JP2010254591A - External preparation for skin and method for producing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an external preparation for the skin, which safely exerts prophylactic or therapeutic effect against dysfunction accompanying skin aging or diseases when applied to the skin. <P>SOLUTION: The external preparation for the skin includes cryptoxanthin capable of activating a retinoic acid receptor (RAR) present on an epidermis or dermal cell, and/or a derivative thereof. The external preparation for the skin is capable of enhancing proliferation of human skin fibroblast and/or enhancing collagen production. The cryptoxanthin and/or the derivative thereof are derived from citrus, particularly satsuma mandarin. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to an external preparation for skin, and more particularly to an external preparation for skin having an activating action of retinoic acid receptor (RAR) present in epidermis or dermal cells.

  The skin is an organ that separates the human body from the outside of the body, and has various functions such as immune functions against external enemies, retention of moisture in the body, and regulation of sensation and body temperature. However, as the skin ages, its function gradually decreases, causing a decrease in lipids in the keratinocytes and breakage of fibers such as collagen. Along with this, there is no decrease in the amount of moisture retained and no skin firmness, and wrinkles and the like are generated. Spots and wrinkles in particular can be a mental burden for women. In addition, when the amount of lipids in the skin of patients with diseases such as atopic dermatitis is measured, it is clear that the lipids related to water retention, such as ceramide, have decreased, although the composition has not changed. Etc. are known to be significantly higher (Non-Patent Document 1).

  Examples of substances for treating these skin abnormalities include tretinoin (Patent Document 1). Tretinoin is another name for all-trans retinoic acid (ATRA), which is known to activate retinoic acid receptor (RAR) in the body, and its action is α, β, γ of RAR expressed in the skin. It is obtained by activating the mold and controlling the activity of the target gene. For example, when ATRA is used, a strong growth promoting effect of epidermal keratinocytes is observed, and by promoting skin turnover, there are whitening and wrinkle-improving effects due to melanin excretion, and in the dermis fibroblasts It has the effect of reducing the expression of matrix metalloproteases (MMPs) that promotes collagen production in cells and decomposes collagen to cause wrinkles and sagging, and also has an improving effect on sagging (Non-Patent Document 2) .

  However, the use of ATRA increases the expression of protein that excretes ATRA out of the body, and when the effect is reduced, it causes side effects associated with fever, dyspnea, pleural effusion, liver failure, renal failure, which is called retinoic acid syndrome, Sometimes life can be lost. Caffeic acid derivatives (patent document 2) and AM80 (trade name: Tamibarotene), which are synthetic retinoids that activate RAR, are also on the market, but there are problems with safety when they are used continuously. ATRA and synthetic retinoids have low absorbability to living organisms, and the dose must be increased in order to obtain the effect. Therefore, although ATRA has been approved for the treatment of some leukemias, it has not been approved for use on the skin in Japan, and it is currently used as a free practice in certain hospitals. .

JP 2002-293746 A WO2005 / 092322 pamphlet

NARDO et al. , Acta Derme Veneol (Stockch) 78, 27-30 (1998) Nicholson et al. , EMBO J 9, 4443-4454 (1990)

  An object of this invention is to provide the skin external preparation which shows the prevention or the treatment effect of the malfunction which accompanies the aging of a skin and a disease safely by apply | coating to skin.

  As a result of intensive studies to solve such problems, the present inventors have found that skin improvement is observed through activation of RAR by containing cryptoxanthin and / or a derivative thereof, The present invention has been reached.

  Specifically, the first aspect of the present invention is a topical skin preparation containing cryptoxanthin having a retinoic acid receptor (RAR) activating action and / or a derivative thereof present in epidermis or dermal cells, It is a skin external preparation having a human skin fibroblast growth promoting action and / or collagen production promoting action, preferably a cryptoxanthin and / or derivative thereof is a skin external preparation derived from citrus, more preferably Citrus is a topical skin preparation that is Unshu mandarin orange and is used to prevent or treat at least one of atopic dermatitis, skin aging, spots, wrinkles, sagging or freckles, or to improve whitening or moisturizing skin quality It is a skin external preparation.

