U.S. patent application number 13/889656 was filed with the patent office on 2013-11-28 for circadian rhythm regulatory agent.
This patent application is currently assigned to National Institute of Advanced Industrial Science & Technology. The applicant listed for this patent is Nisshin Seifun Group Inc., Oriental Yeast Co., Ltd, National Institute of Advanced Industrial Science & Technology. Invention is credited to Shinichi FUKUDOME, Nanako ITOH, Kenji KASUYA, Yosuke KIKUCHI, Koyomi MIYAZAKI, Katsutaka OISHI, Yoshiaki ONISHI, Keiko TANAKA, Tatsunosuke TOMITA.
Application Number | 20130317258 13/889656 |
Document ID | / |
Family ID | 49622103 |
Filed Date | 2013-11-28 |
United States Patent
Application |
20130317258 |
Kind Code |
A1 |
OISHI; Katsutaka ; et
al. |
November 28, 2013 |
CIRCADIAN RHYTHM REGULATORY AGENT
Abstract
A circadian rhythm regulatory agent having as an active
ingredient an alkylresorcinol represented by general formula (I)
below and a circadian rhythm regulatory agent containing as an
active ingredient an alkylresorcinol-containing extract from a
cereal or a nut containing an alkylresorcinol of the following
general formula (I). In the following general formula (I), R.sub.1
is a saturated or unsaturated alkyl group; and R.sub.2 is hydrogen
or methyl. In general formula (I), R.sub.1 is preferably at the
para-position with respect to R.sub.2, and R.sub.1 is preferably a
saturated or unsaturated alkyl group having 15 to 27 carbon atoms.
##STR00001##
Inventors: |
OISHI; Katsutaka; (Ibaraki,
JP) ; MIYAZAKI; Koyomi; (Ibaraki, JP) ;
ONISHI; Yoshiaki; (Ibaraki, JP) ; TOMITA;
Tatsunosuke; (Ibaraki, JP) ; ITOH; Nanako;
(Ibaraki, JP) ; FUKUDOME; Shinichi; (Fujimino-shi,
JP) ; TANAKA; Keiko; (Fujimino-shi, JP) ;
KIKUCHI; Yosuke; (Fujimino-shi, JP) ; KASUYA;
Kenji; (Tokyo, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
& Technology; National Institute of Advanced Industrial
Science
Nisshin Seifun Group Inc.;
Oriental Yeast Co., Ltd; |
|
|
US
US
US |
|
|
Assignee: |
National Institute of Advanced
Industrial Science & Technology
Tokyo
JP
Oriental Yeast Co., Ltd
Tokyo
JP
Nisshin Seifun Group Inc.
Tokyo
JP
|
Family ID: |
49622103 |
Appl. No.: |
13/889656 |
Filed: |
May 8, 2013 |
Current U.S.
Class: |
568/763 |
Current CPC
Class: |
A61K 31/05 20130101;
A61P 25/20 20180101; A61K 2236/33 20130101; C07C 39/08 20130101;
A61P 25/02 20180101; A61K 36/185 20130101 |
Class at
Publication: |
568/763 |
International
Class: |
C07C 39/08 20060101
C07C039/08 |
Foreign Application Data
Date |
Code |
Application Number |
May 15, 2012 |
JP |
2012-111161 |
Nov 1, 2012 |
JP |
2012-241533 |
Claims
1. A circadian rhythm regulatory agent comprising as an active
ingredient an alkylresorcinol represented by general formula (1):
##STR00003## wherein R.sub.1 represents a saturated or unsaturated
alkyl group; and R.sub.2 represents a hydrogen atom or a methyl
group.
2. The circadian rhythm regulatory agent according to claim 1,
wherein R.sub.1 in general formula (I) is at the para-position with
respect to R.sub.2.
3. The circadian rhythm regulatory agent according to claim 1,
wherein R.sub.1 in general formula (I) is a saturated or
unsaturated alkyl group having 15 to 27 carbon atoms.
4. The circadian rhythm regulatory agent according to claim 1,
wherein, in general formula (I), R.sub.1 is a saturated or
unsaturated alkyl group having 15 to 25 carbon atoms, and R.sub.2
is a hydrogen atom.
5. The circadian rhythm regulatory agent according to claim 1,
wherein the active ingredient is an extract which comprises an
alkylresorcinol and is prepared from a cereal or a nut.
