JP2001021551A - Discrimination method of aging prevention and depressing agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new screening method for aging prevention and improvement and a skin medicine for external application that is useful preventing aging and making improvements. SOLUTION: Sugars, proteins, lipid peroxides and secondary products thereof present in an organism are made to coexist with one another and are incubated at a temperature close to that of the organism, and a difference in fluorescent intensity between before and after the incubation is measured. Sugars, proteins, lipid peroxides and secondary products thereof present in an organism are made to coexist with a substance to be tested, and the difference in fluorescent intensity measured under the same condition is compared with the former difference. The degree of depression of an increase in fluorescent intensity during incubation of the substance to be tested is used as an index, and the lipofuscin- like substance formation depression action of the substance to be tested discriminated. Constituents having such action are contained in a cosmetic.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an external preparation for skin which is useful for prevention and improvement of skin aging and a method for designing the same.

[0002]

2. Description of the Related Art A fresh, elastic, fine-textured, white skin has been recognized as a beautiful skin since ancient times, and many people have made various efforts to realize such skin. It is no exaggeration to say that the science for realizing such skin is the origin of cosmetics science. In the young age, even if you have such fresh skin, as you age, it is easy for anyone to lose such beauty and become inelastic, bulky and rough skin It is a phenomenon that must be experienced at least once, but hopefully it will be as late as possible to visit such a phenomenon. For this reason, in the field of cosmetics science, the elucidation of such skin aging phenomenon has become a major theme, and based on many previous research results, active oxygen generated by UV exposure etc. Due to lipid peroxide, reactions such as cross-linking and disruption of tissue structures such as collagen occur,
It is thought that the components of the skin undergo a chemical reaction, causing coloring and insolubilization. Based on such findings, such aging models include screening for agents for preventing and / or improving aging, using the activity of active oxygen and lipid peroxide to inhibit the decomposition of skin components such as collagen and hyaluronic acid as an index. It has been done. On the other hand, as a model of the in vivo reaction, a Maillard reaction, which is a reaction of forming a Schiff base between a sugar and albumin or an amino acid, has been conceived, and a screening for an agent for preventing or improving aging using the inhibitory effect of this reaction as an index. Began to be done. However, even a component which is effective in a screening using such an active oxygen or lipid peroxide as an inhibitory activity of decomposition of skin constituents or an inhibitory activity of Maillard reaction as an index, is not effective in an in vivo drug efficacy assay. There were not many things that were hard to say. That is, it is difficult for any aging researcher to argue that such active oxygen or lipid peroxide alone cannot suppress the degradation of skin constituents or inhibit the Maillard reaction, and thus cannot be an indicator of the prevention or improvement of aging. I think that it is. That is, development of a screening method for prevention and improvement of aging has been desired. From such a point of view, the inventors have continued their research and found that Advanced Glycation End
We have found a screening method that uses products as an index.

On the other hand, the use of a lipofuscin-like substance production inhibitory action as an index for the prevention and improvement of aging can also be achieved by coexisting saccharides, proteins, lipid peroxides and their secondary products present in the body, and maintaining a temperature close to that of a living body. And measure the difference in fluorescence intensity between before and after the incubation.
The protein, lipid peroxide and its secondary product coexist with the test substance, compare the difference in the fluorescence intensity measured under the same conditions, with the degree of suppression of the enhancement of the fluorescence intensity in the incubation of the test substance as an index, No identification of the lipofuscin-like substance production inhibitory action has been carried out, and a substance having such excellent properties is contained in cosmetics, and a skin external preparation such as a cosmetic suitable for prevention and improvement of aging is designed. Nothing was done at all.

[0004]

DISCLOSURE OF THE INVENTION The present invention has been made under such circumstances, and provides a novel screening method for prevention and improvement of aging and application of such technology.
It is an object of the present invention to provide a skin external preparation useful for preventing and improving aging.

