz, CH2), 3.399 (dt, 2, J=6 and 7 Hz,
CH2), 3.862 (s, 3, CH3), 6.836 (d, 1, J=8 Hz, H-5), 7.122–7.287
(m, 5, phenyl), 7.406 (dd, 1, J=8 and 2 Hz, H-6), 7.561 (d, 1,
J=2 Hz, H-2), 7.589 (br. s, 1, NH), and 8.075 (br. s, 1, OH).
N-[2-(4-Hydroxyphenyl)ethyl]ferulamide (11) was synthe-
sized by the coupling of ferulic acid with tyramine in t-BuOH
using dicyclohexylcarbodiimide (DCC), purified by preparative
TLC, and crystallized from acetone/chloroform as needles, mp
142–143 °C (lit. mp 144.5–145 °C (Yoshihara et al., 1981)),
MS m/z: 313.1324 (M+) (calcd. C18H19NO4 =313.1313);
1H NMR (CDCl3 +10% MeOH-d4) δ: 2.762 (t, 2, J=7 Hz,
CH2), 3.546 (t, 2, J=7 Hz, CH2), 3.866 (s, 3, OCH3), 6.197
(d, 1, J=15.4 Hz, CH_CH), 6.771 (d, 2, J=8 Hz, H-2′ and 6′),
6.839 (d, 1, J=8 Hz, H-5), 6.962 (d, 1, J=2 Hz, H-2), 6.997
(m, 1, H-6), 7.027 (d, 2, J=8 Hz, H-3′ and 5′), and 7.474 (d, 1,
J=15.4 Hz, CH_CH). N-(2-Phenylethyl)-4-hydroxycinnama-
mide (13) was also prepared by the DCC-mediated coupling of
p-coumaric acid with 2-phenylethylamine — fine needles from
benzene/hexane, mp 118–121 °C, MS m/z: 267.1236 (M+)
(calcd. C17H17NO2 =267.1258), 1H NMR (CDCl3 +10%
MeOH-d4) δ: 2.869 (t, 2, J=7 Hz, CH2), 3.162 (t, 2, J=7 Hz,
CH2), 6.163 (d, 1, J=16 Hz, CH_CH), 6.799 (d, 2, J=8 Hz,
H-2′ and 6′), 7.195–7.357 (m, 5, Phenyl), 7.343 (d, 2, J=8 Hz,
H-3′ and 5′), and 7.506 (d, 1, J=16 Hz, CH_CH).
N-[(4-Hydroxy-3-methoxyphenyl)methyl]cinnamamide (16)
was synthesized as follows. Vanillylamine hydrochloride (10 g)
was dissolved in 25 ml 5 N NaOH and mixed with 150 ml water.
To the ice-cold mixture was slowly added a solution of 11 g
cinnamic chloride in 2 ml toluene under vigorous stirring. After
stirring at room temperature for 2 h, the mixture was neutralized
with 6 N HCl and then extracted with 800 ml ethyl acetate.
The organic layer was washed with 10% NaHCO3 and con-
centrated by an evaporator. The residue was dissolved in 500 ml
ethanol and mixed with 10 ml hydrazine monohydrate. The
mixture was left at room temperature for 2 h, supplemented with
10.2 ml acetic acid, and then concentrated by an evaporator. The
residue was dissolved in 900 ml ethyl acetate and the solution was
usually worked up to give 15.6 g compound 16 upon evaporation.
Some part of the crude product was crystallized from benzene as
needles, mp 145–147 °C, MS m/z: 283.1150 (M+) (calcd.
C17H17NO3=283.1207), 1H NMR (CDCl3) δ: 3.771 (s, 3, CH3),
4.367 (d, 2, J=7 Hz, CH2), 5.704 (br. s, 1, OH), 6.151 (t, 1,
J=6 Hz, NH), 6.426 (d, 1, J=16 Hz, CH_CH), 6.791 (dd, 1,
J=8 and 2 Hz, H-6), 6.825 (d, 1, J=2 Hz, H-2), 7.322–7.486 (m,
5, phenyl), 7.653 (d, 1, J=16 Hz, CH_CH), and 8.861 (d, 1,
J=8 Hz, H-5).
