WO2020235727A1 - Composition for skin whitening comprising fraction of inula helenium extract or compound isolated therefrom as active ingredient - Google Patents

Abstract

The present invention relates to a cosmetic composition for skin whitening comprising a hexane fraction of an Inula helenium extract, or an alantolactone or isoalantolactone compound isolated therefrom as an active ingredient. The fraction or compound according to the present invention inhibits melanin synthesis, and the compound of the present invention exhibits an effect of inhibiting mRNA or protein expression of a microphthalmia-associated transcription factor (MITF) which is a melanin synthesis promoting factor, tyrosinase, tyrosinase related protein 1, or tyrosinase related protein 2, and thus can be effectively used as a cosmetic composition for skin whitening, a pharmaceutical composition, or a functional health food.

Description

A composition for skin whitening comprising a fraction of the Tomok Hyang extract or a compound isolated therefrom as an active ingredient

The present invention relates to a composition for skin whitening comprising a fraction of the extract or a compound isolated therefrom as an active ingredient.

Melanin, which determines the color of a person's skin, is made in cells called melanocytes and moves to cells called keratinocytes. Here, melanin plays an important role in protecting genes from ultraviolet rays by forming a cap-like structure around the nucleus and protecting proteins in cells by removing free radicals. There is no enzyme that breaks down melanin in the living body, but keratinocytes are removed by breaking away from the skin, like when they break away from the epidermis.

Looking at the process of melanin synthesis, an enzyme called tyrosinase that first uses tyrosine as a substrate in the melanosome in melanocytes generates dopaquinone through DOPA, and it is dopaquinone from dopaquinone through automatic oxidation and enzyme reactions. Through chromium (DOPAchrome), a copolymer, melanin, is produced. The melanin produced in this way is transferred to keratinocytes through the endoplasmic reticulum called melanosomes, where it comes out to the surface of the skin and disappears along with the keratin after undergoing a keratinization process for about 28 days. However, in this process, when melanin is not completely eliminated due to excessive production of melanin and keratinization due to factors that promote melanin production, pigmentation occurs. Therefore, in order to prevent such pigmentation phenomenon, it is necessary to reduce the production of melanin by inhibiting a part of the melanogenesis process.

Currently, in the development of whitening agents for skin beauty, a method of reducing the produced melanin pigment to decolorize and a method of inhibiting the activity of tyrosinase, an enzyme that forms melanin pigment, are known (Korea Patent Registration No. 10-1370113). arc). However, since whitening agents using tocopherols or vitamins used to reduce melanin pigments are known to have insignificant bleaching effect of melanin pigments, many studies on inhibitors that inhibit the production of melanin pigments are being conducted.

In the conventional cosmetic field, as a whitening component, for example, substances that inhibit tyrosinase enzyme activity such as kojic acid and arbutin, hydroquinone, vitamin-C (L-Ascorbic) acid) and their derivatives have been used. However, the use of the whitening ingredients is limited due to poor stability and decomposition in the prescription system, or due to the occurrence of off-flavor, efficacy at the biological level, unclear effects, and safety issues.

Kojic acid inhibits enzymatic activity by adsorbing copper ions present in the active site of tyrosinase, but when formulated into cosmetics, it has been found to cause instability, skin side effects, and liver cancer as a result of recent animal tests, and it was discontinued as a cosmetic ingredient. Vitamin-C and its derivatives are difficult to use as a cosmetic ingredient because of the instability that is well oxidized, and hydroquinone has an excellent whitening effect on the skin, but it causes allergies, toxicity to melanogenic cells, and permanent decolorization on the skin. It is highly irritating to the skin, and it is currently banned as a carcinogenic substance, so only limited concentrations are permitted for each country. In addition, arbutin is a derivative in which glucopyranoside is bound to hydroquinone, and has fewer side effects when using hydroquinone, and has the effect of inhibiting the synthesis of melanin pigment without toxicity to the human body, thereby increasing melanin pigmentation. It has been suggested that it can be used as a therapeutic agent for skin diseases, but it is partially degraded by skin enzymes.

In addition to these whitening substances, studies have been conducted to find whitening active ingredients among natural products, especially plants, and it has been found that a number of plant extracts and herbal extracts inhibit melanin production by acting on tyrosinase, but these are also safety and potential for discoloration. In terms of the like, there are many problems in using more than the effective concentration in cosmetics or pharmaceuticals, and the situation has not yet exhibited excellent effects.

It is an object of the present invention to provide a composition for skin whitening comprising a fraction of a Tomok Hyang extract or a compound isolated therefrom as an active ingredient.

In order to achieve the above object, the present invention provides a cosmetic composition for skin whitening comprising a hexane fraction of an extract of Inula helenium as an active ingredient.

In addition, the present invention provides a cosmetic composition for skin whitening comprising as an active ingredient a compound represented by the following Formula 1 or 2, or a pharmaceutically acceptable salt thereof, isolated from the hexane fraction of the extract of Inula helenium :

[Formula 1]

[Formula 2]

.

In addition, the present invention provides a pharmaceutical composition for skin whitening comprising a hexane fraction of an extract of Inula helenium as an active ingredient.

In addition, the present invention provides a health functional food for skin whitening comprising the hexane fraction of the extract of Inula helenium as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for skin whitening comprising, as an active ingredient, a compound represented by the following Formula 1 or 2, or a pharmaceutically acceptable salt thereof, isolated from the hexane fraction of the extract of Inula helenium . :

[Formula 1]

[Formula 2]

.

In addition, the present invention provides a health functional food for skin whitening comprising, as an active ingredient, a compound represented by the following Formula 1 or 2 separated from the hexane fraction of the extract of Inula helenium or a pharmaceutically acceptable salt thereof. :

[Formula 1]

[Formula 2]

.

