JP2008273922A - External preparation for skin and external preparation for mucosa - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an external preparation for skin exhibiting an effect of lightening the skin 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 skin-lightening effect and an external preparation for mucosa exhibiting an excellent antiallergic action and antihistaminic action by acting on the mucosa. <P>SOLUTION: 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 achieves the effect of more lightening the skin by the 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. The external preparation for mucosa exhibits an excellent antiallergic action and antihistaminic action by acting on the mucosa and is particularly alleviating the symptoms of so-called hayfever such as a runny nose or sneezing by acting on the nasal mucosa. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention allows the skin to permeate into the skin by applying, soaking, rubbing, or touching the skin, reducing the stains, freckles, etc. that have already been formed, and fading the skin. For skin external preparations used to regain the color of skin, mainly used as basic cosmetics such as skin lotions and cosmetic creams, bath preparations, medicinal soaps, etc., and excellent antiallergic action on mucous membranes The present invention relates to a mucosal external preparation that exhibits an antihistamine action.

  Protects the skin from ultraviolet rays that increase due to the recent destruction of the stratospheric ozone layer, prevents aging and blackening of the skin, and eliminates spots, freckles, and dullness to restore the original beauty of the skin Demand for basic cosmetics for use is increasing. In particular, because of the black skin that was popular in the 1990s, women who had been sunburned by irradiating ultraviolet rays began to turn into a whitening fashion, and the removal of stains and freckles that remained after sunburn was subsided. There is a strong demand.

  For whitening cosmetics, it suppresses pigment production due to active oxygen generated by UV irradiation to improve dullness due to skin aging, and tyrosinase activity catalyzes part of the melanogenesis reaction that causes spots Some attempt to reduce spots by inhibiting As the former cosmetics, cosmetics containing an antioxidant such as vitamin C, tocopherol (vitamin E) and superoxide dismutase (SOD) are on the market. However, vitamins such as vitamin C and E are generally poor in stability, and oxidized vitamin A and oxidized vitamin E produced after oxidation with active oxygen have an adverse effect. Absent. Synthetic antioxidants with excellent stability tend to be avoided from recent consumer safety, nature and health consciousness.

  Under such circumstances, in order to meet the demands of consumers who prefer natural cosmetics, natural cosmetics for whitening using herbal extracts or herbal extracts as ingredients useful for skin beautification and whitening have been proposed.

  For example, Patent Document 1 discloses a skin cosmetic containing a herbal extract such as turmeric or clove as a melanin production inhibitor based on tyrosinase activity inhibition. The turmeric extract used here is an extract obtained by extracting the rhizome part of turmeric with a hydrophilic organic solvent such as water, methanol, ethanol or the like.

  In Patent Document 2, herbal extracts such as turmeric, cowpea, horseradish and the like are excellent in unsaturated fatty acid oxidation-preventing action and active oxygen scavenging action, and skin external preparations containing these are superior to vitamin E in improving dullness. Is disclosed. The extract of Banturcum used here is a hot water extract of rhizome, an ethanol extract of whole grass, and an extract of 1,3-butylene glycol of rhizome, and due to the excellent antioxidant action of the extract It is intended to improve wrinkles, firmness and dullness of the skin.

Patent Document 3 includes water-soluble thickeners such as glycerin and sodium hyaluronate, antibacterial alcohols such as ethanol, antioxidant vitamins such as tocopherols, anti-inflammatory agents such as mefenamic acid and tranexamic acid, and whitening ingredients. Cosmetic compositions to which plant extracts such as horn, button, aloe, carrot, turmeric are added are disclosed.
JP-A-6-227960 JP 2002-121144 A JP2002-212052

  The capture of active oxygen by the antioxidant component is intended to suppress the formation of stains and freckles due to ultraviolet irradiation. For the whitening effects such as the reduction, disappearance, and fading of the existing stains and freckles Improvement is desired. In addition to the whitening effect, a skin moisturizing effect to keep the skin moisturized, as well as an effect to prevent and control inflammation, antiallergic diseases, and skin aging can provide a further skin beautifying effect. Development of such an external preparation for skin is strongly desired.

  On the other hand, in recent years, patients with various allergic diseases such as atopic dermatitis and hay fever are increasing, and their treatment has become a social problem. However, at present, there is a great deal of reliance on symptomatic treatment, and while there is an eager desire for an excellent therapeutic agent that can lead to the fundamental treatment of allergic diseases, it must be said that its development is still in the dark. Recently, various antihistamines and drugs that suppress or antagonize the production of SRS-A have been developed. However, it is not sufficient in terms of side effects, and development of therapeutic substances derived from natural products with few side effects. Is strongly desired.

  The present invention has been made in view of such circumstances, and has a whitening effect such as sunburn caused by ultraviolet irradiation, reduction of spots and freckles associated with aging, and rejuvenation of the original color and gloss of the skin by fading and erasing. Along with the removal of active oxygen, moisturizing, prevention of inflammation, anti-allergic diseases, skin aging, etc., it is possible to obtain even more beautifying effects, and it has a high anti-allergic action to treat and relieve allergic dermatitis. It is an object of the present invention to provide a skin external preparation that can be expected, and a mucosal external preparation that exerts an excellent antiallergic action and antihistamine action by acting on the mucous membrane.

