JP7565075B2 - Elastin production promoter - Google Patents

Description

The present invention relates to an agent for promoting elastin production in myofibroblasts, an agent for enhancing elasticity in fascia or aponeurosis, and a composition for improving or preventing ptosis, each of which contains butterfly pea and/or alpine rose.

Myofibroblasts are one of the cells that make up fascia (Non-Patent Document 1). Their presence in aponeurosis has also been confirmed by electron microscopy (Non-Patent Document 2). Myofibroblasts are the main cell population that expresses elastin, and are thought to be important in the construction of elastic fibers (Non-Patent Document 3).

Elastic fibers are important for the elasticity of fascia (Non-Patent Document 4) and contribute to determining the elasticity of the entire tissue (Non-Patent Document 5). Since the amount of elastin in human fascia decreases with age (Non-Patent Document 6), it is thought that the elasticity of the fascia and the elasticity of the entire tissue declines with age.

As a structure for lifting the eyelids, it has been shown that the connective fascia formed by the fusion of the levator aponeurosis, the posterior fascia of the pretarsal orbicularis oculi muscle, and the orbital septum sends elastic fibers to the tarsal plate and works with the orbicularis oculi muscle and Muller muscle to lift the tarsal plate (Non-Patent Document 7). In addition, the amount of elastin in the levator aponeurosis is reduced in the ptosis group, which is a sagging upper eyelid, and in the aging group (Non-Patent Document 8). Therefore, it is considered that the production of elastin in the connective fascia formed by the fusion of the levator aponeurosis, the posterior fascia of the pretarsal orbicularis oculi muscle, and the orbital septum, and the associated increase in elasticity, are important for improving and preventing ptosis. Ptosis is widely treated by surgery (Non-Patent Document 9), but since surgery is painful, non-surgical treatments are required. Known compositions that can be administered as non-surgical treatment methods include oxymetazoline, phenylephrine (Patent Document 1), and pilocarpine (Patent Document 2).

To date, antioxidants (Patent Document 3) and paxillin stimulants (Patent Document 4) that are characterized by containing an extract of Butterfly Pea as an active ingredient are known. In addition, a method for inducing hair growth using an extract of Alpine Rose (Patent Document 5) and an external skin composition for preventing wrinkles (Patent Document 6) are known. However, no agents that promote elastin production in myofibroblasts, agents that enhance elasticity in fascia or aponeurosis, or compositions for improving or preventing ptosis that are characterized by containing Butterfly Pea or Alpine Rose are known.

Special table 2014-506927 Special table 2018-520176 JP2007-16003 Special table 2013-515054 Special table 2015-522083 Special table 2015-178464

REACH, 14-15, 100030 (2019) Acta. Morphol. Acad. Sci. Hung. , 28(1-2), 71-82 (1980) Scientific Reports, 8, 8334 (2018) Rev. Hosp. Clin. Fac. Med. S. Paulo, 57(6), 265-270 (2002) A Practical Guide to Fascial Manipulation 2017, 59-92 Gegenbaurs Morphol. Jahrb. , 136(6), 645-652 (1990) Ophthalmic. Plast. Reconstr. Surg. , 9, 1-10 (1993) Archives of Aesthetic Plastic Surgery, 21(2), 37-42 (2015) Hiroshi Mizuno, Juntendo Medical Journal, 59, 321-326 (2013)

The problem to be solved by the present invention is to find a plant-derived component that enhances elastin production, and to provide an excellent elastin production promoter with a clear site of action, an elasticity enhancer for fascia or aponeurosis, and a composition for improving or preventing ptosis.

As a result of intensive research aimed at solving the above problems, the inventors have found that butterfly pea and/or alpine rose extract has an excellent effect of promoting elastin production in myofibroblasts. Furthermore, the inventors have found that a composition containing butterfly pea or alpine rose extract has an excellent effect of enhancing elasticity in fascia or aponeurosis, and an effect of improving or preventing ptosis, which led to the completion of the present invention.