  The second of the present invention is a method for producing an external preparation for skin, characterized in that a composition containing cryptoxanthin and / or a derivative thereof is obtained from the squeezed citrus plant and the residue is added, or an enzyme is added to the citrus plant Then, an enzyme treatment is carried out to obtain a composition containing cryptoxanthin and / or a derivative thereof, or a method for producing an external preparation for skin, or an organic solvent is added to a citrus plant, and cryptoxanthin is added to the organic solvent. And a method for producing an external preparation for skin, characterized in that a composition containing a derivative thereof is extracted.

  According to the present invention, since cryptoxanthin contains an RAR activating action, an action for promoting skin turnover and an action for promoting collagen production are obtained. Moreover, the action of improving the water retention function in the stratum corneum can be seen by further containing sphingolipid. In addition, the present invention is a composition with high safety because it is derived from citrus that is also used as a cosmetic.

It is a figure which shows the normal human skin fibroblast proliferation activity of the skin external preparation of this invention. It is a figure which shows the collagen production promotion activity of the skin external preparation of this invention. It is a figure which shows the RAR activation effect of the skin external preparation of this invention.

  Hereinafter, the present invention will be described in detail. First, the skin external preparation which is the first present invention will be described. The cryptoxanthin and / or derivative thereof contained in the present invention may be α-type or β-type, and is not particularly limited. Moreover, as a derivative of cryptoxanthin, for example, a fatty acid ester of cryptoxanthin exists, and specifically, derivatives such as palmitoyl ester, myristoyl ester, and lauryl ester are also included.

  The cryptoxanthin and / or derivative thereof used in the present invention is not particularly limited with respect to its origin, but among these, citrus is preferred. Citrus can include plants belonging to the citrus family. More specifically, Iyokan, Wenzhou mandarin orange, Orange, Kabos, Kawabata, Kishumikan, Kiyomi, Kumquat, Grapefruit, Gecki, Sanpokan, Shikuwasa, Jabara, Sweety, Sudachi, Daidai, Tachibana, Dekopon, Natsudaidai, Hassaku, Valencia Orange Illustrative examples include evening shark, hyuga natsu, buntan, ponkan, mandarin orange, yatsushiro, yuzu, lime, lemon, karatachi and the like (including varieties equivalent or similar to these). Among them, Unshu mandarin is desirable because it has a high content of cryptoxanthin and / or its derivatives.

  In the external preparation for skin of the present invention, the amount is not limited as long as cryptoxanthin and / or a derivative thereof is contained. For example, cryptoxanthin and / or a derivative thereof is 0.00001% or more (mass ratio), less than 100%, preferably 0.0001% or more (mass ratio), less than 50%, more preferably 0.01% or more (mass ratio). Ratio) can be blended at a ratio of less than 10%.

  In the external preparation for skin of the present invention, it is preferable to contain sphingolipid in addition to the above-mentioned cryptoxanthin and / or a derivative thereof because it has an effect of improving moisture retention. Here, sphingolipid is a group of compounds in which an amino alcohol consisting of about 14 to 24 carbon atoms called sphingosine or a long-chain amino alcohol having a structure similar to this and an aliphatic acid such as a long-chain fatty acid is amide-bonded. In other words, compounds in which saccharides are bound to them, and compounds in which phosphate groups are bound are also included. The fatty acid may be either a saturated fatty acid or an unsaturated fatty acid, may be linear or branched, and one or more hydroxyl groups may be substituted. In addition, sugar, phosphoric acid, sialic acid, choline, ethanolamine and the like may be bonded to the hydroxyl group of amino alcohol. As the saccharide, glucose, mannose, galactose, lactose or the like may be bound, and those having a phosphate group bound include inositol via a phosphate group, and also sphingolipids bound with a sugar. In the present invention, those having a specific chain length or structure are not intended, but all those called sphingolipids are included.

  As the origin of the above-mentioned sphingolipid, the above-mentioned plant derived from cryptoxanthin and / or a derivative thereof can be preferably used.

  When the sphingolipid is included, the content is 0.00001% or more (mass ratio), less than 100%, preferably 0.001% or more (mass ratio), less than 50%, more preferably 0.1% or more. (Mass ratio) It can mix | blend in the ratio of less than 20%.