  The gist of the topical skin preparation of the present invention is that it contains an extract of leaves and / or stems of the Rosaceae family.

  The gist of the topical preparation for mucosa of the present invention is that it contains a leaf and / or stem extract of the Rosaceae genus Strawberry as an active ingredient.

  The external preparation for skin of the present invention is percutaneously incorporated into the skin and inhibits the activity of tyrosinase, thereby suppressing the production of melanin that causes stains. It is effective as a whitening essence blended in cosmetics and bathing agents because it can be expected to fade and reduce stains and buckwheat. Moreover, since the skin external preparation of this invention shows the outstanding SOD-like activity value with the tyrosinase inhibitory activity effect, it also exhibits the active oxygen scavenging effect. Furthermore, the external preparation for skin of the present invention exhibits an excellent hyaluronidase inhibition rate value as well as a tyrosinase inhibitory activity effect and an active oxygen scavenging effect, so that the moisturizing effect for keeping the skin moisturized, inflammation, antiallergic disease, skin If it is effective in preventing and suppressing aging, a further skin beautifying effect can be obtained. In addition, it can be expected to treat or alleviate allergic dermatitis due to its excellent antiallergic action.

  The external preparation for mucosa of the present invention acts on the mucous membrane to exhibit excellent antiallergic action and antihistamine action, and particularly acts on the nasal mucosa to relieve symptoms of so-called hay fever such as runny nose and crushing.

  The external preparation for skin of the present invention contains an extract of leaves and / or stems of the Rosaceae family.

  As the Rosaceae Dutch strawberry genus plant used in the present invention, various varieties can be used as long as they are Dutch strawberry cultivated for food. For example, plants of various varieties such as Toyonoka, Nyoho, Tochiotome, Akihime, Iberry, Tochihime, Red Pearl, Sachinoka, Amaou, Hosochou, and Red Hoppe are used. be able to. As a plant to be used, it is necessary to belong to the genus Rosaceae of the Rosaceae family, but it is not intended to be limited to the varieties described above.

  The extraction solvent used to extract the extract of plants belonging to the genus Rosaceae in the family Rosaceae may be selected in consideration of the intended purpose of the product to be provided, the type, or the processing performed later. The plant leaf and / or stem extract is preferably an extract extracted with water and / or alcohol. It is because it is thought that it can extract effectively without destroying the component effective for beautiful skin by doing in this way.

  Examples of the alcohol include lower alcohols such as methanol, ethanol, propyl alcohol, isopropyl alcohol, and butanol; polyhydric alcohols such as propylene glycol, 1,3-butylene glycol, and glycerin. These can be used alone or in combination.

  In the extraction, for example, the leaves and / or stems of the Rosaceae Dutch strawberry genus plant may be extracted with water and then extracted with methanol, for example, in order to increase the whitening component content, It is preferable to include an extraction step using a mixture of water and ethanol.

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

  The leaves and / or stems of the Rosaceae Dutch strawberry genus plant can be used as long as they are cultivated as edible fruits. However, the leaves and / or stems after harvesting the fruits are collected and used. preferable. This is because leaves and / or stems after harvesting fruits that have been conventionally discarded are preferably used from the viewpoint of recycling.

  The leaves and / or stems of the above-mentioned Rosaceae, which can be extracted from the cultivated state and extracted with water and / or alcohol in that state, are naturally dried or artificially dried after collection. Then, it is preferable to extract with water and / or alcohol. It is because it is thought that the extract concentrated by drying can be extracted by doing in this way. In addition, by drying, the state of the leaf and / or stem is stabilized, the variation in the state is reduced, the quality of the extracted extract is stabilized, and the storage stability of the leaf and / or stem is also improved. This is because sex is remarkably improved.

  Moreover, the extraction method may extract only with water or only alcohol, and may extract with the liquid mixture of water and alcohol. Moreover, although it can extract by heating, it is preferable to extract at normal temperature. This is because it is considered that an extract that is effective for beautifying skin is effectively extracted because destruction of the extract by heating is reduced.

  The skin external preparation of the present invention may be used by containing the above-mentioned leaf and / or stem extract of Rosaceae in a suitable solvent or base, and using the extract as an essence for cosmetics and pharmaceuticals. It may be added to quasi-drugs. Specifically, skin cleansing agents such as cleansing creams, cleansing lotions, facial cleansing creams, soaps as well as whitening agents; skin conditioners such as moisturizing lotions, soft lotions, astringent lotions; milky lotions, emollient milks, Protective agents such as vanishing cream; activators such as massage creams, massage lotions, foam packs and peel-off packs; bathing agents; In either case, the active ingredient can be percutaneously taken into the skin and can act to fade, reduce or eliminate stains and freckles.

  The content ratio of the leaf and / or stem extract of the Rosaceae in the final product is appropriately selected according to the type of the final product, but in the case of cosmetics, it is preferably about 1 to 80% by mass.