The hexane fraction of the Tomok Hyang extract according to the present invention or the compound represented by Formula 1 or 2 isolated therefrom inhibits melanin synthesis, and the compound is a melanin synthesis promoting factor MITF (microphthalmia-associated transcription factor), tyrosina As it exhibits the effect of inhibiting the mRNA or protein expression of tyrosinase, tyrosinase related protein 1 or tyrosinase related protein 2, cosmetic composition for skin whitening , It can be usefully used as a pharmaceutical composition or health functional food.

1 shows the results of HPLC analysis of a hexane fraction of an extract of Inula helenium .

Figure 2 shows the results of HPLC analysis of the methylene chloride fraction of the Tomok-hyang extract.

Figure 3 shows the results of HPLC analysis of the ethyl acetate fraction of the Tomok-hyang extract.

4 shows the results of HPLC analysis of the butanol fraction of the extract of Tomok-hyang.

FIG. 5 shows the results of separating allantolactone and isoalantolactone from the hexane fraction of the extract of Tomok Hyang.

Figure 6 shows the cell viability when the hexane fraction (IHHF) of the Tomok-hyang extract was treated on Melan-a cells.

7 shows the cell viability when allantolactone (AL), which is a compound derived from a civilization, was treated with Melan-a cells.

FIG. 8 shows the cell viability when isoalantolactone (IAL), which is a compound derived from a civilization, was treated with Melan-a cells.

9 is a result of evaluating the melanin synthesis inhibitory effect of the hexane fraction (IHHF) of the extract of Tomok-hyang

((A): Color development showing the amount of melanin; (B): A graph quantifying the result of (A); NT: negative control (non-treatment); IHHF: hexane fraction of the extract of Tomok Hyang; arbutin: positive control, Arbutin; 4-n-butylresorcinol: positive control, lucinol).

FIG. 10 is a result of evaluating the inhibitory effect of melanin synthesis of allantolactone (AL), a compound derived from civil scent

((A): Color development indicative of the amount of melanin; (B): Graph quantifying the result of (A); NT: negative control (non-treatment); AL: allantolactone derived from civilization; arbutin: positive control, Arbutin; 4-n-butylresorcinol: positive control, lucinol).

11 is a result of evaluating the melanin synthesis inhibitory effect of isoallantolactone (IAL), which is a compound derived from civil society.

((A): Color development showing the amount of melanin; (B): Graph quantifying the result of (A); NT: negative control (non-treatment); IAL: isoallantolactone derived from civil cultivation; AL: civil cultivation Derived allantolactone; arbutin: positive control, arbutin; 4-n-butylresorcinol: positive control, lucinol).

FIG. 12 shows the effect of inhibiting mRNA expression of MITF (microphthalmia-associated transcription factor) of allantolactone (AL), a compound derived from civilization.

13 shows the effect of inhibiting the mRNA expression of the microphthalmia-associated transcription factor (MITF) of isoallantolactone (IAL), a compound derived from a civilization-derived compound.

FIG. 14 shows the effect of suppressing mRNA expression of tyrosinase (TYR) of isoallantolactone (IAL), a compound derived from a civil scent.

FIG. 15 shows the effect of inhibiting mRNA expression of tyrosinase related protein 1 (TRP-1) of isoallantolactone (IAL), a compound derived from civilian gypsy.

FIG. 16 shows the effect of suppressing mRNA expression of tyrosinase related protein 2 (TRP-2) of isoallantolactone (IAL), a compound derived from civil warfare.

FIG. 17 shows MITF, tyrosinase (TYR), tyrosinase-related protein 1 (TRP-1), and tyrosinase-related protein 2 (TRP-2) of allantolactone (AL), which is a compound derived from civil scent. It shows the inhibitory effect of protein expression.

Hereinafter, the present invention will be described in detail.

The present invention provides a cosmetic composition for skin whitening comprising a hexane fraction of an extract of Inula helenium as an active ingredient.

The geomokhyang extract can be prepared by a manufacturing method comprising the following steps:

1) extracting by adding an extraction solvent to a civil scent;

2) filtering the extract of step 1); And

3) A step of drying after concentrating the filtered extract of step 2) under reduced pressure.

In the above method, the civil scent of step 1) may be used without limitation, such as cultivated or commercially available ones.

In the above manufacturing method, the civil scent may include all parts such as leaves, stems, and roots of civil scent, and specifically may be any one or more selected from the group consisting of leaves, stems, and roots. According to a specific embodiment of the present invention, it is most preferable to use the roots of civil scent.

In addition, the extraction solvent of step 1) may be water, alcohol, or a mixed solvent thereof. The alcohol may be a C ₁ to C ₂ lower alcohol, and specifically, the alcohol may be methanol or ethanol. According to a specific embodiment of the present invention, it is most preferable to extract with methanol.

The extraction method of step 1) may be shaking extraction, Soxhlet extraction, reflux extraction, or ultrasonic extraction. At this time, the extraction temperature is preferably 10 to 60 ℃, and more preferably 15 to 35 ℃, but is not limited thereto. In addition, the extraction solvent is preferably extracted by adding 1 to 50 mL per 1 g of the weight of the civil scent used for extraction, and it is more preferable to extract by adding 1 to 20 mL, but is not limited thereto. In addition, the extraction time is preferably 10 minutes to 10 hours, more preferably 10 minutes to 5 hours, but is not limited thereto. In addition, the number of extractions is preferably 1 to 10 times, more preferably 1 to 3 times, but is not limited thereto. In a range outside the conditions of the extraction temperature, extraction time, or number of extractions, extraction may not be sufficiently performed, so that the content of the active ingredient in the extract may be insignificant. Alternatively, the extraction yield may no longer increase, so that the efficiency of the extraction operation may decrease.