6. The circadian rhythm regulatory agent according to claim 5,
wherein the alkylresorcinol-containing extract is a cereal extract
obtained by extracting a cereal with an alcohol.
7. The circadian rhythm regulatory agent according to claim 5,
wherein the cereal is wheat or rye, and the nut is a cashew
nut.
8. The circadian rhythm regulatory agent according to claim 2,
wherein R.sub.1 in general formula (I) is a saturated or
unsaturated alkyl group having 15 to 27 carbon atoms.
9. The circadian rhythm regulatory agent according to claim 2,
wherein, in general formula (I), R.sub.1 is a saturated or
unsaturated alkyl group having 15 to 25 carbon atoms, and R.sub.2
is a hydrogen atom.
10. The circadian rhythm regulatory agent according to claim 2,
wherein the active ingredient is an extract which comprises an
alkylresorcinol and is prepared from a cereal or a nut.
11. The circadian rhythm regulatory agent according to claim 3,
wherein the active ingredient is an extract which comprises an
alkylresorcinol and is prepared from a cereal or a nut.
12. The circadian rhythm regulatory agent according to claim 4,
wherein the active ingredient is an extract which comprises an
alkylresorcinol and is prepared from a cereal or a nut.
13. The circadian rhythm regulatory agent according to claim 6,
wherein the cereal is wheat or rye, and the nut is a cashew
nut.
14. The circadian rhythm regulatory agent according to claim 8,
wherein the active ingredient is an extract which comprises an
alkylresorcinol and is prepared from a cereal or a nut.
15. The circadian rhythm regulatory agent according to claim 9,
wherein the active ingredient is an extract which comprises an
alkylresorcinol and is prepared from a cereal or a nut.
16. The circadian rhythm regulatory agent according to claim 10,
wherein the cereal is wheat or rye, and the nut is a cashew
nut.
17. The circadian rhythm regulatory agent according to claim 11,
wherein the cereal is wheat or rye, and the nut is a cashew
nut.
18. The circadian rhythm regulatory agent according to claim 12,
wherein the cereal is wheat or rye, and the nut is a cashew nut.
Description
TECHNICAL FIELD
[0001] This invention relates to a circadian rhythm regulatory
agent for regulating biological circadian rhythm. The circadian
rhythm regulatory agent of the invention contains an
alkylresorcinol(s) as an active ingredient.
BACKGROUND ART
[0002] There are known many diseases caused by circadian rhythm
disorders. Various types of circadian rhythm regulatory agents have
been proposed to alleviate these diseases.
[0003] For example, a circadian rhythm regulatory agent containing
an endocrine hormone, such as melatonin, is known. However,
treatment with endocrine hormones has a safety problem on account
of the risk of side effects so that a material with high safety has
been sought for.
[0004] Sleep disorders which are now a social issue include
circadian rhythm sleep disorders. To basically remedy circadian
rhythm sleep disorders, a material that regulates the diurnal cycle
has been demanded.
[0005] There are reports on circadian rhythm regulatory agents of
food origin. For example, patent literature 1 below discloses a
composition containing as an active ingredient cryptoxanthin
extracted from satsuma orange (Citrus unshiu) and/or an ester
thereof and having a circadian clock normalizing effect. Patent
literature 2 below discloses a circadian rhythm normalizing
composition having arachidonic acid-containing fat and oil as an
active ingredient.
[0006] Synthetic alkylresorcinols and derivatives thereof and
alkylresorcinol-containing extracts from the hull of cereal grains,
such as wheat and rye, or the shell of nuts, such as cashew nuts,
are reported to have anti-obesity effect, antioxidant effect,
anti-immune effect, and the like. However, it is unknown that
alkylresorcinols exhibit circadian rhythm regulatory effects.
[0007] Alkylresorcinols are reported to be present in a wide range
of plants as resorcinol-lipids that are natural, non-isoterpenoid
phenolic, amphiphilic compounds (see non-patent literature 1
below). The non-patent literature 1 reports that the plants as a
source of resorcinol lipids include those belonging to the families
Anacardiaceae, Ginkgoaceae, Proteaceae, Myrsinaceae, Primulaceae,
Myristicaceae, lridaceae, Araceae, Artemisia of Compositae,
Fabaceae, and Poaceae. With respect to the toxicity of
alkylresorcinols, non-patent literature 2 below reports that no
apparent toxicity is observed in oral administration to rats of 5
g/kg-b.w. of 4-alkylresorcinol having a saturated C15 alkyl group
of cashew nut shell origin.
