KR101795344B1 - Cosmetic and functional food composition comprising gagunin D isolated from Phorbas sp. having whitening activities - Google Patents

Abstract

Disclosed is a functional food and a skin-whitening cosmetic composition containing gagunin D isolated from a Sponge Phorbas species which is a marine natural material. According to the present invention, the composition inhibits biosynthesis of melanin and show excellent whitening efficacy with little irritation and side effects, thereby being highly applicable as functional foods for whitening or pigmentation prevention or cosmetic products with whitening functions.

Description

[0001] Cosmetic and functional food compositions containing furniturein D isolated from sponge Phorbas species exhibiting whitening activity. having whitening activities}

The present invention relates to a whitening cosmetic composition and a cosmetic food comprising Furniturein D isolated from sponge Phorbas species.

In cosmetics, skin whitening is one of the areas of greatest interest among East Asian women, especially in Korea, Japan and China. The most important factor in skin whitening is skin melanin pigment. Originally, melanin is a black pigment found in animals, plants and microorganisms. It is not essential for growth or development, but it enhances survival and competitiveness in a specific environment. Melanin in animals is especially related to skin disease and malignant melanoma , DHN melanin, GDHB melanin, catechol melanin, etc. have been reported in addition to DOPA melanin produced from tyrosine by tyrosinase (Bell, AA and Weeler MH, Ann. Rev. Phytophathol., 24, 411, 1986).

Melanin pigment is a unique stable pigment in living organisms, insoluble in almost all solvents, and it is not easy to extract or chemically treat melanin. Because of this, melanin is poorly identified in terms of chemistry, and progress in research is also sluggish. In mammals it is clear that almost the same melanogenesis occurs and occurs in the organelle called the melanosome of specifically differentiated cells called melanocytes.

Skin color is determined by the content and distribution of melanin. It is related to the number and distribution of melanosomes released from the melanocytes in the cells and released to the outside of the cells. Hyperpigmentation of the skin can be caused by various factors such as hormonal abnormalities in the body, genetic diseases, and ultraviolet irradiation after inflammatory reaction of the skin. The main factor is due to melanin pigment synthesis abnormality and distribution abnormality. The main function of melanin is to protect the skin from damage by removing oxygen radicals, which means that melanin has an effective countermeasure to protect skin from physical and chemical toxicants.

However, excessive melanin deposition is a cause of pathological problems such as skin, but it is recognized as a cosmetic problem such as spots, freckles, dots, and black spots. Therefore, development of a whitening agent that can basically inhibit melanin formation Is required.

Prevention of skin pigmentation can be performed in terms of controlling the activity of tyrosinase, which is the main enzyme of melanin synthesis, and controlling the melanocyte function of synthesizing melanin. Among them, hitherto known inhibitors of tyrosinase include hydroquinone, resorcinol, 4-hydroxyanisole, ascorbic acid and its derivatives, kojic acid Arbutin, glucosamine, oxyreseveratrol, alpha-viniferin, and ferulic acid. However, due to problems of skin safety and formulation stability, arbutin, And kojic acid are used only in a limited amount as an additive to whitening agents.

Until recently, various plants have been studied for the development of natural whitening agents. Among them, it is known that there is a tyrosinase inhibitory activity, such as alfalfa, rhododendron, licorice, green tea, seaweed and gardenia fruit, and some of them are used in products.

Korean Laid-Open Patent Publication No. 2013-0001027 (published in 2013-01-03) discloses a cosmetic composition for skin whitening comprising an extract of Sanchos Herbaceae and a process for producing the same.

In one embodiment, the present invention provides a cosmetic composition or cosmetic for skin whitening characterized by inhibiting melanin production comprising Furniturein D or a cosmetically acceptable salt thereof as an active ingredient.

Furniturein D included in compositions according to the present application is derived from Phorbas sp.

The composition according to the present invention may exhibit a whitening effect by inhibiting melanin biosynthesis through interfering with tyrosinase, particularly including synthesis of tyrosinase enzyme, inhibition of enzyme activity, and / or promotion of degradation.

In another aspect, the present invention also provides a pharmaceutical composition for the treatment of a pigmented skin disease comprising melanin hypercholesterolemia, characterized by inhibiting melanin production comprising furniturein D or a pharmaceutically acceptable salt thereof as an active ingredient do.

Since the composition comprising furniturein D according to the present application can inhibit melanin biosynthesis inhibition and the expression or activity of various enzymes including tyrosinase involved in the biosynthesis of melanin, It is obvious that the present invention can be effectively used for the treatment or prevention of pigmented skin diseases. Such diseases include, for example, scarring or dermatitis including freckles, senile plaques, dullness, liver, spots, brown or black spots, daylight pigment spots, bluish schizophrenia, hyperpigmentation after drug use, pregnancy brown spots, or scratches and burns , ≪ / RTI > post-inflammatory hyperpigmentation due to < RTI ID = 0.0 >

In another embodiment, the present application also provides a pharmaceutical composition comprising furninin D or a pharmaceutically acceptable salt thereof; Or sponge extract of the present invention as an active ingredient. The present invention also provides a health functional food composition for improving skin melanin pigment over-deposition.

In another embodiment, the present application also provides a pharmaceutical composition comprising furninin D or a pharmaceutically acceptable salt thereof; Or sponge extract as an active ingredient, wherein the skin-whitening functional food composition is a skin-whitening functional food composition.

In another embodiment, the present invention also provides a method of melanin-producing promyelandin hypercholesterolemia in Invitro comprising the step of treating the filamentous D with a cell capable of producing melanin.

A skin whitening cosmetic composition comprising Furniturein D, a diterpenoid-based compound isolated from a marine natural product, marine Phorbas species, according to the present invention has excellent whitening efficacy in in vitro experiments and artificial skin models , It is useful as a functional food composition such as a cosmetic having a whitening function or a whitening or inhibiting pigment deposition.

