WO2008059906A1

WO2008059906A1 - External preparation for skin and external preparation for mucosa

Info

Publication number: WO2008059906A1
Application number: PCT/JP2007/072150
Authority: WO
Inventors: Yoshiharu Kawagoe; Masafumi Akatsuka; Yumi Ohara
Original assignee: Nozaki, Katsunori; Kamata, Yasushi
Priority date: 2006-11-14
Filing date: 2007-11-08
Publication date: 2008-05-22

Abstract

It is intended to provide an external preparation for skin capable of obtaining an effect of making the skin more beautiful by exhibiting an effect of removal of active oxygen, moisturization, prevention of inflammation, antiallergy or skin aging or the like as well as having a whitening effect; and an external preparation for mucosa exhibiting an excellent antiallergic action and antihistaminic action by acting on the mucosa. The external preparation for skin contains an extract of leaves and/or stems of a plant belonging to the genus Fragaria in the Rosaceae family as an active ingredient. The external preparation for skin is capable of obtaining an effect of making the skin more beautiful by exhibiting an effect of removal of active oxygen, moisturization, prevention of inflammation, antiallergy or skin aging or the like as well as has a whitening effect which enables the restoration of the original color and gloss of the skin by reducing, fading or eliminating pigmentation or freckles accompanying aging. Further, the external preparation for mucosa exhibits an excellent antiallergic action and antihistaminic action by acting on the mucosa, and is particularly capable of alleviating the symptoms of so-called hayfever such as a runny nose or sneezing by acting on the nasal mucosa.

Description

Skin external preparation and mucosal external preparation

The present invention allows the skin to penetrate the inside of the skin percutaneously by applying, immersing, rubbing or contacting the skin, thereby reducing or fading the stains and soaks that have already been formed. With regard to external preparations for skin used to restore the original color of the skin, external preparations for external use for skin can be used mainly as basic cosmetics such as lotions, cosmetic creams, bath preparations, soaps for medicinal use, etc. The present invention relates to an external preparation for mucous membrane which exerts an excellent antiallergic action and an antihistamine action by acting on mucous membranes. Background art

It aims to protect the skin from ultraviolet rays that increase based on the stratospheric ozone layer damage in recent years, prevent skin aging and blackening, eliminate stains, buckwheat, and dullness, and restore the skin's original beauty. Demand for basic cosmetics for whitening is increasing. In particular, women who were exposed to ultraviolet rays and tanned because of the face black that was popular in the 1990s began to shift to the trend of whitening, and stains that were left after the tanning was sunk, buckwheat There is a strong demand for removing

As cosmetics for whitening, products that suppress pigment production due to active oxygen generated by ultraviolet irradiation and try to improve the dullness associated with aging of the skin, or catalyze some of the melanogenesis reaction that causes the skin. Some attempts to reduce stains by inhibiting the tyrosinase activity. As the former cosmetics, vitamin C, tocopherol (vitamin E), superoxy Cosmetics containing antioxidants such as dodismutase (SOD) are on the market. However, vitamins such as vitamins C and E are generally unstable, and have an adverse effect on oxidized vitamin A and oxidized vitamin E that are produced after oxidation with active oxygen. It is not satisfactory in terms of points. Synthetic antioxidants with excellent stability tend to be reluctant from recent consumer safety, natural and health concerns.

Under these circumstances, natural cosmetics for whitening using herbal extract or herbal extract as a component useful for skin care and whitening should be proposed to meet the needs of consumers who prefer natural cosmetics. ing.

For example, Patent Document 1 discloses, as a melanin production inhibitor based on the inhibition of tyrosinase activity, a skin cosmetic containing a herbal extract such as curcuma versifolium and cucumber. The cucon extract used herein is an extract obtained by extracting the rhizome of cucon with a hydrophilic organic solvent such as water, methanol, or ethanol.

In Patent Document 2, crude drug extracts such as banshu, cowpea, and persimmon are excellent in the antioxidant action against unsaturated fatty acid and active oxygen scavenging action, and the external preparation for skin containing these is superior to vitamin E for the improvement of blur. Is disclosed. The extract of Bankon used in this case is a hot water extract of the rhizome, an ethanol extract of whole grass, an extract of 1, 3-butylene glycol of the rhizome, and the excellent antioxidant of the extract By action, it is intended to improve skin, skin, skin and dullness.

Patent Document 3 discloses water-soluble thickeners such as glycerol and hyaluronic acid Na, antibacterial alcohols such as ethanol, antioxidant vitamins such as tocopherols, and anti-inflammatory agents such as mefenamic acid and tranexamic acid. There is also disclosed a cosmetic composition to which a botan extract, a button, an aloe, a carrot, a plant extract such as curcuma has been added as a whitening component. Patent Document 1 Japanese Patent Application Laid-Open No. 6-2 2 7 9 6 0

Patent Document 2 Japanese Patent Application Laid-Open No. 200002 1 2 1 1 4 4

Patent Document 3 Japanese Patent Application Laid-Open Publication No. 200002-2 0 1 2 0 5 2

Problems that Invention is to Solve

The scavenging of reactive oxygen species by the antioxidant component is intended to suppress the formation of stains and sorrows by ultraviolet irradiation, and against the whitening effects such as reduction, disappearance, and fading of stains and sorrows that have already been formed, Further improvement is desired. In addition to skin lightening effects, it also has a moisturizing effect to keep the skin moist, inflammation, anti-allergy, and skin lightening effects as long as it is effective in preventing and suppressing skin aging. There is a strong demand for development of such external skin preparations.

On the other hand, in recent years, patients with various allergic diseases such as atopic dermatitis and hay fever have been increasing, and their treatment has become a social problem. However, at present, much reliance on symptomatic treatment is required, and it is necessary to say that its development is still in the midst of groping, while excellent therapeutic agents leading to the fundamental treatment of arelarrhei are in great demand. Recently, various anti-histamine drugs and drugs that reduce or antagonize the production of SRS_A have been developed, but are not sufficient in terms of side effects, and treatment with natural products with fewer side effects Development of substances is strongly desired.

