U.S. patent application number 09/768740 was filed with the patent office on 2001-07-05 for flavanone derivatives and composition for preventing or treating blood lipid level-related diseases comprising same.
This patent application is currently assigned to KOREA RESEARCH INSTITUTE OF BIOSCIENCE AND BIOTECHNOLOGY. Invention is credited to Bok, Song-Hae, Choi, Myung-Sook, Choi, Yang-Kyu, Hyun, Byung-Hwa, Jeong, Tae-Sook, Kim, Ju-Ryong, Lee, Chul-Ho, Lee, Sang-Ku, Moon, Surk-Sik.
Application Number | 20010006978 09/768740 |
Document ID | / |
Family ID | 19704103 |
Filed Date | 2001-07-05 |
United States Patent
Application |
20010006978 |
Kind Code |
A1 |
Bok, Song-Hae ; et
al. |
July 5, 2001 |
Flavanone derivatives and composition for preventing or treating
blood lipid level-related diseases comprising same
Abstract
A compound of formula (I) treating or preventing an elevated
blood lipid level-related disease and inhibiting the activities of
acyl-CoA:cholesterol-O-acyltransferase(ACAT) and
3-hydroxy-3-methylglutar- yl CoA(HMG-CoA) reductase: 1 wherein, R'
is R.sup.5 R.sup.6CO group; R.sup.2 is H or R.sup.6CO group;
R.sup.3 is H, CH3, R.sup.5 or R.sup.6CO group; R.sup.4 is H, OH,
OR.sup.5 or R.sup.6COO group; R.sup.5 is a C.sub.2-5 alkyl group
substituted with a phenyl group optionally having one or more
substituents selected from the group consisting of C.sub.1-3 alkyl,
OH, Cl and NO.sub.2; a C.sub.1-5 alkyl group substituted with a
naphthyl group optionally having one or more substituents selected
from the group consisting of C.sub.1-3 alkyl, OH, Cl and NO.sub.2;
a C.sub.10-18 alkyl; or a C.sub.10-18 alkenyl group; and R.sup.6 is
a C.sub.10-18 alkenyl group; or an aryl group optionally having one
or more substituents selected from the group consisting of
C.sub.1-3 alkyl, OH, Cl or NO.sub.2.
Inventors: |
Bok, Song-Hae; (Daejeon,
KR) ; Jeong, Tae-Sook; (Daejeon, KR) ; Lee,
Sang-Ku; (Daejeon, KR) ; Kim, Ju-Ryong;
(Kwangju, KR) ; Moon, Surk-Sik; (Daejeon, KR)
; Choi, Myung-Sook; (Daegu, KR) ; Hyun,
Byung-Hwa; (Daejeon, KR) ; Lee, Chul-Ho;
(Daejeon, KR) ; Choi, Yang-Kyu; (Daejeon,
KR) |
Correspondence
Address: |
SHAHAN ISLAM, ESQ.
ROSENMAN & COLIN LLP
575 Madison Avenue
New York
NY
10022-2585
US
|
Assignee: |
KOREA RESEARCH INSTITUTE OF
BIOSCIENCE AND BIOTECHNOLOGY
|
Family ID: |
19704103 |
Appl. No.: |
09/768740 |
Filed: |
January 24, 2001 |
Current U.S.
Class: |
514/456 ;
549/399 |
Current CPC
Class: |
A23L 13/42 20160801;
A23V 2002/00 20130101; C07D 311/32 20130101; A23L 2/52 20130101;
A23L 33/105 20160801; A23L 27/63 20160801; A23V 2002/00 20130101;
A23V 2250/2116 20130101 |
Class at
Publication: |
514/456 ;
549/399 |
International
Class: |
C07D 311/04; C07D
311/74; C07D 311/76; A61K 031/35; A01N 043/16 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 30, 1999 |
KR |
2000-87185 |
Claims
What is claimed is:
1. A compound of formula (I): 3wherein, R.sup.1 is R.sup.5
R.sup.6CO group; R.sup.2 is H or R.sup.6CO group; R.sup.3 is H,
CH3, R.sup.5 or R.sup.6CO group; R.sup.4 is H, OH, OR.sup.5 or
R.sup.6COO group; R.sup.5 is a C.sub.2-5 alkyl group substituted
with a phenyl group optionally having one or more substituents
selected from the group consisting of C.sub.1-3 alkyl, OH, Cl and
NO.sub.2; a C.sub.1-5 alkyl group substituted with a naphthyl group
optionally having one or more substituents selected from the group
consisting of C.sub.1-3 alkyl, OH, Cl and NO.sub.2; a C.sub.10-18
alkyl; or a C.sub.10-18 alkenyl group; and R.sup.6 is a C.sub.10-18
alkenyl group; or an aryl group optionally having one or more
substituents selected from the group consisting of C.sub.1-3 alkyl,
OH, Cl or NO.sub.2.
2. The compound of claim 1 wherein R' is phenethyl, cetyl, stearyl,
CH.sub.2.dbd.CH(CH.sub.2).sub.9, 1-naphthalenemethyl, oleoyl,
linoleoyl, benzoyl, o-chlorobenzoyl, p-chlorobenzoyl,
o-methoxybenzoyl, p-methoxybenzoyl or 1 -naphthoyl; R.sup.2 is H,
oleoyl, benzoyl, o-chlorobenzoyl, p-chlorobenzoyl,
o-methoxybenzoyl, p-methoxybenzoyl or 1-naphthoyl; and R.sup.3 is
H, CH.sub.3, phenethyl, cetyl, stearyl, oleoyl, benzoyl,
o-chlorobenzoyl, p-chlorobenzoyl, o-methoxybenzoyl,
p-methoxybenzoyl or 1 -naphthoyl; and R.sup.4 is H, OH or
O-oleoyl.
3. A process for preparing the compound of claim 1 which comprises
(a) dissolving naringenin or hesperetin in an organic solvent; (b)
adding 1 to 1.5 equivalents of an alkyl halide and 1 to 3
equivalents of a base to the solution obtained in step (a); and (c)
stirring the mixture obtained in step (b) at a temperature ranging
from 60 to 90.degree.C.
4. The process of claim 3 wherein the organic solvent is selected
from the group consisting of dimethylformamide(DMF),
dimethylsulfoxide(DMSO), tetrahydrofuran(THF), acetone and mixture
thereof.
5. The process of claim 3 wherein the alkyl halide is hexadecanyl
bromide or octadecanyl bromide.
6. A process for preparing the compound of claim 1 which comprises
(a) dissolving naringenin or hesperetin in an organic solvent; (b)
adding 1 to 10 equivalents of triethylamine(Et.sub.3N) to the
solution obtained in step (a); (c) cooling the mixture obtained in
step (b); (d) adding 1 to 10 equivalents of acyl chloride to the
mixture obtained in step (c); and (e) reacting the mixture obtained
in step (c) for 2 to 20 hours.
7. The process of claim 6 wherein the organic solvent is
tetrahydrofuran(THF) or dichloromethane(CH.sub.2Cl.sub.2).
8. The process of claim 6 wherein acyl chloride is selected from
the group consisting of oleoyl chloride, benzoyl chloride,
p-chlorobenzoyl chloride, o-chlolbenzoyl chloride, p-methoxybenzoyl
chloride, o-methoxybenzoyl chloride and 1-naphthoyl chloride.
9. A pharmaceutical composition for treating or preventing an
elevated blood lipid level-related disease in a mammal, which
comprises an effective amount of the compound of claim 1 as an
active ingredient together with a pharmaceutically acceptable
carrier.
10. The composition of claim 9 wherein the disease is
hyperlipidemia, arteriosclerosis, angina pectoris, stroke or fatty
liver.
11. A pharmaceutical composition for inhibiting the activity of
acyl-CoA:cholesterol-O-acyltransferase(ACAT) in a mammal, which
comprises an effective amount of the compound of claim 1 as an
active ingredient together with a pharmaceutically acceptable
carrier.
12. A pharmaceutical composition for inhibiting the activity of
3-hydroxy-3-methylglutaryl CoA(HMG-CoA) reductase in a mammal,
which comprises an effective amount of the compound of claim 1 as
an active ingredient together with a pharmaceutically acceptable
carrier.
13. A food or beverage composition for treating or preventing an
elevated blood lipid level-related disease in a mammal, which
comprises an effective amount of the compound of claim 1 as an
active ingredient together with a pharmaceutically acceptable
carrier.
14. The composition of claim 13 wherein the disease is
hyperlipidemia, arteriosclerosis, angina pectoris, stroke or fatty
liver.
