WO2014034977A1

WO2014034977A1 - Composition for blocking uv rays or reducing skin wrinkles comprising afzelin as active ingredient

Info

Publication number: WO2014034977A1
Application number: PCT/KR2012/006950
Authority: WO
Inventors: Seoung Woo SHIN; Jang-Hyun Kim; Eun Sun Jung; Deok Hoon Park
Original assignee: Bio Spectrum, Inc.; Dermiskin Inc.
Priority date: 2012-08-30
Filing date: 2012-08-30
Publication date: 2014-03-06

Abstract

The present invention relates to a cosmetic composition for blocking UV rays or reducing skin wrinkles, which contains afzelin as an active ingredient. The invention also relates to a pharmaceutical or food composition for reducing skin wrinkles, which contains afzelin as an active ingredient. The composition of the invention has an excellent effect of reducing wrinkles by absorbing UVA and UVB to prevent UV rays from penetrating the skin, increasing collagen synthesis and inhibiting collagenase activity. In addition, because the composition of the invention is derived from natural materials, it is harmless to the human body and can be safely applied to cosmetic, pharmaceutical and food compositions.

Description

COMPOSITION FOR BLOCKING UV RAYS OR REDUCING SKIN WRINKLES COMPRISING AFZELIN AS ACTIVE INGREDIENT

The present invention relates to a composition for blocking UV rays or reducing skin wrinkles, and more particularly to a composition for blocking UV rays or reducing skin wrinkles, comprising, as an active ingredient, afzelin which is derived from natural materials, and thus can be safely used without causing adverse effects on the skin, and also has the effects of increasing the synthesis of collagen and inhibiting the activity of collagenase.

The structure of the skin can be largely divided into the epidermis and the dermis. The epidermis is a thin protective layer which is located in the uppermost layer of the skin and composed of keratinocytes, melanocytes and the like. The dermis is a thick complex layer of fibrous tissue and connective tissue composed of elastic fibers such as collagen and elastin.

Collagen in the dermis is a major structural protein of the skin, which accounts for about 70% of the dermis, gives flexibility to the skin and has the greatest effect on the formation of skin wrinkles.

Polypeptide chains of collagen fiber are synthesized in the granular endoplasmic reticulum of fibroblasts in a state in which they have a telopeptide at both ends. The synthesized polypeptide chains are secreted extracellularly in the form of procollagens combined into a triple helix structure. The telopeptide in the secreted procollagens is cleaved by an enzyme to produce tropocollagens, which are then combined with each other in the extracellular matrix to produce collagen fibers. The synthesis of collagen fibers decreases rapidly as skin aging progresses, and collagen fibers are modified by an external environment while skin wrinkles are formed.

Collagenase is an enzyme that degrades collagen, and the expression and activity thereof are increased by external factors such as UV rays, heat, oxidative stress, or internal factors such as skin aging, so that collagen is degraded to generate wrinkles.

The term “wrinkles” refers to a phenomenon in which collagen fiber and elastic fiber in the dermis are modified by various factors present inside and outside the body, and the moisture content of the skin decreases, so that the elasticity of the skin decreases, whereby the skin is folded.

Wrinkles are natural conditions caused by skin aging. Skin aging is influenced by various factors, including an environment, heredity, facial expression or a skin color. Skin aging can be divided into intrinsic aging and photoaging. Intrinsic aging is a natural aging phenomenon associated with the skin-related changes in the function, structure and shape of the skin and can appear mainly in a skin which was not exposed to sunlight. It is characterized in that relatively fine wrinkles occur and skin elasticity decreases.

Photoaging is caused mainly by UV rays among external environmental factors. UVA penetrates the dermis to increase the activity of collagenase, and collagen degradation is stimulated due to increased collagenase activity, thus generating wrinkles. It is characterized in that relatively thick deep wrinkles occur and there are many fine wrinkles.

As the level of life is increased, interest in appearance is being increased. Thus, various methods for reducing skin wrinkles which are typical aging-related conditions have been suggested.

Antioxidants have the effect of preventing cell aging by inhibiting the generation of reactive oxygen species, and typical examples thereof include vitamin A (retinol). However, vitamin A is sensitive to UV rays, water, heat and oxygen and is unstable, and thus it is difficult to stabilize the formulation of vitamin A. In addition, when it is applied to the skin at high concentration, it can irritate the skin to cause skin exfoliation or erythema, and when it is used at low concentration, the effect thereof will be insignificant.

Skin scaling is a method of peeling the skin using chemicals such as alpha-hydroxyl acid (glycolic acid) or beta-hydroxyl acid (salicylic acid), thereby reducing thin wrinkles formed on the skin and promoting collagen production. It has the effect of reducing skin wrinkles, but can have risks such as burns, and adverse effects such as post-inflammatory pigmentation, wounds and erythema, because chemicals are used directly on the skin.

Botox injection is a method of reducing wrinkles by injecting a specific amount of botulinum toxin into facial muscles. Because it influences expression muscles, it makes pronunciation incorrect or causes an obstacle in eating. In addition, it causes adverse effects, including headache, muscle pain and ptosis.

