U.S. patent application number 14/150461 was filed with the patent office on 2014-05-01 for anti-obesity agent comprising compound containing benzotropolone ring.
This patent application is currently assigned to SUNTORY HOLDINGS LIMITED. The applicant listed for this patent is SUNTORY HOLDINGS LIMITED. Invention is credited to Sumio ASAMI, Yuko FUKUI, Mitsuru MAEDA.
Application Number | 20140121388 14/150461 |
Document ID | / |
Family ID | 43126272 |
Filed Date | 2014-05-01 |
United States Patent
Application |
20140121388 |
Kind Code |
A1 |
FUKUI; Yuko ; et
al. |
May 1, 2014 |
ANTI-OBESITY AGENT COMPRISING COMPOUND CONTAINING BENZOTROPOLONE
RING
Abstract
The object of the present invention is to provide an
anti-obesity agent which contains a tea-derived component and which
is safe and does not compromise the flavor of foods and beverages.
According to the present invention, a safe and palatable
anti-obesity agent can be provided by incorporating a
benzotropolone ring-containing compound which has tea-derived, high
inhibitory activities against lipase and alfa-glucosidase. The
anti-obesity agent of the present invention does not compromise the
flavor of foods and beverage, has palatability, and can be used in
various use applications including foods and beverages intended for
health enhancement such as reduction in triglycerides.
Inventors: |
FUKUI; Yuko; (Mishima-gun,
JP) ; ASAMI; Sumio; (Mishima-gun, JP) ; MAEDA;
Mitsuru; (Mishima-gun, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
SUNTORY HOLDINGS LIMITED |
OSAKA-SHI |
|
JP |
|
|
Assignee: |
SUNTORY HOLDINGS LIMITED
OSAKA-SHI
JP
|
Family ID: |
43126272 |
Appl. No.: |
14/150461 |
Filed: |
January 8, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
13321299 |
Nov 18, 2011 |
8658237 |
|
|
PCT/JP2010/058624 |
May 21, 2010 |
|
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14150461 |
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Current U.S.
Class: |
549/399 |
Current CPC
Class: |
C07D 311/04 20130101;
C07D 311/62 20130101; A61P 43/00 20180101; A23V 2002/00 20130101;
A61P 3/04 20180101; A61K 31/353 20130101; A61P 3/06 20180101; A23L
33/105 20160801; C07C 62/38 20130101; C07D 311/64 20130101; A23F
3/16 20130101; A23L 2/52 20130101; A61K 31/122 20130101; A23V
2002/00 20130101; A23V 2200/332 20130101 |
Class at
Publication: |
549/399 |
International
Class: |
C07D 311/04 20060101
C07D311/04 |
Foreign Application Data
Date |
Code |
Application Number |
May 21, 2009 |
JP |
2009-123585 |
Claims
1. An anti-obesity agent comprising one or more compounds of
Formula (1) (except epitheaflagallin, theaflavin,
theaflavin-3-O-gallate, theaflavin-3'-O-gallate, and
theaflavin-3,3'-O-digallate): ##STR00042## (wherein R.sub.1 is H or
OH; R.sub.2 is H or a group of Formula (2): ##STR00043## wherein
R.sub.4 is OH, a group of Formula (3): ##STR00044## or a group of
Formula (4): ##STR00045## wherein R.sub.3 is H, COOH, a group of
Formula (5): ##STR00046## or a group of Formula (6): ##STR00047##
wherein R.sub.5 is OH, a group of Formula (7): ##STR00048## or a
group of Formula (8): ##STR00049## wherein R.sub.6 is a group of
Formula (9): ##STR00050## or a group of Formula (10): ##STR00051##
wherein R.sub.1' is the same group as R.sub.1 above and R.sub.2' is
a group of Formula (11): ##STR00052## wherein R.sub.4' is the same
group as R above).
2. The antiobesity agent according to claim 1, comprising one or
more compounds in which R.sub.1 is H.
3. The anti-obesity agent according to claim 1, wherein the
compound is a compound of Formula (12): ##STR00053## Formula (13):
##STR00054## Formula (14): ##STR00055## Formula (15): ##STR00056##
Formula (16): ##STR00057## Formula (17): ##STR00058## Formula (18):
##STR00059## Formula (19): ##STR00060## Formula (20): ##STR00061##
Formula (21): ##STR00062## Formula (22): ##STR00063## Formula (23):
##STR00064##
4. The anti-obesity agent according to claim 1, which is a lipase
inhibitor and/or an alfa-glucosidase inhibitor.
5. The anti-obesity agent according to claim 1, which is for
suppressing absorption of diet-derived fat and sugar.
6. The anti-obesity agent according to claim 1, which is in the
form of a food or a beverage.
7. The anti-obesity agent according to claim 6, wherein the food or
beverage is selected from the group consisting of a tea beverage, a
soft drink, and a health food.
8. The anti-obesity agent according to claim 1, which is in the
form of a pharmaceutical composition.
9. A compound of Formula (24): ##STR00065##
Description
TECHNICAL FIELD
[0001] The present invention relates to an anti-obesity agent
containing a benzotropolone ring-containing compound.
BACKGROUND ART
[0002] Obesity is one of the most significant diseases in modern
society and the main factor of obesity is excessive intake of fat.
Excessive intake of fat is known to cause not only obesity but also
diabetes, hyperlipidemia, hypertension, arteriosclerosis, and the
like that are attributable to obesity. The condition in which two
or more of hyperglycemia, hypertension, and hyperlipidemia develop
with visceral fat obesity is called metabolic syndrome (visceral
fat syndrome), which is at high risk of causing cardiac diseases
and stroke and thus has been regarded as a problem in recent years.
As a therapeutic agent for obesity, for example, Xenical.RTM.,
which has a suppressive action on fat absorption from the
gastrointestinal tract due to its lipase inhibitory activity, is
commercially available as an anti-obesity agent; however, its side
effects such as steatorrhea, increased frequency of defecation,
loose stool, diarrhea, and stomachache have been reported and the
agent thus cannot be necessarily safe (Non-Patent Document 1).
[0003] In order to prevent obesity, cutting calories on a
restrictive diet is an effective way. Nevertheless, it is often
hard to practice it in daily life because substantial nutritional
guidance has to be received. Accordingly, suppressing in a safe and
healthy manner the absorption of diet-derived fat into the body is
expected to be a realistic and effective measure for treatment of
obesity and obesity-related diseases or health enhancement.
[0004] Under these circumstances, the development of specified
health foods that are safe and proven effective in humans is
attracting attention. To date, the following food materials which
suppress the elevation of serum triglyceride level after meals are
commercially marketed as specified health foods: globin digest
which suppresses fat absorption by its pancreatic lipase
inhibition; diacylglycerol, which has digestive and absorptive
properties different from triacylglycerol; eicosapentaenoic acid
(EPA) and docosahexaenoic acid (DHA), which are purified from fish
oil; and the like.
[0005] Also, recent interest is focusing on plant-derived lipase
inhibiting active substances, and particularly, the following
polyphenols which have lipase inhibitory activity have been
reported: plant bark-derived tannin; tannins and flavonoids and
glycosides thereof in the legume Cassia nomame; lipid
absorption-inhibiting foods containing epigallocatechin gallate and
epicatechin gallate, which are main components in green tea; lipase
inhibitors comprising water extracts from pepper, shimeji mushroom,
pumpkin, maitake mushroom, seaweed Hizikia fusiformis, green tea,
oolong tea, and the like; flavones and flavonols; hydroxyberizoic
acids (gallic acid), triterpene compounds and derivatives thereof;
anti-obesity agents containing, as an active ingredient,
procyanidin from tamarind; and the like. Further known are lipase
inhibitory action of grape seed extract (Non-Patent Document 2);
lipase inhibitory action from Salacia reticulate-derived
polyphenol, and anti-obesity action in rats (Non-Patent Document
3); oolong tea extract-derived anti-obesity action in mice
(Non-Patent Document 4); and the like. In addition, teas contain a
lot of catechins, many components of which have been separated and
identified (Non-Patent Document 5), and there are reports on lipase
inhibitors containing tea-derived components (Patent Documents 1
and 2). Above, all, theaflavins, known as pigments of black tea and
oolong tea, exhibit strong lipase inhibitory activity in proportion
to the number of gallate groups in a molecule (Patent Document 2,
Non-Patent Document 6). However, the content and proportion of
these theaflavins are not constant among teas.