In particular, the composition according to the present invention comprises tyrosinase and its related enzyme tyrosinase-related protein-1, -2 [Tyrosinase-Related Protein-1, -2 (TRP-1, -2) (MITF), as well as the protein expression of lap 27a (Rab27a), melanophilin, and MyoVa, which are the main factors of melanosome migration, as well as the regulation of microphthalmia-associated transcription factor (GD) of the present invention is a whitening cosmetic composition, a functional food composition for whitening, or a pigment preparation (composition) for whitening, which is a new whitening candidate substance having various targets by exhibiting an effect of controlling the melanin- And the like.

Figure 1 shows the chemical structure of furniturein D extracted from the sea surface here.
FIGS. 2A and 2B show inhibition rates of melanin pigment formation by treatment of the melanocyte cell line Melan-a (2.5, 7.5, 10, and 20 μM) The cell viability at the same concentration and the cell shape on the microscope are shown.
FIGS. 3A and 3B are graphs showing the effect of inhibiting PAX3, SOX10, tyrosinase and TRP-1, -2 protein expression (FIG. 3B) at 24 hours treatment with melanogenesis- 3A). FIG. 3B shows the expression inhibition of MITF expression from 4 hours to 48 hours.
(Fig. 4A), tyrosinase (Fig. 4B), and TRP-1 (Fig. 4A) 4C), PAX3 (Fig. 4D) and SOX10 (Fig. 4E).
Figures 5A and 5B show the inhibition of the Gaussian Reporter Gene activity of the MITF gene (Figure 5A) and tyrosinase gene (Figure 5B) by the concentration of furniturein D (5, 10, Fig.
FIG. 6A and FIG. 6B are graphs (FIG. 6A) showing inhibitory effects on the melanin production of cell-derived tyrosinase and L-Dopa reaction by the concentration of furniturein D (5, 10, 20 μM) (5, 10, and 20 μM), respectively, on the cell surface, and inhibited cell-derived tyrosinase.
7A, 7B and 7C show the decomposition inducing activity of tyrosinase, showing the inhibitory effect on protein expression (Fig. 7A) upon treatment with 20 [mu] M Ninjin D for 4, 8, 12, 24 and 48 hours, / ml of cycloheximide was treated with 20 μM Furniturein D for 0.5, 1, 3, and 6 hours to promote degradation of tyrosinase (FIG. 7B), and FIG. 7C shows that the 26s proteasome inhibitor MG132 , And that the degradation of 20 μM of N-D tyrosinase was due to proteasome-related degradation.
8A and 8B show the inhibitory effect on melanomas migration by D-5 (10, 20 μM). FIG. 8A shows the effect of the marker factors 27a (Rab27a), Melanophilin And MyoVa expression was inhibited in a concentration-dependent manner. FIG. 8B shows the effect of inhibiting protein expression in immunocytochemistry at the time of processing 24-hour-old furniture N.
9A and 9B show that melanin synthesis was significantly inhibited (Fig. 9A) when housinin D at a concentration of 10 mu M was treated for 72 hours in the 3D artificial skin model (Fig. 9A) It is a picture showing no toxicity to skin cells.

This study was carried out to investigate the effect of the inhibitory effect of tyrosinase and its related enzymes on the melanocyte- derived housein D isolated from the sea surface Phorbas species, and to solve the fundamental problem of pigmentation through inhibition of pigmentation and to provide continuous and effective skin whitening Based on the finding that

In one embodiment, the present invention provides a process for preparing 7-acetoxy-8-hydroxy-1-isopropyl-3a, 5a, 9-trimethyl-1,2,3,3a, 4,5,5a, 6 , 7,8,10a, 10b-dodecahydrocyclohepta [e] indene-2,4,6-triyl tributyrate) or a cosmetically acceptable salt thereof as an active ingredient for inhibiting melanin production To cosmetic compositions or cosmetics.

[Chemical Formula 1]

In one embodiment according to the present invention, Furniturein D can be extracted using the method described in the Examples herein, from Phorbas sp.

Also, the synthesis of furniture nin D can be referred to Jang KH et al. (J Nat Prod, 2008 Oct; 71 (10): 1701-7).

The inventors of the present invention have found that N-D is an inhibitory activity of proteins, gene expression and tyrosinase, which is a rate-limiting enzyme of melanin synthesis, and a major transcription factor, MITF-related mechanism studies, Decomposition induction and so on. As a result, tyrosinase and tyrosinase-related protein 1, -2 [tyrosinase-related protein-1 and -2 (TRP-1, -2)], the most important enzymes for melanin synthesis in melanocytes Protein and gene expression. In addition, the microphthalmia-associated transcription factor (MITF) is a transcription factor that plays an important role in the production of melanin, and the expression of protein and gene is decreased by the Gagunin D gene. In addition, it inhibits gene and protein expression of SRY-related HMG-box 10 (SOX10), PAX3 (Paired box 3) and SOX10 (SRY-related HMG-box 10) which are important transcription factors of MITF and proteasomal related degradation degradation of the tyrosinase. In addition, it was shown that the N-D inhibited the enzymatic activity of tyrosinase and inhibited the protein expression of Rab 27a, Melanophilin and MyoVa, which are the main factors of melanoma migration. It is possible to work as a candidate for inhibition of whitening and pigmentation by showing melanin synthesis inhibitory effect in 3D artificial skin model as well as possibility.

As used herein, the term " skin whitening " is intended to inhibit changes in skin color or pigmentation caused by excessive production of pigment in the skin due to external environmental changes such as ultraviolet rays, stress, hormone secretion abnormality and pregnancy. The effect of improving or preventing or preventing dark circles, spots, freckles and dark circles occurring, the effect of increasing the transparency of the skin, the effect of improving the dullness and tightness of the skin by improving the dullness of the skin.

