KR102681333B1 - Steam fermentation extract for preventing pigmentation or protecting uv - Google Patents

Abstract

The present invention relates to a steamed fermented extract for preventing pigmentation or protecting against ultraviolet rays and a composition containing the same. The composition containing the achiote extract of the present invention as an active ingredient has radical scavenging activity, SOD (superoxide dismutase)-like activity, and xanthine oxidation. It has an antioxidant effect through enzyme inhibitory activity and the ability to reduce tyrosinase activity and melanin production.

Description

Steamed fermented extract for preventing pigmentation or protecting against UV rays {STEAM FERMENTATION EXTRACT FOR PREVENTING PIGMENTATION OR PROTECTING UV}

The present invention relates to a steamed fermented extract for preventing pigmentation or protecting against ultraviolet rays and a composition containing the same. More specifically, the extract containing achiote ( Bixa orellana ) is used as an active ingredient to exhibit anti-pigmentation and ultraviolet ray protection properties. It relates to a composition that does.

Skin color is primarily determined by the amount and type of melanin, a brown pigment present in the skin. A small amount of melanin causes a light skin color, while a large amount of melanin causes a dark skin color. Additionally, excessive pigmentation of the skin is caused by overexpression or accumulation of melanin in the skin. As a result, the pathways involved in melanin production have been targeted by many inhibitors aimed at reducing the levels produced. One of the key enzymes involved in the melanin pathway is tyrosinase.

The synthesis of melanin is a hormonally regulated process involving melanocyte-stimulating hormone and adrenocorticotropic hormone peptides, which are produced from the precursor proopiomelanocortin. This is promoted by DNA damage caused by UVB-radiation, and then long-term exposure to the sun can induce many biochemical reactions in the skin, resulting in sunburn and tanning. Other consequences of sun exposure accumulate over time. These changes can cause the development of age spots and result in uneven, mottled skin tone, so the ability to modify the expression of pigment content in the skin, to promote uniform skin tone or lightening of skin tone is required.

Because of this, efforts to develop effective compositions have focused on inhibitors of the activity of tyrosinase. For example, various tyrosinase inhibitors, such as hydroquinone, C, cysteine, cozic acid, arbutin and glutathione, among others, have been proposed for topical compositions. Additionally, various dermatological compositions have been proposed for improving the appearance of pigment disorders such as those observed in melasma, freckles, vitiligo, albinism mottled, phenylketonuria, etc., and/or for cosmetic purposes.

Additionally, the use of skin bleaching compositions is widespread. However, they destroy melanin or inhibit its formation. Many of these contain harmful chemicals such as peroxides, acids or formaldehyde, or thiolated substances.

To combat diseases associated with abnormal pigmentation or to lighten skin tone, various compounds have been proposed that, when applied topically to the skin, can reduce tyrosinase activity and thus limit melanin production, but the irritating side effects of certain topical agents have been problematic. It is emerging.

Meanwhile, achiote ( Bixa orellana ) is a shrub belonging to the Bixaceae family and Bixa genus. The whitening effect of the extract of this shrub in cosmetic applications is described in Japanese Patent No. 08-012563. The use of the oily extract of the seed as a moisturizing agent was described in French Patent No. 2798591. However, nothing has been disclosed or disclosed about achiote ( Bixa orellana ) in relation to anti-pigmentation or ultraviolet ray protection.

Accordingly, the present inventors completed the present invention by confirming that a mixture using achiote ( Bixa orellana ), lace flower ( Ammi majus ), and Glycyrrhiza glabra can produce an anti-pigmentation effect and an ultraviolet ray protection effect.

The object of the present invention is to produce a powder of a steamed fermented extract obtained by inoculating a solid steamed dried product containing achiote ( Bixa orellana ) with fermentation bacteria or a powder containing the extract as an active ingredient to exhibit anti-pigmentation ability and ultraviolet ray protection ability. The purpose is to provide a composition.

The above and other objects of the present invention can all be achieved by the present invention described below.

In order to achieve the above object, the present invention

Powder of steamed fermented extract obtained by inoculating fermented bacteria into solid steamed and dried products using achiote ( Bixa orellana ), lace flower ( Ammi majus ) and Glycyrrhiza glabra Includes the extract as an active ingredient,
The solid steamed dried product is a reconstituted product obtained by washing and drying achiote ( Bixa Orellana ) leaves, lace flower ( Ammi majus ) leaves, and Glycyrrhiza glabra leaves and then steaming and drying them under elevated temperature conditions, with a moisture content of 10% or less. A composition for preventing phosphorus pigmentation or protecting against ultraviolet rays is provided.

The achiote ( Bixa orellana ), lace flower ( Ammi majus ), and sensitive plant ( Glycyrrhiza glabra ) have achiote (a): lace flower (b): sensitive plant (c) in a weight ratio of 1:0.25 to 2:0.5 to 1.0. can be satisfied.

The steamed fermented extract may be a fermented extract obtained by steaming the steamed product at 90 to 110°C for 1 to 48 hours and drying it at 35 to 75°C for 1 to 24 hours to have a moisture content of 10% or less.

The fermentation bacteria include Bifidobacterium longum , Lactobacillus acidophilus, Leuconostoc mesenteroides , Lactobacillus plantarum , and Lactobacillus bulgaricus ( Lactobacillus bulgaricus ), and Enterococcus faecium .

The steamed fermentation extract can be extracted at 70 to 100°C for 2 to 5 hours under reflux using a reflux extraction solvent.

The reflux extraction solvent may include one or more selected from chloroform, ethanol, methanol, water, ethyl acetate, nucleic acid, and diethyl ether in an amount of 5 to 20 times the weight of the fermented product.

According to the present invention, a steamed fermented extract derived from achiote that simultaneously exhibits radical scavenging activity, SOD (superoxide dismutase)-like activity, antioxidant effect due to xanthine oxidase inhibitory activity, tyrosinase activity, and melanin production reduction effect is used as an active ingredient. A composition for preventing pigmentation and protecting against ultraviolet rays can be provided.

