KR102582367B1 - A cosmetic composition for skin whitening comprising Illicium Verum Fruit extracts, fractions thereof or compounds isolated therefrom as an active ingredient - Google Patents

Abstract

The present invention relates to a whitening cosmetic composition comprising star anise fennel extract, a fraction thereof, or a compound isolated therefrom as an active ingredient; Quasi-drug composition; Pharmaceutical compositions for preventing or treating skin pigmentation diseases; and a health functional food composition for preventing or alleviating skin pigmentation diseases.

Description

A cosmetic composition for skin whitening comprising Illicium Verum Fruit extracts, fractions thereof or compounds isolated therefrom as an active ingredient}

The present invention relates to a whitening cosmetic composition comprising star anise fennel extract, a fraction thereof, or a compound isolated therefrom as an active ingredient; Quasi-drug composition; Pharmaceutical compositions for preventing or treating skin pigmentation diseases; and a health functional food composition for preventing or alleviating skin pigmentation diseases.

The chemical action of melanin production, which determines skin color, has been reported in many papers to date. Melanocytes, melanin-producing cells, protect cells from ultraviolet rays within an intracellular organelle called melanosome. After synthesizing melanin, which has a protective function, it is known that the melanin moves from melanocytes to keratinocytes, and it is reported that the melanin moved to keratinocytes determines skin color.

It is known that the precursor required in the melanin biosynthesis process is an amino acid called tyrosine, which is converted to dihydroxyphenylalanine (DOPA), which is then converted to melanin through a substance called dopaquinone. .

In this regard, melanin-forming enzymes include tyrosinase (TYR), tyrosinase-related protein 1 (TRP-1), and tyrosinase-related protein 2 (TRP-2/DOPA, chrome). tautomerase), etc. are known, and the melanin-forming enzyme is known to be regulated by a transcription factor called MITF (microphthalmia-associated transcription factor). Melanin synthesized by tyrosinase darkens skin color, and due to health or beauty requirements, research is actively underway on skin whitening methods to brighten skin color by suppressing melanin synthesis.

Commonly known whitening ingredients include arbutin, kojic acid, azelaic acid, aloesin, 4-butylresorcinol, resveratrol, ceramide, sphingosine-1-phosphate, and sphingosylphosphorylcholine, which inhibit tyrosinase enzyme activity. There are substances such as hydroquinone (Korea Patent Publication No. 2016-0014271). However, some whitening ingredients have limitations in the amount of use due to safety issues such as irritation and redness when applied to the skin, or the effect is so small that no practical effect can be expected. In particular, in the case of hydroquinone, its use was banned due to unwanted side effects such as irreversible bleaching (European Committee 24th Dir 2000/6/EC), and it was replaced by arbutin, a structural analog of hydroquinone. As such, in the field of whitening, it is particularly important not only to have a whitening effect by having a tyrosinase activity inhibitory effect but also to ensure stability, and the development of a material that satisfies all of the above requirements is still required.

Recently, along with changes in lifestyle and interest in well-being, the demand for well-being is gradually increasing. Accordingly, consumer interest in the skin care field is increasing, and various related skin whitening products are being developed, but actual consumer confidence in their efficacy is not very high. In addition, with the recent naturalism trend, preference for products made from natural ingredients is increasing, and the cosmetics market based on technology using natural ingredients is rapidly increasing. Manufacturing cosmetics using natural ingredients has the advantage of increasing the absorption rate and safety of the active ingredients contained in natural ingredients.

Illicium Verum (Anise) Fruit is native to China's Guangdong Province, Guangxi Province, Fujian Province, Hainan Province, India, and Vietnam, and is a member of the Illiciaceae family. It is a fruit, one of the unfamiliar spices in Korea, and a type of Chinese spice. In China and Korea, it is called star anise or star anise, and in English it is called star anise.

Against this background, the inventors of the present invention have made diligent efforts to develop a material for cosmetics with skin whitening function that can substantially help skin whitening efficacy while ensuring safety when applied to the skin using natural ingredients. , it was discovered that star anise fennel extract, fractions thereof, or compounds isolated therefrom had excellent antioxidant efficacy and tyrosinase activity inhibition ability and thus had excellent whitening effect, and by confirming that it could be used for skin whitening purposes, the present invention was completed. .

One object of the present invention is to provide a cosmetic composition for skin whitening containing star anise fennel extract, a fraction thereof, or a compound isolated therefrom.

Another object of the present invention is to provide a quasi-drug composition for skin whitening containing star anise fennel extract, a fraction thereof, or a compound isolated therefrom.

Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of skin pigmentation disease containing star anise extract, a fraction thereof, or a compound isolated therefrom.

Another object of the present invention is to provide a health functional food composition for preventing or alleviating skin pigmentation disease containing star anise fennel extract, fractions thereof, or compounds isolated therefrom.

This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in the present invention may also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. Additionally, the scope of the present invention cannot be considered to be limited by the specific description described below.

One aspect of the present invention for achieving the above object provides a cosmetic composition for skin whitening comprising star anise fennel extract, a fraction thereof, or a compound isolated therefrom.

The present invention discovered that an extract extracted from star aniseed fennel, a natural material, through high temperature and pressure is safe when applied to the skin, has excellent antioxidant ability, and has an excellent tyrosinase activity inhibition ability and has a whitening effect, and sought to utilize it in a composition for skin whitening. It is based on technical ideas.