The vacuum concentration in step 3) may be performed using a vacuum vacuum concentrator or a vacuum rotary evaporator. In addition, the drying may be vacuum drying, vacuum drying, boiling drying, spray drying, or freeze drying.

Fractions of the extract can be prepared by a manufacturing method comprising the following steps:

1) adding an extraction solvent to a civil hyang to obtain a civil hyang extract; And

2) obtaining a fraction by sequentially fractionating the extract of step 1) with hexane, methylene chloride, ethyl acetate and butanol.

The civil scent extract of step 1) can be obtained by performing the above-described method.

Specifically, for the fraction, after suspending the extract obtained in step 1) in a solvent, hexane, methylene chloride, ethyl acetate and butanol were sequentially added to separate each layer, and then each layer was concentrated under reduced pressure and It is preferred to dry to obtain each fraction.

The hexane fraction of the geomokhyang extract can inhibit melanin synthesis.

In a specific embodiment of the present invention, the present inventors obtained fractions by sequentially fractionating the crypt extract with hexane, methylene chloride, ethyl acetate, and butanol, and then treating each fraction with Melan-a, a melanogenic cell, to reduce cytotoxicity. The concentration of each fraction not shown was confirmed (see FIG. 6).

In addition, the present inventors confirmed that among the fractions of the extract of Tomok-hyang, the hexane fraction inhibited melanin synthesis in Melan-a cells (see FIG. 9).

Therefore, the hexane fraction of the extract according to the present invention inhibits melanin synthesis, so it can be usefully used in a cosmetic composition for skin whitening.

In addition, the present invention provides a cosmetic composition for skin whitening comprising as an active ingredient a compound represented by the following Formula 1 or 2, or a pharmaceutically acceptable salt thereof, isolated from the hexane fraction of the extract of Inula helenium :

[Formula 1]

[Formula 2]

.

The compound represented by Formula 1 or 2 can be obtained by performing chromatography and further purifying the hexane fraction of the Geomokhyang extract obtained by performing the extraction and fractionation steps of the Geomokhyang extract according to the present invention. .

The compound represented by Formula 1 or 2 may inhibit melanin synthesis.

The compounds represented by Formula 1 or 2 are microphthalmia-associated transcription factor (MITF), tyrosinase, tyrosinase related protein 1, and tyrosinase-related protein 2 It can inhibit the expression of any one or more selected from the group consisting of related protein 2).

In a specific embodiment of the present invention, the present inventors analyzed the hexane, methylene chloride, ethyl acetate, and butanol fractions of the civil scent extract by HPLC, respectively, and as a result, allantolactone (Formula 1) and iso are effective for whitening in the hexane fraction. Allantolactone (isoalantolactone, Chemical Formula 2) compound was detected (see FIGS. 2 to 4).

In addition, the present inventors separate allantolactone and isoallantolactone compounds from the hexane fraction of the extract of Tomok-hyang, and then treat each compound in Melan-a, a melanogenic cell, to confirm the concentration of each fraction that does not exhibit cytotoxicity. (See Figs. 7 and 8).

In addition, the present inventors confirmed that the allantolactone and isoallantolactone compounds isolated from the hexane fraction of the Tomok Hyang extract inhibit melanin synthesis in Melan-a cells (see FIGS. 10 and 11).

In addition, the present inventors argue that the allantolactone compound isolated from the hexane fraction of the Geomokhyang extract inhibits the mRNA expression of microphthalmia-associated transcription factor (MITF), and the isoallantolactone compound isolated from the hexane fraction of the Geomokhyang extract is MITF (microphthalmia-associated transcription factor), tyrosinase (tyrosinase), tyrosinase related protein 1 (tyrosinase related protein 1) and tyrosinase related protein 2 (tyrosinase related protein 2) mRNA expression inhibition Confirmed (see Figs. 12 to 16).

In addition, the inventors of the present inventors that the allantolactone compound isolated from the hexane fraction of the Tomok Hyang extract is MITF (microphthalmia-associated transcription factor), tyrosinase, tyrosinase related protein 1 (tyrosinase related protein 1), and It was confirmed that protein expression of tyrosinase related protein 2 was suppressed (see FIG. 17).

Therefore, the allantolactone (the above formula 1) or isoallactolactone (the above formula 2) compound isolated from the hexane fraction of the Tomok Hyang extract inhibits melanin synthesis, and is a microphthalmia-associated transcription factor (MITF), which is a melanin synthesis promoter. , Tyrosinase (tyrosinase), tyrosinase-related protein 1 (tyrosinase related protein 1) or tyrosinase-related protein 2 (tyrosinase related protein 2) mRNA or protein expression suppressing effect, It can be usefully used in whitening cosmetic compositions.

The composition of the present invention may further contain one or more skin whitening active ingredients exhibiting the same or similar function to the compound of the present invention. Skin whitening active ingredients include kojic acid and its derivatives, arbutin, ascorbic acid and its derivatives, hydroquinone and its derivatives, lucinol, cycloalkanone, methylenedioxyphenyl alkanol, 2,7-dinitroindazole, vines There are plant extracts such as extracts, rice extracts, and licorice extracts, but are not limited thereto.

The cosmetic composition for skin whitening of the present invention preferably contains the compound of the present invention in an amount of 0.0001 to 90 parts by weight based on the total weight of the composition, but is not limited thereto.

