Newly synthesized oleylgingerol and oleylshogaol activate TRPV1 ion channels

Article abstract of DOI:10.1271/bbb.70187

The oleyl moiety in vanilloids is important in activating vanilloid receptor 1 (TRPV1), but there was no ingredient of ginger containing the oleyl moiety in the natural form. We synthesized oleylgingerol and oleylshogaol and then evaluated their potential to activate a rat TRPV1 channel. Oleylgingerol is a stronger TRPV1 agonist than natural gingerols, but oleylshogaol is a weaker agonist than natural shogaols. The difference in structure between oleylgingerol and oleylshogaol is only the hydroxy group at carbon-5. This hydroxy group might have an important role in activating a TRPV1 channel.

Full text of DOI:10.1271/bbb.70187

Biosci. Biotechnol. Biochem., 71 (9), 2304–2307, 2007
Note
Newly Synthesized Oleylgingerol and Oleylshogaol
Activate TRPV1 Ion Channels
Akihito MORITA, Yusaku IWASAKI, Kenji KOBATA,
y
Hidehiko YOKOGOSHI, and Tatsuo WATANABE
School of Food and Nutritional Sciences and COE Program in the 21st Century,
University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan
Received April 4, 2007; Accepted June 13, 2007; Online Publication, September 7, 2007
[doi:10.1271/bbb.70187]
The oleyl moiety in vanilloids is important in acti-
Previous study of the structure-activity relationship
among capsaicin-related compounds has demonstrated
that olvanil, which has a oleyl moiety, was a potent
vating vanilloid receptor 1 (TRPV1), but there was no
ingredient of ginger containing the oleyl moiety in the
natural form. We synthesized oleylgingerol and oleyl-
shogaol and then evaluated their potential to activate a
rat TRPV1 channel. Oleylgingerol is a stronger TRPV1
agonist than natural gingerols, but oleylshogaol is a
weaker agonist than natural shogaols. The difference in
structure between oleylgingerol and oleylshogaol is only
the hydroxy group at carbon-5. This hydroxy group
might have an important role in activating a TRPV1
channel.
8
,9)
agonist of TRPV1.
ester of vanillyl alcohol and oleic acid, also strongly
We reported that olvanilate, an
9
)
activated TRPV1. Endogenous agonists for TRPV1
were reported to be endovanilloids, such as N-oleoyl-
1
0)
dopamine. One endocannabinoid, oleylethanolamine,
1
1)
also activates TRPV1.
Thus the oleyl moiety in
vanilloids is important in opening the TRPV1 channel.
In nature, there is no gingerol nor shogaol containing the
oleyl moiety. We think that the new synthetic analogs
containing oleyl moiety will prove to be stronger
agonists. In this experiment, we synthesized two new
compounds (oleylgingerol and oleylshogaol), and then
evaluated their potential to activate a rat TRPV1 ion
channel.
Key words: transient receptor potential vanilloid sub-
type 1 (TRPV1); oleylgingerol; oleylsho-
gaol
Ginger (Zingiber officinale ROSCOE) is used tradition-
ally in China and Japan as a food having many
Oleylgingerol was synthesized according to the
methods of Wada et al.,12) with a slight modification.
