U.S. patent application number 12/049647 was filed with the patent office on 2008-07-03 for sesquiterpenoid derivatives having adipocyte differentiation inhibitory effect.
Invention is credited to Munekazu Iinuma, Nobuyasu Matsuura, Hiroto Suzuki, Toshiyuki Tanaka, Makoto Ubukata, Masashi Yamada.
Application Number | 20080160119 12/049647 |
Document ID | / |
Family ID | 19076248 |
Filed Date | 2008-07-03 |
United States Patent
Application |
20080160119 |
Kind Code |
A1 |
Iinuma; Munekazu ; et
al. |
July 3, 2008 |
Sesquiterpenoid Derivatives Having Adipocyte Differentiation
Inhibitory Effect
Abstract
The objective of the present invention is to identify compounds
having an inhibitory effect on adipocyte differentiation, and to
provide pharmaceutical compositions and food compositions for
prevention, improvement, or treatment of obesity or obesity related
diseases. By enthusiastically studying natural products that
inhibit the differentiation induction of adipocyte precursors into
adipocytes, sesquiterpenoid derivatives, which are extracts from a
Compositae plant, Calea, growing in Central and South America, were
demonstrated to have an excellent inhibitory effect on adipocyte
differentiation induction. Calea extract or the sesquiterpenoid
derivatives are expected to be used in pharmaceutical compositions
and food compositions for prevention, improvement, or treatment of
obesity or obesity related diseases.
Inventors: |
Iinuma; Munekazu; (Gifu,
JP) ; Tanaka; Toshiyuki; (Gifu, JP) ; Ubukata;
Makoto; (Hokkaido, JP) ; Matsuura; Nobuyasu;
(Okayama, JP) ; Yamada; Masashi; (Tokyo, JP)
; Suzuki; Hiroto; (Toyama, JP) |
Correspondence
Address: |
SALIWANCHIK LLOYD & SALIWANCHIK;A PROFESSIONAL ASSOCIATION
PO BOX 142950
GAINESVILLE
FL
32614-2950
US
|
Family ID: |
19076248 |
Appl. No.: |
12/049647 |
Filed: |
March 17, 2008 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10485801 |
Sep 13, 2004 |
|
|
|
PCT/JP2002/008178 |
Aug 9, 2002 |
|
|
|
12049647 |
|
|
|
|
Current U.S.
Class: |
424/764 |
Current CPC
Class: |
A61P 1/18 20180101; A61P
9/00 20180101; A61P 9/10 20180101; C07D 307/93 20130101; A61P 1/16
20180101; C07D 493/04 20130101; A61P 9/04 20180101; A61P 43/00
20180101; A61K 36/28 20130101; A61P 3/06 20180101; A61P 19/06
20180101; A61P 9/12 20180101; A61K 31/335 20130101; A23L 33/105
20160801; A61P 3/10 20180101; A61P 3/04 20180101; A61P 19/02
20180101 |
Class at
Publication: |
424/764 |
International
Class: |
A61K 36/28 20060101
A61K036/28; A61P 19/02 20060101 A61P019/02; A61P 9/00 20060101
A61P009/00; A61P 3/10 20060101 A61P003/10 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 15, 2001 |
JP |
2001-246777 |
Claims
1. A method for the prevention, improvement, or treatment of
obesity, or an obesity related disease, wherein said method
comprises administering, to a patient in need of such prevention,
improvement, or treatment, an effective amount of one or more of
the following: a) a Calea extract having an inhibitory effect on
adipocyte differentiation; and b) a sesquiterpenoid derivative
having an inhibitory effect on adipocyte differentiation.
2. The method, according to claim 1, wherein the disease is
selected from the group consisting of heart disease, vascular
disorders, diabetes, gout, hyperlipemia, fatty liver, gallstone,
pancreatitis, osteoarthritis, and hernia.
3. The method, according to claim 1, wherein said Calea is selected
from the group consisting of Calea urticifolia, Calea pinnatifida,
Calea uniflora, Calea axillaris, Calea insignis, Calea
integrifolia, Calea nelsonii, Calea peduncularis, Calea pringlei,
Calea purpusii, Calea sabazioides, Calea savannamm, Calea scabra,
Calea sororia, Calea standleyi, Calea tejadae, and Calea
zacatechichi.
4. The method, according to claim 1, which comprises administering
a compound having any one of structural formulae (1) to (7):
##STR00006## ##STR00007##
Description
TECHNICAL FIELD
[0001] The present invention relates to adipocyte differentiation
inhibitors containing sesquiterpenoid derivatives that have an
inhibitory effect on adipocyte differentiation as active
ingredients, and pharmaceutical compositions and food compositions
for prevention, improvement, or treatment of diseases caused by the
induction of adipocyte differentiation.
BACKGROUND ART
[0002] Obesity is caused by overeating, lack of exercise, abnormal
feeding pattern, genetic predisposition, thermogenesis impairment,
and such. Excess energy resulting from imbalance of intake and
expenditure of energy is stored in adipocytes, and these adipocytes
gather to form adipose tissue. That is, obesity is a condition of
hyperplasia of adipose tissues.