In the present invention, the term "Illicium Verum (Anise) Fruit" belongs to the Illiciaceae family and is the dried fruit. In China and Korea, it is called star anise or star anise, and in English it is called star anise. It is a fruit that is usually composed of 8 pods arranged radially from the center, and each pod is 1 to 2 cm in length, 0.3 to 0.5 cm in width, and 0.6 to 1 cm in height. The outer surface is reddish brown with irregular wrinkles, the upper end is shaped like a bird's beak and the upper part is mostly open, and the inner surface is light brown, smooth and shiny.

The ingredients consist of essential oils, sesquiterpenes, phenylpropanoids, lignans, etc. In particular, the essential oil ingredients include trans-anethole, anisaldehyde, Limonene, estragole, anisyl acetate, feniculin, α-trans-bergamotene (Ghasemi Z, Hassanpour-Ezatti M, Kamalinejas M, Janahmadi M. Fitoterapia. 2011, 82, 750-756.), etc. are known to be included. Among them, trans-anethole is used as an antioxidant, anti-inflammatory treatment, anti-carcinogenic treatment, and anesthetic.

In the present invention, the star anise fennel can be purchased and used commercially, or collected or cultivated in nature, but is not limited thereto.

In the present invention, the term "extract" refers to a result such as a liquid component obtained by immersing the desired material in various solvents and then extracting it at room temperature, low temperature, or heated state for a certain period of time, and a solid component obtained by removing the solvent from the liquid component. means. In addition, it can be comprehensively interpreted to include all diluted solutions of the results, concentrated solutions thereof, dried products obtained by drying the extracts, or their crude products, purified products, etc.

The method for extracting the extract is not particularly limited, and may be extracted according to a method commonly used in the art. Non-limiting examples of the extraction method include solvent extraction, ultrasonic extraction, filtration, and reflux extraction, which may be performed alone or by combining two or more methods.

Additionally, the type of extraction solvent used for extraction is not particularly limited, and any solvent known in the art can be used. Non-limiting examples of the extraction solvent include water, alcohols having 1 to 4 carbon atoms, or mixed solvents thereof, and these may be used alone or in combination of one or more types.

In the present invention, the extract may be a star anise extract, may be obtained through solvent extraction or carbon dioxide supercritical extraction (SC-CO2 Gas Extraction System), and may be extracted under pressurized conditions. The “pressurization” means applying pressure, and in the present invention, the pressure may be 100 bar to 200 bar, but is not limited thereto.

In the present invention, the solvent extraction may specifically mean accelerated solvent extraction.

The extract may be prepared and used in dry powder form after extraction, but is not limited thereto.

In one embodiment of the present invention, a total of eight types of octagonal fennel extract were prepared through the above extraction method, specifically, n-hexane, dichloromethane, acetone, ethyl acetate, 50% ethanol, 100% ethanol, and water were extracted. Seven types of extracts were prepared by accelerated solvent extraction and one type of extract by supercritical extraction with carbon dioxide (Figure 1).

In addition, in one embodiment of the present invention, a 50% ethanol extract was finally determined through evaluation of the yield and tyrosinase inhibition of the eight extracts by solvent (Figure 2), and then the dried powder of the extract was suspended in anhydrous ethanol and As a result of measuring the tyrosinase activity of the centrifuged supernatant in a concentration range of 0.01 to 0.1 mg/g, it was confirmed that the IC50 value was 52.389% at a concentration of 0.01 mg/g (FIG. 3).

In addition, in one embodiment of the present invention, through evaluation of the DPPH radical scavenging activity of the eight types of extracts, it was confirmed that the 50% ethanol extract showed the best activity (Figure 4), and then the dried powder of the extract The supernatant was suspended in anhydrous ethanol and centrifuged to confirm the DPPH radical scavenging activity in the concentration range of 10 to 100 mg/g. As a result, at a high concentration of 40 mg/g or more, it showed a similar level of DPPH radical scavenging activity as the positive control. It was confirmed that the IC50 value was 54.15% at a concentration of 20.0 mg/g compared to the positive control vitamin C aqueous solution (10 mg/ml) (FIG. 5).

In addition, in one embodiment of the present invention, as a result of confirming the cytotoxicity of the 50% ethanol extract of star anise fennel, a cell viability of more than 100% was confirmed in the entire concentration range of 0.01 to 20 mg/ml (FIG. 6).

In the present invention, the star anise fennel extract can be prepared and used as a fraction thereof.

In the present invention, the term “fraction” refers to the result obtained by performing fractionation to separate a specific component or specific component group from a mixture containing various components.

In the present invention, the fractionation method for obtaining the fraction is not particularly limited and may be performed according to a method commonly used in the art. Solvent fractionation performed by treatment with various solvents, ultrafiltration fractionation performed by passing ultrafiltration membranes with a certain molecular weight cut-off value, and various chromatographs (designed for separation based on size, charge, hydrophobicity, or affinity). ), a chromatographic fractionation method, and a combination thereof. In the present invention, the type of solvent used to obtain the fraction is not particularly limited, and any solvent known in the art can be used. Non-limiting examples of the fractionating solvent may include water, organic solvents, or mixed solvents thereof, and the organic solvents include alcohols having 1 to 4 carbon atoms, polar solvents such as ethyl acetate or acetone, hexane, or dichloromethane. A non-polar solvent of methane or a mixed solvent thereof can be used. Additionally, specifically, water, alcohol with 1 to 4 carbon atoms, or a mixed solvent thereof can be used, and more specifically, ethanol can be used.

In the present invention, the fraction of the star anise fennel extract may be a fraction of the 50% ethanol extract of the star anise fennel.