In general, pigmentation of the skin is stimulated by ultraviolet light or hormone imbalance, leading to melanocyte stimulation, which is the main cause of skin deposition of melanin pigment biosynthesized. Therefore, compositions according to the present invention capable of inhibiting melanin biosynthesis in melanocyte, which is a melanin biosynthesis cell, can be used in various applications requiring inhibition of pigment deposition other than the above-mentioned whitening.

The composition according to the present disclosure may also comprise a cosmetically acceptable salt of furninin D. This is useful for acid addition salts formed by free acids. Acid addition salts can be prepared in a conventional manner, for example, by dissolving the compound in an excess amount of an aqueous acid solution and precipitating the salt with a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. The same molar amount of the compound and the acid or alcohol (e.g., glycol monomethyl ether) in water may be heated and then the mixture may be evaporated to dryness, or the precipitated salt may be subjected to suction filtration. As the free acid, organic acids and inorganic acids can be used. As the inorganic acids, hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, tartaric acid and the like can be used. Examples of the organic acids include methanesulfonic acid, p- toluenesulfonic acid, acetic acid, trifluoroacetic acid, The organic acid may be selected from maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, mandelic acid, propionic acid, citric acid, lactic acid, glycollic acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanillic acid and hydroiodic acid can be used. It is not limited.

As used herein, the term cosmetic composition refers to an article used in the human body to brighten or darken the appearance of a human body to improve appearance or to maintain or promote the health of skin and hair, and refers to a slight action on the human body. Commonly used include lotions, creams, oils, cleansing products, and functional cosmetics. Functional cosmetics are physical control, biochemical, and biotechnological techniques that control the body's defense rhythm, cosmetic control, disease control and recovery, And cosmetics which are processed and designed so that the functions are sufficiently expressed in living bodies.

The cosmetic composition or cosmetic according to the present invention may be provided, for example, as a cream, a lotion, an ampoule, a skin, an essence, a soap, (Body lotion, lipstick, mascara, make-up base), or in the form of a mask or pack.

The site to which the cosmetic composition or cosmetic according to the present invention can be applied is not limited to the specific fat tissue of the body, but can be applied to various parts of the skin, especially face, arm, leg, etc., which require inhibition of pigmentation or whitening. The site of application may be administered by a variety of routes of administration, such as systemic or topical, especially topical, transdermal, or injection, particularly the transdermal route.

The cosmetic composition according to the present invention has no particular limitation on other ingredients other than the furniture D of the present invention as essential ingredients in the indicated ratios, and in the case of ordinary cosmetics such as creams, vegetable oils, emulsifiers, thickeners, , An antioxidant, and a UV absorber.

The content of Furniturein D contained in the composition according to the present invention may vary depending on the specific use or formulation of the desired product, but may be about 0.001 to about 50% by weight in the total composition, It is not.

The cosmetic composition according to the present invention may contain conventional adjuvants and carriers such as antioxidants, stabilizers, solubilizers, vitamins, pigments and / or fragrances, in addition to the furnishin D.

The cosmetic composition of the present invention may comprise a water-soluble base material comprising an unsaturated fatty acid or propylene glycol including conjugated linoleic acid, linoleic acid, gamma-linoleic acid, alpha-linoleic acid.

In addition, the cosmetic composition of the present invention may be prepared in any form conventionally produced in the art, and may be prepared, for example, as a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, , Oil, powder foundation, emulsion foundation, wax foundation and spray, but is not limited thereto. More specifically, it can be manufactured in the form of a soft lotion, a nutritional lotion, a nutritional cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a mask, a pack, a spray or a powder.

When the formulation of the present cosmetic composition is a paste, a cream or a gel, an animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide .

When the formulation of the present cosmetic composition is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. In particular, in the case of a spray, a mixture of chlorofluorohydrocarbons, Propane / butane or dimethyl ether.

The cosmetic composition according to the present invention may further comprise a cosmetically / dermatologically acceptable carrier. &Quot; cosmetically / dermatologically acceptable " means suitable for use in contact with a tissue (e.g., skin or hair) without undue toxicity, insolubility, instability, irritation, allergic response,

When the formulation of the composition is a solution or an emulsion, a solvent, a solubilizing agent or an emulsifying agent is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, , 3-butyl glycol, glycerol aliphatic ester, polyethylene glycol or fatty acid esters of sorbitan can be used.

When the formulation of the present composition is a suspension, a carrier such as water, a liquid diluent such as ethanol or propylene glycol, a suspension such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Cellulose, aluminum metahydroxide, bentonite, agar or tracant, etc. may be used.

When the formulation of the composition of the present invention is an interface-active agent-containing cleansing, the carrier component is selected from the group consisting of aliphatic alcohol sulfates, aliphatic alcohol ether sulfates, sulfosuccinic acid monoesters, isethionates, imidazolium derivatives, methyltaurates, sarcosinates, fatty acid amide ethers Alkylamido betaine, aliphatic alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative, or ethoxylated glycerol fatty acid ester. According to a preferred mode of administration, the compositions of the present invention may comprise at least one pharmaceutically acceptable excipient, especially a dermatologically acceptable excipient.

Further, in the composition for dermatological preparation of each of the formulations, components other than the above-mentioned essential components may be selected and mixed without difficulty by those skilled in the art depending on other compositions for dermal use or the purpose of use.

Since the composition of the present invention has little toxicity and side effects, it can be safely used for prolonged use for preventive purposes.

As described above, familin D according to the present invention can inhibit melanin biosynthesis fundamentally and can be used for the treatment of diseases associated with various hyper melanin pigment deposits requiring inhibition of melanin biosynthesis.