The present invention has been made in view of these circumstances, and it has a skin-whitening effect to rejuvenate the skin's original color and wrinkles by reducing sunburn due to ultraviolet irradiation, reducing stains due to aging and fading off. At the same time, it is effective in removing active oxygen, moisturizing, inflammation, anti-allergy, prevention of skin aging, etc., and it is possible to obtain a more beautiful skin effect. An external preparation for skin which can be expected to treat or alleviate eryrgic dermatitis and the like, and an external preparation for mucous membrane which exerts an excellent antiallergic action and an antihistaminic action by acting on mucous membranes To aim. Means to solve the problem

The external preparation for skin of the present invention is characterized in that it contains an extract of leaves and Z or a stem of a plant belonging to the genus Rosaceae of the family Rosaceae as an active ingredient.

The external preparation for mucous membrane of the present invention is characterized by containing extracts of leaves and stems of a plant belonging to the genus Rosaceae of the family Rosaceae as active ingredients. Effect of the invention

The external preparation for skin of the present invention is percutaneously incorporated into the skin to inhibit the activity of tyrosinase, thereby suppressing the formation of melanin which is a cause of the stain, and further along with the turnover of the skin. Since it can be expected to reduce stains and stains that have already been made, it is effective as a whitening essence to be added to cosmetics and bath agents. In addition to the tyrosinase inhibitory activity effect, the external preparation for skin of the present invention also exhibits an active oxygen scavenging effect because it exhibits an excellent SOD-like activity value. Furthermore, the external preparation for skin of the present invention exhibits excellent hyaluronidase inhibition value together with tyrosinase inhibitory activity effect and active oxygen scavenging effect, and thus a moisturizing effect for keeping the skin moist, inflammation, anti-allergy One symptom · A more beautiful skin effect can be obtained if it is effective in preventing and suppressing skin aging. In addition, due to its excellent anti-allergic action, it can also be expected to have a therapeutic or alleviating effect on allergies such as dermatitis.

The external preparation for mucous membrane of the present invention acts on the mucous membrane to exert an excellent anti-allergic action and anti-histamine action, and in particular, acts on the nasal mucous membrane to alleviate the symptoms of so-called pollinosis such as runny nose and sores. Can. Brief description of the drawings

Fig. 1 Fraction of strawberry leaf extract and measurement results of tyrosinase inhibitory activity.

Fig. 2 ¹ H NMR spectrum of C ompound 3.

Figure 3 shows the structural formula of q u e r c t i n — 3 — o — D — g l. U c u r o n i. D e.

Fig. 4 ¹ H NMR spectrum of C ompound 1.

Figure 5 shows the estimated structure of C o m p o u n d 1

Figure 6 ¹ H NMR spectrum of C ompound 2

A ^{1 3} C-NMR spectrum Honoré in FIG 7 C ompound 2.

Fig. 8 Spectrum of ¹³ C- NMR of Casuarictin. Figure 9 shows the structural formula of C asuarictin. BEST MODE FOR CARRYING OUT THE INVENTION

The external preparation for skin of the present invention contains the extract of leaves and Z or stems of a Rosaceae L. plant as a active ingredient.

As the Rosaceae family Dutch strawberry plant used in the present invention, if it is a so-called edible Dutch strawberry, it is possible to use a variety of mulberry species. For example, Toyo Noka, Nyoho, Tochiotome, Chapter princess (Akihime), Iberly, Tochihime, Red pearl, Sachi's power Ama, Treasure early birth, Benihopope, etc. Plants can be used. The plant to be used needs to be a plant belonging to the genus Rosaceae, but not intended to be limited to the above-mentioned varieties.

As an extraction solvent used to extract an extract of the genus Rosaceae L. planta, the purpose of use of the product to be provided, type or processing to be performed later Although it may be selected in consideration of the etc., it is preferable to use the extract of leaves and Z or stem of the above-mentioned Rosaceae Lubifer plant as extract extracted with water and / or alcohol. By doing this, it is considered that it is possible to extract effectively without destroying the effective ingredients for the beautifying skin.

As the above-mentioned alcohol, lower alcohols such as methanol, ethanol, propyl alcohol, isopro pinole alcohol, butanol, etc .; polyhydric alcohols such as propylidene alcohol, 1, 3-butyleneglycolone, glycerin etc. are used. be able to. These can be used alone or in combination.

The extraction may be performed in multiple steps, for example, extraction with leaves of leaves and stems of the genus Rosaceae and the stem with water and then extraction with medanol, but in order to increase the content of the whitening component It is preferable to include an extraction step with a mixture of water and ethanol.

The extract-containing solution extracted with these solvents may be used as it is as a skin external preparation, or may be used after being subjected to processing such as concentration, dilution, filtration, drying and the like as necessary. In addition, after extraction, the alcohol may be volatilized by drying or the like to obtain a powder having a high content of the whitening component.

The leaves and stems of the above-mentioned Rosaceae Lubiferus plants can be used as long as the plants are grown as edible fruits. It is preferred to use. This is because leaves and / or stems after fruit harvest which have been conventionally discarded are effectively used from the viewpoint of recycling.

The leaves and Z or stalks of the above-mentioned Rosaceae L. plant are collected and can be extracted with water and Z or alcohol as they are. Forced after extraction, natural drying or artificial After being dried Extraction with water and / or alcohol is preferred. By doing this, it is considered that the extract concentrated by drying can be extracted. In addition, drying stabilizes the condition of leaves and green leaves or stems, reduces variations in conditions, stabilizes the quality of the extract to be extracted, and improves the shelf life of leaves and Z or stems. This is because the handling is significantly improved.