  The skin external preparation of the present invention is a base, a moisturizer, a thickener, a surfactant, a pH adjuster, an antiseptic, a fragrance, and a moisturizer as long as the effect of the extract is not impaired depending on the final product , UV absorbers, thickeners, dyes, antioxidants, chelating components can be blended.

  Oily bases include beeswax, cacao oil; solid oily raw materials such as carnauba wax, higher fatty acids, solid paraffin, cetyl alcohol, stearyl alcohol; semi-solid oily raw materials such as petrolatum, lanolin; squalane, olive oil, castor oil, palm Oil, peanut oil, liquid paraffin, oleic acid, linoleic acid, octyldodecyl myristate, silicone oil, and the like can be used.

  As an aqueous base, humectants such as glycerin, sorbitol, polyethylene glycol, pyrrolidone carboxylic acid and salts thereof, collagen, hyaluronic acid and salts thereof, chondroitin sulfate and salts thereof; sodium carboxymethylcellulose, polyvinyl alcohol, xanthan gum, carboxyvinyl polymer Thickeners such as hydroxypropyl cellulose and gum arabic are used.

  As the surfactant, nonionic surfactants such as polyethylene glycol isostearate, sorbitan stearate, glycerin fatty acid ester, polyoxyethylene hexyl decyl ether; anionic surfactants such as sodium lauryl sulfate and sodium lauryl phosphate; Cationic surfactants such as stearyldimethylbenzylammonium chloride; natural surfactants such as sucrose fatty acid ester, sodium caseinate, lecithin and collagen are used.

  Examples of the pH adjuster include potassium carbonate, sodium hydrogen carbonate, ammonium hydrogen carbonate and the like.

  In addition to these, UV absorbers such as paradimethylaminobenzoic acid, ethyl urocanate, ethyl diisopropylcinnamate, 2-hydroxy-4-methoxybenzophenone, triethanolamine salicylate; Antiseptics such as butyl paraben; anti-inflammatory agents such as glycyrrhizic acid derivatives, glycyrrhetinic acid derivatives, salicylic acid derivatives, hinokitiol, zinc oxide, allantoin; vitamins A such as retinol palmitate; vitamin Bs such as riboflavin and nicotinamide; Antioxidants such as vitamin D such as calciferol, vitamin E such as dl-α-tocopherol; antioxidants such as sodium erythorbate, sage extract, parahydroxyanisole; royal jelly, cholesterol induction Activators such as various amino acids; .gamma.-oryzanol, blood circulation promoters such as dextran sulfate sodium; and the other plant extracts; titanium yellow, Kasamin, colorant safflower like.

[Preparation of extract samples of Rosa genus Strawberry genus plants]
Sample 1 after harvesting leaves and stems of Dutch strawberry (Akihime and Toyonoka) cultivated for edible use, each dried naturally and then crushed with fingers and crushed to several mm to several cm (Leaf part) and Sample 2 (stem part) were 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 extraction solvent, allowed to stand at room temperature for 1 day, and then filtered to obtain a primary extract. 1 kg of the above sample was immersed in a solution obtained by adding 1 liter of extraction solvent to the primary extract thus obtained, and allowed to stand at room temperature for 1 day, followed by filtration to obtain a secondary extract. 1 kg of the above sample is immersed in a solution obtained by adding 1 liter of extraction solvent to this secondary extract, and left to stand at room temperature for 1 day, followed by filtration to obtain final extracts 1 and 2 (leaf and stem). Got.

  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 make the extract about one-tenth of the original extraction solvent (about Concentration was performed until it became 300 cc), and extract sample 1 (leaf part) and extract sample 2 (stem part) were obtained.

[Tyrosinase activity inhibitory action]
The extract sample 1 (leaf part) and the extract sample 2 (stem part) were examined for the inhibitory action on the thyroxinase activity, which is an enzyme involved in the production of melanin.

  Tyrosinase (EC 1.1. 14.1) is a biological enzyme that oxidizes L-tyrosine to L-DOPA and oxidizes L-DOPA to DOPA quinone. Since DOPAquinone is a causative substance of melanin, which is a causative substance of stains and freckles, it is considered possible to prevent the formation of spots and freckles by inhibiting tyrosinase.

Enzyme solution (mushroom-derived tyrosinase 30U, SIGMA, 20 μL) suspended in a substrate (100 μL) containing 0.1 ML-DOPA in a sample (20 μL) dissolved in a phosphate buffer solution (pH 6.8) containing 20% DMSO Were mixed well and allowed to undergo enzymatic reaction in a constant temperature bath at 37 ° C. for 15 minutes. Thereafter, a 0.1 NHCL aqueous solution (20 μL) was added as a reaction stop solution (S). At the same time, a sample using a phosphate buffer instead of a substrate and a sample (S · B1) and a sample using a phosphate buffer instead of a sample (B1) were prepared. After the enzyme reaction, the absorbance at 475 nm was measured using a phosphate buffer (pH 6.8) as a measurement blank, and the tyrosinase inhibition rate (%) was calculated according to Equation 1. In addition, the density | concentration of the sample was 5 mg / mL (water dilution).
Inhibition rate (%) = B1− (S−S · B1) / B1 × 100 (Equation 1)

[SOD-like activity; active oxygen removal effect]
The tyrosinase described above is produced in cells called melanocytes existing in the basal layer of the skin, triggered by active oxygen generated by ultraviolet rays. In other words, in addition to inhibition of tyrosinase, it is considered that a whitening effect can be expected if active oxygen that causes an increase in tyrosinase activity can be effectively suppressed.