Filler therapy is a method of reducing wrinkles by filling a wrinkle area with a material such as hyaluronic acid. It has less adverse effects compared to Botox injection, but the effect thereof is temporary, and thus it should be repeated.

As described above, the above methods have insufficient effects on winkle reduction, or cause adverse effects and are problematic in terms of stability and safety. Thus, there is a need to develop a composition which is derived from natural materials, has less adverse effects and is safe while having excellent effects on wrinkle reduction.

Meanwhile, ultraviolet (UV) rays are electron radiations having specific wavelengths from sunlight and are divided according to the wavelength into several categories. Specifically, UVA has a wavelength of 320-400 nm, UVB has a wavelength of 280-320 nm, and UVC has a wavelength of 200-280 nm, and the shorter the wavelength, the greater energy it has. However, UVC having the shortest wavelength is mostly absorbed into the atmosphere by the ozone layer, and thus UV rays that directly influence the human body are UVA and UVB.

UV rays are the major cause of skin injury and aging. UVA accounts for about 90% or more of UV rays and penetrates the dermis of the skin to cause skin cancer, melamine formation stimulation, phototoxicity, photoallergic reactions, skin aging and the like. UVB penetrates the epidermis of the skin to cause sunlight burns such as erythema or blisters, pigmentation, cell membrane injury, skin cancer and the like. Thus, various method for preventing skin injury or skin disease from being caused by direct irradiation of the skin with excessive UV rays have been suggested.

Chemical UV blocking agents are based on organic materials such as cinnamic acid or salicylic acid and chemically absorb UV rays to prevent UV rays from penetrating into the skin. Chemical UV blocking agents are applied to UV rays having a narrow range of wavelength and are transparent, and thus have an advantage of a good appearance when they are used. However, they cause skin irritations such as contact dermatitis. In addition, because they have a low molecular weight and are oil-soluble, they are highly likely to penetrate the skin, and thus the organic materials therein can be chemically activated by absorption of UV rays to show toxicity, even though the chemical UV blocking agents themselves have no toxicity.

Physical UV blocking agents are based on inorganic materials such as titanium dioxide or titanium oxide and physically scatter and reflect UV rays to prevent UV rays from penetrating the skin. The physical UV blocking agents cause less skin irritation and less adverse effects on the skin, but are difficult to disperse and dissolve in formulations, thus causing a white cloudy phenomenon, and have poor touch feeling.

However, the above methods have problems in that, because they use chemical products such as organic or inorganic materials are used, they irritate the skin or the touch feel thereof is reduced due to stickiness, glossiness or a white cloudy phenomenon. Accordingly, there is a need to develop a composition which is derived from natural materials, has less adverse effects and is safe while having good touch feeling.

With respect to methods of reducing skin wrinkles using components derived from natural materials, Korean Patent Registration No. 10-1146104 discloses a skin wrinkle-reducing composition containing ginsenosides Rg₃, Rg₆ and Rg₂ as its main components, and Korean Patent Registration No. 10-0868784 discloses a cosmetic composition for reducing wrinkles, which contains an extract of a culture broth of Rhodotus palmatus. Meanwhile, with respect to methods for blocking UV rays using components derived from natural materials, Korean Patent Registration No. 10-1128591 discloses a cosmetic composition containing, as a UV-blocking component, an extract mixture of red algae such as Porphyra tenera and Gelidium amansii, and Korean Patent Registration No. 10-0630466 discloses a UV-absorbing cosmetic composition containing a green tea extract and a Foeniculum vulgare extract.

However, a method of using afzelin to block UV rays or reduce skin wrinkles as described in the present invention has not yet been disclosed.

Accordingly, the present inventors have made extensive efforts a natural material derived composition for blocking UV rays or reducing skin wrinkles, which is based on a natural material, is harmless to the human body and has excellent safety and stability while having excellent effects. As a result, the present inventors have found that an afzelin compound extracted and purified from Cornus macrophylla, Rosa multiflora, Orostachys japonica and Cedrela Sinensis leaves is very effective in blocking UV rays or reducing skin wrinkles, thereby completing the present invention.

It is an object of the present invention to provide a cosmetic composition for blocking UV rays or reducing skin wrinkles, which contains afzelin as an active ingredient.

Another object of the present invention is to provide a pharmaceutical composition for reducing skin wrinkles, which contains afzelin as an active ingredient.

Still another object of the present invention is to provide a food composition for reducing skin wrinkles, which contains afzelin as an active ingredient.

In one aspect, the present invention provides a composition for blocking UV rays or reducing skin wrinkles, which contains afzelin as an active ingredient.

Afzelin of the present invention is a compound belonging to the group of kaempferol glycosides and has an IUPAC name of 5,7-dihydroxy-2(4-hydroxyphenyl)-3-[(2S,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyloxan-2-yl]oxychromen-4-one. It has a structure represented by the following formula 1:

[Formula 1]

The afzelin can be extracted, separated and purified from Cornus macrophylla, Rosa multiflora, Orostachys japonica and the like. Particularly, it was reported that Cedrela Sinensis leaves harvested in June contain 0.12% or less of afzelin (Journal of The Korean Society of Food and Nutrition, vol. 24, pp.578-581, 1995). It is known that the afzelin has anticancer effects (Phytochemistry, 71(5-6), 641-647, 2010) and a function of protecting the renal cell membrane (Exp Toxicol Pathol, 51(1), 9-14, 1999). In addition, it was reported that the afzelin is a candidate which inhibits the activity of HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A) reductase to ameliorate cardiovascular disease (J Agric Food Chem 27, 58(2), 882-6, 2010).