Also, the extraction method may be extraction with water only or alcohol only, or extraction with a mixture of water and alcohol. It can also be extracted by heating, but extraction at normal temperature is preferred. It is because it is thought that the breakage of the extract due to heating is reduced and the effective ingredient for the beautifying skin is effectively extracted.

The external preparation for skin of the present invention may be used by adding the extract of leaves and Z of stem or sock of the Rosaceae family to an appropriate solvent and base, or the extract as an essence, and cosmetics. Or add it to quasi-drugs. More specifically, skin cleansing agents such as cleansing creams, cleansing lotions, facial cleansing creams, soaps, etc. as well as whitening agents; moisturizing lotions, soft lotions, skin conditioning agents such as astringent lotions; Milky lotions Protection agents such as emollient milk and vanishing cream; activators such as massage cream, massage system, foam pack, peel off pack etc .; bath additives; and whitening agents may be added as whitening ingredients. In any case, the active ingredient is incorporated into the skin and can be used to cause stains and buckwheat discoloration, reduction and disappearance.

The content ratio of leaf and Z or stem extract of Rosaceae L. plant in the final product is appropriately selected according to the type of final product, but in the case of cosmetics, about 1 to 80 mass% is preferable .

The external preparation for skin of the present invention does not impair the effect of the extract depending on the final product. In the range, base, moisturizer, thickener, surfactant, pH adjustment agent, preservative, fragrance, moisturizer, UV absorber, thickener, pigment, antioxidant, chelate component can do.

As the oil base, solid oily raw materials such as beeswax, cacao oil; carnaval roux, higher fatty acids, solid paraffin, cetyl alcohol, stearyl alcohol etc .; semi-solid oily raw materials such as wa- seline, lanolin etc; Leave oil, castor oil, palm oil, peanut oil, liquid paraffin, oleic acid, linoleic acid, octoyldodecyl dodecyl myristate, silicone oil, etc. can be used.

As the aqueous base, moisturizing agents such as glycerin, sorbitol, polyethylene glycol, pyrrolidone carboxylic acid and salts thereof, collagen, hyaluronic acid and salts thereof, chondroitin sulfate and salts thereof, and the like; And polyvinyl cinolea / leconole, xanthan gum, ruboxy vinyl polymer, hydroxy propyl cellulose, thickeners such as arabic gum, and the like are used.

As surfactants, non-ionic surfactants such as polyethylene glycol isostearylate, sorbitan stearate, glyceryl fatty acid ester, polyoxyethylene hexyl decyl ether, etc .; sodium lauryl sulfate, lauryl phosphate Anionic surfactants such as sodium phosphate; cationic surfactants such as stearyldimethylbenzyl ammonium; natural surfactants such as sucrose fatty acid ester, sodium caseinate, lecithin, collagen etc. Be

Examples of pH adjusters include carbonated lithium, sodium bicarbonate, ammonium bicarbonate and the like.

Other than these, paradimethylamino benzoic acid, urocanic acid ethyl, diisopropyl cinnamic acid ethyl, and 2-hydric acid may be added as required. UV absorbers such as droxy 4-methoxybenzophenone and salicylate triethanolamine; preservatives such as cetylparaben and butylparaben; glycyrrhizinic acid derivatives, glycyrrhetinic acid derivatives, salicylic acid derivatives, Hinokitiol, zinc oxide, allan Anti-inflammatory agents such as toin etc. Vitamins A such as retinol palmitate, vitamins B such as riboflavin, nicotinic acid amide, vitamins D such as colecalciferol, d 1 − α-tocopherol etc. Antioxidants such as vitamins and cetaceans; antioxidants such as sodium sorbirate sodium, sage extract, parahydroxyl sole, etc .; active agents such as royal jelly, collesterol derivative various amino acids; γ — orizanol , Blood circulation enhancers such as sodium dextran sulfate; It includes other plant extracts; low force Sami down, colorant safflower like. Example

[Preparation of an extract sample of a rose plant of the genus Rosaceae]

After harvesting the leaves and stems of the harvested fruits of Dutch strawberry (Shohime and Tonooka) grown for food, they were naturally dried and crushed with a finger and crushed into several mm to several cm. Sample 1 (leaf) and sample 2

(Stem) was prepared. On the other hand, a 50% water + 50% ethanol solution was prepared as an extraction solvent.

1 kg of each of the above samples was immersed in 1 liter of the above extraction solvent, allowed to stand at room temperature for 1 day, and filtered to obtain a primary extract. 1 kg of the above sample is immersed in the solution obtained by adding 1 liter of extraction solvent to the primary extract thus obtained, and the mixture is allowed to stand at room temperature for 1 day and then filtered to obtain a secondary extract I got The sample solution extraction solvent followed by addition of further 1 liter in the secondary extract was lk _g immersed, similarly final extract 1 and 2 was filtered after standing for 1 day at room temperature (leaves and I got the stem). Each final extract obtained as described above is heated to about 80 ° C. and heated for about 2 hours to evaporate the alcohol and to extract the extract at about 100 ° C. The extract was concentrated to a half (about 300 cc) to obtain extract sample 1 (leaf) and extract sample 2 (stem).

[Tyrosinase activity inhibitory action]

Extract sample 1 (leaf) and extract sample 2 (stem) were examined for their inhibitory effect on tyroxinase activity, an enzyme involved in the formation of melanin. Tyrosinase (EC1.14.18.1) is a bioenzyme that oxidizes L-tyroosine to L-DOPA and oxidizes L-DOPA to DOPA-quinone. Since DOPA quinone is a causative agent of melanine which is a causative agent of stains and freckles, it is considered that inhibiting tyrosinase can prevent the formation of stains and freckles.