Figure 1 is a graph comparing the DPPH and ABTS radical scavenging activities of four types of samples (BAG-1 to BAG-3) obtained in Example 1 and Comparative Examples 1 to 3 (BAG-4). .
Figure 2 is a graph comparing the tyrosinase activity of four types of samples (BAG-1 to BAG-3) obtained in Example 1 and Comparative Examples 1 to 3 (BAG-4).
Figure 3 is a graph comparing the degree of melanin production for four types of samples (BAG-1 to BAG-3) obtained in Example 1 and Comparative Examples 1 to 3 (BAG-4).
Figure 4 is a graph comparing the cytotoxicity of four types of samples (BAG-1 to BAG-3) obtained in Example 1 and Comparative Examples 1 to 3 (BAG-4).

Hereinafter, the present invention will be described in more detail to facilitate understanding of the present invention.

Terms and words used in this specification and claims should not be construed as limited to their common or dictionary meanings, and the inventor may appropriately define the concept of terms in order to explain the invention in the best way. Therefore, it should be interpreted with meaning and concept consistent with the technical idea of the present invention.

As confirmed in the examples and experimental examples below, the present invention provides a method for producing powders or extracts of steamed fermented extracts using achiote ( Bixa orellana ), lace flower ( Ammi majus ), and ginseng herb ( Glycyrrhiza glabra ). A composition for preventing pigmentation and protecting against ultraviolet rays by simultaneously expressing antioxidant ability through radical scavenging activity, SOD (superoxide dismutase)-like activity, xanthine oxidase inhibitory activity, and melanin production reduction ability due to tyrosinase activity, as the powder or extract can be provided.

Ethanol room temperature stirred extracts, ethanol fermented extracts, or steamed extracts did not simultaneously exhibit antioxidant effects due to radical scavenging activity, SOD (superoxide dismutase)-like activity, and xanthine oxidase inhibitory activity, and melanin production reduction effects due to tyrosinase activity. In addition, although not shown in the experimental example below, the total amount of achiote ( Bixa orellana ), lace flower (Ocimum Ocimum basilicum basilicum), and sensitive herb ( Glycyrrhiza glabra ) is 1:0.25 to 2:0.5. Even when the weight ratio of ~1.0 is not satisfied, the antioxidant effect due to radical scavenging activity, SOD (superoxide dismutase)-like activity, and xanthine oxidase inhibitory activity, and the melanin production reduction effect due to tyrosinase activity are not shown at the same time or the effect is reduced. .

The composition of the present invention that simultaneously implements the antioxidant effect due to radical scavenging activity, SOD (superoxide dismutase)-like activity, and xanthine oxidase inhibitory activity, and the effect of reducing melanin production due to tyrosinase activity is provided based on the results of these experiments. , The composition for preventing pigmentation or protecting against ultraviolet rays of the present invention is obtained by mixing achiote ( Bixa orellana ) with lace flower ( Ammi majus ) and sensitive herb ( Glycyrrhiza glabra ) at a weight ratio of 1:0.25 to 2:0.5 to 1.0. It is characterized in that it contains as an active ingredient a steamed fermentation extract obtained by inoculating fermentation bacteria into the steamed and dried solid phase product.

In this description, unless otherwise specified, an active ingredient refers to an ingredient that exhibits the desired activity alone or can exhibit activity in combination with a carrier that is not active on its own.

In this description, unless otherwise specified, extract refers to an extract obtained by using an extraction solvent on the solid material itself, which is the object of extraction, or a fraction obtained by fractionating the extract, and the extraction method refers to the polarity of the active substance, the degree of extraction, and the degree of preservation. Considering this, methods such as cold immersion, reflux, heating, supercritical extraction, and ultra-high pressure extraction can be applied. In the case of fractionated extracts, the extract is suspended in a specific solvent, mixed with a solvent of different polarity, and the obtained fraction and crude extract are adsorbed on a column filled with silica gel, etc., and then a hydrophobic solvent, a hydrophilic solvent, or a mixed solvent thereof is used as the mobile phase. It is meant to include fractions obtained by.

The solid material can be obtained in the form of a solid material after thoroughly washing achiote ( Bixa orellana ), lace flower ( Ammi majus ), and sensitive herb ( Glycyrrhiza glabra ). After being thoroughly washed, some of the moisture is removed by drying in any method such as natural drying (shade drying) or hot air drying, and then it can be shredded and used for steaming.

In the present invention, achiote, lace flower, and sensitive herb refer to those obtained by extracting from various organs or parts (e.g. leaves, branches, flowers, roots, stems, fruits, etc.), unless otherwise specified, and preferably each It refers to an extract obtained from the leaves or fruits of and most preferably refers to an extract obtained from each leaf.

In the present invention, lace flower ( Ammi majus ) is a plant belonging to the Ammi root genus of the Apiaceae family, and its leaves and fruits are known to be effective in treating skin diseases such as vitiligo.

In the present invention, Glycyrrhiza glabra is a plant used as an herbal medicine material and contains triterpene saponin, glycyrrhizin, liquiritin, asparagin, glutamic acid, and glucose. It contains etc. Glycyrrhizin, a 2-glucuronic acid glycoside of glycyrrhizic acid, the sweet component of licorice, is known to have effects such as disinfecting bacteria as well as detoxifying drug addiction, plant poisoning, or poisoning of body metabolites.

In addition, it can be used as a pulverized product or in a step-by-step steamed and then dried state. When used in a step-by-step steamed and then dried state, it can provide storage usefulness and the effect of increasing the effective ingredients.

In the present disclosure, unless otherwise specified, the steamed extract refers to a product steamed at 90 to 110°C, or 95 to 105°C for 1 to 48 hours, or 1 to 10 hours at 35 to 75°C, or 40 to 60°C. for 1 to 24 hours, or 6 to 15 hours It may be a steamed dried product with a moisture content of 10% or less. In this case, it is desirable because it can provide an increased effect of the active ingredient.

