KR20240132172A - Whitening composition containing fermented liquid or fermented extract of Diplotaxis tenuifolia L. - Google Patents

Abstract

The present invention relates to a whitening composition containing a fermented liquid or an extract of a fermented product of wild arugula ( Diplotaxis tenuifolia L.) as an effective ingredient. The fermented liquid or the extract of a fermented product of wild arugula inhibits tyrosinase activity involved in melanin production, and therefore can be usefully used as an effective ingredient of a cosmetic composition, a food composition, a quasi-drug composition, a composition for external use in skin whitening, a health functional food capable of preventing or alleviating skin pigmentation diseases, or a therapeutic agent for skin pigmentation diseases.

Description

Whitening composition containing fermented liquid or fermented extract of Diplotaxis tenuifolia L.

The present invention relates to a whitening composition comprising an extract of wild arugula fermentation liquid or fermented product.

Melanin is produced in melanosomes in the skin and plays a role in protecting the skin from damage caused by ultraviolet rays. However, when exposed to ultraviolet rays for a long time or when external stimuli including environmental pollution and internal stimuli such as hormones and inflammatory factors act simultaneously, melanin is produced excessively and accumulates, causing skin disorders such as blemishes, freckles, spots, liver spots, and hyperpigmentation, and promoting skin aging.

Skin color is genetically determined by the concentration and distribution of melanin in skin cells, but it can also be affected by environmental or physiological conditions such as exposure to ultraviolet rays from the sun, use of female hormones, pregnancy, cosmetics, fatigue, and stress. This can cause melanin pigmentation to be deposited on the skin, darkening the skin and causing spots and freckles.

It increases the production of new melanin through the activity of tyrosinase, an enzyme that produces melanin, and the synthesis of melanosomes. Therefore, if the activity of tyrosinase, an important enzyme in melanin production, is inhibited, a skin whitening effect can be expected.

Patent Publication No. 10-2012-0061733 (June 13, 2012)

Accordingly, in order to develop a whitening agent that inhibits the activity of tyrosinase and thus inhibits melanin production, the present invention was completed by studying various natural materials and confirming that a fermented liquid or an extract of a fermented product of wild arugula inhibits the activity of tyrosinase and melanin production.

In order to achieve the above-mentioned purpose, the purpose of the present invention is to provide a whitening cosmetic composition containing a fermented liquid or an extract of a fermented product of wild arugula ( Diplotaxis tenuifolia L.) as an effective ingredient.

Another object of the present invention is to provide a whitening food composition containing an extract of a fermented liquid or fermented product of wild arugula ( Diplotaxis tenuifolia L.) as an effective ingredient.

Another object of the present invention is to provide a health functional food for preventing or alleviating pigmentation disease, containing a fermented solution or an extract of a fermented product of wild arugula ( Diplotaxis tenuifolia L.) as an effective ingredient.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating pigmentation diseases, containing an extract of a fermented solution or a fermented product of wild arugula ( Diplotaxis tenuifolia L.) as an effective ingredient.

Another object of the present invention is to provide a whitening quasi-drug composition containing a fermented liquid or an extract of a fermented product of wild arugula ( Diplotaxis tenuifolia L.) as an effective ingredient.

Another object of the present invention is to provide a skin external application composition for whitening or preventing or treating pigmentation diseases, containing a fermented solution or an extract of a fermented product of wild arugula ( Diplotaxis tenuifolia L.) as an effective ingredient.

The following describes this specification in more detail.

Each description and embodiment disclosed in the present invention can also be applied to other descriptions and embodiments for each. That is, all combinations of various elements disclosed in the present invention fall within the scope of the present invention. In addition, the scope of the present invention cannot be considered limited by the specific description described below.

Expressions such as “including” as used herein should be understood as open-ended terms implying the possibility of including other embodiments, unless specifically stated otherwise in the phrase or sentence in which the expression is included.

The terms or words used in the description and claims of the present invention should not be interpreted as limited to their usual or dictionary meanings, but should be interpreted as meanings and concepts that conform to the technical idea of the present invention, based on the principle that the inventor can appropriately define the concept of the term in order to explain his or her own invention in the best manner.

As one embodiment to achieve the above object, the present invention provides a whitening cosmetic composition containing a fermented liquid or an extract of a fermented product of wild arugula ( Diplotaxis tenuifolia L.) as an effective ingredient.

In the present invention, the term "wild arugula" has the scientific name Diplotaxis tenuifolia L. The wild arugula may use the whole plant or the above-ground part or the underground part. The above-ground part may be a stem, a branch, a leaf, a flower or a combination thereof. The underground part may be a root.

The above wild arugula may be grown naturally or cultivated artificially. It may be grown by sprouting seeds indoors, or it may be a wild arugula that is commonly sold.

In the present invention, the term "wild arugula" may use raw wild arugula, and raw wild arugula means undried wild arugula, corresponding to dried wild arugula.

The above wild arugula fermentation liquid or wild arugula fermentation product may be obtained by fermentation with sugar or sugar and lactic acid bacteria. Specifically, it may be obtained by fermentation with sugar in an amount of 15 to 25 wt % relative to the weight of raw wild arugula, or by fermentation using sugar in an amount of 15 to 25 wt % relative to the weight of raw wild arugula and lactic acid bacteria.

