U.S. patent application number 09/726338 was filed with the patent office on 2001-12-27 for method of promoting synthesis of nerve growth factor.
Invention is credited to Ito, Hisatomi, Kosaka, Kunio, Miyazaki, Toshitsugu.
Application Number | 20010056121 09/726338 |
Document ID | / |
Family ID | 18506871 |
Filed Date | 2001-12-27 |
United States Patent
Application |
20010056121 |
Kind Code |
A1 |
Kosaka, Kunio ; et
al. |
December 27, 2001 |
Method of promoting synthesis of nerve growth factor
Abstract
A method of promoting the synthesis of nerve growth factor
comprising administering an effective amount of rosemary and/or
sage extracts or carnosic acid and/or carnosol as an effective
ingredient to a subject requiring such promotion. The present
method can safely and efficiently promote the production of NGF in
the living body, without being accompanied by a side effect such as
a loss of a quantitative balance of hormones in the living
body.
Inventors: |
Kosaka, Kunio; (Kobe,
JP) ; Miyazaki, Toshitsugu; (Kobe, JP) ; Ito,
Hisatomi; (Kobe, JP) |
Correspondence
Address: |
JACOBSON HOLMAN PLLC
400 SEVENTH STREET N.W.
SUITE 600
WASHINGTON
DC
20004
US
|
Family ID: |
18506871 |
Appl. No.: |
09/726338 |
Filed: |
December 1, 2000 |
Current U.S.
Class: |
514/570 ;
424/745 |
Current CPC
Class: |
A61K 31/366 20130101;
A61K 31/192 20130101; A61K 36/53 20130101; A61K 36/537 20130101;
A61P 43/00 20180101; A61P 25/28 20180101; A61K 36/53 20130101; A61P
25/00 20180101; A61K 36/537 20130101; A61K 31/366 20130101; A61K
31/192 20130101; A61K 2300/00 20130101; A61K 2300/00 20130101; A61K
2300/00 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
514/570 ;
424/745 |
International
Class: |
A61K 031/19; A61K
035/78 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 2, 1999 |
JP |
376276/1999 |
Claims
1. A method of promoting the synthesis of nerve growth factor
comprising administering an effective amount of at least one plant
extract having an effect of promoting the synthesis of nerve growth
factor selected from the group consisting of rosemary and sage
extracts as an effective ingredient to a subject requiring such
promotion.
2. The method according to claim 1 wherein the rosemary and sage
extracts are those obtained by extracting rosemary and sage with
ethanol or a mixture of water and ethanol, respectively.
3. The method according to claim 1 or 2 wherein the plant extract
is administered in the form of a drug or food composition.
4. A method of producing a plant extract having an effect of
promoting the synthesis of nerve growth factor comprising the steps
of: soaking rosemary or sage in ethanol or an aqueous ethanol
solution having an ethanol concentration of 80% (v/v) to 100% (v/v)
to obtain a first extract; adding water to the first extract so as
to give an ethanol concentration of not greater than 40% (v/v), to
deposit a precipitate having an effect of promoting the synthesis
of nerve growth factor; and separating the precipitate to obtain
the desired plant extract.
5. A method of promoting the synthesis of nerve growth factor
comprising administering an effective amount of at least one
ingredient selected from the group consisting of carnosic acid and
carnosol as an effective ingredient to a subject requiring such
promotion.
6. The method according to claim 5 wherein the ingredient is
administered in the form of a drug or food composition.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a method of promoting the
synthesis of nerve growth factor, more particularly, a method
capable of efficiently promoting the synthesis of nerve growth
factor in the treatment of nerve-denaturing diseases such as
Alzheimer-type dementia and brain ischemia pathologies.
DESCRIPTION OF THE PRIOR ART
[0002] Senile dementia has a tendency to increase with the shift to
an aging society. This tendency has become an extremely large
social problem. A number of diseases are known which are
responsible for senile dementia. These are roughly divided into
dementia attributable to an organic disorder of the brain, dementia
incidental to a disease of other organs than the brain, and
dementia attributable to a physical disease due to stress. In
particular, the dementia attributable to an organic disorder of the
brain, which constitutes the greater part of the causes of
dementia, is divided into cerebrovascular dementia and
Alzheimer-type dementia due to the differences of the causes.