[0006] Alfa-glucosidase inhibiting substances have an inhibitory
action on the elevation of blood glucose level by inhibiting
alfa-glucosidase, which is localized on small intestinal
epithelium, and by suppressing or delaying the decomposition and
absorption of sugar. Accordingly, alfa-glucosidase-inhibiting
substances are useful in various diseases such as diabetes and
obesity, which are derived from the chronicity of high blood sugar
symptoms.
[0007] Since alfa-glucosidase inhibitory activity was discovered in
malt component in 1933, many alfa-glucosidase-inhibiting substances
that are derived from wheat and pulse have been discovered. In
1966, nojirimycin, which has alfa-glucosidase inhibitory activity,
was isolated from a microbial metabolite and its structure was
determined. From mulberry leaf extract, a related compound of
nojirimycin, 1-deoxynojirimycin, was obtained, which is known to
have alfa glucosidase inhibitory activity, and a method of
extraction for keeping the activity froth decreasing is disclosed
(Patent Document 3).
[0008] A compound that contains a 13-membered ring cyclitol
structure having a sulfoxide, which is isolated from the extract of
the root of Salacia reticulate, is reported to have maltase
inhibitory activity (Patent Document 4). Diacylated pelargonidin,
cyanidin, and peonidin 3-sophoroside-5-glucosides are reported to
have maltase inhibitory activity as an anthocyanin compound
isolated from morning glories or the root of purple sweet potato
(Non-Patent Document 7). The maltase inhibitory activity has also
been confirmed in the components contained in tea leaves, such as
theasinensin A, theaflavin derivatives having a galloyl group, and
proanthocyanidins having epiafzelechingallate as a constitutional
unit. Although theaflavin derivatives having a galloyl group have
maltase inhibitory activity, they are contained in tea leaves in
only a small proportion, 0.1 to 0.2% (Patent Document 5, Non-Patent
Document 8).
[0009] Black tea theaflavins and green tea catechins are reported
to have alfa-glucosidase inhibitory activity (Non-Patent Document
8); the activity has been confirmed in catechins having a galloyl
group at their 3 position including epigallocatechin-3-O-gallate
(hereinafter referred to as "EGCG") and epicatechin-3-O-gallate,
and theaflavins including theaflavin-3-O-gallate and
theaflavin-3,3' di-O-gallate. The fractions and the like of black
tea have also been examined for their alfa-glucosidase inhibitory
activity; polymeric fractions formed by fermentation are also known
to have the activity (Non-Patent Document 9).
[0010] On the other hand, it is known that in fermentation process
in production of black tea or oolong tea, polyphenols such as
catechins or gallic acid are condensed into a compound having a
benzotropolone ring through the activity of enzymes such as
polyphenol oxidase in tea leaves (Non-Patent Document 10).
[0011] It is reported that aside from theaflavins, many
benzotropolone ring-containing compounds are present in teas.
Reported are, for example, apoptosis induction caused by
purpurogallin derivatives (Patent Document 6) and a method of
manufacturing epitheaflagallins for use in foods (Patent Document
7), an enzymatic method of manufacturing a theaflavin type trimer,
theadibenzotropolone A, and presence thereof in black tea
(Non-Patent Document 11), and the like. The anti-inflammatory
actions of various benzotropolone ring-containing compounds
(Non-Patent Document 12) are also known. Nevertheless, for
benzotropolone ring-containing compounds other than theaflavins and
epitheaflagallins, nothing is known about their lipase inhibitory
action relating to fat absorption and their alfa-glucosidase
inhibitory action relating to their inhibitory action on the
elevation of blood glucose level.
CITATION LIST
Patent Document
[0012] Patent Document 1: WO2005/077384 [0013] Patent Document 2:
WO 2006/004110 [0014] Patent Document 3: Japanese Patent Public
Disclosure 2007-60908 [0015] Patent Document 4: Japanese Patent
Public Disclosure 2008-137925 [0016] Patent Document 5: Japanese
Patent Public Disclosure 2007-231009 [0017] Patent Document 6:
Japanese Patent Public Disclosure 2004-359576 [0018] Patent
Document 7: WO 2007-141945
Non-Patent Document
[0018] [0019] Non-Patent Document 1: Lancet 1998; 352: 167-172
[0020] Non-Patent Document 2: Nutrition 2003; 19(10): 876-879
[0021] Non-Patent Document 3: J. Nutr. 2002; 132: 1819-1824 [0022]
Non-Patent Document 4: Int. J. Obes. 1999; 23: 98-105 [0023]
Non-Patent Document 5: Journal of the Japanese Society for Food
Science and Technology, Vol. 46, No. 3, pp. 138-147, March 1999
[0024] Non-Patent Document 6: Chem. Pharm. Bull, 2008; 56: 266-272
[0025] Non-Patent Document 7: J. Agric. Food Chem. 2001, 49,
19524956 [0026] Non-Patent Document 8: J. Agric, Food. Chem., 55,
99-105, 2007 [0027] Non-Patent Document 9: Chem. Pharm. Bull.
56(3), 266-272, 2008 [0028] Non-Patent Document 10: Tetrahedron
1973; 29: 125-442 [0029] Non-Patent Document 11: Tetrahedron
Letters 2002; 43: 7129-7133 [0030] Non-Patent Document 12:
Bioorganic & Medicinal Chemistry 2004; 12: 459-467
SUMMARY OF THE INVENTION
Technical Problem
[0031] Even if some effect is found to be obtained from a plant
extract, unless the quantity of the active components contained in
the extract is determined, it is difficult to ensure stable
maintenance of its anti-obesity activity because the extract is of
natural product origin. Further, some of the reported lipase
inhibitors and alfa-glucosidase inhibitors as shown above are not
sufficiently effective.
[0032] A less palatable, vegetable-derived anti-obesity agent, when
used as foods or beverages, is expected to make a negative
influence on their flavor. On the other hand, a more palatable,
tea-derived anti obesity agent can possibly be an effective
material candidate; however, for example, even when we drink
palatable black tea or oolong tea in order to lower lipid, we
cannot obtain its effect without drinking the tea in large amounts,
and thus to practice it in daily life is thus not realistic. Simply
condensed black tea or oolong tea is also not appropriate to drink
as a realistic measure because of its strong bitterness and
astringency and increased caffein.
[0033] Accordingly, an object of the present invention is to
provide an anti-obesity agent which is of natural product origin
and effective.
[0034] Another object of the present invention is to provide a
tea-derived, palatable inhibitor of lipase activity which shows a
highly inhibitory activity against pancreatic lipase and suppresses
absorption of diet-derived fat, and/or contributes to suppression
and prevention of obesity. Still another object is to provide an
alfa-glucosidase inhibitor which suppresses absorption of
diet-derived sugar and contributes to long-term prevention and/or
treatment of diabetes resulting from the chronicity of high blood
sugar symptoms.
[0035] Still another object of the present invention is to provide
foods and beverages which are palatable and intended to lower blood
triglyceride and which enhance health.