[0005]

In view of such a situation, the present inventors have conducted intensive studies for a screening method for prevention and improvement of aging.・ We found that the presence of lipid peroxide under the conditions of endo-product formation caused the formation of a different lipofuscin-like substance, which was further investigated and found to be due to light irradiation, etc. ,)
It has been found that lipofuscin-like substances play an important role as aging reactants in living organisms. Based on this knowledge,
By using such a lipofuscin-like substance as an index,
As a result of finding that it is possible to distinguish skin aging and conducting repeated research for a model, saccharides, proteins, lipid peroxides and their secondary products present in the living body coexist, and incubated at a temperature close to that of the living body Then, the difference in fluorescence intensity before and after the incubation is measured, and the saccharides present in the living body,
The protein, lipid peroxide and its secondary product coexist with the test substance, compare the difference in the fluorescence intensity measured under the same conditions, and use the degree of suppression of the enhancement of the fluorescence intensity in the incubation of the test substance as an index As a result, it was possible to discriminate the test substance from inhibiting the production of a lipofuscin-like substance, and based on this effect, it was possible to screen for an agent for preventing or improving aging, thereby completing the invention. Hereinafter, the present invention will be described in detail focusing on embodiments.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION (1) Method for discriminating the action of inhibiting the production of lipofuscin-like substance of the present invention In the present invention, lipofuscin-like substance is used as an indicator of aging. Here, lipofuscin-like substances refer to proteins, peptides and lipid peroxides and their secondary products (aldehydes, etc.) in vivo.
Means a lipid-peroxide-protein complex that is insoluble in water and is caused by a multiple production reaction with the same. It is a naturally irreversible product and a fluorescent material. As shown in Examples below, the formation of this substance is closely related to skin aging. Therefore, the effect of the substance on aging can be discriminated by using, as an index, the ease with which the substance is produced, the difficulty with which it is produced, and the ease with which it can be suppressed. One of the reasons why this reaction is complicated is that there are many types of substances in the skin that cause the reaction, and lipid peroxidation due to various causes such as ultraviolet rays and inflammation is also an important factor. Because there is. Therefore, such screening requires the presence of the main components and lipid peroxide in the living body. As such participating substances in the living body, if it is a saccharide, hyaluronic acid and / or a salt, glucose and the like can be preferably exemplified, and if it is a substance having an amino group such as a protein, a peptide and an amino acid,
Collagen, elastin and the like can be preferably exemplified. In the case of nucleic acids, nucleosides such as adenosine, guanosine, uridine and thymidine, nucleotides such as ATP and ADP, RNA and DNA can be preferably exemplified. Among them, sugars and / or proteins can be exemplified. Is particularly preferred. In view of the influence of solubility and importance as a reaction site, lipid peroxides and secondary products thereof generated by irradiation of phospholipids such as lecithin with ultraviolet rays can be exemplified. Further, it is preferable to coexist nucleic acids present in the cells. This is mixed, incubated at a temperature close to the living body at 35 ° C. to 45 ° C., the production of the lipofuscin-like substance is measured by fluorescence intensity, the difference in the amount of production before and after the incubation is determined, and the magnitude of this difference is determined by the test substance. The degree to which this difference becomes smaller can be discriminated from the inhibition value of lipofuscin-like substance production, that is, the inhibitory effect of the test substance on lipofuscin-like substance. This degree can be expressed as the absolute value obtained by subtracting the peak area before incubation in the presence of the test substance from the peak area after incubation in the presence of the test substance, or as that of the control. The comparison and display as a ratio, for example, (peak area after incubation in the presence of the test substance-peak area before incubation in the presence of the test substance) is expressed as (post-incubation in the absence of the test substance) Peak area −
(The peak area before incubation in the absence of the test substance) can also be used. this is,
This is because the reproducibility of the screening method of the present invention is good, and there is no problem in treating it as an absolute value. Preferably, the comparison is performed using a relative value compared to a control. This value is more stable. The smaller the value (including the negative value), the greater the effect of inhibiting the production of lipofuscin-like substances. In terms of the strength of such an effect, (peak area after incubation in the presence of the test substance−peak area before incubation in the presence of the test substance) is calculated as (peak area after incubation in the absence of the test substance). Area-
A component having a value of 0.5 or less, more preferably 0.3 or less, divided by the peak area before incubation in the absence of the test substance) has such a strong effect and is considered to be a lipofuscin-like substance production inhibitor. Be identified.