15. A food or beverage composition for inhibiting the activity of
acyl-CoA:cholesterol-O-acyltransferase(ACAT) in a mammal, which
comprises an effective amount of the compound of claim 1 as an
active ingredient together with a pharmaceutically acceptable
carrier.
16. A food or beverage composition for inhibiting the activity of
3-hydroxy-3-methylglutaryl CoA(HMG-CoA) reductase in a mammal,
which comprises an effective amount of the compound of claim 1 as
an active ingredient together with a pharmaceutically acceptable
carrier.
17. A method for treating or preventing an elevated blood lipid
level-related disease in a mammal, which comprises administering an
effective amount of the compound of claim 1 thereto.
18. The method of claim 17 wherein the disease is hyperlipidemia,
arteriosclerosis, angina pectoris, stroke or fatty liver.
19. A food or beverage composition for inhibiting the activity of
acyl-CoA:cholesterol-O-acyltransferase(ACAT) in a mammal, which
comprises administering an effective amount of the compound of
claim 1 thereto.
20. A food or beverage composition for inhibiting the activity of
3-hydroxy-3-methylglutaryl CoA(HMG-CoA) reductase in a mammal,
which comprises administering an effective amount of the compound
of claim 1 thereto.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to novel flavanone
derivatives; a process for preparing same; a pharmaceutical
composition containing same for treating or preventing an elevated
blood lipid level-related disease, inhibiting the activity of
acyl-CoA:cholesterol-O-acyltransferase(ACAT) and inhibiting the
activity of 3-hydroxy-3-methylglutaryl CoA(HMG-CoA) reductase.
BACKGROUND OF THE INVENTION
[0002] In recent years, coronary artery diseases, e.g.,
atherosclerosis and hypercholesterolemia, have increasingly become
a major cause of deaths. It has been reported that an elevated
plasma cholesterol level causes the deposition of fat, macrophages
and foam cells on the wall of blood vessels, such deposit leading
to plaque formation and then to atherosclerosis(Ross, R., Nature,
362, 801-809(1993)). One of the methods for decreasing the plasma
cholesterol level is alimentotherapy to reduce the ingestion of
cholesterol and lipids. Another method is to inhibit the absorption
of cholesterol by inhibiting enzymes involved therein.
[0003] Acyl-CoA:cholesterol-O-acyltransferase(ACAT) promotes the
esterification of cholesterol in blood. Foam cells are formed by
the action of ACAT and contain a large amount of cholesterol ester
carried by low density lipoproteins. The formation of foam cells on
the wall of artery increases with the ACAT activity, and,
accordingly, an inhibitor of ACAT may also be an agent for
preventing atherosclerosis. Further, it has been reported that the
blood level of LDL-cholesterol can be reduced by inhibiting the
ACAT activity(Witiak, D. T. and D. R. Feller(eds.), Anti-Lipidemic
Drugs: Medicinal, Chemical and Biochemical Aspects, Elsevier,
pp159-195(1991)).
[0004] Therefore, numerous efforts have been made to develop
medicines which inhibit ACAT activity; and, as a result, several
compounds isolated from the cultures of various microorganisms have
been reported. Examples of such compounds include pyripyropenes
isolated from the culture of Aspergillus fumigatus(S. Omura et al.,
J. Antibiotics, 46, 1168-1169(1993)) and Acaterin isolated from
Pseudomonas sp.(S. Nagamura et al., J. Antibiotics, 45,
1216-1221(1992)).
[0005] Further, it has been reported that hypercholesterolemia can
be treated effectively by reducing the rate of cholesterol
biosynthesis through the inhibition of HMG-CoA reductase which
mediates the synthesis of mevalonic acid, an intermediate in a
biosynthesis of sterol or isoprenoids(Cardiovascular Pharmacology,
William W. Parmley and Kanu Chatterjee Ed, Wolf Publishing,
pp8.6-8.7, 1994).
[0006] Accordingly, numerous efforts have been made to develop
medicines to inhibit HMG-CoA reductase; and, as a result, several
compounds derived from Penicillium sp. and Aspergillus sp. have
been commercialized. Specifically, Lovastatin.RTM. and
Simvastatin.RTM. developed Merck Co., U.S.A., and Pravastatin.RTM.
developed by Sankyo Co., Japan, have been commercialized(C. D. R.
Dunn, Stroke: Trends, Treatment and Markets, SCRIPT Report, PJB
Publications Ltd., 1995). However, these medicines are very
expensive and a long-term administration thereof is known to induce
an adverse side effect of increasing creatine kinase in the liver.
Accordingly, there has continued to exist a need to develop an
inexpensive and non-toxic inhibitor of HMG-CoA reductase.
[0007] On the other hand, deterioration of hepatic functions may
occur due to an excessive intake of alcohol or foods having a high
lipid content, or an infection of hepatitis B or C virus, and it
may develop into hepatitis, hepatocirrhosis or hepatic cancer. In
particular, the excessive intake of fat-containing foods and
alcohol causes fatty liver wherein a large amount of lipids is
deposited in the liver tissue and the levels of serum
GOT(glutamate-oxaloacetate transaminase), GPT(glutamate-pyruvate
transaminase) and .gamma.-GTP(.gamma.-glutamyl transpeptidase) are
elevated(T. Banciu et al., Med. Interne., 20, 69-71(1982); and A.
Par et al., Acta. Med. Acad. Sci. Hung., 33, 309-319(1976)).
Accordingly, there has continued to exist a need to develop
non-toxic agents for preventing and treating elevated blood lipid
level-related diseases, and hepatic diseases.
[0008] The present inventors have reported that naringenin and
hesperetin, which are the aglycons of naringin and hesperidin found
in lemons, grapefruits, tangerines and oranges(Citrus sinensis),
have activities for inhibiting hyperlipidemia and atherosclerosis
(U.S. Pat. Nos. 5,877,208 and 5,763,414).
[0009] The present inventors have continued to screen compounds
having the flavanone core structure; and have discovered that
certain novel flavanone derivatives have enhanced activity in
treating or preventing elevated blood lipid level-related diseases,
inhibiting an activity of
acyl-CoA:cholesterol-O-acyltransferase(ACAT) and inhibiting an
activity of 3-hydroxy-3-methylglutaryl CoA(HMG-CoA) reductase.
SUMMARY OF THE INVENTION
[0010] Accordingly, it is an object of the present invention to
provide novel flavanone derivatives.
[0011] It is another object of the present invention to provide a
process for the preparation of the inventive flavanone
derivatives.
[0012] It is a further object of the present invention to provide a
pharmaceutical composition for treating or preventing an elevated
blood lipid level-related disease, inhibiting an activity of
acyl-CoA:cholesterol-O-acyltransferase(ACAT) and inhibiting an
activity of 3-hydroxy-3-methylglutaryl CoA(HMG-CoA) reductase.
[0013] It is a further object of the present invention to provide a
method for treating or preventing an elevated blood lipid
level-related disease, inhibiting an activity of
acyl-CoA:cholesterol-O-acyltransferase(ACAT) and inhibiting an
activity of 3-hydroxy-3-methylglutaryl CoA(HMG-CoA) In accordance
with the present invention, there is provided a novel compound of
formula (I): 2
[0014] wherein,
[0015] R.sup.1 is R.sup.5 or R.sup.6CO group;
[0016] R.sup.2 is H or R.sup.6CO group;
[0017] R.sup.3 is H, CH3, R.sup.5 or R.sup.6CO group;
[0018] R.sup.4 is H, OH, OR.sup.5 or R.sup.6COO group;
[0019] R.sup.5 is a C.sub.2-5 alkyl group substituted with a phenyl
group optionally having one or more substituents selected from the
group consisting of C.sub.1-3 alkyl, OH, Cl and NO.sub.2; a
C.sub.1-5 alkyl group substituted with a naphthyl group optionally
having one or more substituents selected from the group consisting
of C.sub.1-3 alkyl, OH, Cl and NO.sub.2; a C.sub.10-18 alkyl; or a
C.sub.10-18 alkenyl group; and
[0020] R.sup.6 is a C.sub.10-18 alkenyl group; or an aryl group
optionally having one or more substituents selected from the group
consisting of C.sub.1-3 alkyl, OH, Cl or NO.sub.2.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] The above and other objects and features of the present
invention will become apparent from the following description of
the invention, when taken in conjunction with the accompanying
drawings, in which:
[0022] FIGS. 1A, 1B and 1C show the arteries of the rabbits
administered with 1% cholesterol(control); 1% cholesterol plus 1
mg/kg Lovastatin.RTM.; and 1% cholesterol plus 0.1% NG5006(compound
4), respectively; and
[0023] FIGS. 2A, 2B and 2C present the microscopic features of the
livers of the rabbits administered with 1% cholesterol(control); 1%
cholesterol plus 1 mg/kg Lovastatin.RTM.; and 1% cholesterol plus
0.1% NG5006, respectively.