CITATION LIST
Patent Literature
[0008] Patent literature 1: JP 2009-084192A [0009] Patent
literature 2: US 2006073187A1
Non-Patent Literature
[0009] [0010] Non-patent literature 1: Arkadiusz Kozubek et. al.,
Chemical Reviews, vol. 99, No. 1, pp. 1-25 (1999) [0011] Non-patent
literature 2: Alastair B. Ross, et al., Nutrition Review, vol. 62,
No. 3, pp. 81-95 (2004)
SUMMARY OF INVENTION
Technical Problem
[0012] The invention relates to a circadian rhythm regulatory agent
of food origin that is safe and secure even when taken for a long
period of time.
Solution to Problem
[0013] As a result of extensive investigations, the present
inventors have found that an alkylresorcinol or a cereal extract
containing alkylresorcinols is influential on expression of
circadian clock gene and circadian activity rhythm.
[0014] Based on the above finding, the invention provides a
circadian rhythm regulatory agent containing alkylresorcinols
represented by general formula (I) shown below as an active
ingredient. The invention also provides a circadian rhythm
regulatory agent containing, as an active ingredient, an
alkylresorcinol-containing extract obtained from a cereal or a nut
containing alkylresorcinols represented by general formula (I).
##STR00002##
[0015] wherein R.sub.1 represents a saturated or unsaturated alkyl
group; and R.sub.2 represents a hydrogen atom or a methyl
group.
Advantageous Effects of Invention
[0016] The circadian rhythm regulatory agent of the invention is
effective in regulating circadian rhythm disorder to normalize
biological diurnal rhythm. The alkylresorcinol or
alkylresorcinol-containing extract as an active ingredient has high
safety with no side effects so that the agent of the invention has
the advantage of involving no problem in use.
[0017] The circadian rhythm regulatory agent of the invention is
effective in treating a circadian rhythm sleep disorder.
BRIEF DESCRIPTION OF DRAWINGS
[0018] FIG. 1 is a graph showing the influence of the circadian
rhythm regulatory agent of Example 1 (synthetic alkylresorcinol) on
expression of circadian clock genes.
[0019] FIG. 2 is a graph showing the influence of the circadian
rhythm regulatory agent of Example 2 (alkylresorcinol-containing
ethanolic extract of a wheat bran prepared by extracting the bran
with ethanol) on expression of circadian clock gene.
[0020] FIG. 3 is a graph showing the influence of the circadian
rhythm regulatory agent of Example 3 (alkylresorcinol-containing
ethanolic extract of rye prepared by extracting rye with ethanol)
on expression of circadian clock gene.
[0021] FIG. 4 is a graph showing the influence of the
alkylresorcinol-containing ethanolic extract of the on drinking
behavior rhythm in mice fed a high-fat diet in Test Example 2.
[0022] FIG. 5 is a graph showing the influence of the
alkylresorcinol-containing ethanolic extract of the bran on
corticosterone secretion in mice fed a high-fat diet in Test
Example 3.
[0023] FIG. 6 is a graph showing the influence of the
alkylresorcinol-containing ethanolic extract of the bran on
wheel-running activity in stressed mice in Test Example 4.
[0024] FIG. 7 is a graph showing the influence of the
alkylresorcinol-containing ethanolic extract of the bran on
drinking behavior rhythm in mice fed a high-fat high-sucrose diet
in Test Example 5.
DESCRIPTION OF EMBODIMENTS
[0025] The circadian rhythm regulatory agent of the invention
contains alkylresorcinols represented by general formula (I) as an
active ingredient. The alkylresorcinols are effective in regulating
circadian rhythm disorders to normalize biological diurnal rhythm.
That is, the alkylresorcinols have a circadian rhythm regulatory
effect.
[0026] In general formula (I), while the saturated or unsaturated
alkyl group as represented by R.sub.1 is not limited by the number
of carbon atoms, the number of carbon atoms of R.sub.1 is
preferably 15 to 27, more preferably 15 to 25.
[0027] Representative examples of the C15-27 saturated alkyl groups
include straight-chain alkyl groups, such as n-pentadecyl,
n-heptadecyl, n-nonadecyl, n-heneicosyl, n-tricosyl, n-pentacosyl,
and n-heptacosyl. Branched or cyclic alkyl groups are also useful.
