JP6681064B2 - Acrolein derivative activity inhibitor - Google Patents

Description

  The present invention relates to an acrolein derivative activity inhibitor that suppresses reactions between acrolein derivatives and biomolecules such as proteins and nucleic acids, and relates to a skin external preparation containing the drug.

  Generally, α, β-unsaturated aliphatic aldehyde compounds such as acrolein, malondialdehyde, and 4-hydroxy-2-nonenal which are produced in the living body are highly reactive and have strong toxicity to the living body and have various symptoms. Or it is known to be associated with a disease. Examples of its production pathway include a peroxidation reaction of biological lipids and an enzymatic reaction of a basic physiologically active substance (Non-patent Documents 1 and 2). Acrolein (CH2 = CHCHO) has the simplest structure among such aldehyde compounds produced in vivo, but malondialdehyde, 4-hydroxy-2-nonenal, etc. also show the same in vivo reaction as acrolein. I know. Therefore, in the present invention, such aldehyde compounds are collectively referred to as "acrolein derivatives", and substances that suppress the reaction between acrolein derivatives and biomolecules are referred to as "activity inhibitors".

  As an example of the harmfulness of an acrolein derivative to the living body, it has been confirmed that acrolein or an acrolein adduct increases in the course of arteriosclerosis or in the blood of stroke patients (Non-patent Documents 3 and 4). It is also known that an acrolein derivative reacts non-specifically and non-enzymatically with proteins of all tissues in the living body to cause carbonylation (Non-Patent Document 5). The presence of such carbonylated proteins has been confirmed throughout the body such as the internal organs, brain, and skin, and it has been reported that carbonylated proteins are also involved in various diseases and symptoms.

  As an example thereof, the presence of carbonylated proteins in the dermis and corneal cells of the skin is recognized. Excessive accumulation of carbonylated protein in the dermis promotes skin color change (yellowish tinge) due to decreased optical permeability and wrinkle formation due to aggravation of actinic elastic fibrosis (Non-patent Documents 6 and 7). In addition, excessive accumulation of carbonylated protein in the stratum corneum causes a skin color change (yellowish tinge) and reduces the water retention function in the stratum corneum (Patent Document 1). It is known that the visual transparency of the skin is reduced by such yellowish tinge, and it has been shown that the skin moisture content and texture may be reduced in the skin with reduced transparency. Patent document 8). Further, it has been reported that cornified cells in patients with atopic dermatitis and psoriasis have excessive accumulation of carbonylated protein (Non-Patent Document 9).

  Further, it has been suggested that an acrolein derivative reacts with not only a protein but also a nucleic acid to form an adduct and damages the nucleic acid, thereby showing mutagenicity (Non-Patent Documents 10 and 11).

  As described above, the acrolein derivative acts on various biomolecules, remarkably lowers biological functions, and accelerates aging. Therefore, if it is possible to suppress the production of acrolein derivatives, the reaction inhibition, and the activity inhibition, it is considered to play a major role in the prevention and alleviation of various diseases and aging.

  In the study of protecting a living body from an acrolein derivative, inventions that inhibit the production of the acrolein derivative have already been made (Patent Documents 2 and 3). However, even by these, the production of acrolein derivative is not completely suppressed, and therefore, there is a strong demand for the development of a drug that suppresses the activity of acrolein derivative.

  With respect to this study, various drugs that inhibit the reaction of acrolein and an amino acid derivative chemically synthesized have been invented (Patent Documents 4 and 5). However, it is unclear whether such a drug is effective even in living cells, and it has been desired to develop a drug capable of suppressing the activity of an acrolein derivative and inhibiting a practical reaction with a biomolecule. As a result of intensive studies aimed at solving this problem, the inventors have reached the present invention.

