JP7539127B2 - Cell proliferation promoters, MMP-2 inhibitors, skin preparations for external use, medicines and internal preparations - Google Patents

Description

The present invention relates to a cell proliferation promoter, an MMP-2 inhibitor, a skin topical agent, a pharmaceutical, and an internal agent.

As we age, the proliferation and division ability of epidermal cells declines, and the epidermal layer itself becomes thinner (Non-Patent Document 1). Biological factors such as epidermal growth factor (EGF) and female hormones (estrogen) promote the proliferation of epidermal cells in the skin, but their secretion declines with age. This decline in the metabolic function of epidermal cells due to aging slows down the turnover rate of the skin, causing rough skin and skin aging. In addition, when keratinocytes that peel off from the surface of the stratum corneum remain, the excretion of melanin in the epidermis is not carried out smoothly, causing pigmentation and dull skin. It is also known that wound healing of the epidermis slows down. In order to prevent or improve the progression of these phenomena, there has been a search for ingredients that promote the proliferation of epidermal cells, and many topical skin preparations have been proposed.

Gelatinase (MMP-2), which belongs to matrix metalloproteinases (hereinafter referred to as MMP), is an enzyme produced by fibroblasts, endothelial cells, cancer cells, etc., and degrades matrices such as collagen, gelatin, and elastin (structural proteins that are special components of elastic tissues such as arteries, tendons, and skin), as well as laminin 5, a major component of basement membrane. It has been revealed that its expression and activity are greatly increased by UV irradiation, and that it is the cause of the decrease in basement membrane components and structural changes in the basement membrane caused by UV rays, which is a major factor in the formation of wrinkles and sagging in the skin (Non-Patent Document 2). Therefore, substances that have inhibitory activity against gelatinase are expected to have an effect of suppressing angiogenesis and metastasis of cancer in cancer tissues as well as preventing wrinkles and sagging in the skin, and are considered to be useful for the prevention and treatment of cancer diseases. Furthermore, it has been reported that MMP-2 is involved in the decomposition of extracellular matrix in various pathological conditions such as ulcer formation, chronic rheumatoid arthritis, osteoporosis, and periodontitis. Therefore, if it has MMP-2 inhibitory activity, it will be useful in treating and improving various diseases caused by increased MMP-2, such as cancer metastasis, ulcer formation, chronic rheumatoid arthritis, osteoporosis, and periodontitis.

It is known that the extract of the whole plant of Agrimonia pilosa, which belongs to the family Rosaceae and the order Rosales, has tyrosinase activity inhibitory effects and active oxygen inhibitory effects, and is used in whitening and anti-aging cosmetics (Patent Document 1), has melanin production inhibitory effects, MMP-1 activity inhibitory effects, and collagen production promoting effects, and is used in skin cosmetics (Patent Document 2), and has dopa oxidase activity inhibitory effects and is used as a whitening agent (Patent Document 3). However, it was not known that Agrimonia has cell proliferation promoting effects and MMP-2 inhibitory effects.

Japanese Patent Application Publication No. 5-97648 JP 2010-65009 A JP 2010-195731 A

Varani J et al. , J Invest Dermatol, Vol. 3, pp 57-60, 1998 Gary J. Fisher et al. , Nature, Vol. 379, No. 25, pp335, 1996

There is a demand for materials that are safe, stable, and have excellent cell proliferation promoting and MMP-2 inhibitory effects, but currently no fully satisfactory materials have been provided.

In light of these circumstances, the inventors conducted extensive research and discovered that an extract from Agrimony has excellent cell proliferation promoting and MMP-2 inhibitory effects, and is also highly stable. Furthermore, they discovered that topical or internal preparations containing the extract are safe and stable, have excellent cell proliferation promoting and MMP-2 inhibitory effects, and can be used as multifunctional beauty and health materials and medicines, leading to the completion of the present invention.

The agrimony used in this invention is a perennial plant of the genus Agrimony, family Rosaceae, order Rosales, which is widely distributed from Japan to Eastern Europe. In Japan, it grows in forests, fields, roadsides, etc. in Honshu, Shikoku, Kyushu, etc. The plant grows to a height of about 1m, and its surface is densely covered with long hairs. From summer to autumn, it produces small spike-shaped yellow flowers at the top of its long stems. It is said that the name "Agrimony" came from the fact that the long, slender spikes of these small yellow flowers resemble "golden mizuhiki". The herbal name is "Sengakusou" or "Ryugasou".