When the afzelin is extracted from natural materials with a solvent, water, an anhydrous or hydrated lower alcohol having 1 to 4 carbon atoms (methanol, ethanol, propanol, butanol and the like), acetone, ethyl acetate, chloroform and 1,3-butylene glycol are preferably used alone or in a mixture of two or more thereof as the extraction solvent, but are not limited thereto.

Moreover, the afzelin can be obtained by a conventional purification process, in addition to the extraction method employing the extraction solvent. For example, the afzelin can also be obtained from fractions obtained through various additional purification methods, including separation with an ultrafiltration membrane having a given molecular weight cut-off, and separation by various chromatography systems (manufactured for separation according to size, charge, hydrophobicity or affinity).

Examples of the afzelin include, in addition to the compound of formula 1, derivatives obtained by adding substituents to the compound of formula 1 or substituting the compound of formula 1 with substituents, which exhibit the effects of blocking UV rays or reducing skin wrinkles. In other words, the scope of the present invention also encompasses not only the compound of formula 1, but also derivatives having the compound of formula 1 as the nucleus and obtained using various substituents known in the art. For example, the scope of the present invention also includes derivatives formed by linking a hydroxyl, halo, nitro or C1-3 alkyl group to the compound of formula 1, but is not limited thereto.

In the present invention, the afzelin exhibits the effect of reducing skin wrinkles caused by external factors such as an environment and/or internal factors such as heredity, but is not limited thereto.

As used herein, the expression “reducing skin wrinkles” means that skin elasticity increases so that skin wrinkles are reduced or eliminated.

In one specific example, the increase rate of collagen synthesis as a function of the concentration of afzelin was examined using human normal fibroblasts, and as a result, it was found that collagen synthesis increased as the concentration of afzelin increased. This suggests that afzelin synthesizes collagen to exhibit the effect of reducing wrinkles.

In another example, human normal fibroblasts were irradiated with UVA, and collagen synthesis and collagenase activity as a function of the concentration of afzelin were examined. As a result, it was shown that, as the concentration of afzelin increased, collagen synthesis reduced by UVA irradiation increased and collagenase activity that increased by UVA irradiation decreased, suggesting that afzelin has the effect of preventing wrinkles.

In still another example, a cosmetic composition containing afzelin was applied to the human skin and the elasticity of the skin was observed. As a result, it was found the skin elasticity increased as the content of afzelin in the cosmetic composition increased, suggesting that afzelin has the effect of reducing wrinkles.

In still another example, afzelin was administered orally into UV-irradiated hairless mice, and the effect thereof was examined. As a result, it was shown that the group administered with afzelin had the effect of preventing wrinkles.

In the present invention, the afzelin exhibits the effect of blocking UV rays by absorbing UVA and UVB.

In one specific example, the UV absorbing effect of afzelin was measured. As a result, it was shown that afzelin absorbs UVA and UVB, suggesting that afzelin has the effect of blocking UV rays.

In addition, whether afzelin has phototoxicity was examined. As a result, it was shown that afzelin had no phototoxicity even at relatively high concentrations, suggesting that afzelin is not likely to cause toxicity even when it absorbs UV rays, and it is safe.

The afzelin of the present invention is contained in the composition in an amount of 0.00001-15 wt%, preferably 0.0001-10 wt%, and more preferably 0.0001-5 wt%, based on the total weight of the composition. If the content of afzelin in the composition is less than 0.00001 wt%, the effect of blocking UV rays or reducing wrinkles will be insignificant, and if the content of afzelin is more than 15 wt%, an increase in the effect, which results from an increase in the content of afzelin, will be insignificant and the stability of a formulation comprising afzelin will not be ensured.

Meanwhile, the inventive composition comprising afzelin as an active ingredient can be used as a pharmaceutical, cosmetic or food composition.

If the inventive composition is used as a cosmetic composition, it comprises, in addition to afzelin as an active ingredient having the effects of blocking UV rays and reducing wrinkles, components which are conventionally used in cosmetic compositions, for example, conventional additives, such as an antioxidant, a stabilizer, a solubilizing agent, vitamins, a pigment and perfume, and carriers.

The cosmetic composition can be formulated as a solution, a suspension, an emulsion, a paste, a gel, a cream, a lotion, powder, soap, a surfactant-containing cleanser, an oil, a powder foundation, an emulsion foundation, a wax foundation, a spray, a nourishing cream, an astringent lotion, a skin softener, an essence, a nourishing gel or a massage cream, but is not limited thereto.

If the formulation of the cosmetic composition is a paste, a cream or a gel, it may contain, as carrier components, animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide.

If the formulation of the cosmetic composition is a powder or a spray, it may contain, as carrier components, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder. Particularly, if it is spray, it may additionally contain a propellant, such as chlorofluorohydrocarbon, propane/butane or dimethyl ether.

