JP2002087975A - Agent for suppressing production of nitrogen monoxide and skin care preparation, cosmetic and quasi-drug formulating the same agent therein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To relate to a nitrogen monoxide production-suppressing agent for suppressing excess production of nitrogen monoxide in vivo and a skin lotion preparation, a cosmetic and a quasi-drug in which the above nitrogen monoxide production-suppressing agent is formulated and provide the nitrogen monoxide production-suppressing agent used as the skin lotion preparation, the cosmetic and the quasi-drug having effect for improving skin disorder caused by ultraviolet light and inflammatory response and excellent in safety and feeling to users. SOLUTION: This nitrogen monoxide production-suppressing agent comprises at least one kind of rosemary extract, carnosol, carnosic acid, coffee bean extract, Pratia nummularia extract, Coptis japonica extract, Phellodendron Bark extract, Glycyrrhiza extract, Rosa Canina extract, Cnidii Rhizoma extract, persicae semen extract, Peonia albiflora extract, coix seed extract and red grape extract.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a nitric oxide production inhibitor for suppressing excessive production of nitric oxide in a living body, a skin external preparation containing the nitric oxide production inhibitor, and a cosmetic. And quasi-drugs.

[0002]

2. Description of the Related Art As is well known, nitric oxide is a nitrogen oxide that causes air pollution and acid rain, but has recently been found to be a physiologically important substance produced in a living body. Have been.

[0003] In 1980, Furchgott et al. Discovered an endothelium-derived vasodilator (EDRF) that functions as a vasorelaxant instead of prostacyclin, and it was discovered by Moncada et al. In 1987 that it was identical to nitric oxide. Proven.

Since then, in fields other than the circulatory system, the role of nitric oxide as a signal transmitting substance in many physiological functions in vivo such as the nervous system and the immune system has been clarified one after another.

[0005]

However, when a large amount of nitric oxide is biosynthesized, it is not nontoxic to living organisms. For example, a large amount of biosynthesized nitric oxide causes relaxation and excessive permeation of vascular smooth muscle. It is also known to cause increased sex and to cause endotoxin shock by significantly lowering blood pressure.

[0006] Production of large amounts of nitric oxide is considered to be one of the causes of many autoimmune diseases such as long-term and short-term inflammations and also type I diabetes.

In view of the above points, the present inventors have conducted intensive studies, and as a result, rosemary extract, carnosol, carnosic acid, coffee bean extract, primrose extract, spinach extract, and oak extract At least one component selected from liquorice extract, liquorice extract, cane rose extract, senkyu extract, tounin extract, peony extract, yokunin extract, and red grape extract, contains nitric oxide produced by ultraviolet irradiation or an inflammatory reaction. Were specifically suppressed, and the skin disorder resulting therefrom was found to be improved, and the present invention was completed.

[0008] The present invention has the effect of improving skin disorders caused by ultraviolet rays and inflammatory reactions, and is excellent in safety and usability, and is used as an external preparation for skin, a cosmetic, and a quasi-drug. An object of the present invention is to provide a production inhibitor.

[0009]

Means for Solving the Problems The present invention has been made to solve such problems, and means for solving the problems are as follows: a nitric oxide production inhibitor, a rosemary extract, carnosol, Carnosic acid, coffee bean extract, primrose extract, spinach extract, oak extract, liquorice extract, inunobara extract, senkyu extract, tonin extract, peony extract, yokinin extract, and red grape extract Is contained.

[0010] Rosemary extract, carnosol, carnosic acid, coffee bean extract, primrose extract,
For skin extract, cosmetics, quasi-drug of spinach extract, oak extract, liquorice extract, ino noibara extract, senkyu extract, tonin extract, peony extract, yokunin extract, red grape extract The blending amount is not particularly limited, but is a dry solid amount,
Desirably, it is 0.0005% to 5.0% by weight based on the total amount.

[0011] When the amount of these components is less than 0.0005% by weight,
The effects aimed at by the present invention are not so satisfactory. On the other hand, even if the content of these compounds exceeds 5.0% by weight, no improvement in the effects commensurate with the increase is observed.

The external preparation for skin, cosmetics and quasi-drug containing the nitric oxide production inhibitor of the present invention may be in the form of lotions, emulsions, creams, packs, ointments and the like. It is possible.

The external preparation for skin, cosmetics and quasi-drugs of the present invention can be appropriately blended with pigments, preservatives, surfactants, fragrances, pigments and the like.

[0014]

Embodiments of the present invention will be described below.

(Example 1) This example is an example of a nitric oxide production inhibitor, in which a rosemary extract was used as the nitric oxide production inhibitor, and nitric oxide from mouse macrophage cells was cultured. A production inhibition test was performed.

[Proliferation of Mouse Macrophage Cells] Mouse macrophage cells (RAW 264.7 cells; manufactured by Dainippon Pharmaceutical Co., Ltd.) were grown according to the following conventional method.

For the culture of RAW cells, 10% fetal bovine serum (manufactured by Sigma) and 50% penicillin-streptomycin were used.
Dulbec with 0unit / ml ・ 500μg / ml (Sigma)
o's MEM (Sigma) medium was used.

The RAW cells were passaged by replacing the medium with a fresh medium every 3 or 4 days.

The RAW cells were suspended in a medium at a concentration of 1.0 × 10 ⁵ cells / ml, and the RAW cells were dispensed into a 24-well plate in 1 ml portions and cultured for 24 hours.

[Preparation of rosemary extract] On the other hand, rosemary extract (manufactured by Maruzen Pharmaceutical Co., Ltd.)
An ol / vol% solution was prepared and sterilized by filtration.

This is diluted to 0.5, 0.3, 0.1 vol / vol%
And

[Addition of Materials and Cell Stimulation] RAW cells were transformed into lipopolysaccharide (hereinafter referred to as LPS; manufactured by Sigma).
To induce production of nitric oxide, and after a certain period of time, the concentration of nitric oxide in the culture supernatant was measured.

LPS was dissolved in a medium to a concentration of 1 mg / ml and sterilized by filtration.

Specifically, the culture supernatant was first removed by aspiration, 450 μl of each concentration solution was added, and then 1 mg / ml LPS was added to stimulate the cells.