[0003] Currently, it has been known that a drug such as a
cerebrovascular dilator exhibits a certain effect on the
cerebrovascular dementia. However, the causes of development of
Alzheimer-type dementia are not known yet, and a pharmacotherapy
and other treating methods suitable for preventing the development
and progression of the dementia are not known yet. Accordingly, it
is greatly desired to develop a drug useful for the treatment of
dementia due to an organic disorder of the brain, in particular,
Alzheimer-type dementia.
[0004] Recently, it has been found that a neurotrophic factor such
as nerve growth factor (NGF) secreted from nerve cells has an
excellent effect on nerve-denaturing diseases and special attention
has been paid to the factor. NGF is a factor necessary and
important to the growth and functional maintenance of the nervous
tissue. NGF is essential for maturation, differentiation and
survival of sensory and sympathetic nerves in the peripheral
nervous system as well as for those of large cell cholinergic
neurons in the central nervous system. Also, NGF exhibits an effect
of preventing denaturing of nerve cells when undergoing a brain
lesion. Accordingly, it is believed that an elevation of the NGF
level in the living body is effective for treating a disorder of
central functions (including Alzheimer-type dementia and
cerebrovascular dementia), a lesion of peripheral nerves, a
diabetic neuropathy and a disorder of peripheral functions
(including amyotrophic lateral sclerosis).
[0005] However, NGF is a protein having a high molecular weight of
about 13,000 in its monomer form and about 26,000 in its dimer
form, and can not pass through the blood-brain barrier.
Accordingly, it is necessary to administer NGF intraventricularly,
for example, when the treatment of a disorder of central functions
is aimed. In addition, it is difficult to prepare NGF in a large
amount. Thus, it is very problematic to use NGF per se.
Consequently, it is very difficult to use NGF per se
clinically.
[0006] A method of administering a substance for promoting the
synthesis of NGF in the living body, instead of NGF, is also known
in the art. For example, Y. Furukawa et al. (FEBS Lett., Vol.208
(1986), p.258 et seq.) discloses that catecholamines (epinephrine,
norepinephrine and dopamine) are used as the substance for
promoting the synthesis of NGF.
[0007] However, they are hormone substances, and therefore, the
administration of them causes a problem of losing a quantitative
balance of hormones in the living body.
[0008] The present invention is addressed to the solution of the
above problems. Thus, the object of the present invention is to
provide a substance for promoting the synthesis of NGF which allows
an effective synthesis of NGF in the living body without being
accompanied by a side effect such as a loss of a quantitative
balance of hormones in the living body. Another object of the
present invention is to provide a method of promoting the synthesis
of NGF in the living body.
SUMMARY OF THE INVENTION
[0009] The present inventors have intensively searched for a
substance having a potent effect of promoting the synthesis of NGF.
As a result, they found that rosemary and sage extracts have such
an effect. Furthermore, they found that carnosic acid of the
formula (I) below and carnosol of the formula (II) below, which are
contained in the rosemary and sage extracts, also have the above
effect. 1
[0010] Thus, the present invention provides a method of promoting
the synthesis of nerve growth factor comprising administering an
effective amount of at least one plant extract having an effect of
promoting the synthesis of nerve growth factor selected from the
group consisting of rosemary and sage extracts as an effective
ingredient to a subject requiring such promotion.
[0011] In a preferred embodiment, the above rosemary and/or sage
extracts are those obtained by extracting rosemary and/or sage with
ethanol or a mixture of water and ethanol.
[0012] Also, the present invention provides a method of producing a
plant extract having an effect of promoting the synthesis of nerve
growth factor comprising the steps of:
[0013] soaking rosemary or sage in ethanol or an aqueous ethanol
solution having an ethanol concentration of 80% (v/v) to 100% (v/v)
to obtain a first extract;
[0014] adding water to the first extract so as to give an ethanol
concentration of not greater than 40% (v/v), to deposit a
precipitate having an effect of promoting the synthesis of nerve
growth factor; and
[0015] separating the precipitate to obtain the desired plant
extract.