Patent No. 4526839 JP, 2002-281999, A JP, 2002-138013, A Patent No. 4944372 Patent No. 5063947

Free Radical Biol Med. 2000 Jun 15; 28 (12): 1685-96. Biochem Soc Trans. 2003 Apr; 31 (2): 371-4. Atherosclerosis. 2010 Aug; 211 (2): 475-9. Mol Nutr Food Res. 2011 Sep; 55 (9): 1332-41. YAKUGAKU ZASSHI 133 (10) 1055-1063 (2013) J Dermatol Sci. 2011 Oct; 64 (1): 45-52. Arch Dermatol Res. 2001 Jul; 293 (7): 363-7. J. Soc. Cosmet. Chem. Jpn. 39 (3) 201-208 (2005) Journal of Drmatology 2010; 37: 693-698. Chem Res Toxicol. 2009 May; 22 (5): 798-806. Mol Nutr Food Res. 2011 Sep; 55 (9): 1291-300. Dental Medicine, 2000, 63 (1): 23-32. About Axim Co., Ltd., measurement of skin elasticity (URL: http://www.med-a.co.jp/upfile/pdf_1273891499.pdf?nocache=1391830910) J. Soc. Cosmet. Chem. Japan, Vol. 23, No. 1,55-57 (1989)

  An object of the present invention is to provide a drug that reduces the activity of an acrolein derivative in vivo and improves or alleviates various negative effects of the acrolein derivative on a living body, and a drug or quasi drug containing the drug. To provide an external preparation for skin such as cosmetics or cosmetics.

  In order to solve the above problems, as a result of intensive studies by the present inventors, the Rosaceae plant fruit extract, particularly the apple fruit extract significantly reduces the activity of the acrolein derivative, and with biomolecules such as intracellular proteins. They have found that they have the effect of significantly suppressing the reaction, and have completed the present invention.

  In the present invention, the effect of suppressing the activity of the acrolein derivative was recognized in the Rosaceae plant fruit extract, particularly in the apple fruit extract. Therefore, the external preparation for skin containing the acrolein derivative activity inhibitor and the agent of the present invention is a vascular disease such as stroke or arteriosclerosis in which the symptoms progress due to the reaction of the acrolein derivative and a biomolecule, and aging of systemic tissues (for example, in the case of skin. It can be expected to have a preventive or improving effect on wrinkle formation, reduction of skin water content, enhancement of dullness, etc.).

  Examples of the fruit of the Rosaceae plant include strawberry, apple, pear, loquat, and karin. The acrolein derivative activity inhibitor of the present invention is preferably a fruit extract of the Rosaceae plant, particularly an apple fruit extract.

  In the apple fruit extract, varieties of apple (Malus pumila) include Fuji, Obayashi, Kodama, Mutsu, Tsugaru, Asahi, Delicious, Jonagold, and sunlight. The extraction site of apple fruit is not particularly limited, such as whole fruit, pulp, skin and seed. Further, the fruit may be in a matured state such as a matured fruit or an immature fruit (young fruit), but an immature fruit (young fruit) is particularly preferable, and the fruit is picked at a time of about 5 to 20 g per piece. Things are especially desirable. You may use the said extraction site | part and the extract of a mature state 1 type or in combination of 2 or more types.

  The apple fruit extract may be a commercially available apple fruit extract or may be extracted from apple fruit, and is not particularly limited. The extraction method is not particularly limited, and a conventionally known method can be adopted. Specifically, it may be as follows. For example, apple fruits are crushed and squeezed to obtain fruit juice, and insoluble components such as pectin contained therein are removed by enzymatic decomposition and filtration to obtain clear fruit juice. Alternatively, apple fruits are mixed with alcohol (ethanol, methanol, etc.) and crushed, and then soaked and squeezed or extracted while heating under reflux, and then alcohol is removed by concentration under reduced pressure, followed by centrifugation and filtration, or an organic solvent ( Examples include a method of obtaining an extract by partitioning with hexane, chloroform, etc.) and filtration. The extraction solvent is not particularly limited.