The culture supernatant was sampled 24 hours after cell stimulation. The measurement of nitric oxide released in the culture supernatant was performed using a grease reagent.

Although nitric oxide is released into the culture supernatant, it is unstable and immediately oxidized to nitrogen dioxide. Therefore,
In practice, it is difficult to directly measure the released amount of nitric oxide. Therefore, the amount of released nitric oxide in each sample was compared by measuring the amount of nitrogen dioxide.

Since nitric oxide is simply oxidized to nitrogen dioxide, by comparing the amount of such nitrogen dioxide, it is possible to objectively compare the liberated amount of nitric oxide in each sample. Can be estimated.

FIG. 1 shows the test results.

In FIG. 1, LPS means that only LPS was added and no rosemary extract was contained. Also, N.I. T. Means that no LPS was added.

As shown in FIG. 1, in mouse macrophage cells to which only LPS was added, the amount of released nitrogen dioxide was 1
Although it was 9.66 nmol / ml, in mouse macrophage cells supplemented with 0.3% rosemary extract together with LPS, the amount of released nitrogen dioxide was reduced to 16.24 nmol / ml, and the release of nitric oxide in the culture supernatant was reduced. It is estimated that was slightly suppressed.

In mouse macrophage cells to which rosemary extract was added at 0.5% together with LPS, the amount of released nitrogen dioxide was considerably reduced to about 13.45 nmol / ml, and mouse macrophage to which rosemary extract was added at 1.0% together with LPS was added. In macrophage cells, the amount of released nitrogen dioxide was remarkably reduced to about 8.55 nmol / ml.

Therefore, it is recognized that as the amount of the rosemary extract increases, the release of nitric oxide into the culture supernatant is suppressed.

Example 2 This example is another example of a nitric oxide production inhibitor. Inhibition of nitric oxide production from cultured mouse macrophage cells using carnosol as the nitric oxide production inhibitor. The test was performed.

The growth of mouse macrophage cells was carried out in the same manner as in Example 1.

[Preparation of Carnosol Solution] Carnosol solution was prepared by diluting a 10 mg / ml DMSO solution with
The solution was sterilized by filtration as M (μmol / l).

This was diluted to 40, 20, 10, 1 μM.

Cell stimulation with LPS to induce production of nitric oxide and measurement of nitric oxide concentration were carried out in the same manner as in Example 1.

In this example, in addition to LPS, interferon γ (hereinafter referred to as IFN-γ; a product of Biosource)
Was used to stimulate the cells, and the concentration of nitric oxide was also measured.

IFN-γ was dissolved in a medium and
l and used after filtration and sterilization.

The concentrations of the carnosol solution used in the case of IFN-γ were 40, 20, 10, and 1 μM, respectively.

FIG. 2 shows the test results by LPS,
FIG. 3 shows the test results by N-γ.

As is clear from FIG. 2, in the mouse macrophage cells to which only LPS was added, the amount of released nitrogen dioxide was 19.18 nmol / ml.
In the mouse macrophage cells to which μM was added, the amount of released nitrogen dioxide was reduced to 17.02 nmol / ml, and it is estimated that the release of nitric oxide in the culture supernatant was slightly suppressed.

In mouse macrophage cells to which the carnosol solution was added at 20 μM, the amount of released nitrogen dioxide was considerably reduced to about 12.56 nmol / ml. In mouse macrophage cells to which the carnosol solution was added at 40 μM, the amount of released nitrogen dioxide was reduced. A remarkable decrease of about 8.81 nmol / ml was observed.

Therefore, it is recognized that as the amount of the carnosol solution increases, the release of nitric oxide into the culture supernatant is suppressed.

In FIG. 3, IFN (-) is
It means that IFN-γ was not added, and 0 means I
Means that FN-γ has been added but no carnosol solution has been added.

As is clear from FIG. 3, in the mouse macrophage cells to which only IFN-γ was added, the release amount of nitrogen dioxide was 39.85 nmol / ml, but the mouse macrophages to which 1 μM of carnosol solution was added together with IFN-γ. In cells, the amount of released nitrogen dioxide was reduced to 33.91 nmol / ml, and in mouse macrophage cells to which 10 μM was added together with IFN-γ, the amount of released nitrogen dioxide was 22.46 nmo.
l / ml.

In mouse macrophage cells to which carnosol solution was added together with IFN-γ at 20 μM, the release amount of nitrogen dioxide was considerably reduced to 10.67 nmol / ml. Further, mouse macrophage cells to which carnosol solution together with IFN-γ was added at 40 μM were added. In addition, the amount of released nitrogen dioxide was remarkably reduced to 5.61 nmol / ml.

Therefore, it is recognized that as the amount of the carnosol solution increases, the release of nitric oxide into the culture supernatant is suppressed.

(Example 3) This example is another example of a nitric oxide production inhibitor, in which carnosic acid was used as the nitric oxide production inhibitor to produce nitric oxide from cultured mouse macrophage cells. An inhibition test was performed.

The growth of mouse macrophage cells was performed in the same manner as in Examples 1 and 2.

The preparation of the carnosic acid solution was performed in the same manner as the preparation of the carnosol solution in Example 2.

However, the concentrations of the carnosic acid solution used in the case of IFN-γ were 10, 5, 2, and 1 μM, respectively.

Further, induction of nitric oxide production by LPS or IFN-γ cell stimulation and measurement of nitric oxide concentration were performed in the same manner as in Examples 1 and 2.

FIG. 4 shows the test results by LPS,
FIG. 5 shows the test results by N-γ.

As is clear from FIG. 4, in the mouse macrophage cells to which only LPS was added, the amount of released nitrogen dioxide was 21.13 nmol / ml. In the mouse macrophage cells to which 10 μM of carnosic acid solution was added together with LPS, The release amount of nitrogen dioxide decreased to 13.75 nmol / ml, and it is estimated that release of nitric oxide into the culture supernatant was slightly suppressed.

Further, a carnosic acid solution was added together with LPS.
The release of nitrogen dioxide was 8.36 nmol / ml in mouse macrophage cells supplemented with 20 μM, and the release of nitrogen dioxide was 4.27 nmol / ml in mouse macrophage cells supplemented with 40 μM of carnosic acid solution together with LPS. Was done.