That is, the present invention relates to an agent for promoting elastin production in myofibroblasts, an agent for enhancing elasticity in fascia or aponeurosis, and a composition for improving or preventing ptosis, which is characterized by containing an extract of butterfly pea and/or alpine rose.

The extract of butterfly pea and/or alpine rose of the present invention had an excellent effect of promoting elastin production in myofibroblasts. In addition, an elastin production promoter, an elasticity enhancer for fascia or aponeurosis, and a composition for improving or preventing ptosis, each of which contains this extract, were safe and had an excellent effect of improving and preventing ptosis.

The myofibroblasts in the present invention are also called activated fibroblasts, and cause the accumulation of extracellular matrix such as fibers, i.e., fibrosis. In the body, fibroblasts are acted upon by cytokines such as transforming growth factor-β1 (TGF-β1), growth factors such as platelet-derived growth factor (PDGF), mechanical stress, etc., and are transdifferentiated into myofibroblasts that express α-smooth muscle actin.

In the present invention, fascia refers to superficial fascia, deep fascia (aponeurotic fascia), epimysium, perimysium, endomysium, etc., and contains elastin fibers that contribute to flexibility, extensibility, and shape memory, and collagen fibers that contribute to strength, in the matrix, and functions as a cushioning material for the sliding of muscle fibers. In addition, fascial tissue in the broad sense also includes high-density flat tissue sheets (septum, joint capsule, aponeurosis, organ capsule, retinaculum), ligaments, and tendons.

In the present invention, the term "tendon" refers to a strong connective tissue fiber bundle that is located at both ends of skeletal muscles and acts as an intermediary for attaching the muscles to bones, and specifically refers to a membranous tendon.

The elastin in the present invention is a fibrous extracellular matrix protein and is the main component of elastic fibers. Elastin is distributed in almost all organs and tissues throughout the body, such as blood vessels, ligaments, lungs, and skin, and is involved in maintaining elasticity. Elastin is synthesized as tropoelastin in myofibroblasts, fibroblasts, or smooth muscle cells, and is produced by self-assembly of tropoelastin and intermolecular interaction with fibrillin.

In this invention, ptosis refers to aponeurotic ptosis, a condition in which the aponeurosis of the levator palpebrae superioris, which should originally extend to the front of the tarsal plate, becomes detached from the tarsal plate with age, and its tip recedes from the upper edge of the tarsal plate, making it difficult to lift the eyelids. This causes proprioception via the mechanoreceptors of the Müller muscle and sympathetic hypertonia, which results in many autonomic symptoms such as eye fatigue, stiff shoulders, headaches, and photophobia.

The butterfly pea used in the present invention is a plant of the genus Clitoria, family Fabaceae, order Fabaceae, and has the scientific name Clitoria ternatea. It is native to Southeast Asia and is distributed in tropical and subtropical regions. The butterfly pea is a creeping annual plant that can grow up to 5 meters long when fully grown. It has elliptical leaflets and 5 to 9 odd-pinnate compound leaves. It is also known as Clitora, butterfly flower, blue butterfly, butterfly pea, and anchan.

In the present invention, the Clitoria ternatea plant is used as the extract of Clitoria ternatea, and the part of the plant that can be used for the extract is a part of the plant such as the flower, legume, leaf, or root, or the whole plant. Callus obtained by tissue culture of the plant may also be used. Preferably, "Clitoria ternatea Flower Extract BG-50" (Koei Kogyo) is commercially available, and this may also be used.

The alpine rose used in the present invention is a plant of the genus Rhododendron, family Ericaceae, order Ericales, and has the scientific name Rhododendron ferrugineum. Alpine rose is one of the Swiss Alpine plants, and is distributed in the European Alps, the Pyrenees, and the Jura Mountains, growing in subalpine zones at altitudes of 1,600-2,200 meters. Alpine rose is a perennial evergreen shrub with characteristic dark pink to purple hermaphroditic flowers and dark green oval leaves.