The cosmetic composition for skin whitening of the present invention may be used by mixing a dermatologically acceptable carrier in addition to the compound of the present invention. The dermatologically acceptable carriers are purified water, oils, waxes, fatty acids, fatty alcohols, fatty acid esters, surfactants, humectants, thickening agents, antioxidants, viscosity stabilizers, chelating agents. , Buffers, preservatives, lower alcohols, and the like are included, but are not limited thereto, and the types and concentrations thereof are various, and modifications and changes are included within the scope of those skilled in the art without departing from the spirit and scope of the present invention. If necessary, whitening agents, moisturizing agents, anti-inflammatory agents, antibacterial agents, antifungal agents, vitamins, sunscreens, antibiotics, anti-acne agents, perfumes, and dyes may be included, and these may be used in amounts commonly used in the cosmetic field to prevent wrinkles or It may be included in the cosmetic composition for improvement or whitening.

Cosmetics prepared from the cosmetic composition for skin whitening of the present invention can be prepared in the form of a general emulsifying formulation and a solubilizing formulation. Cosmetics in emulsified form include nutrient lotion, cream, and essence, and cosmetic products in solubilized form include softening lotion. In addition to the peptoid compound of the present invention, the cosmetic may contain a dermatologically acceptable medium or base, and thus may be prepared in the form of an adjuvant that can be applied topically or systemically used in the field of dermatology.

Suitable cosmetic formulations include, for example, solutions, gels, solid or paste dry products, emulsions obtained by dispersing the oil phase in an aqueous phase, suspensions, microemulsions, microcapsules, microgranules or ionic (liposomes), nonionic It can be provided in the form of a vesicle dispersant, a cream, skin, lotion, powder, ointment, spray or conceal stick. In addition, it may be prepared in the form of a foam or an aerosol composition further containing a compressed propellant.

Products to which the cosmetic composition for skin whitening of the present invention can be added include, for example, astringent lotion, softening lotion, nutritional lotion, various creams, essences, cosmetics such as packs, foundations, and cleansing, face wash, soap, treatment Treatment or essence. Specific formulations include skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nutrition lotion, massage cream, nutrition cream, moisture cream, hand cream, essence, nutrition essence, pack, soap, shampoo, cleansing foam. , Cleansing lotion, cleansing cream, body lotion, body cleanser, emulsion, press powder, loose powder or eye shadow formulations.

In addition, the present invention provides a pharmaceutical composition for skin whitening comprising a hexane fraction of an extract of Inula helenium as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for skin whitening comprising, as an active ingredient, a compound represented by the following Formula 1 or 2, or a pharmaceutically acceptable salt thereof, isolated from the hexane fraction of the extract of Inula helenium . :

[Formula 1]

[Formula 2]

.

In a specific embodiment of the present invention, the present inventors have obtained a fraction by sequentially fractionating the extract of civil scents with hexane, methylene chloride, ethyl acetate and butanol, and then the hexane fraction of the fraction inhibits melanin synthesis in Melan-a cells. Since it was confirmed (see FIG. 9), the hexane fraction of the Tomok Hyang extract according to the present invention can be usefully used in a pharmaceutical composition for skin whitening.

In addition, the present inventors argue that the allantolactone and isoallantolactone compounds isolated from the hexane fraction of the Tomok Hyang extract inhibit melanin synthesis in Melan-a cells (see Figs. 10 and 11), and the melanin synthesis promoting factor MITF ( microphthalmia-associated transcription factor), tyrosinase, tyrosinase related protein 1 or tyrosinase related protein 2 mRNA or protein expression Since it was confirmed (see Figs. 12 to 17), the hexane fraction of the extract according to the present invention can be usefully used in a pharmaceutical composition for skin whitening.

The pharmaceutical composition according to the present invention may contain 10 to 95% by weight of the hexane fraction of the extract or the compound represented by Formula 1 or 2 as an active ingredient based on the total weight of the composition. In addition, the pharmaceutical composition of the present invention may further include one or more active ingredients exhibiting the same or similar functions in addition to the active ingredients.

The pharmaceutical composition of the present invention may contain a carrier, a diluent, an excipient, or a mixture thereof commonly used in biological preparations. Any pharmaceutically acceptable carrier can be used as long as it is suitable for delivering the composition in vivo. Specifically, the carrier is Merck Index, 13th ed., Merck & Co. Inc., saline, sterile water, Ringer's solution, dextrose solution, maltodextrin solution, glycerol, ethanol, or a mixture thereof. In addition, conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added as needed.

When formulating the composition, diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants may be added.

The composition of the present invention may be formulated as an oral or parenteral formulation. Oral formulations may include solid formulations and liquid formulations. The solid preparation may be a tablet, a pill, a powder, a granule, a capsule, or a troche, and the solid preparation may be prepared by adding at least one excipient to the composition. The excipient may be starch, calcium carbonate, sucrose, lactose, gelatin, or a mixture thereof. In addition, the solid preparation may contain a lubricant, examples of which include magnesium stearate and talc. On the other hand, the liquid formulation may be a suspension, a liquid formulation, an emulsion or a syrup. At this time, the liquid formulation may contain excipients such as wetting agents, sweetening agents, fragrances, preservatives, and the like.

The parenteral preparations may include injections, suppositories, powders for respiratory inhalation, aerosols for sprays, powders and creams. The injection may include a sterilized aqueous solution, a non-aqueous solvent, a suspension solvent, an emulsion, and the like. At this time, as the non-aqueous solvent or suspension solvent, vegetable oils such as propylene glycol, polyethylene glycol, and olive oil, or injectable esters such as ethyl oleate may be used.

The composition of the present invention may be administered orally or parenterally according to a desired method. Parenteral administration may include intraperitoneal, rectal, subcutaneous, intravenous, intramuscular or intrathoracic injection.

The composition may be administered in a pharmaceutically effective amount. This may vary depending on the type of disease, the severity, the activity of the drug, the patient's sensitivity to the drug, the administration time, the administration route, the treatment period, and the drugs used at the same time. However, for a desirable effect, the amount of the active ingredient contained in the pharmaceutical composition according to the present invention may be 0.0001 to 1,000 mg/kg, specifically 0.001 to 500 mg/kg. The administration may be several times a day.