(2) Method for Screening Agent for Prevention / Improvement of Aging of the Present Invention
It is characterized in that the degree of the lipofuscin-like substance production inhibitory action is used as an index. That is, as compared with the presence or absence of the test substance, the greater the effect, the older the aging, especially, the greater the suppression of skin aging, and the greater the suppression of lipofuscin-like substance production. The screening method of the present invention distinguishes a substance having an action from a substance that prevents or ameliorates aging. In this index,
In the present invention, a substance which is suppressed to 0.5 or less in the presence of about 0.1% by weight of a test substance as compared with a control in which no test substance is present is distinguished from an excellent preventive / ameliorating agent for aging. I do.
Such an agent for preventing or improving aging according to the present invention can prevent aging of the skin by containing it in an external preparation for skin such as cosmetics and administering it. In addition, it is possible to further suppress the progress of aging and improve the aging state.
By preventing or improving aging in this manner, the effect of substances that supplement the cortical function and barrier function of the skin that has deteriorated due to aging can be further increased, and the skin can be kept more youthful. Thus, it is possible to design a skin external preparation such as a cosmetic which is useful for aging by using the lipofuscin-like substance production-suppressing action and aging-suppressing action of the present invention as an index. It is.

(3) The Aging Inhibitor of the Present Invention The aging inhibitor of the present invention is determined to be effective in the above-mentioned method for discriminating the aging inhibitory action based on the lipofuscin-like substance inhibitory action as an index. (I.e., (peak area after incubation in the presence of the test substance-peak area before incubation in the presence of the test substance)), (peak area after incubation in the absence of the test substance- (The peak area before incubation in the absence of the compound) is 0.5 or less, more preferably 0.3 or less. Here, the term “component” means a generic term for a simple chemical substance, a mixed composition of a crude drug, an extract from plants and animals, and a fractionated purified product thereof. These components inhibit the production of lipofuscin-like substances, thereby preventing the aging of the skin, which loses elasticity and becomes dull due to the deposition of such substances, the rate of their deposition and its The balance of the rate of disappearance due to self-reaction can be improved by leaning in the direction of disappearance. The route of administration is not particularly limited, and may be any of oral administration, external application to the skin, and intravascular administration. External application to the skin is particularly preferable because it is closest to the site of the reaction.

(4) External preparation for skin of the present invention The external preparation for skin of the present invention is characterized by containing the above-mentioned aging inhibitor. The external preparation for skin of the present invention has an effect of suppressing the generation and accumulation of lipofuscin-like substances in the skin, preventing and improving skin aging by the action of the above-mentioned skin aging inhibitor. Further, it is possible to prevent the hair and the like from generating such a substance and causing troubles such as hair loss. These may contain only one kind or two or more kinds in combination.
The content of the skin aging inhibitor in the external preparation for skin of the present invention is preferably 0.01 to 10% by weight, and is preferably 0.05 to 10% by weight.
-5% by weight is more preferred. This is because if the concentration is too high, the percutaneous absorption efficiency may decrease and the effect may reach a plateau, and if the concentration is too low, the effective concentration may not be obtained.
Further, as the type of the external preparation for skin of the present invention, cream,
Emulsion, cosmetics, under makeup cosmetics, bath agent,
Preferred examples include cosmetics such as hair tonics, and external medicines for skin such as antifungal agents, anti-inflammatory agents, and analgesics. In the skin external preparation of the present invention, in addition to the above-mentioned essential ingredient, an agent for inhibiting skin aging, the optional ingredients used in these skin external preparations are contained as long as the effect of the skin external preparation is not impaired. can do. Such optional components include, for example, hydrocarbons such as squalane, petrolatum, and microcrystalline wax, jojoba oil, carnauba wax, esters such as octyldodecyl oleate, triglycerides such as olive oil, tallow, and coconut oil; Fatty acids such as stearic acid, oleic acid and ritinoleic acid, higher alcohols such as oleyl alcohol, stearyl alcohol and octyldodecanol, anionic surfactants such as sulfosuccinates and sodium polyoxyethylene alkyl sulfate, and amphoteric salts such as alkyl betaine salts Surfactants, cationic surfactants such as dialkylammonium salts, sorbitan fatty acid esters, fatty acid monoglycerides, their polyoxyethylene adducts, polyoxyethylene alkyl ethers, polyoxyethylene Nonionic surfactants such as len fatty acid esters, polyhydric alcohols such as polyethylene glycol, glycerin and 1,3-butanediol, thickening / gelling agents, antioxidants, ultraviolet absorbers, coloring agents, preservatives And powder.
Of course, ursolic acid, ursolic acid alkyl esters, benzyl esters and other collagen fiber bundle remodeling agents, and phytosterols, steroids such as phytosteide, etc. preferable. The external preparation for skin of the present invention can be produced by treating these optional and essential components according to a conventional method. The above-mentioned effects can be exerted by administering an appropriate amount of this skin external preparation to the skin once or several times a day.