Therefore, it is recognized that as the amount of the carnosic acid solution increases, the release of nitric oxide into the culture supernatant is suppressed.

As is clear from FIG. 5, IFN
In mouse macrophage cells to which only -γ was added, the release amount of nitrogen dioxide was 39.55 nmol / ml, but IFN-γ
In addition, in the mouse macrophage cells to which 5 μM of the carnosic acid solution was added, the released amount of nitrogen dioxide was 32.16 nmol /
ml of mouse macrophage cells to which IFN-γ was added at 10 μM, the release amount of nitrogen dioxide was 12.85 nmol /
ml.

Therefore, it is recognized that as the amount of the carnosic acid solution increases, the release of nitric oxide into the culture supernatant is suppressed.

Example 4 This example is another example of the nitric oxide production inhibitor. As the nitric oxide production inhibitor, coffee liquid B (Ichimaru Falcos), which is an extract of coffee beans, is used. Was used to conduct a test for inhibiting nitric oxide production from cultured mouse macrophage cells.

The growth of mouse macrophage cells was carried out in the same manner as in Examples 1 to 3.

The coffee liquid B is an extract of coffee beans, comprising 30% by weight of 1,3-butylene glycol,
It contains 0.3% by weight of chlorogenic acid.

This coffee liquid B was diluted with a medium to prepare a 1.0 vol / vol% solution, sterilized by filtration, and further diluted to 0.1, 0.01, 0.001 vol / vol%.

Further, induction of nitric oxide production by cell stimulation by LPS and measurement of nitric oxide concentration were performed in the same manner as in Examples 1 to 3.

FIG. 6 shows the test results by LPS.

As is clear from FIG. 6, in the mouse macrophage cells to which only LPS was added, the amount of released nitrogen dioxide was 23.83 nmol / ml, but the mouse macrophage cells to which 0.1% of the coffee liquid B solution was added together with LPS. In this case, the release amount of nitrogen dioxide decreased to about 16.57 nmol / ml, and it is estimated that release of nitric oxide in the culture supernatant was slightly suppressed.

In addition, coffee liquid B together with LPS
In mouse macrophage cells to which 1.0% solution was added,
The amount of released nitrogen dioxide was remarkably reduced to about 2.87 nmol / ml.

Accordingly, it is recognized that as the amount of the coffee liquid B solution increases, the release of nitric oxide into the culture supernatant is suppressed.

(Example 5) This example is another example of the nitric oxide production inhibitor, in which the extract of primrose was used as a nitric oxide production inhibitor and the monoxide from cultured mouse macrophage cells was used. A nitrogen production suppression test was performed.

The proliferation of mouse macrophage cells was carried out in the same manner as in Examples 1 to 4.

The extract of primrose was used as the extract of primrose (Pr
Atia nummularia) is an extract obtained by immersing stems and leaves in 50% ethanol. Primrose (Pratia nummula)
ria) is a plant belonging to the genus Pratia in the family Pleuronaceae, and in this example, Pratia nummularia made in Taiwan was used.

This Primrose extract was diluted with a medium to prepare a 1.0 vol / vol% solution, sterilized by filtration, and further diluted to 0.5, 0.1 and 0.05 vol / vol%.

Further, induction of nitric oxide production by cell stimulation by LPS and measurement of nitric oxide concentration were performed in the same manner as in Examples 1 to 4.

FIG. 7 shows the test results by LPS.

As is clear from FIG. 7, in the mouse macrophage cells to which only LPS was added, the release amount of nitrogen dioxide was 19.68 nmol / ml, but in the mouse macrophage cells to which 0.05% of the Primrose extract was added together with LPS. The amount of released nitrogen dioxide was reduced to about 17.45 nmol / ml, and the amount of released nitrogen dioxide was reduced to about 15.37 nmol / ml in mouse macrophage cells to which 0.1% primrose extract was added together with LPS. In the mouse macrophage cells supplemented with 0.5% of the solution, the released amount of nitrogen dioxide decreased to about 12.66 nmol / ml,
In mouse macrophage cells to which 1.0% of Primrose extract was added together with PS, the amount of released nitrogen dioxide was about 1%.
It decreased to 0.0 nmol / ml.

Therefore, it is recognized that the release of nitric oxide into the culture supernatant is suppressed as the amount of the Primrose extract increases.

(Example 6) This example is another example of a nitric oxide production inhibitor, in which a spinach extract was used as a nitric oxide production inhibitor to reduce monoxide from mouse keratinocytes. A nitrogen production suppression test was performed.

[Proliferation of Mouse Keratinocytes] One-week-old BALB / c mice (purchased from Japan SLC) were used as mice. The skin collected from this mouse was immersed in a 70% ethanol solution for 30 seconds, and then cut into a strip of about 5 mm × 15 mm to obtain a skin piece.

A piece of skin was treated with a 0.25% trypsin solution (Clonetic
After incubating at 37 ° C. for 3 to 4 hours, the epidermis is separated from the skin piece, and the epidermis is transferred to KGM medium (Clonetics) and pipetted to disperse the keratinocytes. I let it.

This KGM medium is based on a KBM medium (manufactured by Clonetics) comprising various amino acids, vitamins, inorganic salts, glucose, etc., and is supplemented with insulin, hEGF (human EGF), BPE (bovine bovine). Brain extract), hydrocortisone, gentamicin (GA100
0) and the like.

The dispersed liquid was added to a cell strainer (Falcon
), And after the filtration, mouse keratinocytes are 1.5 ×
The filtrate was adjusted to 10 ⁴ cells / ml, and this was added to 24 wells.
Each 1 ml was dispensed to the plate and cultured for 72 hours.

[Preparation of spinach extract] On the other hand, spinach extract (manufactured by Maruzen Pharmaceutical Co., Ltd.) was diluted with a medium to 1.0 vol / vol%.
The solution was prepared and sterilized by filtration.

This was diluted to 0.1 and 0.01 vol / vol%.

[Addition of Materials and Cell Stimulation] Mouse keratinocytes were treated with interferon γ (hereinafter referred to as IFN-γ; Bios
(manufactured by ource) to induce the production of nitric oxide, and the concentration of nitric oxide in the culture supernatant after a certain time was measured.