  The active oxygen suppression effect was measured according to a standard operating method using SOD Activity Detection Kit (manufactured by Wako Pure Chemical Industries, Ltd.). That is, 0.1 mL of a coloring reagent (0.1 M phosphate buffer (pH 8.0) containing 0.4 μM xanthine and 0.24 μM nitroblue tetrazolium) was added to 10 μL of a sample solution dissolved in distilled water in a 20% DMSO aqueous solution. Added and stirred for 1 minute. Subsequently, the enzyme suspension (xanthine oxidase (derived from Butter milk) 0.049 U / mL suspended in 0.1 M phosphate buffer (pH 8.0)) was added to 0.1 mL and in a 37 ° C. constant temperature bath. After reacting for 28 minutes, a reaction stop solution (690 mM sodium dodecyl sulfate, 20 μL) was quickly added and stirred for 5 minutes (S). The sample concentration was 100 μg / mL (water dilution).

At the same time, using the same volume of distilled water instead of the sample (B1), using the blank solution instead of the enzyme solution (S · B1), and using the same volume of distilled water instead of the sample solution, Further, a blank solution (Bl / Bl) was used instead of the enzyme solution. The test solution prepared as described above calculated the active oxygen inhibition rate (%) according to the formula 2 from the absorbance at 560 nm using distilled water as a measurement blank.
Active oxygen inhibition rate (%) = (B1−B1 · B1) − (S · S · B1) / (B1−B1 · B1) × 100 (Equation 2)

[Hyaluronidase inhibition rate]
Using hyaluronidase derived from beef testis, the inhibition of the activation stage of inactive enzyme by compound 48/80 was mainly measured. Enzyme activity was measured by colorimetric determination of the increase in reducing power of tetrasaccharides having N-acetylhexosamine generated by hydrolysis of hyaluronic acid as a reducing end by absorbance at 585 nm (Eisho Journal, Vol. 31, Vol. 233). 237 pages, 1990).

First, the following were prepared as reagents.
・ 0.1M acetate buffer (pH 4.0)
Hyaluronidase compound 48/80
-Sodium hyaluronate solution-0.4N sodium hydroxide solution-p-DAB reagent: 10 g of p-DAB, 12.5 ml of 10N hydrochloric acid solution and 87.5 ml of acetic acid were mixed, and diluted 10 times with acetic acid immediately before use.
Boric acid solution: 50 ml of water was added to 4.95 g of boric acid, and the pH was adjusted to 9.1 with a 1N sodium hydroxide solution, and then water was added to make 100 ml.

And the hyaluronidase inhibition rate was measured by the following method.
1) 200 μl of a sample solution diluted to 5 mg / ml with water and 100 μl of hyaluronidase (400 units) were mixed and incubated at 37 ° C. for 20 minutes.
2) Compound 48/80 was added in an amount of 200 μl (final concentration 0.1 mg / ml) and incubated at 37 ° C. for 20 minutes.
3) 500 μl of sodium hyaluronate solution (final concentration 0.4 mg / ml) was added and incubated at 37 ° C. for 40 minutes.
4) 200 μl of 0.4N sodium hydroxide solution was added and placed on ice to stop the reaction.
5) 200 μl of boric acid solution was added and treated at 100 ° C. for 3 minutes.
6) 140 μl of the reaction solution and 600 μl of the p-DAB solution were mixed and incubated at 37 ° C. for 20 minutes.
7) The absorbance at 585 nm was measured.

Samples of Sample, Control, S-Blank, and C-Blank prepared according to Table 1 below were prepared, and their respective absorbances were measured. The hyaluronidase inhibition rate (inhibitory activity) was calculated from Equation 3 below. In addition, x mark shows having prepared using the said acetate buffer instead of the solution of the left column.
Hyaluronidase inhibition rate (%) = (C · C · BL) − (S · S · BL) / (S · S · BL) × 100 (Equation 3)
S: Absorbance of Sample C: Absorbance of Control S · BL: Absorbance of S-Blank C · BL: Absorbance of C-Blank

The measurement results of the tyrosinase inhibition rate, SOD-like activity, and hyaluronidase inhibition rate measured as described above are shown in Table 2 below. As a comparative example, the measurement result about the extract which extracted the leaf part of bitter gourd similarly is shown collectively.

  As can be seen from Table 2 above, the examples (extract samples 1 and 2) have extremely high and favorable values for tyrosinase inhibition rate, SOD-like activity, and hyaluronidase inhibition rate. On the other hand, the comparative example hardly inhibits the hyaluronidase inhibition rate, and takes much lower values for the tyrosinase inhibition rate and the SOD-like activity.

  In addition, since it exhibits high hyaluronidase inhibitory activity and excellent antiallergic activity, it can be expected to treat or alleviate allergic dermatitis.