The composition of the present invention may be administered alone or in combination with other therapeutic agents. When administered in combination, administration may be sequential or simultaneous.

In addition, the present invention provides a health functional food for skin whitening comprising the hexane fraction of the extract of Inula helenium as an active ingredient.

In addition, the present invention provides a health functional food for skin whitening comprising, as an active ingredient, a compound represented by the following Formula 1 or 2 separated from the hexane fraction of the extract of Inula helenium or a pharmaceutically acceptable salt thereof. :

[Formula 1]

[Formula 2]

.

In a specific embodiment of the present invention, the present inventors have obtained a fraction by sequentially fractionating the extract of civil scents with hexane, methylene chloride, ethyl acetate and butanol, and then the hexane fraction of the fraction inhibits melanin synthesis in Melan-a cells. Since it was confirmed (see FIG. 9), the hexane fraction of the extract according to the present invention can be usefully used in a health functional food for skin whitening.

In addition, the present inventors argue that the allantolactone and isoallantolactone compounds isolated from the hexane fraction of the Tomok Hyang extract inhibit melanin synthesis in Melan-a cells (see Figs. 10 and 11), and the melanin synthesis promoting factor MITF ( microphthalmia-associated transcription factor), tyrosinase, tyrosinase related protein 1 or tyrosinase related protein 2 mRNA or protein expression Since it was confirmed (see FIGS. 12 to 17), the hexane fraction of the geomokhyang extract according to the present invention can be usefully used in a health functional food for skin whitening.

The hexane fraction of the extract of the present invention or the compound represented by Formula 1 or Formula 2 may be added to food as it is, or may be used together with other foods or food ingredients. At this time, the content of the active ingredient to be added may be determined according to the purpose, and generally may be 0.01 to 90 parts by weight of the total food weight.

The form and type of the health functional food is not particularly limited. Specifically, the health functional food may be in the form of tablets, capsules, powders, granules, liquids, and pills. The health functional food may contain various flavoring agents, sweetening agents, or natural carbohydrates as additional ingredients. The sweetener may be a natural or synthetic sweetener, and examples of the natural sweetener include taumatin and stevia extract. Meanwhile, examples of synthetic sweeteners include saccharin and aspartame. In addition, the natural carbohydrates may be monosaccharides, disaccharides, polysaccharides, oligosaccharides and sugar alcohols.

In addition to the above-described additional ingredients, the health functional food of the present invention includes nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pexane and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives. , Glycerin, alcohol, and the like may be further included. These ingredients can be used independently or in combination. The proportion of the additive may be selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

Hereinafter, the present invention will be described in detail by the following examples.

However, the following examples are only illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

<Example 1> Preparation of Geomokhyang extract

The dry powder (1 g) of the roots of Inula helenium was added to 10 mL of methanol and extracted for 1 hour at 25° C. by ultrasonic extraction, and this was repeated three times to obtain a methanol extract of civil scent.

<Example 2> Preparation of a fraction of the extract of Tomokhyang

The methanol extract obtained in Example 1 was filtered and concentrated under reduced pressure with a reduced pressure concentrator to remove the solvent. After suspending the dried extract in 25 mL of water, extraction was performed three times with 25 mL of hexane, followed by extraction of methylene chloride, ethyl acetate, and butanol three times in order of 25 mL, and the fractions of the civil scent extract (hexane fraction, methylene chloride fraction , An ethyl acetate fraction and a butanol fraction) were obtained.

<Experimental Example 1> Analysis of the whitening active ingredient of the fraction of the extract

In order to measure the content of allantolactone and isoalantolactone, the main active ingredients of whitening, the fractions of the extract obtained in Example 2 were concentrated under reduced pressure, and the concentration was 1000 μg/mL. As much as possible, HPLC analysis was performed by re-dissolving in methanol.

For HPLC, HITACHI L-6200 pump, Shimadzu SIL-9A autoinjector, and Hitachi UV detector L-7400 were used. Water was used as solvent A and acetonitrile was used as solvent B, and 20-60% solvent B from 0 to 30 minutes, 60-100% solvent B from 30 to 35 minutes, and 20% solvent for the last 5 minutes in a gradient manner Flowed with B at 1 mL/min. Individual fractions were separated using a reversed-phase INNO C18 column (150 mm x 4.6 mm, 5 μm particle size, Youngjin biochrom, Seongnam, Korea), and the absorption wavelength of the UV detector was set to 220 nm.

As a result of confirming allantolactone and isoallantolactone from the fractions of the Geomokhyang extract using HPLC-UV, allantolactone (the following Formula 1) and isoallantolactone (the following Formula 2) only in the hexane fraction of the Geomokhyang extract Was detected, and was not detected in the methylene chloride, ethyl acetate, and butanol fractions (see FIGS. 1 to 4).

[Formula 1]

[Formula 2]

.

<Example 3> Separation of allantolactone and isoallantolactone from civil scent fraction

Based on the results confirmed in Experimental Example 1, it was attempted to separate allantolactone and isoallantolactone from the hexane fraction of the Geomok-hyang extract.

HSCCC TBA-300A (Tauto Biotech, Shanghai, China) chromatography equipped with a polytetrafluoroethylene (PTFE) tube column having an inner diameter of 1.8 mm and a total volume of 260 mL was used. A mixed solvent of hexane:acetonitrile:methanol:water (5:3:1:2 ratio) was prepared in advance the day before, and the upper layer was divided into a stationary phase, and the lower layer was divided into a mobile phase, and dissolved gas was removed before use. First, fill the column wound in a coil with the solvent to be used as a stationary bed, and then rotate the column at a centrifugal speed of 850 rpm (TBE-300A). I spilled it. When equilibration between the stationary phase and the mobile phase is achieved in the column, 200 mg of the hexane fraction of the extract obtained in Example 2 is dissolved in 20 mL of a two-phase (fixed and mobile phase) solvent mixed at a volume ratio of 1:1 and injected into the column. I did. The lower mobile phase was flowed for 500 minutes at a flow rate of 1.0 mL/min, and the eluent of HSCCC was detected using a UV detector (220 nm) and fractionated using a fraction collector.