DETAILED DESCRIPTION OF THE INVENTION
[0024] Among the compounds of the present invention, the preferred
are those wherein R.sup.1 is phenethyl, cetyl, stearyl,
CH.sub.2.dbd.CH(CH.sub.2).sub.9--, 1-naphthalenemethyl, oleoyl,
linoleoyl, benzoyl, o-chlorobenzoyl, p-chlorobenzoyl,
o-methoxybenzoyl, p-methoxybenzoyl or 1-naphthoyl; R.sup.2 is H,
oleoyl, benzoyl, o-chlorobenzoyl, p-chlorobenzoyl,
o-methoxybenzoyl, p-methoxybenzoyl or 1-naphthoyl; and R.sup.3 is
H, CH.sub.3, phenethyl, cetyl, stearyl, oleoyl, benzoyl,
o-chlorobenzoyl, p-chlorobenzoyl, o-methoxybenzoyl,
p-methoxybenzoyl or 1-naphthoyl; and R.sup.4 is H, OH or
O-oleoyl.
[0025] More preferred are compounds of formula (I) having R.sup.1,
R.sup.2, R.sup.3 and R.sup.4 groups shown in Table I:
1TABLE I No. R.sup.1 R.sup.2 R.sup.3 R.sup.4 1 phenethyl H H H 2
phenethyl H phenethyl H 3 phenethyl H CH.sub.3 OH 4 cetyl H H H 5
cetyl H CH.sub.3 OH 6 cetyl H cetyl H 7 stearyl H H H 8 stearyl H
stearyl H 9 stearyl H CH.sub.3 OH 10 CH.sub.2CH(CH.sub.2).sub.9 H H
H 11 1-naphthalenemethyl H H H 12 oleoyl H H H 13 oleoyl H oleoyl H
14 oleoyl oleoyl oleoyl H 15 oleoyl H CH.sub.3 OH 16 oleoyl H
CH.sub.3 O-oleoyl 17 linoleoyl H H H 18 benzoyl H H H 19 benzoyl H
CH.sub.3 OH 20 benzoyl benzoyl benzoyl H 21 p-chlorobenzoyl
p-chlorobenzoyl p-chlorobenzoyl H 22 o-chlorobenzoyl
o-chlorobenzoyl o-chlorobenzoyl H 23 o-methoxybenzoyl
o-methoxybenzoyl o-methoxybenzoyl H 24 p-methoxybenzoyl
p-methoxybenzoyl p-methoxybenzoyl H 25 1-naphthoyl H H H 26
1-naphthoyl H CH.sub.3 OH 27 1-naphthoyl 1-naphthoyl 1-naphthoyl
H
[0026] Most preferred compound of the inventive compounds is
naringenin 7-O-cetyl ether.
[0027] Ether-type flavanone derivatives of the inventive compounds
may be prepared by a process which comprises dissolving naringenin
or hesperetin in an organic solvent; adding 1 to 1.5 equivalents of
an alkyl halide such as hexadecanyl bromide or octadecanyl bromide
and 1 to 3 equivalents of a base such as sodium carbonate to the
solution obtained above; and stirring the mixture at a temperature
ranging from 60 to 90.degree.C. for 3 to 20 hours.
[0028] Exemplary organic solvents that may be used in the present
may include dimethylformamide(DMF), dimethylsulfoxide(DMSO),
tetrahydrofuran(THF), acetone and a mixture thereof.
[0029] After the reaction, the resulting solution is diluted with
ethylacetate(EtOAc) and washed with water to remove the organic
solvent. The resulting solution is washed with physical saline
water and dried, filtered and the filtrate is concentrated to
obtain an ether-type flavanone derivative.
[0030] Further, ester-type flavanone derivatives of the inventive
compounds may be prepared by the conventional method. For example,
naringenin or hesperetin is dissolved in an organic solvent such as
tetrahydrofuran(THF) and dichloromethane(CH.sub.2Cl.sub.2) and 1 to
10 equivalents of triethylamine(Et.sub.3N) is added thereto. The
mixture is cooled with ice water and 1 to 10 equivalents of acyl
chloride is added thereto and then, reacted for 2 to 10 hours. The
acyl chloride which may be used in the above reaction is oleoyl
chloride, benzoyl chloride, p-chlorobenzoyl chloride,
o-chlorobenzoyl chloride, p-methoxybenzoyl chloride,
o-methoxybenzoyl chloride or 1-naphthoyl chloride. The resulting
solution is diluted with ethylacetate(EtOAc), followed by washing
with saturated sodium hydrogen carbonate solution and saline water.
The resulting extract is dried, filtered and the filtrate is
concentrated to obtain an ester-type flavanone derivative. Further,
the compound of the present invention may be purified by
triturating with an insoluble solvent or subjecting to
chromatography such as silica gel chromatography and C-18 HPLC.
[0031] The flavanone derivatives of the present invention exert
inhibitory effects on: elevated blood lipid level-related diseases,
e.g., hyperlipidemia, arteriosclerosis, angina pectoris, stroke and
hepatic diseases; the ACAT activity; and the HMG-CoA reductase
activity. Further, in spite of their potent efficacies, the
inventive flavanone derivatives exhibit no toxicity or mitogenicity
in tests using mice.
[0032] A pharmaceutical formulation may be prepared by using the
compositions of the invention in accordance with any of the
conventional procedures. In preparing the formulation, the active
ingredient is preferably admixed or diluted with a carrier, or
enclosed within a carrier which may be in the form of a capsule,
sachet or other container. When the carrier serves as a diluent, it
may be a solid, semi-solid or liquid material acting as a vehicle,
excipient or medium for the active ingredient. Thus, the
formulations may be in the form of a tablet, pill, powder, sachet,
elixir, suspension, emulsion, solution, syrup, aerosol, soft and
hard gelatin capsule, sterile injectable solution, sterile packaged
powder and the like.
[0033] Examples of suitable carriers, excipients, and diluents are
lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum
acacia, alginates, gelatin, calcium phosphate, calcium silicate,
cellulose, methyl cellulose, microcrystalline cellulose,
polyvinylpyrrolidone, water, methylhydroxybenzoates,
propylhydroxybenzoates, talc, magnesium stearate and mineral oil.
The formulations may additionally include fillers,
anti-agglutinating agents, lubricating agents, wetting agents,
flavoring agents, emulsifiers, preservatives and the like. The
compositions of the invention may be formulated so as to provide
quick, sustained or delayed release of the active ingredient after
their administration to a mammal by employing any of the procedures
well known in the art.
[0034] The pharmaceutical composition of the present invention can
be administered via various routes including oral, transdermal,
subcutaneous, intravenous and intramuscular introduction. In case
of human, a typical daily dose of the flavanone derivative of
formula (I) may range from about 0.1 to 50 mg/kg body weight,
preferably 1 to 10 mg/kg body weight, and can be administered in a
single dose or in divided doses.
[0035] However, it should be understood that the amount of the
active ingredient actually administered ought to be determined in
light of various relevant factors including the condition to be
treated, the chosen route of administration, the age, sex and body
weight of the individual patient, and the severity of the patient's
symptom; and, therefore, the above dose should not be intended to
limit the scope of the invention in any way.
[0036] Moreover, the flavanone derivative of formula (I) can be
incorporated in foods or beverages, as an additive or a dietary
supplement, for the purpose of treating or preventing elevated
blood lipid level-related diseases, and inhibiting the ACAT and the
HMG-CoA reductase activities. The foods or beverages may include
meats; juices such as a vegetable juice(e.g., carrot juice and
tomato juice) and a fruit juice(e.g., orange juice, grape juice,
pineapple juice, apple juice and banana juice); chocolates; snacks;
confectionery; pizza; foods made from cereal flour such as breads,
cakes, crackers, cookies, biscuits, noodles and the likes; gums;
dairy products such as milk, cheese, yogurt and ice creams; soups;
broths; pastes, ketchups and sauces; teas; alcoholic beverages;
carbonated beverages such as Coca-Cola.RTM. and Pepsi-Cola.RTM.;
vitamin complexes; and various health foods.
[0037] In this case, the content of the flavanone derivative of
formula (I) in a food or beverage may range from 0.01 to 20% by
weight, preferably 0.1 to 10% by weight.
[0038] The following Examples are intended to further illustrate
the present invention without limiting its scope.