In the present invention, the extract of agrimony is made from agrimony (scientific name: Agrimonia pilosa) of the family Rosaceae in the order Rosalis, and is extracted from parts of the plant such as flowers, fruits, seeds, leaves, stems, and roots, or from the whole plant. The extraction method is not particularly limited, and may be, for example, extraction by heating or extraction at room temperature. For extraction, the plant may be used as it is, or may be processed by drying, crushing, shredding, etc.

The extraction method is not particularly limited, but may be carried out by stirring or column extraction using water or hot water, or a mixed solvent of water and an organic solvent. Examples of the extraction solvent include water, lower alcohols (methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, etc.), liquid polyhydric alcohols (1,3-butylene glycol, propylene glycol, glycerin, etc.), ketones (acetone, methyl ethyl ketone, etc.), acetonitrile, esters (ethyl acetate, butyl acetate, etc.), hydrocarbons (hexane, heptane, liquid paraffin, etc.), and ethers (diethyl ether, tetrahydrofuran, propyl ether, etc.). Preferably, polar solvents such as water, lower alcohols, and liquid polyhydric alcohols are good, and more preferably, water, ethanol, 1,3-butylene glycol, and propylene glycol are good. These solvents may be used alone or in combination. Particularly preferred extraction solvents include water or a mixed polar solvent of water-ethanol. There is no particular limit to the amount of solvent used; for example, it is sufficient to use at least 10 times, and preferably at least 20 times, the amount of the whole plant (dry weight) of Agrimony, but it is preferable to use no more than 100 times for convenience of operations when concentrating or isolating after extraction. In addition, the extraction temperature and time can be appropriately selected depending on the type of solvent used, the pressure during extraction, etc.

The above extract may be used as it is in the form of an extracted solution, but if necessary, it may be used after being subjected to processes such as concentration (vacuum concentration, membrane concentration, etc.), dilution, filtration, decolorization with activated carbon, deodorization, ethanol precipitation, etc., within the scope of the effects of the present invention. Furthermore, the extracted solution may be concentrated to dryness, spray-dried, freeze-dried, etc., and used as a dried product.

In the present invention, the above extract may be used as is, or may contain ingredients such as fats and oils, waxes, hydrocarbons, fatty acids, alcohols, esters, surfactants, metal soaps, pH adjusters, preservatives, fragrances, moisturizers, powders, UV absorbers, thickeners, pigments, antioxidants, whitening agents, chelating agents, excipients, coating agents, sweeteners, acidulants, and other ingredients used in cosmetics, quasi-drugs, medicines, and foods, within the scope of the extract's effects.

The present invention can be used in any of cosmetics, quasi-drugs, medicines, and foods, and examples of the dosage forms include lotions, creams, milky lotions, gels, aerosols, essences, packs, cleansers, bath products, foundations, dusting powders, lipsticks, ointments, poultices, tablets, capsules, chocolates, gums, candies, beverages, powders, granules, tablets, sugar-coated tablets, capsules, syrups, pills, suspensions, liquids, emulsions, suppositories, and injectable solutions.

When used externally, the content of the above extract used in the present invention is preferably 0.0001% by weight or more, and more preferably 0.001 to 10% by weight, calculated as solid matter. Furthermore, 0.01 to 5% by weight is most preferable. If it is less than 0.0001% by weight, it is difficult to expect a sufficient effect. If it exceeds 10% by weight, it is difficult to see an increase in effect, and it is uneconomical.

When taken internally, the dosage varies depending on age, body weight, symptoms, therapeutic effect, administration method, treatment time, etc. In general, the daily dosage for an adult is preferably 5 mg or more, more preferably 10 mg to 5 g. Furthermore, 20 mg to 2 g is most preferable.

In the following, in order to explain the present invention in detail, examples of production, experiment and formulation of the extract used in the present invention are given as examples, but the present invention is not limited to these. The percentages shown in the production examples are % by weight, and the parts of the content shown in the formulation examples are parts by weight.

Production Examples of Agrimony Extracts Agrimony extracts were produced as follows: In Production Examples 1 to 4, the whole plant of Agrimony was used as the extraction material.

(Production Example 1) Preparation of hot water extract of agrimony 200 mL of water was added to 10 g of dried agrimony, and extraction was carried out for 2 hours at 95 to 100° C. The obtained extract was filtered, and the filtrate was concentrated and freeze-dried to obtain 0.9 g of a hot water extract of agrimony.

(Production Example 2) Preparation of 50% ethanol extract of agrimony 10 g of dried agrimony was immersed in 200 mL of 50% ethanol aqueous solution at room temperature for 7 days to perform extraction. The obtained extract was filtered and then concentrated to dryness using an evaporator to obtain 1.0 g of 50% ethanol extract of agrimony.