At this time, a normal steaming device can be used for the steaming, a normal drying device such as a hot air dryer can be used for drying, and a normal drying device such as a freeze dryer can be used for drying to control the moisture content. .

The steamed extract of the present invention is preferably obtained by adjusting the moisture content to 20% or less through drying pretreatment and using the steamed drying.

The steam drying can be performed in multiple stages, if necessary. For example, the first steaming and drying can be performed by setting the steaming time to 6 to 15 hours, the second steaming and drying by setting the steaming time to 8 to 15 hours, and the third steaming and drying by setting the steaming time to 8 to 12 hours. .

The steamed dried product is inoculated with fermentation bacteria.

In the present invention, the fermentation strain is a mixture of Bifidobacterium longum (KACC 20597), Lactobacillus acidophilus (KACC 12419), and Leuconostoc mesenteroides (KACC 12312) used in the examples below. To provide a composition for preventing pigmentation and protecting against ultraviolet rays. In addition, Lactobacillus plantarum , Lactobacillus bulgaricus , and Enterococcus faecium can also be used as substitutes or combinations, and the amount used is 10 ⁴ to 10 ⁷ CFG. /g, as a specific example, may be in the range of 10 ⁵ to 10 ⁶ CFG/g.

In the present invention, the culture after inoculation may be performed at 15 to 45°C, or 25 to 45°C. If the culture temperature is lower than the above range, the fermentation speed may be slowed or unwanted by-products may be generated. Even if the culture temperature is higher than the above range, the fermentation speed may be slowed or unwanted fermentation by-products may be formed due to the death of fermentation microorganisms. . Cultivation time may be 1 to 7 days, or 2 to 5 days.

Additionally, the properties of the active ingredient may change depending on the temperature and pressure conditions of extraction. For example, if the steamed fermentation extract is extracted under low temperature conditions, the solubility of the active ingredient decreases, resulting in a high concentration of sugar in the extract, and even when extracted under high temperature conditions suitable for the active ingredient extraction conditions, the extract at the beginning of the extraction is fractionated and separated separately. If you do this, the concentration of active ingredients will be higher compared to the extract in the second half. In this extraction method, it is possible to control the free fatty acid content and the active ingredient content by adjusting the fractionation point. In order to obtain an extract with a low free fatty acid content, that is, a low acid value, a fraction with a low free fatty acid content of the raw material or a high content of the active ingredient is used. There is a way to lower the degree of concentration by mixing sections.

The free fatty acid content of raw materials varies depending on the country of origin or crop conditions, and raw materials with low acid value are usually sold at high prices, making them less competitive as raw materials. In addition, since there are no raw materials without free fatty acids, free fatty acids are concentrated in the extract concentration fraction section, making it easy to meet the acid value standards while meeting the content of active ingredients above a certain concentration even if a standard is set and a product is made using raw materials with low acid value. It's not work. Accordingly, in order for the process of producing a high-concentration fraction using a steamed fermentation extract to have industrial stability, a technology to remove free fatty acids through repeated extraction (also known as reflux extraction) under reflux is required.

The reflux extraction may be repeated at 70 to 100°C, or at 85 to 95°C for 12 hours or less, or 3 to 10 hours. The number of repeated extractions may be 5 times or less, for example, 2 to 3 times.

In the case of the reflux extraction, it is preferable to increase treatment efficiency by supplying an extraction solvent to the fermented product. In this case, the extraction solvent that can be used is one or more selected from chloroform, ethanol, methanol, water, ethyl acetate, nucleic acid, and diethyl ether. You can use it. At this time, the extraction solvent may be included in an amount of 5 to 20 times, or 8 to 15 times, the weight of the fermented product.

A purification process is required that removes the above-mentioned free fatty acids but does not cause loss of active ingredients and does not affect efficacy. For this purpose, a method of removing free fatty acids by filtration using a fine filtration membrane and then concentrating under reduced pressure is preferred, resulting in better efficacy. To provide an effect, it is more preferable to use a fine filtration membrane of 1.0 ㎛ or less, preferably 0.45 ㎛ or less.

An effective extract can be obtained through the above-described purification process.

In the present disclosure, the effective extract includes a concentrated liquid extract or solid extract from which the reflux extraction solvent has been removed by methods such as freeze-drying, vacuum drying, hot air drying, spray drying, or reduced-pressure concentration.

In the present disclosure, the form of the resulting extract is not particularly limited and may be in the form of pills, granules, powder, powder, or other extract forms.

The extract manufacturing method according to the present disclosure includes, for example, washing achiote ( Bixa orellana ), lace flower ( Ammi majus ), and sensitive herb ( Glycyrrhiza glabra ) thoroughly and then steaming and drying the obtained solid or dried product; and inoculating the steamed dried product with fermentation bacteria.

In the present description, the achiote ( Bixa orellana ), lace flower ( Ammi majus ), and sensitive herb ( Glycyrrhiza glabra ) can be obtained in solid form after being thoroughly washed. After being thoroughly washed, some of the moisture is removed by drying in any method such as natural drying (shade drying) or hot air drying (in the form of pulverized material so that the moisture content of the solid is less than 10% (w/w)). It can be crushed to 50 mesh, specifically 30 to 50 mesh to increase the surface area, and then used for fermentation pretreatment.

If necessary, it can be dried at 95°C or lower, or 50 to 90°C for more than 36 hours, or more than 48 hours, and then pulverized.

The solid may be subjected to a fermentation extraction step prior to reflux extraction.

There is no particular limitation on the solid material used in this description, and the fermentation extraction step is used to remove harmful substances such as microorganisms, residual pesticides, and preservatives on the surface of the solid material or its dried material (also referred to as 'solid dried material'). Treatment, treatment to increase extraction area, etc.

In the case of fermentation extraction, fermentation bacteria include Bifidobacterium longum , Lactobacillus acidophilus, Leuconostoc mesenteroides , and Lactobacillus plantarum . , Lactobacillus bulgaricus , and Enterococcus faecium can be used.