  The amount of the extract according to the present invention to be mixed with various skin external preparations can be varied depending on the embodiment of the skin external preparation, the usage form of the skin external preparation, and the like, and is not particularly limited. For example, it is 0.00005 to 1 mass%, more preferably 0.0005 to 0.1 mass%. In addition, these values are mass ratios as a dried product.

  When the acrolein derivative activity inhibitor of the present invention is used as a skin external preparation, after dissolving the Rosaceae fruit extract in a water-soluble solvent, it can be applied as an aqueous solution diluted with water, or a drug or a pharmaceutical part. It can also be applied in a shape that is generally used as an external product or a cosmetic product.

  The form of the external preparation for skin according to the present invention is not particularly limited. For example, it can be applied in various forms such as cream, emulsion, lotion, pack, face wash, sol, gel, powder, spray and solid.

  Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited thereto.

(1) Suppression of acrolein activity by drugs [Preparation of samples containing various materials and acrolein solution]
Apple extract, was used APPLEPHENONE C-100 (the Asahifu Doandohe Healthcare Ltd. apple polyphenol) as a sample. As comparative materials, L (+)-ascorbic acid ( manufactured by Wako Pure Chemical Industries ), which is known to have high antioxidant power, and its derivative, ascorbyl magnesium phosphate ( manufactured by ITO), are used. I was there.
Each of these three materials was dissolved in pure water to a concentration of 1% and serially diluted to prepare sample solutions of various concentrations. Acrolein Monomer ( manufactured by Tokyo Kasei Kogyo Co., Ltd. ) was used as an acrolein reagent, and this was dissolved in pure water to a concentration of 0.005%.

[Preparation of Acrolein Detection Reagent]
4-Hydrazono-7-nitrobenzofurazan Hydrazine (NBD-H; Tokyo Kasei Kogyo Co., Ltd.), which is a carbonyl group detection reagent, was dissolved in N, N-dimethylformamide (DMF; Wako Pure Chemical Industries, Ltd.) to a concentration of 25 mM. Then, the NBD-H-containing DMF solution was diluted to 0.25 mM with pure water containing 0.05% of Trifluoroacetic acid (TFA; Nacalai Tesque).

[Evaluation of acrolein activity inhibition ability of various materials]
25 μL of the material-containing solution and 50 μL of the acrolein-containing solution were added to each well of the 96-well plate, and the plate was shaken for 1 minute. Then, in order to prevent the evaporation of the solution in the well, the plate surface was sealed and sealed at 37 ° C. for 2 hours. Then, the seal was peeled off, 25 μL of the above-mentioned acrolein detection reagent was added to each well, and the plate was shaken for 1 minute. After that, the plate was left at room temperature in the dark for 30 minutes, and then the fluorescence intensity was measured with a microplate reader (excitation wavelength: 470 nm, fluorescence wavelength: 550 nm). The experimental results are shown in FIG.

[Data processing]
In each of the three materials, the fluorescence intensity value under the condition that each material was not added was defined as the acrolein residual rate of 100%. The residual rate of acrolein was calculated by dividing the fluorescence intensity value when the material was added by this fluorescence intensity value. The acrolein deactivation rate shown in the graph of FIG. 1 was calculated by taking the difference in the acrolein residual rate from 100%.

It is a graph which showed the acrolein activity inhibitory ability evaluation result of various materials.

  From the results shown in FIG. 1, apple fruit extract strongly reacts with aldehyde groups which show reactivity with various molecules as compared with L (+)-ascorbic acid and magnesium ascorbyl phosphate having high antioxidant power, and thus acrolein It was suggested that the effect of deactivating was extremely high.

(2) Inhibition of carbonylated protein production by drugs in acrolein-treated living cells [Preparation of apple fruit extract and acrolein-containing medium]
APPLEPHENONE C-100 as the apple fruit extract (Asahifu Doandohe Healthcare Ltd. apple polyphenol), the acrolein reagent was used Acrolein Monomer (manufactured by Tokyo Kasei Kogyo). Acrolein was added to Dulbecco's modified Eagle medium "Nissui" (DMEM, Nissui Pharmaceutical Co. , Ltd. ) containing 5% fetal bovine serum (FBS) so that the final concentration was 0.0005%. Apple fruit extracts were dissolved in this acrolein-containing medium at various concentrations to prepare apple fruit extract- and acrolein-containing culture solutions.