  The above-mentioned skin external preparation of the present invention is a component generally used for external preparations within the range where the effect is not further impaired, oils, waxes, hydrocarbons, fatty acids, alcohols, esters, interfaces. Components such as activators, metal soaps, pH adjusters, preservatives, fragrances, moisturizers, powders, ultraviolet absorbers, thickeners, dyes, antioxidants, whitening agents, chelating agents and the like can be blended.

  The topical skin preparation of the present invention has an action of promoting the proliferation of human skin fibroblasts and / or promoting the production of collagen through the activation action of RAR when applied to the skin. As a result, atopic dermatitis, At least one prevention or treatment of skin aging, spots, wrinkles, sagging and freckles, or skin quality improving effects such as whitening and moisturizing properties can be obtained.

  As a method for applying the external preparation for skin of the present invention, the external preparation for skin may be used by dispersing it alone in a solvent, or may be used in combination with other functional components.

  Next, a method for producing the above-mentioned external preparation for skin will be described. Such a production method includes squeezing a citrus plant and obtaining a skin external preparation containing cryptoxanthin and / or a derivative thereof from the residue, adding an enzyme to the citrus plant, treating the citrus plant, and treating the cryptoxanthin And / or a method of obtaining a skin external preparation containing a derivative thereof, and a method of adding an organic solvent to a citrus plant and extracting the skin external preparation containing cryptoxanthin and / or a derivative thereof into the organic solvent. . Hereinafter, these methods will be described sequentially.

  The residue obtained by squeezing citrus plants, that is, squeezed rice cake, is obtained by squeezing the fruits of citrus plants with an inline squeezing machine, chopper helper squeezing machine, brown squeezing machine, etc., and then using a paddle type or screw type finisher. It is prepared by collecting the juice residue from which the fruit juice has been prepared by filtration or sieving or centrifugation. The squeezed residue of the fruits of the citrus plant thus prepared may be as it is, or may be obtained by subjecting them to physical treatments such as grinding, crushing, crushing, heating, dehydration, and drying.

  In the enzyme treatment, the fruit of the citrus plant is used as it is, or after the physical treatment such as grinding, crushing, crushing, heating, dehydration, drying, etc., and the enzyme is added to the juice residue obtained as described above. Is done.

  The enzyme used for the enzyme treatment is not particularly limited as long as it is capable of degrading organic substances contained in citrus plants, particularly biopolymers constituting cell walls, etc. Amylase, glucoamylase, cellulase, hemicellulase Pectinase, mannanase, xylanase, protease, peptidase, lipase, malation enzyme (cell wall degrading enzyme) and the like are used. Among these, cellulase, hemicellulase, pectinase, mannanase, xylanase, and malation enzyme, which are carbohydrate hydrolases, are desirable because of their high efficiency of concentrating cryptoxanthin and / or its derivatives as active ingredients.

  As the enzyme agent to be added, these purified enzymes may be used, or microbial cells or cultures exhibiting these activities, or crudely purified products thereof may be used. These enzymes may be used alone, or two or more kinds of enzymes may be mixed and used. The amount of the enzyme to be added is not particularly limited, and may be added according to the reactivity of the enzyme. For example, when pectinase is used, it is preferably 1 to 100,000 units, more preferably 10 to 10,000 units, based on 100 g of the extract.

  After the enzyme is added, the enzyme and the extract are uniformly mixed by stirring or the like to allow the enzyme reaction to proceed. As the reaction temperature at this time, it is desirable to carry out the reaction under such conditions that the enzyme is not inactivated and does not easily rot, and the cryptoxanthin and / or its derivatives are not lost. Specifically, the temperature is 0 to 90 ° C, preferably 0 to 80 ° C, more preferably 0 to 70 ° C. Needless to say, the pH of the reaction is preferably carried out under the optimum conditions of the enzyme, and it is preferably pH 2 to 12, preferably pH 3 to 8. Although the reaction time depends on the squeezed residue used and the amount of enzyme, it is usually preferable to set the reaction time to 1 to 48 hours. In the reaction, the reaction may be performed while stirring the reaction product, or may be a stationary reaction.