[0003] In addition, obesity is thought to be a major risk factor
for heart diseases (angina pectoris, myocardial infarction, cardiac
hypertrophy, heart failure, etc.), vascular disorders
(hypertension, arteriosclerosis, cerebral thrombosis, cerebral
infarction, etc.), diabetes, gout, hyperlipemia, fatty liver,
gallstone, pancreatitis, osteoarthritis, hernia, and such.
[0004] Adipocytes are produced from mesenchymal cells and are
formed by differentiation induction from adipocyte precursors.
Therefore, adipocyte formation can be suppressed by inhibiting
differentiation induction of adipocyte precursors to form
adipocytes. When the formation of adipocyte is suppressed, adipose
tissue will not be formed. Thus, compounds that inhibit
differentiation of adipocytes are considered to have preventive or
therapeutic effects against obesity related diseases.
DISCLOSURE OF THE INVENTION
[0005] An objective of the present invention is to identify
compounds having inhibitory effects on adipocyte differentiation,
and to provide pharmaceutical compositions and food compositions
for prevention, improvement, or treatment of obesity or obesity
related diseases.
[0006] In order to achieve the above-mentioned objective, the
present inventors made every effort to study natural products that
inhibit the differentiation induction of adipocyte precursors to
adipocytes. As a result, sesquiterpenoid derivatives extracted from
a Compositae plant, Calea, growing in Central and South America,
were found for the first time to possess an excellent inhibitory
effect on the induction of adipocyte differentiation. Therefore,
there is a great expectation that these sesquiterpenoid derivatives
may be used in pharmaceutical compositions and food compositions
for prevention, improvement, or treatment of obesity or obesity
related diseases.
[0007] More specifically, the present invention relates to an
adipocyte differentiation inhibitor containing a sesquiterpenoid
derivative that has an inhibitory effect on adipocyte
differentiation as an active ingredient, and also to pharmaceutical
compositions and food compositions for prevention, improvement, or
treatment of obesity or obesity related diseases, and more
specifically provides:
[0008] [1] a Calea extract having an inhibitory effect on adipocyte
differentiation;
[0009] [2] the Calea extract according to [1], wherein said Calea
is selected from the group consisting of Calea urticifolia, Calea
pinnatifida, Calea uniflora, Calea axillaris, Calea insignis, Calea
integrifolia, Calea nelsonii, Calea peduncularis, Calea pringlei,
Calea purpusii, Calea sabazioides, Calea savannarum, Calea scabra,
Calea sororia, Calea standleyi, Calea tejadae, and Calea
zacatechichi;
[0010] [3] a sesquiterpenoid derivative having an inhibitory effect
on adipocyte differentiation;
[0011] [4] a sesquiterpenoid derivative of any one of structural
formula (1) to (4):
##STR00001##
[0012] [5] a sesquiterpenoid derivative having an inhibitory effect
on adipocyte differentiation of any one of structural formula (1)
to (4) of [4] and (5) to (7):
##STR00002##
[0013] [6] an adipocyte differentiation inhibitor containing the
Calea extract according to [1] or [2], or the sesquiterpenoid
derivative according to [3] or [5] as an active ingredient;
[0014] [7] a pharmaceutical composition for prevention,
improvement, or treatment of obesity or obesity related diseases,
containing the Calea extract according to [1] or [2], or the
sesquiterpenoid derivative according to [3] or [5] as an active
ingredient;
[0015] [8] the pharmaceutical composition according to [7], wherein
the disease is selected from the group consisting of heart disease,
vascular disorder, diabetes, gout, hyperlipemia, fatty liver,
gallstone, pancreatitis, osteoarthritis, and hernia; and
[0016] [9] a food composition for prevention or improvement of
obesity or obesity related diseases, containing the Calea extract
according to [1] or [2], or the sesquiterpenoid derivative
according to [3] or [5] as an active ingredient.
[0017] The present inventors discovered that the Calea plant
extract, sesquiterpenoid derivatives, have an inhibitory effect on
the induction of adipocyte differentiation. Therefore, the present
invention provides Calea extract having adipocyte differentiation
inhibitory effect.
[0018] Calea is a plant belonging to Compositae and grows mainly in
Central and South America. The Calea of the present invention
includes Calea urticifolia, Calea pinnatifida, Calea uniflora,
Calea axillaris, Calea insignis, Calea integrifolia, Calea
nelsonii, Calea peduncularis, Calea pringlei, Calea purpusii, Calea
sabazioides, Calea savannarum, Calea scabra, Calea sororia, Calea
standleyi, Calea tejadae, and Calea zacatechichi, but is not
particularly limited to these species or variations.