[Cultivation of immortalized human epidermal cell line and culture with apple fruit extract and acrolein-containing culture medium]
Immortalized human epidermal cell line HaCaT was seeded on a 96-well plate at 5 × 10 4 / well, and cultured at 37 ° C. in a 5% CO 2 incubator for 24 hours. The culture solution was discarded, the cells were washed with PBS (-) solution, 100 μL of the apple fruit extract- and acrolein-containing medium was added to each well, and the cells were further cultured for 24 hours.

[Evaluation of intracellular carbonylation protein amount]
The culture solution was discarded, the cells were washed with PBS (-) solution, 100 μL of cold methanol at -20 ° C was added to each well, and the cells were fixed at -20 ° C for 5 minutes. After discarding methanol and washing the cells with PBS (−) solution, 100 μL of 0.1 M MES-Na (pH 5.5) containing 20 μM FTSC was added to each well, and the mixture was incubated at 37 ° C., 5% CO 2 incubator for 1 hour. I let it stand. The solution in the well was discarded, 100 μL of PBS (−) solution was added to each well, and fluorescence measurement was performed with a microplate reader (excitation wavelength: 492 nm, fluorescence wavelength: 516 nm).

[Evaluation of total intracellular protein amount]
The solution in the well was discarded, and 50 μL of 0.5% octylphenol ethoxylate (Wako Pure Chemical Industries) solution was added to each well to dissolve the intracellular protein. Thereafter, 50 μL of a preparation solution of TaKaRa BCA Protein Assay Kit (Takara Bio) was added to the wells, incubated at 37 ° C. for 10 minutes, and the absorbance was measured with a microplate reader (absorption wavelength: 550 nm). The amount of protein was calculated in terms of BSA (bovine serum albumin, SIGMA). Table 1 shows a summary of the experimental data.

[Data processing]
Table 1 shows the ratio (ratio%) of the intracellular total protein amount and the t-test value (p) when acrolein and various concentrations of apple fruit extracts were allowed to act on HaCaT cells. Table 2 shows the ratio (ratio%) and the t-test value (p) of the ratio of the average value of the amount of carbonylated protein to the total amount of intracellular protein when acrolein and apple fruit extracts of various concentrations were allowed to act on HaCaT cells. Show. That is, it is the ratio of the value obtained by [Evaluation of intracellular carbonylation protein amount] described above divided by the value obtained by [Evaluation of total intracellular protein amount]. The presence or absence of a significant difference between the groups was determined using the Student's t-test value (p) similar to that in Table 1, and p <0.05 was defined as a significant difference (*).

  From the results shown in Table 1, no toxicity was observed in cells treated with acrolein and apple fruit extract at the concentrations examined this time. Further, as shown in Table 2, the apple fruit extract resulted in a significant reduction in the ratio of carbonylated protein to the total intracellular protein amount.

  From the above results, the apple fruit extract had an effect of remarkably suppressing the activity of the acrolein derivative, and showed an effect of significantly inhibiting the carbonylation reaction of intracellular protein caused by the acrolein derivative. That is, it was suggested that the apple fruit extract is a drug having a very high effect of inhibiting the reaction between the acrolein derivative and various biomolecules at a concentration capable of ensuring high safety for the living body. Therefore, it is considered that the same effect can be expected in the skin external preparation containing this drug.

  Formulation examples of the external preparation for skin using the present invention will be given below.