[0036] Still another object of the present invention is to provide
a pharmaceutical composition which suppresses absorption of
diet-derived fat and elevation of blood triglyceride.
Solution to Problem
[0037] As a result of intensive studies to solve the above
problems, the present inventors have found a component from tea
leaves which potently inhibits pancreatic lipase, essential for fat
absorption, and which has high alfa-glucosidase inhibitory
activity. Specifically, the inventors have assayed the inhibitory
activities of various polyphenols present in tea leaves against
lipase and alfa-glucosidase, and found out that the benzotropolone
ring-containing compounds of Formula (1) have strong lipase
inhibitory activity and strong alfa-glucosidase inhibitory
activity. These compounds are oxides of catechins and polyphenols
which are contained in black tea, and thus are superior in flavor
and safety and can be taken for long periods of time. From these
findings, the inventors have found that it is possible to provide
foods and beverages, to which a lipase inhibitor and an
alfa-glucosidase inhibitor are added, which are intended to
suppress absorption of diet-derived fat and sugar and elevation of
blood triglyceride and to prevent and/or treat diabetes resulting
from the chronicity of high blood sugar symptoms is in the long
run. The present invention has been thus accomplished.
[0038] More specifically, the present invention is defined by [1]
to [9] below.
[0039] [1] An anti-obesity agent comprising one or more compounds
of Formula (1) (except epitheaflagallin,
epitheaflagallin-3-O-gallate, theaflavin, theaflavin-3-O-gallate,
theaflavin-3'-O-gallate, and theaflavin-3,3'-O-digallate):
##STR00001##
(wherein R.sub.1 is H or OH;
[0040] R.sub.2 is H or a group of Formula (2):
##STR00002##
wherein R.sub.4 is OH or a group of Formula (3):
##STR00003##
or a group of Formula (4):
##STR00004##
[0041] wherein R.sub.3 is H, COOH, a group of Formula (5):
##STR00005##
or a group of Formula (6):
##STR00006##
wherein R.sub.5 is OH, a group of Formula (7):
##STR00007##
or a group of Formula (8):
##STR00008##
wherein R.sub.1 is a group of Formula (9):
##STR00009##
or a group of Formula (10):
##STR00010##
wherein R.sub.1' is the same group as R.sub.1 above and R.sub.1' is
a group of Formula (11):
##STR00011##
wherein R.sub.4' is the same group as R.sub.4 above).
[0042] [2] The anti-obesity agent according to [1], comprising one
or more compounds in which R.sub.1 is H.
[0043] [3] The anti-obesity agent according to [1], wherein the
compound is a compound of Formula (12):
##STR00012##
[0044] Formula (13):
##STR00013##
[0045] Formula (14):
##STR00014##
[0046] Formula (15):
##STR00015##
[0047] Formula (16):
##STR00016##
[0048] Formula (17):
##STR00017##
[0049] Formula (18):
##STR00018##
[0050] Formula (19):
##STR00019##
[0051] Formula (20):
##STR00020##
[0052] Formula (21):
##STR00021##
[0053] Formula (22):
##STR00022##
[0054] or Formula (23):
##STR00023##
[0055] [4] The anti-obesity agent according to any one of [1] to
[3], which is a lipase inhibitor and/or an alfa-glucosidase
inhibitor.
[0056] [5] The anti-obesity agent according to any one of [1] to
[4], which is for suppressing absorption of diet-derived fat and
sugar.
[0057] [6] The anti-obesity agent according to any one of [1] to
[5], which is in the form of a food or a beverage.
[0058] [7] The anti-obesity agent according to [6], wherein the
food or beverage is selected from the group consisting of a tea
beverage, a soft drink, and a health food.
[0059] [8] The anti-obesity agent according to any one of [1] to
[5], which is in the form of a pharmaceutical composition.
[0060] [9] A compound of Formula (24):
##STR00024##
Advantageous Effects of the Invention
[0061] The lipase inhibitor that serves as the anti-obesity agent
of the present invention contains a tea-derived benzotropolone
ring-containing compound and thus exhibits superior lipase
inhibitory activity. The lipase inhibitor of the present invention
does not compromise the flavor of foods and beverages, has
palatability, and can be used in various use applications including
foods and beverages intended for reduction in triglycerides and
health enhancement. It is desirable to take this inhibitor together
with meals for suppression of dietary fat absorption, and
therefore, beverages containing tea-derived, enhanced active
ingredients are of great significance. Particularly, enhancing
these ingredients enabled us to provide a beverage intended for
anti-obesity action and health enhancement.
[0062] The alfa-glucosidase inhibitor that serves as the
anti-obesity agent of the present invention can suppress
decomposition of sugar derived from diet-derived starch and
polysaccharides and absorption of the sugar, in less amount than
conventionally known, natural product-derived alfa-glucosidase
inhibitor. Further, all of the compounds are oxides of catechins
and polyphenols contained in teas and are thus superior in flavor
and safety, which enables long-term intake of the compounds.
[0063] In addition, the anti-obesity agent of the present invention
contains a benzotropolone ring-containing compound, a component
derived from teas, which are widely used in diet, and are thus safe
and can be also used as a pharmaceutical composition with reduced
side effects.
BRIEF DESCRIPTION OF THE DRAWINGS
[0064] FIG. 1 shows the .sup.13C NMR spectrum of Compound 12.
[0065] FIG. 2 shows the .sup.1H NMR spectrum of Compound 12,
DESCRIPTION OF EMBODIMENTS
[0066] The embodiments of the present invention are described in
detail below,
[0067] Anti-Obesity Agents
[0068] The present invention is an anti-obesity agent containing a
benzotropolone ring-containing compound of Formula (1) as an active
ingredient:
##STR00025##
Particularly, the following is preferred as the active ingredient:
purprogallin of Formula (12) below:
##STR00026##
[0069] purprogallin carboxylic acid of Formula (13):
##STR00027##
[0070] theaflavanin 3-O-gallate of Formula (14):
##STR00028##
[0071] EGCG-catechol of Formula (15):
##STR00029##
[0072] theaflavate A of Formula 16
##STR00030##
[0073] theadiberrzotropolone A of Formula (17):
##STR00031##
[0074] theaflavin digallate trimer 1(TFdiGA-tri1) of Formula
(18):
##STR00032##
[0075] theaflavin digallate trimer 2 (TFdiGA-tri2) of Formula
(19):
##STR00033##
[0076] epitheaflavic acid of Formula (20):
##STR00034##
[0077]
3,4,6-trihydroxy-1-((2R,3R)-3,5,7-trihydroxychroman-2-yl)-5H-benzo[-
7]annulen-5-one of Formula (21):
##STR00035##
[0078] epitheaflavic acid-3-O-gallate of Formula (22):
##STR00036##
[0079] or
(2R,3R)-2-(3,4-dihydroxyphenyl)-5,7-dihydroxychroman-3-yl2,3,4,6-
-tetrahydroxy-5-oxo-5H-benzo[7]annulen-8-carboxylate of Formula
(23):
##STR00037##
[0080] The above benzotropolone ring-containing compounds, which
are active ingredients of the anti-obesity agent of the present
invention, can be obtained by solvent extraction from natural
materials such as green tea, black tea, and oolong tea or also by
chemical synthesis or enzymatic synthesis of the materials.
[0081] With respect to the natural materials which are extraction
raw materials, tea leaves may be used as is or in ground form. The
solvent for use in extraction may be water, an organic solvent,
mixtures of these solvents, or the like, and preferably, hot water.
The extract obtained can be separated and purified by using an
adequate carrier for separation. Any carrier can be used if the
carrier can absorb the above benzotropolone ring-containing
compound and separate it with an adequate solvent for separation.