In one embodiment of the present invention, the extract was subdivided into 10 small fractions by polarity using MPLC (Isolera, Biotage, US), and Cosmosil C18 resin was loaded on a separation cartridge to perform gradient elution of water and MeOH (Figure 8).

In the present invention, the star anise extract and its fractions may include compounds isolated therefrom.

In one embodiment of the present invention, compounds 1 and 2 were separated from the octagonal fennel extract and its fractions using various columns (FIG. 9), and the compounds were verimol B and trans anethole, respectively. -anethole) (Figure 10).

In the present invention, the term "whitening" refers to a method of increasing the brightness of skin whose brightness has been reduced due to excessive pigment such as melanin or maintaining the brightness of the skin at a certain level, skin with increased brightness formed by the above method, etc. It is meant to encompass, and may specifically mean skin whitening. The "skin whitening" can be understood as a result of inhibition of tyrosinase activity. Specifically, as tyrosinase activity is inhibited, the production of melanin is inhibited, and it can be understood as an improvement in symptoms resulting from an increase in melanin, such as spots and freckles. .

In one embodiment of the present invention, as a result of measuring the tyrosinase inhibitory activity of isolated compounds 1 and 2, the tyrosinase inhibitory activity of compounds 1 and 2 compared to the positive control group (50 mg/ml arbutin) was each at a concentration of 1 mg/ml. It was confirmed that it had excellent tyrosinase inhibitory activity at 54.029% and 59.243% (Figure 11).

The cosmetic composition for skin whitening of the present invention may contain compounds known to have a skin whitening effect or natural extracts to increase or reinforce the skin whitening effect, in addition to the active ingredient star anise extract, fractions thereof, or compounds isolated therefrom. there is. Here, compounds or natural extracts known to have a whitening effect include mercaptosuccinic acid, mercaptodextran, teprenone, dihydroxy-isoquinoline, indomethacin, 3-hydroxymanul, vitamin K, thiazolidone, and quinu. Examples include, but are not limited to, rennin, lemon extract, cucumber extract, mulberry extract, rosemary extract, acerola cherry extract, ginkgo extract, and geranium extract.

The cosmetic composition of the present invention may contain 0.0001 to 50% by weight (w/w) of star anise extract, fractions thereof, or compounds isolated therefrom, based on the total weight of the composition, and specifically, 0.001 to 5% by weight (w/w). w), but is not particularly limited thereto.

In the present invention, if the star anise extract, its fraction, or compound isolated therefrom is less than 0.0001% by weight based on the total weight of the composition, it may not be desirable because the originally intended activity cannot be sufficiently achieved, and if it exceeds 15% by weight, it may not be desirable. In this case, it may be uneconomical because the effect is not expected to increase as clearly as the content increases.

The cosmetic composition includes softening lotion, astringent lotion, nourishing lotion, nourishing cream, massage cream, essence, pack, skin adhesive patch, skin adhesive gel, powder, ointment, paste, gel, suspension, emulsion, spray, serum, or capsule. It may be formulated as, but is not particularly limited thereto.

The cosmetic composition of the present invention may additionally include one or more cosmetically acceptable carriers that are blended with general skin cosmetics, and common ingredients include, for example, oil, water, surfactant, moisturizer, lower alcohol, and thickener. , chelating agents, pigments, preservatives, fragrances, etc. may be appropriately mixed, but are not limited thereto. Cosmetically acceptable carriers included in the cosmetic composition of the present invention vary depending on the formulation.

When the formulation of the present invention is an ointment, paste, cream or gel, the carrier ingredient may be animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide or Mixtures of these can be used.

When the formulation of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silcate, polyamide powder, or mixtures thereof may be used as the carrier ingredient, and especially in the case of a spray, chloro May contain propellants such as fluorohydrocarbons, propane/butane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, solubilizing agent or emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-Butylglycol oil may be used, in particular cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol aliphatic esters, polyethylene glycol or fatty acid esters of sorbitan. there is.

When the formulation of the present invention is a suspension, the carrier ingredients include water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, and polyoxyethylene sorbitan ester, and miso. Crystalline cellulose, aluminum metahydroxide, bentonite, agar, or tracant may be used.

On the other hand, when the formulation of the present invention is a capsule, it may be formulated in the form of an alginate capsule, agar capsule, gelatin capsule, wax capsule, or double capsule. It is not limited to this.

In addition, the cosmetic composition of the present invention contains, in addition to star anise extract, fractions thereof, or compounds isolated therefrom, commonly used auxiliaries such as hydrophilic or lipophilic gelling agents, hydrophilic or lipophilic activators, preservatives, antioxidants, solvents, fragrances, It may contain fillers, blocking agents, pigments, odorants and dyes.

Another aspect of the present invention for achieving the above object provides a quasi-drug composition for skin whitening comprising star anise fennel extract, a fraction thereof, or a compound isolated therefrom.

The terms “star anise”, “extract”, “fraction”, “compound” and “skin whitening” are as defined above.

The whitening quasi-drug composition may have the function of helping to lighten the color of melanin pigment deposited on the skin, and prevents excessive deposition of melanin pigment on the skin by inhibiting tyrosinase activity, thereby suppressing the occurrence of skin pigmentation disease. By doing so, it can have a function that helps whiten the skin.

In addition to the above ingredients, the quasi-drug composition of the present invention may further include pharmaceutically acceptable carriers, excipients, or diluents as needed. The pharmaceutically acceptable carrier, excipient, or diluent is not limited as long as it does not impair the effect of the present invention, and includes, for example, fillers, extenders, binders, wetting agents, disintegrants, surfactants, lubricants, sweeteners, fragrances, preservatives, etc. It can be included.