IFN-γ was dissolved in the medium and
l and used after filtration and sterilization.

Specifically, the culture supernatant was first removed by aspiration, 450 μl of each concentration solution was added, and then 50 μl of 1000 unit / ml IFN-γ was added to stimulate the cells.

The culture supernatant was sampled 24 hours after cell stimulation. The measurement of nitric oxide released in the culture supernatant was performed using a grease reagent.

FIG. 8 shows the test results.

As shown in FIG. 8, in the mouse keratinocytes to which only IFN-γ was added, the amount of released nitrogen dioxide was 17.04 nmo.
l / ml, but the spinach extract with IFN-γ
In mouse keratinocytes to which 0.01 vol / vol% was added, the amount of released nitrogen dioxide was reduced to 14.13 nmol / ml.

In mouse keratinocytes to which 0.1 vol / vol% of the spinach extract was added together with IFN-γ, the release amount of nitrogen dioxide was reduced to 10.3 nmol / ml, and 1 vol / vol of the spinach extract together with IFN-γ was added. In mouse keratinocytes to which vol% was added, the release amount of nitrogen dioxide was reduced to 9.18 nmol / ml.

Accordingly, it is recognized that the release of nitric oxide from the culture supernatant is suppressed as the amount of the spinach extract increases.

Example 8 This example is another example of a nitric oxide production inhibitor, in which an oak extract was used as a nitric oxide production inhibitor, and monoxide from mouse keratinocytes was used. A nitrogen production suppression test was performed.

The growth of mouse keratinocytes was carried out in the same manner as in Example 6.

[0094] In addition, the oak extract (the oak extract-
J; manufactured by Maruzen Pharmaceutical Co., Ltd.) was performed in the same manner as in the preparation of the spinach extract of Example 6.

Further, induction of nitric oxide production by IFN-γ cell stimulation and measurement of nitric oxide concentration were carried out in the same manner as in Example 6.

FIG. 9 shows the test results.

As shown in FIG. 9, in mouse keratinocytes to which only IFN-γ was added, the amount of released nitrogen dioxide was about 17.15 n
mol / ml, but in mouse keratinocytes to which 0.01 vol / vol% of the oat extract was added together with IFN-γ, the release amount of nitrogen dioxide was reduced to about 13.86 nmol / ml.

Further, in mouse keratinocytes to which 0.1 vol / vol% of the oat extract was added together with IFN-γ, the release amount of nitrogen dioxide decreased to about 8.12 nmol / ml, and 1 vol of the oat extract together with IFN-γ was added. In mouse keratinocytes to which / vol% was added, the amount of released nitrogen dioxide was about 11.2 nmol / ml.

[0099] Therefore, it is recognized that the release of nitric oxide into the culture supernatant is suppressed as the amount of the oak extract increases, but when the oak extract is added at 1 vol / vol%, 0.1% It increased slightly compared to the case where vol / vol% was added.

(Example 8) This example is another example of a nitric oxide production inhibitor, in which a licorice extract was used as a nitric oxide production inhibitor to remove monoxide from mouse keratinocytes. A nitrogen production suppression test was performed.

The growth of mouse keratinocytes was performed in the same manner as in Examples 6 and 7.

In addition, liquorice extract (liquorice extract-
(BG; Maruzen Pharmaceutical) was prepared in the same manner as in the preparation of the spinach extract of Example 6 and the extract of oak of Example 7.

Further, induction of nitric oxide production by cell stimulation by IFN-γ and measurement of nitric oxide concentration were performed in the same manner as in Examples 6 and 7.

FIG. 10 shows the test results.

As shown in FIG. 10, in the mouse keratinocytes to which only IFN-γ was added, the amount of released nitrogen dioxide was about 14.97 n.
Although it was moL / mL, in the mouse keratinocytes to which liquorice extract was added together with IFN-γ at 1.0 vol / vol%, the release amount of nitrogen dioxide was reduced to about 11.82 nmol / ml.

(Example 9) This example is another example of the nitric oxide production inhibitor. As the nitric oxide production inhibitor, Falcolex Novara E, which is an extract of Ino noibara, manufactured by Ichimaru Falcos Was used to conduct a test for inhibiting nitric oxide production from mouse keratinocytes.

The proliferation of mouse keratinocytes was carried out in the same manner as in Examples 6 to 8.

The preparation of Falcolex Novara E was performed in the same manner as in the spinach extract of Example 6, the oak extract of Example 7, and the liquorice extract of Example 8.

Further, induction of nitric oxide production by cell stimulation by IFN-γ and measurement of nitric oxide concentration were performed in the same manner as in Examples 6 to 8.

FIG. 11 shows the test results.

As shown in FIG. 11, in mouse keratinocytes to which only IFN-γ was added, the release amount of nitrogen dioxide was 13.05 nmo.
However, in mouse keratinocytes to which 1.0 vol / vol% of Falcolex Novara E was added together with IFN-γ, the amount of released nitrogen dioxide was reduced to about 5.71 nmol / ml.

(Example 10) This example is an example of a nitric oxide production inhibitor. In this example, the extract of Senkyu was used as a nitric oxide production inhibitor to inhibit nitric oxide production from human macrophage cells. The test was performed.

[Proliferation of Human Macrophage Cells] As the human macrophage cells, those obtained by culturing and proliferating THP-1 (Dainippon Pharmaceutical), a monocyte, according to a conventional method were used.

THP-1 cells were cultured using 10% fetal bovine serum (Sigma) and penicillin / streptomycin.
Dulbec with 500unit / ml ・ 500μg / ml (Sigma)
co's MEM (manufactured by Sigma) medium was used.

The THP-1 cells were passaged by replacing the medium with a fresh medium every 3 or 4 days.

The THP-1 cells were suspended in a medium at 1.0 × 10 ⁵ cells / ml, and 100 nM PMA (phorbol-
1,2-myristate-1,3-acetate, Gibco) solution was added, and the solution was dispensed into a 24-well plate in 1 ml portions.
The culture was performed for 72 hours.

[Preparation of Senshu Extract] On the other hand, Senshu extract (manufactured by Maruzen Pharmaceutical Co., Ltd.)
An ol% solution was prepared and sterilized by filtration.