Preferred of them are C15-25 saturated alkyl groups, with C15-25
straight-chain saturated alkyl groups being more preferred.
[0028] The C15-27 unsaturated alkyl groups include those
corresponding to the above recited C15-27 saturated alkyl groups.
The number and position of the unsaturated bonds in the unsaturated
alkyl group are not particularly limited.
[0029] In general formula (I), R.sub.2 is preferably a hydrogen
atom. R.sub.1 is preferably at the para-position to R.sub.2.
[0030] Specific examples of the alkylresorcinol represented by
general formula (I) that can be used as an active ingredient of the
circadian rhythm regulatory agent of the invention are listed
below: [0031] 1,3 -Dihydroxy-5-n-pentadecylbenzene (C15:0) [0032]
1,3 -Dihydroxy-5-n-heptadecylbenzene (C17:0) [0033]
1,3-Dihydroxy-5-n-nonadecylbenzene (C19:0) [0034]
1,3-Dihydroxy-5-n-heneicosylbenzene (C21:0) [0035]
1,3-Dihydroxy-5-n-tricosylbenzene (C23:0) [0036]
1,3-Dihydroxy-5-n-pentacosylbenzene (C25:0) [0037]
1,3-Dihydroxy-5-n-heptacosylbenzene (C27:0)
[0038] Particularly preferred of the alkylresorcinols of general
formula (I) are those in which R.sub.1 is a C15-25 saturated alkyl
group, and R.sub.2 is hydrogen atom. Especially preferred are
1,3-dihydroxy-5-n-pentadecylbenzene (C15:0),
1,3-dihydroxy-5-n-heptadecylbenzene (C17:0),
1,3-dihydroxy-5-n-nonadecylbenzene (C19:0),
1,3-dihydroxy-5-n-heneicosylbenzene (C21:0),
1,3-dihydroxy-5-n-tricosylbenzene (C23:0), and
1,3-dihydroxy-5-n-pentacosylbenzene (C25:0).
[0039] The alkylresorcinol of general formula (I) can be
synthesized in a usual manner or may be a commercially available
product. An extract prepared from a plant in a usual manner is also
useful. Plants containing alkylresorcinols include those of the
families Anacardiaceae, Ginkgoaceae, Proteaceae, Myrsinaceae,
Primulaceae, Myristicaceae, Iridaceae, and Araceae, the genus
Artemisia of the family Compositae, and the families Fabaceae and
Poaceae. Inter alia, plants of the family Poaceae have been under
researches with respect to alkylresorcinols as an edible effective
component and are therefore suitable as a source of the active
ingredient for use as a circadian rhythm regulatory agent of the
invention. Of the plants of the family Poaceae particularly
preferred as a source are wheat and rye because of their high
contents of the alkylresorcinols of general formula (I) ranging
from about 0.015% of 0.3% by mass based on the whole grain
weight.
[0040] Nuts, such as cashew nuts, are also suitable as
alkylresorcinols source, the active ingredient of the circadian
rhythm regulatory agent of the invention.
[0041] In using an alkylresorcinol-containing extract prepared from
cereals or nuts as an active ingredient, an alcoholic extract is
preferred.
[0042] The manner for extracting with an alcohol is not
particularly limited, including, for example, a method in which the
gramineous plant seeds are immersed, stirred, or refluxed in an
alcohol or a supercritical fluid extraction method.
[0043] Examples of alcohols used for extraction include those that
are liquid at room temperature, such as lower alcohols, e.g.,
methanol, ethanol, n-propanol, isopropyl alcohol, and n-butanol,
and polyhydric alcohols, e.g., 1,3-butlene glycol, propylene
glycol, and glycerol. Ethanol is preferred in terms of convenience
of use and environmental friendliness. The alcohols include those
containing an aqueous component, namely, aqueous alcohols. The
alcohol content of the aqueous alcohols is usually 70 vol % or
more, preferably 80 vol % or more, more preferably 90 vol % or
more. The extraction may be carried out in the usual manner.
[0044] The circadian rhythm regulatory agent of the invention
contains the alkylresorcinols of general formula (I) as an active
ingredient and, optionally, other pharmaceutically acceptable
ingredients including carriers, excipients, stabilizers, and other
additives and ingredients. The circadian rhythm regulatory agent of
the invention may be formulated into preparations in a usual
manner. Specifically, it can be formulated into dosage forms
including oral dosage forms, such as tablets, powders, granules,
and capsules, and parenteral dosage forms, such as injectable
solutions that are prepared by dissolving the alkylresorcinol of
general formula (I) in sterilized distilled water or sterilized
physiological saline together with a solubilizing agent and sealing
the solution in an ampule. As other ingredients, substances having
other medicinal effects, various vitamins, crude drugs, and
minerals may be added appropriately in amounts that do not impair
the circadian rhythm regulating effects of the invention.