As used herein, the term " hyperpigmentation ", including melanotic hypercholinism, refers to darkening or darkening of the skin or other areas of the hand or toenails due to excessive increase of melanin at certain sites. The melanin pigment hyperpigmentation disorder may be selected from the group consisting of freckles, dullness, senile spots, liver, spots, brown or black spots, daylight pigmentation, cyanicmelasma, hyperpigmentation after drug use, gravidic chloasma, And post-inflammatory hyperpigmentation due to burns or dermatitis, including burns, and the like.

Pharmaceutically acceptable salts herein refer to those which are physiologically acceptable and do not cause an allergic reaction or similar reaction that is common in humans, and the salt is preferably an acid addition salt formed by free acid Do. The free acid may be an organic acid or an inorganic acid. The organic acids include, but are not limited to, citric, acetic, lactic, tartaric, maleic, fumaric, formic, propionic, oxalic, trifluroacetic, benzoic, gluconic, methosulfonic, glycolic, succinic, Glutaric acid and aspartic acid. In addition, the inorganic acid includes, but is not limited to, hydrochloric acid, bromic acid, sulfuric acid, and phosphoric acid. In one embodiment according to the present disclosure, the pharmaceutically acceptable salt may be present as an acid addition salt, wherein the furniturein D compound according to the present invention forms a salt with the free acid. Also, furninin D according to the present application may include not only pharmaceutically acceptable salts, but also all salts, hydrates, solvates which may be prepared by conventional methods.

As used herein, the term " treatment " refers to any action that improves or alleviates melanotic hypercholesterolemia with administration or application of the composition. Those skilled in the art will be able to ascertain, by reference to the data provided by the Korean Medical Association, the precise criteria of the disease for which the composition of the present invention is effective, .

A pharmaceutical composition comprising Furniturein D or a spongy extract comprising the same according to the present disclosure may be formulated with suitable carriers, diluents, preservatives, stabilizers, wetting agents, emulsifiers, solubilizers, sweeteners, colorants, Antioxidants, and the like. Specific examples include lactose, dextrose, sucrose, sorbitol, mannitol xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone Water, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium, stearate, mineral oil and the like.

The method of administration of the pharmaceutical composition comprising Furniturein D or a spongy extract comprising the same according to the present invention can be readily selected according to the formulation and can be administered to a human in various routes. For example, it may be formulated in the form of powders, tablets, pills, granules, dragees, hard or soft capsules, liquids, emulsions, suspensions, syrups, elixirs, external preparations, suppositories, sterilized injection solutions, Orally or parenterally. In particular, oral administration is preferred.

Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin, . In addition to simple excipients, lubricants such as magnesium stearate talc are also used. Examples of the liquid formulations for oral administration include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, which are commonly used diluents, various excipients such as wetting agents, sweeteners, have.

Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the non-aqueous solvent and suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As a base for suppositories, witepsol, macrogol, tween 61, cacao paper, laurin, glycerol, gelatin and the like can be used.

Further, the pharmaceutical compositions comprising the extract according to the present invention may be prepared using the appropriate methods known in the art or by methods disclosed in Remington's Pharmaceutical Science (recent edition), Mack Publishing Company, Easton PA) And the like.

The dosage of the pharmaceutical composition comprising the extract according to the present invention or the active ingredient compound isolated therefrom may vary depending on the patient's body weight, age, sex, health condition, diet, administration time, administration method, excretion rate, The effective dose of the extract is usually about 1 to 100 g / day, especially about 10 to 50 g / day, more preferably about 30 g / day for an adult (60 kg). It will be apparent to those skilled in the art that doses may be additive or subtracted, as the dosage can vary depending on various conditions, and thus the dose is not intended to limit the scope of the invention in any way.

The number of administrations can be administered once or several times a day within a desired range, and the administration period is not particularly limited. In addition, the extract of the present invention or a composition comprising the active ingredient compound isolated therefrom may be added to any food in addition to oral administration as it is, and may be routinely ingested.

The pharmaceutical composition comprising Furniturein D or a spongy extract containing the same according to the present invention not only provides an excellent therapeutic effect on pigment deposition through inhibition of melanin biosynthesis but also has no toxicity and side effects caused by drugs, Can be used.

Accordingly, Furniturein D according to the present invention or a sea surface extract containing the same can be used as a main ingredient, a minor ingredient, a food additive for food, or a health functional food material such as a whitening functional food.

As used herein, the term " food " means a natural or processed product containing one or more nutrients, preferably a state of being ready to be eaten through a certain degree of processing, It is intended to include food, food additives, functional foods and beverages as a meaning.

As used herein, the term " functional food " refers to a food group that has been imparted with added value to function or express the function of the food by physical, biochemical, or biotechnological techniques, , And the body control function of disease prevention and recovery is designed to be fully expressed in the living body, and in particular, includes "health functional food".

As used herein, the term " health supplement food " is a food prepared by using raw materials or ingredients having useful functions in the human body, and is useful for health and other purposes such as controlling nutrients and physiological actions on the structure and function of the human body For the purpose of health assisting to obtain the effect, it refers to a food which is made by processing a specific ingredient as a raw material or by extracting, concentrating, refining, mixing, etc. ingredients contained in a food raw material.

The functional food and the health supplement food may further include a food-acceptable food supplementary additive, and may further comprise suitable carriers, excipients and diluents conventionally used in the production of a functional food.

Examples of the foods to which the noodle D or spongy extract according to the present invention can be added include various foods, beverages, gums, tea, vitamin complexes, and functional foods. In addition, in the present invention, the food may contain special nutritional foods (e.g., crude oil, spirits, infant food, etc.), meat products, fish products, tofu, jelly, noodles (Such as soy sauce, soybean paste, hot pepper paste, mixed sauce), sauces, confectionery (eg snacks), dairy products (eg fermented milk, cheese), other processed foods, kimchi, pickled foods But are not limited to, fruits, vegetables, beverages, fermented beverages, etc.), natural seasonings (e.g., ramen soup, etc.). The food, beverage or food additive may be prepared by a conventional production method.