[0019] The term "Calea extract" herein refers to a substance
extracted from all or a portion of the Calea plant. The Calea
extract of the present invention is typically in a liquid form (for
example, Calea extract solution) but is not limited thereto, and
includes also powdered substances made upon drying the extracted
solution, or substance obtained by cutting or grinding an arbitrary
portion of Calea. The Calea extract of the present invention can be
obtained by extracting all or an arbitrary portion of the Calea
plant. However, extract from the entire plant is especially
preferable.
[0020] The Calea extract of the present invention can be obtained
according to the method for preparing plant extract solutions
generally performed by those skilled in the art. For example, the
Calea extract solution can be obtained according to the following
method. First, the collected plant is cut and is dried in a cool
place away from direct sunlight. Then, following the production of
powder using a grinder, a variety of appropriate organic solvents,
water, or their mixed solutions are used for extraction by heated
reflux. Next, the extracted solution is filtered, and concentrated
under reduced pressure to obtain a concentrated extract.
[0021] Whether the Calea extract obtained by the above-mentioned
method has an inhibitory effect on adipocyte differentiation or not
can be evaluated, for example, by measuring acetic acid
(.sup.14C--CH.sub.3COOH) incorporation activity, or by glucose
(.sup.14C-2-deoxyglucose) incorporation activity described later in
the Examples. Examples of biochemical evaluation methods include
measurements of glucose-6-phosphate dehydrogenase activity, neutral
fat releasing activity, and such. In addition, regarding detection
at the mRNA level, evaluation can be conducted by detecting
adipocyte differentiation markers, such as the aP2 gene.
[0022] Furthermore, the present inventors discovered that
sesquiterpenoid derivatives contained in Calea extract have an
inhibitory effect on adipocyte differentiation. Therefore, the
present invention provides sesquiterpenoid derivatives having
adipocyte differentiation inhibitory effect.
[0023] The sesquiterpenoid derivative of the present invention
having adipocyte differentiation inhibitory effect may be a natural
compound (for example, a compound extracted from plants other than
Calea) or an artificially synthesized compound.
[0024] Compounds having the characteristics (chemical formula,
structural formula, molecular weight, etc.) indicated in the
following (1) to (7) may be cited as examples of the
sesquiterpenoid derivatives of the present invention having
adipocyte differentiation inhibitory effects.
##STR00003## ##STR00004## ##STR00005##
[0025] The above-mentioned compounds 1, 4, 7, and 12 are compounds
found for the first time in the present invention. The present
invention also provides these novel compounds. Furthermore, the
sesquiterpenoid derivative of the present invention includes
analogs of the above-mentioned compounds, or compounds produced as
derivatives based on these compounds so long as they possess an
inhibitory effect on adipocyte differentiation. For example, a
compound wherein a functional group of the above-mentioned compound
is replaced with another functional group is included in the
compound of the present invention so long as it has an inhibitory
effect on adipocyte differentiation. Furthermore, some compounds
are known to become pro-drugs (a drug that shows activity only
after being metabolized in vivo due to modification of the chemical
structure) by esterification. Such chemically modified
sesquiterpenoid derivatives (such as pro-drug, etc.) are also
included in the present invention.
[0026] In addition, the present invention provides Calea extract
having adipocyte differentiation inhibitory effect, or adipocyte
differentiation inhibitor containing sesquiterpenoid derivatives as
the active ingredient. Adipocytes are produced from mesenchymal
cells and are formed by differentiation induction from adipocyte
precursors. Compounds having an inhibitory effect on adipocyte
differentiation suppress the formation of adipocytes, and as a
result, adipose tissue formation is expected to be suppressed.
Therefore, such compounds are expected to be not only useful as
reagents for suppressing adipocyte differentiation, but also
effective in prevention, improvement, or treatment of obesity or
obesity related diseases. Thus, the present invention provides
Calea extract, or pharmaceutical compositions containing
sesquiterpenoid derivatives as the active ingredient for
prevention, improvement, or treatment of obesity or obesity related
diseases.
[0027] The term "prevention" herein includes not only prevention
before having the disease, but also prevention against recurrence
of the disease after treatment. Specifically, "obesity related
diseases" that are the targets of prevention, improvement, or
treatment of the present invention include, for example, heart
diseases (angina pectoris, myocardial infarction, cardiac
hypertrophy, heart failure, etc.), vascular disorders
(hypertension, arteriosclerosis, cerebral thrombosis, cerebral
infarction, etc.), diabetes, gout, hyperlipemia, fatty liver,
gallstone, pancreatitis, osteoarthritis, hernia, and such, but are
not particularly limited to these diseases.
[0028] When using the adipocyte differentiation inhibitor, the
anti-obesity drug for prevention, improvement, or treatment of
obesity, and the pharmaceutical compositions for prevention,
improvement, or treatment of obesity related diseases of the
present invention, it is prepared as a generic medical formulation.