[Effects as cosmetics]
200 cc of extract sample 1 (leaf part) was prepared and mixed with 400 cc of glycerin to prepare a cosmetic.

  When this cosmetic was used for 8 months as a substitute for cosmetics to be applied after face washing at night, the following results were obtained.

Paneler 1 (a 51-year-old woman): I feel a firmness when I get up in the morning and wash my face. This made it easier to make up, and made it easier for the makeup to break. You can stay up all day without taking any care during the holidays.
Paneler 2 (65-year-old woman): A stain of about 10 yen has been broken. Moreover, it has very high moisturizing properties and the skin becomes finer, and the elongation of the foundation is more than doubled.
Paneler 3 (64-year-old woman): It is said that the overall stain due to age has faded and the skin has become whiter. Moisturizing that you have never experienced before.
Paneler 4 (40-year-old woman): Dried skin, but after using the sample, she can feel moist without care. In addition, breakout due to allergic constitution has decreased.
Paneler 5 (a 36-year-old woman): The improvement in the single use was felt because the pores were large and the makeup was bad. Moreover, the darkening around the eyes has started to come out.
Paneler 6 (34-year-old woman): Just using it once, the difference in makeup was clear. The whole face like Kusumi has disappeared. In addition, it is moisturizing and has been moisturized without any action after cleansing.
Paneler 7 (51-year-old man): Some buckwheat have become thinner and smaller. I noticed it in about 10 days.
Paneler 8 (a 65-year-old man): The skin was greasy and I was worried, but now I started to use the sample, the greasy was improved and I was not worried about anything. Moreover, when the remaining sample is used only for the left hand, it turns white, and the difference in color from the right hand is clearly seen and surprised.

  As can be seen from the results of panelists 1 to 8, the cosmetic of the present invention has a very high moisturizing property and is effective in decoloring, reducing, and erasing stains and freckles. In addition, most of the participants commented that their skins seemed to be soft and rejuvenated. Also, there were no side effects such as itchiness and irritation.

[Evaluation of tyrosinase inhibitory activity of fraction samples]
<Sample preparation>
A sample of the Dutch strawberry leaves that had been air-dried and then crushed with a finger and crushed to a few millimeters to a few centimeters (87.6 g) was immersed in a 50% aqueous ethanol solution (1.8 l) and extracted at room temperature for 3 days. . The obtained extract was filtered, and the sample was added to a solution obtained by adding the same amount of 50% aqueous ethanol solution again, followed by extraction at room temperature for 3 days. The combined extracts after filtration were concentrated under reduced pressure until the volume reached 50 cc, thereby obtaining a Dutch strawberry leaf 50% ethanol extract sample solution.

<Tyrosinase inhibitory activity test>
Enzyme solution (mushroom-derived tyrosinase 30U, SIGMA, 20 μl) suspended in a substrate (100 μl) containing 0.1 ML-DOPA in a sample (20 μl) dissolved in a phosphate buffer solution (pH 6.8) containing 20% DMSO Were mixed well and allowed to undergo enzymatic reaction in a constant temperature bath at 37 ° C. for 15 minutes. Thereafter, a 0.1 NHCL aqueous solution (20 μl) was added as a reaction stop solution (S). At the same time, a sample using a phosphate buffer instead of the substrate and the sample (S · B1) and a sample using a phosphate buffer instead of the sample (B1) were prepared. After the enzyme reaction, the absorbance at 475 nm was measured using phosphate buffer (pH 6.8) as a measurement blank, and the tyrosinase inhibition rate (%) was calculated according to the following formula 4. In addition, the density | concentration of the sample was 5 mg / mL (water dilution).
Tyrosinase inhibition rate (%) = B1− (SSB1) / B1 × 100 (Equation 4)

  In the extract sample solution described above, 81.1% tyrosinase inhibitory activity was observed. The extract (about 20.4 g) was 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 water fraction showed 79.5% tyrosinase inhibitory activity, which was higher than the 47.8% ethyl acetate fraction.

  Therefore, the above water fraction was fractionated by DIANON HP-20 column chromatography, and the water fraction (about 3.9 g), 25% methanol aqueous solution elution fraction (about 1.9 g), 50% methanol aqueous solution elution fraction. Minute (about 1.3 g), 75% methanol aqueous solution elution fraction (about 134.0 mg), and methanol elution fraction (about 33.9 mg). Each tyrosinase inhibitory activity is as follows: water fraction 20.1%, 25% methanol aqueous solution elution fraction 87.8%, 50% methanol aqueous solution elution fraction 75.5%, 75% methanol aqueous solution elution fraction. The fraction eluted with 55.1% and methanol was 18.2%, and a high tyrosinase inhibitory activity was observed in the fraction eluted with 25% aqueous methanol solution.