1
H21NO3=311.1520), H NMR (CDCl3) δ: 1.869 (q, 2, J=7 Hz,
CH2), 2.681 (t, 2, J=7 Hz, CH2), 3.338 (dt, 2, J=7 and 6 Hz,
CH2), 3.903 (s, 3, OCH3), 6.501 (d, 1, J=16 Hz, CH_CH), 6.836
(d, 1, J=8 Hz, H-5), 7.048 (dd, 1, J=8 and 2 Hz, H-6), 7.161 (d, 1,
J=2 Hz, H-2), 7.13–7.305 (m, 6, phenyl and NH), 7.445 (d, 1,
J=16 Hz, CH_CH), and 7.985 (br. s, 1, OH); N-[2-(3-indolyl)
ethyl]ferulamide(9) — fine needles from ethanol, mp 163–165 °C
(lit. mp 163–165 °C (Vecchietti et al., 1979)), MS m/z 336.1424
1
(M+) (calcd. C20H20N2O3=336.1473); H NMR (CDCl3+10%
MeOH-d4) δ: 3.047 (t, 2, J=7 Hz, CH2), 3.722 (t, 2, J=7 Hz,
CH2), 3.892 (s, 3, OCH3), 6.161 (d, 1, J=15.7 Hz, CH_CH),
6.859 (d, 1, J=8 Hz, H-5), 6.965 (d, 1, J=2 Hz, H-2), 7.012 (dd, 1,
J=8 and 2 Hz, H-6), 7.079 (s, 1, H-2′), 7.121 (m, 1, H-5′ or 6′),
7.205 (m, 1, H-6′ or 5′), 7.395 (m, 1, H-7′ or 4′), 7.486 (d, 1,
J=15.7 Hz, CH_CH), and 7.628 (m, 1, H-4′ or 7′); 2-(3-indolyl)
ethyl ferulate (10) — fine needles from benzene, mp 73–74 °C,
MS m/z 337.1314 (M+) (calcd. C20H19NO4=337.1313); 1H NMR
(CDCl3) δ: 3.185 (dt, 2, J=7 and 1 Hz, CH2), 3.930 (s, 3, OCH3),
4.496 (t, 2, J=7 Hz, CH2O), 5.862 (br. s, 1, OH), 6.302 (d, 1,
J=16 Hz, CH_CH), 6.920 (d, 1, J=8 Hz, H-5), 7.018 (d, 1,
J=2 Hz, H-2), 7.069 (dd, 1, J=8 and 2 Hz, H-6), 7.097 (d, 1,
J=2 Hz, H-2′), 7.147 (ddd, 1, J=8, 8, and 1 Hz, H-5′ or 6′), 7.218
(ddd, 1, J=8, 8, and 1 Hz, H-6′ or 5′), 7.385 (dd, 1, J=8 and 1 Hz,
H-7′ or 4′), 7.611 (d, 1, J=16 Hz, CH_CH), 7.685 (dd, 1, J=8
and 1 Hz, H-4′ or 7′), and 8.038 (br. s, 1, NH).
The following reference compounds (12, 14, and 15) were
analogously prepared using the corresponding acids (3,4-di-O-
acetylcaffeic acid, cinnamic acid, or 4-O-acetylvanillic acid) and
phenylethylamine or phenylbutylamine: N-(2-phenylethyl)caffea-
mide (12) — fine needles from ethanol/benzene/hexane, mp 154–
155 °C, MS m/z 283.1202 (M+) (calcd. C17H17NO3=283.1207),
1H NMR (acetone-d6) δ: 2.847 (t, 2, J=7 Hz, CH2), 3.534 (dt, 2,
J=8 and 7 Hz, CH2), 6.420 (d, 1, J=16 Hz, CH_CH), 6.827 (d,
1, J=8 Hz, H-5), 6.943 (dd, 1, J=8 and 2 Hz, H-6), 7.060 (d, 1,
J=2 Hz, H-2), 7.16–7.32 (m, 6, phenyl+NH), 7.404 (d, 1,
J=16 Hz, CH_CH), and 8.216 (br. s, 2, OH×2); N-(2-
phenylethyl)cinnamamide (14) — needles from benzene/ethyl
acetate, mp 122–123 °C, MS m/z 251.1306 (M+) (calcd. C17H17
NO=251.1309), 1H NMR (CDCl3) δ: 2.927 (t, 2, J=7 Hz, CH2),
3.699 (dt, 2, J=7 and 6 Hz, CH2), 5.667 (br. s, 1, OH), 6.347 (d, 1,
J=16 Hz, CH_CH), 7.244–7.592 (m, 10, phenyl×2), and 7.650
(d, 1, J=16 Hz, CH_CH); N-(4-phenylbutyl)-2-hydroxy-3-
methoxybenzamide (15) — needles from benzene/ethyl acetate/
hexane, mp 142–143 °C, MS m/z 299.1520 (M+) (calcd.
Cell culture
Mouse 3T3-L1 cell line was obtained from Health Science
Research Resources Bank (Sennan-shi, Japan), and cultured in
DMEM (Sigma) containing 10% fetal bovine serum (FBS).
Cells were precultured for 3 days, collected with an EDTA-
trypsin solution, and resuspended in the medium at the rate of
6×104 cells/ml. The suspension was divided into wells of 96-
well plates (for ELISA) and 12- or 24-well plates (for PCR) by
0.2 ml and 1.5 or 2 ml, respectively. The plates were precoated
1
C18H21NO3=299.1520), H NMR (acetone-d6) δ: 1.644 (m, 4,

Y. Yamazaki et al. / Life Sciences 82 (2008) 290–300
293
Table 1
pounds such as 7 at their optimum concentrations) was found in
3T3-L1 cells as well as in human preadipocytes by microscopic
examination just before the recovery of medium.