In one embodiment, the functional beverage is made of a functional beverage. The functional beverage includes a beverage group to which the added value is imparted so as to function and express the function of the beverage to a specific purpose, physical or biochemical, Defensive rhythm control, disease prevention and recovery, etc., means a beverage which is designed to sufficiently express the body control function against the living body, and particularly refers to a drink for improving skin elasticity and promoting collagen synthesis.

The food composition of the present invention can be used as a flavoring agent such as a variety of nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, colorants and heavies (cheese, chocolate, etc.), pectic acid and its salts, Organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like. These components can be used independently or in combination.

It is to be understood that both the foregoing general description and the following detailed description of the present invention are exemplary and explanatory and are intended to be illustrative of the invention and are not intended to limit the scope of the claims. It will be apparent to those skilled in the art that such variations and modifications are within the scope of the appended claims.

Example

Example  1. Sponges Phorbas sp . from Untreated  Extract preparation and Furniture nin  Of D detach

The phytoplankton was collected from 20 to 25 meters of the coast of Kagajo coast in South Korea, and then freeze-dried after collecting the sponges Phorbas sp. (Kyoung Hwa Jang et al. (2008). Polyoxygenated diterpenes from the sponge Phorbas sp . J. Nat. Prod., 71 , 1701-1707). To summarize, the crude extracts were repeatedly extracted with methanol and dichloromethane, and the active compounds were screened by solvent partitioning and C18 reverse phase vacuum chromatography. Then, the active material was separated by reversed phase silica HPLC, and the structure was confirmed by comparing with the physical properties described in the existing literature (FIG. 1).

Furniture nin  D ( gagunin  D)  (Fig. 1)

7-acetoxy-8-hydroxy-1-isopropyl-3a, 5a, 9-trimethyl-1,2,3,3a, 4,5,5a, 6,7,8,10a, 10b-dodecahydrocyclohepta [ 2,4,6-triyl tributyrate

: Amorphous solid;

[?] _D + 51.3 FAB-MS m / z: 607.3 (M + H)

^{1 H-NMR (400 MHz,} CDCl3) δ5.47 (1H, d, J = 5.9 Hz, H-2), 5.42 (1H, d, J = 10.3 Hz, H-6), 5.14 (1H, dt, 4.9, 4.4 Hz, H-12), 4.96 (1H, dd, J = 10.3,1.5Hz, H-5), 4.92 (1H, dd, J = 12.2, 3.9Hz, H- (1H, br s, H-4), 3.50 (1H, dd, J = 9.8,5.9 Hz, H- 2.34 (1H, d, J = 12.8, 7.3 Hz, H-2b '''), 2.22 (1H, m, H-15), 1.88 (1H, br d, J = H-11), 1.84 (1H, br s, H-15), 1.82 (1H, dd, J = 9.8,4.4 Hz, H- H-3 ''''), 1.60 (2H, m, H-3 '', H-3 '''), 1.56 (1H, dd, J = 12.7, 3.9 Hz, (d, J = 12.7 Hz, H-8b), 1.06 (3H, s, H-20) (3H, t, J = 7.3 Hz, H-4 ''), 0.96 (3H, d, J = 5.4 Hz, Hz, H-4 '''), 0.86 (3H, d, J = 5.9 Hz, H-16);

^{13 C-NMR (100 MHz,} CDCl3) δ: 173.5 (s, C-1 ''''), 173.5 (s, C-1 '''), 172.7 (s, C-1''), 170.0 ( C-4), 138.1 (d, C-2), 131.6 (d, C-3), 78.1 ), 74.5 (d, C-4), 72.6 (d, C-5), 55.0 , 36.5 (t, C-2 '''), 36.5 (t, C-2) (Me, C-18), 24.0 (Me, C-15), 23.6 (Me, C-20) C-17), 21.9 (Me, C-16), 21.2 (Me, C-2 '), 18.6 13.8 (Me, C-4 '''), 13.8 (Me, C-3''), 14.1 4'').

Example  2: Melanogenesis inhibitory efficacy analysis

The melanin production inhibitory effect of the sample obtained in Example 1 was analyzed as follows.

2-1. Cell culture

The Melan-a cell line, which is a melanocyte derived from an animal cell line used in the present invention, was purchased from Dr. DC Bennett (University of London, UK), and 2 mM glutamine, 100 (100 μg / ml streptomycin, Gibco, USA), 10% fetal bovine serum (Gibco, USA), 100 nM TPA (12-O-tetradecanoylphorbol (Gibco, USA) medium supplemented with 10% CO2 and 90% air in a CO ₂ incubator at 37 ° C was used for the experiment .

2-2. Melanin synthesis inhibition efficacy and cytotoxicity evaluation

The test method was used to compare the amount of melanin produced in the cell with the amount of the blank. Gagunin D (GD) was added to the culture medium of melan-a cells, a melanocyte of mice, to test the effect of improving whitening at the cellular level.

More specifically, Melan-a cells, which are melanocytes of mice, were divided into 6-well plates at a concentration of 3 × 10 ⁴ cells / well and cultured for 48 hours, , And then treated with furniture N at a final concentration of 2, 5, 7.5, 10, 20 μM and cultured for 48 hours. Cultured cells were washed twice with phosphate buffered saline (DPBS) containing no calcium chloride (CaCl ₂ ) or magnesium chloride (MgCl ₂ ) and treated with trypsin (Gibco, USA) The same number of cells per group were collected. The cell precipitate was washed with phosphate buffered saline (DPBS) containing no calcium chloride (CaCl ₂ ) or magnesium chloride (MgCl ₂ ), and 100 μl of 1 normal concentration added with 10% dimethylsulfoxide (DMSO) (N) solution of sodium hydroxide (NaOH), treated at 60 DEG C for 30 minutes, absorbance was measured at 475 nm, and the amount of melanin was converted from a standard curve made of synthetic melanin.