[0016] Furthermore, the present invention provides a method of
promoting the synthesis of nerve growth factor comprising
administering an effective amount of at least one ingredient
selected from the group consisting of carnosic acid of the above
formula (I) and carnosol of the above formula (II) as an effective
ingredient to a subject requiring such promotion.
DETAILED DESCRIPTION OF THE INVENTION
[0017] In the present invention, the rosemary and/or sage extracts
as well as carnosic acid and/or carnosol are generally administered
in the form of a preparation for promoting the synthesis of nerve
growth factor.
[0018] The first preparation according to the present invention
contains a rosemary extract or a sage extract. The rosemary and
sage extracts used in the present invention are obtained from
rosemary (Rosmarinus officinalis L.) and sage (Salvia officinalis
L.). Rosemary and sage are plants having a high safety and are
widely used for a long time in the West as a herb for medicinal,
fragrant and cooking purposes. In the present invention, the
rosemary and sage extracts may be used in admixture with each
other.
[0019] The content of the rosemary and sage extracts contained in
the first preparation as an effective ingredient is preferably
about 0.0001% to 100% by weight, more preferably about 0.1% to 100%
by weight, per 100% by weight of the first preparation. If the
content of the extracts is less than 0.0001% by weight, the
preparation can not produce an sufficient amount of NGF.
[0020] The rosemary or sage extract may be obtained in the
following manner, for example. Firstly, the whole plant, leaves
and/or petals of rosemary or sage are soaked in an extraction
solvent, or refluxed with the extraction solvent. There is no
limitation in the type of the extraction solvent used. Examples of
the extraction solvent which may be used are organic solvents such
as methanol, ethanol, propanol, butanol, propylene glycol,
1,3-butylene glycol, glycerin, acetone, methyl ethyl ketone, ethyl
acetate, ethers, chloroform and dichloromethane as well as water.
These solvents may be used alone or in combination with one
another. In the present invention, it is preferable to use
methanol, ethanol, ethyl acetate or a mixture of these solvents
with water. More preferably, ethanol or a mixture of water and
ethanol is used, in the light of the safety (low toxicity) in the
living body.
[0021] Preferably, the rosemary and sage extracts are obtained in
the following manner, respectively. Firstly, rosemary or sage are
soaked in ethanol or an aqueous ethanol solution having an ethanol
concentration of 80% (v/v) to 100% (v/v), preferably 90% (v/v) to
100% (v/v) to obtain a first extract.
[0022] There is no limitation in the extraction temperature and
time to obtain the first extract, and those skilled in the art may
determine suitable conditions. Typical conditions include a
temperature of about 20.degree. C. to about 50.degree. C. and an
extraction time of about 24 hrs to about 72 hors.
[0023] Then, water is added to the first extract so as to give an
ethanol concentration of not greater than 40% (v/v), preferably not
greater than 30% (v/v). By doing so, it is possible to deposit a
precipitate having an effect of promoting the synthesis of nerve
growth factor from the first extract. Alternatively, the above
first extract may be concentrated to one-half to one-twentieth of
its original volume, and then, water may be added to the
concentrate to deposit the rosemary or sage extract.
[0024] There is no limitation in the deposition temperature and
time to obtain the rosemary or sage extract from the first extract,
and those skilled in the art may determine suitable conditions.
Typical conditions include a temperature of about 0.degree. C. to
about 25.degree. C. and a deposition time of about 16 hrs to about
48 hors.
[0025] By repeating several times the step of adding ethanol or
water to the extract so as to give a particular ethanol
concentration, the step of dissolving or depositing a precipitate,
and the step of filtration, it is possible to obtain the extract
described below containing carnosic acid of the formula (I) and
carnosol of the formula (II) in a large amount. By using the
extract obtained by such extraction procedures, it is possible to
remarkably increase the ability to produce NGF.
[0026] The first preparation according to the present invention may
be made up into suitable forms such as food or drug compositions.
Also, the first preparation according to the present invention may
be used for both of oral administration and parenteral
administration.