This was diluted to 0.1 and 0.01 vol / vol%.

[Addition of Materials and Stimulation of Cells] THP-1 cells were stimulated with lipopolysaccharide (hereinafter, referred to as LPS; manufactured by Sigma) to induce the production of nitric oxide, and the cells in the culture supernatant after a certain period of time. The nitric oxide concentration was measured.

LPS was dissolved in a medium to a concentration of 1 mg / ml and sterilized by filtration.

Specifically, first, the culture supernatant was removed by aspiration, 450 μl of each concentration solution was added, and then 1 mg / ml LPS was added to stimulate the cells.

The culture supernatant was sampled 24 hours after cell stimulation. The measurement of nitric oxide released in the culture supernatant was performed using a grease reagent.

FIG. 12 shows the test results.

As shown in FIG. 12, in human macrophage cells to which only LPS was added, the amount of released nitrogen dioxide was about 7.
Although the concentration was 46 nmol / ml, in human macrophage cells to which 0.01% of the extract was added together with LPS, the amount of released nitrogen dioxide was slightly reduced to about 7.2 nmol / ml.
In human macrophage cells to which 0.1% of Senghu extract was added together with S, the released amount of nitrogen dioxide was about 6.6 nmol /
ml, and human macrophage cells to which 1.0% of Sengkyu extract was added together with LPS,
The released amount of nitrogen dioxide decreased slightly to about 6.0 nmol / ml.

Therefore, the release amount of nitrogen dioxide gradually decreases with an increase in the amount of the extract of Sengyu, and the release of nitric oxide in the culture supernatant is increased with the increase of the amount of the extract of Senghu. Is considered to be suppressed.

(Example 11) This example is an example of a nitric oxide production inhibitor, and uses a tonin extract as a nitric oxide production inhibitor to inhibit nitric oxide production from human macrophage cells. The test was performed.

The growth of human macrophage cells is described in the Examples
In the same manner as in Example 10, THP-1 was used.

The preparation of the tonin extract was prepared in Example 10.
Was performed in the same manner as in the preparation of the extract of Sengoku.

Further, induction of nitric oxide production by cell stimulation by LPS and measurement of nitric oxide concentration were performed in the same manner as in Example 10.

FIG. 13 shows the test results.

As shown in FIG. 13, in the human macrophage cells to which only LPS was added, the released amount of nitrogen dioxide was 7.96.
nmol / ml, but the tonin extract was combined with LPS.
In human macrophage cells to which 0.01% was added, the amount of released nitrogen dioxide decreased to about 6.14 nmol / ml. In human macrophage cells to which 0.1% of tonin extract was added together with LPS, the amount of released nitrogen dioxide was about 6.78 nmol / ml. However, in human macrophage cells to which 1.0% of tonin extract was added together with LPS, the amount of released nitrogen dioxide was about 6.
It decreased to 13 nmol / ml.

(Example 12) This example is an example of a nitric oxide production inhibitor, and uses a peony extract as a nitric oxide production inhibitor to inhibit nitric oxide production from human macrophage cells. The test was performed.

The growth of human macrophage cells is described in the Examples
10 and in the same manner as in Example 11.

The preparation of the peony extract was carried out in the manner described in Example.
The preparation was carried out in the same manner as in the preparation of 10 extract extracts and the tonin extract of Example 11.

Further, induction of nitric oxide production by cell stimulation by LPS and measurement of nitric oxide concentration were performed in the same manner as in Examples 10 and 11.

FIG. 14 shows the test results.

As shown in FIG. 14, in the human macrophage cells to which only LPS was added, the amount of released nitrogen dioxide was 8.15.
In human macrophage cells to which 0.01% of a peony extract was added together with LPS, the release amount of nitrogen dioxide was slightly reduced to 7.03 nmol / ml.
In addition, in the human macrophage cells to which 0.1% of the peony extract was added, the release amount of nitrogen dioxide was about 6.45 nmol / m2.
The amount of released nitrogen dioxide was slightly reduced to about 6.18 nmol / ml in human macrophage cells to which 1.0% of the peony extract was added together with LPS.

Therefore, the release amount of nitrogen dioxide gradually decreases as the amount of the peony extract increases. Therefore, the release of nitric oxide in the culture supernatant is increased with the increase of the amount of the peony extract. Is considered to be suppressed.

(Example 13) This example is an example of a nitric oxide production inhibitor, and uses a quininine extract as a nitric oxide production inhibitor to inhibit nitric oxide production from human macrophage cells. The test was performed.

The growth of human macrophage cells is described in the Examples.
Performed in the same manner as in Examples 10 to 12.

Further, the preparation of a yokuinin extract (Maruzen Pharmaceutical Co., Ltd.) was carried out in the same manner as in the preparation of the extract of Senkyu in Example 10, the extract of tonin from Example 11, and the extract of peonies of Example 12.

Further, induction of nitric oxide production by cell stimulation by LPS and measurement of nitric oxide concentration were performed in the same manner as in Examples 10 to 12.

The test results are shown in FIG.

As shown in FIG. 15, in human macrophage cells to which only LPS was added, the amount of released nitrogen dioxide was about 8.
Although the concentration was 19 nmol / ml, in human macrophage cells to which 0.01% of ioquinin extract was added together with LPS, the amount of released nitrogen dioxide was slightly reduced to about 7.3 nmol / ml.
In human macrophage cells supplemented with 0.1% of yoquinin extract together with S, the released amount of nitrogen dioxide was about 6.73 nmol.
/ ml, but in human macrophage cells to which 1.0% of yoquinin extract was added together with LPS,
The released amount of nitrogen dioxide was about 7.01 nmol / ml.

(Example 14) This example is an example of a nitric oxide production inhibitor, in which a red grape extract was used as the nitric oxide production inhibitor to inhibit nitric oxide production from human macrophage cells. The test was performed.

The growth of human macrophage cells is described in the Examples.
Performed in the same manner as in Examples 10 to 13.

The red grape extract (Red grape extract-BG; Maruzen Pharmaceutical Co., Ltd.) was prepared according to the method described in Example 10 including the extract of Senkyu, the extract of Tonin from Example 11, the extract of Peony in Example 12, and the extract of Example 13. The preparation was carried out in the same manner as in the preparation of the yokunin extract.