Subsequently, the fermented extract can be subjected to repeated reflux extraction under reflux using a reflux extraction solvent. At this time, extraction may be repeated at 70 to 100°C, specifically at 85 to 95°C for 12 hours or less, for example, 1 to 6 hours. The number of repeated extractions may be 5 times or less, for example, 2 to 3 times.

The steamed fermented extract can be purified and concentrated under reduced pressure as described above to remove the reflux extraction solvent and separate the effective extract.

The composition of the present invention may be prepared by including skin whitening ingredients, skin wrinkle improvement ingredients, skin elasticity improvement ingredients, ultraviolet ray protection ingredients, skin moisturizing ingredients, etc. known in the art in order to reinforce or add skin-related activities in addition to the active ingredients. You can.

Examples of the skin whitening ingredients include arbutin, niacinamide, ascorbyl glucoside, alpha-bisabolol, and oil-soluble licorice extract.

The skin wrinkle improvement ingredients include retinol, retinyl palmitate, adenosine, and polyethoxylated amide.

The UV protection ingredients include complexes such as drometrizole, drometrizole trisiloxane, digalloyltrioleate, dimethycodiethylbenzalmalonate, diethylhexylbutamidotriazone, and pine bark extract, phosphatidylserine, and finger. Examples include root extract powder, complex extracts of red ginseng and cornelian cherry, etc.

The moisturizing ingredients include AP collagen enzymatically degraded peptide, collagen peptide, N-acetylglucosamine, konjac potato extract, complex extracts such as dandelion, rice bran extract, corn germ extract, low molecular weight collagen peptide, agar extract powder, phosphatidylserine, hyaluronic acid, etc. I can hear it.

For other skin whitening ingredients, wrinkle improvement ingredients, UV protection ingredients, and skin moisturizing ingredients, the Functional Cosmetics Code according to the Cosmetics Act (Ministry of Food and Drug Safety Notification “Functional Cosmetics Standards and Test Methods”) and the Health Functional Food Code under the Health Functional Foods Act. (Please refer to the Ministry of Food and Drug Safety Notice “Standards and Specifications for Health Functional Foods”). One or more of these ingredients may be included in the composition of the present invention together with the active ingredient.

The composition of the present invention may contain the active ingredient in any amount (effective amount) as long as it can exhibit anti-pigmentation effect, ultraviolet ray protection effect, etc. depending on the product type, product formulation, etc., and the effective amount is the total weight of the composition. As a standard, it can be determined within the range of 0.001 to 15 wt%.

In the present invention, unless otherwise specified, the effective amount is the anti-pigmentation effect, ultraviolet protection effect, etc. when the composition of the present invention is administered to the subject of application, a mammal, preferably a human, during the administration period as recommended by a medical expert, etc. It refers to the amount of active ingredient contained in the composition of the present invention that can exhibit the intended medical/cosmetic effect, etc. Such effective amounts can be determined experimentally within the scope of the ordinary ability of those skilled in the art.

In a specific aspect, the composition of the present invention can be considered a cosmetic composition.

Even if the composition of the present invention is recognized as a cosmetic composition, the cosmetic composition may be classified as an arbitrary product in terms of its use and by law, and may specifically be a functional cosmetic with uses such as hair growth effect, a non-functional general cosmetic, etc. .

The product form can take any product form, specifically solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax. It can be formulated as foundation, spray, etc. Specific product forms may include softening lotion, nourishing lotion, nourishing cream, massage cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, pack, spray, or powder.

In addition to the active ingredient, the cosmetic composition of the present invention may include ingredients commonly used in cosmetic compositions, such as stabilizers, solubilizers, surfactants, vitamins, auxiliaries such as pigments and flavorings, and carriers.

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

When the formulation of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder can be used as the carrier ingredient. In particular, when the formulation is a spray, chlorofluorohydrocarbon and propane may be used as carrier ingredients. /May contain propellants such as butane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, solubilizing agent, or emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 , 3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol, fatty acid ester of sorbitan, etc. can be used.

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

When the formulation of the present invention is a surfactant-containing cleansing agent, the carrier ingredients include aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, and fatty acid amide. Ether sulfate, alkylamidobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative, or ethoxylated glycerol fatty acid ester can be used.

The cosmetic composition of the present invention is a cosmetic composition commonly performed in the art, except that it contains active ingredients that exhibit radical scavenging activity, SOD (superoxide dismutase)-like activity, xanthine oxidase inhibitory activity, hair melamine synthesis, etc. It can be manufactured according to the manufacturing method.

In another specific aspect, the composition of the present invention can be considered a food composition.

The food composition of the present invention can be manufactured in any form, for example, beverages such as tea, juice, carbonated beverages, and isotonic drinks, processed oils such as milk and yogurt, gums, rice cakes, Korean snacks, bread, cookies, noodles, etc. It can be manufactured into health functional food preparations such as foods, tablets, capsules, pills, granules, liquids, powders, flakes, pastes, syrups, gels, jellies, bars, etc.

In addition, the food composition of the present invention may have any product classification in terms of functionality as long as it complies with the enforcement laws and regulations at the time of manufacture/distribution. For example, it may be a health functional food according to the Act on Health Functional Foods, or a special purpose food according to each food type according to the Food Code of the Food Sanitation Act (Ministry of Food and Drug Safety Notification, Food Standards and Specifications).

The food composition of the present invention may contain food additives in addition to the active ingredients. Food additives are generally understood as substances that are added to, mixed with, or infiltrated into food when manufacturing, processing, or preserving food. Since they are taken with food every day and for a long period of time, their safety must be guaranteed. The Food Additive Code (Notice of the Ministry of Food and Drug Safety, Food Additive Standards and Specifications) under the Food Sanitation Act provides limited regulations on food additives with guaranteed safety, dividing them into chemical compounds, natural additives, and mixed preparations.

In terms of functionality, these food additives can be divided into sweeteners, flavors, preservatives, emulsifiers, acidulants, thickeners, etc.

Sweeteners can be used in amounts to give foods an appropriate sweet taste and can be natural or synthetic. Preferably, a natural sweetener is used, and natural sweeteners include sugar sweeteners such as corn syrup solids, honey, sucrose, fructose, lactose, and maltose.