[0027] In case of making up into food compositions, the above
rosemary extract and/or sage extract are mixed with suitable
materials which may be commonly used as food materials. Examples of
the food materials are rice, wheat, corn, potato, sweet potato,
soybean, sea tangle, wakame (Undaria pinnatifida), or agar weed;
starch syrup; lactose; glucose; fructose; sucrose; mannitol; and
combinations of these materials. In addition, flavoring agents,
coloring agents, sweetening agents, edible oils, vitamins and the
like may be added to the food compositions. These materials and
additives may be used alone or in combination with one another.
Also, the food compositions may be made up into a desired shape, if
necessary, by adding water.
[0028] In case of making up into drug compositions, the above
rosemary extract and/or sage extract are mixed with suitable
additives. Examples of the additives are surfactants, excipients,
coloring agents, preservatives, coating aids and combinations of
these additives. These additives may be those commonly used in the
production of drug compositions and are not limited to particular
ones. More specific examples of the additives are lactose, dextrin,
sucrose, mannitol, corn starch, sorbitol, crystalline cellulose,
polyvinylpyrrolidone and combinations of these additives. Also,
flavoring agents, sweetening agents and the like may be added to
the drug compositions. In addition, other drugs may be added to the
drug compositions, if necessary.
[0029] There is no limitation in dosage forms of the drug
compositions and they may be produced in suitable dosage forms
according to a conventional process. For oral administration, in
particular, the compositions may be prepared in the forms of
capsules, tablets, powder, slow-releasing agents and the like. For
parenteral administration, the compositions may be prepared in the
forms of injections, infusions and the like.
[0030] There is no limitation in the content of the above suitable
materials and additives and the compositions may be produced
depending on the content of the above rosemary extract and/or sage
extract.
[0031] The second preparation according to the present invention
contains carnosic acid of the above formula (I) or carnosol of the
above formula (II). In the present invention, carnosic acid of the
formula (I) and carnosol of the formula (II) may be used in
admixture with each other.
[0032] The content of carnosic acid of the formula (I) and carnosol
of the formula (II) contained in the second preparation as an
effective ingredient is preferably about 0.00001% to 100% by
weight, more preferably about 0.001% to 100% by weight, per 100% by
weight of the second preparation. If the content of the above
ingredients is less than 0.00001% by weight, the preparation can
not promote the production of NGF sufficiently.
[0033] Although carnosic acid of the above formula (I) and carnosol
of the above formula (II) may be prepared by chemical synthesis, in
general, they may be prepared by isolation from a plant extract,
preferably from the above rosemaryor sage extract. Typically,
carnosic acid of the formula (I) and carnosol of the formula (II)
are prepared by removing impurities from the above rosemary or sage
extract through various column chromatographic means. Those skilled
in the art can easily identify by well known means such as
.sup.1H--NMR or .sup.13C--NMR that the substance thus obtained is
carnosic acid of the formula (I) or carnosol of the formula
(II).
[0034] Similar to the above first preparation, the second
preparation according to the present invention may also be made up
into suitable forms such as food or drug compositions, and may be
used for both of oral administration and parenteral
administration.
EXAMPLES
[0035] The present invention is illustrated in more detail by the
following examples, but it is not limited thereto.
Example 1
[0036] Rosemary (whole plant, 60 g) was soaked in aqueous 90%
ethanol solution (300 ml), and extracted at 40.degree. C. for 48
hrs. The resultant solution was concentrated to a volume of 100 ml.
After the concentration, the concentrate was filtered to remove
insoluble materials. Purified water (200 ml) was added to the
filtrate and the mixture was allowed to stand overnight at
4.degree. C. Subsequently, the mixture was again filtered to obtain
a rosemary extract (dry weight 3.5 g) which is insoluble part.
[0037] Human glioblastoma cells (T98G, Cell line) were seeded in a
MEM medium containing fetal bovine serum (10%; Gibco), sodium
pyruvate (x 1; Gibco) and nonessential amino acids (x 1; Gibco) in
wells of a flat-bottom 96-well plate (Corning), in a cell density
of 2.times.10.sup.4/well, and cultivated in a CO.sub.2 incubator
(37.degree. C., 5% CO.sub.2) for 3 days. The medium was then
replaced by an Opti-MEM medium (containing 5 mg/ml of bovine serum
albumin). The cultivation was continued for further 6 days with
replacing the medium at intervals of 3 days.