If the formulation of the cosmetic composition is a solution or an emulsion, it may contain, as carrier components, a solvent, a solubilizing agent or an emulsifying agent, for example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan fatty acid ester.

If the formulation of the cosmetic composition is a suspension, it may contain, as carrier components, a liquid diluent, such as water, ethanol or propylene glycol, and a suspending agent, such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tragacanth.

If the formulation of the cosmetic composition is a surfactant-containing cleanser, it may contain, as carrier components, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic monoester, isethionate, imidazolium derivatives, methyl taurate, sarcosinate, fatty acid amide ether sulfate, alkyl amido betaine, aliphatic alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanoline derivatives, or ethoxylated glycerol fatty acid ester.

When the composition of the present invention is used as a pharmaceutical composition, it comprises, in addition to afzelin as an active ingredient having the effects of blocking UV rays and reducing wrinkles, a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier that is contained in the pharmaceutical composition of the present invention is one commonly used in formulations, and examples thereof include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, potassium phosphate, arginate, gelatin, potassium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methyl cellulose, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, and mineral oil. The pharmaceutical composition of the present invention may comprise, in addition to the above-described components, a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative and the like. Suitable pharmaceutically acceptable carriers and preparations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition may be administrated orally or prenterally, preferably orally, and more preferably by topical application.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. As used herein, the term "pharmaceutically effective amount" refers to an amount sufficient to treat or prevent diseases, at a reasonable benefit/risk ratio applicable to any medical treatment or prevention. The effective dosage level of the composition may be determined depending on various factors, including the disease’s type, the disease severity, the activity of the drug, the patient’s age, bodyweight, health, gender, the patient’s sensitivity to the drug, the time of administration of the extract used, the route of administration, the excretion rate of the extract, the duration of treatment, a drug which is combined or used simultaneously with the extract, and other factors known in the medical field. Generally, the afzelin of the present invention can be administered to an adult at a dosage of 0.1-1000 mg/kg/day, preferably 10-100 mg/kg/day, once or several times a day.

The pharmaceutical composition may be formulated using a pharmaceutically acceptable carrier and/or excipient according to a method known to those skilled in the art, so that it can be prepared as a unit dosage form or included in a multi-dosage container. According to the intended therapeutic purpose, the pharmaceutical composition may be formulated into pharmaceutical preparations common in the pharmaceutical field. For example, the formulations include tablets, capsules, powders, granules, suspensions, emulsions, syrups, emulsions in water, plasters, ointments, sprays, oils, gels, spirits, tinctures, baths, liniments, lotions, patches, pads and creams. Topical formulations are preferably used for direct application of the composition to a desired area of the external surface of the skin. Preferred topical formulations include ointments, lotions, sprays and gels. Topical formulations may be contained in a support base or matrix directly applicable to a desired area of the skin. Examples of the support base include gauze or bandages.

When the inventive composition is used as a food composition, it may comprise, in addition to afzelin as an active ingredient having the effects of blocking UV rays and reducing wrinkles, components which are generally added in the preparation of foods. For example, it includes proteins, carbohydrates, fats, nutrients, seasoning agents and flavoring agents. The carbohydrate may be, for example, a monosaccharide, e.g., glucose, fructose, etc., a disaccharide, e.g., maltose, sucrose, oligosaccharide, etc., a polysaccharide, e.g., dextrin, cyclodextrin, etc., or a sugar alcohol such as xylitol, sorbitol, erythritol, etc. The flavor may be a natural flavor (thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)) or a synthetic flavor (saccharin, aspartame, etc.).

There is no particular limit to the kind of food composition. Examples of foods to which afzelin can be added include meats, sausages, bread, chocolates, candies, snacks, confectionery, pizza, noodles, gum, dairy products including ice cream, various soups, beverages, teas, drinks, alcoholic beverages and multi-vitamin preparations. The health foods include all health foods in a conventional sense.

When the health food of the present invention is a health beverage, it may additionally contain various sweetening agents or natural carbohydrates as in conventional beverages. The natural carbohydrates include monosaccharides, such as glucose and fructose, disaccharides, such as maltose and sucrose, natural sweeteners, such as dextrin and cyclodextrin, and synthetic sweeteners, such as saccharin and aspartame. The natural carbohydrates are used in an amount of about 0.01-0.04 g, and preferably about 0.02-0.03 g, based on 100 ml of the health beverage of the present invention.

In addition, the health food of the present invention may contain various nutrients, vitamins, electrolytes, flavoring agents, colorants, pectic acid or its salt, alginic acid or its salt, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated drinks, etc. Additionally, the health food of the present invention may contain fruit flesh for the preparation of natural fruit juices, fruit juice beverages and vegetable juices. These components may be used alone or in combination. Although not critical, these additives are used in an amount of 0.01-0.1 parts by weight based on 100 parts by weight of the health food of the present invention.

When the composition comprising afzelin as an active ingredient is used, it can exhibit the effect of blocking UV rays or reducing skin wrinkles by absorbing UVA and UVB, increasing collagen synthesis and reducing collagenase activity.