In brief, oleylgingerol was obtained by aldol addition of
zingerone in potassium t-butoxide with oleylaldehyde
prepared by oxidation of oleyl alcohol with pyridinium
chlorochromate. Oleylshogaol was synthesized by de-
hydration of oleylgingerol with p-toluenesulfonic acid
under heating. The structures of these compounds were
confirmed by spectroscopic data as follows: oleylgin-
1
)
biological activities. The components to which the
pungency of ginger can be attributed are gingerols and
shogaols. Gingerols are present in raw ginger and
2
)
mainly comprise [6]-, [8]-, and [10]-gingerols. Sho-
gaols, dehydration products of gingerols, are present in
steamed ginger and mainly comprise [6]-, [8]-, and [10]-
shogaols.2 These compounds are phenolic substances
and possess a vanillyl moiety (see Fig. 1); therefore,
these are types of vanilloids. Recently, the receptor for
vanilloids was cloned and named TRPV1 (transient
receptor potential vanilloid subtype 1), which is a
)
þ
gerol; HR-FABMS m=z [M ] calcd for C H O
2
9
48
4
460.3554, found 460.3557; IR ꢀmax (film) 3431, 3003,
2924, 2854, 1705, 1605, 1516, 1462, 1371, 1271, 1153,
ꢀ1
1124, 1036, 814, 723 cm ; UV ꢁmax (MeOH) 282 nm
3
)
1
nonselective cation channel. The most famous vanil-
loid is capsaicin, a pungent ingredient of red pepper
(" ¼ 2600), 210 nm (" ¼ 7000); H NMR (CDCl3,
399.65 MHz, TMS as the internal standard) ꢂ 6.82
0
(
channel and then increases the intracellular Ca
Capsicum fruits). Capsaicin strongly activates a TRPV1
2
(1H, d, J ¼ 7:8 Hz, H-5 ), ꢂ 6.68 (1H, d, J ¼ 2:0 Hz, H-
þ
0
0
2 ), ꢂ 6.65 (1H, dd, J ¼ 7:8, 2.0 Hz, H-6 ), ꢂ 5.53 (1H,
4)
0
concentration. [6]-Gingerol and [8]-gingerol activate
a native or recombinant TRPV1.5,6) We have reported
that other natural gingerols and shogaols also activated
TRPV1 channels, but their potency is not so strong as
that of capsaicin.7
bs, 4 -OH), ꢂ 5.34 (2H, m, H-13, 14), ꢂ 4.02 (1H, m, H-
5), ꢂ 3.87 (3H, s, OCH3), ꢂ 2.93 (1H, bs, 5-OH), ꢂ 2.83
(2H, t, J ¼ 7:2 Hz, H-1), ꢂ 2.73 (2H, t, J ¼ 7:2 Hz, H-2),
ꢂ 2.56 (1H, dd, J ¼ 17:6, 2.8 Hz, H-4a), ꢂ 2.48 (1H, dd,
J ¼ 17:6, 8.8 Hz, H-4b), ꢂ 2.01 (4H, m, H-12, 15), ꢂ 1.2
)
y
To whom correspondence should be addressed. Fax: +81-54-264-5550; E-mail: watanbt@u-shizuoka-ken.ac.jp
Abbreviation: TRPV1, transient receptor potential vanilloid subtype 1

Newly Synthesized Oleylgingerol Is a More Potent TRPV1 Agonist
2305
Fig. 1. Chemical Structure of Vanilloids.
The four gingerols and four shogaols used in this experiment were illustrated.
to 1.5 (24H, H-6 to 11, 16 to 21), ꢂ 0.88 (3H, t, J ¼
Table 1. Hydrophobicity and TRPV1 Activation Potency among
Capsaicinoids, Gingerols and Shogaols
1
3
6
:8 Hz, H-22); C NMR (CDCl3, 100.40 MHz) ꢂ 211.4
0 0 0
(
1
5
C-3), ꢂ 146.5 (C-3 ), ꢂ 144.0 (C-4 ), ꢂ 132.6 (C-1 ), ꢂ
EC50
0
30.0 (C-13), ꢂ 129.8 (C-14), ꢂ 120.7 (C-6 ), ꢂ 114.4 (C-
0
Compounds
Log P
(
mM)
0
0
), ꢂ 111.0 (C-2 ), ꢂ 67.7 (C-5), ꢂ 55.9 (3 -OCH3), ꢂ 49.4
ꢂꢂ
[6]-gingerol
capsaicin
2.718
3.622
4.374
4.612
5.891
6.111
7.540
9.182
10.40
11.93
4.55
0.0819
(
2
(
1
C-4), ꢂ 45.5 (C-2), ꢂ 36.5 (C-6), ꢂ 31.9, 29.8, 29.8, 29.5,
9.5, 29.5, 29.3, 29.3, 29.3, 29.2, 27.2, 27.2, 25.5, 22.7
C-1, 7 8, 9, 10, 11, 12, 15, 16, 17, 18, 19, 20 or 21), ꢂ
ꢂ
ꢂꢂ
[8]-gingerol
[6]-shogaol
[10]-gingerol
2.09
0.611
ꢂꢂ
ꢂꢂ
þ
4.1 (C-22): oleylshogaol; HR-FABMS m=z [M ] calcd
1.99
0.711
0.874
ꢂꢂ
ꢂꢂ
[
[
8]-shogaol
10]-shogaol
for C29H46O3 442.3448, found 442.3467; IR ꢀmax (film)
3
1
8
552, 3433, 3003, 2926, 2854, 1670, 1628, 1516, 1462,
365, 1271, 1236, 1207, 1151, 1120, 1036, 978, 852,
ꢂ
olvanil
oleylgingerol
oleylshogaol
0.00686
0.264
4.17
ꢀ1
14, 723, 628.7, 557.3 cm ; UV ꢁmax (MeOH) 280 nm
1
(
3
" ¼ 2500), 227 nm (" ¼ 16200); H NMR (CDCl3,
Hydrophobicity was evaluated by reversed-phase HPLC. TRPV1 activation
potency was evaluated as the change of the intracellular Ca2þ concentration
99.65 MHz, TMS as the internal standard) ꢂ 6.82
0
in HEK293 cells heterologously expressing rat TRPV1 channels. Each
change of the intracellular Ca2 concentration was normalized to the
(
6
1H, d, J ¼ 8:0 Hz, H-5 ), ꢂ 6.81 (1H, dt, J ¼ 16:4.