Flavoring agents can be used to improve taste or aroma, and both natural and synthetic ones can be used. Preferably, natural products are used. When using natural products, they can serve the purpose of enhancing nutrition in addition to flavor. Natural flavoring agents may be obtained from apples, lemons, tangerines, grapes, strawberries, peaches, etc., or may be obtained from green tea leaves, coriander leaves, bamboo leaves, cinnamon, chrysanthemum leaves, jasmine, etc. You can also use products obtained from ginseng (red ginseng), bamboo shoots, aloe vera, and ginkgo nuts. Natural flavoring agents may be liquid concentrates or solid extracts.

Synthetic flavoring agents may also be used, and synthetic flavoring agents may include esters, alcohols, aldehydes, terpenes, etc.

Preservatives include calcium sodium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, sodium benzoate, potassium benzoate, and EDTA (ethylenediaminetetraacetic acid), and emulsifiers include acacia gum, carboxymethyl cellulose, and xanthan gum. , pectin, etc.

As acidulants, acidic acid, malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartaric acid, ascorbic acid, acetic acid, phosphoric acid, etc. may be used. In addition to improving taste, acidulants may be added to ensure that the food composition has an appropriate acidity for the purpose of suppressing the growth of microorganisms.

As a thickening agent, a suspending agent, settling agent, gel forming agent, bulking agent, etc. may be used.

In addition to the food additives described above, the food composition of the present invention may contain bioactive substances or minerals known in the art and whose safety is guaranteed as food additives for the purpose of supplementing and reinforcing functionality and nutrition.

Such physiologically active substances include catechins contained in green tea, vitamins such as vitamin B1, vitamin C, vitamin E, and vitamin B12, tocopherol, dibenzoylthiamine, etc., and minerals include calcium preparations such as calcium citrate and magnesium stearate. Magnesium preparations such as iron citrate, iron preparations such as chromium chloride, potassium iodine, selenium, germanium, vanadium, zinc, etc. are included.

The food composition of the present invention may contain the above-described food additives in an appropriate amount to achieve the purpose of addition depending on the product type. Regarding other food additives that may be included in the food composition of the present invention, reference may be made to the Food Code or the Food Additive Code.

In specific embodiments, the composition of the present invention can be considered a pharmaceutical composition.

The pharmaceutical composition of the present invention contains a pharmaceutically acceptable carrier in addition to the active ingredient and can be prepared into an oral formulation or a parenteral formulation depending on the route of administration by a conventional method known in the art.

In the present invention, pharmaceutically acceptable means that, unless otherwise specified, the activity of the active ingredient is not inhibited and the subject of application (prescription) does not have toxicity beyond what is acceptable.

When the pharmaceutical composition of the present invention is prepared as an oral dosage form, it can be prepared as powder, granules, tablets, pills, sugar-coated tablets, capsules, solutions, gels, syrups, suspensions, and wafers along with a suitable carrier according to methods known in the art. It can be manufactured in a dosage form such as:

Suitable pharmaceutically acceptable carriers include, for example, sugars such as lactose, glucose, sucrose, dextrose, sorbitol, mannitol, and xylitol, starches such as corn starch, potato starch, and wheat starch, cellulose, methylcellulose, and ethyl. Cellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate, mineral oil, malt, gelatin, talc, polyol , vegetable oil, etc. If necessary, it can be formulated with diluents and/or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

When the pharmaceutical composition of the present invention is prepared as a parenteral formulation, it can be formulated in the form of injections, transdermal administration, nasal inhalation, and suppositories along with a suitable carrier according to methods known in the art.

When formulated as an injection, suitable carriers include sterile water, ethanol, polyols such as glycerol or propylene glycol, or mixtures thereof, preferably Ringer's solution, phosphate buffered saline (PBS) containing triethanol amine, or sterile for injection. Isotonic solutions such as water or 5% dextrose can be used.

When formulated for transdermal administration, it can be formulated in the form of ointments, creams, lotions, gels, topical preparations, paste preparations, liniment preparations, and aerol preparations.

In the case of nasal inhalation, it can be formulated in the form of an aerosol spray using a suitable propellant such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, and carbon dioxide.

The bases of suppositories include witepsol, tween 61, polyethylene glycols, cacao fat, laurel paper, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, and sorbitan fatty acid esters. can be used

Regarding the formulation of pharmaceutical compositions, it is known in the art, and specifically, references can be made to the literature [Remington's Pharmaceutical Sciences (19th ed., 1995)]. The above documents are considered part of this specification.

The preferred dosage of the pharmaceutical composition of the present invention ranges from 0.001 mg/kg to 10 g/kg per day, preferably from 0.001 mg/kg, depending on the patient's condition, weight, gender, age, severity of the patient, and route of administration. It may be in the range of 1 g/kg. Administration can be done once a day or divided into several times. These dosages should not be construed as limiting the scope of the invention in any respect.

The cosmetic composition, food composition, and pharmaceutical composition of the present invention can be prepared according to the method for producing the composition commonly performed in the art, except that it contains active ingredients known in the art.

In describing the composition for preventing pigmentation or protecting against ultraviolet rays of the present invention, it is stated that other conditions or equipment not explicitly described can be appropriately selected within the range commonly practiced in the art and are not particularly limited.

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

[Example]

<Example 1> Preparation of steamed fermented achiote extract

(1) Achiote ( Bixa orellana ) leaves, lace flower ( Ammi majus ) leaves, and Glycyrrhiza glabra ( Glycyrrhiza glabra ) leaves are thoroughly washed, dried at 70 ℃ for 48 hours, steamed at 100 ℃ for 6 hours, and then at 50 ℃ for 12 hours. It was dried naturally for some time.

(2) The dried raw materials were ground to a size of 40 mesh or less. The pulverized raw materials were mixed at a constant weight (100 g: 100 g: 100 g).