For example, styrene- or dextran-based synthetic adsorbents can be
used for the separation and purification. After loading of the
above extract on such a carrier, an adequate solvent is used to
separate the above benzotropolone ring-containing compound. More
specifically, the above benzotropolone ring-containing compound for
use in the anti-obesity agent of the present invention can be
obtained in accordance with the descriptions in Example 1 of the
present specification. The thus obtained benzotropolone
ring-containing compound above may be used in condensed form or as
a powder obtained by methods such as lyophilization.
[0082] The above benzotropolone ring-containing compounds can be
synthesized with enzymes such as polyphenol oxidase (PPO) and
peroxidase (POD), or with oxidants such as potassium ferricyanide
(K.sub.3[Fe(CN).sub.6]) as a catalyst by using, as raw materials,
catechins (epigallocatechin-3-O-gallate epicatechin-3-O-gallate
epicatechin epigallocatechin, and the like) and gallic acid.
Specifically, the synthesis can be performed in accordance with the
descriptions in, for example, Examples 2 and 7 of the present
specification.
[0083] The anti-obesity agent of the present invention has strong
inhibitory action against lipase, especially, pancreatic lipase.
The lipase inhibitory activity can be measured by any method of the
lipase activity assays described in the related applications shown
in the Background Art section. For example, the assay can be
achieved by using the oleate ester of fluorescent
4-methylumbelliferone as a substrate to measure the fluorescence of
4-methylumbelliferone produced by reaction with lipase. Exemplary
is the method described in Example 6, which allows a measurement of
lipase inhibitory activity. Lipase inhibitory activity can be
expressed, for example, as IC.sub.50, a sample volume which
produces 50% inhibition.
[0084] The anti-obesity agent of the present invention have potent
alfa glucosidase inhibitory activity. The activity can be measured
by any method of the activity assays described in the related
applications shown in the Background Art section. Exemplary is the
method described in Example 11, which allows a measurement of
alfa-glucosidase inhibitory activity. Alfa-glucosidase inhibitory
activity can be expressed, for example, as IC.sub.50, a sample
volume which produces 50% inhibition.
[0085] The purified products or partially purified products of the
above benzotropolone ring-containing compounds can be used alone as
an anti-obesity agent, or can be combined with a solvent or a
carrier so as to be used as an anti-obesity agent. It is preferred
that the solvent or the carrier can be used safely as a food or a
pharmaceutical product, in prospect of use as the foods and
beverages below and/or pharmaceutical products. The anti-obesity
agent of the present invention has various use applications;
exemplary is use of the agent as foods and beverages or
pharmaceutical compositions which are used for an experimental
study and intended for prevention of triglyceride accumulation.
[0086] Foods and Beverages
[0087] The anti-obesity agent of the present invention may be in
the form of food and beverage that suppresses undesirable elevation
of blood triglyceride associated with fat intake from meals.
Preferred examples of the food and beverage include those taken on
a daily basis; for example, green tea, barley tea, oolong tea,
black tea, coffee, sports drinks, drinking water, seasoning, and
dressing. However, the foods and beverages also may be those that
are commonly consumed: soft drinks, cocktails, beer, whiskey,
distilled spirit (shochu), wine, sake, seasoning, dressing,
flavored rice, processed foods, instant foods, retort foods,
chocolate, fresh cream, Western confectionery, dairy products,
health foods, supplements, and the like.
[0088] In the anti-obesity agent of the present invention which is
in the form of food and beverage, the benzotropolone
ring-containing compound of Formula (1) is contained so that the
intake of the compound is 0.1 mg to 10 g per meal, preferably, 0.5
mg to 5 g. However, the compound of Formula (1) used in the
anti-obesity agent of the present invention is highly safe because
it is derived from food, and thus there is no substantial upper
limit on the amount of addition to foods and beverages.
[0089] Pharmaceutical Composition
[0090] The anti-obesity agent of the present invention which serves
as a lipase inhibitor can also be used as a pharmaceutical
composition intended to suppress diet-derived fat absorption and to
prevent undesirable increase of blood triglyceride and/or to lower
increased blood triglyceride. The anti-obesity agent which serves
as an alfa-glucosidase inhibitor can also be used as a
pharmaceutical composition intended to suppress diet-derived fat
absorption and to prevent undesirable increase of blood
triglyceride and/or to lower increased blood triglyceride.
Preferred are agents intended for oral administration; examples of
the agent include drinks, tablets, capsules, granules, powders,
candies, drops, and the like. The agents contain a benzotropolone
ring-containing compound of Formula (1) in amounts of 0.1 mg to 10
g per dose, preferably, 0.5 mg to 5 g.
[0091] The pharmaceutical composition of the present invention is
safe for long-term dosage due to high safety of lipase and
alfa-glucosidase activity of the inhibitory components.
Accordingly, the composition can also be taken on a daily basis to
prevent or resolve obesity as a lifestyle-related disease.
[0092] Novel Compound
[0093] Further, the present invention provides a compound shown
below as a novel compound present in black tea.
##STR00038##
[0094] This compound is useful as an anti-obesity material.
[0095] The present invention is more specifically described in
Examples below, but is not limited thereto.
Example 1
Separation on LH-20
[0096] Black tea leaves originated in Assam, India were extracted
with hot water at 90.degree. C. and lyophilized, and distilled
water was added to the resulting product followed by warming and
dissolution to obtain a solution of concentration 10 mg/ml. While
heating the solution to 60 to 70.degree. C. in a hot-water bath, 15
ml of the same was loaded on 60 ml of Sephadex LH-20 (GE Healthcare
Biosciences, Ltd.) After washing with 45 ml of 20% acetone
(acetone:distilled water=2:8, v/v) and subsequent washing with 60
ml (1 column volume) of the same, elution was conducted with 4
column volumes each of the following solvents; 30% acetone
(acetone:distilled water=3:7, v/v); 50% acetone (acetone:distilled
water=1:1, v/v); and 60% acetone (acetone:distilled water=6:4,
v/v), and the resulting solution was fractionated into 1 column
volume for each fraction. Two milliliters each taken from a total
of fourteen 60 ml fractions was concentrated under vacuum and then
0.1 ml of a 50% aqueous dimethylsulfoxide solution
(dimethylsulfoxide:distilled water=1:1, v/v) was prepared, and the
solution was subjected to a measurement of lipase inhibitory
activity. In addition, each fraction was concentrated under vacuum
and lyophilized, and the recovery volume was calculated from the
resulting solids; the calculations were used for the component
analysis conducted in Example 5. The weights and the recovery rates
of lipase inhibitory activity are shown in Table 1.
TABLE-US-00001 TABLE 1 Location of Activity in LH20 Fractions
Abundance Abundance Ratio of Ratio of Fra No. Fraction Activity (%)
Yield (%) 1 20% acetone_1 0.0005 5.0 30.6 54.3 2 20% acetone_2 5.01
23.7 3 30% acetone_1 4.72 20.1 3.7 16.3 4 30% acetone_2 6.30 4.7 5
30% acetone_3 5.63 5.1 6 30% acetone_4 3.48 2.8 7 50% acetone_1
14.12 70.9 4.8 23.6 8 50% acetone_2 33.59 11.2 9 50% acetone_3
17.08 5.1 10 50% acetone_4 6.15 2.5 11 60% acetone_1 2.53 3.9 1.3
5.8 12 60% acetone_2 1.05 1.8 13 60% acetone_3 0.20 1.2 14 60%
acetone_4 0.13 1.5
[0097] As a result of the measurement of lipase inhibitory activity
in accordance with the method described in Example 6, 20%
acetone.sub.--1 and 20% acetone.sub.--2 fractions, which exhibited
high yield, were each found to have contained polysaccharides and
caffeins and have had almost no activity. On the other hand, in 50%
acetone.sub.--2 fraction, which exhibited the next highest yield
(11.2% by weight), activity of 316% was eluted; in the subsequent
entry, 50% acetone.sub.--3 fraction, activity of 17.1% was eluted.