In addition, the afzelin of the present invention has little or no toxicity and adverse effects, and thus can be safely used for a long period of time. Accordingly, it can be safely applied to the above-described cosmetic, pharmaceutical and food compositions.

In another aspect, the present invention provides a method of blocking UV absorption into the skin of a subject or reducing skin wrinkles by administering to the subject the above composition for blocking UV rays or reducing skin wrinkles.

As used herein, the term “subject” means mammals, including humans, for example, dogs, pigs, horses and cattle, in which UV rays are to be blocked or skin wrinkles are to be reduced.

As used herein, the term “administration” means introducing a specific substance into a subject by any suitable method. The composition of the present invention may be administered orally or parenterally by any general route, as long as it can reach a target tissue. In addition, the composition of the present invention can be administered by any device which can deliver the active ingredient to a target site, for example, a cell.

From the foregoing and examples described below, it will be obvious to those skilled in the art that the disorders, diseases, abnormalities or skin wrinkles caused by UV rays in a subject can be ameliorated, improved, prevented or treated by administering to the subject the above composition for blocking UV rays or reducing skin wrinkles.

As described above, the inventive composition comprising afzelin as an active ingredient has the effect of blocking UV rays or reducing skin wrinkles by absorbing UVA and UVB, increasing collagen synthesis and reducing collagenase activity.

In addition, the inventive composition comprising afzelin as an active ingredient has no cytotoxicity, causes no adverse effects on the skin, and thus can be safely used as a cosmetic, pharmaceutical or food composition.

FIG. 1 shows the synthesis of collagen as a function of the concentration of afzelin when human normal fibroblasts were irradiated with UVA.

FIG. 2 shows collagenase activity as a function of the concentration of afzelin when human normal fibroblasts were irradiated with UVA.

FIG. 3 shows the degrees of absorption of UVA and UVB by afzelin.

FIG. 4 shows the degree of phototoxicity as a function of the concentration of afzelin when Balb/c 3T3 fibroblasts were irradiated with UVA.

Hereinafter, the present invention will be described in further detail with reference to examples. It is to be understood, however, that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Example 1: Measurement of wrinkle-reducing effect of afzelin

1-1: Preparation of samples

In order to measure the increase in collagen synthesis caused by afzelin, human normal fibroblasts (the Department of Dermatology, Ajou University) were seeded in a 24-well plate containing DMEM medium at a density of about 2 x 10⁵ cells per well, and were then cultured in a 5% CO₂ incubator at 37 ℃ for 24 hours. Then, the medium was removed from each well of the plate, and each well was treated with 10, 50 and 100 μM of afzelin. Then, the cells were incubated for 24 hours, after which the cell culture media were collected, thereby preparing samples. As a control, a sample prepared under the same conditions as above without treatment with afzelin was used.

1-2. Measurement of the increase rate of collagen synthesis caused by afzelin

The amounts of collage synthesis in the samples prepared in Example 1-1 were determined by measuring the amount of procollagen type I C-peptide (PICP) using a procollagen type I C-peptide EIA kit (MK101; Takara, Kyoto, Japan). Table 1 below shows the results of measuring the increase rate of collagen synthesis in the afzelin-treated group compared to the group not treated with afzelin.

Table 1

Test item	Afzelin (10 μM)	Afzelin (50 μM)	Afzelin (100 μM)
Increase rate of collagen synthesis (%)	8	16	21

The results of the test showed that afzelin increased collagen synthesis in human normal fibroblasts. In addition, collagen synthesis increased as the concentration of afzelin increased, suggesting that the increase in collagen synthesis was dependent on the afzelin concentration.

Example 2: Wrinkle-preventing effect of afzelin

2-1: Preparation of samples

In order to measure collagen synthesis induced by afzelin, human normal fibroblasts (the Department of Dermatology, Ajou University) were seeded into a 24-well plate containing DMEM medium at a density of 2 x 10⁵ cells per well, and were then cultured in a 5% CO₂ incubator at 37 ℃ for 24 hours. Then, the cells were pretreated with 10, 50 and 100 μM of afzelin, and after 4 hours, the cells were irradiated with UVA at a dose of 5J using a UV irradiator (Luzchem, Ottawa, Canada). Then, the UVA-irradiated cells were incubated for 24 hours, after which the cell culture media were collected, thereby preparing samples. As a control, a sample prepared under the same conditions as above without treatment with afzelin, and a sample neither treated with afzelin nor irradiated with UVA were used.

2-2: Measurement of collagen synthesis induced by afzelin

The amounts of collagen synthesis in the samples prepared in Example 2-1 were determined by measuring the amount of procollagen type I C-peptide (PICP) using a procollagen type I C-peptide EIA kit (MK101; Takara, Kyoto, Japan). The measured standard values were expressed as mean ± standard deviation, and significance was analyzed by t-test using SPSS/PC+ program. The results of the measurement are shown in FIG. 1.

The results of the test showed that the amount of collagen synthesis which was reduced by UVA was increased by afzelin and that collagen synthesis increased as the concentration of afzelin increased, suggesting that the increase in collagen synthesis was dependent on the concentration of afzelin.