The above lactic acid bacteria may be at least one selected from the group consisting of Lactiplantibacillus genus, Limosilactobacillus genus, and Pediococcus genus.

Specifically, the lactic acid bacteria may be at least one species selected from the group consisting of Lactiplantibacillus plantarum , Limosilactobacillus fermentum , and Pediococcus pentosaceus .

The above wild arugula fermentation liquid may be a supernatant of fermented wild arugula, and may be a supernatant of wild arugula fermented with 15 to 25 wt % of sugar based on the weight of raw wild arugula, or may be a supernatant of wild arugula fermented using lactic acid bacteria and 15 to 25 wt % of sugar based on the weight of raw wild arugula.

The above term "wild arugula ferment" refers to the solid portion corresponding to the remaining supernatant after separating the supernatant from fermented wild arugula.

The extract of the above wild arugula fermentation product may be obtained by extracting the wild arugula fermentation product with water, an alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof. Specifically, the extract may be obtained by extracting the wild arugula fermentation product with water, an alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof, from a wild arugula fermentation product fermented with 15 to 25 wt % of sugar based on the weight of the raw wild arugula or a wild arugula fermentation product fermented using lactic acid bacteria and 15 to 25 wt % of sugar based on the weight of the raw wild arugula.

In the present invention, the term "extract" includes the extract itself and all formulations that can be formed using the extract, such as an extract obtained by extraction treatment, a diluted or concentrated extract, a dried product obtained by drying the extract, a controlled or purified product of the extract, or a mixture thereof.

In the present invention, drying of the extract can be carried out by a known method within a range where useful components from the collected plant are not destroyed, and for example, can be carried out by a natural drying method in a shaded area. In addition, crushing or pulverizing can be carried out by crushing or pulverizing to a degree that useful components of the plant can be sufficiently extracted during the subsequent extraction process to make it powdered. The drying and crushing or pulverizing processes can be carried out in reverse order or repeatedly as needed.

In the extraction of the present invention, the extraction method is not particularly limited, and extraction can be performed according to a method commonly used in the relevant technical field. Non-limiting examples of the extraction method include hot water extraction, ultrasonic extraction, filtration, and reflux extraction, and these can be performed alone or in combination of two or more methods.

More specifically, the extract of the wild arugula fermentation product may be an ethanol extract of the wild arugula fermentation product.

The above ethanol extract can be obtained by separating the supernatant from fermented wild arugula, adding ethanol to the remaining residue, performing reflux extraction at 70 to 80°C for 1 to 4 hours, filtering the resulting extract, and then concentrating under reduced pressure. The ethanol can use 30 to 90% (v/v) of ethanol.

The above wild arugula fermentation can proceed for 1 to 28 days, and specifically for 2 to 10 days.

In the present invention, “active ingredient” means an ingredient that exhibits the desired activity alone or can exhibit the activity together with a carrier that is inactive on its own.

In addition, the term "whitening" used in the present invention comprehensively means a method of increasing the brightness of skin whose brightness has decreased due to an excess of pigments such as melanin, or a method of maintaining the brightness of skin at a certain level, skin with increased brightness formed by the method, etc., and can specifically mean skin whitening. "Skin whitening" is understood as a result of inhibiting the production of melanin, and specifically can be understood as an improvement in symptoms resulting from an increase in melanin, such as improvement of blemishes, freckles, and skin aging, as the production of melanin is inhibited.

The whitening cosmetic composition of the present invention may contain, in addition to the extract of wild arugula fermentation liquid or fermented product, which is an effective ingredient, a compound or natural extract known to have a skin whitening effect so as to increase or reinforce the skin whitening effect. Here, the compounds or natural extracts known to have a whitening effect include, but are not limited to, mercaptosuccinic acid, mercaptodextran, teprenone, dihydroxy-isoquinoline, indomethacin, 3-hydroxy-limumen, vitamin K, thiazolidone, kynurenine, lemon extract, cucumber extract, odi extract, rosemary extract, acerola cherry extract, ginkgo extract, geranium extract, and the like.

Meanwhile, in the whitening cosmetic composition of the present invention, the effective ingredient may be included in any amount (effective amount) depending on the intended use, formulation, mixing purpose, etc., as long as it can exhibit skin whitening activity. A typical effective amount may be included within a range of 0.001 wt % to 99.99 wt % based on the total weight of the composition. Here, the "effective amount" refers to the amount of the effective ingredient that can induce a whitening effect. This effective amount can be experimentally determined within the normal ability range of a person skilled in the art.

The whitening cosmetic composition of the present invention can be manufactured in various forms. For example, the cosmetic composition of the present invention can be manufactured in any formulation commonly manufactured in the art, and can be formulated as, for example, a solution, a suspension, an emulsion, a paste, a gel, a cream, a lotion, a powder, a soap, a cleansing, an oil, a powder foundation, an emulsion foundation, a wax foundation, and a spray, but is not limited thereto. In addition, specifically, the composition may have a formulation selected from the group consisting of 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, milk, lipstick, makeup base, foundation, pressed powder, and loose powder, but is not limited thereto.

The whitening cosmetic composition of the present invention may contain, in addition to its effective ingredient, a carrier acceptable in cosmetic formulations. Here, the term "acceptable carrier in cosmetic formulations" refers to a compound or composition already known and used, or a compound or composition to be developed in the future, which may be included in cosmetic formulations and which does not have toxicity, instability, or irritation beyond what the human body can adapt to when in contact with the skin. The carrier may be included in the whitening cosmetic composition of the present invention in an amount of about 1 wt % to about 99.99 wt % based on the total weight of the composition, preferably about 5 wt % to about 99 wt % of the weight of the composition.