(Preparation of external preparation for skin)
The amount in the table in Example 3 and Comparative Example 3 below shows the mass% unless otherwise specified.
Preparation of skin cream (Table 3, Example 3)
(1) Liquid paraffin 6.0 (mass%)
(2) Isopropyl myristate 3.0
(3) Cyclopentasiloxane 2.5
(4) Dimethicone 2.0
(5) Behenyl alcohol 3.4
(6) Vaseline 2.0
(7) Sorbitan stearate 1.5
(8) Setes-20 3.4
(9) Tocopherol 0.02
(10) Citric acid 0.05
(11) 1,3-butylene glycol 1.0
(12) Methylparaben 0.15
(13) Propylparaben 0.07
(14) Dipropylene glycol 1.9
(15) Apple immature fruit extract 0.003
(16) Purified water balance

(Production method)
The above raw materials (1) to (9) were mixed, shielded from light, and heated and stirred at 80 ° C. ... Oil phase On the other hand, (10) to (16) were mixed and heated and stirred at 80 ° C. ... Water phase The water phase was added while stirring the oil phase at 80 ° C in the light-shielded state, and the mixture was uniformly emulsified with a homogenizer. It is desirable to carry out the entire process in a light-shielded manner, and it is desirable to store it in a light-shielded manner. The immature fruit extract of apple was colored in the dark because it is easily colored, but it can be performed without light.

(Preparation of Comparative Cream (Table 3, Comparative Example 3))
A comparative cream (Table 3, Comparative Example 3) was prepared in exactly the same manner as the preparation of the skin cream described above, except that the immature apple extract was not added. The shading at the time of production and storage was the same as in the example.

(Effect confirmation test of external preparation for skin)
Volunteer women in their 20's to 50's gave Example 15 (average age 35 years) and Comparative Example 14 (Example 3 and Comparative Example 3 (control group)) Average age 36 years).

  The same makeup as usual was applied except that the prepared cream of Example 3 and the cream of Comparative Example 3 were applied to the lower part of both eyes, and the same life as usual was continued for 8 weeks. However, we avoid behaviors such as intentional sunbathing that are extremely exposed to ultraviolet rays.

Regarding the firmness of the skin, the repulsive force of the skin under the eyes was measured before and after the test, and the areas surrounded by the hysteresis curves were compared (Non-Patent Documents 12 and 13).
Regarding the texture of the skin, after the test, the outermost layer of the stratum corneum under both eyes was collected by the tape stripping method (Non-Patent Document 14), and the state change before and after was examined.
Although the panel explained the purpose of the test and publicized precautions such as avoiding sunbathing, it was not clear whether each member corresponded to an example or a comparative example.

Although it is difficult to clearly distinguish the elasticity and elasticity of the skin, the resilience to the minimum amount of fine indentation was measured as elasticity. It is judged that the smaller the area surrounded by the hysteresis curve is, the firmer the elasticity is.
For each person, the area ratio before and after the test was calculated, and the average was calculated.

The texture of the skin was evaluated as follows.
The observation area of the stratum corneum sample was 2.4 cm 2.
From each non-overlapping horny layer cell, 5 cells were selected from the largest one per sample, and the total area was measured. For each person, the ratio of sums before and after the test was calculated. Generally, it is judged that the larger the stratum corneum area, the better the texture.

The results are shown in Table 3.
In Example 3 and Comparative Example 3, the effect of improving the firmness and texture of the skin characteristics was recognized. Moreover, no negative difference was observed.

The acrolein derivative activity inhibitor of the present invention makes it possible to suppress the reaction between an acrolein derivative and various biomolecules at a concentration that can ensure high safety for living bodies. By applying this to an external preparation for skin, it can be expected to have an effect of improving skin firmness and texture, and when applied to food and the like, it can be expected to be effective against various diseases such as arteriosclerosis and stroke. Therefore, this drug is expected to be widely applicable to amelioration of symptoms and diseases related to acrolein derivatives.

Claims (2)

With apple polyphenol extracted from immature apple fruit as an active ingredient,
By suppressing the production of carbonylated proteins
An agent that suppresses the generation of yellowish skin on the skin . The content of apple polyphenol is 0.0005 to 0.1% by mass,
The agent for suppressing skin yellowing according to claim 1.

Images