[0045] The content of the active ingredient in the circadian rhythm
regulatory agent of the invention is not particularly limited and
may be selected as appropriate to the dosage form, the symptoms,
age, and sex of a subject to be given the agent, and other factors.
In administering the agent to a human, a recommended dosage of the
active ingredient of the circadian rhythm regulatory agent of the
invention is usually 0.01 to 10 g per day for an adult.
EXAMPLES
[0046] The invention will now be illustrated in greater detail with
reference to Examples and Test Examples, but it should be
understood that the invention is not limited thereto.
Example 1
[0047] Synthetic 1,3-Dihydhroxy-5-n-pentadecylbenzene (C15:0)
manufactured by Researchem and purchased from Namiki Shoji Co.,
Ltd. was used as a circadian rhythm regulatory agent.
Example 2
[0048] An alkylresorcinol-containing ethanolic bran extract was
prepared from bran according to the following extraction method.
The extract was used as a circadian rhythm regulatory agent. The
alkylresorcinols contained in the extract were found to be composed
mainly of 1,3-dihydroxy-5-n-pentadecylbenzene (C15:0), 1,3
-dihydroxy-5-n-heptadecylbenzene (C17:0), 1,3-dihydroxy-5
-n-nonadecylbenzene (C19:0), 1,3-dihydroxy-5-n-heneicosylbenzene
(C21:0), 1,3-dihydroxy-5-n-tricosylbenzene (C23:0), and
1,3-dihydroxy-5-n-pentacosylbenzene (C25:0).
Extraction Method:
[0049] To the bran was added five times by mass the amount of
ethanol, and the mixture was shaken at 150 rpm for 2 hours at room
temperature to extract. The mixture was centrifuged at 3500 rpm for
15 minutes at room temperature, and the supernatant was dried in a
centrifugal concentrator. The resulting concentrate was weighed and
dissolved in ethanol in a concentration of 420 mg/ml to provide an
alkylresorcinol-containing ethanolic bran extract.
Example 3
[0050] An alkylresorcinol-containing ethanolic rye extract was
prepared in the same manner as in Example 2, except for replacing
the bran with rye and dissolving the concentrate in ethanol in a
concentration of 110 mg/ml. The extract was used as a circadian
rhythm regulatory agent. The alkylresorcinols contained in the
extract were composed mainly of 1,3-dihydroxy-5-n-pentadecylbenzene
(C15:0), 1,3-dihydroxy-5-n-heptadecylbenzene (C17:0),
1,3-dihydroxy-5-n-nonadecylbenzene (C19:0),
1,3-dihydroxy-5-n-heneicosylbenzene (C21:0),
1,3-dihydroxy-5-n-tricosylbenzene (C23:0), and
1,3-dihydroxy-5-n-pentacosylbenzene (C25:0).
Example 4
[0051] An alkylresorcinol-containing ethanolic bran extract was
prepared from a wheat bran according to the following extraction
method. The extract was used as a circadian rhythm regulatory
agent. The alkylresorcinols contained in the extract were composed
mainly of 1,3-dihydroxy-5-n-pentadecylbenzene (C15:0),
1,3-dihydroxy-5-n-heptadecylbenzene (C17:0),
1,3-dihydroxy-5-n-nonadecylbenzene (C19:0),
1,3-dihydroxy-5-n-heneicosylbenzene (C21:0),
1,3-dihydroxy-5-n-tricosylbenzene (C23:0), and
1,3-dihydroxy-5-n-pentacosylbenzene (C25:0).
Extraction Method:
[0052] To the bran was added five times by mass the amount of
ethanol and stirred at 600 rpm for 16 hours at room temperature to
extract. The mixture was filtered to remove unnecessary matter to
give an ethanolic extract. Ethanol was removed by evaporation to
give a solvent extract.
[0053] The resulting ethanolic bran extract was purified by
medium-pressure chromatography under the conditions below. Peak
components appearing during the period of 31 to 36 minutes from the
start of elution were collected, and the solvent was removed by
evaporation to give an alkylresorcinol-containing ethanolic bran
extract.