Adiponectin productive activity in cultured preadipocytes
Compounds 3T3-L1 cells
Human preadipocytes
Emax (relative to
D50
Emax (relative to
D50
PGZ)
(μM)
PGZ)
(μM)
RT-PCR
PGZ
Curcumin
Gingerol
Capsaicin
4
5
1
b0.1
0.8
b0.1
b0.1
0.2
0.5
nd
68
nd
nd
5
1
0.04
0.01
b0.01
–
–
nda
42
76
nd
–
Cells in the 12- or 24-well plates were washed with Ca- and
Mg-free Dulbecco's phosphate-buffered saline and treated with a
phenol reagent (Isogen, Wako Pure Chemicals, 0.3 or 0.2 ml/well).
The extracted RNA was purified from proteins and DNA, and
precipitated with 2-propanol according to the manufacturer's
instructions. The total RNA was converted to cDNA by a reverse
transcriptase system (Invitrogen). Quantitative PCR was per-
formed in a Corbett Rotor-Gene 2000 Real-Time Cycler using the
cDNA, QuantiTect SYBR Green PCR system (Quiagen), and the
following primers: 5′-AAGGACAAGGCCGTTCTCT-3′ and 5′-
TATGGGTAGTTGCAGTCAGTTGG-3′ for mouse adiponectin,
and 5′-GACGAGGCCCAGAGCAAGAGA-3′ and 5′-TAGAT
GGGCACAGTGTGGGTGA-3′ for β-actin. Cycling conditions
were 94 °C — 1 min, 64 °C — 1 min, and 72 °C — 1 min for
adiponectin, and 94 °C — 30 s, 62 °C — 30 s, and 72 °C — 30 s
for β-actin.
–
6
7
8
9
10
11
12
13
14
15
16
0.2
16.4
5.4
11.0
0.3
9.2
0.1
b0.1
0.6
1.8
1.3
67
86
72
59
28
118
nd
nd
49
54
77
0.04
0.81
–
0.35
75
77
–
77
–
N180
–
–
–
N0.23b, a
–
–
–
–
–
–
–
–
The dose-production dependency was studied in quadruplicate wells with 0.1,
0.3, 0.5, 0.7, 1, 2, and 4 μM PGZ, 1, 2, 4, 10, 20, 30, 50, 70, and 100 μM
curcumin, 1, 3, 10, 30, 50, 70, 100, and 150 μM compound 4, 5, and 12, 0.1, 0.3,
1, 3, 10, 30, 50, 70, 100, and 200 μM compound 10, and 10, 30, 50, 70, 100, 150,
200, and 300 μM other compounds. Emax (maximal adiponectin production) and
D50 (half-maximal dose) were determined from the plots, and the maximal
adiponectin production was given as relative to the maximum by PGZ (92 or
62 ng/ml at 0.7 or 0.9 μM) in the case of 3T3-L1 cells or to the amount by 4 μM
PGZ (6 or 15 ng/ml) in the case of human preadipocytes. Values are given as the
mean of two separate experiments. nd, not determined. –, not tested.
Reporter plasmids and PPARγ activation assay
A firefly luciferase reporter plasmid with 3 copies of a
PPAR-responsive element (PPRE) in rat acyl-CoA oxidase
(ACO-PPREx3-Luc) was generated, as described previously
(Heim et al., 2002). To generate protein expression vectors
containing a full-length human PPAR (peroxisome proliferator-
a
Plot did not reach the maximum at the largest concentration tested.
Value at 300 μM.
b
with a collagen coating solution (Toyobo). After 3 days in-
cubation, the medium was changed to a fresh medium (0.2, 1.5,
or 2 ml/well) containing 0.1–4 μM PGZ or 10–50 μM DHA, if
necessary. Ethanol solutions (2–6.7 μl) containing 0.1–30 mM
test compounds or the corresponding volume of ethanol (as
control) were added to the culture and the plates were incubated
for 10–14 days before harvesting the medium and cells. The
stock solution of PGZ (10 mM) was prepared in dimethyl
sulfoxide (DMSO) and further diluted with ethanol before
addition to the culture.