The sample (20 μL) dissolved in phosphate buffer (pH 6. 8) containing 20% DM SO was suspended on a substrate (100; z L) containing 0.1 ML-DO. The turbid enzyme solution (mushroom-derived tyrosinase 30 U, manufactured by SIGMA, 20 μL ·) was thoroughly mixed and subjected to enzyme reaction for 15 minutes in a thermostat bath at 37 ° C. After that, 0.1 N aqueous solution of HCl (20 μL) was added as a reaction stop solution (S). At the same time, one was prepared using a phosphate buffer instead of the substrate and the sample (S · B 1) and one using a phosphate buffer instead of the sample (B 1). After the enzyme reaction, the phosphate buffer (pH 6.8) was used as a measurement blank, and the absorbance at 45 nm was measured, and the tyrosinase inhibition rate (%) was calculated according to formula 1. The sample concentration was 5 mg zml (water dilution).

Inhibition rate (%) = B 1-(S-S · B 1) / B 1 X 1 0 0-. '(Equation 1) [S OD like activity; active oxygen scavenging effect]

The previously mentioned tyrosinase is triggered by active oxygen generated by ultraviolet light and is produced in cells called melanosite present in the basal layer of the skin. Be That is, in addition to the inhibition of tyrosinase, if it is possible to effectively suppress the active oxygen causing the activity of tyrosinase, it is considered that a whitening effect can be expected.

The reactive oxygen inhibition effect was measured according to a standard operation method using S O D A c t i t i d i d t e i t i t i o i K i t (manufactured by Wako Pure Chemical Industries, Ltd.). That is, a coloring reagent (containing 0.5 μm of xanthine and 0.42 μm of nitro bluet triazolium) in 10 μl of a sample solution dissolved in distilled water containing a 20% aqueous solution of DM SO. 1 phosphate buffer (ρ Η 8. 0)) 0. lm L was added and stirred for 1 minute. Subsequently, an enzyme suspension (a suspension of 0.10 4 9 U ml of Xanthine Oxidase (derived from Buttermilk) in 0.1 M phosphate buffer (pH 8. 0)) 0.1 m In addition to L, the mixture was allowed to react for 28 minutes in a 37 ° C constant temperature bath, and then the reaction stopping solution (69 O mM sodium dodecyl sulfate, 20 L) was added rapidly and stirred for 5 minutes (S ). The concentration of the sample was 100 μg / mL (water dilution).

At the same time, use the same volume of distilled water instead of the sample (B 1), use the blank solution instead of the enzyme solution (S · B 1), and use the same volume of distilled water instead of the sample solution. A solution (B1.B1) was prepared using a blank solution instead of the enzyme solution. The test solution thus prepared used distilled water as a measurement blank, and the active oxygen inhibition rate (%) was calculated according to the equation 2 from the absorbance at 5600 nm.

Reactive oxygen inhibition rate (%) = (B 1-B 1 · B 1)-(S-S · B 1) / (B 1-B 1 · B 1) X 1 0 0 · · · · (Equation 2)

[Heal mouth nidase inhibition rate]

Using the hyaluronic acid-derived calf testis, the inhibitory effect on the activation step of the inactive enzyme by compound 48/80 was mainly measured. The enzyme activity is N-acetyl hexoside produced by hydrolysis of hyaluronic acid. The increase in reducing power of tetrasaccharides having a reducing end with samin was measured by colorimetric determination with absorbance at 585 nm (Goery Journal, 31: 23-3237, 19) 9 0).

First, the following were prepared as reagents.

· 0.1 M acetate buffer (pH 4.0)

■ · Hyanoleronidase

• compound 4 8 / ^/ 8 0

'Sodium hyaluronate solution

• 0.4 N sodium hydroxide solution

· P-DA reagent: 10 g of p-DAB, 2.5 m 1 of 10 N hydrochloric acid solution, and 8 7.5 m 1 of acetic acid were mixed and diluted 10-fold with acetic acid immediately before use.

• Boric acid solution: 50m1 of water was added to 4.95g of boric acid, adjusted to pH 9.1 with a 1 N sodium hydroxide solution, and then water was added to make it 100m1. And, the hyaluronidase inhibition rate was measured by the following method.

1) The sample solution 200 μl diluted with water was mixed with 5 mg 1 and hyaluronidase 100 / il (40 0 n i t s) mixed and cultured at 37 ° C. for 20 minutes.

2) 200 μl (final concentration 0.1 mg / ml) of comonomer was added and the mixture was incubated at 37 ° C for 20 minutes.

3) An aqueous solution of sodium hyaluronate 500.mu. 1 (final concentration: 0.4 mg Zm.sup.1) was added and the mixture was incubated at 37.degree. C. for 40 minutes.

4) 0.20 μl of 0.4 N sodium hydroxide solution was added and placed on ice to stop the reaction.

5) 200 μl of boric acid solution was added and treated for 3 minutes at 100 ° C.

6) Reaction solution 1401 and p-DAB solution 600 AZ 1 were mixed and incubated at 37 ° C. for 20 minutes. 7) Absorbance at 585 nm was measured.

The measurement samples of S amp 1 e, C ontro 1, S-B lank and C-B lank prepared according to Table 1 below are prepared and their respective absorbances are measured, and the hyaluronidase inhibition rate (inhibition from Activity) was calculated. The X mark indicates that the above-mentioned acetate buffer was used instead of the solution in the left column.