þ
0
.8 Hz, H-5), ꢂ 6.72 (1H, d, J ¼ 1:6 Hz, H-2 ), ꢂ 6.68
ꢂ
response activated by 10 mM capsaicin. Indicates data reported by Morita et
0
(
1H, dd, J ¼ 8:0, 1.6 Hz, H-6 ), ꢂ 6.09 (1H, dt, J ¼ 16:4,
al.9) ꢂꢂIndicates data reported by Iwasaki et al.7)
0
1
1
2
6
1
.6 Hz, H-4), ꢂ 5.48 (1H, bs, 4 -OH), ꢂ 5.34 (2H, m, H-
3, 14), ꢂ 3.87 (3H, s, OCH3), ꢂ 2.85 (4H, m, H-1, 2), ꢂ
.19 (2H, qd, J ¼ 6:8, 1.6 Hz, H-6), ꢂ 2.01 (4H, q, J ¼
:0 Hz, H-12, 15), ꢂ 1.44 (2H, quint, J ¼ 6:8 Hz, H-7),
.2 to 1.4 (20H, H-8 to 11, 16–21), ꢂ 0.88 (3H, t, J ¼ 6:4
The hydrophobicity of the compounds used in this
experiment was evaluated from relative log P values, as
1
3)
described previously. The log of the octanol/water
partition coefficient (log P) of capsaicin, N-vanillylbu-
tanamide, and N-vanillylhexanamide was measured
directly using an octanol-water partition system. Other
relative log P values were calculated from the retention
time in reverse-phase HPLC (J’sphere ODS-H80,
150 mm ꢁ 4.6 i.d., YMC, Kyoto, Japan, using 80%
methanol). The hydrophobicity of vanilloids depends on
the length of the carbon chain and the presence of
hydroxy-moiety in the side chain. The most hydrophilic
compound was [6]-gingerol, and the most hydrophobic
compound was the newly synthesized oleylshogaol
(Table 1). In the case of the same alkyl carbon length,
the hydrophobicity of the shogaols was observed to be
higher than that of the gingerols.
1
3
Hz, H-22); C NMR (CDCl , 100.40 MHz) ꢂ 199.7 (C-
3
0
0
3
), ꢂ 147.8 (C-5), ꢂ 146.4 (C-3 ), ꢂ 143.9 (C-4 ), ꢂ 133.3
0
(
C-1 ), ꢂ 130.3 (C-4), ꢂ 130.0 (C-13), ꢂ 129.7 (C-14), ꢂ
0 0 0 0
1
20.8 (C-6 ), ꢂ 114.3 (C-5 ), ꢂ 111.1 (C-2 ), ꢂ 55.9 (3 -
OCH3), ꢂ 42.0 (C-2), ꢂ 32.5, 3.19, 29.9, 29.8, 29.7, 29.5,
2
6
9.3, 29.3, 29.3, 29.2, 29.2, 28.1, 27.2, 27.2, 22.7 (C-1,
, 7 8, 9, 10, 11, 12, 15, 16, 17, 18, 19, 20 or 21), ꢂ 14.1
(
C-22). The purities of oleylgingerol and oleylshogaol
by HPLC–UV (280 nm) were 94.3 and 99.7% respec-
tively. Three gingerols ([6], [8], and [10]-gingerols) and
three shogaols ([6], [8], and [10]-shogaols) were
obtained from Professor K. Kubota of Ochanomizu
7
)
University, Tokyo, Japan. Capsaicinoids were pur-
chased or synthesized as described previously.9 Other
chemicals were purchased from Sigma (St. Louis, MO,
USA) or Wako Pure Chemical Industries (Osaka,
Japan).
)
To compare the potential of each compound with
regard to TRPV1 activation, we measured the intra-
2
þ
cellular Ca concentration in HEK293 cells hetero-

2306
A. MORITA et al.
Fig. 2. Relationship between Hydrophobicity and TRPV1 Activation Potency.