In the 50% acetone elution portions including these fractions, it
was found that 70.9% of the activity was eluted. In the assay
described in Example 5, many of active benzotropolone
ring-containing compounds were eluted with 50% acetone, which is
well consistent with the high activity found in 50% acetone elution
portions.
Example 2
Syntheses of Test Compounds
[0098] 1. purprogallin 2. purprogallin carboxylic acid 3.
epitheaflagallin 4. epitheaflagallin-3-O-gallate 5. theaflavate A
6. theadibenzotropolone A 7. theaflavin digallate trimer 1
(TFdiGA-tri1) 8. theaflavin digallate trimer 2 (TFdiGA-tri2) 9.
epitheaflavic acid 10. theaflavanin 11. epitheaflavic
acid-3-O-gallate 12.
(2R,3R)-2-(3,4-dihydroxyphenyl)-5,7-dihydroxychroman-3-yl2,3,4,6-tetrahyd-
roxy-5-oxo-5H-benzo[7]annulen-8-carboxylate
Syntheses of epitheaflagallin (3) and epitheaflagallin-3-O-gallate
(4)
[0099] To epigallocatechin (0.2 mmol) (EGC; Wako Pure Chemical
Industries, Ltd.) or epigallocatechin-3-O-gallate (0.2 mmol) (EGCG;
Wako Pure Chemical Industries, Ltd.), potassium ferricyanide (0.8
mmol) and NaHCO.sub.3 (0.8 mmol) were added to prepare 150 ml of an
aqueous solution, and the solution was chilled on ice. Fifty
milliliters of an aqueous pyrogallol solution (0.2 mmol) was
dripped into the solution and stirring the mixture were continued
for one hour. The reaction solution was loaded on 20 ml volume of
Sep-pak C18 (Waters Corp.), and after washing with 60 ml of water
and the subsequent washing with 5% acetonitrile/water, 50%
acetonitrile/water containing 0.1% formic acid was used to elute a
reaction product. The eluted product was lyophilized and purified
by preparative HPLC shown below.
[0100] The reaction product of EGC and pyrogallol was loaded on YMC
Pak Polymer C-18 (20.times.300 mm, YMC Co., Ltd.) and an elution
with a linear gradient of 30-50% acetonitrile (6 ml/min, 60
minutes) was performed in the presence of 0.1% formic acid. The
component eluted at between 48 and 50 minutes was lyophilized to
obtain 4 mg of a brown solid (epitheaflagallin (3)). Also, the
component eluted at between 68 and 70 minutes in this chromatogram
was lyophilized to obtain 2 mg of a brown solid (purpurogallin
(1)).
[0101] The reaction product of EGCG and pyrogallol was loaded on
Develosil C30-UG-5 (20 nm.times.250 nm, Nomura Chemical Co., Ltd.)
and an elution with a linear gradient of 15-50% acetonitrile (6
ml/min, 60 minutes) was performed in the presence of 0.1% formic
acid, The component eluted at between 44 and 46 minutes was
lyophilized to obtain 6 mg of a brown solid
(epitheaflagallin-3-O-gallate (4)). Also, the component eluted at
between 48 and 50 minutes in this chromatogram was lyophilized to
obtain 3 mg of a brown solid (purpurogallin (1)).
[0102] In the same manner as the above reactions, 800 mg of gallic
acid was subjected to a reaction to obtain 65 mg of purprogallin
carboxylic acid (2), which is produced by polymerization of two
gale acid molecules. In addition, 100 mg of epicatechin-3-O-gallate
(ECG) was subjected to a reaction to obtain 3 mg of theaflavate A
(5), which is produced by polymerization of two ECG molecules.
Further, 400 mg each of epicatechin (EC) and EGCG were subjected to
a reaction to obtain a main product, theaflavin-3-O-gallate as well
as 2 mg of a by-product, theadibenzotropolone A (6). In the same
manner as the above reaction, 200 mg each of
epicatechin-3-O-gallate (ECG) and EGCG were subjected to a
reaction, and 12 mg of the resulting theaflavin-3,3'-digallate and
20 mg of ECG were subjected to a reaction to obtain 1.7 mg of
TFdiGA-tri1 (7) and 0.6 mg of TFdiGA-tri2 (8). The following
compounds were also obtained: 48 mg of epitheaflavic acid (9)
obtained by reaction of 330 mg of gallic acid and 101 mg of EC; 12
mg of theaflavanin (10) obtained by reaction of 869 mg of
pyrogallol and 400 mg of EC; and 37 mg of epitheaflavic
acid-3-O-gallate (11) and 3.3 mg of
(2R,3R)-2-(3,4-dihydroxyphenyl)-5,7-dihydroxychroman-3-yl2,3,4,6-tetrahyd-
roxy-5-oxo-5H-benzo[7]annulen-8-carboxylate (12) which were
simultaneously obtained by reaction of 470 mg of gallic acid and
221 mg of ECG.
Example 3
Structural Analyses of Compounds
[0103] MS and NMR measurements of the compounds obtained in Example
2 were performed. Their mass spectra were determined with Q-TOF
Premier (Micromass Co., Ltd., UK) using Z-Spray ESI ion source, in
negative, V mode. Cone voltage: 45V, Capillary voltage: 3 KV,
Source Temp.: 80.degree. C., Desolvation Temp.: 180.degree. C. Mass
correction was performed with LockSpray, and leucine enkepharine
(m/z 554.2615 [M-H].sup.-) was used as a reference.
[0104] The accurate mass, molecular formula, and theoretical mass
value of each compound are shown in Table 2.
TABLE-US-00002 TABLE 2 Mass Spectrometric Results of Compounds m/z
Com- Actual ma/z pound Meas. Molecular Calc. No. Compound Value
Formula Value 1 Purprogallin 219.0285 C11H8O5 219.0293 2
purprogallin carboxylic 263.0183 C12H8O7 263.0192 acid 3
epitheaflagallin 399.0701 C20H16O9 399.0716 4 epitheaflagallin-3-O-
551.0818 C27H20O13 551.0826 gallate 5 theaflavateA 851.1471
C43H32O19 851.1460 6 theadibenzotropoloneA 973.1832 C50H38O21
973.1827 7 TFdiGA-tri1 1279.2185 C64H46O29 1279.2203 8 TFdiGA-tri2
1279.2218 C64H46O29 1279.2203 9 epitheaflavic acid 427.0666
C21H16O10 427.0665 10 theaflavanin 383.0764 C20H16O8 383.0767 11
epitheaflavic acid-3-O- 579.0778 C28H20O14 579.0775 gallate 12
(2R,3R)-2-(3,4- 535.0883 C27H20O12 535.0877 dihydroxyphenyl)-
5,7-dihydroxy- chroman-3-yl 2,3,4,6- tetrahydroxy-5-oxo-5H-
benzo[7]annulene- 8-carboxylate 14 theaflavanin-3-O-gallate
535.0871 C27H20O12 535.0877 15 EGCG-catechol 535.0885 C27H20O12
535.0877
Example 4
Quantification in Tea Leaf Extract
[0105] The LC-MS/MS measurements were performed with 4000 Q TRAP
(Applied Biosystems Inc.) using TurboIonSpray in negative mode
under the following conditions: Collision energy: 46 eV (nega.);
Ionspray voltage: 4500V; Temperature: 450.degree. C.
[0106] The elution time and measurement channels of each compound
in MRM (multiple reaction monitoring) mode are as shown in Table
3.