In the present invention, the alpine rose extract is made from the alpine rose plant. Callus obtained by tissue culture of the plant may also be used. Preferably, "Alpine Rose Active" (H. Holstein), which is commercially available, can be used.

The method of extracting butterfly pea and alpine rose used in the present invention is not particularly limited, and may be, for example, heated extraction or room temperature extraction. In addition, the plant body may be used as it is for extraction, or may be processed by drying, crushing, shredding, etc. Examples of extraction solvents 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 (ethyl ether, tetrahydrofuran, propyl ether, etc.). Polar solvents such as water, lower alcohols, and liquid polyhydric alcohols are preferable, and water, ethanol, 1,3-butylene glycol, and propylene glycol are particularly preferable. These solvents may be used alone or in combination of two or more. Particularly preferred extraction solvents include water or a water-ethanol mixed polar solvent. There are no particular limitations on the amount of solvent used, and it is sufficient to use, for example, 10 times or more, preferably 20 times or more, the plant body (dry weight) of butterfly pea and alpine rose, but it is preferable to use 100 times or less 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 extract of butterfly pea and alpine rose may be used as the extracted solution, but if necessary, it may be used after processing such as concentration (vacuum concentration, membrane concentration, etc.), dilution, filtration, decolorization with activated carbon, deodorization, ethanol precipitation, etc., within the range that achieves 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, etc., which are used in pharmaceuticals, quasi-drugs, cosmetics, foods, etc., within the scope of the invention that does not impair the effects of the extract.

The present invention can be used in any of medicines, quasi-drugs, cosmetics, and foods, and examples of dosage forms include ointments, poultices, tablets, capsules, eye drops, lotions, creams, milky lotions, gels, aerosols, essences, packs, cleaning agents, bath agents, foundations, dusting powders, lipstick, chocolate, gum, candy, beverages, powders, granules, tablets, sugar-coated tablets, 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, manufacturing examples, formulation examples and experimental examples of the extracts used in the present invention are given as examples, but the present invention is not limited to these. The percentages shown in the manufacturing examples are % by weight, and the parts of the content shown in the formulation examples are parts by weight.

The butterfly pea extract of the present invention can be produced by the following extraction method as an example.

(Production Example 1) Preparation of 50% 1,3-butylene glycol extract of butterfly pea 5 g of dried butterfly pea flowers were immersed in 50 mL of 50% 1,3-butylene glycol aqueous solution at room temperature for 7 days to perform extraction. The obtained extract was filtered to obtain 49.8 g of 1,3-butylene glycol extract of butterfly pea.

(Production Example 2) Preparation of a hot water extract of Clitoria ternatea 50 mL of water was added to 5 g of dried Clitoria ternatea flowers, 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 1.3 g of a hot water extract of Clitoria ternatea.

(Production Example 3) Preparation of 50% ethanol extract of Clitoria ternatea 5 g of dried aerial parts of Clitoria ternatea was immersed in 50 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 1.18 g of 50% ethanol extract of Clitoria ternatea.

(Production Example 4) Preparation of Clitoria ternatea ethanol extract 5 g of dried Clitoria ternatea aerial part was immersed in 50 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 1.15 g of Clitoria ternatea ethanol extract.

(Formulation Example 1) Ointment Formulation Content (parts)
1. 50% 1,3-butylene glycol extract of butterfly pea (Production Example 1) 0.5
2. Polyoxyethylene cetyl ether (30 E.O.) 2.0
3. Glyceryl monostearate 10.0
4. Liquid paraffin 5.0
5. Cetanol 6.0
6. Methyl parahydroxybenzoate 0.1
7. Propylene glycol 10.0
8. Make the total volume 100 with purified water. [Manufacturing method] Heat and dissolve ingredients 2-5, mix, and keep at 70°C to make the oil phase. Heat and dissolve ingredients 1 and 6-8, 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.

Comparative Formulation Example 1: Conventional Ointment A conventional ointment was prepared by replacing the 50% 1,3-butylene glycol extract of Clitoria ternatea in Formulation Example 1 with purified water.