As a result, isoallantolactone and allantolactone were obtained at 280-310 min and 330-380 min, respectively (FIG. 5).

<Experimental Example 2> Confirmation of the cell viability of the Melan-a cell line according to the treatment of the fraction of the extract of Tomokhyang and the compound derived from Tomokhyang

It was attempted to confirm the cell viability for the Melan-a cell line (pigmented melanocytes), which are melanogenic cells of allantolactone and isoallantolactone, which are the civil hyang fraction obtained in Example 2 and the civil hyang-derived compounds obtained in Example 3.

The Melan-a cell line was first Dr. It was isolated from C57BL/6 mice by the Bennett team (Cancer Research Center, London, England), and the present inventors used it by pre-sale (Bennett et al., 1987, Int. J. Cancer).

The Melan-a cell line was inoculated into a 6-well culture vessel by 7×10 ⁵ cells, and cultured for 24 hours under conditions of 37°C and 10% CO ₂ . As a culture medium, 10% fetal bovine serum (FBS) (Gibco, Australiaorigin), 1% antibiotics (antibiotic-antimyocotics, A/A) (Gibco, USA) and 200 nM of TPA (Phorbol 12-myristate 13-) acetate) (Cayman, USA) was added to RPMI 1640 medium (GE Healthcare Life Sciences, USA). Then, the civil scent fractions obtained in Example 2 were concentrated under reduced pressure and dissolved in DMSO, and allantolactone and isoallantolactone, which were compounds derived from civil scent obtained in Example 3, were also dissolved in DMSO, respectively, and then Melan-a The cell line was treated.

Fractions of the Tomok Hyang extract were 0 to 50 μg/mL, and allantolactone and isoallantolactone were treated at a concentration of 0 to 20 μM on Melan-a cells and cultured for 72 hours, and then 0.5 mg/mL of MTT ( 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was added and incubated for 3 hours. The supernatant was removed, treated with 2 mL of DMSO to dissolve the formed formazan, and shaken on a shaker for 1 hour, and then the absorbance (540 nm) was measured using an ELISA reader. CV ₈₀ (concentrations that reduce cell viability by 20%), which is the concentration at which the cell viability is 80%, was calculated (Table 1).

	CV 80 (in μg/mL for fraction 1 , in μM for compound 2 )
Hexane fraction 1	7.93 ± 0.01	91% CV at 5 μg/mL
Methylene chloride fraction 1	25.26 ± 0.30	84% CV at 25 μg/mL
Ethyl acetate fraction 1	> 50	96% CV at 50 μg/mL
Butanol fraction 1	> 50	98% CV at 50 μg/mL
Allantolactone 2	> 15	CV 87% at 15 μM
Isoallantolactone 2	> 20	CV 93% at 20 μM

As shown in Table 1, the CV ₈₀ of the hexane fraction of the Tomok Hyang extract is 7.93 ± 0.01 μg/mL (see Figure 6), the methylene chloride fraction is 25.26 ± 0.30 μg/mL, and the ethyl acetate and butanol fractions are 50 μg/mL. It was ideal. The CV ₈₀ of allantolactone derived from civil scent was 15.52 ± 0.06 μM (FIG. 7), and isoallantolactone did not show toxicity in the range of 0 to 20 μM (see Table 1). In a subsequent experiment, the fractions, allactolactone and isoalactolactone of the extract of Tomok-hyang were performed within the maximum concentration (CV ₈₀ ) that did not show significant toxicity to the Melan-a cell line.

<Experimental Example 3> Confirmation of the melanin synthesis inhibitory effect of the Melan-a cell line according to the treatment of the fraction of the Tomok Hyang extract and the compound derived from Tomok Hyang

To confirm the inhibitory effect of melanin synthesis on the melanocytes Melan-a cell line (pigmented melanocytes) of allantolactone and isoallantolactone, the fractions of the Tomok Hyang extract obtained in Example 2 and the Tomok Hyang-derived compounds.

Specifically, the Melan-a cell line was dispensed into a 6-well culture vessel by 7×10 ⁵ cells and cultured for 24 hours at 37°C and 10% CO ₂ . Then, the fractions of the civil scent extract obtained in Example 2 and allantolactone and isoallantolactone, which are compounds derived from civil scents obtained in Example 3, were dissolved in DMSO and treated within the maximum concentration (CV ₈₀ ) where no significant toxicity appears. Incubated for 72 hours.

After incubation, the medium was removed, washed with 1 mL of DPBS (Dulbecco's Phosphate BufferedSaline, DPBS) (Sigma-Aldrich, USA), and treated with 800 μL of trypsin-EDTA (Gibco, USA) at 37° C. for 2 minutes. The separated cells were collected and centrifuged at 5,000 rpm for 3 minutes at 4° C. to recover the cells. After washing the cell pellet with DPBS and adding 100 μL of 1N NaOH (including 10% DMSO), it was left at 60° C. for 15-30 minutes to extract melanin. After dissolving the extracted melanin, the absorbance was measured at a wavelength of 490 nm using an ELISA reader to confirm the degree of intracellular melanin synthesis. IC ₅₀ (The half maximal inhibitory concentration), which is the concentration at which melanin synthesis is inhibited by 50% compared to the negative control (non-treatment, NT), was calculated (Table 2).