(3) Bifidobacterium longum (KACC 20597), Lactobacillus acidophilus (KACC 12419), and Leuconostoc mesenteroides (KACC 12312) were inoculated into the mixed steamed dried product at a concentration of 10 ⁶ CFU/g and fermented at 37°C for 3 days. The supernatant was taken by centrifugation.

(4) Then, 10 times the total weight of the steamed fermentation product was added with 70% ethanol, and reflux extraction was performed three times for 8 hours at 90°C.

(5) The obtained extract was filtered through a 0.45 ㎛ membrane filter, and then ethanol was removed using a reduced pressure concentrator to prepare a steam-fermented achiote extract (sample BAG-4).

<Comparative example>

<Comparative Example 1> Preparation of achiote extract stirred at room temperature

(1') Achiote ( Bixa orellana ) leaves, lace flower ( Ammi majus ) leaves, and Glycyrrhiza glabra leaves were thoroughly washed and dried at 70°C for 48 hours.

(2) The dried raw materials were ground to a size of 40 mesh or less. The pulverized raw materials were mixed at a constant weight (100 g: 100 g: 100 g).

(4') 70% ethanol was added 10 times the total weight of the solid dried material and extracted at room temperature at 200 rpm for 12 hours, a total of 3 times.

(5) The obtained extract was filtered through a 0.45 ㎛ membrane filter and then concentrated under vacuum and normal pressure to prepare a room temperature stirred achiote extract (sample BAG-1).

<Comparative Example 2> Preparation of ultrasonic achiote extract

(1') Achiote ( Bixa orellana ) leaves, lace flower ( Ammi majus ) leaves, and Glycyrrhiza glabra leaves were thoroughly washed and dried at 70°C for 48 hours.

(2) The dried raw materials were ground to a size of 40 mesh or less. The pulverized raw materials were mixed at a constant weight (100 g: 100 g: 100 g).

(4”) 70% ethanol, 10 times the total weight of the solid dried material, was added and ultrasonic extraction was performed at 40 kHz for 2 hours.

(5) The obtained ultrasonic extract was filtered through a 0.45 ㎛ membrane filter and then concentrated under vacuum and at room temperature to prepare an ultrasonic achiote extract (sample BAG-2).

<Comparative Example 3> Preparation of steamed achiote extract

(1) Achiote ( Bixa orellana ) leaves, lace flower ( Ammi majus ) leaves, and Glycyrrhiza glabra ( Glycyrrhiza glabra ) leaves are thoroughly washed, dried at 70 ℃ for 48 hours, steamed at 100 ℃ for 6 hours, and then at 50 ℃ for 12 hours. It was dried naturally for some time.

(2) The dried raw materials were ground to a size of 40 mesh or less. The pulverized raw materials were mixed at a constant weight (100 g: 100 g: 100 g).

(4') 70% ethanol 10 times the total weight of the steamed dried material was added, and reflux extraction was performed three times for 3 hours at 70°C.

(5) The obtained extract was filtered through a 0.45 ㎛ membrane filter and then concentrated in vacuum and at room temperature to prepare a steamed achiote extract (sample BAG-3).

<Experimental example>

<Experimental Example 1> Antioxidant activity test 1

ABTS method and DPPH method radical scavenging ability tests were performed to evaluate the radical scavenging ability of the samples through antioxidant tests on the samples obtained in Example 1 and Comparative Examples 1 to 3 (BAG-1 to 4).

(1-1) ABTS radical scavenging activity

Samples (BAG-1 to BAG-4) are each diluted in water to prepare test solutions with concentrations of 100 ㎍/mL, 250 ㎍/mL, and 500 ㎍/mL. Mix 7mM ABTS and 2.45mM potassium persulfate and react at room temperature for 12 hours in the dark to form ABTS cations. Afterwards, ethanol was added to adjust the absorbance value to 0.70 ± 0.02 at 734 nm.

Add 100 ㎕ of the test solution and 100 ㎕ of the prepared ABTS solution to a 96-well plate, react at room temperature for 7 minutes, and measure at 734 nm. Compared to the blank test solution, the ABTS removal rate was calculated as a percentage (%) as follows and is shown in Figure 1 below.

ABTS radical scavenging ability (%) = [Control - (Sample - Blank)]/Control Х 100

(However, Control: Absorbance of ABTS reagent, Sample: Absorbance of Sample + ABTS reagent, Blank: Absorbance of Sample + Blank)

(1-2) DPPH radical scavenging activity

Samples (BAG-1 to BAG-4) are each diluted in water to prepare test solutions with concentrations of 100 ㎍/mL, 250 ㎍/mL, and 500 ㎍/mL. Add 100 ㎕ of the test solution and 100 ㎕ of 0.2 mM DPPH to a 96-well plate, and after 30 minutes, measure the absorbance at 517 nm using a microplate reader. Compared to the blank test solution, the DPPH radical scavenging rate was calculated as a percentage (%) as follows and is shown in Figure 1 below.

DPPH radical scavenging ability (%) = [Control - (Sample - Blank)]/Control Х 100

(Control: Absorbance of DPPH reagent, Sample: Absorbance of Sample + DPPH reagent, Blank: Absorbance of Sample + Blank)

As shown in Figure 1 below, as a result of ABTS radical analysis, ascorbic acid, the control group, was confirmed to be the highest at 98.2% at 500 μg/mL, followed by the sample (BAG-4) according to Example 1 at 72.3% at 500 μg/mL. It was confirmed to be high, followed by the sample according to Comparative Example 3 (BAG-3), the sample according to Comparative Example 2 (BAG-2), and the sample according to Comparative Example 1 (BAG-1) at 500 μg/mL. showed 68.3%, 64.3%, and 59.4%, respectively.

Additionally, the DPPH radical analysis results showed the same trend as the ABTS analysis results described above. Specifically, the sample according to Example 1 (BAG-4), the sample according to Comparative Example 3 (BAG-3), the sample according to Comparative Example 2 (BAG-2), and the sample according to Comparative Example 1 (BAG-1) ) In the case of 500 ㎍/mL, it was 75.2%, 71.2%, 67.2%, and 63.8%, respectively. At this time, ascorbic acid, the control group, was analyzed at 97.9% at 500 ㎍/mL.