Human subcutaneous preadipocytes were purchased from
Cambrex Bio Science Walkersville, Inc. (Walkersville, MD)
and cultured according to the supplier's manual with PBM
(Cambrex) as the basal medium. The precultured cells were
suspended in PBM containing 2 mM glutamine and 10% FBS at
the rate of 4.7×104 cells/ml. The cell suspensions were added
by 0.2 ml into each well of 96-well plates precoated with the
collagen solution. After 3 days incubation, the medium was
changed to a fresh one (0.2 ml/well) containing 0.01 mg/ml
insulin, 0.1 μM dexamethasone, and if necessary, 10–50 μM
DHA. Samples were added in the same way as described above,
and the plates were incubated for 14 days before collecting the
medium. Adiponectin in the medium was determined by ELISA
(mouse/rat or human adiponectin ELISA Kit, Ohtuska Pharma-
ceuticals, Tokyo, Japan). In addition, accumulation of lipid
droplets in many cells (40–50% or more for the potent com-
Fig. 2. Adiponectin production in mouse 3T3-L1 cells cultured with the
ferulamides and ferulates. Cells (1.2×104/well containing 0.2 ml medium in 96-
well plates) were incubated for 14 days without or with 0.1, 0.3, 0.5, 0.7, 1, 2, or
4 μM PGZ (open circle), 10, 20, or 30 μM curcumin (open triangle), or 10, 30, 50,
70, 100, 150, 200, or 300 μM capsaicin (open square), [6]-gingerol (closed
square), 6 (open diamond), 7 (closed circle), or 11 (closed triangle), and
adiponectin concentration in the medium was determined by ELISA. Data shown
are the means S.D. of quadruplicate samples in a representative experiment that
⁎
was repeated twice with comparable results. represents pb0.05 vs. control by
Dunnett's t-test for each compound.

294
Y. Yamazaki et al. / Life Sciences 82 (2008) 290–300
Fig. 3. Adiponectin gene expression in mouse 3T3-L1 cells. Cells (9.4×104 in each well containing 2 ml medium in 12-well plates) were incubated for 10 days without
or with 10, 30 or 100 μM 6, 7, 9, or 11, and then treated with Isogen for RNA extraction. The medium was also collected for adiponectin determination by ELISA (see
⁎
inset). Ratio of the expressed adiponectin gene vs. β-actin gene was determined by quantitative RT-PCR. Data shown are the means S.D. (n=3). represents pb0.05
vs. control by Dunnett's t-test for each compound.
activated receptor) γ1 or RXR (retinoid X receptor) α (pcDNA-
PPARγ1, pcDNA-RXRα), the corresponding cDNAs from
human cDNA libraries were amplified by PCR and subcloned
into the pcDNA3.1(+) mammalian expression plasmid (Invitro-
gen). CV-1 cells were cultured in 24-well plates and transfected
with ACO-PPREx3-Luc, pcDNA-PPARγ1 and pcDNA-
RXRα, using LipofectAMINE and PLUS reagents (Invitrogen).
Twenty four hours after the transfection, the cells were treated
with test compounds (dissolved in DMSO) for another 24 h.
The luciferase activity in the cells was determined using Dual-
Luciferase Reporter Assay System (Promega).
Fig. 5. Activation of PPARγ/RXRα heterodimers by the ferulamide 7. Dose–
response curves of 7 for luciferase activity in CV-1 cells in the absence (open
circle) or presence (closed circle) of 3 μM GW9662 and that of rosiglitazone
(triangle, as reference) are shown. The cells were transfected with a PPRE-
driven luciferase gene and cDNAs encoding PPARγ and RXRα. The values
were normalized to the mean luciferase activity in the absence of test
Fig. 4. Adiponectin production in human preadipocytes cultured with the present
ferulamides. Human preadipocytes (9.4×103 in each well containing 0.2 ml
medium in 96-well plates) were cultured in quadruplicate wells for 14 days
without or with 0.1, 0.3, 0.5, 0.7, 1, 2, or 4 μM PGZ (open circle) or 10, 30, 50,
70, 100, 150, 200, or 300 μM capsaicin (open square), 7 (closed circle), or 9
(closed triangle). Adiponectin in the medium was determined by ELISA. Data
shown are the means S.D. (n=4) in a representative experiment that was
⁎
compounds, and expressed as means S.E.M. (n=4). represents pb0.05 vs.
control by Dunnett's t-test, and # represents pb0.05 vs. reaction of 7 without
GW9662 by Dunnett's T3 test for the control, control+GW9662, 100 μM 7, and
100 μM 7+GW9662.
⁎
repeated twice with comparable results. represents pb0.05 vs. control by
Dunnett's t-test for each compound.

Y. Yamazaki et al. / Life Sciences 82 (2008) 290–300
295
Aliquots (9 μl) of the blood were mixed with 50 mg/ml heparin
solution (3 μl each) and the blood cells were removed by
centrifugation (10,000 rpm, 10 min). The plasma was analyzed by
SDS-PAGE (1 μg protein/lane) under the condition of Laemmli
with 10–20% density gradient gels. Western blot was performed
with Hybond-P membranes and adiponectin was detected with
mouse anti-adiponectin, mouse monoclonal antibody (Chemicon,
MAB3608) and rabbit anti-mouse Ig G1-HRP conjugate (Zymed,
61-0120), and ECL (Plus) detection kit (Amersham Biosciences).