For example, the medicament of the present invention is prescribed
in a form suited for oral administration or parenteral
administration as a formulation, such as pharmaceutical
compositions or tablet, pill, powder, granule, encapsulated
formulation, troche, syrup, solution, emulsion, suspension,
injection obtained by mixing with carriers (excipient, binder,
disintegrant, corrigent for taste, corrigent for smell, emulsifier,
diluent, adjuvant, etc.) acceptable for formulation.
[0029] Examples of excipient include lactose, cornstarch, white
sugar, glucose, sorbitol, plasma cellulose, and such. Examples of
binders include polyvinyl gum arabia, tragacanth, gelatin, shellac,
hydroxypropylcellulose, hydroxypropyl starch, polyvinylpyrrolidone,
and such.
[0030] Examples of disintegrants include starch, agar, gelatin
powder, crystalline cellulose, calcium carbonate, sodium
bicarbonate, calcium citrate, dextran, pectin, and such. Examples
of lubricants are magnesium stearate, talc, polyethylene glycol,
silica, hardened vegetable oil, and such. Further, coloring agents
acceptable for addition into medicaments may be used. Example of
corrigents for taste and smell include cocoa powder, l-menthol,
aromatic acids, peppermint oil, Borneo camphor, cinnamon powder,
and such. These tablets and granules may be appropriately coated as
necessary with sugar coating, gelatin coating, and so on.
[0031] When preparing an injection, pH regulator, buffer,
stabilizer, preservative, and such, are added as necessary, and is
made into a subcutaneous, intramuscular, or intravenous injection
by conventional methods. The injection can be a formulation that is
prepared just before use by storing the solution into a container,
then producing a solid formulation by freeze drying and such. A
single dose can be stored into a container, and a dose can be
stored into the same container.
[0032] The dosage of the adipocyte differentiation inhibitor, the
anti-obesity drug for prevention, improvement, or treatment of
obesity, and the pharmaceutical composition for prevention,
improvement, or treatment of obesity related diseases of the
present invention is determined by considering the type of dosage
form, administration method, age and weight of the subject (mammals
including humans), condition of the subject, and such.
Specifically, for an adult patient, for example, 0.01 to 600 mg can
be administered orally per day as 1 to several doses. Examples of
doses are more preferably 0.1 to 400 mg/day, and even more
preferably 1 to 200 mg/day. Although these doses vary depending on
the weight and age of the patient, and on the administration
method, one skilled in the art can appropriately select the correct
dose. It is also preferable to appropriately determine the
administration period depending on the healing course of the
patient, and such.
[0033] A preferred embodiment of the above-mentioned pharmaceutical
composition of the present invention may be an oral composition.
Specifically, the oral composition may be, for example, in a dry
powdered form of the Calea extract of the present invention. The
dry powder may be prepared, for example, according to the method
selected from (a) and (b) mentioned below:
(a) freeze-drying method: The extract solution is suspended in
water, and frozen. Then, the frozen solution is dried by subliming
water by reduced pressure to powderize the extract solution; and
(b) spray-drying method: The extract solution or suspension is
sprayed into hot air and is dried instantaneously to obtain a
spherical dried substance.
[0034] In addition, the present invention provides food
compositions for prevention or improvement of obesity or obesity
related diseases that contain Calea extract, or sesquiterpenoid
derivatives as active ingredients. Examples of the food
compositions of the present invention are health food, functional
food, specified health food, nutritional supplements, enteral
nutrition, and such, but are not limited to these foods as long as
it has the effect of preventing or improving obesity or obesity
related diseases. The food compositions of the present invention
may be used favorably in the form of the above-mentioned oral
composition. Further, the present invention includes the use of the
dried powder or the extract for producing oral compositions for
improvement, or prevention, or otherwise prevention of recurrence
of diseases caused by adipocyte differentiation induction in
mammals, including humans. The production method of the oral
compositions is a well known, frequently used technology for those
skilled in the art. More specifically, the sesquiterpenoid
derivative of the present invention or Calea extract solution or
dried Calea powder containing the sesquiterpenoid derivative can be
processed into health food, functional food, specified health food,
nutritional supplements, enteral nutrition, and such, by mixing
compositions that are acceptable in terms of food sanitation. For
example, compositions, such as stabilizers, preservatives, coloring
agents, fragrances, and vitamins, can be added appropriately to the
above-mentioned sesquiterpenoid derivatives, or to the Calea
extract solution and dried Calea powder containing the
sesquiterpenoid derivatives, mixed, and then formed appropriately
by conventional methods for oral compositions, such as tablet,
particulate, granule, powder, capsule, liquid, cream, beverage, and
such.
[0035] Any patents, patent applications, and publications cited
herein are incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0036] FIG. 1 is a diagram showing the inhibitory effect of Calea
extract on adipocyte differentiation induction. The ordinate shows
.sup.14C--CH.sub.3COOH incorporation (cpm). "-Ins.", "+Ins.", and
"Ins. +" on the abscissa indicate no addition of insulin, addition
of insulin, and addition of both insulin and Calea extract to the
sample, respectively.