(Production Example 3) Preparation of ethanol extract of agrimony 10 g of dried agrimony was immersed in 200 mL of ethanol at room temperature for 7 days to perform extraction. The obtained extract was filtered and then concentrated to dryness using an evaporator to obtain 0.2 g of ethanol extract of agrimony.

(Production Example 4) Preparation of 1,3-butylene glycol extract of agrimony 10 g of dried agrimony was immersed in 200 mL of 1,3-butylene glycol at room temperature for 7 days to perform extraction. The obtained extract was filtered to obtain 190 g of 1,3-butylene glycol extract of agrimony.

(Formulation Example 1) Lotion Formulation Content (parts)
1. Hot water extract of agrimony (Production Example 1) 2.0
2. 1,3-butylene glycol 8.0
3. Glycerin 2.0
4. Xanthan gum 0.02
5. Citric acid 0.01
6. Sodium citrate 0.1
7. Ethanol 5.0
8. Methyl parahydroxybenzoate 0.1
9. Polyoxyethylene hydrogenated castor oil (40 E.O.) 0.1
10. Fragrance, appropriate amount 11. Add purified water to make the total amount 100 [Manufacturing method] Dissolve ingredients 1-6 and 11 and ingredients 7-10 uniformly, mix the two, and filter to make the product.

(Comparative Formulation Example 1) Conventional lotion A conventional lotion was prepared by replacing the hot water extract of agrimony in Formulation Example 1 with purified water.

(Formulation Example 2) Cream Formulation Content (parts)
1. 50% ethanol extract of agrimony (Preparation Example 2) 1.0
2. Squalane 5.5
3. Olive oil 3.0
4. Stearic acid 2.0
5. Beeswax 2.0
6. Octyldodecyl myristate 3.5
7. Polyoxyethylene cetyl ether (20 E.O.) 3.0
8. Behenyl alcohol 1.5
9. Glyceryl monostearate 2.5
10. Fragrance 0.1
11. Methyl parahydroxybenzoate 0.2
12. 1,3-butylene glycol 8.5
13. Make up to 100% with purified water [Manufacturing method] Heat and dissolve ingredients 2-9, mix, and maintain at 70°C to make the oil phase. Heat and dissolve ingredients 1 and 11-13, mix, and maintain at 75°C to make the water phase. Add the water phase to the oil phase to emulsify, cool with stirring, add ingredient 10 at 45°C, and further cool to 30°C to make the product.

(Comparative Formulation Example 2) Conventional Cream A conventional cream was prepared by replacing the 50% ethanol extract of Agrimony in Formulation Example 2 with purified water.

(Formulation Example 3) Emulsion Formulation Content (parts)
1. Ethanol extract of agrimony (Production Example 3) 0.01
2. Squalane 5.0
3. Olive oil: 5.0
4. Jojoba oil 5.0
5. Cetanol 1.5
6. Glyceryl monostearate 2.0
7. Polyoxyethylene cetyl ether (20 E.O.) 3.0
8. Polyoxyethylene sorbitan monooleate (20 E.O.) 2.0
9. Fragrance 0.1
10. Propylene glycol 1.0
11. Glycerin 2.0
12. Methyl parahydroxybenzoate 0.2
13. Make up to 100% with purified water [Manufacturing method] Heat and dissolve ingredients 1-8, mix, and keep at 70°C to make the oil phase. Heat and dissolve ingredients 10-13, mix, and keep at 75°C to make the water phase. Add the water phase to the oil phase to emulsify, cool with stirring, add ingredient 9 at 45°C, and cool further to 30°C to make the product.

(Formulation Example 4) Gel Formulation Content (parts)
1. 1,3-butylene glycol extract of agrimony (Production Example 4) 1.0
2. Ethanol 5.0
3. Methyl parahydroxybenzoate 0.1
4. Polyoxyethylene hydrogenated castor oil (60 E.O.) 0.1
5. Fragrance (appropriate amount) 6. 1,3-butylene glycol 5.0
7. Glycerin 5.0
8. Xanthan gum 0.1
9. Carboxyvinyl polymer 0.2
10. Potassium hydroxide 0.2
11. Add purified water to make the total volume 100. [Manufacturing method] Dissolve ingredients 2 to 5, 1, and 6 to 11 uniformly, and mix the two to make the product.