[0010]

EXAMPLES Hereinafter, the present invention will be described with reference to Examples.
Although described in more detail, it goes without saying that the present invention is not limited to only these examples.

Example 1 A 50% ethanol extract of leaves of various plants shown in Table 1 was concentrated to produce a plant essence. These extract concentrates were examined for lipofuscin-like substance production inhibitory action. That is, a 50 mM phosphate buffer (pH 7.4) solution of sodium hyaluronate (10
0 mg / 40 ml), ultraviolet rays (BLB lamp;
1 ml of irradiated and peroxidized lecithin liposome and 500 μl of elastin (Sansei Pharmaceutical Co., Ltd.)
l, glucose 20 mg, 50 mM phosphate buffer (pH
7.4) Weigh 4 ml, stir well, dilute 1/10 with the above phosphate buffer, add 1 m to 10 ml of this solution
g of plant essence, 40 ° C in a shaking incubator,
Incubated for one week. Before and after the incubation, the fluorescence intensity (Ex: 360 nm, Em: 400 to 600)
nm), and the value of this difference (after-before) was determined and defined as d1. For the control, the same operation was performed without adding the plant essence, and the value of the difference (after-before) was obtained and defined as d2. D1 / d2 which is an index of inhibition of skin aging according to the present invention was determined. This is shown in Table 1.

[0012]

[Table 1]

Example 2 A group of 5 hairless mice was used to examine the effect of ultraviolet radiation on the aging inhibition in a photoaging model. That is, the hairless mouse was applied with 0.1 ml of the above-mentioned plant essence and pre-treated, and then 1/3 of MED.
Irradiation with twice the amount of ultraviolet light (BLB lamp; manufactured by Toshiba Corporation) was performed. This operation was performed 8 times at a rate of 5 times / week for 8 weeks, and the state of elasticity of the skin was compared with the average level of the control group not subjected to ultraviolet irradiation and sample administration, ++: very elastic, + : Elasticity, ±: Slight loss of elasticity,-: Based on the remarkable loss of elasticity, and the dullness of the skin color is similarly ++: Almost no dullness, +: Dullness is suppressed, ± Table 2 shows the results of the evaluation based on the criteria of:: Slight dullness is suppressed,-: Dullness is not suppressed. Thus, the lipofuscin-like substance production inhibitor (aging inhibitor) of the present invention, wherein the value of d1 / d2 is 0.5 or less,
It can be seen that in the photoaging model, there is an effect of preventing aging due to light. This tendency is even stronger when the value of d1 / d2 is 0.3 or less. It can also be seen that these effects are correlated with the small value of d1 / d2.

[0014]

[Table 2]

Example 3 Using a group of five hairless mice, the effect of ultraviolet light on the aging inhibition in a photoaging model was examined. That is, 1/3 times the amount of ultraviolet light (BL
B lamp (manufactured by Toshiba Corporation) was applied, and 0.1 ml of the above-mentioned plant essence was applied and post-treated. This work was performed 5 times / week for 8 weeks, and the state of skin elasticity was
++: very elastic, +: elastic, ±: slightly less elastic,-: significantly less elastic compared to the average level of the control group without UV irradiation and sample administration Also, the dullness of the skin color was evaluated in the order of ++: almost no dullness, +: dullness was suppressed, ±: slightly dulled, −: dullness was not suppressed. Table 3 shows the results. This indicates that the aging inhibitor of the present invention inhibits aging even after administration after ultraviolet irradiation. From this, it can be seen that the effect of Example 2 is not an effect due to the ultraviolet absorbing function or the like, but is a substantial anti-aging effect.

[0016]

[Table 3]

<Examples 4 to 11> Lotions were prepared according to the following formulations. That is, the ingredients are stirred at room temperature.
Solubilized to obtain a lotion. For these cosmetics, a group of three panelists suffering from dullness were used twice a day for one month in the morning and evening to evaluate the improvement of dullness. The evaluation criteria are rating 2: significant improvement, rating 1: obvious improvement, rating 0.5: slight improvement, 0: no improvement. Table 4 shows the results. From this, it was confirmed that the cosmetic for preventing and improving aging of the present invention has an effect of improving aging such as dullness. Plant essence * 1 part by weight 1,3-butanediol 5 parts by weight Glycerin 3 parts by weight Sodium citrate 0.1 part by weight Methylparaben 0.2 parts by weight Ethanol 8 parts by weight Water 82.7 parts by weight * Details are shown in Table 4. Show.