The quasi-drug composition of the present invention may include, but is not limited to, disinfectant cleaner, shower foam, ointment, wet tissue, coating agent, etc., and the formulation method, dosage, usage method, and components of the quasi-drug are commonly known in the technical field. It can be appropriately selected from the techniques of.

Another aspect of the present invention for achieving the above object provides a pharmaceutical composition for the prevention or treatment of skin pigmentation disease, comprising an octagonal fennel extract, a fraction thereof, or a compound isolated therefrom.

The terms “star anise”, “extract”, “fraction” and “compound” are as defined above.

The term "skin pigmentation disease" in this proclamation may mean a disease that occurs locally on the skin due to increased synthesis of melanin pigment, specifically, freckles, senile spots, liver spots, melasma, brown or black spots, and solar pigment spots. It may be one or more diseases selected from the group consisting of cyanic melasma, hyperpigmentation after drug use, gravidic chloasma, hyperpigmentation due to wounds or dermatitis, including scratches and burns, but Not limited.

The term “prevention” of the present invention may refer to any action that suppresses or delays the onset of skin pigmentation disease by administering the pharmaceutical composition according to the present invention.

As used herein, the term “treatment” may refer to any action in which the symptoms of an individual suspected of or affected by skin pigmentation are improved or beneficially changed by administration of the pharmaceutical composition.

The pharmaceutical composition of the present invention can be administered orally or parenterally during clinical administration, but is usually administered topically directly to the skin in the form of an external formulation.

The pharmaceutical composition of the present invention may include the star anise extract, its fraction, or a compound isolated therefrom in an amount of 0.0001 to 50% by weight (w/w) based on the total weight of the composition, specifically 0.001. It may contain from 5% by weight (w/w), but is not limited thereto.

In addition, the pharmaceutical composition may further include a pharmaceutically acceptable carrier, excipient, or diluent commonly used in the preparation of the pharmaceutical composition, and the carrier may include a non-naturally occurring carrier. there is. The carriers, excipients, and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, and microcrystalline. Examples include cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

In addition, the pharmaceutical composition can be manufactured into tablets, pills, powders, granules, capsules, suspensions, oral solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and transdermal preparations according to conventional methods. It can be formulated and used in the form of an absorbent, gel, lotion, ointment, cream, patch, cataplasma, paste, spray, skin emulsion, skin suspension, transdermal delivery patch, drug-containing bandage, or suppository. Specifically, when formulating, it may be prepared using commonly used diluents or excipients such as fillers, weighting agents, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include, but are not limited to, tablets, pills, powders, granules, capsules, etc. These solid preparations may be prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose, lactose, gelatin, etc. Additionally, in addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. In addition to oral liquid and liquid paraffin, it can be prepared by adding various excipients, such as wetting agents, sweeteners, fragrances, and preservatives. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, wethepsol, macrogol, Tween 61, cacao, laurin, glycerogelatin, etc. can be used.

Another aspect of the present invention for achieving the above object is skin pigmentation disease comprising administering a pharmaceutical composition containing star anise extract, a fraction thereof, or a compound isolated therefrom to an entity other than a human. Provides prevention or treatment methods.

The terms "star anise", "extract", "fraction", "compound", "skin pigmentation disease", "prevention" and "treatment" are as described above.

In the present invention, the term “individual” may refer to any animal, including humans, that has or is affected by the skin pigmentation disease of the present invention. By administering the pharmaceutical composition of the present invention to an individual, prevention and treatment effects of skin pigmentation disease can be obtained.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount.

In the present invention, the term “administration” refers to introducing the pharmaceutical composition of the present invention into a subject by any appropriate method, and the administration route may be oral or parenteral, as long as it can reach the target tissue.

The pharmaceutical composition can be appropriately administered to the subject according to the purpose or need according to the conventional method, administration route, and dosage used in the art. Examples of routes of administration include oral, parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal administration; parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In addition, the appropriate dosage and number of administrations may be selected according to methods known in the art, and the amount and number of administrations of the pharmaceutical composition of the present invention actually administered may vary depending on the type of symptom to be treated, route of administration, gender, health condition, It can be appropriately determined by various factors such as diet, age and weight of the individual, and severity of the disease.

The term "pharmaceutically effective amount" means an amount sufficient to inhibit or alleviate increased vascular permeability with a reasonable benefit/risk ratio applicable to medical use, and the effective dose level is determined by the type and severity of the subject, age, gender, It can be determined based on factors including the activity of the drug, sensitivity to the drug, administration time, route of administration and excretion rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field. For example, the star anise extract, fractions thereof, or compounds isolated therefrom can be administered at a dose of 0.01 to 500 mg/kg per day, specifically 10 to 100 mg/kg, and the administration is done once a day. Can be administered once or in divided doses

The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. And it can be administered single or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve maximum effect with the minimum amount without side effects, and can be easily determined by a person skilled in the art.

The composition of the present invention can be used alone to prevent or treat skin pigmentation, and can be used in combination with methods using surgery, hormone therapy, drug therapy, and biological response regulators.

Another aspect of the present invention for achieving the above object provides a health functional food composition for preventing or alleviating skin pigmentation disease, comprising star anise fennel extract, a fraction thereof, or a compound isolated therefrom.

The terms "star anise", "extract", "fraction", "compound", "skin pigmentation disease" and "prevention" are as described above.

