KR20110119271A - Composition for skin whitening comprising extracts of microalgae culture medium as an active ingredient - Google Patents

Abstract

PURPOSE: A composition containing microalgae culture liquid extract for skin whitening is provided to remarkably suppress melanogenesis and to ensure anti-inflammation without cytotoxicity. CONSTITUTION: A cosmetic composition for skin whitening contains microalgae culture liquid extract as an active ingredient. The microalgae are Chlorella kessleri, Synechocystis sp. PCC 6803, or Botryococcus braunii. A method for preparing the culture liquid comprises: a step of culturing microalgae at 24°C and 100 uE/m2/s for ten days to obtain culture liquid; a step of centrifuging the culture liquid to remove cells; and a step of filtering the culture liquid and isolating supernatant. The microalgae culture liquid extract is isolated using hexane, chloroform, or butanol. A pharmaceutical composition for skin whitening contains the microalgae culture liquid extract as an active ingredient.

Description

Composition for skin whitening comprising extracts of Microalgae culture medium as an active ingredient}

The present invention relates to a composition for skin whitening containing the microalgal culture extract as an active ingredient.

Melanin, which determines human skin color, is made from skin cells called melanocytes and migrates to epidermal cells called keratinocytes. Here, melanin plays an important role such as forming a hat-like structure around the nucleus to protect genes from ultraviolet rays and to remove free radicals to protect intracellular proteins. There are no enzymes in the body that break down melanin, but they are removed by falling off the skin as keratinocytes fall off the epidermis.

In the melanin synthesis process, in the melanoma in melanocytes, tyrosine is first used as a substrate to generate an enzyme called tyrosinase through dopa (DOPA) to produce dopaquinone (DOPAquinone). DOPAchrome produces melanin, a copolymer. The melanin thus produced is transferred to keratinocytes through a endoplasmic reticulum called melanosome, which, after about 28 days of keratinization, exits the skin surface and disappears with the keratin. However, in this process, the melanin is excessively produced by the factor that promotes melanin production and keratinization is not smoothly performed, so that the melanin is not completely disappeared, the pigmentation phenomenon occurs. Therefore, in order to prevent such a pigmentation phenomenon, it is necessary to inhibit a part of melanogenesis process to reduce melanin production.

In the development of a whitening agent for skin beauty, the characteristic of a composition having a conventional skin whitening effect is a method of reducing melanin by reducing the melanin pigment produced by targeting melanin, which is the main cause of coloring, and of tyrosinase for reducing melanin synthesis. Methods of inhibiting activity are known. Skin whitening ingredients used to reduce pigmentation of melanin include substances that inhibit tyrosinase enzyme activity such as kojic acid and arbutin, hydroquinone, and vitamin-C (L-Ascorbic). acid) and their derivatives and various plant extracts have been used. However, the skin whitening ingredients are poor in stability in the prescription system, and are degraded or colored, or their use is limited due to the occurrence of off-flavor, efficacy at the biological level, unclear effect and safety issues. In addition, skin whitening agents using tocopherol, vitamins, and the like used to reduce the melanin pigment are known to have a slight decolorizing effect of the melanin pigment, and many studies have been conducted on inhibitors that inhibit the production of melanin pigment.

In addition, it has been found that a number of plant extracts and herbal extracts act on tyrosinase to inhibit melanin production. However, in terms of stability and discoloration, there are many problems in using them in effective concentrations over pharmaceuticals. The situation is not effective.

Recently, as interest in health has increased, interest in functional biological materials has also increased. Most of these functional biomaterials have been researched on microorganisms, but recently, various bioactive substances have been found in microalgae at home and abroad, and high-value bioactive substances produced by microalgae have been actively researched to use them as resources. There is a growing interest in. Microalgae are photosynthetic microorganisms that have been a supplier of oxygen since the time of the Earth's time and are the producers of many useful substances. Recently, microalgae are being researched as a new functional biological material, and industrial use of microalgae has been used in applications such as biochemicals, as well as alternative energy sources, dietary supplements, aquaculture feeds and pharmaceutical raw materials.

In particular, chlorella is one of the microorganisms that are attracting attention for its various functionalities.