However, since the above ratio varies depending on the form in which the cosmetic of the present invention is manufactured as described above, as well as its specific application site (face or hands) and its desired application amount, the above ratio should not be understood as limiting the scope of the present invention in any way.

Meanwhile, examples of the carrier include alcohol, oil, surfactant, fatty acid, silicone oil, humectant, moisturizer, viscosity modifier, emulsion, stabilizer, UV blocker, colorant, fragrance, etc. Compounds/compositions that can be used as the carrier, such as alcohol, oil, surfactant, fatty acid, silicone oil, humectant, moisturizer, viscosity modifier, emulsion, stabilizer, UV blocker, colorant, fragrance, etc. are already known in the art, and therefore, a person skilled in the art can select and use an appropriate corresponding substance/composition.

In another aspect, the present invention provides a whitening food composition containing a fermented solution or an extract of a fermented product of wild arugula ( Diplotaxis tenuifolia L.) as an effective ingredient.

In the present invention, the description of wild arugula ( Diplotaxis tenuifolia L.), fermented wild arugula ( Diplotaxis tenuifolia L.), fermented wild arugula (Diplotaxis tenuifolia L.), fermented wild arugula ( Diplotaxis tenuifolia L.), extract of the fermented wild arugula, and whitening are as described above.

The above whitening food composition may have a function of helping to lighten the color of melanin pigment deposited on the skin, and may have a function of helping to whiten the skin by preventing excessive deposition of melanin pigment on the skin and thereby suppressing the occurrence of skin pigmentation disease.

The food composition of the present invention may include forms such as pills, powders, granules, infusions, tablets, capsules or liquids, and there is no particular limitation on the types of foods to which the extract of the wild arugula fermented liquid or fermented product of the present invention can be added. Examples of foods to which the above substance can be added include dairy products including meat, sausages, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, beverages, tea, drinks, alcoholic beverages and vitamin complexes.

In addition to the extract of wild arugula fermentation liquid or fermented product, other ingredients may be added to the above food composition, and the types thereof are not particularly limited. For example, as with ordinary foods, various herbal extracts, food science-acceptable food additives, or natural carbohydrates may be contained as additional ingredients, but are not limited thereto.

In the present invention, the term "food supplement additive" means a component that can be added to food as an auxiliary, and can be appropriately selected and used by those skilled in the art as added in the manufacture of food of each formulation. Examples of food supplement additives include various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and fillers, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, and the like, but the types of food supplement additives of the present invention are not limited by the above examples.

Examples of the above natural carbohydrates include monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; and polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. In addition to the above, natural flavoring agents (thaumatin, etc.), stevia extracts (rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used.

The food composition of the present invention may include a health functional food. The term "health functional food" used in the present invention refers to a food manufactured and processed in the form of tablets, capsules, powders, granules, liquids, and pills using raw materials or ingredients having functionality useful to the human body. Here, functionality means obtaining a useful effect for health purposes such as regulating nutrients for the structure and function of the human body or physiological effects. The health functional food of the present invention can be manufactured by a method commonly used in the art, and can be manufactured by adding raw materials and ingredients commonly added in the art during the manufacturing process. In addition, unlike general drugs, it has the advantage of not having side effects that may occur when taking drugs for a long period of time by using food as a raw material, and can be highly portable.

The amount of the active ingredient mixed can be appropriately determined depending on the purpose of use (prevention, health or therapeutic treatment). Generally, when manufacturing a food, the active ingredient of the present invention can be added in an amount of 1 to 50 wt%, preferably 5 to 10 wt%, of the raw material composition, but is not limited thereto. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount can be used below the above range.

In another aspect, the present invention provides a health functional food for preventing or alleviating pigmentation disease, containing a fermented solution or an extract of a fermented product of wild arugula ( Diplotaxis tenuifolia L.) as an effective ingredient.

In the present invention, the description of wild arugula ( Diplotaxis tenuifolia L.), fermented wild arugula ( Diplotaxis tenuifolia L.), fermented wild arugula ( Diplotaxis tenuifolia L.), extract of the fermented wild arugula (Diplotaxis tenuifolia L.), and health functional food is as described above.

The health functional food of the present invention may have a function of helping to lighten the color of melanin pigment deposited on the skin, and may have a function of helping to whiten the skin by preventing excessive deposition of melanin pigment on the skin and thereby suppressing the occurrence of skin pigmentation diseases.

In the present invention, the pigmentation disease is a skin pigmentation disease, and may include, but is not limited to, freckles, senile spots, liver spots, lentigines, brown or black spots, solar pigment spots, cyanic melasma, hyperpigmentation after drug use, gravidic chloasma, or post-inflammatory hyperpigmentation due to wounds or dermatitis including abrasions and burns.

In another aspect, the present invention provides a pharmaceutical composition for preventing or treating pigmentation diseases, containing a fermented solution or an extract of a fermented product of wild arugula ( Diplotaxis tenuifolia L.) as an effective ingredient.