The star anise fennel extract, fractions thereof, or compounds isolated therefrom according to the present invention exhibit excellent tyrosinase inhibitory activity, so they can be included in food compositions for the purpose of preventing or alleviating skin pigmentation diseases, and the food compositions are consumed on a daily basis. Since it is possible, high effectiveness can be expected in preventing or alleviating skin diseases caused by pigmentation.

In the present invention, the term “alleviation” may mean suppressing a non-specific systemic syndrome by reducing the biological response caused by an injurious stimulus above a certain level.

The term "health functional food" in the present invention refers to food manufactured and processed using raw materials or ingredients with functionality useful to the human body in accordance with Act No. 6727 on Health Functional Food, and "functionality" refers to the structure of the human body. It means controlling nutrients for functions or obtaining useful effects for health purposes such as physiological effects. Meanwhile, health food refers to food that has a more active health maintenance or promotion effect compared to general food, and health supplement refers to food for health supplement purposes. In some cases, the terms health functional food, health food, and health supplement food are used. can be used interchangeably.

The star anise fennel extract, fractions thereof, or compounds isolated therefrom of the present invention can be added as is or used together with other foods or food ingredients, and can be used appropriately according to conventional methods.

The food of the present invention can be manufactured by methods commonly used in the industry, and can be manufactured by adding raw materials and ingredients commonly added in the industry. Specifically, the food composition may further include a physiologically acceptable carrier. The type of carrier is not particularly limited, and any carrier commonly used in the art can be used. In addition, the food composition contains food additives such as preservatives, disinfectants, antioxidants, colorants, coloring agents, bleaches, seasonings, sweeteners, flavorings, leavening agents, strengthening agents, emulsifiers, thickening agents, coating agents, gum base agents, foam suppressants, solvents, and improvers. It can be included. The additives can be selected depending on the type of food and used in an appropriate amount.

Additionally, the food formulation can be manufactured without limitation as long as it is a formulation recognized as a food. The food composition of the present invention can be manufactured in various types of formulations, and unlike general drugs, it is made from food as a raw material and has the advantage of not having side effects that may occur when taking the drug for a long period of time and is highly portable, so the present invention Foods can be consumed as supplements to improve the prevention or alleviation effect of skin diseases caused by pigmentation.

The star anise fennel extract of the present invention, its fractions, or compounds isolated therefrom may be included in various weight percentages in food compositions if they can show the effect of preventing or alleviating skin diseases caused by pigmentation. Specifically, it may be included in an amount of 0.00001 to 100% by weight or 0.01 to 80% by weight based on the total weight of the food composition, but is not limited thereto. When consumed for a long period of time for health and hygiene purposes, the content may be below the above range. Since there is no problem in terms of safety, the active ingredient may be used in amounts above the above range.

The cosmetic composition containing the star anise fennel extract, its fraction, or a compound isolated therefrom of the present invention as an active ingredient has excellent antioxidant efficacy and has an effect of inhibiting tyrosinase activity, so it can be safely and usefully used for skin whitening purposes. You can.

Figure 1 shows a total of eight types of star anise fennel extracts using various extraction solvent compositions, extraction temperatures, and extraction pressures.
Figure 2 shows the tyrosinase inhibitory activity (n=5) of seven types of Illicium Verum (Anise) Fruit Extract by solvent.
Figure 3 shows the tyrosinase inhibitory activity (n=5) of star anise fennel 50% ethanol extract at different concentrations (0.01 mg/ml to 0.1 mg/ml).
Figure 4 shows the DPPH radical scavenging ability (n = 3) of star anise fennel extract for each solvent.
Figure 5 shows the DPPH radical scavenging ability (n=5) by concentration (10 mg/g~100 mg/g) of the 50% ethanol extract of star aniseed fennel (C: Control(-), V: 10 mg/g Ascorbic acid, A: 10 mg/g, B: 20 mg/g, C: 30 mg/g, D: 40 mg/g, E: 50 mg/g, F: 60 mg/g, G: 70 mg/g , H: 80 mg/g, I: 90 mg/g, J: 100 mg/g).
Figure 6 shows the cell survival rate of HaCaT cells, which are skin keratinocytes, of 50% ethanol extract of star aniseed fennel.
Figure 7 shows a schematic diagram of manufacturing 50% ethanol extract of star aniseed fennel.
Figure 8 shows the MPLC chromatogram of the 50% ethanol extract of star aniseed fennel.
Figure 9 is a schematic diagram showing the separation of compounds 1 and 2 from 50% ethanol extract of star aniseed fennel.
Figure 10 shows the structures of compounds 1 and 2 isolated from 50% ethanol extract of star aniseed fennel.
Figure 11 shows the melanin production inhibition effect of compounds isolated from star anise fennel extract (C: Control, A: 50 mg/ml arbutin, T: Trans-anethole, V: Verimol B (Verimol) B).

Hereinafter, the present invention will be described in more detail through examples. These examples are for illustrating the present invention in more detail, and the scope of the present invention is not limited by these examples.

Example 1. Extraction of bioactive ingredients from star anise fennel

1-1. Experimental methods and materials

In order to prepare an extract showing maximum physiological activity from star anise fennel and derive useful substances from it, an octagon fennel extract was prepared by applying various extraction conditions. We attempted to analyze the economic feasibility of the extract by comparing the physiological activity by extraction condition and calculating the extraction yield at the same time. The star aniseed fennel used in the extraction was purchased from Duson Ae Herb (Country of Origin: Vietnam), and the dried octagonal fennel was ground into powder with a blender and passed through a 2.38 mm sieve, and the passed sample was used in the experiment.