Next, fraction purification of the components was carried out by HPLC (liquid chromatography) for the fraction eluted with 25% aqueous methanol solution (about 866.9 mg). That is, according to the HPLC chromatogram previously performed on the same sample, the Fr. 1 (about 525.8 mg), Fr. 2 (about 44.8 mg), Fr. 3 (about 11.9 mg), Fr. 4 (about 34.8 mg), Fr. 5 (about 48.3 mg) was fractionated into 5 fractions. The HPLC analysis conditions are as follows.
Model: SIMAZU SCL-10AVP
Column: Unison UK-C18 (Imtakt)
Elution solvent: 18% acetonitrile Flow rate: 1 mL / min
Detector: UV detector 254 nm, 330 nm

  Each tyrosinase inhibitory activity is Fr. 1 is 27.0%, Fr. 2 is inactive, Fr. 3 is 96.4%, Fr. 4 is inactive, Fr. 5 is 27.9%, and Fr. 3 showed a high tyrosinase inhibitory activity. These results are shown in FIG.

[Evaluation of SOD-like activity of fraction samples]
<SOD-like activity test>
The active oxygen suppression effect was measured according to a standard operating method using SOD Activity Detection Kit (manufactured by Wako Pure Chemical Industries, Ltd.). That is, 0.1 mL of a coloring reagent (0.1 M phosphate buffer (pH 8.0) containing 0.4 μM xanthine and 0.24 μM nitroblue tetrazolium) was added to 10 μL of a sample solution dissolved in distilled water in a 20% DMSO aqueous solution. Added and stirred for 1 minute. Subsequently, the enzyme suspension (xanthine oxidase (derived from Butter milk) 0.049 U / mL suspended in 0.1 M phosphate buffer (pH 8.0)) was added to 0.1 mL and in a 37 ° C. constant temperature bath. After reacting for 28 minutes, a reaction stop solution (690 mM sodium dodecyl sulfate, 20 μL) was quickly added and stirred for 5 minutes (S). The sample concentration was 100 μg / mL (water dilution).

At the same time, using the same volume of distilled water instead of the sample (B1), using the blank solution instead of the enzyme solution (S · B1), and using the same volume of distilled water instead of the sample solution, Further, a blank solution (Bl / Bl) was used instead of the enzyme solution. The test solution prepared as described above calculated the active oxygen inhibition rate (%) according to the following formula 5 from the absorbance at 560 nm using distilled water as a measurement blank.
Active oxygen inhibition rate (%) = (B1−B1 · B1) − (S · S · B1) / (B1−B1 · B1) × 100 (Equation 5)

  Fr. 2 and Fr. When SOD-like activity was measured for No. 4, Fr. 2 is 90.8%, Fr. 4 showed a high value of 91.4%.

[Structural analysis]
Next, the Fr. 2, Fr. 3 and Fr. 4 was Compound 1, Compound 2 and Compound 3, respectively, and structural analysis was performed by mass spectrometry (FAB-MS), nuclear magnetic resonance spectroscopy (NMR), and the like.

<Compound 3>
First, the measurement result of Mass Spectrometry (FAB-MS) and Nuclear Magnetic Resonance Spectroscopy (NMR) for Compound 3 is shown below. A ¹ H-NMR spectrum of Compound 3 is shown in FIG.

(1) FAB-MS: m / z = 479 [M + H] ⁺
(2) ¹ H-NMR (CD ₃ OD) δ (ppm)
3.43-3.77 (4H, m, C2 ″, C3 ″, C4 ″, C5 ″), 5.33 (1H, d, J = 7.4 Hz, C1 ″), 6.19 (1H, d , J = 1.8 Hz, C6), 6.38 (1H, d, J = 1.8 Hz, C8), 6.84 (1H, d, J = 9.0 Hz, C5 ′), 7.62 (2H) , C2 ′, C6 ′)
(3) ¹³ C-NMR (CD ₃ OD) δ (ppm)
73.6 (C4 ″), 76.2 (C2 ″), 77.8 (C5 ″), 78.4 (C3 ″), 95.5 (C8), 100.7 (C6), 105.0 ( C1 ″), 106.4 (C10), 116.8 (C5 ′), 118.0 (C2 ′), 123.6 (C1 ′), 124.3 (C6 ′), 136.2 (C3), 146.7 (C3 ′), 150.7 (C4 ′), 159.2 (C9), 159.8 (C2), 163.8 (C5), 166.8 (C7), 173.1 (C6 ″) ), 180.0 (C4)

From the results of the above analysis and literature values, Compound 3 was identified as quercetin-3-o-D-glucuronide. The structure is shown in FIG.
Reference: K.K. R. Price, I.D. J. et al. Colquhoun, K.M. A. Barnes, M .; J. et al. C. Rhodes, J. et al. Agric. Food Chem. 46.4898-4903 (1998)

<Compound 1>
Next, measurement results of Nuclear Magnetic Resonance Spectroscopy (NMR) for Compound 1 are shown below. Further, ¹ H-NMR spectrum of Compound 1 is shown in FIG.