Conditions of Medium-Pressure Chromatography:
[0054] Column: silica gel (Inject Column 3L+Hi-Flash Column 5L;
pore size: 60 .ANG.; particle size: 40 .mu.m; available from
Yamazen Corp.) [0055] Mobile phase: hexane/ethyl acetate=90/10 (by
volume) for 9 minutes; 80/20 for 15 minutes; and 60/40 for 16
minutes [0056] Detection wavelength: 254 nm
Test Example 1
[0057] The circadian rhythm regulatory agents of Examples 1 to 3
(i.e., 1,3-dihydroxy-5-n-pentadecylbenzene,
alkylresorcinol-containing ethanolic bran extract, and
alkylresorcinol-containing ethanolic rye extract) were evaluated
for the influence on expression of circadian clock gene in
accordance with the following test method. The results obtained are
shown in FIGS. 1 to 3, respectively.
[0058] As is apparent from FIG. 1, addition of
1,3-dihydroxy-5-n-pentadecylbenzene delays the circadian rhythm
phase of Bmal1 expression and shortens the cycle by 0.5 hours.
[0059] As is apparent from FIG. 2, addition of the
alkylresorcinol-containing ethanolic bran extract increases the
amplitude of the circadian rhythm of Bmal1 expression. As is
apparent from FIG. 3, addition of the alkylresorcinol-containing
ethanolic rye extract delays the phase of the circadian rhythm of
Bmal1 expression and increases the amplitude of the rhythm.
Test Method:
[0060] To prepare a recombination vector, the promoter regions of a
clock gene Bmal1 (-97 to +27 regions including the RORE sequence
and the transcription initiation site) were inserted into a
luciferase luminescence vector containing a PEST sequence serving
as a signal for rapid degradation of expressed proteins (pGL3-dLuc,
from Promega). About 5.times.10.sup.5 cells of mouse fibroblast
cell line NIH3T3 were inoculated into a 35 mm diameter culture
dish. After culturing for 24 hour, the recombination vector was
transfected into the cells.
[0061] Screening of candidate substances was performed using the
culture cells. After 24 hours from the transfection, the cells were
cultured under stimulation in a medium containing 100 nM
dexamethasone for 2 hours so as to synchronize the rhythmic phase
of each cell. Then, a test substance was added to a medium
containing a luciferin, (a luminescence substrate of the NIH3T3
cell line), and chemiluminescence by the reporter genes was
measured in real-time while continuing the cell culture. The test
substances were 1,3-dihydroxy-5-n-pentadecylbenzene, the
alkylresorcinol-containing ethanolic bran extract, and the
alkylresorcinol-containing ethanolic rye extract. The
1,3-dihydroxy-5-n-pentadecylbenzene was dissolved in ethanol in a
concentration of 25 mM, and the solution was added to the medium in
a concentration of 12.5 .mu.M. Each of the
alkylresorcinol-containing ethanolic bran extract and the
alkylresorcinol-containing ethanolic rye extract was dissolved in
ethanol in a concentration of 420 mg/ml and 110 mg/ml,
respectively, and the solution was added to the medium in a
concentration of 1.68 mg/ml and 0.33 mg/ml, respectively. As a
control, ethanol, which is the solvent of the test substance, was
added at the same timing, and the changes in amount of luminescence
were compared. Luminescence was measured for one minute at 10
minute interval using AB-2550 Kronos Dia from ATTO Corp.
Test Example 2
[0062] The circadian rhythm regulatory agent of Example 4 (i.e.,
the alkylresorcinol-containing ethanolic bran extract) was
evaluated for the influence on drinking activity rhythm in mice fed
a high-fat diet in accordance with the following test method. The
drinking activity data were collected for every 6 hours (L1: first
part of a light period; L2: second part of a light period; D1:
first part of a dark period; D2: second part of a dark period). The
results are shown in FIG. 4, in which ND refers to a group of mice
fed a normal diet; HF refers to a group of mice fed a high-fat
diet; and HFAR refers to a group of mice fed a high-fat diet
containing 0.5 mass % alkylresorcinol-containing ethanolic bran
extract.