1-2. Preparation of star anise fennel extract

A total of 8 types of star aniseed fennel extracts were prepared through accelerated solvent extraction (Accelerated Solvent Extractor) and carbon dioxide supercritical extraction (SC-CO2 Gas Extraction System) by applying various extraction solvent composition, extraction temperature, and extraction pressure ( Figure 1). Accelerated solvent extraction used a pressurized solvent extraction device (Speed Extractor E-916, Buchi). Specifically, a 20 ml cell for extraction was filled with fat free quartz sand (0.3 ~ 0.9 mm) and celite and octagonal fennel were added. The powder was mixed in equal amounts 1:1, a total of 4 g was loaded, fat free quartz sand was filled on top, and extraction was performed (Figure 7).

In order to elute the polar components of octagonal fennel, n-hexane, dichloromethane, acetone, and ethyl acetate were used as extraction solvents to prepare four types of extracts by extracting them at 30°C for 15 minutes, and 50% alcohol that can be used as a cosmetic raw material. Using ethanol and 100% ethanol as extraction solvents, two types of alcohol extracts were prepared by extraction for 15 minutes at 70°C, which is below the bp point of ethanol. And the hot water extract was extracted using purified water at a temperature of 100°C for 15 minutes. The seven types of extracts extracted with the accelerated solvent extraction device were extracted in one cycle by adding 200 ml of extraction solvent each for 15 minutes at a pressure of 100 bar, and are shown in Table 1 below.

No. Group Extract solvent Time (min) Temperature(℃) Pressure(bar) One Organic solvent extract n -hexane 15 30 100 2 Dichloromethane 3 Acetone 4 Ethyl Acetate 5 spirit extract 50% EtOH 70 6 100% EtOH 7 thermal water extract DDW 100

Supercritical extraction was performed using a supercritical extraction device (ISA-SCCO-S-1000-500, Ilshin). 400 g of octagonal fennel powder was loaded into the supercritical extraction cell and extracted in 1 cycle for 1 hour under a pressure of 200 bar.

1-3. Key results

The color, aroma, and viscosity of the octagonal fennel extract prepared by the above method were compared, and the extraction yield was calculated for each extraction condition.

In the case of extracts extracted with an accelerated solvent extraction device, relatively low extraction yields of 6.23 to 8.83% were shown when organic solvents were used as extraction solvents, and alcohol extracts of 50% ethanol and 100% ethanol showed 25.50% and 28.37%, respectively. , in the case of hot water extract, the extraction yield of hot water extract was found to be the highest at 28.97% (Table 2). However, in the case of the hot water extract, it was cloudy and precipitates were generated. When evaluating activity, all tests were conducted through filtration. In the case of supercritical extract, a total of 29 ml of liquid extract was obtained from 400 g of star aniseed fennel powder.

No. Extraction Methods Extract solvent Star anise fennel (g) Extract (mg) transference number (%) One Accelerated solvent extraction n-hexane 2 124.6 6.23 2 Dichloromethane 175.4 8.77 3 Acetone 176.6 8.83 4 Ethyl acetate 157.0 7.85 5 50% EtOH 510.0 25.50 6 100% EtOH 567.4 28.37 7 DDW 579.4 28.97 8 supercritical extract CO2 gas 400 29 (ml) -

Example 2. Activity evaluation of eight types of extracts of star aniseed fennel in each solvent

2-1. Star anise fennel extract in vitro Evaluation of tyrosinase inhibitory activity

The whitening activity of star anise fennel extract was evaluated through L-tyrosinase assay. Tyrosinase is an enzyme that mediates the production of melanin pigment from tyrosine. By measuring the degree of inhibition of the activity of the tyrosinase enzyme, we attempted to indirectly evaluate the melanin production inhibitory activity (whitening activity) of star aniseed fennel extract. The experimental method was conducted in accordance with the guidelines of the Ministry of Food and Drug Safety.

Specifically, the dried powder obtained by freeze-drying the extract of star aniseed fennel for each solvent was suspended in anhydrous ethanol to a concentration of 10 mg/g, centrifuged at 13,000 rpm for 15 minutes, the insoluble pellet was removed, and the supernatant was removed. was recovered. To evaluate the tyrosinase inhibitory activity of the recovered supernatant for each solvent, 220 ul of 0.1 M Sodium phosphate Buffer (pH 6.5), 20 ul of octagonal fennel extract, and 1.5 mM L-tyrosine were added to each well of a 96-well plate using the method described above. 40 ul of solution was added in order. Here, 20 ul of each tyrosinase solution prepared at a concentration of 1500 U/ml (Sigma, USA) was added and shaken for 3 seconds at 1 minute intervals for 15 minutes at 37°C to prevent melanin pigment from aggregating, and then measured in a Microplate spectrophotometer ( Absorbance was measured at 490 nm using BIORAD xMarkTM, USA). As a control, 0.1 M Sodium phosphate Buffer (pH 6.5) was added instead of tyrosinase enzyme as a blank, and anhydrous ethanol for extract preparation was added instead of star anise fennel extract as a control. Instead of star anise fennel extract, it was notified as a whitening raw material by the Ministry of Food and Drug Safety. Arbutin at a concentration of 20 mg/g (Ministry of Food and Drug Safety standard) was used as a positive control.