Serum albumin in the fresh gels (cut out after electrophoresis) was
detected by Coomassie brilliant blue staining. The band
intensities were digitized with a scanner and a computer program
(Gel Pro Analyzer, Bio-Rad Laboratories) to calculate the relative
adiponectin level against serum albumin. Triglyceride in the
plasma was determined with an enzymatic assay kit (Triglyceride
E-Test Wako). In the experiment with DHA, diets were prepared
by mixing 250 g low fat diet (A-13251, Nippon Clea) with 14 ml
corn oil or 2.7 ml corn oil plus 11.3 ml DHA enriched oil [DHA
oil; DHA70, Nippon Kagaku Shiryo, Hakodate, Japan; DHA and
eicosapentaenoic acid (EPA) were contained in 70% and 4% of
the total fatty acids, respectively] and 3.75 ml ethanol containing 0
or 0.75 g ferulamide 7. The mixed powder was divided into
screw-capped small tubes (32 g each), in which the air was
replaced with nitrogen using a vacuum apparatus. The tubes were
tightly stoppered and stored in a refrigerator until use. The diets
were changed every day (8 g for one mouse a day). The residual
diet in the feed box was collected and weighed for calculating the
intake. Blood was taken on day 31 and 37, and treated in the same
way as described above, but the plasma adiponectin level and
glucose concentration were determined with the ELISA and an
enzymatic assay kit (Glucose C-II test Wako), respectively.
Fig. 6. Synergistic enhancement of adiponectin production by coaddition of the
ferulamide 7 with PGZ or DHA in 3T3-L1 cells. Cells were incubated for
11 days (A and B) or 14 days (C and D) in triplicate wells in the same way as
described before without or with 10 μM 7 and/or 0, 0.1, or 1 μM PGZ (A and B),
or without or with 10 or 30 μM 7 and/or 30 μM DHA (C and D) and adiponectin
in the medium was determined by ELISA. Data shown are the means S.D.
(n=3–9). Means without a common letter differ, pb0.05. The calculated sum
values (Calcd. sum) were obtained from the data for PGZ, DHA, or 7 alone. The
amplification rates (combination data/calculated sum) are 11.6, 3.6, 3.0, and 4.4
for the data of A, B, C, and D, respectively.
Animal studies
The experimental protocol was approved by the Institutional
Animal Care and Use Committee of AIST. Thirteen male KK-Ay/
Ta Jc1 mice (5 weeks old) were purchased from Nippon Clea
(Tokyo, Japan) and housed in individual cages in a temperature,
humidity, and light-controlled room (22–23 °C, 50%, and 12 h
light/dark cycle). Diets containing 0 or 1% ferulamide 7 were
prepared by mixing 120 g of a normal chow (CE-2, Nippon Clea)
with 6 ml ethanol containing 0 or 1.2 g 7 and an autoclaved
solution of 2.4 g starch in 48 ml water. Each mixture was firmly
packed in a dish (15 cm in diameter) and dried at room
temperature for a few days. The mice took these cakes and
autoclaved tap water ad libitum. The body weight and food intake
of each mouse was measured every weekday. On day 34, blood
was taken from the tail end of the mice anesthetized with ethyl
ether in a container and blood glucose was directly measured with
a glucose sensor (Sanwa Kagaku Kenkyusho, Nagoya, Japan).
Fig. 7. Synergistic enhancement of adiponectin production by coaddition of
ferulamide 7 with DHA in human preadipocytes. Cells (9.6×103/well containing
0.2 ml medium in 96-well plates) were incubated in triplicate wells for 14 days
without or with 10, 30, 50, or 70 μM ferulamide 7 and/or 10, 30, or 50 μM DHA, or
1, 2, 3, or 4 μM PGZ. Data shown are the means S.D. (n=3) in a representative
experiment that was repeated twice with comparable results. represents pb0.05
vs. control by Dunnett's t-test for each compound or for each group of the same
DHA concentration.
⁎

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Y. Yamazaki et al. / Life Sciences 82 (2008) 290–300
Statistical analysis
compounds and curcumin, [6]-gingerol, capsaicin, and PGZ were
tested on adiponectin production at different concentrations in
mouse 3T3-L1 cells by determining adiponectin in the culture
medium by ELISA. From the dose-adiponectin production plots,
the intrinsic activity or maximal efficacy (Emax) and potency (D50)
were determined. The results are summarized in Table 1 and the
plots for the typical compounds are shown in Fig. 2. Curcumin
and capsaicin showed a marginal Emax and [6]-gingerol showed
only an Emax comparable to that for PGZ (cf. inset of Fig. 2). In
contrast to these known compounds, four ferulamides (7, 8, 9, and
11) showed 5 to 16 times stronger activity than PGZ. The
phenethylferulamide 7 was the most active among them. These
ferulamides were designed to have a hydrophobic aromatic ring in
the N-substituent. Another ferulamide with a long alkyl group (5)
was less active than PGZ. The ferulates 4, 6, and 10 and analogs of
7 with no guaiacol functional groups (12, 13, and 14) were all less
active than PGZ. The amides 15 and 16, which were guaiacol
derivatives but different from ferulamides, were slightly more
All experimental measurements are expressed as means
standard deviation (S.D.). Statistical comparisons between two
groups were evaluated by two-tailed Student's t-test and those
among more than three groups were done by one-way ANOVA
followed by Dunnett's t-test, Tukey's test, or Dunnett's T3 test
using SPSS 15.0 J software, unless otherwise stated. Differences
with pb0.05 were considered to be significant.