In the present invention, the description of wild arugula ( Diplotaxis tenuifolia L.), fermented wild arugula ( Diplotaxis tenuifolia L.), fermented wild arugula ( Diplotaxis tenuifolia L.), extract of the fermented wild arugula (Diplotaxis tenuifolia L.), and pigmentation disease is as described above.

In the present invention, preventing or treating skin pigmentation disease may mean not only preventing melanin pigment from being deposited on the skin by applying a composition containing a wild arugula fermented liquid or an extract of the fermented product to hyperpigmented skin, but also all actions of removing melanin already deposited on the skin.

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

In addition, when formulating the pharmaceutical composition of the present invention, it can be formulated using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrating agents, and surfactants that are commonly used.

Solid preparations for oral administration include tablets, pills, powders, granules, and capsules, and these solid preparations are prepared by mixing the active ingredient of the present invention with at least one excipient, such as starch, calcium carbonate, sucrose, lactose, and gelatin. In addition to simple excipients, lubricants may also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups, and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, may be included. Preparations for parenteral administration include topical preparations such as creams, lotions, ointments (semi-solid external medicines), microemulsions, gels, pastes, and transdermal preparations (TTS).

The pharmaceutical composition of the present invention is usually administered at a daily dosage of 0.001 to 150 mg/kg body weight, and may be administered once or in several divided doses. However, the actual dosage of the pharmaceutical composition of the present invention is determined in light of various related factors such as the administration route, the patient's age, sex, body weight, and the patient's severity, and therefore the dosage should not be understood as limiting the scope of the present invention in any way.

In another aspect, the present invention provides a whitening quasi-drug composition containing a fermented liquid or an extract of a fermented product of wild arugula ( Diplotaxis tenuifolia L.) as an effective ingredient.

In the present invention, the description of wild arugula ( Diplotaxis tenuifolia L.), fermented wild arugula ( Diplotaxis tenuifolia L.), fermented wild arugula (Diplotaxis tenuifolia L.), fermented wild arugula ( Diplotaxis tenuifolia L.), extract of the fermented wild arugula, and whitening are as described above.

The term "quasi-drug" used in the present invention refers to products that are used for the purpose of diagnosing, treating, improving, alleviating, managing or preventing diseases of humans or animals, and have a milder effect than a pharmaceutical product. For example, according to the Pharmaceutical Affairs Act, quasi-drugs are products other than those used for the purpose of pharmaceutical products, and include products used for the treatment or prevention of diseases of humans or animals, products that have a mild effect on the human body or do not act directly, etc.

The above-described pharmaceutical composition of the present invention can be manufactured in a formulation selected from the group consisting of body cleansers, disinfectant cleaners, detergents, kitchen detergents, cleaning detergents, toothpaste, gargles, wet tissues, detergents, soaps, hand washes, hair cleansers, hair softeners, humidifier fillers, masks, ointments, and filter fillers, but is not limited thereto.

In another aspect, the present invention provides a skin external application composition for whitening or preventing or treating pigmentation diseases, containing a fermented solution or an extract of a fermented product of wild arugula ( Diplotaxis tenuifolia L.) as an effective ingredient.

In the present invention, the description of wild arugula ( Diplotaxis tenuifolia L.), fermented wild arugula ( Diplotaxis tenuifolia L.), fermented wild arugula ( Diplotaxis tenuifolia L.), extract of the fermented wild arugula (Diplotaxis tenuifolia L.), and pigmentation disease is as described above.

The term "external preparation" of the present invention refers to a preparation provided for external use, and includes external powders, external tablets, external solutions, ointments, warnings, suppositories, etc., and the skin external preparation of the present invention particularly includes, without limitation, a preparation that acts on the skin externally.

The skin external preparation according to the present invention may be a parenteral administration preparation formulated in a solid, semi-solid or liquid form by adding commercially available inorganic or organic carriers, excipients and diluents. The preparation for parenteral administration may be a transdermal administration formulation selected from the group consisting of drops, ointments, lotions, gels, creams, patches, sprays, suspensions and emulsions, but is not limited thereto.

Carriers, excipients and diluents that may be included in the above external preparations include lactose, dextrose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

In addition to the extract of the wild arugula fermented liquid or fermented product of the present invention, a person skilled in the art can appropriately select and mix other ingredients for the external skin preparation composition according to the formulation or intended use of the external skin preparation without difficulty. In this case, a synergistic effect may occur when applied simultaneously with other raw materials.

The skin external preparation composition of the present invention may contain an effective ingredient at 0.0001 to 30% (w/v) based on the total weight of the composition. If it is contained at less than 0.0001% (w/v), the whitening effect of the skin or the prevention or treatment of pigmentation diseases cannot be expected in practice. On the other hand, if it is contained at more than 30% (w/v), the overall processability of the composition, such as solubility in a solvent, may be reduced, which may limit its use in various purposes, such as formulations.

The present invention relates to a whitening composition containing a fermented liquid or an extract of a fermented product of wild arugula ( Diplotaxis tenuifolia L.) as an effective ingredient. The fermented liquid or the extract of a fermented product of wild arugula inhibits tyrosinase activity involved in melanin production, and therefore can be usefully used as an effective ingredient of a cosmetic composition, a food composition, a quasi-drug composition, a composition for external use in skin whitening, a health functional food capable of preventing or alleviating skin pigmentation diseases, or a therapeutic agent for skin pigmentation diseases.