Hyalid nidase inhibition rate (%) = (C-C-B L)-(S-S-B L) / (S-S-B L) X I O O-· '(Equation 3)

S: Absorbance of S a m p 1 e

C: Absorbance of C o n t r o 1

S * B L: Absorbance of S _ B l a n k

C-B L: Absorbance of C-B l a n k Table 1

The measurement results of the tyrosinase inhibition rate, the S OD-like activity, and the hyaluronidase inhibition rate measured as described above are shown in Table 2 below. As a comparative example, measurement results of extracts obtained by similarly extracting the leaves of Niugori are also shown. Table 2 Sample Tyrosinase inhibition rate (%) SOD-like activity (3⁄4) Hyaluronidase inhibition rate (%) Strawberry leaf 81.1 99.5 97.0

Strawberry stem 75.3 100.0 94.6

As shown in Table 2 above, Examples (Extract samples 1 and 2) have extremely high values of tyrosinase inhibition rate, SOD-like activity, and hyaluronidase inhibition rate. On the other hand, the comparative example hardly inhibits the hyaluronidase inhibition rate, and has a significantly low value for the tyrosinase inhibition rate and the SOD-like activity.

In addition, since it exhibits high hyaluronidase inhibitory activity and has excellent antiallergic activity, it can also be expected to have a therapeutic or alleviating effect on dermatitis etc. of allergies.

[Effect as cosmetics]

Extract sample 1 (leaf) was prepared at 200 c c and mixed and diluted in 400 c c glycerol to prepare a cosmetic.

The following results were obtained when this cosmetic was used for eight months as a substitute for a cosmetic applied after face-washing at night for one month. Paneler 1 (5 1 year old woman): I feel firmness when I wake up and wash my face in the morning, I feel very smooth. As a result, the effort to put on makeup has become easier, and it has become difficult to lose makeup. You can stay there all day without taking care of your vacation.

Paneler 2 (woman of 65 years old): The stain of about 10 yen hardening has broken. In addition, it has very high moisturizing properties and fine-grained skin, and there is more than double the extension of the foundation.

Paneler 3 (a woman of 64 years old): The overall age spots became pale, and it was said that the skin became white. I have no experience so far It is moisturizing.

Paneler 4 (40-year-old woman): Although it is dry skin, after using the sample products, it became possible to feel a sense of security without taking any care. In addition, the number of blowouts due to the quality of allergies has decreased.

Paneler 5 (36-year-old woman): It was felt that the pores became large and cosmetic makeup was bad after one use. Moreover, I was getting darker around my eyes.

Paneler 6 (34-year-old woman): I found that the difference in cosmetic makeup was clear when used only once. Things like Kusumi in the whole face have disappeared. In addition, after being well moisturized, I have not been doing anything until now.

Paneler 7 (Male, 51 years old): Some buckwheat have become thinner and smaller. I noticed in about 10 days.

Paneler 8 (65-year-old man): The skin was oily and bothersome, but the sample became used and the oiliness was improved and nothing bothered. In addition, when the remaining sample is used only in the left hand, it becomes white, and I am surprised that the difference between the right hand and the color is clearly seen.

As can be seen from the results of panelists 1 to 8, the cosmetic composition of the present invention is very moisturizing, and is effective in decolorization, reduction, and elimination of Shiva, etc. Also, many of the participants commented that most of the participants said, "The skin seems to have revived." Also, there was no such thing as side effects such as itching or irritation.

[Evaluation of tyrosinase inhibitory activity of fractionated sample]

Sample preparation>

After drying the leaves of the Dutch strawberry naturally, the sample (87. 6 g) crushed by hand with a finger and crushed to several mm to several cm is mixed with 50% aqueous ethanol solution (1.8 1) Immersed in 1) and extracted at room temperature for 3 days. After the obtained extract was filtered, the above sample was added to a solution to which the same amount of 50% aqueous ethanol solution was added again, and extraction was performed for 3 days at normal temperature. The extracts after filtration were combined and concentrated under reduced pressure until the volume became 50 cc to obtain a solution solution of 50% ethanol extract of Dutch strawberry leaves.

The sample (20 μM) dissolved in phosphate buffer (pH 6. 8) containing 20% DM 2 SO was suspended in a substrate containing 0.1 μM-DOPA (μM buffer) The enzyme solution (mushroom-derived tyrosinase 30 U, manufactured by SIGMA, 2 μM) was mixed well and subjected to enzyme reaction in a thermostat at 37 ° C. for 15 minutes. After that, 0.1 NHCL aqueous solution (20 μl) was added as a reaction stop solution (S). At the same time, those using phosphate buffer instead of substrate and sample (S · B 1) and those using phosphate buffer instead of sample (B 1) were prepared. After the enzyme reaction, absorbance was measured at 45 nm using phosphate buffer (pH 6. 8) as a measurement blank, and tyrosinase inhibition rate (%) was calculated according to the following equation 4. . The sample concentration was 5 mg / mL (water dilution). Tyrosinase inhibition rate (%) = B 1-(S-S · Β 1) / Β 1 X I 0 0 · · · · (Equation 4)

The above-described extract sample solution showed 81. 1% tyrosinase inhibitory activity. Also, the above extract (about 20. 4 g) is liquid-liquid partitioned between water and ethyl acetate to obtain a water fraction (about 11. 4 g) and an ethyl acetate fraction (about 2.6 g). The The water fraction showed 79.5% tyrosinase inhibitory activity, which was higher than 47.8% of the ethyl acetate fraction.

Therefore, the above water fraction was fractionated with DI ANON HP-20 column chromatography, and the water fraction (about 3.9 g), the fraction eluted with 25% methanol aqueous solution (about 1. 9 g), 50% methanol aqueous solution elution fraction (about 1.3 g), The fraction eluted with 75% aqueous methanol solution (about 134.0 mg) and the fraction eluted with methanol (about 33.9 mg) were obtained. The respective tyrosinase inhibitory activity was 20. 1% in water fraction, 87.8% in 25% methanol aqueous solution eluted fraction,

The fraction eluted with 50% methanol aqueous solution is 75.5%, the fraction eluted with 75% methanol aqueous solution is 55.1%, and the fraction eluted with methanol is 18.2%,

A high tyrosinase inhibitory activity was observed in the 5% methanol aqueous solution eluted fraction.