[0038] After removing the medium, an Opti-MEM medium (containing 5
mg/ml of bovine serum albumin) containing 5 ug (microgram)/ml of
the above rosemary extract was added to each well in an amount of
50 ul (microliter) /well, and the cultivation was continued for
further 4 days. After the cultivation, the supernatant was taken as
a sample solution.
[0039] On the other hand, 1 ug/ml of an anti-NGF antibody (Promega)
solution (50 ul) was added to each well of a 96-well microplate
(Nunc), and the plate was allowed to stand overnight at 4.degree.
C. After washing the plate with PBS(-) (Nissui Pharmaceuticals), 1%
of a bovine serum albumin (Sigma) solution (100 ul) was added to
each well of the plate and the plate was allowed to stand at room
temperature for 4 hrs. Subsequently, the plate was washed with PBS
(-) and the above sample solution (50 ul) was added to each well of
the plate. After the reaction at room temperature for one hour, the
plate was washed with PBS (-).
[0040] Subsequently, 0.4 unit/ml of an beta-galactosidase-labeled
anti-NGF antibody (Boehringer Mannheim) solution (50 ul) was added
to each well of the plate, and allowed to react at room temperature
for one hour. After washing the plate with PBS (-), 0.5 mg/ml of a
4-methylumbelliferyl-beta-- D-galactoside solution (200 ul) was
added to each well of the plate, and allowed to react overnight at
room temperature. The fluorescence intensity of
4-methylumbelliferone produced was measured on a fluorescence plate
reader, and the content of NGF contained in the sample solution was
determined using a standard curve obtained from a standard solution
(human beta-NGF; PEPRO TECH EC). The results are shown in Table 1
below.
Example 2
[0041] The content of NGF contained in the sample solution was
determined as described in Example 1, except that an Opti-MEM
medium (containing 5 mg/ml of bovine serum albumin) containing 10
ug/ml of the rosemary extract of Example 1 was used in an amount of
50 ul/well, instead of the Opti-MEM medium containing 5 ug/ml of
the rosemary extract. The results are shown in Table 1 below.
Comparative Example 1
[0042] The content of NGF contained in the sample solution was
determined as described in Example 1, except that an Opti-MEM
medium (containing 5 mg/ml of bovine serum albumin) not containing
the rosemary extract of Example 1 was used in an amount of 50
ul/well, instead of the Opti-MEM medium containing 5 ug/ml of the
rosemary extract. The results are shown in Table 1 below.
1 TABLE 1 NGF Content Concent- in sample Relative Effective ration
solution NGF ingredient (ug/ml).sup.*1 (pg/ml).sup.*2
content.sup.*3 Example 1 Rosemary 5 12.9 .+-. 1.7 2.1 extract
Example 2 Rosemary 10 15.6 .+-. 3.3 2.5 extract Comparative none 0
6.2 .+-. 0.5 1.0 example 1 .sup.*1: Concentration off effective
ingredient in medium .sup.*2: NGF Content shown by mean
measurements .+-. standard deviation .sup.*3: Ratio of example
solutions to comparative example solution
Example 3
[0043] Sage (whole plant, 60 g) was soaked in ethanol (300 ml), and
the mixture was allowed to stand overnight at room temperature. The
resultant solution was then concentrated to a volume of 100 ml. The
concentrated solution was filtered to remove insoluble materials.
Purified water (600 ml) was added to the filtrate and the mixture
was allowed to stand overnight at 4.degree. C. Subsequently, the
mixture was again filtered to obtain a sage extract (dry weight 4
g) which is insoluble part. The content of NGF contained in the
sample solution was determined as described in Example 1, except
that the sage extract was used instead of the rosemary extract. The
results are shown in Table 2 below.