As a result of calculating the activity value using the formula below, the activity was more than 50% in all test sections for each solvent, and in particular, the dichloromethane and 50% ethanol extracts showed the highest activity of 81.982% and 75.664% (Figure 2). . In the case of whitening activity, the dichloromethane extract was about 1.08 times higher than the 50% ethanol extract, but the extract yield was about 1.31 times higher for the 50% ethanol extract than the dichloromethane extract, so in the tyrosinase inhibition evaluation, the 50% ethanol extract was finally decided. Then, tests were performed for each concentration.

The number of samples in all tests was set to 5, and the degree of inhibition of tyrosinase activity of the samples was calculated as follows.

Inhibition on tyrosinase activity (%) =

100 - {(Test - blank)/(Control-blank)}

Test: Absorbance after reaction of star anise fennel extract treatment group

Control: Absorbance after reaction of group treated with anhydrous ethanol instead of star anise fennel extract

Blank: Absorbance after reaction of the group treated with buffer instead of tyrosinase enzyme

The dried powder obtained by freeze-drying the 50% ethanol extract of star aniseed fennel was suspended in absolute ethanol to a concentration of 100 mg/g, centrifuged at 13,000 rpm for 15 minutes, the insoluble pellet was removed, and the supernatant was used. The supernatant was prepared by diluting it in increments of 0.02 mg/g in the range of 0.01 mg/g to 0.1 mg/g. As a result of measuring the tyrosinase inhibitory activity using a microplate spectrophotometer (BIORAD xMarkTM, USA), a value of 52.389%, which is the range of IC50 at 0.01 mg/g, was confirmed (Figure 3).

2-2. Evaluation of antioxidant power of star anise fennel extract

The DPPH radical scavenging ability test for evaluating antioxidant power measured the electron donating ability (EDA) by DPPH (1,1-diphenyl-2-picryl hydrazyl, Sigma, USA).

Specifically, 0.2 ml of star anise extract (10 mg/g) for each solvent was added to 0.8 ml of a 0.2 mM DPPH solution and mixed, then reacted at room temperature and in the dark for 30 minutes, and then the absorbance was measured at 517 nm. Antioxidant power was determined by electron donating ability (%) using the formula [1-(absorbance of sample added/absorbance of no sample added)]Х100. For the control group, ethanol was added instead of the sample, and 10 mg/g L-ascorbic acid aqueous solution was added. It was used as a positive control group.

As a result of measuring the DPPH radical scavenging activity of octagonal fennel extract in various solvents, as shown in Figure 4, dichloromethane showed activity of 23.148%, 50% ethanol extract 31.050%, and 100% ethanol extract showed activity of 28.224%. , the extracts of the remaining solvents showed less than 10% activity. Therefore, in order to examine the antioxidant activity of 50% ethanol extract at different concentrations, the 100 mg/g 50% ethanol extract sample used in the whitening activity evaluation was diluted with absolute ethanol in a concentration range of 10 mg/g to 100 mg/g in 10 mg/g units. The test was performed by diluting with .

As a result, as shown in Figure 5, the result of 52.07649%, which is the IC50 range at 20 mg/g, was confirmed. In order to ensure the reliability of this value, we commissioned a test from an accredited institution and confirmed that the IC50 range was 20 mg/ml compared to the final target value of vitamin C aqueous solution (10 mg/ml).

Example 3. Cytotoxicity evaluation of star anise fennel extract

3-1. Cytotoxicity evaluation on HaCaT cells, skin keratinocytes

cell culture

HaCaT cells, a human-derived skin keratinocyte cell line to be used in the experiment, were added to DMEM medium with 10% FBS (Gibco, Rockville, MD, USA), 100 U/ml Penicillin (Invitrogen, Carlsbad, CA, USA), and 100 ug/ml streptomycin. Cultured in one medium, maintained at 37°C with 5% CO ₂ .

Cytotoxicity evaluation

HaCaT cells were distributed on 96-well plates (1Х10 ⁴ cells/well), cultured in a 5% CO ₂ incubator at 37°C for 24 hours, and star anise extract was added at various concentrations (0 to 20 mg/ml in FBS). treated with and cultured for 24 hours. Remove the medium and replace it with medium containing 10% MTT solution (5 mg/ml) in each well, react at 37°C for 2 hours, measure absorbance at 540 nm using an ELISA reader, and control Cell viability was measured through comparison with .

As a result, as shown in Figure 6, it was confirmed that the star anise extract did not exhibit toxicity at all concentrations.

Example 4. Isolation and identification of active ingredients from star anise fennel extract

4-1. Preparation of star anise fennel extract for isolation of active ingredients

In order to identify functional substances that exhibit whitening activity from star anise fennel, star anise fennel extract was prepared in the same manner as the preparation of the first extract.

Specifically, to produce star anise fennel extract, a pressurized solvent extraction device (Speed Extractor E-916, Buchi) was used, and the extraction cell (capacity of 20 ml) was filled with fat free quartz sand (0.3 ~ 0.9 mm) and then extracted into the cell. Light and octagonal fennel powder were mixed 1:1, a total of 4 g was loaded, fat free quartz sand was filled on top, and extraction was performed. 50% ethanol, which is an extraction solvent condition confirmed to have excellent tyrosinase inhibitory activity, was set as the extraction solvent, and extraction was performed for 15 minutes at 100 bar and 70°C to prepare a 50% ethanol extract of star anise fennel (FIG. 7).

The 50% ethanol extract of star anise fennel was dissolved in a small amount of ethanol to remove the solvent using a vacuum concentrator, transferred to a vial, and then the solvent was removed using a nitrogen concentrator. The dried extract was left at -20°C for more than 3 hours and then moisture was completely removed using a freeze dryer.