2-3: Measurement of collagenase activity in afzelin-treated samples

Collagenase activity in the samples prepared in Example 2-1 was measured using a type I collagenase assay kit (Amersham Biosciences, RPN2629). The measured standard values were expressed as mean ± standard deviation, and significance was analyzed by t-test using SPSS/PC+ program. The results of the measurement are shown in FIG. 2.

The results of the test indicated that collagenase activity which was increased by UVA was inhibited by afzelin and that collagenase activity was more inhibited as the concentration of afzelin increased, suggesting that the inhibition of collagenase activity was dependent on the concentration of afzelin.

Example 3: Measurement of wrinkle-reducing effect of afzelin-containing cosmetic composition

3-1: Preparation of nourishing cream containing afzelin

In order to measure the wrinkle-reducing effect of afzelin by a clinical test, a nourishing cream having the components and contents shown in Table 2 below was prepared. Specifically, an aqueous phase including purified water, triethanolamine and propylene glycol was dissolved by heating to 70 ℃. And an oily phase including a fatty acid, oily components, an emulsifier and a preservative was dissolved by heating to 70 ℃ and added to the aqueous phase. Then, the resulting solution was cooled to 45 ℃, and afzelin was added in amounts of 0.01, 0.05 and 1 wt% based on the total weight of the composition and was dispersed, followed by cooling to 30 ℃. As a control, a nourishing cream containing purified water in place of afzelin was used.

Table 2

Component	Content(wt%)
Afzelin	0.01, 0.05 or 1
Jojoba oil	5.0
Liquid paraffin	7.0
Cetylaryl alcohol	2.0
Polyglyceryl-3 methyl glucose distearate	2.0
Glyceryl stearate	0.5
Squalane	3.0
Propylene glycol	4.0
Glycerin	5.0
Triethanolamine	0.3
Carboxyvinyl polymer	0.3
Tocopheryl acetate	0.2
Preservative and fragrance	Trace
Purified water	Balance
Total
	100

3-2. Measurement of wrinkle-reducing effect of nourishing cream containing afzelin

Each of the nourishing creams containing 0.01, 0.05 and 1 wt % of afzelin, prepared in Example 3-1, and a nourishing cream containing no afzelin, was applied to the face of each of 30 healthy women (21-42 years old) twice a day for 3 months. The effect on wrinkle reduction was evaluated by measuring a change in skin elasticity. The measurement of skin elasticity was performed with Cutometer SEM 474 (Courage+Khazaka, Cologne, Germany) under the conditions of constant temperature of 24 to 26 ℃ and constant humidity of 38-40%. The evaluation criteria were scaled from 0 (no skin elasticity) to 5 (highest skin elasticity), and the results of comparison of the relative values are shown in Table 3 below.

Table 3

Afzelin (wt%)	0	0.01	0.05	1
Skin elasticity	2.64	2.75	2.90	3.21

The results of the test indicated that the use of the nourishing creams containing afzelin increased skin elasticity compared to the use of the nourishing cream containing no afzelin and that the skin elasticity increased as the concentration of afzelin increased, suggesting that the increase in skin elasticity was dependent on the concentration of afzelin.

Example 4: Effect of wrinkle prevention by oral administration

In order to measure the wrinkle-preventing effect of afzelin, 6-7-week-old hairless mice (Skh: HR-1) were divided into a control group, a UV group and a UV/afzelin group, each consisting of 8 animals, and were bred during the test period. The control group and the UV group were administered orally with 0.5 ml of saline, and the UV/afzelin group was administered orally with a solution of 500 mg/kg afzelin (on a solid basis) in 0.5 ml of saline.

The above-prepared samples were administered orally at the same point of time for 5 days a week for a total of 5 weeks. From 2 weeks to 5 weeks after oral administration, the UV group and the UV/afzelin group were irradiated with UV light three times a week such that the total dose of UV radiation reached 600 mJ/cm².

To measure the wrinkle-preventing effect of afzelin, skin replicas were taken from the back of the hairless mice using a silicon polymer before autopsy. In the same manner, after 5 weeks, skin replicas were taken from the groups treated with each of the samples. Using the taken skin replicas, the antiaging effect (i.e., wrinkle-reducing effect) of each sample was measured, and the results of the measurement are shown in Table 4 below.

Table 4

R-parameter	Control group	UV group	UV/afzelin group
R1 value	0.43+0.0120	0.53+0.0130	0.45+0.0151
R2 value	0.32+0.0111	0.38+0.0170	0.33+0.0157
R3 value	0.23+0.0035	0.28+0.0037	0.22+0.0098
R4 value	0.09+0.0020	0.17+0.0040	0.08+0.0044
R5 value	0.22+0.0132	0.31+0.0120	0.24+0.0115

R1 value: Skin roughness, R2 value: Maximum roughness, R3 value: Average roughness, R4 value: Smoothness depth, R5 value: Arithmetic average roughness (International Journal of Cosmetic Science, 2005 Jun; 27(3):155-60).

The results of the test indicated that, when the hairless mice were irradiated with UV light, the R1, R2, R3, R4 and R5 values indicative of wrinkles all increased. However, it was observed that, when afzelin was administered, the R1, R2, R3, R4 and R5 values which were increased by UV irradiation were all reduced to values close to the normal levels.