(Formulation Example 2) Ointment Formulation Content (parts)
1. Alpine rose extract (H. Holstein) 0.5
2. Polyoxyethylene cetyl ether (30 E.O.) 2.0
3. Glyceryl monostearate 10.0
4. Liquid paraffin 5.0
5. Cetanol 6.0
6. Methyl parahydroxybenzoate 0.1
7. Propylene glycol 10.0
8. Make the total volume 100 with purified water. [Manufacturing method] Heat and dissolve ingredients 2-5, mix, and keep at 70°C to make the oil phase. Heat and dissolve ingredients 1 and 6-8, 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 3) Cream Formulation Content (parts)
1. 50% ethanol extract of Butterfly Pea (Production Example 3) 0.1
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.

(Formulation Example 4) Cream Formulation Content (parts)
1. Alpine Rose Extract (H. Holstein) 0.1
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.

(Formulation Example 5) Lotion Formulation Content (parts)
1. Hot water extract of butterfly pea (Production Example 2) 0.1
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.

(Formulation Example 6) Emulsion Formulation Content (parts)
1. Ethanol extract of Clitoria ternatea (Production Example 4) 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 the total volume 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 7) Gel Formulation Content (parts)
1. 50% 1,3-butylene glycol extract of butterfly pea (Production Example 1) 0.05
2. Alpine Rose Extract (H. Holstein) 0.05
3. Ethanol 5.0
4. Methyl parahydroxybenzoate 0.1
5. Polyoxyethylene hydrogenated castor oil (60 E.O.) 0.1
6. Fragrance (appropriate amount) 7. 1,3-butylene glycol 5.0
8. Glycerin 5.0
9. Xanthan gum 0.1
10. Carboxyvinyl polymer 0.2
11. Potassium hydroxide 0.2
12. Add purified water to make the total volume 100. [Manufacturing method] Dissolve ingredients 2 to 6, 1, and 7 to 12 uniformly, and mix the two to make the product.

(Formulation Example 8) Pack formulation Content (parts)
1. Hot water extract of butterfly pea (Production Example 2) 0.05
2. Alpine Rose Extract (H. Holstein) 0.05
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 9) Foundation Formulation Content (parts)
1. 50% ethanol extract of Butterfly Pea (Production Example 3) 0.1
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 agent (as needed) 19. Add purified water to make the total 100 [Manufacturing method] Heat and dissolve ingredients 2-8, and keep at 80°C to make the oil phase. Allow ingredient 9 to swell well in ingredient 19, then add ingredients 1 and 10-13 and mix uniformly. Add ingredients 14-17, 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 this 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 10) Bath additive Formulation Content (parts)
1. Ethanol extract of Clitoria ternatea (Production Example 4) 0.1
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 11) Powder Formulation Content (parts)
1. Hot water extract of butterfly pea (Production Example 2) 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 12) Tablets Formulation Content (parts)
1. Ethanol extract of Clitoria ternatea (Production Example 4) 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 13) Tablets Formulation Content (parts)
1. Ethanol extract of Clitoria ternatea (Production Example 4) 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. Purified water 0.1
[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 14) Beverage Formulation Content (parts)
1. Hot water extract of butterfly pea (Production Example 2) 0.05
2. Stevia 0.05
3. Malic acid 5.0
4. Fragrance 0.1
5. Purified water 94.8
[Production method] Dissolve ingredients 2 and 3 in a small amount of water. Then add ingredients 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: Effect of butterfly pea and alpine rose extract on elastin expression in myofibroblasts FBN2 (fibrillin 2) and EMILIN2 (elastin microfibril interface 2) are glycoproteins involved in the formation of elastic fibers, and their increased expression promotes elastin production. Therefore, the expression levels of FBN2 and EMILIN2 mRNA were measured. Human dermal fibroblasts (NB1RGB) were seeded in a 6-well plate and cultured in DMEM medium containing 10% FBS under conditions of 37°C and 5% _CO2 . When the cells reached a semi-confluent state, the medium was replaced with DMEM medium containing 10% FBS adjusted to a final concentration of 2 ng/mL TGF-β1, and differentiated into myofibroblasts. Three days later, a butterfly pea extract (Production Example 1) or an alpine rose extract (H. Holstein) dissolved in FBS-free DMEM medium was added to the cells so that the final concentration was 200 μg/mL as a solid concentration. Total RNA was extracted 24 hours after the addition. Total RNA was extracted from the cells using RNAiso Plus (Takara Bio), and the amount of total RNA was determined by absorbance at 260 nm using a spectrophotometer (NanoDrop). The amount of mRNA expression 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 (Applied Biosystems) 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 levels of FBN2 and EMILIN2 mRNA were calculated as a ratio to the expression level of β-actin mRNA, which is an internal standard. The FBN2 and EMILIN2 expression promotion rate was calculated as the ratio of the expression levels of FBN2 and EMILIN2 mRNA in the sample-added group to the expression levels of FBN2 and EMILIN2 mRNA in the control (sample-free) group of human fibroblasts and myofibroblasts. The primers used to measure the expression level of each gene are as follows.