4-2. Isolation of functional compounds from star anise fennel extract

Preparation of extracts by polarity using MPLC

In order to separate the functional substance showing tyrosinase inhibitory activity from the star anise fennel extract, the 50% ethanol extract of star anise fennel prepared above was subdivided into 10 subfractions according to polarity using MPLC (Isolera, Biotage, US). Cosmosil C18 resin was loaded into the separation cartridge, and gradient elution was performed with water and MeOH.

Isolation and purification of compounds 1 and 2 from star anise fennel extract

Compounds 1 and 2 were separated from fractions 5 and 6 among the 10 subfractions using HPLC (Figure 9). The peak resolution for each fraction was compared using various columns (Shsheido Capcell Pak, Phenomenex Synergi 4μ RP80A, Thermo Acclaim ^TM Advantage ±, Phenomenex Gemini 5μ C18 110A), and the column with the best resolution was used for the separation of compounds 1 and 2. were used respectively. The HPLC system used to separate compounds was a Thermo Scientific DIONEX Ultimate 3000 (Thermo scientific, Germany), which consists of a binary pump (LPG-3400SD) and a diode array detector (DAD-3000).

Isolation and purification of compound 1

HPLC conditions for separation of compound 1 are as follows. Shiseido Capcell Pak C18, 4.6Х250 mm was used as the column, water:MeOH = 55:45 (isocratic) was used as the mobile phase, and analysis was performed for 50 minutes at a flow rate of 1.0 ml/min. The detection wavelength was set at 254 nm.

Column Shiseido Capcell Pack Flow rate 1.0ml/min Injection volume 30 μl (diluted in MeOH) Mobile phase 45% MeOH (isocratic)

Isolation and purification of compound 2

HPLC conditions for separation of compound 2 are as follows. The column used was Phenomenex Synergi 4μ RP80A, 4.6Х250 mm, water: MeOH = 47:53 (isocratic) was used as the mobile phase, and analysis was performed for 40 minutes at a flow rate of 1.0 ml/min. The detection wavelength was set at 254 nm.

Column Phenomenex Synergi 4μ RP80A Flow rate 1.0ml/min Injection volume 50 μl (diluted in MeOH) Mobile phase 53% MeOH (isocratic)

4-3. Identification of chemical structures of compounds 1 and 2 isolated from star aniseed fennel

In order to determine the three-dimensional structure of compounds 1 and 2 isolated from octagonal fennel, information on hydrogen and carbon in the molecule was obtained through 1H and 13C-NMR spectra, and planar structure was obtained through 2D-NMR experiments (COSY, TOCSY, HSQC, HMBC). The structure was determined. The three-dimensional structure of the compound was determined through the ROESY experiment, and the molecular weight and molecular formula of the compound were confirmed through MS analysis. Afterwards, as a result of comparison with literature values, it was confirmed that Compound 1 was Verimol B and Compound 2 was Trans-anethole (FIG. 10).

Example 5. Identification of isolated compounds, analysis of content contained in star anise fennel extract, and confirmation of its whitening effect

5-1. Identification of isolated compounds, analysis of content contained in star anise fennel extract

In order to identify functional ingredients from a 50% ethanol extract of star aniseed fennel, which exhibits excellent tyrosinase inhibitory activity, it was separated into small fractions using MPLC, and then the two functional ingredients were successfully separated by setting various columns and solvent conditions using HPLC. and purified. As described above, Compound 1 was identified as Verimol B, and Compound 2 was identified as Trans-anethole. The chemical structure of the isolated compound was determined through 1D and 2D-NMR experiments, and the compound Content analysis was conducted for 1 and 2 using LC and GC-MS.

Compound 1 was analyzed for content using LC, and was found to contain 8.901 mg/ml in the 50% ethanol extract of star anise fennel. Compound 2 was analyzed for content using GC, and was found to be contained at a concentration of 159.493 mg/ml. It was confirmed that it was done.

5-2. Tyrosinase inhibitory activity for isolated compounds 1 and 2

Whitening activity was evaluated for the isolated compounds 1 and 2.

It was carried out in the same manner as in Example 2-1, and the treatment concentration of the sample was the final mg/ml concentration of compounds 1 and 2, and 50 mg/ml, which is the maximum concentration of arbutin notified by the Ministry of Food and Drug Safety, as a positive control. used.

As a result, as shown in Figure 11, 50 mg/ml arbutin, which is a positive control, was 96.431%, isolated compound 2, trans-anethole (1 mg/ml), was 59.243%, and compound 1, varimol B (1 mg/ml), excellent tyrosinase inhibitory activity was confirmed at 54.029%.

From the above description, those skilled in the art to which the present invention pertains will understand that the present invention can be implemented in other specific forms without changing its technical idea or essential features. In this regard, the embodiments described above should be understood in all respects as illustrative and not restrictive. The scope of the present invention should be construed as including the meaning and scope of the patent claims described below rather than the detailed description above, and all changes or modified forms derived from the equivalent concept thereof are included in the scope of the present invention.

Claims (8)

A cosmetic composition for skin whitening containing Verimol B, a compound isolated from star anise fennel extract. delete delete delete The cosmetic composition of claim 1, wherein the whitening is achieved by inhibiting tyrosinase activity. A quasi-drug composition for skin whitening containing Verimol B, a compound isolated from star anise fennel extract. A pharmaceutical composition for preventing or treating skin pigmentation disease, comprising Verimol B, a compound isolated from star anise fennel extract. A health functional food composition for preventing or alleviating skin pigmentation disease containing Verimol B, a compound isolated from star anise fennel extract.