(Formulation Example 5) Pack formulation Content (parts)
1. Hot water extract of agrimony (Production Example 1) 1.0
2. 1,3-butylene glycol extract of agrimony (Production Example 4) 5.0
3. Polyvinyl alcohol 12.0
4. Ethanol 5.0
5. 1,3-butylene glycol 8.0
6. Methyl parahydroxybenzoate 0.2
7. Polyoxyethylene hydrogenated castor oil (20 E.O.) 0.5
8. Citric acid 0.1
9. Sodium citrate 0.3
10. Fragrance (as needed) 11. Add purified water to make a total of 100 parts. [Manufacturing Method] Dissolve ingredients 1 to 11 uniformly to make the product.

(Formulation Example 6) Foundation Formulation Content (parts)
1. 50% ethanol extract of agrimony (Preparation Example 2) 1.0
2. Stearic acid 2.4
3. Polyoxyethylene sorbitan monostearate (20 E.O.) 1.0
4. Polyoxyethylene cetyl ether (20 E.O.) 2.0
5. Cetanol 1.0
6. Liquid Lanolin 2.0
7. Liquid paraffin 3.0
8. Isopropyl myristate 6.5
9. Sodium carboxymethylcellulose 0.1
10. Bentonite 0.5
11. Propylene glycol 4.0
12. Triethanolamine 1.1
13. Methyl parahydroxybenzoate 0.2
14. Titanium dioxide 8.0
15. Talc 4.0
16. Bengala 1.0
17. Yellow iron oxide 2.0
18. Flavoring, appropriate amount 19. Make the total volume 100 with purified water [Manufacturing method] Heat and dissolve ingredients 2 to 8, and keep at 80°C to make the oil phase. Allow ingredient 9 to swell well in ingredient 19, then add ingredients 1 and 10 to 13 and mix uniformly. Add ingredients 14 to 17, which have been ground and mixed in a grinder, and stir with a homomixer to make the water phase, keeping the temperature at 75°C. Add the water phase to the oil phase while stirring, and emulsify. Then cool, add ingredient 18 at 45°C, and cool to 30°C while stirring to make the product.

(Formulation Example 7) Bath additive Formulation Content (parts)
1. Ethanol extract of agrimony (Production Example 3) 1.0
2. Sodium bicarbonate 50.0
3. Yellow No. 202 (1) appropriate amount 4. Fragrance appropriate amount 5. Sodium sulfate to make the total amount 100 [Manufacturing method] Mix ingredients 1 to 5 uniformly to make the product.

(Formulation Example 8) Ointment Formulation Content (parts)
1. Hot water extract of agrimony (Production Example 1) 5.0
2. 1,3-butylene glycol extract of agrimony (Production Example 4) 1.0
3. Polyoxyethylene cetyl ether (30 E.O.) 2.0
4. Glyceryl monostearate 10.0
5. Liquid paraffin 5.0
6. Cetanol 6.0
7. Methyl parahydroxybenzoate 0.1
8. Propylene glycol 10.0
9. Make the total volume 100 with purified water. [Manufacturing method] Heat and dissolve ingredients 3-6, mix, and keep at 70°C to make the oil phase. Heat and dissolve ingredients 1, 2, and 7-9, mix, and keep at 75°C to make the water phase. Add the water phase to the oil phase to emulsify, and cool to 30°C while stirring to make the product.

(Formulation Example 9) Powder Formulation Content (parts)
1. Hot water extract of agrimony (Production Example 1) 1.0
2. Dried cornstarch 39.0
3. Microcrystalline cellulose 60.0
[Manufacturing method] Mix ingredients 1 to 3 to make a powder.

(Formulation Example 10) Tablets Formulation Content (parts)
1. Ethanol extract of agrimony (Production Example 3) 5.0
2. Dried cornstarch 25.0
3. Calcium carboxymethylcellulose 20.0
4. Microcrystalline cellulose 40.0
5. Polyvinylpyrrolidone 7.0
6. Talc 3.0
[Manufacturing method] Components 1 to 4 are mixed, and then an aqueous solution of component 5 is added as a binder to form granules. Component 6 is added to the formed granules and compressed into tablets. Each tablet weighs 0.52 g.

(Formulation Example 11) Tablets Formulation Content (parts)
1. Ethanol extract of agrimony (Production Example 3) 2.0
2. Dry cornstarch 49.8
3. Erythritol 40.0
4. Citric acid 5.0
5. Sucrose fatty acid ester 3.0
6. Fragrance 0.1
7. Add purified water to make up to 100%
[Manufacturing method] Mix ingredients 1 to 4 and 7 and mold into granules. Add ingredients 5 and 6 to the molded granules and compress into tablets. Each tablet weighs 1.0 g.