Further, induction of nitric oxide production by cell stimulation by LPS and measurement of nitric oxide concentration were performed in the same manner as in Examples 10 to 13.

FIG. 16 shows the test results.
Shown in

As shown in FIG. 16, in human macrophage cells to which only LPS was added, the amount of released nitrogen dioxide was about 6.
18 nmol / ml, but human macrophage cells to which 0.01% red grape extract was added together with LPS, and 0.1%
% Of human macrophage cells to which% spiked was added, the release of nitrogen dioxide was considerably reduced to about 4.08 nmol / ml, and that of human macrophage cells to which red grape extract was added 1.0% together with LPS, the amount of released nitrogen dioxide was about 3.46 nmol / ml. And further reduced.

Accordingly, as the amount of red grape extract increases, the amount of released nitrogen dioxide gradually decreases. Therefore, the amount of nitric oxide released into the culture supernatant increases with the amount of red grape extract. Is considered to be suppressed.

(Example 15) In this example, RT-PCR
Was used to measure mRNA for inducible nitric oxide synthase.

[Extraction of RNA] The cells from which the culture supernatant was sampled were washed twice with 1 ml of PBS (manufactured by Dainippon Pharmaceutical), and RNA in the cells was extracted according to SV TotaL RNA Isolation System (manufactured by Promega).

[RT-PCR] RT-PCR was performed using TaKaRa.
RNA PCR Kit (AMV) Ver.2.1 (Takara Shuzo) was used.

The cDNA obtained by reverse transcription of the extracted RNA was increased by RT-PCR.

The PCR was performed under the conditions of a denaturation temperature of 94 ° C., an annealing temperature of 65 ° C., and an extension reaction temperature of 72 ° C. for 30 cycles.

[Agarose gel electrophoresis] PCR reaction solution
To 50 μl, 10 μl of loading buffer (Nippon Gene) was added and mixed.

[0158] 15 µl of this solution was electrophoresed on a 1.2% agarose gel.

After completion of the electrophoresis, the agarose gel was immersed in a 0.5 μg / ml ethidium bromide solution for 30 minutes for staining.

Ultraviolet rays were irradiated using a transilluminator and photographs were taken.

[Numericalization of Fluorescent Bands] Numericalization of the fluorescence intensity of each band was performed using an ATTO Lane & Spot Analyzer (manufactured by ATTO Corporation).

From these fluorescence intensities, the ratio (iNOS / GAPDH) between the amount of mRNA for iNOS, which is an enzyme producing nitric oxide, and the amount of mRNA for GAPDH, which is an enzyme in the glycolytic system of cells, was determined. Was.

GAPDH is an enzyme that is constantly expressed, and therefore, the amount of mRNA for this enzyme is constant. Therefore, by determining iNOS / GAPDH, iNOS
The degree of suppression of the expression of the enzyme and thus the degree of suppression of release of nitric oxide can be estimated.

FIG. 17 shows the result.

In FIG. 17, LPS means that only LPS was added, and Non Treat. Means that no LPS was added.

Her.A means a product to which herbimycin A, which is an antibiotic, is added.

As shown in FIG. 17, the value of iNOS / GAPDH was 1.344 in cells to which only LPS was added, while the value of iNOS / GAPDH was 0.586 in cells to which herbimycin A was added together with LPS. Become
Furthermore, in cells to which rosemary extract was added together with LPS, the value of iNOS / GAPDH was further reduced to 0.125, and in cells to which carnosol solution was added together with LPS, the value of iNOS / GAPDH was similarly reduced to about 0.146, In the cells to which the carnosic acid solution was added together with LPS, the value of iNOS / GAPDH was 0.

Therefore, in a system to which a rosemary extract, a carnosol solution and a carnosic acid solution were added, iNOS
It can be presumed that the release of nitric oxide is suppressed by suppressing the expression of the enzyme.

(Examples 16 to 18 and Comparative Examples 1 to 3) In this example, the effect of improving the inflammation of rosemary extract, one of the above-mentioned nitric oxide production inhibitors, was tested.

The concentration of the rosemary extract relative to the total amount was
Mix well with the ointment base material Plastibase (Taisho Pharmaceutical Co., Ltd.) to give 0.01, 0.5 and 1.0% by weight.
16, Example 17, and Example 18.

On the other hand, indomethacin was well mixed with the plastic base so that the concentration with respect to the total amount was 1% by weight, and this was used as a positive control (Comparative Example 1).

Comparative Example 2 shows the case where only the base was applied.
The case of no application was designated as Comparative Example 3.

[0173] Tests of the following inflammation-improving effects were conducted on the nitric oxide production inhibitors of Examples 16 to 18 and Comparative Examples 1 to 3 as follows.

(Test Method) The samples of Examples 16 to 18 and Comparative Examples 1 to 3 were continuously applied to the back of a guinea pig for 3 days and irradiated with ultraviolet rays. The skin color at the ultraviolet irradiation site was measured using a color difference meter. Erythema on the skin was calculated by comparing a case of Comparative Example 3 where no base was applied with 100 as a *.
Indicated by value.

Table 1 shows the results.

[0176]

[Table 1]

As is clear from Table 1, erythema was suppressed in Comparative Example 1 containing indomethacin, which was a positive control, but good results were not shown in Comparative Example 2 containing only the base.

On the other hand, the ointments of Examples 16 to 18 showed generally good results, though not as good as Comparative Example 1, which was a positive control. In particular, rosemary extract is 1.0% by weight
In Example 18, an effect comparable to that of indomethacin, which generally has an excellent skin erythema inhibitory effect, was obtained.

(Examples 19 to 21 and Comparative Example 4) In this example, the formulation in which rosemary extract, one of the above-mentioned nitric oxide production inhibitors, was blended into a cream as an example of cosmetics was used. For example, the cream was subjected to a whitening test.

The preparation of the cream was carried out as follows.

Squalene, cetyl isooctanoate,
After heating and dissolving the microcrystalline wax, add the clay mineral, POE glycerol triisostearate (surfactant), adjust to 70 ° C, and disperse uniformly.
Upon dissolution, an oily gel was obtained.