[0039] As described above, flavanone derivatives can be used as an
effective, non-toxic pharmaceutical agent for treating or
preventing elevated blood lipid level-related diseases, inhibiting
the ACAT activity and/or inhibiting the HMG-CoA reductase
activity.
[0040] Further, percentages given below for solid in solid mixture,
liquid in liquid, and solid in liquid are on a wt/wt, vol/vol and
wt/vol basis, respectively, and all the reactions were carried out
at room temperature, unless specifically indicated otherwise.
Example 1: Preparation and Analysis of naringenin 7-O-cetyl
ether(Compound 4, NG5006)
[0041] 10 g(36.73 mmol) of naringenin was dissolved in a mixture of
100 ml of acetone and 100 ml of dimethylformamide(DMF). 13.5 ml of
hexadecanyl bromide and 4.70 g of sodium carbonate were added to
the mixture and stirred in a water bath at 80.degree.C. for 12
hours. The resulting solution was cooled, and then, 100 ml of water
and 800 ml of EtOAc were added thereto and the mixture was
extracted with EtOAc. The extract thus obtained was washed with
water and concentrated under a reduced pressure. The solid formed
was filtered using a glass filter and dried under a reduced
pressure to give 10.1 g of naringenin 7-O-cetyl ether. Further, the
residue was concentrated and subjected to silica gel column
chromatography(45 mm.times.150 mm, 70-230 mesh, eluent:
hexane/EtOAc(8:2)) to obtain an additional 3g of naringenin
7-O-cetyl ether as a pale yellow solid (Yield: 72%).
[0042] melting point(m.p.): 114-117.degree.C.
[0043] .sup.1H NMR (CDCl.sub.3).delta. 12.0 (s, 1H), 7.32 (d, J=8.4
Hz, 2H), 6.87 (d, J=8.4 Hz, 2H), 6.04 (d, J=2.0 Hz, 1H), 6.02 (d,
J=2.0 Hz, 1H), 5.33 (dd, J=13.2, 2.8 Hz, 1H), 3.95 (t, J=6.8 Hz,
2H), 3.07 (dd, J=17.2, 13.2 Hz, 1H), 2.77 (dd, J= 17.2, 2.8 Hz,
1H), 1.75 (quin, J=6.8 Hz, 2H), 1.44-1.36 (m, 2H), 1.34-1.22 (m,
24H), 0.87 (t, J=6.8 Hz, 3H) ppm.
[0044] .sup.13C NMR (CDCl.sub.3).delta. 195.8, 167.6, 164.0, 162.8,
156.0, 130.6, 127.9, 115.6, 103.0, 95.6, 94.6, 78.9, 68.6, 43.2,
32.0, 29.75-29.69 (six carbons), 29.62, 29.57, 29.4, 29.3, 28.9,
25.9, 22.7, 14.2 ppm.
[0045] 1D NOESY: NOE contacts were observed between H (6.04 and
6.02 ppms) and H (3.95 ppm).
[0046] Example 2: Preparation and analysis of naringenin
7-O-stearyl ether(compound 7)
[0047] 10 g(36.73 mmol) of naringenin was dissolved in a mixture of
20 ml of acetone and 20 ml of DMF. 1.5 ml of octadecanyl bromide
and 470 mg of sodium carbonate were added to the mixture and
stirred in a water bath at 80.degree.C. for 19 hours. The resulting
solution was cooled, and then, 20 ml of water and 200 ml of EtOAc
were added thereto and the mixture was extracted with EtOAc. The
extract thus obtained was washed with water and concentrated under
a reduced pressure. A small amount of MeOH was added to the solid
thus formed and the mixture was stirred with glass stick. The solid
thus obtained was filtered and washed with EtOAc to give 1.25g of
naringenin 7-O-stearyl ether as a very pale yellow solid.
[0048] m.p.: 117-119.degree.C
[0049] .sup.1H NMR (CDCl.sub.3).delta. 12.0 (s, 1H), 7.32 (d, J=8.4
Hz, 2H), 6.87 (d, J=8.4 Hz, 2H), 6.04 (d, J=2.0 Hz, 1H), 6.02 (d,
J=2.0 Hz, 1H), 5.34 (dd, J=12.8, 2.8 Hz, 1H), 3.94 (t, J=6.8 Hz,
2H), 3.07 (dd, J=17.2, 12.8 Hz, 1H), 2.77 (dd, J=17.2, 2.8 Hz, 1H),
1.75 (quin, J=6.8 Hz, 2H), 1.44-1.36 (m, 2H), 1.34-1.22 (m, 28H),
0.89 (t, J=6.8 Hz, 3H) ppm.
[0050] .sup.13CNMR(CDCl.sub.3) .delta. 195.8, 167.5, 164.0, 162.7,
156.0, 130.6, 127.9, 115.6, 103.0, 95.5, 94.6, 78.9, 68.6, 43.2,
32.0, 29.74-29.65 (8 carbons), 29.62, 29.57, 29.4, 29.3, 28.9,
25.9, 22.7, 14.2 ppm.
[0051] 1D NOESY: NOE contacts were observed between H (6.04 and
6.02 ppms) and H (3.94 ppm).
[0052] Example 3: Preparation and analysis of hesperetin 7-cetyl
ether(compound 9)
[0053] 100 mg of hesperetin and 40 mg of sodium carbonate were
dissolved in a dryed DMSO, and then, 0.15 ml of hexadecanyl bromide
was added thereto. The mixture was stirred at 80.degree.C. for 18
hours. The resulting solution was cooled and diluted with EtOAc,
and then, washed with water and saline. The resulting solution thus
obtained was dried with anhydrous magnesium sulfate and
concentrated. The solid thus formed was triturated with
EtOAc/hexane to give 180 mg of hesperetin 7-cetyl ether as a very
pale yellow solid.
[0054] m.p.: 106-108.degree.C.
[0055] .sup.1H NMR (CDC1.sub.3) .delta. 12.0 (s, 1H), 7.04 (d,
J=2.0 Hz, 1H), 6.93 (dd, J=8.4, 2.4 Hz, 1H), 6.88 (d, J=8.4 Hz,
1H), 6.05 (d, J=2.0 Hz, 1H), 6.02 (d, J=2.0 Hz, 1H), 5.32 (dd,
J=13.2, 3.2 Hz, 1H), 3.95 (t, J=6.4 Hz, 2H), 3.07 (dd, J=17.2, 13.2
Hz, 1H), 2.78 (dd, J=17.2, 3.2 Hz, 1H), 1.75 (quin, J=7.2 Hz, 2H),
1.40 (m, 211), 1.32-1.20 (m, 24H), 0.87 (t, J=6.4 Hz, 3H) ppm.
[0056] 1D NOESY : NOE contacts were observed between H (6.05 and
6.02 ppms) and H (3.95 ppm).
[0057] Example 4: Preparations and analyses of naringenin 7-O-oleic
acid monoester(compound 12) and 7,4'-O-oleic acid diester(compound
13)
[0058] 500 mg of naringenin and 0.2 ml of triethylamine were
dissolved in 7 ml of dried tetrahydrofuran(THF) and the mixture was
cooled with an ice water. 0.6 ml of oleoyl chloride was added
dropwise to the resulting solution. The mixture thus obtained was
stirred at room temperature for 3 hours. The resulting solution was
diluted with EtOAc and washed with a saturated sodium hydrogen
carbonate solution, followed by washing with saline. The resulting
solution was dried with anhydrous magnesium sulfate, filtered, and
concentrated. The residue was subjected to silica gel column
chromatography(25 mm.times.150 mm, 70-230 mesh, eluent:
hexane/EtOAc (9: 1)) to give 527 mg of 7-O-oleic monoester and 57
mg of 7,4'-O-oleic acid diester as colorless liquids.
[0059] 7-O-oleic acid monoester
[0060] Viscous liquid
[0061] .sup.1H NMR (CDC1.sub.3) .delta. 11.9 (s, 1H), 7.28 (d,
J=8.8 Hz, 2H), 6.85 (d, J=8.8 Hz, 2H), 6.28 (d, J=2.0 Hz, 1H), 6.26
(d, J =2.0 Hz, 1H), 5.38-5.31 (m, 3H), 3.11 (dd, J=17.2, 13.2 Hz,
1H), 2.80 (dd, J=17.2, 2.8 Hz, 1H), 2.53 (t, J=7.6 Hz, 2H), 2.00
(m, 4H), 1.71 (quin, J=7.2 Hz, 2H), 1.40-1.20 (m, 22H), 0.87 (t,
J=6.0 Hz, 3H) ppm.