  After completion of the enzyme treatment, the enzyme-treated reaction product may be used as it is, or a product that has undergone some processing may be used. Specifically, a residue obtained by solid-liquid separation of the reaction product, a product obtained by drying the residue obtained by solid-liquid separation, or a product obtained by drying the reaction product without solid-liquid separation may be used. Moreover, you may use what extracted the component etc. using the solvent, water, supercritical carbon dioxide, etc. Further, impurities may be subsequently removed. Examples of the impurity removal method include water washing, organic solvent washing, silica gel column, resin column, reverse phase column and the like, activated carbon treatment, partitioning with solvents of different polarity, recrystallization method, vacuum distillation method and the like. . In particular, the water washing in which the enzyme-treated reaction product is solid-liquid separated and then water is again added to the solid content and stirred, followed by solid-liquid separation is a preferable method because reactive organisms such as sugar produced by the enzyme treatment can be easily removed. It is.

  As a solvent used in a method for adding an organic solvent to a citrus plant and extracting a skin external preparation containing cryptoxanthin and / or a derivative thereof and a sphingolipid in the organic solvent, a citrus plant as a raw material or a processed product thereof Any fraction can be used as long as a fraction having an RAR activating effect is obtained and the present invention is not impaired. One kind of solvent may be used alone, or a plurality of solvents may be mixed and used. Examples of such solvents include alcohols such as methanol, ethanol, propanol and butanol, polyhydric alcohols such as ethylene glycol, propylene glycol, butylene glycol and glycerin, ketones such as acetone and methyl ethyl ketone, methyl acetate and ethyl acetate. Esters such as tetrahydrofuran, ethers such as diethyl ether, halogenated hydrocarbons such as dichloromethane, dichloroethane and chloroform, aliphatic hydrocarbons such as hexane and pentane, aromatic hydrocarbons such as toluene, polyethylene glycol Polyethers such as pyridine, supercritical carbon dioxide and the like can be used. Of these, acetone, ethanol, hexane, and supercritical carbon dioxide are preferably extracted from cryptoxanthin and / or its derivatives.

  Moreover, when extracting with these organic solvents, in order to raise extraction efficiency, additives, such as water and surfactant, can be added in the range which does not impair the effect of this invention, for example. Furthermore, in addition to the extraction using the organic solvent, a supercritical extraction method which has recently been attracting attention can be used.

  The skin external preparation of the present invention can be used for any of cosmetics, quasi drugs, and pharmaceuticals, and the dosage form is not particularly limited, and examples thereof include emulsions, creams, lotions, packs, Detergent, makeup cosmetics, scalp / hair products, dispersion, ointment, solution, aerosol, patch, patch, liniment, oil, lip, lipstick, foundation, eyeliner, blusher, mascara, eye shadow, nail polish Pedicure (and remover), shampoo, rinse, hair treatment, permanent agent, hair dye, shaving agent, gel agent, essence agent, cleaning agent, bath preparation, dusting powder, soap (hand soap, body soap, face wash), Any dosage form such as dentifrice, mouthwash, etc. may be used. Also included are those that may come into direct contact with humans and animals, impregnated in kitchen / bathroom / toilet detergent, glass cleaner, glasses / contact lens cleaner, automotive detergent, sanitary products, wet paper, etc. Also included.

  Hereinafter, the present invention will be specifically described by way of examples. In addition, this invention does not limit the range by this Example.

  In the examples, the content of β-cryptoxanthin and / or its derivative was measured by high performance liquid chromatography (HPLC). That is, LC-10A (manufactured by Shimadzu Corporation) was used as an HPLC apparatus, a Resolve C18 (φ3.9 × 150 mm, manufactured by Waters) column was connected, and a sample to which an equal amount of methanol was added was introduced. The mobile phase was analyzed with methanol: ethyl acetate = 7: 3, a column temperature of 30 ° C., a flow rate of 1.0 ml / min, and a detection wavelength of 450 nm.