Next, the rosemary extract was dissolved in purified water of a predetermined concentration, adjusted to 70 ° C., and slowly added to the oily gel with sufficient stirring.

After uniformly mixing with a homomixer, degassing
After filtration, the mixture was cooled to 30 ° C. to obtain a cream.

The compositions and compounding ratios of the obtained creams of Examples 19 to 21 are as follows.

(Example 19) Composition Composition ratio (% by weight) Squalene 20% Cetyl isooctanoate 8.5% Microcrystalline wax 1% Clay mineral 1.3% POE glycerol triisostearate 0.2% Rosemary extract 0.001% Water residue amount

(Example 20) Composition Composition ratio (% by weight) Squalene 20% Cetyl isooctanoate 8.5% Microcrystalline wax 1% Clay mineral 1.3% POE glycerol triisostearate 0.2% Rosemary extract 0.1% Water residue amount

(Example 21) Composition Composition ratio (% by weight) Squalene 20% Cetyl isooctanoate 8.5% Microcrystalline wax 1% Clay mineral 1.3% POE glycerol triisostearate 0.2% Rosemary extract 1% Water residue amount

A whitening test was performed using the creams of Examples 19 to 21 thus prepared.

The test method is as follows.

The creams of Examples 19 to 21 were applied twice a day for 3 months to 25 female subjects (25 to 45 years old).

The evaluation was made based on the number of persons who answered that the skin became white.

On the other hand, the composition of the cream base was as described in Example 19 above.
A similar test as Comparative Example 4 was performed on a cream that was the same as No. 21 but did not contain the rosemary extract.

The test results are shown in Table 2.

[0194]

[Table 2]

As is clear from Table 2, Examples 19 to 21
The number of persons who answered that the degree of whitening improvement was good was about twice or more as compared with Comparative Example 4.

(Examples 22 to 24 and Comparative Example 5) In this example, the rosemary extract was formulated into a lotion as an example of a cosmetic. Was done.

The preparation of the lotion was carried out as follows.

A lotion base containing the surfactant POE (20) oleyl alcohol ether, the thickener methylcellulose, quince seed and ethanol was prepared, and a predetermined concentration of a rosemary extract was added.

The compositions and compounding ratios of the obtained lotions of Examples 22 to 24 are as follows.

(Example 22) Composition Mixing ratio (% by weight) POE (20) oleyl alcohol ether 0.5% Methyl cellulose 0.2% Quince seed 0.1% Ethanol 10% Rosemary extract 0.001% Water remaining amount

Example 23 Composition Composition Ratio (% by Weight) POE (20) Oleyl Alcohol Ether 0.5% Methylcellulose 0.2% Quince Seed 0.1% Ethanol 10% Rosemary Extract 0.1% Water Remaining

(Example 24) Composition Mixing ratio (% by weight) POE (20) oleyl alcohol ether 0.5% Methyl cellulose 0.2% Quince seed 0.1% Ethanol 10% Rosemary extract 1% Water Remaining amount

A whitening test was carried out using the lotions of Examples 22 to 24 thus prepared.

The test method is as follows.

The lotions of Examples 22 to 24 were applied twice a day for three months to 25 female subjects (25 to 45 years old).

The evaluation was made by the number of persons who answered that the skin became white.

On the other hand, the composition of the lotion base was as described in Examples 22 to
The same test as Comparative Example 5 was performed on a lotion that was the same as 24 but did not contain the rosemary extract.

Table 3 shows the test results.

[0209]

[Table 3]

As is clear from Table 3, in Examples 22 to 24, the number of persons who answered that the skin became white was about 1.5 to 2 times as large as that in Comparative Example 5.

[Examples 25 and 26, Comparative Examples 6 and 7] Sensory test (feeling of use)

This example is a test on the feeling of use of a cosmetic (cream and lotion) containing a rosemary extract (sensory test).

Example 25 is a sample of Example 21, Comparative Example 6 is a sample of Comparative Example 4, Example 26 is a sample of Example 24, Comparative Example 7
Using each of the samples of Comparative Example 5, 20 subjects evaluated the characteristics of the sample after using the sample for one week.

The evaluation was made based on the number of respondents who answered that "the skin adapts well" in the questionnaire.

The test results are shown in Table 4.

[0216]

[Table 4]

As shown in Table 4, in Examples 25 and 26,
Compared to Comparative Examples 6 and 7, about twice as many people answered that "the skin fits well".

(Examples 27 to 29 and Comparative Example 4) In this example, a formulation example in which the extract of primrose, one of the above-mentioned nitric oxide production inhibitors, was added to a cream as an example of a cosmetic. The cream was subjected to a whitening test.

The preparation of the cream was carried out in the same manner as in Examples 19 to 21.

The compositions and compounding ratios of the creams of Examples 27 to 29 are as follows.

(Example 27) Composition Composition ratio (% by weight) Squalene 20% Cetyl isooctanoate 8.5% Microcrystalline wax 1% Clay mineral 1.3% POE glycerol triisostearate 0.2% Primrose extract 0.01% Water remaining amount

(Example 28) Composition Composition ratio (% by weight) Squalene 20% Cetyl isooctanoate 8.5% Microcrystalline wax 1% Clay mineral 1.3% POE glycerol triisostearate 0.2% Primrose extract 0.1% Water Remaining amount

Example 29 Composition Composition (% by weight) Squalene 20% Cetyl isooctanoate 8.5% Microcrystalline wax 1% Clay mineral 1.3% POE glycerol triisostearate 0.2% Primrose extract 1% Water Remaining amount

Using the creams of Examples 27 to 29, a whitening test was performed.

The test was performed on female subjects (25 to 45 years old).
The test was performed on humans in the same manner as in Examples 19 to 21 described above.

On the other hand, using the cream of Comparative Example 4 above,
A similar test was performed on the same female subjects as in Examples 27 to 29.

Table 5 shows the test results.

[0228]

[Table 5]

As is clear from Table 5, Examples 27 to 29
The number of persons who answered that the degree of improvement in whitening was good was about 1.5 to 2.5 times that of Comparative Example 4.

(Examples 30 to 32 and Comparative Example 5) This example is a formulation example in which the above Primrose extract was blended with a lotion as an example of a cosmetic. went.