(1) ¹ H-NMR (CD ₃ OD) δ (ppm)
3.17-3.94 (m, sugar), 4.75, 5.59 (2H, anomalous proton), 6.17 (1H, brs), 6.36 (1H, brs), 6.86 (1H) , D, J = 8.0 Hz), 7.62 (2H)
(2) ¹³ C-NMR (CD ₃ OD) δ (ppm)
67.3, 71.7, 73.4, 75.5, 77.7, 78.4, 82.4, 95.4, 100.6, 101.7, 106.0, 106.5, 116. 8, 118.0, 123.8, 124.3, 135.6, 146.8, 150.5, 159.1, 159.2, 163.8, 166.5, 172.9, 180.0

¹ H-NMR of Compound ¹ is very similar to Compound 3, and a characteristic peak of flavonoid was detected at 6.2 to 7.6 ppm of ¹ H-NMR. Of these, the meta-position coupling of the aromatic ring can be inferred from the trisubstituted benzene and the two doublets of 6.2 ppm and 6.4 ppm from the peaks of 7.6 ppm and 6.8 ppm. In addition, there is a sugar-derived peak in the vicinity of 3 to 6 ppm, and of these, two peaks considered to be hydrogen derived from the sugar anomeric position are observed at 4.7 ppm and 5.6 ppm. It is done.

Next, when the ¹³ C-NMR of Compound 1 compared to the ¹³ C-NMR of Compound 3, 67.3ppm, 71.7ppm, 82.4ppm , the peaks other than 101.7ppm almost coincided with Compound 3.

  From the above results, Compound 1 can be presumed to be a compound obtained by adding a saccharide having 4 carbon atoms to Compound 3 (quercetin-3-ο-D-glucuronide). The estimated structure of Compound 1 is shown in FIG.

<Compound 2>
Next, measurement results of Compound 2 by nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (FAB-MS) are shown below. In addition, FIG. 6 shows a ¹ H-NMR spectrum of Compound 2 and FIG. 7 shows a ¹³ C-NMR spectrum.

(1) ¹ H-NMR (CD ₃ OD) δ (ppm)
3.39 (dd, J = 4.2, 9.1 Hz), 3.81 (d, J = 12.9), 4.59 (dd, J = 5.5, 10.2 Hz), 5.04 −5.5 (m), 6.34 (d, J = 15.0 Hz), 6.35 (d, J = 0.8 Hz), 6.42 (s), 6.48 (d, J = 3) .8 Hz), 6.52 (d, J = 15.0 Hz), 6.59 (d, J = 3.8 Hz), 6.69 (s), 6.81 (d, J = 2.0 Hz), 7.24 (s), 7.38 (d, J = 2.0 Hz)
(2) ¹³ C-NMR (CD ₃ OD) δ (ppm)
64.5, 69.8, 70.4, 72.2, 72.4, 75.8, 77.2, 77.4, 92.2, 92.7, 108.3, 108.6, 109. 0, 109.1, 109.3, 109.6, 109.7, 111.1, 112.5, 113.3, 115.7, 115.9, 115.9, 116.2, 116.3, 116.7, 117.1, 117.3, 117.7, 120.9, 126.3, 126.6, 126.8, 127.0, 127.2, 127.5, 138.0, 138. 0, 138.2, 138.3, 138.4, 138.5, 141.2, 142.0, 143.1, 145.1, 145.5, 145.5, 145.6, 145.8, 146.5, 146.5, 146.6, 146.6, 146.7, 148.1, 149.1, 166.2, 166.4, 69.8,169.9,170.3,170.5,170.7,171.2,171.4
(3) FAB-MS: m / z = 935 [M−H] ⁻
(Negative)

When the FeCl ₃ color was developed by TLC, it turned blue. Therefore, it is suggested that Compound 2 is a compound having many hydroxyl groups. Further, the ¹³ C-NMR spectrum of Compound 2 shown in FIG. 7, are very similar when comparing the spectra of ¹³ C-NMR of Casuarictin shown in FIG. The structural formula of Casuarictin is shown in FIG.

  From the above results, Compound 3 is very likely to be a hydrolyzable tannic acid derivative, and 4-5 gallic acids appear to be ester-linked around a sugar having 6 carbons such as Casuarictin. It can be inferred that this is a structure with something attached. However, Compound 2 has as many as 66 carbon atoms, suggesting the possibility that two types of compounds are mixed as isomers.

Table 3 below shows the results of measuring tyrosinase inhibition rate and SOD-like activity for Compound 1, Compound 2 and Compound 3 in the same manner as described above. In addition, Table 4 below shows the results of measuring the tyrosinase inhibition rate of arbutin and the SOD-like activity of (+)-catechin as activity indicator substances.

  Thus, Compound 2 showed high tyrosinase inhibitory activity even compared with arbutin, which is an activity indicator substance. Compounds 1 and 3 showed almost the same SOD-like activity as the activity indicator substance (+)-catechin. From the above results, it can be said that Compounds 1-3 are physiologically active substances contained in strawberry leaves.

[Alleviating symptoms of hay fever]
When the above-mentioned Dutch strawberry leaf extract was used by 8 panelists suffering from hay fever when symptoms occurred, the following results were obtained.