Hydrophobicity and the TRPV1 activation potency were measured as described in Table 1. The log P and EC50 values were plotted for each
compound of the gingerols (closed circle) and the shogaols (closed square), and the curve were fitted using Prizm4 software.
logously expressing rat TRPV1 ion channels, as describ-
ed previously.9) All test compounds were dissolved in
oleylshogaol (4.17 mM) was higher than those of other
shogaols.
ꢃ
dimethyl sulfoxide and stored at ꢀ20 C until experi-
To clarify the relationship between hydrophobicity
(the length of the alkyl chain) and the potential for
TRPV1 activation, the relative log P values and the EC50
values were plotted (Fig. 2). The EC50 values decreased
with increases in the carbon chain length of the gingerol-
related compounds (Fig. 2). The longer the alkyl chain
length of the gingerol, the higher the potential to activate
TRPV1 channels. Capsaicin-related compounds also
exhibited the same relationship between hydrophobicity
and the potential to activate TRPV1 channels, in pre-
ments were done. Fura-2 AM, a calcium sensitive dye,
was obtained from Molecular Probes (Eugene, OR,
USA). Curve fitting and parameter estimation were
carried out using Prism4 (Graph Pad Software, San
Diego, CA, USA).
Table 1 indicates the EC50 values of the tested com-
pounds. The responses of all the compounds tested were
inhibited by pretreatment with capsazepine, a specific
antagonist for TRPV1 (data not shown), and no com-
2
þ
9)
pound increased the intracellular Ca concentration in
parent HEK293 cells (data not shown), indicating that
vious experiments. On the other hand, the EC values
5
0
of the shogaols increased with increases in the carbon
chain length (Fig. 2). The longer the alkyl chain length
of the shogaol, the lower the potential to activate TRPV1
channels.
all compounds increased the intracellular Ca2 concen-
þ
tration via activation of TRPV1.
Capsaicin increased the intracellular Ca2 concen-
tration in HEK293 cells expressing the rat TRPV1
channels. The values of EC50 for capsaicin and olvanil
þ
In this experiment, oleylgingerol proved to be a
stronger agonist than natural gingerols, although its
potential to activate TRPV1 was observed to be lower
than that of capsaicin. On the other hand, oleylshogaol
was a weaker agonist than natural shogaols. Among the
gingerols, there was the positive relationship between
hydrophobicity and potential with regard to the activa-
tion of TRPV1 channels, but there was a negative re-
lationship among the shogaols. The reason for this
opposite relationship remains obscure. The TRPV1
channel shares structural similarities with Kv-type
potassium channels, including the same six-transmem-
(
others. [6]-, [8]-, and [10]-Gingerols also increased the
Table 1) were almost the same as those reported by
8)
2
þ
intracellular Ca
were lower than that of capsaicin. The EC50 value for
6]-gingerol was 4.55 mM (Table 1), almost the same
concentration, but their potencies
[
6
)
value as in a previous report. The newly synthesized
oleylgingerol (EC50 ¼ 0:264 mM) was more potent than
other gingerols. In addition to gingerol-related com-
pounds, [6]-, [8]-, and [10]-shogaols were more potent
TRPV1 agonists than [6]-gingerol (Table 1). Un-
expectedly, the EC50 value for the newly synthetic
7
)
1
4)
brane topology. Assuming helix packing for TRPV1

Newly Synthesized Oleylgingerol Is a More Potent TRPV1 Agonist
2307
similar to that for potassium channels, the acyl moiety of
capsaicin may bind to transmembrane domains 2 and
5) Dedov, V. N., Tran, V. H., Duke, C. C., Connor, M.,
Christie, M. J., Mandadi, S., and Roufogalis, B. D.,
Gingerols: a novel class of vanilloid receptor (VR1)
agonists. Br. J. Pharmacol., 137, 793–798 (2002).
1
4)
3
on the channel-lipid interface.
The presence of
the oleyl moiety may be suitable for the binding of van-
niloids to the TRPV1 channel, and may increase the
6)
7)
8)
Witte, D. G., Cassar, S. C., Masters, J. N., Esbenshade,
T., and Hancock, A. A., Use of a fluorescent imaging
plate reader-based calcium assay to assess pharmaco-
logical differences between the human and rat vanilloid
receptor. J. Biomol. Screen., 7, 466–475 (2002).