Results
We chose ferulic acid as the synthon for the guaiacol unit and
coupled it with some alcohols and amines to form 3 species of
ferulic esters (4, 6, and 10) and 5 species of ferulamides (5, 7, 8, 9,
and 11) (Fig. 1). In addition, three analogs lacking a guaiacol unit
(12, 13, and 14) and two guaiacol derivatives of no ferulamide
skeleton (15 and 16) were synthesized for comparison. These
Fig. 8. Experiment with KK-Ay/Ta Jc1 mice fed diets without or with ferulamide 7 (1%). Number of mice was 6 in the control group and 7 in the 7-fed group. (A)
Average body weight of control (open circle) and 7-fed group (closed circle); (B) average daily food intake of the control (open bar) and 7-fed group (closed bar) in the
1st, 3rd, and 5th week; (C) Western blots for adiponectin in the plasma collected on day 34 from the control mice (6 bands on the left side) and the 7-fed mice (7 bands
on the right side); (D) Coomassie brilliant blue-stained serum albumin bands in the same polyacrylamide gel as electrophoresed for the adiponectin detection; (E)
plasma adiponectin level calculated from relative intensities of the bands in panels C and D; (F) blood glucose concentration; and (G) plasma triglyceride
⁎
⁎⁎
concentration. Data shown are the means S.D. (n=6 or 7 for A and E, n=42 or 49 for B, n=11 or 14 for F, and n=18 or 21 for G). and represent pb0.05 and
pb0.01 vs. control, respectively.

Y. Yamazaki et al. / Life Sciences 82 (2008) 290–300
297
active than the non guaiacol analogs (12, 13, and 14). These facts
indicate that compounds having a guaiacol group linked with an
aromatic ring via an alkylamide chain such as ferulamide 7 are
promising as the strong adiponectin inducer. The stimulation of
adiponectin gene expression by the ferulamides was confirmed by
quantitative RT-PCR (Fig. 3). The patterns of gene expression and
protein production of adiponectin with different compounds and
concentrations were nearly parallel with each other (see the inset).
The ferulamides 7, 9, and 11 and the ferulate 6 were further
studied on the adiponectin production in human preadipocytes
(Fig. 4 and Table 1). The maximal efficacy (Emax) of curcumin,
[6]-gingerol, and capsaicin was less than 5% of the adiponectin
amount produced by 4 μM PGZ, but the Emax of ferulamide 7
approximated to the value by PGZ. Thus, the activity of
adiponectin induction in human cells by the natural guaiacol
derivatives such as curcumin was also markedly improved by
adopting the ferulamide structure. The activities of the
ferulamides 7 and 9 were stronger than that of the ferulate 6,
and the long alkyl amide 5 was only slightly active (data not
shown), being consistent with the result in 3T3-L1 cells.
Gene expression of adiponectin is mainly controlled by a
transcription factor PPARγ1 in a heterodimeric complex with
another transcription factor RXRα (Iwaki et al., 2003). To ex-
amine the functional significance of PPARγ in the adiponectin
induction by the ferulamides, we studied the effect of 7 on the
luciferase activity in CV-1 cells transfected with a PPRE-driven
luciferase gene and cDNAs encoding PPARγ and RXRα. The
ferulamide 7 increased the luciferase activity of the CV-1 cells in a
dose-dependent manner, suggesting that this compound stimu-
lated transcriptional activity of PPARγ/RXRα heterodimers
(Fig. 5). However, a competitive PPARγ antagonist GW9662
(2-chloro-5-nitro-N-phenylbenzamide) (Schopfer et al., 2005) did
not clearly shift the dose-response curve to the high concentration
side, though the maximal response to 7 was attenuated in the
presence of GW9662. On the other hand, the adiponectin
production by 7 in 3T3-L1 cells was increased by simultaneous
addition of the PPARγ-specific ligand, PGZ, to a higher level than
the sum of the responses to either compound alone, especially at
their low concentrations (Fig. 6A, B). A similar effect of co-
addition was found with another PPARγ agonist DHA (Krey
et al., 1997) in 3T3-L1 cells (Fig. 6C, D) as well as in human
preadipocytes (Fig. 7).
Finally we studied the biological activity of the ferulamide 7
in vivo. Diabetic male KK-Ay/Ta mice were fed a diet containing
1% 7 for 5 weeks. The adiponectin level in the 7-fed mice was
not significantly different from that of the control mice, while the
Fig. 9. Experiment with KK-Ay/Ta Jc1 mice fed diets containing ferulamide 7 (0.3%) and/or DHA oil (4%), or none of them. Number of mice was 4 in each group.