TABLE-US-00003 TABLE 3 LC-MSMS Quantitative Parameters Compound
Compound R.T. Q1/Q3 1 purprogallin 10.99 219.0/191.0 2 purprogallin
carboxylic acid 8.01 263.0/190.9 3 epitheaflagallin 8.64
399.1/233.0 4 epitheaflagallin-3-O-gallate 11.54 551.1/233.1 5
theaflavateA 14.46 851.2/579.1 6 theadibenzotropoloneA 11.49
973.2/227.1 7 TFdiGA-tri1 15.01 1277.2/579.1 8 TFdiGA-tri2 17.37
1277.2/697.1
[0107] Column: YMC-Polymer C18, S-6 .mu.m (YMC Co., Ltd., 2
mm.phi..times.150 mm)
[0108] Flow Rate: 0.2 mL/min
[0109] Column Temp.: 40.degree. C.
[0110] Mobile Phase A; 0.1 V/V % HCOOH/H.sub.2O
[0111] Mobile Phase B: 0.1 V/V % HCOOH/CH.sub.3CN
[0112] Gradient Program: A/B=91/9 (0 min)--->A/B=40/60 (17
min)--->A/B=15/85 (17.1 min)--->A/B=15/85 (17.1 to 19
min)
[0113] The above conditions were used to assay a hot water extract
of Indian Assam CTC tea. Compounds 2 to 8 were found to have been
present in the tea leaves. The quantitative values are shown in
Table 4.
TABLE-US-00004 TABLE 4 Quantitative Values of Black Tea Components
Compound Compoun .mu.g/g extract 1 purprogallin 0.15 2 purprogallin
carboxylic acid 166.00 3 epitheaflagallin 224.00 4
epitheaflagallin-3-O-gallate 369.0 5 theaflavateA 165.5 6
theadibenzotropoloneA 35.95 7 TFdIGA-trI1 67.20 8 TFdIGA-trI2
61.55
Example 5
Distribution of Each Compound in Tea Leaf Fraction
[0114] LC-MS/MS measurements were performed with Q-TOF Premier
(Micromass Co., Ltd., UK) using Z-Spray ESI ion source, in
negative, V mode. Cone voltage: 33V Capillary voltage: 3 KV, Source
Temp.: 150.degree. C., Desolvation Temp.: 250.degree. C., Mass
correction was performed with LockSpray, and leucine enkepharine
(m/z 554.2615 [M-H].sup.-) was used as a reference.
[0115] Column: YMC-Polymer C18, S-6 .mu.m (YMC Co., Ltd., 2
mm.phi..times.150 mm)
[0116] Flow Rate: 0.2 mL/min
[0117] Column Temp.: 40.degree. C.
[0118] Mobile Phase A: 0.1V/V % HCOOH/H.sub.2O
[0119] Mobile Phase B: 0.1V/V % HCOOH/CH.sub.3CN
[0120] Gradient Program: A/B=70/30--->A/B=50150 (20
min)--->A/B=50/50 (25 min)
[0121] The above conditions were used to analyze each of the
fractions of tea leaves originated in Assam, India, which were
obtained by fractionation in Example 1. Compound 2 (m/z 263.02,
R.T.=9.29 min) was detected in 30% acetone.sub.--1 fraction,
Compound 3 (m/z 399.07, R.T.=9.83 min) was in 50% acetone.sub.--2
and 50% acetone.sub.--3 fractions, Compound 4 (m/z 551.08,
R.T.=13.48 min) was in 50% acetone.sub.--3 and 50% acetone.sub.--4
fractions, Compound 5 (m/z 851.15, R.T.=17.50 min) was in 50%
acetone.sub.--4 fraction, Compound 9 (m/z 427.07, R.T.=8.60 min)
was in 50% acetone.sub.--3 fraction, Compound 10 (m/z 383.08,
R.T.=9.65 min) was in 30% acetone 3 and 30% acetone 4 fractions,
Compound 11 (m/z 579.08, R.T.=13.86 min) was in 50% acetone.sub.--2
fraction, and Compound 12 (ink 535.09, R.T.=13.90 min) was in 50%
acetone.sub.--2 fraction.
Example 6
Measurement of Lipase Inhibitory Activity
[0122] Measurement samples were synthesized and purified in
accordance with the method in Example 2.
1. purprogallin 2. purprogallin carboxylic acid 5. theaflavate A 6.
theadibenzotropolone A 7. theaflavin digallate trimer 1
(TFdiGA-tri1) 8. theaflavin digallate trimer 2 (TFdiGA-trig) 9.
epitheaflavic acid 10. theaflavanin 11. epitheaflavic
acid-3-O-gallate 12.
(2R,3R)-2-(3,4-dihydroxyphenyl)-5,7-dihydroxychroman-3-yl2,3,4,6-tetrahyd-
roxy-5-oxo-5H-benzo[7]annulen-8-carboxylate
13. EGCG (Wako Pure Chemical Industries, Ltd.)
[0123] Method of Measurement
[0124] The measurement of lipase activity was performed by using as
a substrate the oleate ester of fluorescent 4-methylumbelliferone
(4-MUO; Sigma-Aldrich Corp) to measure the fluorescence of
4-methylumbelliferone produced by a reaction. In the measurement,
13 mM Tris-HCl (pH8.0) containing 1.50 mM NaCl and 1.36 mM
CaCl.sub.2 was used as a buffer. The following were subjected to an
enzymatic measurement: the substrate 4-MUO which was dissolved into
a 0.1M DMSO solution followed by dilution of the solution 4000-fold
with the above buffer; and as a lipase, porcine pancreatic lipase
(Sigma-Aldrich Corp.) which was prepared as a 400 U/ml solution by
using the above buffer likewise,
[0125] An enzymatic reaction was initiated by the following steps
under 25.degree. C. condition: adding to a 96-well microplate 50
.mu.l of a 4-MUO buffer solution and 25 .mu.l of distilled water
(or an aqueous sample solution) for each well; mixing them; and
then adding 25 .mu.l of a lipase buffer solution to each well.
After a 30-minute reaction, 100 .mu.l of a 0.1M citric acid buffer
(pH4.2) was added to terminate the reaction, and the fluorescence
of 4 methylumbelliferone (excitation wavelength: 355 nm,
fluorescence wavelength: 460 nm) produced by the reaction was
measured with a fluorescence plate reader (Fluoroskan Asent CF from
Labsystems, Inc.)
[0126] The inhibitory activity of test samples were determined as
IC.sub.50 (.mu.M), a sample volume which produces 50% inhibition,
relative to the activity of a control (distilled water).
[0127] Results
[0128] The lipase inhibitory activity of Compounds 1, 2, and 5 to
14 is shown in Table 5.
TABLE-US-00005 TABLE 5 Lipase Inhibitory Activity per mole Compound
IC50 No. Compound .mu.M -- pyrogallol 17.273 1 purprogallin 0.413 2
purprogallin carboxylic acid 4.165 5 theaflavateA 0.202 6
theadibenzotropoloneA 0.178 7 TFdiGA-tri1 0.135 8 TFdiGA-tri2 0.138
9 epitheaflavic acid 4.626 10 theaflavanin 0.984 11 epitheaflavic
acid 3-O-gallate 0.266 12 (2R,3R)-2-(3,4-dihydroxyphenyl)- 0.207
5,7-dihydroxychroman-3-yl 2,3,4,6-tetrahydroxy-5-oxo-5H-
benzo[7]annulene-8-carboxylate 13 EGCG 0.349
[0129] All of the benzotropolone ring-containing compounds
exhibited lipase inhibitory activity. In
(-)-epigallocatechin-3-O-gallate (EGCG; Compound 13), which is
known to exhibit strong lipase inhibition, its IC.sub.50 was 0.349
.mu.M. The compounds other than purprogallin carboxylic acid (2)
and epitheaflavic acid (9) which have a free carboxylic acid
residue exhibited activity equivalent to or stronger than EGCG,
which showed that their benzotropolone ring contributes to their
lipase inhibitory activity. A carboxylic acid residue was found to
serve to reduce the activity. Accordingly, in Formula (1), R.sub.3
is preferably a group other than COOH.