[0063] As is apparent from FIG. 4, the group fed a normal diet had
a larger amount of activity in the first part of the dark period
than in the second part of a dark period (morningness) and that the
group fed a high-fat diet had a larger amount of activity in the
second part of the dark period than in the first part of the dark
period (eveningness). The diet containing 0.5 mass %
alkylresorcinol-containing ethanolic bran extract proves to change
the chronotype of the mice fed a high-fat diet in drinking behavior
from eveningness to morningness.
[0064] The mice were then housed under constant dark conditions to
record the drinking behavior rhythm cycles. As is apparent from
Table 1, the cycle is shortened in the group given the circadian
rhythm regulatory agent (alkylresorcinol-containing ethanolic bran
extract) with significance compared with the group fed a normal
diet and the group fed a high-fat diet. The suppression of the
increase in amount of activity in the second part of the dark
period is related to the shortening of the circadian rhythm cycle.
Thus, the tendency to eveningness caused by a high-fat diet loading
can be reformed through a series of the tests.
[0065] Seeing that the cycle is significantly shortened by the
uptake of the circadian rhythm regulatory agent compared with the
intake of ND or HF, the uptake of the circadian rhythm regulatory
agent proves effective in promoting waking early and alleviating
delayed sleep-phase syndrome caused by a long circadian clock
cycle.
Test Method:
[0066] The normal diet (ND) was D12450B (rodent diet with 10% (kcal
%) fat, from Research Diets, Inc.). The high-fat diet (HF) was
D12492 (rodent diet with 60% (kcal %) fat, from Research Diets,
Inc.). The alkylresorcinol-containing ethanolic bran extract was
added to the high-fat diet in an amount of 0.5 mass %.
[0067] The mice were individually housed throughout the testing.
The drinking activity data of the mice were collected using
Chronobiology Kit software from Stanford Software Systems,
California.
[0068] Four-week-old male C57BL/6J Jms Slc mice (from Japan SLC,
Inc.) were acclimated to light/dark cycles (12-hour light period
and 12-hour dark period; lighting from 0:00 to 12:00) for two
weeks. After the acclimation, the mice were divided into three
groups: a group fed a normal diet (n=6), a group fed a high-fat
diet (n=6), and a group fed a high-fat diet containing the
alkylresorcinol-containing ethanolic bran extract (n=5), and fed ad
lib. for 10 weeks. After the 10-week feeding, the mice were kept
under constant dark conditions, and the drinking activity data were
collected.
TABLE-US-00001 TABLE 1 ND HF HFAR Period (hr) 23.94 .+-. 0.03 23.91
.+-. 0.03 23.83 .+-. 0.00* *P < 0.05 vs. ND and HF diet
Test Example 3
[0069] The circadian rhythm regulatory agent of Example 4
(alkylresorcinol-containing ethanolic bran extract) was evaluated
for the influence on corticosterone secretion in mice fed a
high-fat diet according to the following test method. The results
are shown in FIG. 5.
[0070] As is apparent from FIG. 5, the corticosterone level in
blood varies diurnally in the normal diet-fed group, showing the
highest peak immediately before the dark period. It is seen that
the high-fat diet-fed group shows a delay of the peak (phase delay)
and a decrease of the amplitude of the diurnal variation. The diet
containing 0.5 mass % alkylresorcinol-containing ethanolic bran
extract proves to be effective in counteracting the phase delay and
the decrease of the amplitude in blood corticosterone rhythm.
Test Method:
[0071] The normal diet (ND) was D12450B (rodent diet with 10% (kcal
%) fat, from Research Diets, Inc.). The high-fat diet (HF) was
D12492 (rodent diet with 60% (kcal %) fat, from Research Diets,
Inc.). The alkylresorcinol-containing ethanolic bran extract was
added to the high-fat diet in an amount of 0.5 mass %.
[0072] Four-week-old male C57BL/6J Jms Slc mice (from Japan SLC,
Inc.) were acclimated to light/dark cycles (12-hour light period
and 12-hour dark period; lighting from 0:00 to 12:00) for two
weeks. After the acclimation, the mice were divided into three
groups each consisting of 24 mice: a group fed a normal diet, a
group fed a high-fat diet, and a group fed a high-fat diet
containing the alkylresorcinol-containing ethanolic bran extract,
and fed ad lib. for 10 weeks.
[0073] Four mice per group were sacrificed every 4 hours from
10:00, their whole blood samples were collected, and the blood
plasma was separated. The plasma samples were stored at -80.degree.
C. in their frozen state. The blood corticosterone level was
determined using a commercially available kit (Assay Pro, St.