[0018]

[Table 4]

Example 12 According to the following formulation,
An emulsion (external skin medicine) was prepared. That is, the prescription ingredient a,
(B) and (c) were heated to 80 ° C., and (b) was gradually added to the mixture while stirring to emulsify. Further, (c) was gradually added to neutralize the mixture. B) Squalane 10 parts by weight Cetanol 3 parts by weight Sorbitan sesquistearate 2 parts by weight Polyoxyethylene (20) behenyl ether 2 parts by weight Vitamin A acid 1 part by weight b) 1,3-butanediol 5 parts by weight Yukinoshita essence 0. 1 part by weight Carboxyvinyl polymer 0.3 part by weight Water 40 parts by weight C) Water 36.4 parts by weight Potassium hydroxide 0.2 parts by weight

[0020]

According to the present invention, it is possible to provide a novel screening method for prevention and improvement of aging and to provide a skin external preparation useful for prevention and improvement of aging by applying such a technique.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61K 31/00 617 A61K 31/00 617 35/78 35/78 C 45/00 45/00 G01N 21/78 G01N 21/78 C (72) Inventor Masaki Okada 27-1 Takashimadai, Kanagawa-ku, Yokohama-shi, Kanagawa Prefecture F-term in the Yokohama Research Laboratories, Ltd. F-term (reference) 2G054 AA06 AB10 BB13 CA21 EA03 JA01 4C083 AA112 AB032 AC022 AC072 AC102 AC122 AC182 AC302 AC442 AC482 AD092 AD622 CC04 CC05 DD27 DD31 EE50 4C084 AA30 BA34 BA36 CA13 MA17 MA22 MA63 NA14 ZA891 ZA921 ZB351 ZC801 4C088 AB03 AB12 AB40 AB51 AB52 AB66 AB80 AC05 BA08 MA17 MA22 MA63 NA14 ZA89 ZAZZA92

Claims (9)

[Claims] Claims: 1. A saccharide present in a living body, in which a saccharide, a protein, a lipid peroxide and a secondary product thereof present in the living body are allowed to coexist, incubated at a temperature close to that of the living body, and a difference in fluorescence intensity between before and after the incubation is measured. , Proteins, lipid peroxides and their secondary products coexist with the test substance, compare the difference in fluorescence intensity measured under the same conditions, the degree of suppression of the enhancement of the fluorescence intensity in the incubation of the test substance as an index A method for identifying the inhibitory action of a test substance on the production of lipofuscin-like substances. 2. As the saccharides present in the living body, one or two or more selected from glucose, hyaluronic acid and salts thereof.
The method for identifying a lipofuscin-like substance production inhibitory action according to claim 1, wherein at least one species is used. 3. The method according to claim 1, wherein collagen and / or elastin is used as an index as a protein present in a living body. 4. As a lipid peroxide and a secondary product thereof,
The method for identifying a lipofuscin-like substance production-suppressing action according to claim 1, wherein a phospholipid which has been peroxidized by ultraviolet light or the like is present. 5. A method for screening a skin aging inhibitor, characterized by using a lipofuscin-like substance production inhibitory action as an index, which is distinguished by the method according to any one of claims 1 to 4. 6. The method for screening an inhibitor according to claim 5, wherein (peak area after incubation in the presence of the test substance−peak area before incubation in the presence of the test substance) is determined by (test A skin aging inhibitor comprising a component having a value divided by 0.5 or less (peak area after incubation in the absence of a substance-peak area before incubation in the absence of a test substance) of 0.5 or less. 7. An external preparation for skin for preventing and improving skin aging, comprising the skin aging inhibitor according to claim 5. 8. The external preparation for skin according to claim 7, which is a cosmetic. 9. It is discriminated by the method for discriminating the action of inhibiting lipofuscin-like substance production according to any one of claims 1 to 4,
A method for designing an external preparation for skin for preventing and improving aging using the degree of lipofuscin-like substance production inhibitory action as an index.