Next, with respect to the fraction eluted with 25% methanol aqueous solution (about 869. 9 mg), the components were fractionated and purified with HP LC (liquid chromatography). That is, according to the HPLC chromatography pattern previously performed for the same sample, F r. 1 (about 52 5.8 mg), F r. 2 (about 4 4.8 mg), based on the elution time of the main component. Fractions of F r. 3 (about 1 1.9 mg), F r. 4 (about 3 4.8 mg) and F r. 5 (about 4 8.3 mg) were made. The analysis conditions of H P L C are as follows.

Model: S I MA Z U S C L-1 0 AV P

Column: U n i so n UK — C 18 (I m t a k t)

Dissolution solvent: 18% acetonitrile

Flow rate: l ml ^ / min

Detector: UV detector 2 5 4 n m, 3 3 0 n m

Each tyrosinase inhibitory activity was 27.0% for F r. 1 and inactive for F r. 2, F r, 3 force S 9 6.4%, F r. 4 for inactive, F r. 5 The strength was S 2 7. 9%, and F r. 3 showed high tyrosinase inhibitory activity. These results are shown in FIG.

[SOD-like activity evaluation of fractionated sample]

S S OD like activity test>

Reactive oxygen suppression effect is S OD A ctivity D etection It measured according to the standard procedure using Kit (made by Wako Pure Chemical Industries, Ltd.). That is, the color reagent (Xanthine 0.4 M and NitroBluete trazolium 0.24 μm contained) in 10 μL of the sample solution dissolved in distilled water containing 20% aqueous DM 2 SO 4. 1 M phosphate buffer (ρ Η 8. 0)) 0. lm L was added and stirred for 1 minute. Enzyme suspension (Xanthine oxidase (derived from Buttermilk) 0. OS UZmL suspended in 0. IM acid buffer (pH 800) 0. 1 ml added to 0. 7 ml 37 ° The reaction was allowed to proceed for 28 minutes in a thermostatic bath C. Then, a reaction termination solution (690 mM sodium dodecyl sulfate) was rapidly added, and the mixture was stirred for 5 minutes (S). The concentration was μg / mL (water dilution).

At the same time, use the same volume of distilled water instead of the sample (B 1), use the blank solution instead of the enzyme solution (S · B 1), and use the same volume of distilled water instead of the sample solution. In addition, using the blank solution instead of the enzyme solution (B 1 · B 1) was prepared. The test solution thus prepared used distilled water as a measurement blank, and the active oxygen inhibition rate (%) was calculated according to the following equation 5 from the absorbance at 5600 nm.

Reactive oxygen inhibition rate (%) = (B 1-B 1 · B 1)-(S-S · B 1) / (B 1-B 1 · B 1) X 1 0 0 · · · · (Equation 5)

Measurement of the SOD-like activity for the above F r. 2 and F r. 4 showed high values of F r. 2 force S 9. 0 8%, F r. 4 force; 9 1. 4%.

[Structural analysis]

Next, the above-mentioned F r. 2, F r. 3 and F r. 4 are respectively C ompound 1, C ompound 2 and C ompound 3, mass spectrometry (F AB-MS), nuclear magnetic resonance spectroscopy ( Structural analysis was performed by NMR) and the like. C ompound 3>

First, the measurement results of mass spectrometry (FAB-MS) and nuclear magnetic resonance spectroscopy (NMR) for C ompound 3 are shown below. Also, ¹ H-NMR spectrum of C ompound 3 is shown in FIG.

(1) F AB-MS: m / z = 4 7 9 [M + H] ⁺

(2) ¹ H-NMR (CD a OD) δ (ppm)

3. 4 3-3. 7 7 (4 H, m, C 2 ", C 3", C 4 ", C 5,,), 5. 3

3 (1H, d, J = 7.4 Hz, CI, 6. 1 9 (1 H, d, J = 1. 8 H z, C 6), 6. 38 (1 H, d, J = 1. 8 H z, C 8), 6. 8

4 (1 H, d, J = 9.0 H z, C 5 '), 7. 6 2 (2 H, C 2 C 6, 6) (3) ^{1 3} C NMR (CD 3 OD) δ (ppm) )

7 3.6 (C 4 "), 7 6.2 (C 2"), 7 7. 8 (C 5 "), 7 8.4 (C 3,,), 9 5.5 (C 8), 1 0 0. 7 (C 6), 1 0 5. 0 (C 1 ,,), 1 0 6. 4 (C 1 0), 1 1 6. 8 (C 5,), 1 1 8. 0 C 2 '), 1 2 3. 6 (C 1,), 1 2 4.3 (C 6'), 1 3 6. 2 (C 3), 1 4 6. 7 (C 3 '), 1 5 0.7 (C 4,), 1 59.2 (C 9), 1 59.8 (C 2), 1 6 3.8 (C 5), 1 6 6. 8 (C 7), 1 7 3.1 (C 6 "), 1 8 0. 0 (C 4)

From the results of the above analysis and the literature values, C o m p o u n d 3 was identified as q u e r c e t i n-3-o-D-g 1 u c u r o n i d e. The structure is shown in Fig.3.

References: KR Price, I. J. Co. 1 quhoun, K. A. Barnes, M. J. C. Rhodes, J. A. gric .. Food C. Hem. 4 6. 4 8 9 8 — 4 9 0 3 (1 9 9 8) <C ompound 1>

Next, the measurement results of nuclear magnetic resonance spectroscopy (NMR) for C ompound 1 are shown below. Also, ¹ H—NMR spectrum of C ompound 1 is shown in FIG.