Example 5: Measurement of UV-absorbing effect of afzelin

In order to measure the UV-absorbing effect of afzelin, the UV absorbance of afzelin was measured using a UV/VIS spectrophotometer in the UV wavelength range of 280-400 nm. The results of the measurement are shown in FIG. 3.

The results of the test indicated that afzelin absorbed both UVA and UVB and the maximum absorption wavelength was 346 nm.

Example 6: Measurement of whether afzelin has phototoxicity

In order to measure whether afzelin has phototoxicity, Balb/c 3T3 fibroblasts were seeded into a 24-well plate at a density of 2 x 10⁵ cells per well, and then cultured in a 5% CO₂ incubator at 37 ℃ for 24 hours. Then, the cells were pretreated with 2.5, 5, 10, 20, 40, 100 and 200 μg/mL of afzelin. After 4 hours, the cells were irradiated with UVA at a dose of 5 J using a UV irradiator (Luzchem, Ottawa, Canada). Then, the UVA-irradiated cells were washed with PBS and further incubated for 24 hours.

The incubated cells were washed, and neutral red solution was added thereto. Then, the cells were incubated in a 5% CO₂ incubator at 37 ℃ for 3 hours, after which the medium was removed and the cells were washed with PBS. Next, 500 ㎕ of an extraction solution consisting of 1% acetic acid and 50% ethyl alcohol was added to each well to extract the neutral red from the cells, and then the absorbance at 540 nm was measured using a spectrophotometer. The results of the measurement are shown in FIG. 4. As a negative control, a sample treated under the same conditions as above without irradiation with UVA was used, and as a positive control, a sample treated with chlorpromazine (CPZ) in place of afzelin was used.

Phototoxicity was evaluated by calculating the PIF (photo irritation factor) using the following equation 1, and when the PIF value was 5 or less, it was determined that there was no possibility of inducing phototoxicity. In addition, when the test sample had no toxicity so that the IC₅₀ value could not be determined, the PIF value appeared to be 1 and the test sample had no possibility of inducing phototoxicity.

[Equation 1]

PIF (photo irritation factor) = IC₅₀(-UVA)/ IC₅₀(+UVA)

(IC₅₀ = concentration at which cells are inhibited by 50%)

The results of the test showed that afzelin had no toxicity so that the IC₅₀ value could not be determined in both the group irradiated with UVA and the group not irradiated with UVA, even when it was used at relatively high concentrations. In addition, the PIF value was 1, suggesting that afzelin has no phototoxicity. However, when chlorpromazine as the comparative group was used, it was shown that the PIF value was 30.23, suggesting that chlorpromazine has phototoxicity.

Example 7: Test for confirming the safety of afzelin for human skin

7-1: Preparation of skin external formulation containing afzelin

In order to confirm whether afzelin is safe for use on human skin, an afzelin-containing skin external formulation having the components and contents shown in Table 5 below was prepared, and then a skin safety verification test was carried out. Specifically, purified water, glycerin, and butylene glycol were mixed and dissolved at a temperature of 70 ℃ (aqueous phase). The remaining components except for the above three components and trimethanolamine were dissolved at a temperature of 70 ℃ (oil phase). The oil phase was added to the aqueous phase, and stirred with a homomixer (Tokushu Kika, Japan) to prepare an emulsion, and trimethanolamine was added thereto. Then, bubbles produced in the mixture were removed, after which the mixture cooled to room temperature, thereby preparing skin external formulations.

Table 5

Component	Content (wt%)
Component	Control	Test group	Comparative group
Purified water	72	71	71
Glycerin	8.0	8.0	8.0
Butylene glycol	4.0	4.0	4.0
Afzelin	0	1.0	0
Vitamin C	0	0	1.0
Caprylic/capric triglyceride	8.0	8.0	8.0
Squalane	5.0	5.0	5.0
Cetearyl glucoside	1.5	1.5	1.5
Sorbitan stearate	0.4	0.4	0.4
Cetearyl alcohol	1.0	1.0	1.0
Trimethanolamine	0.1	0.1	0.1
Total	100	100	100

7-2: Cumulative irritation test

Each of the skin external formulations prepared in Example 7-1 was applied every other day to the forearms of 30 healthy adults and allowed to stand for 24 hours, and this was repeated so that each subject was treated with 9 fresh patches in total, so as to confirm whether afzelin irritates the skin. As a test group, a skin external formulation containing 1 wt% of afzelin based on the total weight of the skin external formulation was used, and as a comparative group, a skin external formulation containing 1 wt% of vitamin C based on the total weight of the skin external formulation was used.

The patch test was performed using a Finn chamber (Epitest Ltd, Finland). The external formulations were added dropwise to the chamber in an amount of 15 ul per patch. At every round of the patch application, the response of the skin was scored using the following equation 2. The results are shown in Table 6 below.

[Equation 2]

Average response degree = [[Response index x Response degree / Total number of subjects x Highest score (4 points)] x 100] / Number of examinations

In regard to the response degree, 1 point was provided for ±, 2 points for +, and 4 points for ++. When the average response degree was less than 3, the composition was determined to be safe for use on the skin.