As shown in FIG. 2, the inhibitory rate of melanin production was 11.1% at 2.5 μM, 27.3% at 7.5 μM, 45.3% at 10 μM and 70.0% at 20 μM compared with the control. (IC ₅₀ , 12.7 μM). In particular, a 20 μM concentration of furniturein D showed a stronger inhibitory effect on melanin formation than 1 mM arbutin. In addition, the inhibition of the melanin synthesis of the filamentous D (GD) was not cytotoxic, as demonstrated by the MTT cytotoxicity assay (Fig. 2 (B)).

2-3. Melanosite  In a cell Melano  Assessment of inhibitory effect on biosynthesis-related protein expression

Melan-a cells were cultured in 100-mm culture dishes and washed twice with phosphate buffered saline (DPBS) containing no calcium chloride (CaCl ₂ ) or magnesium chloride (MgCl ₂ ) Were treated to final concentrations of 5, 10 and 20 μM and cultured for 24 hours. After washing twice with DPBS, the plate was incubated with boiling 2 × sample loading buffer (250 mM Tris-HCl (pH 6.8), 4% SDS, 10% glycerol, 0.006% bromophenol blue, 2% 5 mM sodium orthovanadate), and the cells were disrupted at 100 ° C for 5 minutes. After cooling, the solution was stored at -20 ° C. The solution was dissolved at 37 ° C just before use and used for protein quantification and electrophoresis. 80 μg of the protein was electrophoresed at 138 V for 2 hours using 5-12% SDS-polyacrylamide gel (# 456-1036, Bio-Rad, Hercules, Calif.) And the separated proteins were transferred to PVDF membrane (ISEQ15150, Millipore , Bedford, MA) for 1 hour at 100 V, washed twice with TBST, and incubated in a blocking buffer (5% Albumin from Bovine Serum in PBS containing 0.1% Tween-20 (TBST) Lt; / RTI > After washing with TBST 3 times for 5 minutes, the monoclonal antibody was diluted with TBST in 3% Albumin from Bovine Serum (BSA (A9647, Sigma)) and reacted with membrane at 4 ° C for 18 hours. After washing the membranes (Membrane, ISEQ 1S150, Millipore, Bedford, MA) three times for 5 minutes with TBST, the HRP (horseradish peroxidase) -binding secondary antibody was diluted with TBST to a ratio of 1: 1,000-1: And reacted with the membrane at room temperature for 3 to 4 hours. After washing with TBST three times for 5 minutes, the western blotting substrate (WEST-ZOL PLUS (16021, Intron)) was treated to confirm protein expression level on LAS-3000 (Fuji Film Corp., Japan).

As a result, tyrosinase, tyrosinase related protein 1, -2, tyrosinase related protein 1, -2, and microphthalmia-associated transcription factor (MITF), the most important transcription factors involved in melanin synthesis, Protein expression of PAX3 (Paired box 3) and SOX10 (SRY-related HMG-box 10), transcription factors of MITF, decreased in a concentration-dependent manner as shown in Fig.

As shown in FIG. 3 (B), MITF expression was analyzed from time 4 to 48 hours after treatment of melanoma cells with 20 μM of N-D, and the expression of MITF was decreased in a time- Respectively.

2-4. Furniture nin  Of D Melano biosynthesis  related MITF , Tyrosinase , TRP-1, PAX3  And SOX10  Evaluation of gene expression inhibition efficacy

A gene for MITF, tyrosinase, tyrosinase related protein (TRP) -1, PAX3, and SOX10 in melanocytes, which are melanocytes of mice, For the evaluation of the inhibitory effect on the expression of the protein, the melanin A cells were treated with DIN at a concentration of 12 hours in a 100 mm dish. After the cells were washed twice with PBS, the cells were harvested using TRI reagent (TRIZol (# 15596-026, Invitrogen)), and the RNA was extracted with CHCl ₃ and precipitated with isopropyl alcohol. The RNA precipitate was washed with 70% ethanol, dried in air and dissolved in nuclease-free water (AM9937, Ambion). Heated at 55 占 폚 for 10 minutes, and heated at 70 占 폚 for 5 minutes so that RNA was present in a single stranded state. After the Total RNA quantified using a NanoDrop (ND-1000, Dae Myung Sceince Co., Ltd) diluted to 1μg / μl avian myeloblastosis virus (AMV ) reverse transcriptase (M5108, Promega) and the oligo (dT) ₁₅ primer ( C110B, Promega). iQ ^TM SYBR  The target gene was amplified by MiniOpticon ^™ Real-time PCR Detection System (CFB-3120, Bio-Rad) using Green Supermix (# 170-8880, Bio-Rad) and target gene-specific primers [Table 1]. Real-time PCR conditions were denaturation at 95 ° C for 20 seconds, followed by denaturation at 95 ° C for 20 seconds, annealing at 56 ° C for 20 seconds, and elongation at 72 ° C for 40 cycles Followed by a final step at 95 ° C for 1 min and at 55 ° C for 1 min. The Ct value was determined by the method of Leak et al. (Livak KJ et al (1996) Method 25: 402-408) using MJ Opticon Monitor software (Opticon Monitor 3.1.32, Bio-rad) , And it was confirmed that gene expression of MITF, tyrosinase, TRP-1, PAX3 and SOX10 was decreased by the N-D of the house.

In detail, MITF decreased by 35% at 5 μM, 40% at 10 μM and 80% at 20 μM, 21% at 10 μM and 42% at 20 μM for tyrosinase and 17% at 10 μM for TRP- , And 32% at 20 [mu] M (Fig. 4A, 4B, 4C). In addition, gene expression of PAX3 (Paired box 3) and SOX10 (SRY-related HMG-box 10), which are factors regulating MITF expression, was also dose-dependently reduced (FIG. 4D, 4E). In detail, PAX3 decreased 23% at 5 μM, 23% at 10 μM and 46% at 20 μM compared to the control, while SOX10 inhibited gene expression at 4% at 10 μM and 31% at 20 μM.