(1) ¹ H-NMR (CD ₃ OD) δ (ppm)

3. 1 7-3. 9 4 (m, sugar), 4. 7 5, 5. 5 9 (2 H, an omericproton), 6. 1 7 (1 H, brs), 6. 3 6 (1 H) , brs), 6. 8 6 (1 H, d, J = 8.0 H z), 7. 6 2 (2 H)

(2) ¹³ C-NMR (CD ₃ OD) δ (ppm)

6 7. 3, 7 1. 7, 7 3. 4, 5 5. 5, 7 7. 7, 7 8.4, 8 2.4, 9 5. 4, 1006, 10 1. 7, 1 0 6. 0, 1 0 6. 5, 1 1 6. 8, 1 1 8. 0, 1 2 3. 8, 1 2 4. 3, 1 3 5. 6, 1 4 6. 8, 1 5 0. 5, 1 5 9. 1, 1 5 9. 2, 1 6 3. 8, 1 6 6. 5, 1 7 2. 9, 1 8 0. 0

¹ H-NMR of C ompound ¹ is very similar to C ompound 3, and characteristic flavonoid peaks were detected at 6.2 to 7. 6 ppm of NMR. Of these, the meta position coupling of the aromatic ring can be inferred from the tri-substituted benzene and the doublets of 6.2 ppm and 6.4 ppm from the peaks at 7.6 ppm and 6.8 ppm. In addition, since there is a sugar-derived peak in the vicinity of 3 to 6 ppm, of which a peak thought to be hydrogen derived from a sugar anomer position is recognized as a double peak at 4.7 ppm and 5. 6 ppm. Is considered to exist.

Next, when the ^{1 3} C-NMR of C ompound 1 compared with ^{1 3} C-NMR of C ompound 3, 6 7. 3 ppm , 7 1. 7 ppm, 8 2. 4 ppm, 1 0 1. 7 ppm Other than peak power S almost C ompound 3 Matched with.

From the above results, it can be inferred that C o m p o u n d 1 is a compound obtained by adding a sugar having 4 ash to C o m o p o u n d 3 (q u e r c e t i n-3-o _ D-g 1 u c u r o n i d e). The estimated structure of Com po u n d 1 is shown in Fig.5. C o m p o u n d 2

Next, the measurement results of nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (FAB-MS) for C ompound 2 are shown below. Also, ¹ H NMR spectrum of C ompound 2 is shown in FIG. 6, and ¹³ C NMR spectrum / re is shown in FIG.

(1) ^X H-NMR (CD 3 OD) δ (ppm)

3 3 9 (d d, J = 4.2, 9.1 H z), 3. 8 1 (d, J = = 1 2.

9), 4.59 (dd, J = 5.5, 10.2 Hz), 5.04. 5 (m), 6.33 4 (d, J = 15.0 Hz) , 6. 3 5 (d, J = 0.8)

Hz), 6 • 4 2 (s), 6.48 (d, J = 3.8 Hz), 6. 52 (d,

J = 15.0 Hz), 6. 5 9 (d, J = 3.8 Hz), 6. 6 9 (s),

6 8 1 (d, J = 2.0 Hz), 7. 2 4 (s), 7. 38 (d, J =

2.0 H z)

(2) ¹³ C-NMR (CD a OD) δ (ppm)

6 4 5 6 9 8. 7 0 0.4 7 2 2. 7 2 4 7 5 8. 7 7

2, 7 7 4, 9 2 2, 9 2 7, 1 0 8. 3, 1 0 8 6, 1 0 9.

0, 1 0 9 1, 1 0 9. 3, 1 0 9. 6, 1 0 9 • 7, 1 1 1 1,

1 1 2 5 1 1 3 5 3 1 1 5. 7 1 1 5. 9 1 1 5. 9 1

6-2, 1 1 6. 3, 1 1 6. 7, 1 1 7 1, 1 1 7 3, 1 1 7.

7, 1 2 0 9, 1 2 6. 3, 1 2 6.6, 1 2 6. 8, 1 2 7. 0, 2 7.2, 1 2 7.5, 1 3 8.0, 1 3 8.0, 1 3 8.2, 1 3 8 • 3, 1

3 8.4, 1 3 8.5, 1 4 1.2, 1 4 2.0, 1 4 3.1, 1 4 5 1. 1

4 5.5, 1 4 5. 5, 1 4 5. 6, 1 4 5. 8, 1 4 6. 5, 1 4 6.

5, 1 4 6. 6, 1 4 6.6, 1 4 6. 7, 1 4 8. 1, 1 4 9. 1, 1

6 6. 2, 1 6 6. 4, 1 6 9 .8, 1 6 9 9. 9, 1 7 0. 3, 1 7 0.

5, 1 7 0. 7, 1 7 1. 2, 1 7 1. 4

(3) F A B-M S: m / z = 9 3 5 [M-H]

(N eg g at i v e)

It was subjected to F e C 1 ₃ color by TLC, and blue coloration. Therefore, it is suggested that C ompound 2 is a compound having many hydroxyl groups. Further, the ¹³ C-NMR spectrum of C ompound 2 shown in FIG. 7 is very similar to the ¹³ C- NMR spectrum of C asuarictin shown in FIG. 8. Figure 9 shows the structural formula of Casuarictin.

From the above results, C ompound 3 is very likely to be a hydrolyzable derivative of tannic acid, and 4 to 5 gallic acid esters are present around a sugar having 6 carbons such as C asuarictin. It can be inferred that the structure has something like a bond. However, because the carbon number of C o m p o u n d 2 is as large as 66, it is suggested that the two types of compounds may be mixed as an isomer.

The results of measuring tyrosinase inhibition rate and SOD-like activity for C ompound 1., C ompound 2 and C ompound 3 in the same manner as described above are shown in Table 3 below. The results obtained by measuring the tyrosinase inhibition rate of arbutin and the S OD-like activity of (+) — tekin are shown in Table 4 below. Table 3

Treatment concentration Tyrosinase inhibitory activity test 1 mg / ml

SOD like activity test 50 g / ml Table 4

Activity indicator substance>

Treatment concentration Tyrosinase inhibitory activity test 5 mg / ml

SOD like activity test 50 i / ml

― ■ ■ ■ Not Test

Thus, Compo n d 2 exhibited high tyrosinase inhibitory activity as compared to the activity indicator substance, albumin. C o m p o u n d 1 and 3 showed an S OD-like activity almost similar to that of the activity indicator (+)-force tekin. From the above results, it can be said that C o m p o u n d 1 to 3 are physiologically active substances contained in strawberry leaves.