Figure 1 shows the inhibition of tyrosinase activity of the 50% EtOH extract of raw wild arugula prepared in Example 1, the ethanol extract (50% EtOH) of the 3-day and 6-day wild arugula natural fermentation product fermented with sugar in Example 2, and the wild arugula natural fermentation solution (supernatant) in mouse B16-F10 melanoma cells.
Figure 2 shows the inhibition of tyrosinase activity of the 50% EtOH extract of raw wild arugula prepared in Example 1, the ethanol extract (50% EtOH) of the 3-day and 6-day wild arugula fermentation product fermented with sugar and LF in Example 3, and the wild arugula fermentation solution (supernatant) in mouse B16-F10 melanoma cells.
Figure 3 shows the inhibition of tyrosinase activity of the 50% EtOH extract of raw wild arugula prepared in Example 1, the ethanol extract (50% EtOH) of the 3-day and 6-day wild arugula fermentation product fermented with sugar and LP in Example 4, and the wild arugula fermentation solution (supernatant) in mouse B16-F10 melanoma cells.
Figure 4 shows the inhibition of tyrosinase activity of the 50% EtOH extract of raw wild arugula prepared in Example 1, the ethanol extract (50% EtOH) of the 3-day and 6-day wild arugula fermentation product fermented with sugar and PP in Example 5, and the wild arugula fermentation solution (supernatant) in mouse B16-F10 melanoma cells.
Figure 5 shows a calibration curve using catechin as a standard substance.

Hereinafter, embodiments of the present invention will be described in detail with reference to the attached drawings so that those skilled in the art can easily implement the present invention. However, the present invention may be implemented in various different forms and is not limited to the embodiments described herein.

Manufacturing Examples 1-5

ingredient

The main ingredient for fermentation, wild arugula, was grown organically in the Indoor farm of Next On, an agricultural corporation ( Diplotaxis tenuifolia L.). Brown sugar (Cheil Jedang) was used as the sugar for fermentation. The part used for wild arugula fermentation was the leaves, and the fermentation vessel was a glass vessel (Bormioli Rocco, 500 mL) that was sterilized in boiling water and cooled sufficiently in a clean bench before use. Fermentation was performed using a digital incubator (Coatech Korea, HQ-DI 156).

The strains used in the following examples for fermentation were provided by the Kimchi Microorganism Resource Bank of the Korean Collection for Kimchi Microorganisms (KCKM).

The three lactic acid bacteria strains used were Limosilactobacillus fermentum (KCKM 0729, hereinafter referred to as “LF” strain), Lactiplantibacillus plantarum (KCKM 0531, hereinafter referred to as “LP” strain), and Pediococcus pentosaceus (KCKM 0941, hereinafter referred to as “PP” strain).

Example 1.

A 10-fold volume of 50% ethanol was added to raw wild arugula leaves, and reflux extraction was performed at 70 to 80°C for 1 hour. After filtering the extract, the filtrate was concentrated under reduced pressure to obtain a 50% ethanol (EtOH) extract of wild arugula.

Example 2. Production of wild arugula natural fermentation supernatant and ethanol extract of fermented product using sugar (20 wt%) (natural fermentation)

150 g of raw wild arugula and 30 g of brown sugar (20% by weight of raw wild arugula) were weighed and placed in a fermentation vessel, the lid was closed and sealed, and fermentation was performed in an incubator set to 30℃. On the 3rd and 6th days of the fermentation period, the fermented wild arugula pulp and wild arugula supernatant were separated and collected, respectively.

The supernatant of wild arugula naturally fermented with 20 wt% sugar based on the weight of the wild arugula was used as the natural fermentation liquid. In addition, 10 times the volume of 50% ethanol was added to the dried part of the fermented wild arugula and refluxed for 1 hour at 70-80℃. After filtering the extract, the filtrate was concentrated under reduced pressure to obtain an ethanol extract of the fermented wild arugula.

Example 3. Production of wild arugula fermentation supernatant and ethanol extract of wild arugula fermentation product using sugar (20%) and LF lactic acid bacteria (LF fermentation)

Limosilactobacillus fermentum KCKM distributed by KCKM 0729 was diluted in MRS broth medium, spread on MRS agar plate, and cultured at 30℃ for 24 hours before use for inoculation. 150g of raw wild arugula and 30g of brown sugar (20% by weight compared to the weight of raw wild arugula) were weighed and placed in a fermentation vessel, and Limosilactobacillus fermentum KCKM produced on the agar plate Two 1 mm sized single colonies of 0729 were placed in a tube containing 1 mL of sterilized distilled water and inoculated into a fermentation vessel. After rinsing the tube with 1 mL of distilled water again, it was injected into the fermentation vessel, sealed with a lid, and fermentation was performed in an incubator set to 30℃. On the 3rd and 6th days of the fermentation period, the fermented wild arugula stems and wild arugula supernatant were separated and collected, respectively.

The supernatant of wild arugula fermented with sugar (20%) and LF lactic acid bacteria was used as the fermentation solution. In addition, 10-fold volume of 50% ethanol was added to the dried portion of wild arugula fermented with sugar (20%) and LF lactic acid bacteria, and reflux extraction was performed at 70-80℃ for 1 hour. After filtering the extract, the filtrate was concentrated under reduced pressure to obtain an ethanol extract of the fermented wild arugula.