The same experiment was performed three times to calculate the standard deviation of each analysis result. For the experimental results, the p-value was calculated using the student's t-test, and for p<0.05, it was marked as *, for p<0.01, it was marked as **, and for p<0.001, it was marked as *** to indicate significance.

As a positive control, lucinol (4-n-butylresorcinol) (Sigma-Aldrich, USA) and arbutin (Sigma-Aldrich, USA) used in the evaluation of whitening efficacy were used. Lucinol (4-n-butylresorcinol) was treated at a concentration of 100 μM and arbutin at a concentration of 1 mM.

	IC 50 (fraction 1 is μM/mL, compound 2 is μM)
Hexane fraction 1	5.16 ± 0.02	-
Methylene chloride fraction 1	> 25	-
Ethyl acetate fraction 1	> 50	8% inhibition at 50 μg/mL
Butanol fraction 1	> 50	8% inhibition at 50 μg/mL
Allantolactone 2	11.77 ± 0.29	-
Isoallantolactone 2	17.77 ± 0.38	-

As shown in Table 2, the hexane fraction of the Tomok Hyang extract, allantolactone and isoallantolactone, which are compounds derived from Tomok Hyang, inhibited melanin synthesis (see FIGS. 9 to 10). In particular, the hexane fraction of the Tomok Hyang extract exhibited excellent whitening effect at low concentration with an IC ₅₀ of 5.16 ㎍/mL. In addition, in the case of allantolactone and isoallantolactone, IC ₅₀ was 11.77 μM and 17.88 μM, respectively, and suppressed melanin synthesis better than 100 μM of 4-n-butylresorcinol and 1 mM arbutin, which are positive controls. On the other hand, in the methylene chloride fraction, ethyl acetate fraction, and butanol fraction excluding the hexane fraction of the Tomok Hyang extract, significant inhibition of melanin synthesis at the maximum treatment concentration (methylene chloride fraction 25 μg/mL, ethyl acetate and butanol fraction 50 μg/mL) There was no effect (see Table 2).

<Experimental Example 4> Confirmation of the inhibitory effect on the mRNA expression level of the melanin synthesis promoting factor of the Melan-a cell line according to the treatment of allantolactone and isoallantolactone, which are compounds derived from civil scent

Microphthalmia-associated transcription factor (MITF), tyrosinase, and tyrosinase-related protein 1 (Tyrosinase), which are factors that promote melanin synthesis of allantolactone and isoallantolactone, which are compounds derived from civil scent obtained in Example 3 Related protein 1, TRP-1) and tyrosinase-related protein 2 (Tyrosinase related protein 2, TRP-2) were subjected to the following experiments to determine the inhibitory effect of mRNA expression levels.

Specifically, the Melan-a cell line was dispensed into a 6-well culture vessel by 7×10 ⁵ cells and cultured for 24 hours at 37°C and 10% CO ₂ . Then, allantolactone and isoallantolactone obtained in Example 3 were dissolved in DMSO, treated at 0, 5, 10, 15 and 20 μM concentrations, and cultured for 24 hours. After obtaining the cultured cells, total RNA was extracted according to the manufacturer's protocol using TRIzol (Invitrogen, USA) reagent. The isolated RNA was quantified and then treated with amfiRivert cDNA master mix (GenDEPOT, USA) for 5 minutes at 25°C, 60 minutes at 42°C, and 15 minutes at 70°C according to the manufacturer's protocol to synthesize cDNA.

The synthesized cDNA was applied with a primer set (Bioneer, Korea) and a SYBR green working solution (iTaqTM Universal SYBR Green Supermix, BIO-RAD, USA) at the level of 40 cycles by Applied Biosystems Prism 7300 Real-Time PCR (Applied Biosystems, USA). Performed. Initial denaturation was performed at 95°C for 30 seconds, after that, at 95°C for 15 seconds (denaturation), and at 60°C for 6 seconds at 40 cycles (annealing, extension and plate read). The obtained Ct value was normalized to a value relative to β-actin, and the mRNA gene expression level was analyzed. Primers used for Real-Time PCR are shown in Table 3 below.

Sequence number	gene	order
One	MITF	Forward primer	5’-CTAAGTGGTCTGCGGTGTCTC-3’
2		Reverse primer	5’-GGTTTTCCAGGTGGGTCTG-3’
3	Tyrosinase	Forward primer	5’-CACCCTGAAAATCCTAACTTACTCA-3’
4		Reverse primer	5’-CTCTTCTGATCTGCTACAAATGATCT-3’
5	TRP-1	Forward primer	5’-TGGGAACACTTTGTAACAGCA-3’
6		Reverse primer	5’-ACTGCTGGTCTCCCTACATTTC-3’
7	TRP-2	Forward primer	5’-GGCTACAATTACGCCGTTG-3’
8		Reverse primer	5’-CACTGAGAGAGTTGTGGACCAA-3’
9	β-actin		Forward primer	5′-CCCACTCCTAAGAGGAGGATG-3’
10		Reverse primer	5′-AGGGAGACCAAAGCCTTCAT-3’

As a result, it was found that allantolactone, a compound derived from Tomok-hyang, inhibits the mRNA expression level of MITF, a melanin synthesis promoter (Fig. 12), and isoallantolactone is a melanin synthesis promoter MITF and tyrosinase. (Tyrosinase), TRP-1 and TRP-2 were found to inhibit the mRNA expression levels (Figs. 13 to 15).

<Experimental Example 5> Confirmation of the inhibitory effect of the protein expression level of the melanin synthesis promoting factor in the Melan-a cell line according to the treatment of allantolactone, which is a compound derived from civil scent

Microphthalmia-associated transcription factor (MITF), tyrosinase, and tyrosinase-related protein 1, TRP, which are melanin synthesis promoters of allantolactone, a compound derived from a civil scent obtained in Example 3 -1) and tyrosinase-related protein 2 (Tyrosinase-related protein 2, TRP-2) was carried out as follows to investigate the effect of inhibiting the protein expression level.