[The symptom alleviation effect of hay fever]

The above-mentioned Dutch strawberry leaf extract was used by eight panelists suffering from hay fever when symptoms appeared, and the following results were obtained. Paneler 1 (doctor, woman): I was having trouble with daily life with severe runny nose and sneezing due to hay fever. Of course, we have used a lot of remedies, but none of them have the expected effect. However, when I received the "strawberry leaf extract" and applied it to the nasal cavity at the time of severe symptoms, in 4 to 5 minutes, the runny nose and Kushami will stop wonderfully and the effect also It lasted for a long time. Then, I use about twice a day, but I can feel comfortable everyday life without feeling any side effect.

Paneler 2 (Housewife 4 and 7 years old): My hobby is gardening, but when pollen was about to fly, I was plagued by hay fever. "If I apply it to the nasal cavity," I have a strawberry leaf extract, and if I apply it with a cotton swab, I get a squeeze once and only blow my nose. I could do gardening comfortably until the evening.

Paneler 3 (Housewife 3 to 8 years old): Every time I washed the laundry out because of hay fever, I had a hard time thinking. I applied the strawberry leaf extract and was convinced that if it was a strawberry, I applied it to the nasal cavity with a cotton swab, and I was happy because the bad runny nose stopped and it worked for a long time. I can not let go.

Paneler 4 (Instructor 2 6 years old): I went to work by bicycle, but my nose was blocked, sore, and my runny nose stopped working. It was in the state where I could not do makeup. One day, I applied it to my nasal cavity and got a sample of fig leaf extract, and I used it immediately, and I had a runny nose, sneezing, and a bitter stuffy nose. Please sell products that are effective against rhinitis so quickly.

Paneler 5 (A full-time housewife 5 4 years old): I really appreciate the strawberry leaf extract. I went to the doctor and gave me various medicines, but I also had problems with my stomach and getting worried. The strawberry leaf extract smells better, and I prefer house dust than hay fever, but the mucous membranes become loose and exfoliate, nosebleeds, stuffy nose, and runny nose does not stop. Was in the middle of the year. Because of that, thanks to the strawberry leaf extract, the mucous membranes are normal, the nose is not clogged, and the nasal discharge is not much. A quick product for many suffering people.

Paneler 6 (unemployed old man 7 2 years old): I go to the pool every day, Even if you get older, you suddenly have hay fever. At first I went to the doctor's office thinking that I had a cold, and I was diagnosed with hay fever. I thought this age, but I got a medicine from a doctor, but it didn't work very well. A clerk from the pool told me, “I put a strawberry leaf extract on a cotton swab and applied it to the nose hole”. After about 15 minutes of painting, the runny nose and sneezing stopped. If it is good, I will have you kindly when I raise a sample. I am now happy to be in the pool. It has immediate effect, and it smells good even if it's painted in the nose, so I spend my time in good mood. Please make it a product soon.

Paneler 7 (38-year-old housewife): Even though I took measures against pollinosis such as injection for a month or more and burning the mucous membranes of the nose in anticipation of flying pollen, the painful symptoms did not change from the previous year. The When I applied the strawberry leaf extract to the back of my nose with a cotton swab, it became very easy. As conventional medicines cause stomach upset and side effects such as having to take stomach medicines appear, I would like you to use strawberry leaf extract as a product.

Paneler 8 (64-year-old housewife): Pollinosis history 20 years. If this time was this, I gave up, but when I applied the strawberry leaf extract to the back of the nasal cavity, in the moment Kushami went out five times in a row, and it became very easy from now on, when I had a runny nose. I can breathe suussu with my nose. Also, it was easier for my eyes to be crushing. In the past, it was difficult to make the nasal mucous membrane a power subta, and when I took it I got nosebleed, and then I made a physical subta and repeated it, but the strawberry leaf extract was repeated 3 times a day. I have been able to spend an easy day by painting four times. Hay fever is the most important nose to breathe, so I thought that I would be glad if I could apply for this medicine, because I had been suffering for many years. Anyway, it was very easy with strawberry leaves. Thus, it acts on the mucous membrane to exert excellent anti-allergic and antihistamine actions, and in particular it acts on the nasal mucous membrane to alleviate the symptoms of so-called pollinosis such as runny nose and mucilage, etc. It can be used as an external preparation for mucous membranes such as mucous membranes. In addition, due to the excellent antiallergic action, therapeutic and alleviating effects such as allergic dermatitis can be expected. Industrial applicability

Since the external preparation for skin of the present invention has a high tyrosinase activity inhibitory action and also an antibacterial activity, it is an external preparation for skin care intended for the prevention of acne and stains and treatment including skin lightening cosmetics. Can be used. It can also be used as an external preparation for mucous membranes such as nasal mucous membranes.

Claims

Claims 1. An external preparation for skin comprising an extract of leaves and Z or stems of a plant belonging to the genus Rosaceae of the genus Rosaceae as an active ingredient.

2. The external preparation for skin according to claim 1, wherein the extract of leaves and or stems of the plant of the genus Rosaceae of the family Rosaceae is an extract extracted with water and / or alcohol.

3. An external preparation for mucous membranes comprising an extract of leaves and Z or stems of a plant belonging to the genus Rosaceae of the genus Rosaceae as an active ingredient.

4. The external preparation for external layer according to claim 3, wherein the extract of leaves and or stems of the plant of the genus Rosaceae of the family Rosaceae is an extract extracted with water and / or alcohol.