(Formulation Example 12) Beverage Formulation Content (parts)
1. Hot water extract of agrimony (Production Example 1) 0.05
2. Stevia 0.05
3. Malic acid 5.0
4. Fragrance 0.1
5. Add purified water to make the total volume 100. [Production method] Dissolve components 2 and 3 in a small amount of water. Next, add components 1, 4, and 5 and mix.

Next, experimental examples will be presented to explain the effects of the present invention in detail.

Experimental Example 1 Cell proliferation promotion test HaCaT cells were seeded in a 96-well plate at 5 x ¹⁰³ cells per well in DMEM culture medium containing 0.1% FBS, and each sample was added and then cultured for 4 days under conditions of 37°C and 5% _CO2 . The cell count was measured by staining. That is, after the culture was completed, the culture medium was removed and the cells were fixed using methanol. Then, 0.1% methylene blue was added and the cells were stained for 1 hour. After drying, 100 μL of 0.1N HCl was added to each well and stirred well, and the absorbance at 650 nm was measured using a microplate reader.

The results of these experiments are shown in Table 1. As a result, the hot water extract (Production Example 1), 50% ethanol extract (Production Example 2) and ethanol extract (Production Example 3) of Agrimony of the present invention exhibited excellent cell proliferation promoting activity.

Experimental Example 2 Measurement of MMP-2 mRNA expression level Human fibroblasts NB1RGB were seeded at 1×10 ⁵ cells on a φ60 mm dish, and when the cells became confluent, a sample was added to the cells so that the final concentration was 1 μg/mL. A solvent in which the sample was diluted was added to the control. After 24 hours of culture, total RNA was extracted. Total RNA was extracted from the cells using RNAiso Plus (Takara Bio), and the total RNA amount was determined by absorbance at 260 nm using a spectrophotometer (NanoDrop). The mRNA expression level was measured by real-time RT-PCR based on the total RNA extracted from the cells. For the real-time RT-PCR, High Capacity RNA-to-cDNA Kit (Applied Biosystems) and SYBR Select Master Mix (Life Technologies) were used. That is, 500 ng of total RNA was subjected to reverse transcription reaction, followed by PCR reaction (95°C: 15 seconds, 60°C: 60 seconds, 40 cycles). Other operations were performed according to the prescribed method, and the expression level of MMP-2 mRNA was calculated as a ratio to the expression level of β-actin mRNA, which is an internal standard. The MMP-2 expression rate was calculated as the ratio of the expression level of MMP-2 mRNA in the sample addition group to the expression level of MMP-2 mRNA in the control. The primers used to measure the expression level of each gene are as follows.

Primer set for MMP-2 CCGTCGCCCATCATCAA (SEQ ID NO: 1)
CTTCTGCATCTTCTTTAGTGTGTCCTT (SEQ ID NO: 2)
Primer set for β-Actin: CACTCTTCCAGCCTTCCTTCC (SEQ ID NO: 3)
GTGTTGGCGTACAGGTCTTTG (SEQ ID NO: 4)

The results of these experiments are shown in Table 2. As a result, it was found that the extract of agrimony of the present invention had a significant MMP-2 expression suppressing effect (MMP-2 inhibiting effect). Furthermore, the 50% ethanol extract of agrimony (Production Example 2) had a particularly high MMP-2 expression suppressing effect.

Experimental Example 5: Use test A one-month use test was conducted on five subjects (ages 27 to 65) with wrinkles and sagging skin using the lotion of Formulation Example 1 and the conventional lotion of Comparative Formulation Example 1. After use, the extent of wrinkles and sagging skin was assessed by questionnaire.

As a result, wrinkles and sagging skin were reduced by using the topical skin preparation containing the extract of the present invention. Furthermore, during the test period, not a single subject experienced skin troubles, and there were no safety issues. There were also no problems with deterioration of the prescribed ingredients.

From the above, the agrimony extract of the present invention has excellent cell proliferation promoting effects and MMP-2 inhibitory effects, and is also highly stable. Therefore, the agrimony extract of the present invention can be used not only in the cosmetic field, such as skin aging, but also in the medical field, such as suppressing functional decline due to aging, and preventing and treating cancer, and is expected to be applied to foods, cosmetics, quasi-drugs, pharmaceuticals, etc.

Claims (1)

An oral keratinocyte proliferation promoter (excluding those for improving wrinkles or sagging conditions) characterized by containing an extract of agrimony using ethanol or aqueous ethanol containing 50% by weight or more of ethanol .