[Table 1] Real-time PCR primer

2-5. Furniture nin  By D MITF  And Tyrosinase  Gene-introduced Gaussian Lucy Gaussian luciferase ) Active inhibition efficacy

Melan-a cells were transfected with a firefly luciferase control vector (pFL) to correct the transfer efficiency with the Gaussia luciferase reporter plasmid pMITF-Gluc or pTyr-Gluc, MITF and tyrosinase plasmid. In particular, the tyrosinase receptor gene activity, which is the most important enzyme system in Melano genesis, was significantly decreased with respect to the concentration of NN (FIG. 5).

2-6. Furniture nin  Of D Cell-derived Tyrosinase  Active inhibition efficacy

Cell-free tyrosinase activity analysis was performed using melanin-a cell lysate containing tyrosinase, in order to examine the activity against tyrosinase, a rate-limiting enzyme in the synthesis of melanin. As a result of adding GD to the cell lysate by the concentration and treating the melanin substrate L-Dopa, the enzyme activity was inhibited in a concentration-dependent manner as shown in Fig. 6 (A). In addition, Melan-a cells were treated with N-terminal D for 24 hours, and cell lysates were obtained. Cellular tyrosine kinase assays were also performed to inhibit tyrosinase activity in a concentration-dependent manner as shown in Fig. 6 (A) (Fig. 6B).

2-7. Furniture nin  Of D Tyrosinase Induce decomposition  efficacy

Analysis of protein expression of tyrosinase over time in the mouse melanocyte for 48 hours showed that tyrosinase protein expression was effectively reduced in the case of N-terminal D-treatment compared to the control (Fig. 7A). In FIG. 7B, when cycloheximide inhibiting protein synthesis was treated and the expression of tyrosinase was analyzed for 6 hours, the tyrosinase degradation was promoted at the same time as that of the control group . And the 26s proteasome inhibitor MG132 was treated to examine the expression of tyrosinase protein. As a result, it was found that the N-D increased the degradation of tyrosinase through proteasomal degradation (Fig. 7C) .

2-8. Furniture nin  Of D Melano  move ( Melanosome  transfer inhibition efficacy

In the analysis of the expression of Rab 27a, Melanophilin and MyoVa, which are the main factors of melanoma migration in Melan-a cells, the expression of protein for major factors related to melanoma migration . In particular, melanophilin and MyoVa showed remarkably decreased protein expression as the concentration of the filamentous D was increased (FIG. 8A). Immunocytochemistry analysis also confirmed the inhibition of protein expression of lap 27a (Rab27a), melanophilin and MyoVa in the processing of the furninin D at a concentration of 20 μM (FIG. 8B).

2-9. In 3D artificial skin model Furniture nin  Evaluation of efficacy and toxicity of inhibiting melanin synthesis after D treatment

A 3D artificial skin model (Neoderm ^® -ME) containing human epidermis and melanocytes was treated with 10 μM of Furniturein D for 72 hours, resulting in a significant inhibition of melanin synthesis (FIG. 9A). This was also demonstrated by the MTT cytotoxicity assay that the inhibition of melanin synthesis in NN was not due to toxicity (FIG. 9B).

Hereinafter, a cream composition, a massage cream composition, a lotion composition, a skin lotion composition, an essence composition, a pack composition and a cleansing foam composition are exemplified as the formulations of the present invention. However, the formulation including the cosmetic composition of the present invention is not limited thereto no. In addition, Furniturein D, a diterpenoid-based compound isolated from the marine natural product, marine Phorbas species, as an active substance, is not limited to the following composition when it is used as a cosmetic composition or a cosmetic food composition.

Formulation Example  1. Cream composition

The oil phase and water phase are heated and mixed at 75 DEG C respectively with the composition and content (weight%) shown in the following table, and then cooled to room temperature.

Formulation Example  2. Cream composition

The oil phase and water phase are each heated to dissolve and mix at 75 ° C with the composition and content (% by weight) shown in the following table, and then cooled to room temperature.

Formulation Example  3. lotion composition

The oil phase and water phase are mixed and emulsified at 75 ° C with the composition and content (% by weight) shown in the following table, and then cooled to room temperature.

Formulation Example  4. Skin lotion composition

The water phase and the ethanol phase are prepared and mixed respectively with the composition and the content (% by weight) described in the following table, and then filtered.

Formulation Example  5. Essence composition

The water phase and the ethanol phase are prepared and mixed respectively with the composition and the content (% by weight) described in the following table, and then filtered.

Formulation Example  6. Pack composition

The water phase and the ethanol phase are dispersively dissolved and mixed in the composition and the content (weight%) shown in the following table, and then cooled to room temperature.

Formulation Example  7. Cleansing Foam  Composition

The water phase and oil phase are dispersively dissolved and mixed with each other in the composition and the content (weight%) shown in the following table, and then cooled to room temperature.

In addition, examples of formulations for beauty food of the present invention include chewing gum, candy, biscuit, ice cream, sausage, chocolate, ordinary beverage, and whitening probiotic lactic acid bacteria beverage formulation. However, the formulation including the cosmetic food composition of the present invention is not limited thereto no.

Manufacturing example  One : Of chewing gum (A)  Produce

Gum base 20 wt%

Sugar 76.5 wt%

Furniture N D Containing Sponges Phorbas Species extract 0.5 wt%

Fruit flavor 1 wt%

Water 2 wt%

Chewing gum was prepared in a conventional manner with the above composition and content.

Manufacturing example  2 : Of chewing gum (B)  Produce

Gum base 25 wt%

Sorbitol 72 wt%

Furniture N D Containing Sponges Phorbas Species extract 0.5 wt%

Peppermint Lever 2.5 wt%

Chewing gum was prepared in a conventional manner with the above composition and content.