[0037] FIG. 2 is a diagram showing the inhibitory effect of Calea
extract on adipocyte differentiation induction. The ordinate shows
.sup.14C-2-deoxyglucose incorporation (cpm) "-Ins.", "+Ins.", and
"Ins. +" on the abscissa indicate no addition of insulin, addition
of insulin, and addition of both insulin and Calea extract to the
sample, respectively.
[0038] FIG. 3 is a diagram showing the isolation process of
compounds (sesquiterpenoid derivatives) having an inhibitory effect
on adipocyte differentiation induction.
[0039] FIG. 4 is a diagram showing the inhibitory effect of
sesquiterpenoid derivatives on adipocyte differentiation induction.
The ordinate shows .sup.14C--CH.sub.3COOH incorporation (cpm/mg
protein). "-Ins.", and "+Ins." on the abscissa indicate no addition
of insulin, and addition of insulin to the sample, respectively. 1,
3, 4, 6, 7, 11, and 12 on the abscissa indicate samples to which
compounds 1, 3, 4, 6, 7, 11, and 12, respectively, have been added
in combination with insulin.
BEST MODE FOR CARRYING OUT THE INVENTION
[0040] Hereinafter, the present invention will be described more
specifically by way of Examples, but the present invention is not
limited to these Examples.
Example 1
Evaluation of the Inhibitory Effect of Calea Extract Solution on
Adipocyte Differentiation Induction
[0041] Whether Calea extract solution has an inhibitory activity on
adipocyte differentiation induced by the addition of insulin was
investigated. First, the aerial portion of Calea (170 g) was
ground, and extracted by heated reflux using acetone (24
hours.times.2 times) to produce Calea extract solution. The
extracted solution was filtered, and then concentrated under
reduced pressure to yield a concentrated extract. Mouse adipocyte
precursors (3T3-L1) obtained from Human Science Research Resources
Bank was used as the adipocyte in the present experiment. Normal
successive cultivation was performed using Dulbecco's modified
Eagle MEM medium containing 10% calf serum (CS) at 37.degree. C. in
the presence of 5% CO.sub.2. To evaluate the inhibitory effect on
differentiation induction into adipocytes, the above-mentioned
cells were removed from a dish by trypsin treatment to obtain cell
suspension, and then after separation by centrifugation at 1,000
rpm for 3 minutes at 4.degree. C., the concentration was adjusted
to 3.5.times.10.sup.4 cells/ml, and 200 .mu.l aliquots were placed
into a 96-well plate to prepare an examination plate. After 72
hours, 2 .mu.l each of a sample dissolved in 10% DMSO was placed
into each of the examination plate wells. Simultaneously,
dexamethasone and isobutylmethyl xanthine were added to a final
concentration of 20 .mu.M and 10 mM, respectively. After 48 hours,
the media was removed, 200 .mu.l of fresh media was added, and the
same concentration of sample was added again. Furthermore, insulin
was added to a final concentration of 17 .mu.M. Subsequently, media
exchange and addition of accompanying reagents and insulin were
carried out at 3-day intervals. 8 to 10 days later, media were
removed, cells were washed with PBS (-), immobilized using 10%
formalin solution, adipose granules stored in the cells were
stained with Oil Red-O/60% isopropanol saturated solution for 1
hour at room temperature. After staining, excess Oil Red O solution
was removed using PBS (-). Observation of the stained adipose
granules under a light microscope confirmed that the cells had
undergone differentiation induction.
[0042] In a system for evaluating the inhibitory activity against
adipocyte differentiation induced by insulin addition, acetic acid
(.sup.14C--CH.sub.3COOH) incorporation activity, which is used as
an index for differentiation induction activity, and incorporation
activity of glucose (.sup.14C-2-deoxyglucose), which is known not
to be metabolized in cells, were used as indices.
[0043] In the system where acetic acid incorporation activity was
used as an index, first the cells were placed into a 24-well plate
at a density of 4.2.times.10.sup.4 cell/well. After 72 hours, Calea
extract solution, IBMX (10 mM), and dexamethazone (20 .mu.M) were
added, and was cultivated for another 48 hours. Then, the media was
exchanged to a fresh media, and Calea extract solution and insulin
(17 .mu.M) were added. The media was exchanged to a fresh media at
3-day intervals, and Calea extract solution and insulin were added
each time. After 9 days of cultivation following insulin addition,
.sup.14C-sodium acetate was added to a concentration of 1
.mu.Ci/ml, and was cultivated for 3 hours. Then, the media was
removed, the cells were washed twice using 2 ml of PBS(-) and then
dried for a few minutes on a clean bench. Next 1 ml isopropanol was
gently added, left standing for a few minutes, and then isopropanol
was collected and transferred to a scintillation vial. Isopropanol
extraction was performed 2 more times. Using a liquid scintillation
counter, radioactivity of the isopropanol fraction (adipose
component) was measured. Next, upon completely drying the
isopropanol extracted cells, the cells were dissolved using 200
.mu.l of 0.1 N NaOH, and then were neutralized with 200 .mu.l of 1
M Tris-HCl pH7.5. Then, protein quantification was performed
according to the Bradford dye-binding method. For the adipose
synthesis activity, .sup.14C--CH.sub.3COOH incorporation activity
per amount of protein was used as an index. Upon analysis,
approximately 50% and nearly 100% incorporation inhibitory
activities were indicated at concentrations of 4 .mu.g/ml and 8
.mu.g/ml extract solutions, respectively (FIG. 1).