Example 4. Production of wild arugula fermentation supernatant and ethanol extract of wild arugula fermentation product using sugar (20%) and LP lactic acid bacteria (LP fermentation)

Lactiplantibacillus plantarum KCKM 0531 provided by KCKM was diluted in MRS broth medium, spread on MRS agar plates, and used for inoculation after culturing at 30℃ for 24 hours. 150 g of raw wild arugula and 30 g of brown sugar (20 wt% of the weight of raw wild arugula) were weighed and added to a fermentation vessel, and two 1 mm-sized single colonies of Lactiplantibacillus plantarum KCKM 0531 grown on the agar plate were dissolved in a tube containing 1 mL of sterilized distilled water and inoculated into the fermentation vessel. After rinsing the tube with 1 mL of distilled water again, it was injected into the fermentation vessel, closed and sealed, and fermentation was performed in an incubator set to 30℃. On the 3rd and 6th days of the fermentation period, the fermented wild arugula pulp and the wild arugula supernatant were separated and collected, respectively.

The supernatant of wild arugula fermented with sugar (20%) and LP lactic acid bacteria was used as the fermentation solution. In addition, 10-fold volume of 50% ethanol was added to the dried portion of wild arugula fermented with sugar (20%) and LP lactic acid bacteria, and reflux extraction was performed at 70-80℃ for 1 hour. After filtering the extract, the filtrate was concentrated under reduced pressure to obtain an ethanol extract of the fermented wild arugula.

Example 5. Production of wild arugula fermentation supernatant and ethanol extract of wild arugula fermentation product using sugar (20%) and PP lactic acid bacteria (PP fermentation)

Pediococcus pentosaceus KCKM 0941, received from KCKM, was diluted in MRS broth medium, spread on MRS agar plates, and used for inoculation after culturing at 30℃ for 24 hours. 150 g of raw wild arugula and 30 g of brown sugar (20 wt% of the weight of raw wild arugula) were weighed and added to a fermentation vessel, and two 1 mm-sized single colonies of Pediococcus pentosaceus KCKM 0941 grown on the agar plate were dissolved in a tube containing 1 mL of sterilized distilled water and inoculated into the fermentation vessel. After rinsing the tube with 1 mL of distilled water again, it was injected into the fermentation vessel, closed and sealed, and fermentation was performed in an incubator set to 30℃. On the 3rd and 6th days of the fermentation period, the fermented wild arugula pulp and the wild arugula supernatant were separated and collected, respectively.

The supernatant of wild arugula fermented with sugar (20%) and PP lactic acid bacteria was used as the fermentation solution. In addition, 10-fold amount of 50% ethanol was added to the dried portion of wild arugula fermented with sugar (20%) and PP lactic acid bacteria, and reflux extraction was performed at 70-80℃ for 1 hour. After filtering the extract, the filtrate was concentrated under reduced pressure to obtain an ethanol extract of the fermented wild arugula.

Experimental Example 1. Tyrosinase Inhibitory Activity of Natural Fermentation Supernatant and Fermentation Extract of Wild Arugula

B16-F10 melanoma cells were seeded at 1x10 cells/well in a 24-well plate and cultured for 24 hours. The cells were treated with 25 μg/mL of the 50% EtOH extract of raw wild arugula prepared in Example 1, the ethanol extract (50% EtOH) of the 3rd and 6th day wild arugula natural fermentation product fermented with sugar in Example 2, and the wild arugula natural fermentation solution (supernatant) in DMEM medium not containing FBS and antibiotics, and cultured for another 24 hours. After removing the medium treated with the samples, the cells were washed with phosphate-buffered saline (PBS, pH 6.8), and PBS containing 1% Triton X-100 was added to the wells. The cells attached to the wells were suspend in a 1.5 mL tube. The sample with destroyed cell membrane was centrifuged at 15,000 rpm for 10 minutes, and the supernatant was used as an enzyme source for measuring tyrosinase activity and a sample for measuring protein. Protein was quantified using a protein quantification kit (BCA protein kit), and tyrosinase activity was measured by adding 100 ㎕ of 10 mM L-DOPA and 40 ㎕ of tyrosinase enzyme source (supernatant obtained from cells), incubating at 37℃ for 1 hour, and then measuring the absorbance at 405 nm. 25 ㎕/mL kojic acid was used as a positive control.

Figure 1 shows the inhibition of tyrosinase activity of the 50% EtOH extract of raw wild arugula prepared in Example 1, the ethanol extract (50% EtOH) of the 3-day and 6-day wild arugula natural fermentation product fermented with sugar in Example 2, and the wild arugula natural fermentation solution (supernatant) in mouse B16-F10 melanoma cells.

As a result, the 50% EtOH extract of raw wild arugula of Example 1, the extract (50% EtOH) of the natural fermentation of wild arugula fermented with sugar in Example 2, and the wild arugula natural fermentation liquid (supernatant) showed excellent tyrosinase activity inhibition. In particular, the extract (50% EtOH) of the natural fermentation of wild arugula prepared in Example 2 showed significantly superior tyrosinase inhibition activity compared to kojic acid, the positive control.