The preparation of the lotion was carried out in the same manner as in Examples 22 to 24.

The compositions and compounding ratios of the lotions of Examples 30 to 32 are as follows.

The compositions and compounding ratios of the obtained lotions of Examples 30 to 32 are as follows.

(Example 30) Composition Mixing ratio (% by weight) POE (20) oleyl alcohol ether 0.5% Methyl cellulose 0.2% Quince seed 0.1% Ethanol 10% Primrose extract 0.01% Water remaining amount

Example 31 Composition Composition Ratio (% by Weight) POE (20) Oleyl Alcohol Ether 0.5% Methylcellulose 0.2% Quince Seed 0.1% Ethanol 10% Primrose Extract 0.1% Water Remaining

(Example 32) Composition Mixing ratio (% by weight) POE (20) oleyl alcohol ether 0.5% Methylcellulose 0.2% Quince seed 0.1% Ethanol 10% Primrose extract 1% Water remaining amount

A whitening test was conducted using the lotions of Examples 30 to 32.

The test was performed on female subjects (25 to 45 years old).
The test was performed on humans in the same manner as in Examples 22 to 24 above.

On the other hand, using the lotion of Comparative Example 5, the same test was performed on the same female subjects as in Examples 30 to 32.

Table 6 shows the test results.

[0241]

[Table 6]

As is clear from Table 6, in Examples 30 to 32, the number of persons who answered that the skin became white was about 1.3 to 2.3 times as large as that of Comparative Example 5.

[Examples 33 and 34, Comparative Examples 6 and 7] Sensory test (feeling of use)

This example is a test on the feeling of use of a cosmetic (cream and lotion) containing a Primrose extract (sensory test).

Example 33 is the sample of Example 29, Comparative Example 6 is the sample of Comparative Example 4, Example 34 is the sample of Example 32, and Comparative Example 7
Using each of the samples of Comparative Example 5, 20 subjects evaluated the characteristics of the sample after using the sample for one week.

As in the case of Examples 25 and 26, the evaluation was based on the number of respondents who answered that "skin familiarity was good" in the questionnaire.

The test results are shown in Table 7.

[0248]

[Table 7]

As shown in Table 7, in Examples 33 and 34,
Compared to Comparative Examples 6 and 7, about twice as many people answered that "the skin fits well".

[0250]

As described above, in the present invention, a nitric oxide production inhibitor having an excellent nitric oxide production inhibitory effect could be provided.

As a result, by incorporating such a nitric oxide production inhibitor, a skin cosmetic which has an effect of improving skin disorders caused by ultraviolet rays and inflammatory reactions, and which is excellent in safety and usability, A quasi-drug or a skin external preparation could be provided.

[Brief description of the drawings]

FIG. 1 is a graph showing the correlation between the concentration of a rosemary extract added to LPS and the amount of released nitrogen dioxide.

FIG. 2 is a graph showing the correlation between the concentration of carnosol added to LPS and the amount of released nitrogen dioxide.

FIG. 3 is a graph showing the correlation between the concentration of carnosol added to IFN-γ and the amount of released nitrogen dioxide.

FIG. 4 is a graph showing the correlation between the concentration of carnosic acid added to LPS and the amount of released nitrogen dioxide.

FIG. 5 is a graph showing the correlation between the concentration of carnosic acid added to IFN-γ and the amount of released nitrogen dioxide.

FIG. 6 is a graph showing the correlation between the concentration of coffee liquid added to LPS and the amount of released nitrogen dioxide.

FIG. 7 is a graph showing the correlation between the concentration of the Primrose extract added to LPS and the released amount of nitrogen dioxide.

FIG. 8 is a graph showing the correlation between the concentration of a spinach extract added to IFN-γ and the release amount of nitrogen dioxide.

FIG. 9 is a graph showing the correlation between the concentration of oat extract added to IFN-γ and the amount of released nitrogen dioxide.

FIG. 10 is a graph showing the correlation between the concentration of a liquorice extract added to IFN-γ and the release amount of nitrogen dioxide.

FIG. 11 is a graph showing the correlation between the concentration of Falcolex Novara E added to IFN-γ and the amount of released nitrogen dioxide.

FIG. 12 is a graph showing a correlation between the concentration of the extract extract added to LPS and the release amount of nitrogen dioxide.

FIG. 13 is a graph showing the correlation between the concentration of a tonin extract added to LPS and the amount of released nitrogen dioxide.

FIG. 14 is a graph showing the correlation between the concentration of a peony extract added to LPS and the amount of released nitrogen dioxide.

FIG. 15 is a graph showing the correlation between the concentration of a yoquinin extract added to LPS and the amount of released nitrogen dioxide.

FIG. 16 is a graph showing the correlation between the concentration of red grape extract added to LPS and the amount of released nitrogen dioxide.

FIG. 17 is a graph showing the results of measuring mRNA for inducible nitric oxide synthase by RT-PCR.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61K 35/78 A61K 35/78 N Q U 7/00 7/00 K N M X 7/48 7/48 A61P 17/00 A61P 17/00 17/16 17/16 43/00 111 43/00 111 (72) Inventor Kazuhiko Hamada 64-5 Kamigamo Toyota-cho, Kita-ku, Kyoto F term (reference) 4C083 AA111 AA112 AB442 AC012 AC102 AC182 AC352 AC422 AC851 AC852 AD262 AD352 CC04 CC05 CC19 DD31 EE12 EE13 EE16 EE17 4C088 AB12 AB14 AB30 AB32 AB38 AB40 AB51 AB52 AB56 AB60 AB62 AB77 AC01 BA08 MA17 MA28 MA63 NA14 ZA89 ZC20

Claims (4)

[Claims] 1. Rosemary extract, carnosol, carnosic acid, coffee bean extract, primrose extract, spinach extract, oak extract, licorice extract, inuinobara extract, senkyu extract, tonin extract, A nitric oxide production inhibitor comprising at least one of a peony extract, a yokinin extract, and a red grape extract. 2. An external preparation for skin, comprising the nitric oxide production inhibitor according to claim 1. 3. A cosmetic comprising the nitric oxide production inhibitor according to claim 1. 4. A quasi-drug comprising the nitric oxide production inhibitor according to claim 1.