[0044] On the other hand, in a system wherein glucose incorporation
activity is used as an index, cells were placed into a 24-well
plate at a density of 4.2.times.10.sup.4 cells/well. After 72
hours, Calea extract solution, IBMX (10 mM), and dexamethazone (20
.mu.M) were added, and cells were cultivated for another 48 hours.
Then, the media was exchanged to a fresh media, and Calea extract
solution and insulin (17 .mu.M) were added. The media was exchanged
to a fresh media at 3-day intervals, and Calea extract solution and
insulin were added each time. After 9 days of cultivation after
insulin addition, the media was replaced with 500 .mu.l/well of
glucose deficient DMEM media, and was cultivated for 15 minutes.
Then, .sup.14C-2-deoxyglucose (Amersham Pharmacia Biotech) was
added to a final concentration of 0.25 .mu.Ci/ml, and was
cultivated for 15 minutes. Then, the cells were washed three times
with ice cold PBS(-) containing 10 mM glucose, were removed from
the dish using trypsin, and were transferred to a scintillation
vial. Using a liquid scintillation counter, radioactivity of
.sup.14C-2-deoxyglucose incorporated into the cells was measured.
Upon analysis, approximately 25% and approximately 15% of
incorporation inhibitory activities were indicated at extract
solution concentrations of 4 .mu.g/ml and 8 .mu.g/ml, respectively
(FIG. 2).
Example 2
Identification of Compounds Having Adipocyte Differentiation
Induction Inhibitory Activity in Calea Extract Solution
[0045] From Example 1, a compound existing in the Calea extract
solution was thought to inhibit differentiation induction of
adipocytes. Consequently, compounds having adipocyte
differentiation induction inhibitory activity were identified.
[0046] Specifically, first, the above-mentioned compound was
isolated by the process indicated in FIG. 3. The aerial portion of
Calea (170 g) was ground, and was extracted by heated reflux (24
hours.times.2 times) using acetone. After concentrating the
extracted solution under reduced pressure (9.8 g), the solution was
distributed into chloroform and water. Upon concentrating the
chloroform fraction under reduced pressure (7.1 g), the
concentrated extract was dissolved in CHCl.sub.3, and was
chromatographed through a silica gel column (50.times.200 mm: 400
ml). Practically, 1200 ml each of solvents composed of
CHCl.sub.3:MeOH=30:1 and 20:1 were used for elution in order. 6
fractions, each containing 400 ml, were obtained. Active fraction 2
was concentrated under reduced pressure (2.1 g). Next, the
concentrated extract of the active fraction was dissolved in 5 ml
of MeOH, and was chromatographed on a reverse-phase silica gel
column (38.times.130 mm: 150 ml Cosmosil 140C18-OPEN (Nakarai)).
Specifically, the sample was added to a column material
equilibrated with 40% MeOH, and was eluted using 40 and 65% MeOH in
order. The active fraction eluted with 65% MeOH was concentrated
under reduced pressure, and 1.14 g of active extract was obtained.
Furthermore, using reverse-phase HPLC, the active ingredient was
separated. Upon analysis, 7 compounds having adipocyte
differentiation induction inhibitory activity were isolated under
the following conditions: column, Mightysil RP-18 20.times.250 mm;
column temperature, 40.degree. C.; flow rate, 11.342 ml/minute;
detector, 210 nm; solvent, 42% CH.sub.3CN. Following the
concentration under reduced pressure, each compound was
recrystallized from Et.sub.2O. The retention times of the isolated
compounds were, compound 1: 13.5 minutes; compound 3: 18.1 minutes;
compound 4: 20.2 minutes; compound 6: 23.3 minutes; compound 7:
25.3 minutes; compound 11: 35.1 minutes; and compound 12: 38.3
minutes.
[0047] Next, to identify the above-mentioned compounds, their
physico chemical properties were investigated (Table 1).
TABLE-US-00001 TABLE 1 1 3 4 + 5 6 7 11 12 m.p. (.degree. C.)
142-144 133-135 170-172 150-153 99-101 decomp. 98-100 EI/MS 406 406
450 432 434 cation FAB/MS 433 449 [.alpha.].sub.0.sup.25 (c 0.001,
184.2 192.2 92.1 154 168.7 -- 195.4 CHCl.sub.3)
[0048] Furthermore, by .sup.1H (Table 2, Table 3) and .sup.13C-NMR
analysis (Table 4), as well as various two-dimensional NMR,
relative configuration of each compound was determined and
respective structural formula was determined. As a result, all of
these compounds having the inhibitory activity on the induction of
adipocyte differentiation were revealed to be sesquiterpenoid
derivatives. In particular, compounds 1, 4, 7, and 12 were novel
sesquiterpenoid derivatives.