[0062] .sup.13C NMR(CDCl.sub.3) .delta. 197.2, 171.4, 163.1, 162.3,
158.3, 156.3, 130.0, 129.7, 129.6, 127.8, 115.6, 106.1, 103.1,
101.7, 79.0, 43.3, 34.4, 31.9, 29.8, 29.7, 29.5, 29.3, 29.2, 29.13,
29.07, 29.0, 27.23, 27.16, 24.8, 22.7, 14.2 ppm.
[0063] 1D NOESY: NOE contacts were observed between H (6.28 and
6.26 ppms) and H (2.53 ppm).
[0064] 7,4'-O-oleic acid diester
[0065] Viscous liquid
[0066] .sup.1H NMR (CDCl.sub.3) .delta. 11.8 (s, 1H), 7.45 (d,
J=8.8 Hz, 2H), 7.14 (d, J=8.8 Hz, 2H), 6.30 (d, J=2.0 Hz, 1H), 6.28
(d, J=2.0 Hz, 1H), 5.45 (dd, J=13.6, 2.8 Hz, 1H), 5.40-5.32 (m,
4H), 3.10 (dd, J=17.2, 13.6 Hz, 1H), 2.86 (dd, J=17.2, 2.8 Hz, 1H),
2.54 (m, 4H), 2.02 (m, 8H), 1.76 (m, 4H), 1.40-1.20 (m, 44H), 0.87
(t, J=6.0 Hz, 6H) ppm.
[0067] .sup.13C NMR (CDCl.sub.3) .delta. 196.6, 172.1, 171.0,
163.2, 162.0, 158.4, 151.0 135.3, 130.0, 129.6, 127.2, 122.1,
106.1, 103.4, 101.7, 78.7, 43.6, 34.4, 31.9, 29.7-29.1(16 carbons),
27.3, 27.2, 24.9, 24.8, 22.7, 14.3, 14.2 ppm.
[0068] Example 5: Preparation and analysis of naringenin
5,7,4'-O-oleic acid triester(compound 14)
[0069] 100 mg of naringenin and 1 ml of oleoyl chloride were
dissolved in 7 ml of dried THF and the mixture was cooled with an
ice water. 0.5 ml of triethylamine was added dropwise to the
resulting solution and the mixture was stirred at room temperature
for 5 hours. The resulting solution was diluted with EtOAc and
washed with a saturated sodium hydrogen carbonate solution,
followed by washing with saline. The resulting solution was dried
with anhydrous magnesium sulfate, filtered, and concentrated. The
concentrate was subjected to silica gel column chromatography(25
mm.times.150 mm, 70-230 mesh, eluent: hexane/EtOAc(9:1)) to give
203 mg of naringenin 5,7,4'-O-oleic acid triester as coloress
viscous liquid.
[0070] .sup.1H NMR (CDCl.sub.3) .delta. 7.44 (d, J=8.8 Hz, 2H),
7.13 (d, J=8.8 Hz, 2H), 6.76 (d, J=2.0 Hz, 1H), 6.50 (d, J=2.0 Hz,
1H), 5.47 (dd, J=13.6, 2.8 Hz, 1H), 5.40-5.30 (m, 6H), 3.00 (dd,
J=17.2, 13.6 Hz, 1H), 2.75 (dd, J=17.2, 2.8 Hz, 1H), 2.66 (m, 2H),
2.55 (m, 4H), 2.00 (m, 12H), 1.74 (m, 6H), 1.40-1.20 (m, 66H), 0.87
(m, 9H) ppm.
[0071] Example 6: Preparation and analysis of naringenin
5,7,4'-O-benzoic acid triester(compound 20)
[0072] The procedure of Example 5 was repeated by using 100 mg of
naringenin and 0.21 ml of benzoyl chloride to obtain 209 mg of
naringenin 5,7,4'-O-benzoic acid triester as a white solid.
Finally, the solid was triturated with hexane.
[0073] m.p.: 126-129.degree.C.
[0074] .sup.1H NMR (CDCl.sub.3) .delta. 8.24-8.16 (m, 6H),
7.68-7.60 (m, 3H), 7.54-7.49 (m, 8H), 7.29 (d, J=8.8 Hz, 2H), 6.99
(d, J=2.0 Hz, 1H), 6.83 (d, J=2.0 Hz, 1H), 5.58 (dd, J=13.6, 2.8
Hz, 1H), 3.09 (dd, J=16.8, 13.6 Hz, 1H), 2.81 (dd, J=16.8, 2.8 Hz,
1H) ppm.
[0075] .sup.13C NMR (CDCl.sub.3) .delta. 188.7, 164.9, 164.8,
163.8, 163.2, 156.2, 151.5,151.1, 135.7, 134.1, 133.7, 133.5,
132.0, 130.3, 129.4, 129.2, 128.7, 128.55,128.52, 127.4, 127.3,
122.2, 112.0, 111.1, 109.4, 79.1,45.2 ppm.
[0076] Example 7: Preparation and analysis of naringenin
5,7,4'-O-(p-chlorobenzoic acid) triester(compound 21)
[0077] The procedure of Example 5 was repeated by using 100 mg of
naringenin and 0.21 ml of p-chlorobenzoyl chloride to obtain 316 mg
of naringenin 5,7,4'-O-(p-chlorobenzoic acid) triester as a white
solid. Finally, the solid was triturated with EtOAc/hexane.
[0078] m.p.: 189-191.degree.C.
[0079] .sup.1H NMR (CDCl.sub.3) .delta. 8.15 (d, J=8.8 Hz, 2H),
8.13 (d, J=8.8 Hz, 2H), 8.10 (d, J=8.8 Hz, 2H), 7.53-7.48 (m, 8H),
7.28 (d, J=8.8 Hz, 2H), 6.98 (d, J=2.0 Hz, 1H), 6.81 (d, J=2.0 Hz,
1H), 5.58 (dd, J=13.6, 2.4 Hz, 1H), 3.07 (dd, J=16.4, 13.6 Hz, 1H),
2.80 (dd, J=16.4, 2.8 Hz, 1H) ppm.
[0080] .sup.13CNMR(CDC1.sub.3) 6 188.7, 164.1, 164.0, 163.2, 163.0,
155.9, 151.2, 150.9, 140.8, 140.3, 140.1, 135.7, 131.8, 131.7,
131.6, 131.5, 129.3, 129.1, 128.94, 128.92, 127.8, 127.6, 127.4,
126.9, 122.1, 111.9, 110.9, 109.5, 79.1, 45.2 ppm.
[0081] Example 8: Preparation and analysis of naringenin
5,7,4'-O-(o-chlorobenzoic acid) triester(compound 22)
[0082] The procedure of Example 5 was repeated by using 100 mg of
naringenin and 0.233 ml of o-chlorobenzoyl chloride to obtain 100
mg of naringenin 5,7,4'-O-(o-chlorobenzoic acid) triester as a
white solid. Finally, the solid was triturated with
EtOAc/hexane.
[0083] m.p.: 121-124.degree. C.
[0084] .sup.1H NMR (CDCl.sub.3) .delta. 8.29 (dd, J=8.0, 1.2 Hz,
1H), 8.05 (dd, J=8.0, 0.8 Hz, 1H), 8.03 (dd, J=8.0, 0.8 Hz, 1H),
7.56-7.48 (m, 8H), 7.45-7.38 (m, 3H), 7.33 (d, J=8.8 Hz, 2H), 7.03
(d, J=2.4 Hz, 1H), 6.86 (d, J=2.4 Hz, 1H), 5.59 (dd, J=13.6, 2.4
Hz, 1H), 3.10 (dd, J=16.8, 13.6 Hz, 1H), 2.84 (dd, J=16.8, 2.4 Hz,
1H) ppm.
[0085] .sup.13C NMR (CDC1.sub.3) .delta. 188.8, 163.8, 163.13,
163.09, 162.5, 155.8, 151.0, 150.9, 135.8, 134.7, 134.6, 134.4,
133.7, 133.3, 133.2, 132.6, 132.0, 131.9, 131.5, 131.3, 131.1,
128.9, 128.7, 128.1, 127.4, 126.8, 126.7, 122.1, 112.1, 110.9,
109.6, 79.1, 45.2 ppm.
[0086] Example 9: Preparation and analysis of naringenin
5,7,4'-O-(o-methoxybenzoic acid) triester(compound 23)
[0087] The procedure of Example 5 was repeated by using 100 mg of
naringenin and 0.233 ml of o-methoxybenzoyl chloride to obtain 100
mg of naringenin 5,7,4'-O-(o-methoxybenzoic acid) triester as a
white solid. Finally, the solid was subjected to silica gel
chromatography(25 mm.times.150 mm, 70-230 mesh, eluent:
EtOAc/hexane(3:7)).