Example 7
(1) Preparations of Compounds
Syntheses of Compounds 12 and 14 (theaflvanin 3-O-gallate) with
Peroxidase
[0130] Used were horseradish peroxidase from Zymed Laboratories,
Inc. as a peroxidase, epicatechin 3-O-gallate (ECG) of 90% purity
or higher, which was purified by reversed-phase HPLC from tea
extract, and pyrogallol from Nacalai Tesque, Inc. (99.0%
purity).
[0131] (2) Reactions
[0132] In 10 ml of a 0.058 M acetic acid buffer, 4.3 mg of the
horseradish peroxidase was dissolved, and to this solution, 250 mg
of ECG (0.566 mmol) dissolved in 500 .mu.l of acetone and 192.8 mg
of pyrogallol (1.53 mmol) dissolved in 500 .mu.l of acetone were
added followed by stirring. Under 30.degree. C. condition, 450
.mu.l of a 3% (w/v) hydrogen peroxide solution was added to
initiate a reaction. For improvement of reaction efficiency, 450
.mu.l of a 3% (w/v) hydrogen peroxide solution was added twice,
i.e., after 10 and 20 minutes of the reaction initiation. Added
were 192.8 mg of pyrogallol (1.53 mmol) and 450 .mu.l of a 3%
hydrogen peroxide solution after 30 minutes of the reaction
initiation, and then reacted for another 30 minutes.
[0133] After 60 minutes of the reaction initiation, the reaction
solution was loaded on a reversed-phase stationary phase (Waters
Corp., Sep-Pak, C18-Vac 20 cc (5 g)) followed by washing with 40 ml
of distilled water. Consecutive elutions were then performed with
20 ml of a 20% (v/v) aqueous acetonitrile solution and then with 40
ml of a 70% (v/v) aqueous acetonitrile solution. The 70%
acetonitrile eluate was concentrated and lyophilized to obtain 68.0
mg of a fraction containing Compounds 12 and 14 (theaflavanin
3-O-gallate).
[0134] The mixture containing Compounds 12 and 14 (theaflavanin
3-O-gallate) was purified by HPLC under the conditions below.
[0135] The mixture was loaded on YMC-Pak Polymer C48 (20.times.300
mm, YMC Co., Ltd.), and in the presence of 0.1% formic acid, a 30
minute isocratic elution with 30% acetonitrile and then an elution
with a linear gradient of 30-45% acetonitrile (6 ml/min, 150
minutes) were performed. The component eluted at between 144 and
148 minutes and that eluted at between 158 and 162 minutes were
lyophilized to obtain 3.9 mg of the compound identical to Compound
1'' shown in Example 2 and 3.0 mg of Compound 14 (theaflavanin
3-O-gallate). Further, the component eluted at between 108 and 113
minutes in this chromatogram was lyophilized to obtain 36 mg of a
brown solid (Compound 1 in Example 2: purprogallin).
Example 8
Instrumental and Structural Analyses of Reaction Products
[0136] Compounds 12 and 14, which were obtained in Example 7, were
subjected to MS and NMR measurements.
[0137] Their mass spectra were determined with Q-TOF Premier
(Micromass Co., Ltd., UK) using Z-Spray ESI ion source, in
negative, V mode. Cone volt.: 33 V, Capillary voltage: 2.7 kV,
Source Temp.: 80.degree. C., Desolvation Temp.: 180.degree. C. Mass
correction was performed with LockSpray, and leucine enkepharine
(m/z 554.2615 [M-H].sup.-) was used as a reference. The collision
energy was set at 4 eV at the time of MS measurement and at 22 eV
at the time of MS/MS measurement.
[0138] Compounds 12 and 14 produced molecular ions of m/z 535.0883
and 535.0871 [M-H]--, respectively, and their molecular formula was
determined as C.sub.27H.sub.20O.sub.12 (theoretical value:
535.0877). As a result of the MS/MS measurement in which collision
energy was set at 22 eV, fragment ions of 263.07 and 219.07 were
detected in Compound 12 and those of 383.08 and 169.01 were in
Compound 14, Compound 14 was found to be a known compound,
theaflavanin 3-O-gallate.
[0139] An NMR measurement was performed to confirm the structure of
Compound 12. The .sup.13C NMR and .sup.1H NMR spectra of Compound
12 are shown in FIGS. 1 and 2, respectively. The NMR measurement
was performed under the conditions below. In CD.sub.3OH, 3 mg of
Compound 12 obtained in Example 7 was dissolved, and the residual
peaks of protons and .sup.13C of CD.sub.3OH, .delta.3.30 and
.delta.48.97, were set as internal standards. Measurements in
accordance with the following methods were performed with a DMX-750
spectrometer (BRUKER BIOSPIN, Germany): .sup.1H NMR, .sup.13C NMR,
.sup.1H{.sup.13C}-HSQC, .sup.1H{.sup.13C}-HMBC, TOCSY, DQF-COSY
NOES and ROESY. As a result, Compound 12, which was obtained in
Example 7, was confirmed to have the structure shown below.
##STR00039##
[0140] The numbering of each atom is shown in the above structural
formula.
[0141] The NMR measurement results and the signal assignments are
shown below.
TABLE-US-00006 TABLE 6 .sup.1H .sup.13C .delta. J (Hz) .delta.
A(C)-ring C-2 5.09 brs 78.23 C-3 5.58 dd 71.75 C-4 2.94 dd 17.4,
1.9 26.48 A-5 3.04 dd 17.4, 4.5 157.07 A-6 6.02 d 2 96.75 A-7 OH
157.91 A-8 5.97 d 2 95.8 A-8a 158.07 C-4a 99.07 B-ring B-1 131.25
B-2 6.95 d 1.4 114.76 B-3 OH 146.25 B-4 OH 146.11 B-5 6.72 d 8
116.04 B-6 6.83 dd 8, 1.4 118.87 benzotropolon a C.dbd.O 184.35 b
OH 154.63 c 7.51 d 1 114.45 d 125.12 e 8.03 brs 139.62 f 6.91 s
114.76 g OH 152.62 h OH 138.44 i OH 153.73 j 116.29 k 131.96
[0142] The structure of Compound 14 is also shown below.
##STR00040##
Example 9
Synthesis of Compound 15
[0143] To 229.2 mg of epigallocatechin-3-O-gallate (0.5 mmol)
(EGCG; Wako Pure Chemical Industries, Ltd.), 3.293 g of potassium
ferricyanide (10 mmol) (Nacalai Tesque, Inc.) and 0.84 g of
NaHCO.sub.3 (10 mmol) were added to prepare 400 ml of an aqueous
solution, and the solution was chilled on ice. Into the solution,
100 ml of an aqueous solution of 275.3 mg of catechol (2.5 mmol)
was dripped over one hour, and the mixture was kept stirred. The
reaction solution was loaded on 300 mL of Sephadex LH-20 (GE
Healthcare Biosciences, Ltd.) and elutions were performed with 1 L
of 40% acetone/water, 1.2 L of 45% acetone/water, and 900 mL of 50%
acetone/water, consecutively, followed by lyophilization.
Consequently, 77 mg of a 45% fraction containing EGCG-catechol was
obtained, The fraction was purified by preparative HPLC shown
below.