Example 4
[0044] The content of NGF contained in the sample solution was
determined as described in Example 1, except that an Opti-MEM
medium (containing 5 mg/ml of bovine serum albumin) containing 10
ug/ml of the sage extract of Example 3 was used in an amount of 50
ul/well, instead of the Opti-MEM medium containing 5 ug/ml of the
rosemary extract. The results are shown in Table 2 below.
Comparative Example 2
[0045] The content of NGF contained in the sample solution was
determined as described in Example 1, except that an Opti-MEM
medium (containing 5 mg/ml of bovine serum albumin) not containing
the sage extract of Example 3 was used in an amount of 50 ul/well,
instead of the Opti-MEM medium containing 5 ug/ml of the rosemary
extract. The results are shown in Table 2 below.
2 TABLE 2 NGF Content Concent- in sample Relative Effective ration
solution NGF ingredient (ug/ml).sup.*1 (pg/ml).sup.*2
content.sup.*3 Example 3 Sage 5 17.4 .+-. 1.6 2.6 extract Example 4
Sage 10 28.4 .+-. 1.6 4.2 extract Comparative none 0 6.8 .+-. 0.3
1.0 example 2 .sup.*1: Concentration of effective ingredient in
medium .sup.*2: NGF Content shown by mean measurements .+-.
standard deviation .sup.*3: Ratio of example solutions to
comparative example solution
[0046] As shown in Tables 1 and 2, it is evident that the content
of NGF in the sample solution increases in the systems containing
the rosemary or sage extract according to the present invention
(Examples 1 to 4), as compared with the systems not containing any
effective ingredient (Comparative Examples 1 and 2). It is also
evident that the higher the concentration of the effective
ingredient used, the more the amount of NGF produced.
Example 5
[0047] Rosemary (whole plant, 5 kg) was soaked in ethanol (20 L),
and extracted at 40.degree. C. for 72 hrs. The resultant solution
was concentrated to a volume of 1 L. After the concentration, the
concentrate was filtered to remove insoluble materials. Purified
water (2 L) was added to the filtrate and the precipitate (105 g)
deposited at this time was filtered. The precipitate was dissolved
in ethyl acetate, and separated and purified through a silica gel
column chromatography (developing solvent; ethyl acetate:hexane=1:4
(v/v)). The crystals obtained by removing the solvent were
recrystallized in hexane to obtain pale yellow crystal 1 (1.5 g)
and white crystal 2 (0.8 g). .sup.13C--NMR and .sup.1H--NMR spectra
(CDCl.sub.3) of crystals 1 and 2 are shown in the following Tables
3 and 4, respectively.
3TABLE 3 NMR spectra of crystal 1 .sup.13C, .delta. (ppm) .sup.1H,
.delta. (ppm) 34.4(t); 20.3(t); 1.24(1H, m) and. 3.29(1H, m);
41.8(t); 34.4(s); 1.60(1H, m) and 1.75(1H, m); 54.0(d); 18.9(t);
1.32(1H, m) and 1.50(1H, dt); 31.5(t); 129.0(s); 1.57(1H, dd);
122.1(s); 48.7(s); 1.86(1H, m) and 2.36(1H, m); 142.1(s); 141.4(s);
2.85(2H, m); 6.64(1H, s); 133.8(s); 119.4(d); 3.17(1H, m); 1.21(3H,
d); 27.2(d); 22.1(q); 1.20(3H, d); 0.89(3H, s); 22.5(q); 32.6(q);
1.00(3H, s); 6.56(2H, s); 21.7(q); 183.1(s) 7.36(1H, s)
[0048]
4TABLE 4 NMR spectra of crystal 2 .sup.13C, .delta. (ppm) .sup.1H,
.delta. (ppm) 30.0(t); 19.9(t); 2.81(1H, m) and 2.51(1H, td);
42.2(t); 35.2(s); 1.97(1H, m) and 1.533(1H, m); 46.5(d); 30.8(t);
1.27(1H, m) and 1.474(1H, m); 78.1(d); 123.7(s); 1.64(1H, dd);
133.2(s); 49.2(s); 1.77(1H, m) and 2.144(1H, m); 144.0(s);
144.1(s); 5.28(2H, dd); 6.64(1H, s); 134.7(s); 112.1(d); 3.22(1H,
m); 1.18(3H, d); 27.2(d); 23.0(q); 1.17(3H, d); 0.87(3H, s);
23.1(q); 20.1(q); 0.83(3H, d); 32.2(q); 175.2(s) 7.7-7.2(2H,
OH)
[0049] From the results of NMR spectra, it was found that crystal 1
is carnosic acid of the above formula (I) and crystal 2 is carnosol
of the above formula (II).