[0088] m.p.: 84-87.degree.C.
[0089] .sup.1H NMR (CDCl.sub.3) .delta. 8.23 (dd, J=7.6, 1.6 Hz,
1H), 8.02 (dd, J=7.6, 2.0 Hz, 1H), 7.97 (dd, J=8.4,2.0 Hz, 1H),
7.58-7.52 (m, 3H), 7.50 (d, J=8.4 Hz, 2H), 7.29 (d, J=8.4 Hz, 2H),
7.09-7.02 (m, 6H), 7.33 (d, J=8.8 Hz, 2H), 6.97 (d, J= 2.0 Hz, 1H),
6.81 (d, J=2.0Hz, 1H), 5.55 dd, J=13.6, 2.4 Hz, 1H), 3.93 (s, 9H),
3.07 (dd, J=16.4, 13.6 Hz, 1H), 2.80 (dd, J=176.4, 2.4 Hz, 1H)
ppm.
[0090] .sup.13C NMR (CDCl.sub.3) .delta. 188.9, 164.1, 163.4,
163.0, 162.8, 160.0, 159.9, 159.8, 156.2, 151.4, 151.1, 135.6,
134.8, 134.4, 134.3, 132.8, 132.3, 132.2, 127.2, 122.3, 120.21,
120.18, 120.14, 118.7, 118.6, 118.0, 112.1, 112.0, 111.9, 111.4,
109.3, 79.1, 56.04, 56.02, 45.3 ppm.
[0091] Example 10: Preparation and analysis of naringenin
5,7,4'-O-P-methoxybenzoic acid) triester(compound 24)
[0092] The procedure of Example 5 was repeated by using 100 mg of
naringenin and 0.273 ml of p-methoxybenzoyl chloride to obtain 100
mg of naringenin 5,7,4'-O-(p-methoxybenzoic acid) triester as a
white solid. Finally, the solid was triturated with hexane.
[0093] m.p.: 113-117.degree.C.
[0094] .sup.1H NMR (CDCl.sub.3) .delta. 8.17 (d, J=8.8 Hz, 2H),
8.15 (d, J=8.8 Hz, 2H), 8.12 (d, J=8.8 Hz, 2H), 7.51 (d, J=8.8 Hz,
2H), 7.27 (d, J=8.8 Hz, 2H), 7.01-6.95 (m, 7H), 6.80 (d, J=2.0 Hz,
1H), 5.56 (dd, J=13.6, 2.8 Hz, 1H), 3.892 (s, 3H), 3.890 (s, 3H),
3.888 (s, 3H), 3.07 (dd, J=16.4, 13.6 Hz, 1H), 2.78 (dd, J=16.4,
2.8 Hz, 1H) ppm.
[0095] .sup.13C NMR (CDC1.sub.3) .delta. 188.8, 164.6, 164.5,
164.2, 163.9, 163.8, 163.5, 163.1, 156.3, 151.6, 151.2, 135.6,
132.4, 132.3, 127.3, 122.2, 121.8, 121.5, 120.8, 113.9, 111.9,
111.2, 109.2, 55.6, 55.55, 55.53, 45.3 ppm.
[0096] Example 11: Preparation and analysis of naringenin
5,7,4'-O-(1-naphtoxic acid) triester(compound 27)
[0097] The procedure of Example 5 was repeated by using 100 mg of
naringenin and 0.35 ml of 1-naphtoxic acid to obtain 100 mg of
naringenin 5,7,4'-O-(1-naphtoxic acid) triester as a white solid.
Finally, the solid was triturated with EtOAc/hexane.
[0098] m.p.: 165-167.degree. C.
[0099] .sup.1H NMR (CDCl.sub.3) .delta. 8.44 (br s, 1H), 8.79 (br
s, 1H), 8.78 (br d, J=0.8 Hz, 1H), 8.23 (dd, J=8.8, 2.0 Hz, 1H),
8.19 (dd, J 8.8, 2.0 Hz, 1H), 8.16 (dd, J=8.8, 2.0 Hz, 1H),
8.02-7.91 (m, 9H), 7.67-7.55 (m, 8H), 7.35 (d, J=8.4 Hz, 2H), 7.09
(d, J=2.4 Hz, 111), 6.95 (d, J=2.4 Hz, 1H), 5.63 (dd, J=13.6, 2.8
Hz, 1H), 3.12 (dd, J=16.8, 13.6 Hz, 1H), 2.85 (dd, J=16.8, 2.8 Hz,
1H) ppm.
[0100] .sup.13CNMR (CDCl.sub.3) .delta. 188.8, 165.1, 165.0, 164.0,
163.2, 156.3, 151.6, 151.2, 135.9, 135.82, 135.78, 135.7, 132.5,
132.38, 132.34, 132.3, 132.2, 132.0, 129.54, 129.50, 129.4, 129.4,
128.9, 128.7, 128.6, 128.5, 128.38, 128.36, 127.80, 127.78, 127.74,
127.4, 127.0, 126.8, 126.62, 126.60, 126.4, 125.7, 125.5, 125.3,
125.2, 122.2, 112.2, 111.1, 109.5, 79.2,45.3 ppm.
[0101] Example 12: Decrease of Plasma Cholesterol, HDL-Cholesterol
and Triglyceride Lipid Levels in Compound 4(NG5006)-Fed Rats
[0102] (Step 1) Administration of NG5006 to rats
[0103] 30 four-week-old white Sprague-Dawley rats (Korea Institute
of Science and Technology, Korea), each weighing about 90 to 120 g,
were evenly divided into three dietary groups by a randomized block
design. The rats of the three groups were fed with three different
high-cholesterol diets, i.e., AIN-76 laboratory animal diet(ICN
Biochemicals, Cleveland, Ohio, U.S.A.) containing 1%
cholesterol(Control group), 1% cholesterol plus 0.02% naringenin
and 1% cholesterol plus 0.036% NG5006, respectively. The
compositions of the diets fed to the three groups are shown in
Table II.
2 TABLE II Control Naringenin NG5006 Dietary group group group
group Component (n = 10) (n = 10) (n = 10) Casein 20 20 20
D,L-methionine 0.3 0.3 0.3 Corn starch 15 15 15 Sucrose 49 48.98
48.964 Cellulose powder 5 5 5 Mineral mixture.sup.*1 3.5 3.5 3.5
Vitamin mixture.sup.*1 1 1 1 Choline bitartrate 0.2 0.2 0.2 Corn
oil 5 5 5 Cholesterol 1 1 1 Naringenin.sup.*2 -- 0.02 -- NG5006 --
-- 0.036 Total 100 100 100 .sup.*1Purchased from TEKLAD premier Co.
(Madison, WI, U.S.A.) .sup.*2Purchased from Sigma Chemical Co. (St.
Louis, MO, U.S.A.)
[0104] The rats were allowed to feed freely on the specified diet
together with water for six weeks, the ingestion amount was
recorded daily and the rats were weighed every 7 days, and then the
record was analyzed. All rats showed a normal growth rate and there
were observed no significant difference among the three groups in
terms of the feed ingestion amount and the weight gain. (Step 2)
Determination of total cholesterol, HDL-cholesterol and
triglyceride contents in blood
[0105] The effects of administering naringenin or NG5006 to rats on
the plasma cholesterol and triglyceride contents were determined as
follows.
[0106] Blood samples were taken from the rats of the three dietary
groups and total cholesterol level was determined by using Sigma
Diagnostic Kit Cat. No. C0534(Sigma Chemical Co., U.S.A.). HDL
fractions were separated therefrom by using HDL-cholesterol
reagent(Sigma Chemical Co., Cat. No. 352-1) and HDL-cholesterol
level was determined by using Sigma Diagnostic Kit Cat. No.
C9908(Sigma Chemical Co., U.S.A.). Triglyceride level was
determined by using Sigma Diagnostic Kit Cat. No. 336-10(Sigma
Chemical Co., U.S.A.). The result is shown in Table III.