Manufacturing example  3: Preparation of candy (A)

54.5 wt%

45% by weight of starch syrup

Furniture N D Containing Sponges Phorbas Species extract 0.4 wt%

0.1% by weight of orange flavor

Candies were prepared by a conventional method with the above composition and content.

Manufacturing example  4: Preparation of candy (B)

Maltitol 53.6 wt%

Reduced starch syrup 45%

Furniture N D Containing Sponges Phorbas Species extract 0.5 wt%

Natural herb fragrance 0.14 wt%

Candies were prepared by a conventional method with the above composition and content.

Manufacturing example  5: Biscuit  Produce

First class Flour 88 kg

Grade 1 flour 76.4 kg

16.5 kg

Salt 2.5 kg

Glucose 2.7 kg

Palm Shortening 40.5 kg

Ammonium 5.3 kg

0.6 kg of soy sauce

0.55 kg of sodium bisulfite

Rice flour 5.0 kg

Vitamin B ₁ 0.003 kg

Vitamin B ₂ 0.003 kg

Milk flavor 0.16 kg

Water 71.1 kg

Whole milk powder 4 kg

1 kg of replacement milk powder

Calcium Phosphate 0.1 kg

Spray salt 1 kg

Spray oil 25 kg

Furniture N D Containing Sponges Phorbas Species extract 1.7 kg

Biscuits were prepared by the conventional method with the above composition and content.

Manufacturing example  6: Manufacture of ice cream

10% by weight milk fat

Non-oil solid content 10.8 wt%

Sugar 12.0 wt%

3.0% by weight of starch syrup

0.5% by weight of an emulsion stabilizer (span, span)

Perfume (Strawberry) 0.15 wt%

Water 63.05 wt%

Furniture N D Containing Sponges Phorbas Species extract 0.5 wt%

Ice cream was prepared in a conventional manner with the above composition and content.

Manufacturing example  7: Sausage  Produce

Pork 63.6%

Chicken meat 27.5%

Starch 3.5%

Soy protein 1.7%

Salt 1.62%

Glucose 0.5%

Other additives (glycerin) 1.34-1.46%

Furniture N D Containing Sponges Phorbas Species extract 0.12-0.24%

The sausage having the whitening effect was prepared by mixing as described above

Manufacturing example  8 : Of chocolate  Produce

Sugar 34.5 wt%

Cocoa Butter 34 wt%

Cocoa mass 15 wt%

Cocoa powder 15 wt%

Lecithin 0.5 wt%

Vanilla flavor 0.5%

Furniture N D Containing Sponges Phorbas Species extract 0.5 wt%

Chocolate was prepared in a conventional manner with the above composition and content.

Manufacturing example  9. Intestinal health  Whitening Probiotics  Production of lactic acid bacteria beverage

Furniture N D Containing Sponges Phorbas Species extract 5 wt%

Probiotics 19.885 wt%

12% by weight isomalt

Powdered crystalline glucose 12 wt%

3% by weight of chicory extract powder

10% by weight of mixed skim milk powder

10% by weight vegetable cream

Xylitol 9 wt%

9% by weight of fructooligosaccharide

Fish collagen 5 wt%

Milk flavor powder 1 wt%

Yoghurt flavor powder 1 wt%

1% by weight of silicon dioxide

1% by weight of magnesium stearate

0.5% by weight of vitamin C

Dry egg yolk powder 0.5 wt%

Enzymatically treated Stevia 0.09 wt%

0.02% by weight of vitamin B2

0.005 wt% folic acid

Manufacturing example  10. Manufacture of whitened beverages

Furniture N D Containing Sponges Phorbas Species extract 1000 mg

Citric acid 1000 mg

100 g of oligosaccharide

Plum concentrate 2 g

Taurine 1 g

Purified water was added to a total of 900 ml

The above components were mixed according to a conventional health drink manufacturing method, and the mixture was heated at 85 DEG C for about 1 hour with stirring, and the solution thus prepared was filtered to obtain a sterilized 2-liter container, which was sealed and sterilized, ≪ / RTI >

Although the composition ratio is a mixture of the components suitable for the preferred beverage as a preferred embodiment, the compounding ratio may be arbitrarily varied depending on the regional and national preferences such as the demand level, the demanding country, and the intended use.

Claims (8)

Claims 1. A cosmetic composition for skin whitening characterized by inhibiting melanin production comprising Furniturein D or a cosmetically acceptable salt thereof as an active ingredient.
The cosmetic composition for skin whitening according to claim 1, wherein the furniturein D is separated from the sponge ( Phorbas sp.).
The cosmetic composition for skin whitening according to claim 1, wherein the inhibition of melanogenesis is by at least one of inhibition of tyrosinase expression, inhibition of tyrosinase activity, or degradation of tyrosinase.
A pharmaceutical composition for the treatment of a pigmented skin disease comprising melanin hypercholesterolemia, characterized by suppressing the production of melanin comprising Furniturein D or a pharmacologically acceptable salt thereof as an active ingredient.
5. The method of claim 4,
The pigmented skin disease may include scarring or dermatitis including freckles, senile spots, dullness, liver, spots, brown or black spots, daylight pigment spots, bluish schistosomiasis, hyperpigmentation after drug use, Wherein the hyperpigmentation is hyperpigmentation after inflammation due to inflammation.
A health functional food composition for improving melanin pigment over-deposition in skin characterized by inhibiting the production of melanin comprising Furniturein D or a pharmaceutically acceptable salt thereof as an active ingredient.
A functional food composition for skin whitening characterized by inhibiting the production of melanin comprising Furniturein D or a pharmaceutically acceptable salt thereof as an active ingredient.
A method of inhibiting melanin production in an Invitro comprising the step of treating the melanin cells with a filamentous D.

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