Primer set for FBN2 CCCAGTACAAAAAAGATATGTCCTC (SEQ ID NO: 1)
AGGGTCTATACAAGAGGTTCCACTG (SEQ ID NO: 2)
Primer set for EMILIN2: AAAGCCACAGATAATGAACCC (SEQ ID NO: 3)
GAACCTTCTCCTCTAGCACC (SEQ ID NO: 4)
Primer set for β-actin CACTCTTCCAGCCTTCCTTCC (SEQ ID NO:5)
GTGTTGGCGTACAGGTCTTTG (SEQ ID NO: 6)

The experimental results on FBN2 mRNA expression are shown in Table 1. The amount of FBN2 mRNA expression in myofibroblasts was increased by the Clitoria ternatea and alpine rose extracts. Furthermore, the amount of expression was significantly increased in myofibroblasts compared to human fibroblasts. Furthermore, the Clitoria ternatea extracts (Production Examples 2, 3 and 4) had a similar effect.

The experimental results on EMILIN2 mRNA expression are shown in Table 2. The expression level of EMILIN2 mRNA in myofibroblasts was increased by the Clitoria ternatea and alpine rose extracts. Furthermore, the expression level was significantly increased in myofibroblasts compared to human fibroblasts. Furthermore, the Clitoria ternatea extracts (Production Examples 2, 3 and 4) had a similar effect.

Thus, butterfly pea and alpine rose extracts promoted elastin expression in myofibroblasts.

Experimental Example 2 Usage test (Improvement of ptosis)
A use test was carried out using each of the ointments obtained in Formulation Examples 1 and 2 and Comparative Formulation Example 1. The samples were applied to the eyelids of 20 female subjects twice a day, morning and evening, for three consecutive months, and then the degree of eye opening was measured. The degree of eye opening was calculated from the ratio of the size of the pupil when the eye was normally opened to that when the eye was wide open.

As a result, when the ointments of Formulation Examples 1 and 2 were used, the degree of eye opening increased significantly before and after continuous use compared to when the ointment of Comparative Formulation Example 1 was used. Therefore, the extracts of Butterfly Pea and Alpine Rose related to the present invention showed excellent effects in improving ptosis. Furthermore, during the test period, not a single subject experienced skin trouble, and there were no safety issues.

The elastin production promoter, the elasticity enhancer for fascia or aponeurosis, and the composition for improving or preventing ptosis according to the present invention, each of which contains an extract of butterfly pea and/or alpine rose, exert excellent improving effects for the purpose of each agent. Therefore, it is possible to provide medicines, quasi-drugs, cosmetics, and foods for the purpose of improving or preventing ptosis.

Claims (3)

An agent for promoting elastin production in myofibroblasts, characterized by containing butterfly pea and/or alpine rose. An elasticity enhancer for fascia or aponeurosis, characterized by containing butterfly pea and/or alpine rose. A composition for improving or preventing ptosis, characterized by containing butterfly pea and/or alpine rose.