Experimental Example 2. Tyrosinase Inhibitory Activity of Fermented Supernatant and Extract of Fermented Product of Wild Arugula Using Sugar and LF Lactic Acid Bacteria

B16-F10 melanoma cells were seeded at 1x10 cells/well in a 24-well plate, cultured for 24 hours, and treated with 25 μg/mL of the 50% EtOH extract of raw wild arugula prepared in Example 1, the ethanol extract (50% EtOH) of the 3-day and 6-day wild arugula fermentation product fermented with sugar and LF in Example 3, and the wild arugula fermentation broth (supernatant) in DMEM medium not containing FBS and antibiotics, and cultured for further 24 hours. After removing the medium treated with the samples, the cells were washed with phosphate-buffered saline (PBS, pH 6.8), and PBS containing 1% Triton X-100 was added to the wells. The cells attached to the wells were suspend in water and collected in a 1.5 mL tube. The sample with destroyed cell membrane was centrifuged at 15,000 rpm for 10 minutes, and the supernatant was used as an enzyme source for measuring tyrosinase activity and a sample for measuring protein. Protein was quantified using a protein quantification kit (BCA protein kit), and tyrosinase activity was measured by adding 100 ㎕ of 10 mM L-DOPA and 40 ㎕ of tyrosinase enzyme source (supernatant obtained from cells), incubating at 37℃ for 1 hour, and then measuring the absorbance at 405 nm. 25 ㎕/mL kojic acid was used as a positive control.

Figure 2 shows the inhibition of tyrosinase activity of the 50% EtOH extract of raw wild arugula prepared in Example 1, the ethanol extract (50% EtOH) of the 3-day and 6-day wild arugula fermentation product fermented with sugar and LF in Example 3, and the wild arugula fermentation solution (supernatant) in mouse B16-F10 melanoma cells.

As a result, the 50% EtOH extract of raw wild arugula in Example 1, the extract (50% EtOH) of the wild arugula fermented with sugar and LF in Example 3, and the wild arugula fermented liquid (supernatant) showed excellent tyrosinase activity inhibition. In particular, the extract (50% EtOH) of the wild arugula fermented with sugar and LF in Example 3 showed significantly superior tyrosinase inhibition activity compared to kojic acid, the positive control.

Experimental Example 3. Tyrosinase Inhibitory Activity of Fermented Supernatant and Extract of Fermented Product of Wild Arugula Using Sugar and LP Lactic Acid Bacteria

B16-F10 melanoma cells were seeded at 1x10 cells/well in a 24-well plate, cultured for 24 hours, and treated with 25 μg/mL of the 50% EtOH extract of raw wild arugula prepared in Example 1 in DMEM medium not containing FBS and antibiotics, the ethanol extract (50% EtOH) of the 3-day and 6-day wild arugula fermentation product in Example 4, and the wild arugula fermentation broth (supernatant) and cultured for further 24 hours. After removing the medium treated with the samples, the cells were washed with phosphate-buffered saline (PBS, pH 6.8), and PBS containing 1% Triton X-100 was added to the wells. The cells attached to the wells were suspend in water and collected in a 1.5 mL tube. The sample with destroyed cell membrane was centrifuged at 15,000 rpm for 10 minutes, and the supernatant was used as an enzyme source for measuring tyrosinase activity and a sample for measuring protein. Protein was quantified using a protein quantification kit (BCA protein kit), and tyrosinase activity was measured by adding 100 ㎕ of 10 mM L-DOPA and 40 ㎕ of tyrosinase enzyme source (supernatant obtained from cells), incubating at 37℃ for 1 hour, and then measuring the absorbance at 405 nm. 25 ㎕/mL kojic acid was used as a positive control.

Figure 3 shows the inhibition of tyrosinase activity of the 50% EtOH extract of raw wild arugula prepared in Example 1, the ethanol extract (50% EtOH) of the 3-day and 6-day wild arugula fermentation product fermented with sugar and LP in Example 4, and the wild arugula fermentation solution (supernatant) in mouse B16-F10 melanoma cells.

As a result, the 50% EtOH extract of raw wild arugula in Example 1, the extract (50% EtOH) of the wild arugula fermented with sugar and LP in Example 4, and the wild arugula fermented liquid (supernatant) showed excellent tyrosinase activity inhibition. In particular, the extract (50% EtOH) of the wild arugula fermented with sugar and LP in Example 4 showed significantly superior tyrosinase inhibition activity compared to kojic acid, the positive control.

Experimental Example 4. Tyrosinase Inhibitory Activity of Fermented Supernatant and Extract of Fermented Product of Wild Arugula Using Sugar and PP Lactic Acid Bacteria

B16-F10 melanoma cells were seeded at 1x10 cells/well in a 24-well plate, cultured for 24 hours, and treated with 25 μg/mL of the 50% EtOH extract of raw wild arugula prepared in Example 1, the ethanol extract (50% EtOH) of the 3-day and 6-day wild arugula fermentation product fermented with sugar and PP in Example 5, and the wild arugula fermentation broth (supernatant) in DMEM medium not containing FBS and antibiotics, and cultured for further 24 hours. After removing the medium treated with the samples, the cells were washed with phosphate-buffered saline (PBS, pH 6.8), and PBS containing 1% Triton X-100 was added to the wells. The cells attached to the wells were suspend in water and collected in a 1.5 mL tube. The sample with destroyed cell membrane was centrifuged at 15,000 rpm for 10 minutes, and the supernatant was used as an enzyme source for measuring tyrosinase activity and a sample for measuring protein. Protein was quantified using a protein quantification kit (BCA protein kit), and tyrosinase activity was measured by adding 100 ㎕ of 10 mM L-DOPA and 40 ㎕ of tyrosinase enzyme source (supernatant obtained from cells), incubating at 37℃ for 1 hour, and then measuring the absorbance at 405 nm. 25 ㎕/mL kojic acid was used as a positive control.