TABLE-US-00002 TABLE 2 1 3 4 6 7 2 4.21 (d, 4.1 Hz, 1H) 6.50 (d,
12.0, 1H) 3.08 (dd, 16.0, 8.0 Hz, 1H) 4.30 (d, 4.4 4.27 (d, 4.1 Hz,
1H) Hz, 1H) 3.60 (dd, 16.0, 9.5 Hz, 1H) 3 3.30 (dd, 9.4, 4.3 Hz,
1H) 5.98 (dd, 12.0 5.92 (m, 1H) 3.34 (dd, 9.4, 3.35 (dd, 9.4, 4.1
Hz, 1H) and 12.0, 1H) 4.3 Hz, 1H) 4 1.45: assigned from H--H COSY
3.05 (m, 1H) 1.45: assigned 1.45: assigned from H--H COSY from H--H
COSY 5 1.46, 1.90 (each m, 1H) 1.38 (m, 1H) 2.77 (dd, 15.0, 4.0 Hz,
1H) 1.50, 1.96 1.51, 1.95 (each m, 1H) (each m, 1H) 1.80 (m, 1H)
2.86 (dd, 15.0, 3.6 Hz, 1H) 6 4.80 (dd, 12.2, 4.5 Hz, 1H) 4.5 5
(dd, 11.7, 4.95 (m, 1H) 4.84 (dd, 12.1, 4.86 (dd, 12.1, 4.3 Hz, 1H)
4.9 Hz, 1H) 4.5 Hz, 1H) 7 2.34 (br s, 1H) 2.60 (s, 1H) 2.68 (m, 1H)
2.38 (br s, 1H) 2.39 (br s, 1H) 8 5.66 (dd, 10, 1.7 Hz, 1H) 5.55
(s, 1H) 5.90 (dd, 10.5, 1.5 Hz, 1H) 5.75 (dd, 9.7, 5.72 (dd, 9.9,
1.7 Hz, 1H) 2.0 Hz, 1H) 9 5.73 (d, 10 Hz, 1H) 5.55 (s, 1H) 5.70 (d,
8.1 Hz, 1H) 5.85 (d, 12.0 5.82 (d, 9.9 Hz, 1H) Hz, 1H) 13 5.81,
6.31 (each m, 1H) 5.80, 6.25 (each 5.72 (s, 1H) 5.84, 6.34 5.85 (d,
1.0 Hz, 1H) d, 1.1, 1H) (each s, 1H) 6.30 (d, 3.4 Hz, 1H) 6.34 (s,
1H) 14 1.19 (d, 7.1 Hz, 3H) 1.05 (d, 6.3, 3H) 1.86 (s, 3H) 1.23 (d,
6.1 1.21 (d, 6.1 Hz, 3H) Hz, 3H) 15 1.45 (s, 3H) 1.30 (s, 3H) 1.35
(s, 3H) 1.46 (s, 3H) Acetyl .sup. 2' 1.95 (s, 3H) Meacr .sup. 3'
5.56, 6.03 (each m, 1H) 5.45, 5.95 (each 5.60, 6.00 (each m, 1H)
5.51 (m, 1H) 5.58 (m, 1H) s, 1H) 5.97 (s, 1H) 6.05 (s, 1H) .sup. 4'
1.83 (br s, 1H) 1.78 (s, 3H) 1.87 (s, 3H) 1.78 (s, 3H) 1.83 (s, 3H)
Acetyl 2'' 2.03 (s, 3H) 2.06 (s, 3H) Meacr 3'' 5.66 (m, 1H) 6.16
(s, 1H) 4'' 1.90 (s, 3H) iBu 2'' 2.57 (dq, 7.1, 7.1 Hz, 1H) 3''
1.10 (d, 7.1 Hz, 3H) 4'' 1.16 (d, 7.1 Hz, 3H) 10-OH 3.89 (br s, 1H)
4.28 (br s, 1H) 4.05 (br s, 1H)
TABLE-US-00003 TABLE 3 11 12 12 in d 6-benzene 6.03 (dd, 12.0, 11.5
5.95 (dd, 12.0, 11.5 4.95 (dd, 11.8, 11.5 Hz, Hz, 1H) Hz, 1H) 1H)
6.64 (d, 12.0 Hz, 1H) 6.54 (d, 12.0 Hz, 1H) 6.02 (d, 11.8 Hz, 1H)
3.13 (m, 1H) 3.06 (m, 1H) 2.80 (m, 1H) 1.45, 1.83 (each m, 1.38,
1.79 (each m, 0.95, 1.25 (each m, 1H) 1H) 1H) 4.63 (dd, 12.0, 4.9
4.55 (dd, 11.7, 4.5 4.38 (dd, 11.7, 4.9 Hz, Hz, 1H) Hz, 1H) 1H)
2.66 (s, 1H) 2.56 (s, 1H) 2.48 (br s, 1H) 5.69 (s, 1H) 5.58 (s, 1H)
6.00 (dd, 9.8, 2.1 Hz, 1H)) 5.69 (s, 1H) 5.58 (s, 1H) 5.73 (d, 9.8
Hz, 1H) 5.84 (d, 1.2 Hz, 1H) 5.76 (d, 1.2 Hz, 1H) 5.25 (d, 1.5 Hz,
1H) 6.33 (s, 1H) 6.25 (br s, 1H) 6.18 (d, 1.0 Hz, 1H) 1.15 (d, 6.3
Hz, 3H) 1.08 (d, 7.1 Hz, 3H) 0.58 (d, 6.3 Hz, 3H) 1.34 (s, 3H) 1.25
(s, 3H) 1.09 (s, 3H) 5.61 (br s, 1H) 5.47 (br s, 1H) 5.19 (m, 1H)