[0144] The fraction containing EGCG-catechol was loaded on YMC-Pak
Polymer C-18 (20.times.300 mm, YMC Co., Ltd.), and at a flow rate
of 6 ml/rain in the presence of 0.1% formic acid, an elution with a
linear gradient of 25-45% acetonitrile (75 minutes) was performed,
and then a 30-minute isocratic elution was maintained with 45%
acetonitrile. The component eluted at between 92 and 95 minutes was
lyophilized to obtain 24 mg of a brown solid (Compound 15:
EGCG-catechol). The structure of Compound 15 is shown below.
##STR00041##
Example 10
Measurement of Lipase Activity
[0145] Method of Measurement
[0146] The measurement of lipase activity was performed by using as
a substrate the oleate ester of fluorescent 4-methylumbelliferone
(4-MUO; Sigma-Aldrich. Corp.) to measure the fluorescence of
4-methylumbelliferone produced by a reaction. In the measurement,
13 mM Tris-HCl (pH8.0) containing 150 mM NaCl and 136 mM CaCl.sub.2
was used as a buffer. The following were subjected to an enzymatic
measurement: the substrate 4-MUO which was dissolved into a 0.01M
DMSO solution followed by dilution of the solution 667-fold with
the above buffer; and as a lipase, porcine pancreatic lipase
(Sigma-Aldrich Corp.: Type VI-S) which was prepared as a 400 U/ml
solution by using the above buffer likewise.
[0147] An enzymatic reaction was initiated by the following steps
under 25.degree. C. condition: adding to a 96-well microplate 50 of
4-MUO buffer solution and 25 .mu.l of distilled water (or aqueous
sample solution) for each well; mixing them; and then adding 25
.mu.l of to lipase buffer solution to each well. This measurement
was designed to increase the solubility of the substrate to enable
inhibitory activity to be measured more accurately, because the
concentration of the substrate was 7.5 .mu.M at the time of the
reaction, which showed its dilution as compared with the
concentration 12.5 .mu.M achieved in Example 6 and the DMSO
concentration was increased. After a 30-minute reaction, 100 .mu.l
of 0.1M citric acid buffer (pH4.2) was added to terminate the
reaction, and the fluorescence of 4-methylumbelliferone (excitation
wavelength: 355 nm, fluorescence wavelength: 460 nm) produced by
the reaction was measured with a fluorescence plate reader
(Fluoroskan Asent CF from Labsystems, Inc.).
[0148] The inhibitory activities of test samples were determined as
IC.sub.50 (M), a sample volume which produces 50% inhibition,
relative to the activity of a control (distilled water).
[0149] The lipase inhibitory activity of Compounds 3, 4, 12, 13,
14, and 15 was measured. The results are shown in Table 7.
TABLE-US-00007 TABLE 7 Lipase Inhibitory Activity per Mole IC50
Compound Compound .mu.M 3 epitheaflagalin 0.956 4 epitheaflagalin
3-O-gallate 0.125 12 (2R,3R)-2-(3,4-dihydroxyphenyl)- 0.096
5,7-dihydroxychroman-3-yl 2,3,4,6-tetrahydroxy-5-oxo-5H-
benzo[7]annulene-8-carboxylate 13 EGCG 0.349 14 theaflavanin
3-O-gallate 0.168 15 EGCG-catechol 0.104
[0150] Compounds 12, 14, and 15 all had stronger activity than a
positive control, EGCG, and exhibited activity equivalent to or
stronger than Compound 4 (Japanese Patent Public Disclosure
2009-114079), which is known as a lipase inhibitor.
[0151] The structures of Compounds 14 and 15 are shown as
antioxidants and anti-inflammatory pharmaceutical agents in
Japanese Patent Public Disclosure 2007-504168, but their lipase and
alfa-glucosidase inhibitions were not known.
[0152] Compounds 12 and 14 can be synthesized by using polyphenol
oxidase (PPO) or oxidants such as potassium ferricyanide besides
the enzyme shown in Examples. Also, the compounds can be
synthesized not only by a combination of ECO and pyrogallol but
also by reaction with gallic acid.
Example 11
Measurement of Alfa-glucosidase Inhibitory Activity of Various
Benzotropolone Ring-Containing Compounds
[0153] A 1M sodium phosphate buffer was prepared by mixing a 0.1M
NaH.sub.2PO.sub.4-2H.sub.2O and a 0.1M Na.sub.2HPO.sub.4-12H.sub.2O
and adjusting the mixture to pH7.0, and thereto 2 g/L of bovine
serum albumin (Nacalai Tesque, Inc., F-V, 015.2, 96% purity) and
0.2 g/L of NaN.sub.3(Nacalai Tesque, Inc., a special grade reagent)
were added. To prepare an enzyme solution, alfa-glucosidase (Wako
Pure Chemical Industries, Ltd., yeast-derived, 100 units/mg) was
dissolved in the above buffer so as to achieve 0.5 units/mg
protein, ml (100 .mu.g/20 ml). To prepare a substrate solution,
p-nitrophenyl-alfa-D-glucopyranoside (Nacalai Tesque, Inc., a
special grade reagent) was dissolved in the above buffer so as to
achieve 5 mM concentration (7.525 mg/5 ml).
[0154] Among the samples used for the assays,
epigallocatechin-3-O-gallate (Compound 13: EGCG), a positive
control, was a product from Wako Pure Chemical Industries, Ltd.,
and Compounds 1, 3, 4, 5, 11, 12, 14, and 15 were products that
were synthesized and purified in Example 1, 2, or 7,
[0155] These samples were adjusted so as to obtain 10 mg/ml of DMSO
and the solution was diluted 2-fold in 6 steps. Using a 96-well
microplate, 45 .mu.L of the enzyme solution was added to 10 .mu.L
of the sample solution. After preincubation at 37.degree. C. for 5
minutes, 45 .mu.L of the substrate solution was added and
absorbance at 405 nm (A405 nm) was measured. After incubation at
37.degree. C. for 5 minutes, the absorbance A405 nm was measured.
Percent inhibition was calculated as a difference in A405 nm from a
solution in which only DMSO was added as a control instead of a
sample, and two consecutive measurements were performed for
activity of each compound.
[0156] As a result, the alfa glucosidase inhibitory activity of
each Compound, when indicated by IC.sub.50 value, is as shown in
Table 8; Compounds 12 and 14 exhibited particularly strong
activity.
TABLE-US-00008 TABLE 8 alfa-glucosidase inhibitory activity IC50
Compound Compound (mM) 1 purpurogallin 1.237 3 epitheaflagallin No
4 epitheaflagallin 3-O-gallate 0.180 5 theaflavate A 0.116 11
epitheaflavic acid-GA 0.287 12 (2R,3R)-2-(3,4-dihydroxyphenyl)-
0.159 5,7-dihydroxychroman-3-yl 2,3,4,6-tetrahydroxy-5-oxo-5H-
benzo[7]annulene-8-carboxylate 13 EGCG 0.485 14 theaflavanin
3-O-gallate 0.196 15 EGCG-catechol 0.103
[0157] From these results, together with the results on lipase
inhibitory activity, these benzotropolone ring containing compounds
were found to have strong inhibitory activities against digestive
enzymes. Among all, Compound 12, which exhibited its strong
inhibitory activities against both the two enzymes, was confirmed
to be present also in black tea; to date, the compound has not been
known, but was found to be useful as an anti-obesity material.
INDUSTRIAL APPLICABILITY
[0158] The anti-obesity agent of the present invention contains a
tea-derived benzotropolone ring-containing compound and thus
exhibits superior inhibitory activities against lipase and
alfa-glucosidase. The agent does not compromise the flavor of foods
and beverages, has palatability, and can be used in various use
applications including foods and beverages intended for health
enhancement such as reduction in triglycerides.
* * * * *