Figure 4 shows the inhibition of tyrosinase activity of the 50% EtOH extract of raw wild arugula prepared in Example 2, the ethanol extract (50% EtOH) of the 3-day and 6-day wild arugula fermentation product fermented with sugar and PP in Example 5, and the wild arugula fermentation solution (supernatant) in mouse B16-F10 melanoma cells.

As a result, the 50% EtOH extract of raw wild arugula of Example 1, the extract (50% EtOH) of the wild arugula fermented with sugar and PP in Example 5, and the wild arugula fermented liquid (supernatant) showed excellent tyrosinase activity inhibition. In particular, the extract (50% EtOH) of the wild arugula fermented with sugar and PP in Example 5 showed excellent tyrosinase inhibition activity compared to kojic acid, the positive control.

Experimental Example 5. Comparison of flavonoid contents in fermented supernatant and fermented extract of wild arugula

The total flavonoid content was measured as follows by comparing the 50% EtOH extract of wild arugula of Example 1 and the extracts (50% EtOH) of the 3rd and 6th day fermented wild arugula natural, LF, LP, and PP prepared in Examples 2 to 5 and the wild arugula natural, LF, LP, and PP fermented solutions (supernatants).

5% sodium nitrite, 10% aluminum chloride, and 1 M sodium hydroxide aqueous solutions were prepared, respectively. Catechin was prepared as a standard material for preparing a calibration curve at concentrations of 0, 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, and 220 μg/ml. After that, the 50% EtOH extract of wild arugula and the extracts (50% EtOH) of wild arugula natural, LF, LP, and PP fermented products on the 3rd and 6th days and the wild arugula natural, LF, LP, and PP fermented solutions (supernatants) were prepared at a concentration of 1 mg/ml, respectively.

After dispensing 25 μl of each sample into a test tube, 125 μl of DW and 8 μl of sodium nitrite were dispensed, mixed, and stirred for 5 minutes. Next, 15 μl of aluminum chloride was dispensed into each test tube, and stirred for 6 minutes. After that, 50 μl of sodium hydroxide was dispensed into each test tube, and 27 μl of DW were dispensed and mixed well. The absorbance was measured at 510 nm using a UV spectrophotometer. The obtained calibration curve is shown in Fig. 5, and the total flavonoid content was calculated from it, and is shown in Table 1 below.

As shown in Table 1, the total flavonoid content was higher in the ethanol extracts of the fermented product than in the fermented liquid (supernatant) of wild arugula. In addition, the total flavonoid content was higher in the ethanol extracts of the 6-day fermented product than in the 3-day fermented product of wild arugula. In particular, the ethanol extracts of the 6-day LF, LP, and PP fermented products of wild arugula had higher total flavonoid contents than the 50% EtOH extract of wild arugula.

Although the preferred embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and various modifications and improvements made by those skilled in the art using the basic concept of the present invention defined in the following claims also fall within the scope of the present invention.

Claims (13)

A whitening cosmetic composition containing a fermented liquid or an extract of a fermented product of wild arugula ( Diplotaxis tenuifolia L.) as an active ingredient. In the first paragraph,
A whitening cosmetic composition, wherein the above wild arugula fermented liquid or wild arugula fermented product is obtained by fermenting with sugar or sugar and lactic acid bacteria. In the first paragraph,
A whitening cosmetic composition, wherein the lactic acid bacteria is at least one selected from the group consisting of Lactiplantibacillus genus, Limosilactobacillus genus, and Pediococcus genus. A whitening cosmetic composition in claim 1, wherein the wild arugula fermented liquid is a supernatant of fermented wild arugula. A whitening cosmetic composition in claim 1, wherein the extract of the wild arugula fermentation product is extracted with water, an alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof. A whitening cosmetic composition, wherein in claim 1, the extract of the wild arugula fermentation product is an ethanol extract of the wild arugula fermentation product. A whitening cosmetic composition in claim 1, wherein the wild arugula fermentation is performed for 1 to 28 days. A whitening cosmetic composition in claim 1, wherein the composition has a formulation selected from the group consisting of 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, milk, lipstick, makeup base, foundation, pressed powder, and loose powder. A whitening food composition containing a fermented liquid or an extract of a fermented product of wild arugula ( Diplotaxis tenuifolia L.) as an effective ingredient. A health functional food containing a fermented liquid or an extract of a fermented product of wild arugula ( Diplotaxis tenuifolia L.) as an effective ingredient for preventing or alleviating pigmentation diseases. A pharmaceutical composition for preventing or treating pigmentation diseases, containing a fermented solution or an extract of a fermented product of wild arugula ( Diplotaxis tenuifolia L.) as an active ingredient. A whitening quasi-drug composition containing a fermented liquid or an extract of a fermented product of wild arugula ( Diplotaxis tenuifolia L.) as an active ingredient. A composition for external application to the skin for whitening or preventing or treating pigmentation diseases, containing a fermented liquid or extract of a fermented product of wild arugula (Diplotaxis tenuifolia L.) as an active ingredient.