6.13 (s, 1H) 5.95 (s, 1H) 6.23 (br s, 1H) 1.88 (s, 3H) 1.74 (s, 3H)
1.83 (br s, 3H) 5.48 (br s, 1H) 5.94 (5, 1H) 1.76 (s, 3H) 2.46 (dq,
7.1, 7.1 2.34 (dq, 7.1, 7.1 Hz, Hz, 1H) 1H) 1.01 (d, 7.1 Hz, 3H)
0.98 (d, 7.1 Hz, 3H) 1.08 (d, 7.1 Hz, 3H) 0.98 (d, 7.1 Hz, 3H) 4.13
(br s, 1H) 4.04 (br s, 1H)
TABLE-US-00004 TABLE 4 12 in d 1 3 4 6 7 11 12 6-benzene C-1 206.0
204.7 210.7 206.1 206.0 204.9 205.1 204.9 2 55.6 125.3 36.0 55.7
55.7 148.1 148.4 148.2 3 62.8 148.2 121.5 62.8 62.7 125.4 125.7
125.5 4 26.0 28.3 136.6 26.0 25.9 28.3 28.6 28.3 5 38.8 40.2 35.9
38.8 38.7 40.3 40.6 40.5 6 74.6 76.3 76.6 74.7 74.7 76.4 76.4 76.2
7 40.9 41.2 42.0 40.9 40.9 41.2 41.7 41.9 8 73.9 74.4 67.0 73.8
73.8 74.3 74.3 75.1 9 71.5 73.8 72.5 71.7 70.8 74.0 73.2 73.9 10
79.7 79.2 80.3 79.9 79.8 79.4 79.7 79.8 11 134.3 134.5 134.1 134.3
134.2 134.6 134.9 135.6 12 168.3 168.7 168.1 168.4 168.0 168.8
169.1 168.6 13 126.8 126.6 124.4 126.7 126.8 126.5 127.0 126.0 14
18.6 19.7 25.4 18.6 18.5 19.7 20.0 19.6 15 24.5 23.4 22.3 24.6 24.4
23.6 23.9 23.8 .sup. 1' 165.2 170.3 165.2 165.4 165.0 166.6 165.6
165.8 .sup. 2' 134.8 20.3 135.1 134.8 134.8 134.7 135.2 135.9 .sup.
3' 127.3 126.9 127.0 127.3 127.8 127.6 127.4 .sup. 4' 18.1 18.1
18.0 18.0 18.0 18.4 18.4 1'' 170.4 165.3 170.4 166.7 176.0 165.4
176.8 176.6 2'' 20.2 134.9 20.4 134.5 33.9 134.9 34.3 34.4 3''
127.1 128.2 18.6 126.9 19.0 19.0 4'' 18.0 18.0 18.8 18.1 19.3
19.3
Example 3
Examination of the Relationship Between Structure and Activity of
Calea Extract
[0049] Structure-activity relationships of the 7 compounds obtained
in Example 2 were examined. Inhibitory activities of respective
compounds on the differentiation induction of 3T3-L1 cells into
adipocytes were compared in the system described in Example 1 that
uses acetic acid incorporation activity as the index. As a result,
high incorporation inhibitory activity was confirmed for compounds
11 and 6. Ranking the compounds in the order of higher inhibitory
activity, the order was: compound 11>6>4>3=12>1>7
(FIG. 4).
INDUSTRIAL APPLICABILITY
[0050] The present invention provides sesquiterpenoid derivatives
having adipocyte differentiation inhibitory effect. Furthermore,
the present invention provides pharmaceutical compositions and food
compositions for prevention, improvement, or treatment of obesity
or obesity related diseases that contain Calea extract or the
sesquiterpenoid derivative as an active ingredient. The use of
these compositions presents great expectations for effective
prevention, improvement, or treatment of obesity or obesity related
diseases.
* * * * *