<Experimental Example 2> Antioxidant activity test 2

SOD-like activity and xanthine oxidase inhibitory activity tests were performed as antioxidant tests on the samples obtained in Example 1 and Comparative Examples 1 to 3 (BAG-1 to 4).

(2-1) SOD-like activity

Samples (BAG-1 to BAG-4) are each diluted in water to prepare test solutions with concentrations of 100 ㎍/mL, 250 ㎍/mL, and 500 ㎍/mL. Add 2.6 mL of tris-HCl buffer corrected to pH 8.5 and 0.2 mL of 7.2 mM pyrogallol to 0.2 mL of the sample solution and react at 25°C for 10 minutes. Add 0.1 mL of 1 N HCl to the reaction solution to stop it. The amount of oxidized pyrogallol is determined by measuring absorbance at 420 nm. Compared to the blank test solution, the SOD-like activity was calculated as a percentage (%) and is shown in Table 1 below.

(2-2) Xanthine oxidase inhibitory activity

Samples (BAG-1 to BAG-4) were respectively diluted in water to prepare screening concentrations of 100 μg/mL, 250 μg/mL, and 500 μg/mL. Add 0.6 mL of 0.1 M potassium phosphate buffer (pH 7.5) and 0.2 mL of 1 mM xanthine to 1.0 mL of the sample solution. Afterwards, 0.1 mL of 0.2 U/mL xanthine oxidase was added to stop the reaction. The produced uric acid is determined by measuring the absorbance at 292 nm. Compared to the blank test solution, the xanthine oxidase inhibitory activity was calculated as a percentage (%) and is shown in Table 1 below.

Extract SOD-like activity (%) Xanthin oxidase inhibition (%) 100 μg/mL 250 μg/mL 500 μg/mL 100 μg/mL 250 μg/mL 500 μg/mL Control 94.2±3.8 95.1±3.3 96.9±4.3 98.1±4.1 98.9±3.7 99.1±3.5 BAG-1 11.3±1.8 17.4±1.2 21.8±1.1 14.1±1.8 19.1±2.9 25.2±2.8 BAG-2 12.6±1.5 19.5±2.7 24.3±2.9 16.2±1.9 20.5±1.7 26.5±3.0 BAG-3 15.7±1.7 21.1±1.9 29.4±2.1 19.2±2.1 22.4±1.7 28.4±2.7 BAG-4 19.2±2.6 24.5±2.3 32.1±2.5 22.7±1.1 26.1±2.1 31.4±2.6

*Control: Ascorbic acid

As shown in Table 1, as a result of SOD-like activity analysis, the sample according to Example 1 (BAG-4) was confirmed to be the highest at 32.1% at 500 μg/mL, followed by the sample according to Comparative Example 3 (BAG-4). 3), the sample according to Comparative Example 2 (BAG-2), and the sample according to Comparative Example 1 (BAG-1) showed 29.4%, 24.3%, and 21.8% at 500 μg/mL in that order. The scavenging activity of all test substances was analyzed in concentration dependence, and all were found to be lower than the control group, ascorbic acid.

In addition, as a result of the analysis of xanthine oxidase inhibitory activity, the sample according to Example 1 (BAG-4) was confirmed to be the highest at 31.4% at 500 μg/mL, followed by the sample according to Comparative Example 3 (BAG-3) , the sample according to Comparative Example 2 (BAG-2), and the sample according to Comparative Example 1 (BAG-1) were analyzed to show 28.4%, 26.5%, and 25.2% at 500 μg/mL in that order, and the above-described SOD The same tendency as the analysis result of similar activity was confirmed.

<Experimental Example 3> Melamine test 1

Tyrosinase activity experiments were performed on the samples obtained in Example 1 and the samples (BAG-1 to 4) obtained in Comparative Examples 1 to 3. Tyrosinase is an enzyme involved in melanin biosynthesis, and whitening ingredients have a mechanism of action that inhibits this enzyme.

Specifically, malignant melanoma cells (B16F10, ATCC) were dispensed at 1Х10 ⁴ cell/ml and cultured for 24 h at 37°C under 5% CO ₂ conditions and then replaced with medium containing 100 nM α-MSH. Afterwards, samples of each concentration (BAG-1 to BAG-4) and control group (water) were added, cultured for 3 days, washed with 10mM PBS, and suspended in 10mM PBS containing 1% Triton X-100. , centrifuge for 5 minutes and use the supernatant as an enzyme solution for measuring activity. 40 ㎕ of this enzyme solution was added to a 96 well plate, and 100 ㎕ of 3,4-Dihydroxyphenylalanine (L-dopa), a substrate, at a concentration of 2 mg/mL was added. After the reaction proceeded at 37°C for 1 hour, absorbance was measured at 405 nm using an ELISA reader. The activity of tyrosinase was calculated as a percentage of the absorbance of the control group and is shown in Figure 2 below.

As shown in Figure 2 below, as a result of tyrosinase activity analysis, the sample (BAG-4) according to Example 1 was confirmed to be the highest at 54.1% at 500 μg/mL, followed by the sample (BAG-3) according to Comparative Example 3. ), the sample according to Comparative Example 2 (BAG-2), and the sample according to Comparative Example 1 (BAG-1) showed 57.5%, 64.2%, and 65.6% at 500 μg/mL in that order. All test substances inhibited the activity of tyrosinase in a concentration-dependent manner, and all were found to be reduced compared to the control group.

<Experimental Example 4> Melanin test 2

An experiment was performed to confirm the degree of melanin production for the samples obtained in Example 1 and the samples (BAG-1 to 4) obtained in Comparative Examples 1 to 3. Melanin is produced by exposure to ultraviolet rays or external stimuli and causes skin disorders and aging such as acne, freckles, and age spots. Melanin is produced through biosynthesis of the tyrosinase enzyme, and melanin production can be suppressed by inhibiting the activity of tyrosinase.