3 TABLE III Group Control Naringenin NG5006 Lipid Conc. group group
group Total-C (mg/dl) 226.8 .+-. 14.0 190.6 .+-. 7.6 183.5 .+-. 6.4
HDL-C (mg/dl) 30.6 .+-. 2.0 35.8 .+-. 1.5 36.7 .+-. 1.3 HDL-C 13.5
.+-. 1.2 18.8 .+-. 0.9 20.4 .+-. 0.7 -------(%) Total-C TG(mg/dl)
153.0 .+-. 8.9 143.7 .+-. 9.4 118.4 .+-. 3.4 AI 6.4 .+-. 0.5 4.3
.+-. 0.3 4.0 .+-. 0.2 * Total-C: Total-cholesterol * HDL-C:
HDL-cholesterol * TG: Triglyceride * AI: Atherosclerosis Index (AI
= (Total-C - HDL-C)/HDL-C)
[0107] As can be seen in Table III, the total plasma cholesterol
level is lower by 19% and 16% in NG5006 and the naringenin groups,
respectively, than in the Control group. Further, the triglyceride
level is lower by 33% and 6% in the NG5006 and the naringenin
groups, respectively, than in the control group. This result
demonstrates that NG5006 is superior to naringenin in lowering
blood lipid level.
[0108] Example 13: Inhibitions of ACAT and HMG-CoA Reductase
Activities in NG5006-Fed Rats
[0109] (Step 1) Preparation of microsomes
[0110] To determine the effect of feeding NG5006 to rats on the
activities of ACAT and HMG-CoA reductase, microsomes were separated
from liver tissues to be used as an enzyme source.
[0111] 1 g each of the livers taken from each group of rats of
Example 12 was homogenized in 5 ml of homogenization medium(0. 1 M
KH.sub.2PO.sub.4, pH 7.4, 0.1 mM EDTA and 10 mM
.beta.-mercaptoethanol). The homogenate was centrifuged at 3,000xg
for 10 min. at 4.degree.C. and the supernatant thus obtained was
centrifuged at 15,000xg for 15 min. at 4.degree.C. to obtain a
supernatant. The supernatant was put into an ultracentrifuge
tube(Beckman) and centrifuged at 100,000xg for 1 hour at
4.degree.C. to obtain microsomal pellets, which were then suspended
in 3 ml of the homogenization medium and centrifuged at 100,000xg
for 1 hour at 4.degree.C. The pellets thus obtained were suspended
in 1 ml of the homogenization medium. The protein concentration of
the resulting suspension was determined by Lowry's method and then
adjusted to 4 to 8 mg/ml. The resulting suspension was stored in a
deep freezer(Biofreezer, Forma Scientific Inc.).
[0112] (Step 2) ACAT assay
[0113] 6.67 .mu.l of 1 mg/ml cholesterol solution in acetone was
mixed with 6 .mu.l of 10% Triton WR-1339(Sigma Co.) in acetone and
then, acetone was removed from the mixture by evaporation under a
nitrogen flow. Distilled water was added to the resulting mixture
to adjust the concentration of cholesterol to 30 mg/ml.
[0114] Added to 10 .mu.l of the resulting aqueous cholesterol
solution were 10 .mu.l of 1 M KH.sub.2PO.sub.4(pH 7.4), 5 .mu.l of
0.6 mM bovine serum albumin(BSA), 10 .mu.l of microsome solution
obtained in (Step 1) and 55 .mu.l of distilled water(total 90
.mu.l). The mixture was pre-incubated in a water bath at
37.degree.C. for 30 min.
[0115] 10 .mu.l of [1-.sup.14C] oleyl-CoA solution(0.05 .mu.Ci,
final concentration: 10 .mu.M) was added to the pre-incubated
mixture and the resulting mixture was incubated in a water bath at
37.degree.C. for 30 min. Added to the mixture were 500 .mu.l of
isopropanol:heptane mixture(4:1(v/v)), 300 .mu.l of heptane and 200
.mu.l of 0.1 M KH.sub.2PO.sub.4(pH 7.4), and the mixture was mixed
vigorously using a vortex mixer and then allowed to stand at room
temperature for 2 min.
[0116] 200 .mu.l of the resulting supernatant was put in a
scintillation bottle and 4 ml of scintillation fluid(Lumac Co.) was
added thereto. The mixture was assayed for radioactivity with 1450
Microbeta liquid scintillation counter(Wallac Co., Finland). ACAT
activity was calculated as picomoles of cholesteryl oleate
synthesized per min. per mg protein(pmoles/min/mg protein). The
result is shown in Table IV.
[0117] (Step 3) HMG-CoA reductase assay
[0118] The activity of HMG-CoA reductase was determined by
employing [.sup.14C]HMG-CoA, in accordance with the method of
Shapiro et al.(Biochemica et Biophysica Acta, 370, 369-377(1974))
as follows.
[0119] The enzyme in the supernatant containing the microsome
obtained in (Step 1) was activated at 37.degree.C. for 30 min.
Added to a reaction tube were 20 .mu.l of HMG-CoA reductase assay
buffer(0.25M KH.sub.2PO.sub.4(pH 7.0), 8.75 mM EDTA, 25 mM DTT,
0.45 M KCl and 0.25 mg/ml BSA), 5 .mu.l of 50 mM NADPH, 5 .mu.l of
[14C]HMG-CoA(0.05.mu. Ci/tube, final conc. 120.mu.M), and 10 .mu.l
of activated microsomal enzyme(0.03-0.04 mg), and the mixture was
incubated at 37.degree.C. for 30 min. The reaction was terminated
by adding 10 .mu.l of 6 M HCl to the mixture, and the mixture was
incubated at 37.degree.C. for 15 min. to allow complete
lactonization of the product(mevalonate). The precipitate was
removed by centrifugation at 10,000.times.g for 1 min. and the
supernatant was applied to a Silica gel 60G TLC plate(Altech, Inc.,
Newark, U.S.A.) and then developed with benzene:acetone(1:1, v/v).
A region having a Rf value ranging from 0.65 to 0.75 was removed by
scraping with a disposable cover slips and assayed for
radioactivity with 1450 Microbeta liquid scintillation
counter(Wallac Co., Finland). Enzyme activities were calculated as
picomoles mevalonic acid synthesized per min. per mg
protein(pmoles/min/mg protein). The result is shown in Table
IV.
4TABLE IV Control Naringenin NG5006 Group Group group group ACAT
activity 173.7 .+-. 6.3 153.3 .+-. 6.9 149.7 .+-. 5.1 (pmole/min/mg
protein) HMG-CoA reductase activity 112.4 .+-. 8.7 87.7 .+-. 5.9
81.6 .+-. 6.0 (pmole/min/mg protein)
[0120] As can be seen from Table IV, the control group rats showed
relatively high ACAT and HMG-CoA reductase activities, while the
ACAT and HMG-CoA activities observed in the NG5006-fed rat group
are lower than that of the control group by 14% and 27.5%,
respectively.
[0121] Example 14: Inhibition of Arteriosclerosis in NG5006-Fed
Rabbits
[0122] (Step 1) Administration of NG5006 to rabbits
[0123] 22 three-month-old New Zealand White rabbits(Yeonam
Horticulture and Animal Husbandry College, Korea) each weighing
about 2.0 to 2.3 kg were bred under a condition of temperature
20.+-.2.degree.C., relative humidity 55.+-.10%, and photoperiod
12L/12D. The rabbits were divided two groups of 6 rabbits and a
group of 10 rabbits, and the rats of three groups were fed with
three different diets, i.e., RC4 diet(Oriental Yeast Co., Japan)
containing 1% cholesterol(Control group); 1% cholesterol plus 1
mg/kg Lovastatin.RTM. (Merck, U.S.A.)(Comparative group); and 1%
cholesterol plus 0.1% NG5006, respectively. RC4 diet comprises 7.6%
moisture, 22.8% crude protein, 2.8% crude fat, 8.8% crude ash,
14.4% crude cellulose and 43.6% soluble nitrogen-free substances.
The rabbits were bred for 8 weeks while being allowed free access
to the diets and water.
[0124] (Step 2) Analysis for fatty streak in the main artery
[0125] The rabbits bred in Step 1 were sacrificed and their chest
were incised. The main artery was cut out therefrom in a length of
about 5 cm downward from the site 1 cm above the aortic valve and
the fat surrounding the main artery was removed. The main artery
was incised in the middle along the longitudinal axis and fixed in
10% neutral buffered formalin for 24 hours, and then, pinned to a
dish. The moist artery was photographed and, then, staining of
fatty streak was carried out in accordance with the method of
Esper, E., et al. (J. Lab. Clin. Med., 121, 103-110(1993)) as
follows.
[0126] A part of the incised main artery was washed three times by
2 min. with anhydrous propylene glycol and stained for 30 min. with
a saturated solution of Oil Red O(ORO, Sigma Co.) dissolved in
propylene glycol. Thereafter, the artery was washed twice by 3 min.
with 85% propylene glycol to remove remaining staining solution
and, then washed with physical saline. The artery was photographed
and the photograph was traced. The area of stained legion(fatty
streak legion) was determined with an image analyzer(LEICA, Q-600,
Germany) and its proportion(%) to the total arterial area was
calculated. The result is shown in Table V. The results were tested
by student t-test by using Microsoft excel(version 7.0)
program.