Example 1
Sumiteam PX (manufactured by Shinnippon Chemical Co., Ltd., 5,000 units / pectinase), a pectinase enzyme agent for processing foods and drinks, is obtained after squeezing fruit juice from Wenzhou mandarin orange (mandarin orange juice lees, moisture content about 90%) g, 1 unit of arabanase 90 units / g) and 1 g of cellulase Y-NC (cellulase 30,000 units / g, manufactured by Yakult Pharmaceutical Co., Ltd.) which is a cellulase / hemicellulase enzyme agent, stir well and let stand at room temperature for 8 hours. Reaction was performed. After centrifuging the reaction solution to remove the supernatant, water was added and stirred, and the supernatant was again removed by centrifugation. This precipitate was dried by a freeze dryer, and pulverized and powdered by a mixer type pulverizer. This powder contained 0.5% by mass of β-cryptoxanthin (free form equivalent).
The obtained Wenzhou mandarin orange powder was extracted with three times the weight of ethanol, and the resulting extract was concentrated under reduced pressure using an evaporator. The residue after concentration was used as a skin external preparation of the present invention. This external preparation for skin contained 1% (converted as a free form) of β-cryptoxanthin.

Test Example 1 [Proliferation promoting effect of human skin fibroblasts]
Normal human skin fibroblasts (NHDF: Kurabo Industries KF-4109) were seeded on a 24 well plate (IWAKI) so as to be 5.0 × 10 ⁴ cells / well and cultured for 24 hours. By mixing and exchanging the obtained skin external preparation with a medium (M-106-500S manufactured by Kurabo Industries Co., Ltd.) to 0.01, 0.05, 0.1%, the action of the reagent on the cells Is detected. The medium was changed every 24 h, and after culturing until 72 h, the cell growth promoting activity was measured using CCK8 (Doujin Chemical Co., Ltd.). 50 μl of CCK8 was added to 500 μl of the medium of each well, and the reaction was performed for 2 hours in a CO 2 incubator in which culture was performed. Thereafter, the number of viable cells was determined in proportion by measuring the wavelength of 450 nm with a plate reader (Dainippon Sumitomo Pharma Co., Ltd.).
The obtained results are shown in FIG. The cell growth promoting activity was significantly increased compared to the control.

Test Example 2 [Collagen production promoting effect]
In addition, the amount of collagen in normal human skin fibroblasts was measured using Sircol Collagen Assay Kit (manufactured by Biocolor) in the same manner as in the test of Test Example 1. The cell culture medium after the culture is removed, 1 ml of Sircol Dye reagent is added, and the reaction is performed at room temperature for 30 minutes. After the reaction, the unreacted solution is removed and Alkali reagent is added to extract the staining solution. Collagen was measured by dispensing 100 μl of the extracted solution into a 96-well plate (manufactured by IWAKI) and measuring the absorbance at 540 nm with a plate reader (manufactured by Dainippon Sumitomo Pharma Co., Ltd.).
The obtained results are shown in FIG. Regarding the collagen production promoting activity, the production was promoted, and the collagen production promoting activity of the external preparation for skin of the present invention was revealed.

  In addition, in Test Examples 1 and 2, when the skin external preparation of the present invention was used in combination with LE540 (manufactured by Wako Pure Chemical Industries, Ltd.) which is an RAR activation inhibitor, the cell proliferation promoting activity was significantly reduced. Moreover, the collagen production promoting activity was not different from the control.

Test Example 3 [RAR activation promoting effect]
About the skin external preparation of this invention, the RAR activation promotion effect was measured. Using a RARα type and γ type activity measurement kit (manufactured by Mycrosystems), RARα or γ activation activity was tested as follows. For the measurement of activation, ATRA, which is a positive control, was used to confirm that the experimental system was established. That is, RARα or γ is mixed with 10 ml of the attached 0.1 M ice-cold buffer A, and this RARα or γ solution is dispensed at 100 μl / well to immobilize RARα or γ on the plate. Let stand at 2-8 ° C. overnight and wash with ice-cold buffer A 120 μl / well three times. Thereto was added 100 μl / well of TIF2-BAP solution, and 1 μl of the external preparation for skin obtained in Example 1 was added so that the final concentrations were 0.01, 0.05, and 0.1%. Let stand for 1 hour. Thereafter, ice-cold buffer B is added to 120 μl / well, washed three times, and the substrate solution is added to 100 μl / well, and reacted at 37 ° C. for 3 to 5 hours. The reaction was terminated by adding 25 μl / well of 0.5N sodium hydroxide, and the absorbance at 405 nm was measured with a plate reader (Dainippon Sumitomo Pharma Co., Ltd.).
As a result, the result of FIG. 3 was obtained. This revealed that the composition of Example 1 has an action of activating RARα type and γ type in a concentration-dependent manner.