Specifically, dilute the test substances (BAG-1 to 4) in water to prepare test solutions with concentrations of 100 ㎍/mL, 250 ㎍/mL, and 500 ㎍/mL. Malignant melanoma cells (B16F10, ATCC) were dispensed at 1Х10 ⁴ cell/ml and cultured for 24 hr at 37°C under 5% CO ₂ conditions and then replaced with medium containing 100 nM α-MSH. Afterwards, test substances and control (water) were added at each concentration, cultured for 3 days, washed with 10mM PBS, suspended in 10mM PBS containing 1% Triton do. Afterwards, 200 ㎕ of 1 N NaOH was added and left at 55°C for 2 hr to dissolve melanin. The absorbance was measured at 405 nm to calculate the amount of melanin produced (%), which is shown in Figure 3 below.

As shown in Figure 3 below, as a result of analyzing the amount of melanin produced, the sample (BAG-4) according to Example 1 was found to be the lowest at 43.2% compared to the control group at 500 ㎍/mL, and the sample (BAG-3) according to Comparative Example 3 ), the sample according to Comparative Example 2 (BAG-2), and the sample according to Comparative Example 1 (BAG-1) showed 46.4%, 50.2%, and 52.1% at 500 μg/mL in that order.

It was found that all samples showed a decrease in melanin production compared to the control group, and the analysis results were confirmed to be consistent with the trend of tyrosinase inhibitory activity described above.

<Experimental Example 5> safety testing

In order to evaluate the safety of the samples obtained in Example 1 and Comparative Examples 1 to 3 (BAG-1 to 4), the cytotoxicity of the test substances (BAG-1 to 4) to mouse melanoma cells was tested. To confirm, MTS analysis was performed. The concentration of the test substance was set at 100 to 500 ㎍/mL, and the cytotoxicity of the test substance was confirmed by culturing mouse melanoma cells (B16F10, ATCC) cells.

Specifically, mouse melanoma cells (B16F10, ATCC) were dispensed at 1Х10 ⁴ cell/ml, cultured for 24 hr under conditions of 37°C and 5% CO _2, and then incubated with 100 nM melanocyte-stimulating hormone (α-MSH) and 100 to 100 mg/ml. Samples (BAG-1 to 4) and control (water) at a concentration of 500 μg/mL were added and cultured for additional 24 hours. Afterwards, 20 ㎕ of MTS reagent was added and incubated for 2 hours, and then the absorbance was measured with an ELISA reader (Epoch ^TM 2, BioTek, USA) at 570 nm using a microplate reader.

Cell viability was calculated using Equation 1 below, and the results are shown in Figure 4 below.

[Equation 1]

Cell viability (%) = [(Exp. - Blank)/Control] Х 100

(In Equation 1 above, Exp: Absorbance of extract containing cells, Blank: Absorbance of extract without cells, Control: Absorbance of distilled water containing cells.)

As shown in Figure 4 below, as a result of cytotoxicity analysis in mouse melanoma cells, it was confirmed that the cell viability was more than 95% at all concentrations.

Therefore, in the sample according to Example 1 (BAG-4), the sample according to Comparative Example 3 (BAG-3), the sample according to Comparative Example 2 (BAG-2), and the sample according to Comparative Example 1 (BAG-1) It can be seen that there is no cytotoxicity at concentrations of 100 to 500 μg/mL.

In conclusion, powder of steamed fermented extract obtained by inoculating fermentation bacteria on solid steamed dried material using achiote ( Bixa orellana ), lace flower ( Ammi majus ), and ginseng herb ( Glycyrrhiza glabra ) or a powder containing the extract as an active ingredient In this case, the obtained composition has excellent ABTS, DPPH free radical scavenging activity and SOD-like activity, so it has excellent antioxidant effect, and is suitable for applications that require anti-pigmentation effect and ultraviolet ray protection effect due to tyrosinase activity and reduction of melanin production. was able to confirm.

Claims (6)

Powder of steamed fermented extract obtained by inoculating fermented bacteria into solid steamed and dried products using achiote ( Bixa orellana ), lace flower ( Ammi majus ) and Glycyrrhiza glabra Includes the extract as an active ingredient,
The solid steamed dried product is a reconstituted product obtained by washing and drying achiote ( Bixa Orellana ) leaves, lace flower ( Ammi majus ) leaves, and Glycyrrhiza glabra leaves and then steaming and drying them under elevated temperature conditions, with a moisture content of 10% or less. Composition for preventing phosphorus pigmentation or protecting against ultraviolet rays. According to paragraph 1,
The achiote ( Bixa orellana ), lace flower ( Ammi majus ), and sensitive plant ( Glycyrrhiza glabra ) have achiote (a): lace flower (b): sensitive plant (c) in a weight ratio of 1:0.25 to 2:0.5 to 1.0. A composition for preventing pigmentation or protecting against ultraviolet rays that satisfies the following. According to paragraph 1,
The steamed fermented extract is heated at 90 to 110°C for 1 to 48 hours. Steamed product at 35 to 75°C for 1 to 24 hours A composition for preventing pigmentation or protecting against ultraviolet rays, which is a fermented extract of dried, steamed dried product with a moisture content of 10% or less. According to paragraph 1,
The fermentation bacteria include Bifidobacterium longum , Lactobacillus acidophilus, Leuconostoc mesenteroides , Lactobacillus plantarum , and Lactobacillus bulgaricus ( Lactobacillus bulgaricus ), and Enterococcus faecium ( Enterococcus faecium ). A composition for preventing pigmentation or protecting against ultraviolet rays. According to paragraph 1,
A composition for preventing pigmentation or protecting against ultraviolet rays, wherein the steamed fermented extract is extracted under reflux at 70 to 100° C. for 2 to 5 hours using a reflux extraction solvent. According to clause 5,
The reflux extraction solvent is a composition for preventing pigmentation or protecting against ultraviolet rays, comprising at least one selected from chloroform, ethanol, methanol, water, ethyl acetate, nucleic acid, and diethyl ether in an amount of 5 to 20 times the weight of the fermented product.