Paneler 1 (doctor, female): I was having problems with daily runny and sneezing due to hay fever. Of course, we have used many improvers so far, but there was no drug that had the expected effect. However, when the received “strawberry leaf extract” was applied to the nasal cavity when the symptoms were severe, the runny nose and sneezing stopped brilliantly in 4 to 5 minutes, and the effect also lasted for a long time. And I use it twice a day, but I can feel my daily life comfortably without any side effects.
Paneler 2 (Housewife 47 years old): I like gardening, but when it was time for pollen to fly, I was suffering from hay fever. When I applied to the nasal cavity, I got a strawberry leaf extract, and when I applied it with a cotton swab, I got a squeak and once I bite my nose. Just gardening was pleasant until the evening.
Paneler 3 (Housewife 38 years old): Because of hay fever, I was having a hard time every time I hang laundry. I picked up the strawberry leaf extract, and I was convinced that if it was a strawberry, I applied it with a cotton swab to the nasal cavity. I can't let go.
Paneler 4 (Instructor 26 years old): I go to work by bicycle, but my nasal congestion and sneezing and my runny nose stopped. I couldn't make up. One day, I applied to the nasal cavity and received a sample of strawberry leaf extract. After I used it immediately, my nasal discharge, sneezing, and stuffy nose clogged up. Please sell quickly so effective for rhinitis.
Paneler 5 (Full-time housewife 54 years old): Thank you very much for the strawberry leaf extract. I went to the doctor and asked me for some medicine, but I was worried because I made my stomach worse. Strawberry leaf extract smells better, and I had better house dust than hay fever, but the mucous membranes became rugged and peeled, nosebleed, nasal congestion, and runny nose did not stop all year round was. Thanks to the strawberry leaf extract, the mucous membrane became normal and the nose was not clogged. Make a product early for many suffering people.
Paneler 6 (72-year-old unemployed person): I go to the pool every day, but I suddenly get hay fever even at this age. I first went to the clinic with Kushami and a runny nose, but I was diagnosed with hay fever. I thought I was at this age, but I got some medicine from my doctor, but it wasn't very effective. The pool clerk told me to apply the strawberry leaf extract to a cotton swab and apply it to my nostrils. After 15 minutes of painting, my runny nose and sneezing stopped. If you like it, I kindly gave you a sample. Thanks to you, I started to put it in the pool. Because it has an immediate effect and smells good even if it is applied to the nose, I spend a good time every day. Please make the product as soon as possible.
Paneler 7 (38-year-old housewife): Even though I had predicted hay fever and took hay fever countermeasures such as injections and burning the nasal mucosa for more than a month, the painful symptoms remained the same as usual. . When I applied strawberry leaf extract to the back of my nose with a cotton swab, it became very easy. The conventional medicines have stomach problems and have side effects such as having to take stomach medicines, so I would like you to commercialize the strawberry leaf extract as soon as possible.
Paneler 8 (64-year-old housewife): 20 years of hay fever. I gave up that this time would be like this, but when I applied the strawberry leaf extract to the back of the nasal cavity, the moment the crushing came out 5 times and biting the runny nose, it became very easy and I can breathe Susuu. Also, it was easier for my eyes to be crispy. In the past, it was painful because nasal mucosa was formed on the mucous membrane of the nose, and when it was removed, nosebleeds were formed, and casabuta was formed again and again, but the strawberry leaf extract was applied 3 to 4 times a day. Now you can have a good time. As hay fever causes the most important nose to breathe, I thought that I would have been happy if I had been able to apply this medicine. Anyway, the strawberry leaf extract made it very easy.

  In this way, it acts on the mucous membrane and exhibits excellent antiallergic action and antihistamine action, especially on the nasal mucosa and can alleviate the symptoms of so-called hay fever such as runny nose and shampoo. It can be used as a mucosal external preparation for mucous membranes. In addition, it can be expected to treat or alleviate allergic dermatitis due to its excellent antiallergic action.

  Since the skin external preparation of the present invention has a high tyrosinase activity inhibitory activity and antibacterial activity, it should be used as a skin external preparation for the purpose of whitening cosmetics, prevention of acne and stains, and treatment. Can do. It can also be used as a mucosal external preparation for mucous membranes such as nasal mucosa.

The figure which shows the measurement result of the fraction and tyrosinase inhibitory activity of a strawberry leaf extract. ^{1 is} a ¹ H-NMR spectrum of Compound 3; The structural formula of quercetin-3-ο-D-glucuronide is shown. 1 is a ¹ H-NMR spectrum of Compound 1; The estimated structure of Compound 1 is shown. ^{1 is} a ¹ H-NMR spectrum of Compound 2. It is a ¹³ C-NMR spectrum of Compound 2. It is a ¹³ C-NMR spectrum of Casuarictin. The structural formula of Casuarictin is shown.

Claims (4)

  An external preparation for skin, comprising an extract of leaves and / or stems of the genus Rosaceae belonging to the family Rosaceae as an active ingredient.   The topical skin preparation according to claim 1, wherein the leaf and / or stem extract of the Rosaceae family is a extract extracted with water and / or alcohol.   An external preparation for mucous membranes containing an extract of leaves and / or stems of the genus Rosaceae belonging to the family Rosaceae as an active ingredient.   The mucosa topical preparation according to claim 3, wherein the leaf and / or stem extract of the Rosaceae genus Strawberry is an extract extracted with water and / or alcohol.

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