(A) Average body weight of control (open circle), 7-fed group (triangle), 7 and DHA oil-fed group (closed circle), and DHA oil-fed group (square); (B) average daily
food intake of the control (open bar), 7-fed group (grey bar), 7 and DHA oil-fed group (closed bar), and DHA oil-fed group (light grey bar) in the 1st, 3rd, and 5th
week; (C) relative adiponectin level in plasma; (D) plasma glucose concentration; and (E) plasma triglyceride concentration. The blood was taken on day 31 (open bar)
and day 37 (closed bar). Data shown are the means S.D. (n=4 for A, n=28 for B, n=8–16 for C, and n=12 for D and E). Means without a common letter differ,
pb0.05, but letters without underlines are applied to comparison of the day-31 samples and underlined letters are to day-37 samples.

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Y. Yamazaki et al. / Life Sciences 82 (2008) 290–300
blood glucose and plasma triglyceride concentrations were 27–
38% lower in the 7-fed group than in the control group (Fig. 8).
The animal experiment was repeated with DHA oil and 7, but the
latter was used at a reduced rate (0.3%). Ferulamide 7 alone at
this rate showed little effect on the plasma adiponectin level, but
the level increased when DHA oil was supplemented (Fig. 9C).
Multiple comparisons indicate that the adiponectin level of 7+
DHA group was significantly different from those of the control
and the 7 alone group in both day-31 and day-37 samples. The
adiponectin level of the DHA alone group was not significantly
different from either that of the 7+DHA group or that of the
control group in the day-37 samples. The plasma glucose and
triglyceride levels were lowered by the coadministration of 7 and
DHA oil. From a statistical analysis by multiple comparison, the
glucose and triglyceride levels of the 7+DHA group were sig-
nificantly different from those of the control and the 7 alone
group (but with some exceptions). However, no significant
difference was detected between the 7+DHA group and the
DHA alone group. In addition, the mice fed the diets containing
1% 7, 7+DHA oil, or DHA oil alone showed 2–9% smaller
body weight than the control in the 2nd–5th weeks, but the body
weight of any mice increased constantly during the experimental
period (Figs. 8A and 9A).
ferulamides were 20–200 times more potent and at least 10 times
more potent than curcumin, [6]-gingerol, or capsaicin in 3T3-L1
cells and in human preadipocytes, respectively, in the view of
efficacy. Further improvement of the activity needs understanding
of the structure–activity relationship on the level of molecular
interaction between the compounds and the binding domain of
their receptors.
Initially, we expected that the ferulamide derivatives would
stimulate the adiponectin expression through binding to the tran-
scription factor PPARγ since the PPARγ-specific ligands thia-
zolidinediones induced adiponectin (Eguchi et al., 2007; Iwaki
et al., 2003), and since curcumin, with which we had found the
adiponectin inductive activity, was reported to activate PPARγ
(Nishiyama et al., 2005). The activation of the PPARγ–RXRα
heterodimers by the ferulamide 7 was clear from the reporter
assay, but the experiment using GW9662 did not confirm that it
was performed only through binding to PPARγ. The synergistic
enhancement of adiponectin production by the combination of 7
with PGZ suggests that 7 might activate the heterodimeric partner
RXRα. In fact, many species of RXR-specific ligands have been
reported to promote adipogenesis either singly or in combination
with PPARγ ligands (Sato et al., 2001; Sewter et al., 2002;
Leibowitz et al., 2006). However, the true mechanism by which 7
induces adiponectin remains to be elucidated in the future.
The present compounds were prepared by chemical synthesis,
but they are also natural products. The ferulamide 7 is reportedly
contained in a garden plant, petunia (Jassey et al., 1982), and the
indolylferulamide 9 is present in safflower seeds (Sato et al.,
1985), sweet corn kernels (Ehmann, 1974), and bark of a tropical
tree (Vecchietti et al., 1979). p-Hydroxyphenethylferulamide 11 is
contained in pepper root (Yoshihara et al., 1981) and beet (Kujala
et al., 2002), and as an anti-inflammatory constituent in some
herbs (Yokozawa et al., 2001). In addition, several ferulamides
including 5 and 11 were shown to stimulate insulin secretion in
cultured pancreatic cells (Nomura et al., 2003). However, the
adiponectin-inducing activity has not been reported for these
ferulamides (except for some parts of our studies disclosed in a
patent application (Yamazaki et al., 2005)).
The activities of these compounds are considerably high as
compared with those of the known active compounds (i.e.,
curcumin, [6]-gingerol, and capsaicin) that are currently used in
food or traditional medicine. Although the in vivo activity in
increasing adiponectin level was not significant, the effective-
ness in decreasing blood glucose and triglyceride levels was
exhibited by the ferulamide 7 in diabetic mice (Fig. 8F, G).