Table 6

The results of the test showed that, in the control group, the subjects corresponding to ±, +, and ++ numbered 1, 0 and 0, respectively, in the first patch, and no skin response appeared after the second patch. In the test group to which afzelin was applied, the subjects corresponding to ±, +, and ++ numbered 2, 0 and 0, respectively, in the first patch, and no skin response appeared after the second patch. In the case of the comparative group containing vitamin C, no skin response appeared. In addition, the average response degree was calculated to be 0.09 for the control group and 0.19 for the test group, which are all less than 3, suggesting that the composition of the present invention is safe for use on human skin.

Preparation Example 1: Preparation of cosmetic formulations

1-1: Preparation of skin softener

Using the components and contents shown in Table 7 below, a skin softener containing afzelin as an active ingredient was prepared according to a conventional method.

Table 7

Component	Content (wt%)
Afzelin	0.01
Glycerin	3.0
Butylene glycol	2.0
Propylene glycol	2.0
Carboxyvinyl polymer	0.1
Ethanol	10.0
Triethanolamine	0.1
Preservative, pigment, fragrance, and purified water	Balance
Total	100.0

1-2: Preparation of nourishing softener

Using the components and contents shown in Table 8 below, a nourishing softener containing afzelin as an active ingredient was prepared according to a conventional method.

Table 8

Component	Content (wt%)
Afzelin	0.01
Beeswax	4.0
Polysorbate 60	1.5
Sorbitan sesquioleate	0.5
Liquid paraffin	5.0
Squalane	5.0
Caprylic/capric triglyceride	5.0
Glycerin	3.0
Butylene glycol	3.0
Propylene glycol	3.0
Carboxyvinyl polymer	0.1
Triethanolamine	0.2
Preservative, pigment, fragrance, and purified water	Balance
Total	100.0

1-3: Preparation of nourishing cream

Using the components and contents shown in Table 9 below, a nourishing cream containing afzelin as an active ingredient was prepared according to a conventional method.

Table 9

Component	Content (wt%)
Afzelin	0.01
Beeswax	10.0
Polysorbate 60	1.5
Sorbitan sesquioleate	0.5
Liquid paraffin	10.0
Squalane	5.0
Caprylic/capric triglyceride	5.0
Glycerin	5.0
Butylene glycol	3.0
Propylene glycol	3.0
Triethanolamine	0.2
Preservative, pigment, fragrance, and purified water	Balance
Total	100.0

1-4: Preparation of massage cream

Using the components and contents shown in Table 10 below, a massage cream containing afzelin as an active ingredient was prepared according to a conventional method.

Table 10

Component	Content (wt%)
Afzelin	0.01
Beeswax	10.0
Polysorbate 60	1.5
Sorbitan sesquioleate	0.8
Liquid paraffin	40.0
Squalane	5.0
Caprylic/capric triglyceride	4.0
Glycerin	5.0
Butylene glycol	3.0
Propylene glycol	3.0
Triethanolamine	0.2
Preservative, pigment, fragrance, and purified water	Balance
Total	100.0

1-5: Preparation of pack

Using the components and contents shown in Table 11 below, a pack containing afzelin as an active ingredient was prepared according to a conventional method.

Table 11

Component	Content (wt%)
Afzelin	0.01
Polyvinyl alcohol	13.0
Sodium carboxymethyl cellulose	0.2
Allantoin	0.1
Ethanol	5.0
Nonyl phenyl ether	0.3
Preservative, pigment, fragrance, and purified water	Balance
Total	100.0

Preparation Example 2: Preparation of pharmaceutical formulations

2-1: Preparation of powder formulation

The components shown in Table 12 below were mixed with each other and then filled in a sealed bag, thereby preparing a powder formulation containing afzelin as an active ingredient.

Table 12

Component	Content (g)
Afzelin	2
Lactose	1

2-2: Preparation of tablet formulation

The components shown in Table 13 below were mixed with each other and then compressed to a tablet according to a conventional method, thereby preparing a tablet formulation containing afzelin as an active ingredient.

Table 13

Component	Content (mg)
Afzelin	100
Corn starch	100
Lactose	100
Magnesium stearate	2

2-3: Preparation of capsule formulation

The components shown in Table 14 below were mixed with each other and then filled into a gelatin capsule according to a conventional method, thereby preparing a capsule formulation containing afzelin as an active

Table 14

Claims

A cosmetic composition for blocking UV rays or reducing skin wrinkles, which contains afzelin as an active ingredient.
The cosmetic composition of claim 1, wherein the content of afzelin is 0.00001-15 wt% based on the total weight of the composition.
The cosmetic composition of claim 1, wherein the cosmetic composition has a formulation selected from the group consisting of a solution, a suspension, an emulsion, a paste, a gel, a cream, a lotion, a powder, a soap, a surfactant-containing cleanser, an oil, a powder foundation, an emulsion foundation, a wax foundation, a spray, a nourishing cream, an astringent lotion, a skin softener, an essence, a nourishing gel and a massage cream.
A pharmaceutical composition for reducing skin wrinkles, which contains afzelin as an active ingredient.
A food composition for reducing skin wrinkles, which contains afzelin as an active ingredient.