Specifically, the Melan-a cell line was dispensed into a 6-well culture vessel by 7×10 ⁵ cells and cultured for 24 hours at 37°C and 10% CO ₂ . Then, the allantolactone obtained in Example 3 was dissolved in DMSO, treated with 0, 5, 10, and 15 μM concentrations, and cultured for 24 hours. After incubation, the medium was removed, washed with 1 mL of DPBS (Dulbecco's Phosphate BufferedSaline, DPBS) (Sigma-Aldrich, USA), and then recovered by scraping the cells, and lysis buffer (20 mM HEPES [pH 7.6], 350 mM NaCl, 20 % Glycerol, 0.5 mM EDTA, 0.1 mM EGTA, 1% NP-40, 50 mM NaF, 0.1 mM DTT, 0.1 mM PMSF) 80 [mu]l were added, and then stored on ice. The cells were shaken once every 10 minutes using a shaking mixer, a total of 3 times, and then centrifuged at 4° C. for 10 minutes at a rotation speed of 15,000 rpm to obtain a cell lysate.

20 μg of protein was electrophoresed on an 8% SDS-PAGE gel at 100 V for 2 hours, and the separated protein was electrically transferred to a nitrocellulose membrane. After blocking the membrane with 5% skim milk at room temperature for 1 hour, the primary antibody against MITF (Santacruz, USA, Cat No. sc-52938), Tyrosinase (Santacruz, USA, Cat No. sc-20035), TRP- The first antibody against 1 (Santacruz, USA, Cat No. sc-166857) and the primary antibody against TRP-2 (Santacruz, USA, Cat No. sc-74439) were diluted 1:3000 respectively to the membrane and 4 It was reacted at °C for 20 hours. After washing the membrane three times for 10 minutes with TBST buffer, HRP-binding anti-mouse (Santacruz, USA, Cat No. sc-2005) as a secondary antibody was diluted at a ratio of 1:3000 and reacted for 1 hour at room temperature. Then, it was washed three times for 10 minutes with TBST buffer. A chemiluminescent substrate reagent (ECL western solution, DoGen, Korea) was treated, and the generated luminescence was detected using LAS-1000 (Fujifilm, Japan) to confirm the protein expression level. The expression level of β-actin protein was measured as a control for comparing the level of expression. Specifically, the same method as the above method was performed using an anti-β-actin antibody as the primary antibody at a ratio of 1:5000. Expression of β-actin was confirmed.

Allantolactone, a compound derived from Tomok-hyang, was shown to inhibit the protein expression levels of MITF, tyrosinase, TRP-1 and TRP-2, which are melanin synthesis promoting factors (Fig. 17), especially melanin synthesis. The protein expression of MITF, an important transcriptional regulator of the process, was completely inhibited even when allantolactone was treated with 10 μM.

The hexane fraction of the Tomok Hyang extract according to the present invention or the compound represented by Formula 1 or 2 isolated therefrom inhibits melanin synthesis, and the compound is a melanin synthesis promoting factor MITF (microphthalmia-associated transcription factor), tyrosina As it exhibits the effect of inhibiting the mRNA or protein expression of tyrosinase, tyrosinase related protein 1 or tyrosinase related protein 2, cosmetic composition for skin whitening , It can be usefully used as a pharmaceutical composition or health functional food.

Claims (12)

1. A cosmetic composition for skin whitening comprising the hexane fraction of the extract of Inula helenium as an active ingredient.
2. The cosmetic composition for skin whitening according to claim 1, wherein the civil hyang extract is extracted using water, C ₁ to C ₂ alcohol, or a mixed solvent thereof.
3. The cosmetic composition for skin whitening according to claim 1, wherein the Geomok Hyang extract is an extract obtained from the root of Geomok Hyang.
4. The cosmetic composition for skin whitening according to claim 1, wherein the hexane fraction is a hexane fraction of the fractions obtained by sequentially fractionating the civil scent extract into hexane, methylene chloride, ethyl acetate and butanol.
5. The cosmetic composition of claim 1, wherein the hexane fraction inhibits melanin synthesis.
6. A cosmetic composition for skin whitening comprising, as an active ingredient, a compound represented by the following Formula 1 or 2 isolated from the hexane fraction of the extract of Inula helenium or a pharmaceutically acceptable salt thereof:

   [Formula 1]

   

   [Formula 2]

   

   .
7. The cosmetic composition for skin whitening according to claim 6, wherein the compound inhibits melanin synthesis.
8. According to claim 6, wherein the compound is MITF (microphthalmia-associated transcription factor), tyrosinase (tyrosinase), tyrosinase related protein 1 (tyrosinase related protein 1) and tyrosinase related protein 2 (tyrosinase related protein 2) To inhibit the expression of any one or more selected from the group consisting of protein 2), a cosmetic composition for skin whitening.
9. Tomokhyang ( Inula helenium ) a pharmaceutical composition for skin whitening comprising the hexane fraction of the extract as an active ingredient.
10. A health functional food for skin whitening comprising the hexane fraction of the extract of Inula helenium as an active ingredient.
11. A pharmaceutical composition for skin whitening comprising as an active ingredient a compound represented by the following formula 1 or 2 isolated from the hexane fraction of the extract of Inula helenium or a pharmaceutically acceptable salt thereof:

    [Formula 1]

    

    [Formula 2]

    

    .
12. A health functional food for skin whitening comprising a compound represented by the following formula 1 or 2 isolated from the hexane fraction of the extract of Inula helenium or a pharmaceutically acceptable salt thereof as an active ingredient:

    [Formula 1]

    

    [Formula 2]

    

    .

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