Iwasaki, Y., Morita, A., Iwasawa, T., Kobata, K.,
Sekiwa, Y., Morimitsu, Y., Kubota, K., and Watanabe,
T., A nonpungent component of steamed ginger-[10]-
shogaol-increases adrenaline secretion via the activation
of TRPV1. Nutr. Neurosci., 9, 169–178 (2006).
Appendino, G., Minassi, A., Morello, A. S., De
Petrocellis, L., and Di Marzo, V., N-Acylvanillamides:
development of an expeditious synthesis and discovery
of new acyl templates for powerful activation of the
vanilloid receptor. J. Med. Chem., 45, 3739–3745
8
–11)
TRPV1-activating potential,
but we report here the
first evidence that the addition of the oleyl moiety to
shogaol decreases the TRPV1-acrtivating potential. We
confirmed the trans-configuration of the double bond at
carbon-4 of oleylshogaol, suggesting that the conforma-
tion of the oleyl moiety as between oleylgingerol and
oleylshogaol is similar. The difference in structure is
only the hydroxy group at carbon-5. This hydroxy group
may have an important role in activating a TRPV1 ion
channel.
Acknowledgments
(
2002).
This work was supported in part by a Grant-in-aid for
Scientific Research from the Ministry of Education,
Culture, Sports, Science and Technology of Japan. We
are grateful to Dr. M. Sugiyama of University of
Shizuoka for supporting the measurement of FAB-MS
spectrum data and for confirmation of the structures of
newly synthesized compounds. We thank Professor K.
Kubota of Ochanomizu University for the gift of three
gingerols ([6], [8], and [10]-gingerols) and three
shogaols ([6], [8], and [10]-shogaols).
9
)
Morita, A., Iwasaki, Y., Kobata, K., Iida, T., Higashi, T.,
Oda, K., Suzuki, A., Narukawa, M., Sasakuma, S.,
Yokogoshi, H., Yazawa, S., Tominaga, M., and
Watanabe, T., Lipophilicity of capsaicinoids and cap-
sinoids influences the multiple activation process of rat
TRPV1. Life Sci., 79, 2303–2310 (2006).
1
0) Chu, C. J., Huang, S. M., De Petrocellis, L., Bisogno, T.,
Ewing, S. A., Miller, J. D., Zipkin, R. E., Daddario, N.,
Appendino, G., Di Marzo, V., and Walker, J. M., N-
Oleoyldopamine: a novel endogenous capsaicin-like
lipid that produces hyperalgesia. J. Biol. Chem., 278,
1
3633–13639 (2003).
References
11) Smart, D., Jonsson, K. O., Vandevoorde, S., Lambert, D.
M., and Fowler, C. J., ‘Entourage’ effects of N-acyl
ethanolamines at human vanilloid receptors. Comparison
of effects upon anandamide-induced vanilloid receptor
activation and upon anandamide metabolism. Br. J.
Pharmacol., 136, 452–458 (2002).
1
)
Chrubasik, S., Pittler, M. H., and Roufogalis, B. D.,
Zingiberis rhizoma: a comprehensive review on the
ginger effect and efficacy profiles. Phytomedicine, 12,
684–701 (2005).
2
)
)
Govindarajan, V. S., Ginger: chemistry, technology, and
quality evaluation: part 1. CRC Crit. Rev. Food Sci.
Nutr., 17, 1–96 (1982).
Caterina, M. J., Schumacher, M. A., Tominaga, M.,
Rosen, T. A., Levine, J. D., and Julius, D., The capsaicin
receptor: a heat-activated ion channel in the pain
pathway. Nature, 389, 816–824 (1997).
Tominaga, M., Caterina, M. J., Malmberg, A. B., Rosen,
T. A., Gilbert, H., Skinner, K., Raumann, B. E.,
Basbaum, A. I., and Julius, D., The cloned capsaicin
receptor integrates multiple pain-producing stimuli.
Neuron, 21, 531–543 (1998).
12) Wada, T., Muranishi, S., Masuda, H., Manabe, H., and
Izumi, K., Japan Kokai Tokkyo Koho, 08-040970
(Feb. 13, 1996).
13) Iida, T., Moriyama, T., Kobata, K., Morita, A.,
Murayama, N., Hashizume, S., Fushiki, T., Yazawa, S.,
Watanabe, T., and Tominaga, M., TRPV1 activation and
induction of nociceptive response by a non-pungent
capsaicin-like compound, capsiate. Neuropharmacology,
44, 958–967 (2003).
3
4
)
14) Jordt, S. E., and Julius, D., Molecular basis for species-
specific sensitivity to ‘‘hot’’ chili peppers. Cell, 108,
421–430 (2002).