[0050] Carnosic acid of the formula (I) obtained as described above
was used as an effective ingredient. The content of NGF contained
in the sample solution was determined as described in Example 1,
except that an Opti-MEM medium (containing 5 mg/ml of bovine serum
albumin) containing carnosic acid of the formula (I) at a
concentration of 20 uM was used in an amount of 50 ul/well, instead
of the Opti-MEM medium containing 5 ug/ml of the rosemary extract.
The results are shown in Table 5 below.
Example 6
[0051] The content of NGF contained in the sample solution was
determined as described in Example 1, except that an Opti-MEM
medium (containing 5 mg/ml of bovine serum albumin) containing
carnosic acid of the formula (I) at a concentration of 100 uM was
used in an amount of 50 ul/well, instead of the Opti-MEM medium
containing 5 ug/ml of the rosemary extract. The results are shown
in Table 5 below.
Example 7
[0052] Carnosol of the formula (II) obtained in Example 5 was used
as an effective ingredient. The content of NGF contained in the
sample solution was determined as described in Example 1, except
that an Opti-MEM medium (containing 5 mg/ml of bovine serum
albumin) containing carnosol of the formula (II) at a concentration
of 20 uM was used in an amount of 50 ul/well, instead of the
Opti-MEM medium containing 5 ug/ml of the rosemary extract. The
results are shown in Table 5 below.
Example 8
[0053] The content of NGF contained in the sample solution was
determined as described in Example 1, except that an Opti-MEM
medium (containing 5 mg/ml of bovine serum albumin) containing
carnosol of the formula (II) at a concentration of 100 uM was used
in an amount of 50 ul/well, instead of the Opti-MEM medium
containing 5 ug/ml of the rosemary extract. The results are shown
in Table 5 below.
Comparative Example 3
[0054] The content of NGF contained in the sample solution was
determined as described in Example 1, except that an Opti-MEM
medium (containing 5 mg/ml of bovine serum albumin) not containing
any effective ingredient was used in an amount of 50 ul/well,
instead of the Opti-MEM medium containing 5 ug/ml of the rosemary
extract. The results are shown in Table 5 below.
5 TABLE 5 NGF Content Concent- in sample Relative Effective ration
solution NGF ingredient (uM).sup.*1 (pg/ml).sup.*2 content.sup.*3
Example 5 Carnosic acid 20 46.0 .+-. 7.4 7.2 of formula (I) Example
6 Carnosic acid 100 158.5 .+-. 16.6 24.8 of formula (I) Example 7
Carnosol of 20 35.4 .+-. 6.9 5.5 formula (II) Example 8 Carnosol of
100 39.9 .+-. 2.3 6.2 formula (II) Comparative none 0 6.4 .+-. 0.3
1.0 example 3 .sup.*1: Concentration of effective ingredient in
medium .sup.*2: NGF Content shown by mean measurements .+-.
standard deviation .sup.*3: Ratio of example solutions to
comparative example solution
[0055] As shown in Table 5, it is evident that the content of NGF
in the sample solution increases in the systems containing carnosic
acid of the formula (I) (Examples 5 and 6) or carnosol of the
formula (II) (Examples 7 and 8) according to the present invention,
as compared with the system not containing any effective ingredient
(Comparative Example 3). It is also evident that the higher the
concentration of the effective ingredient used, the more the amount
of NGF produced.
[0056] According to the present method, it is possible to
efficiently promote the synthesis of NGF. The present method can
promote the production of NGF in the living body with a high
safely, without being accompanied by a side effect such as a loss
of a quantitative balance of hormones in the living body. It is
expected by the increase of NGF in the living body that
nerve-denaturing diseases such as Alzheimer-type dementia and brain
ischemia pathologies are prevented and treated.
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