Charls, Mo.).
Test Example 4
[0074] The circadian rhythm regulatory agent of Example 4
(alkylresorcinol-containing ethanolic bran extract) was evaluated
for the influence on wheel running activity in stressed mice
according to the test method below. The results are shown in FIG.
6.
[0075] As is apparent from FIG. 6, the diet containing 0.5 mass %
alkylresorcinol-containing ethanolic bran extract proves to be
effective in suppressing the increase in amount of activity in the
light period in the mice having stress-related sleep disorder.
Test Method:
[0076] A powdered diet AIN-93M (from Oriental Yeast Co., Ltd.) was
blended with 0.5 mass% alkylresorcinol-containing ethanolic bran
extract, and the blend diet was pelletized to prepare a test diet.
Pelletized AIN-93M was used as a control.
[0077] The mice were individually housed in a cage equipped with a
running wheel (SW-15s, from Melquest, Ltd.) throughout the testing.
The activity data of the mice were collected using Chronobiology
Kit software (Stanford Software Systems, CA).
[0078] Four-week-old male S1c:B6C3F1 mice (from Japan SLC, Inc.)
were bred in light/dark cycles (12-hour light period and 12-hour
dark period; lighting from 0:00 to 12:00). Mice were fed ad lib.
for 4 weeks (non-stress period) with AIN-93M containing the
alkylresorcinol-containing ethanolic bran extract in the test group
(alkylresorcinol group) consisting of 12 mice and with AIN-93M in
the control group consisting of 11 mice.
[0079] After the non-stressed feeding experience, the mice were
physically inhibited from getting out of the wheel thereby to
continuously induce a stress-related sleep disorder for 2 weeks
(stress period). The mice having the stress-related sleep disorder
show a rhythm disorder that can be extrapolated to general sleep
disorders. For example, an increase in activity in the daytime
(light period), which is naturally the inactive period for mice as
the nocturnal animal, and a decrease in activity in the night (dark
period), which is the active period for mice, are observed.
Test Example 5
[0080] The circadian rhythm regulatory agent of Example 4 (i.e.,
the alkylresorcinol-containing ethanolic bran extract) was
evaluated for the influence on drinking behavior rhythm in mice fed
a high-fat high-sucrose diet in accordance with the following test
method. The diurnal drinking activity data were collected for every
6 hours (L1: first part of a light period; L2: second part of a
light period; D1: first part of a dark period; D2: second part of a
dark period). The results are shown in FIG. 7, in which ND refers
to a group of mice fed a normal diet; HFHSD refers to a group of
mice fed a high-fat high-sucrose diet; and HFHSDAR refers to a
group of mice fed a high-fat high-sucrose diet containing 0.5 mass
% alkylresorcinol-containing ethanolic bran extract.
[0081] As is apparent from FIG. 7, although the group fed a
high-fat high-sucrose diet had an increased amount of activity in
the light period compared with the group fed a normal diet, the
increase in activity in the light period in the high-fat
high-sucrose diet-fed mice is suppressed by the blend diet with 0.5
mass % alkylresorcinol-containing ethanolic bran extract.
Furthermore, the phenomenon of increasing activity in D2 compared
with D1 in the high-fat high-sucrose diet-fed mice (the tendency to
eveningness) is reformed by the alkylresorcinol-containing
ethanolic bran extract with significance.
Test Method:
[0082] AIN-93M (containing milk casein, available from Oriental
Yeast Co., Ltd.) was used as the normal diet. F2HFHSD (from
Oriental Yeast) was used as the high-fat high-sucrose diet (HFHSD).
The high-fat high-sucrose diet was blended with 0.5 mass %
alkylresorcinol-containing ethanolic bran extract.
[0083] The drinking activity data of the mice were collected
throughout the testing using Chronobiology Kit software (Stanford
Software Systems, CA).
[0084] Four-week-old male C57BL/6J Jms Slc mice (from Japan SLC,
Inc.) were acclimated to light/dark cycles (12-hour light period
and 12-hour dark period; lighting from 0:00 to 12:00) for two
weeks. After the acclimation, the mice were divided into three
groups: a group fed a normal diet (9 cages), a group fed a high-fat
high-sucrose diet (9 cages), and a group fed a high-fat
high-sucrose diet containing the alkylresorcinol-containing
ethanolic bran extract (9 cages) and fed ad lib. for 6 weeks.
* * * * *