These facts seemed to imply the usefulness of 7. However, the
concentration required to induce adiponectin in the cultured
human preadipocytes was over 10-fold higher than that of PGZ,
and the induced amount of adiponectin by 30 μM 7 was very
smaller (approximately 1/20) than the amount by 3–4 μM PGZ
(Fig. 7). This fact made us apprehensive about whether the
adiponectin-inducing activity of these ferulamides was still too
weak to be applied in human health care. We noted the en-
hancement of adiponectin production by combining 7 with PGZ,
but PGZ is a toxic pharmaceutical with side effects. Then, we
carried out the experiment using DHA with the expectation that
application of the foodstuff DHA instead of PGZ would safely
Discussion
The present results validated our initial molecular design for
new adiponectin inducers (Fig. 1) based on the structures of
curcumin, [6]-gingerol, and capsaicin, with which we had found
stimulation of adiponectin induction. We decided to connect the
guaiacol group with the hydrophobic moiety using an ester or
amide bond for synthetic convenience, and hence we employed
ferulic acid as the synthon. We prepared 8 species of ferulic
derivatives and several related analogs to study the structure–
activity relationship. Among the synthesized compounds, N-
arylalkylferulamides (7–9 and 11) showed the strong activity
(Emax) in 3T3-L1 cells. The strongest compound was phene-
thylferulamide 7, which completely fulfilled our design. The fact
that analogs 12–14 were remarkably less active than the above
ferulamides indicates the importance of 4-hydroxy-3-methoxy
functional groups for the present activity. This relation is sup-
ported by the moderate activity of two guaiacol derivatives 15 and
16 that lack the ferulamide skeleton. The p-hydroxyphenethyl
derivative 11 showed weaker activity (smaller Emax and larger
D50) than the phenethyl derivative 7, implying that the hydro-
phobicity of the N-substituent is another important point for the
present activity. A comparison between the data of 5 and 7 shows
that a long alkyl group in the N-substituent reduces the efficacy
(Emax) but increases the potency (D50) as compared with the
aromatic ring. The compound 16 is an aryl analog of capsaicin,
and their Emax values are in accord with the above consideration.
As the linker between the guaiacol unit and the hydrophobic
moiety, the amide linkage gave markedly more potent compounds
than the ester linkage as shown by the pairs of 6 and 7 or 9 and 10.
This effect might be related to the stronger polarity of the amide
bond as compared with the ester bond. Thus we reached the potent
compounds 7–9 and 11 according to our initial hypothesis. These

Y. Yamazaki et al. / Life Sciences 82 (2008) 290–300
299
strengthen the activities of the ferulamides. The result is prom-
Acknowledgments
ising as the combination of 30 μM 7 with 50 μM DHA amplified
the adiponectin amount to more than 15 times that given by
30 μM 7 alone, resulting in approaching a level of the value by
3 μM PGZ (Fig. 7). In the animal experiment, the group fed 7+
DHA oil showed the tendency that the adiponectin level increased
and the glucose and triglyceride levels decreased as compared
with other groups (Fig. 9C, D, E). However, the significant
difference was only detected against the control or the 7 alone
group. The 7+DHA group was not significantly different from
the DHA alone group in any case. DHA oil itself was reported to
increase plasma adiponectin level in mice (Flachs et al., 2006).
The result of the multiple comparisons suggests that the effect of
the combination of 7 with DHA on blood glucose and triglyceride
levels should be mainly due to DHA in this animal experiment.
Nevertheless, the contribution of 7 would not be completely
excluded, since the adiponectin level in day-37 samples (Fig. 9C,
closed bars) showed no significant difference between the DHA
group and the control in contrast to that between the 7+DHA
group and the control. Optimization of the dose and ratio of 7 to
DHA in the diets would make their combination effect more clear.
On the other hand, further improvement of the efficacy of the
ferulamide is undoubtedly required, because the present dose of 7
(0.4 to 1.4 g/kg/day as estimated from the content and average
food intake) is too high even as a supplement to health food. If
these problems are solved, the prominent plasma triglyceride-
loweringeffect bythe combination of DHAwith a ferulamidesuch
as 7 will have a practical importance for treatment of dyslipidemia.
DHA is known to improve the hypertriglyceridemic condition by
the regulation of lipoprotein metabolism (Lombardo et al., 2007),
while the ferulamide (or the DHA partner) would promote
the effect of DHA on the level involving different transcription
factors (Duval et al., 2007; Chu et al., 2006; Zhang et al., 2006). In
addition, the combination with other compounds will result in
reducing the necessary amount of DHA so that the bad smell of the
oxidation products from DHA oil is diminished.
We thank Drs. Yoji Sato and Hiromi Yoshida (National
Institute of Health Sciences) for conducting the PPRE-driven
reporter assay of compound 7.
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adiponectin-inducing activities in vitro than the known active
compounds, curcumin, [6]-gingerol, or capsaicin, and these
activities are further enhanced by coaddition with DHA. The
most active ferulamide 7 can suppress the blood glucose and
triglyceride levels in diabetic mice. The present ferulamides are
useful lead compounds in developing more potent agents, nat-
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