Example 2
Using the extract obtained in Example 1, the following formulation example was prepared.

Formulation Example 1 Latex Extract 1.00%
SURFHOPE (R) SE COSME C-1216 3.00%
Glycerin 12.00%
Squalane 6.00%
TORAY (R) SH 200 (6cs) 24.00%
Polypropylene glycol 1.00%
CARBOPOL (R) 940 0.06%
Deionized water 47.62%
Preservative 0.20%
Ethanol 5.00%
0.1% aqueous sodium hydroxide solution 0.12%

Formulation Example 2 Sunscreen Cream Extract 1.00%
SURFHOPE (R) SE COSME C-1216 1.00%
SURFHOPE (R) SE COSME C-1816 1.00%
Deionized water 2.00%
90% glycerin 4.00%
DPPG (R) 5.00%
LIPONATE (R) NPGC-2 5.00%
PARSOL (R) 1789 1.00%
PARSOL (R) MCX 5.00%
PARSOL (R) 340 0.50%
EUSOLEX (R) 6300 2.00%
Preservative 0.50%
OXYLESS (R) CLEAR 0.10%
DOW CORNING (R) 1501 FLUID 1.00%
PARSOL (R) HS 4.00%
CARBOPOL (R) ULTREZ20 0.50%
EDTA 0.10%
Deionized water 62.90%
Triethanolamine 99% 3.20%
Fragrance 0.20%

Formulation Example 3 Moisturizing cream extract 1.00%
Deionized water 67.80%
SOLANACE STARCH 0.80%
Glycerin 2.00%
SURFHOPE (R) SE COSME C-1815 1.50%
Kisanta Gum 0.20%
SURFHOPE (R) SE COSME C-1805 3.50%
DPPG (R) 6.00%
MOD (R) 3.00%
ISIS (R) 4.00%
Isononyl isononanoate 5.00%
Preservative 0.50%
Cetostearyl alcohol 0.50%
DOW CORNING (R) 345 FLUID 2.00%
Flour extract 2.00%
Fragrance 0.20%

Test Example 4 [Sensory test]
Using the emulsion of Prescription Example 1 and the emulsion extracted from Prescription Example 1 (placebo) for 36 women (22 to 45 years old) using a one-month double-blind test went. During the test period, intake of foods rich in β-cryptoxanthin, such as mandarin oranges, persimmons, and mangoes, was prohibited, and application was performed at night. After the trial period, the presence or absence of improvement effects such as skin wrinkles, sagging and spots, freckles and the like was determined by a questionnaire.

  Table 1 shows the results. Many people felt that wrinkles, sagging, spots and freckles were all improved by using the external preparation for skin of the present invention. There were no skin problems or deterioration of prescription ingredients during the test period.

Claims (10)

An external preparation for skin comprising cryptoxanthin and / or a derivative thereof. The skin external preparation according to claim 1, which has an action of activating retinoic acid receptor (RAR) present in epidermis or dermal cells. The external preparation for skin according to claim 2, which has a human skin fibroblast growth promoting action and / or a collagen production promoting action. The skin external preparation according to any one of claims 1 to 3, wherein the cryptoxanthin and / or a derivative thereof is derived from citrus. The topical skin preparation according to claim 4, wherein the citrus fruit is Unshu mandarin. The external preparation for skin according to any one of claims 1 to 5, which is used for the prevention or treatment of at least one of atopic dermatitis, skin aging, spots, wrinkles, sagging or freckles. The skin external preparation according to any one of claims 1 to 5, which is used for whitening or moisturizing skin quality improvement. The method for producing an external preparation for skin according to any one of claims 5 to 7, wherein a composition containing cryptoxanthin and / or a derivative thereof is obtained from the residue obtained by squeezing a citrus plant. The method for producing an external preparation for skin according to any one of claims 5 to 7, wherein an enzyme is added to a citrus plant and subjected to enzyme treatment to obtain a composition containing cryptoxanthin and / or a derivative thereof. The preparation of a skin external preparation according to any one of claims 5 to 7, wherein an organic solvent is added to the citrus plant, and a composition containing cryptoxanthin and / or a derivative thereof is extracted into the organic solvent. Method.