5 TABLE V Control Lovastatin .RTM. NG5006 Group Group Group Group
(n = 6) (n = 6) (n = 10) Fatty Streak lesion (%) 61.8 .+-. 14.6
18.0 .+-. 9.5 12.3 .+-. 6.2
[0127] As can be seen from Table V, NG5006 and Lovastatin.RTM.
groups significantly inhibit the formation of fatty streak as
compared to the control group, while 15 NG5006 group shows more
superior result than Lovastatin.RTM. group.
[0128] FIGS. 1A, 1B and 1C show the arteries of the rabbits
administered with 1% cholesterol(control group); 1% cholesterol
plus 1 mg/kg Lovastatin.RTM. (comparative group); and 1%
cholesterol plus 0.1% NG5006, respectively. As shown in FIGS. 1A,
1B and 1C, a thick layer of fatty streak was observed on the
arterial endothelium of the rabbit administered with 1%
cholesterol, while no or very thin layers of macrophage-lipid
complex were observed on the arterial endotheliums of the rabbits
administered with 1% cholesterol plus 1 mg/kg Lovastatin.RTM. and
1% cholesterol plus 0.1% NG5006, respectively.
[0129] Accordingly, it is concluded that NG5006 strongly inhibits
the arteriosclerosis even when the blood cholesterol level is very
high.
[0130] (Step 3) Histologic observation of the organs
[0131] Portions of the main artery, heart, lung, liver, kidney and
muscle were taken from each of the rabbits sacrificed in step 2 and
visually examined to confirm that no pathogenic abnormality was
found. One half of each portion of the organs was deep freezed and
the other half was fixed in 10% neutral buffered formalin for more
than 24 hours. The fixed organ piece was washed sufficiently with
tap water, dehydrated stepwise with 70%, 80%, 90% and 100% ethanol
and, then, embedded in a paraffin by employing SHANDON7(Histocentre
2, U.S.A.). The embedded organ piece was sectioned in 4 .mu.m
thickness with a microtome(LSICA, RM2045, Germany) and stained with
hematoxylin and eosin. The stained organ specimen was made
transparent with xylene, mounted with permount, and then observed
under a microscope to look for the presence of lesions. No lesion
was observed in any of the organ specimen.
[0132] Example 15: Prevention of Hepatic Diseases
[0133] In order to evaluate the effects of feeding a high
cholesterol diet with NG5006 on liver tissues, the liver specimens
taken from the sacrificed rabbit in Step 2 of Example 14 were
treated in accordance with the procedure disclosed in Fogt F. and
Nanji A., Toxicology and Applied Pharmacology, 136, 87-93, 1996;
and Keegan A., et al., Journal of Hepatology, 23, 591-600, 1995,
and observed under a microscope to be classified into four grades,
i.e., 1+(0-25%), 2+(26-50%), 3+(51-75), 4+(76-100%) based on the
proportion of abnormal fat-containing cells around the central vein
in the liver acinus. The result is shown in Table VI. The results
were tested by student t-test by using Microsoft excel(version 7.0)
program.
6TABLE VI Control Lovastatin .RTM. NG5006 Group Group Group Group
Proportion of abnormal 3.0 .+-. 0.7 3.3 .+-. 1.1 2.2 .+-. 0.9
fat-containing liver cells
[0134] As can be seen in Table VI, NG5006 significantly inhibits
the formation of fatty liver as compared to the Control and the
Lovastatin.RTM. group.
[0135] FIGS. 2A, 2B and 2C present the microscopic features of the
livers of the rabbits administered with 1% cholesterol(control); 1%
cholesterol plus 1 mg/kg Lovastatin.RTM.; and 1% cholesterol plus
0.1% NG5006, respectively. In FIGS. 2A and 2B, many cells
containing excessive fat were observed around the central vein. In
contrast, almost all liver cells are of a normal shape in FIGS. 2C,
which suggested that NG5006 can significantly inhibit the formation
of fatty liver.
[0136] As can be seen from the above, the administration of NG5006
can inhibit the development of fatty liver.
[0137] Example 16: Inhibition of ACAT Activity in NG5006-Fed
Rabbits
[0138] The effect of feeding NG5006 to rabbits on the activities of
ACAT was determined using the rabbits bred in Step 1 of Example 14
in accordance with procedure disclosed in step 1 of Example 13. The
result is shown in Table VII.
7TABLE VII Control Lovastatin .RTM. NG5006 Group Group Group Group
ACAT activity 256.9 .+-. 39.6 246.1 .+-. 52.5 215.2 .+-. 30.3
(pmole/min/mg protein) % Inhibition on ACAT 0 4.2 16.2 Activity
[0139] As can be seen from Table VIII, the ACAT activity observed
in the NG5006-fed rat group is lower than that of the control group
by 16.2%.
[0140] Example 17: Toxicity of Orally Administered NG5006
[0141] 4 week-old, specific pathogen-free ICR female mice(12 heads)
and male mice(12 heads) were bred under a condition of temperature
22.+-.3.degree.C., moisture 55.+-.10% and photoperiod 12L/12D.
Fodder(Cheiljedang Co., mouse and rat fodder) and water were
sterilized and fed to the mice.
[0142] NG5006 was dissolved in 0.5 % Tween 80 to a concentration of
100 mg/ml. The solution thus obtained was orally administered to
the mice in amounts of 0.2 ml(1 g/kg), 0.4 ml(2 g/kg) and 0.8
ml(4g/kg) per 20 g of mouse body weight, respectively. The solution
was administered once and the mice were observed for 7 days for
signs of adverse effects or death according to the following
schedule: 1, 4, 8, and 12 hours after the administration and, every
12 hours thereafter. The weight changes of the mice were recorded
every day to examine the effect of NG5006. Further, on the 7th day,
the mice were sacrificed and the internal organs were visually
examined.
[0143] All the mice were alive at day 7 and NG5006 showed no
toxicity. The autopsy revealed that the mice did not develop any
pathological abnormality, and no weight loss was observed during
the 7 day test period. Accordingly, it was concluded that NG5006 is
not toxic when orally administered to an animal.
[0144] Formulation 1: Preparation of Pharmaceutical Formulation
[0145] Hard gelatin capsules were prepared using the following
ingredients:
8 Quantity (mg/capsule) Active ingredient 20 (NG5006) Starch, dried
160 Magnesium Stearate 20 Total 200
[0146] The above ingredients were mixed thoroughly and filled in a
hard gelatin capsule.
[0147] Formulation 2: Foods containing flavanone derivatives
[0148] Foods containing NG5006 obtained in Example 1 were prepared
as follows.
[0149] (1) Preparation of tomato ketchup and sauce
[0150] 0.2 wt % of NG5006 was added to a tomato ketchup or sauce to
obtain a health-improving tomato ketchup or sauce.
[0151] (2) Preparation of wheat flour foods
[0152] 0.5 wt % of NG5006 was added to wheat flour and breads,
cakes, cookies, crackers and noodles were prepared by using the
mixture to obtain health-improving foods.
[0153] (3) Preparation of soups and gravies
[0154] 0.1 wt % of NG5006 was added to soups and gravies to obtain
health-improving soups and gravies.
[0155] (4) Preparation of ground beef
[0156] 10 wt % of NG5006 was added to ground beef to obtain a
health-improving ground beef.
[0157] (5) Preparation of dairy product
[0158] 5 wt % of NG5006 was added to milk and various dairy
products such as butter and ice cream were prepared by using the
milk.
[0159] However, in case of cheese preparation, NG5006 was added to
the coagulated milk protein; and, in case of yogurt preparation,
NG5006 was added to 2 0 the coagulated milk protein obtained after
the fermentation.
[0160] Formulation 2: Beverages containing flavanone
derivatives
[0161] (1) Preparation of vegetable juice
[0162] 25 5 g of NG5006 was added to 1000 ml of a tomato or carrot
juice to obtain a health-improving vegetable juice.
[0163] (2) Preparation of fruit juice
[0164] 1 g of NG5006 was added to 1000 ml of an apple or grape
juice to obtain a health-improving fruit juice.
[0165] While the invention has been described with respect to the
above specific embodiments, it should be recognized that various
modifications and changes may be made to the invention by those
skilled in the art which also fall within the scope of the
invention as defined by the appended claims.
* * * * *