Chlorella is a green algae, single-celled plant that grows in freshwater, and belongs to the genus Chlorophycea, Chlorococcum, Chlorella. They usually grow in fresh water, such as ponds and lakes, and are spherical single-cell algae with a diameter of 2 to 10 um, one cell visible only under a microscope. Reproductive proliferation is abiotic reproduction and biologically the most important feature is that if there is plant growth factors such as water, air, nitrogen and phosphoric acid, independent nutritional growth and growth using light and carbon dioxide, which is the most important in the industrial mass culture process This is considered. Chlorella is a high-protein food with a good essential amino acid composition. Its total amino acid content is much higher than beef, and it is nutritionally balanced in terms of protein, lipids, dietary fiber, vitamins and minerals. Chlorella is currently used for primary and secondary purposes in feeds and foods, confirming its nutritional excellence, and actively searching for functionality as a tertiary food other than anticancer activity. Examples include the release of environmental hormone dioxin into the body, inhibition and excretion of heavy metals in the body, biodegradation of environmental toxic substances, inhibition of arteriosclerosis and hepatic disorders, anticancer activity, enhanced immune function, cell reactivation and food flavor enhancement, There have been reports of moisturizing effects, and research has been reported to use microalgae as a foodstuff for aquaculture in addition to foodstuffs or as a functional material for the removal of mercury from wastewater. There is a bar.

Until now, research on microalgal cultures has reported that chlorella cultures have anti-wrinkle and anti-cancer activity, but no activity on whitening effects has been reported.

Thus, the present inventors have three kinds of microalgae ( Chlorella kessleri , cinecosistis species. PCL 6803 ( Synechocystis sp . PCC 6803) , and Botryococcus brownies braunii )}, the supernatant from which the cells were removed by centrifugation and then the supernatant from which the cells were removed was extracted by adding an organic solvent. By confirming that the extract can be used as an active ingredient of the skin whitening composition and cosmetic composition, the present invention was completed.

An object of the present invention is to provide a composition for skin whitening containing the microalgal culture extract as an active ingredient.

In order to achieve the above object, the present invention provides a cosmetic composition for skin whitening containing the microalgal culture extract as an active ingredient.

The present invention also provides a pharmaceutical composition for skin whitening containing the microalgal culture extract as an active ingredient.

The present invention also provides a health food for skin whitening containing the microalgal culture extract as an active ingredient.

The microalgae culture extract of the present invention has almost no cytotoxicity and exhibits remarkable activity in anti-inflammatory activity and melanin synthesis inhibition compared to arbutin known as a general whitening substance, and thus as a cosmetic composition, pharmaceutical composition or health food for skin whitening. It can be usefully used.

Hereinafter, the present invention will be described in detail.

 The present invention provides a cosmetic composition for skin whitening containing the microalgal culture extract as an active ingredient.

The microalgal culture extract is preferably prepared by a manufacturing method comprising the following steps, but not always limited thereto:

1) culturing the microalgae;

2) separating the supernatant from the culture solution of step 1);

3) filtering the supernatant of step 2);

4) extracting by adding an extraction solvent to the filtered supernatant of step 3); And

5) concentrating the extract of step 4).

The microalgae are Chlorella kessleri , cinecosistis species. PCL 6803 ( Synechocystis sp . PCC 6803) , and Botryococcus brownies braunii )} is preferably any one selected from the group consisting of, but is not limited thereto.

In the above method, the microalgal culture of step 1) is preferably cultured using a photobioreactor, but is not limited thereto, and general microalgal culture methods may be used.

In the above method, the microalgal culture of step 1) is preferably an N-8 medium, but any medium used for microalgal culture is possible, and it is preferable to incubate at 20 to 30 ° C, but at 22 to 25 ° C. It is more preferable to incubate, and most preferably, but not limited thereto, at 24 ° C. Oxygen conditions are preferably from 0.05 to 0.5 vvm, more preferably from 0.1 to 0.3 vvm, most preferably incubated under 0.2 vvm oxygen conditions, but is not limited thereto, preferably incubated for 1 to 15 days, 5 More preferably, the culture is carried out for 12 to 12 days, most preferably for 10 days, but is not limited thereto. The amount of light is preferably 50 to 200 μE / m 2 / s, more preferably 90 to 150 μE / m 2 / s, and most preferably irradiated at 100 μE / m 2 / s, but is not limited thereto.

In the above method, the separation of the supernatant of step 2) is preferably carried out by centrifugation after culturing, but not limited thereto, and general cell separation methods may be used.

In the above method, the filtration of step 3) is preferably filtered with a filter paper, but is not limited thereto.

In the above method, it is preferable to use water, a lower alcohol of C ₁ to C ₄ or a mixed solvent thereof, a solvent selected from butanol, hexane and chloroform as the extraction solvent of step 4), and butanol, hexane and chloroform It is more preferable to use. As the extraction method, it is preferable to use shaking extraction or reflux extraction, but not always limited thereto. The extraction solvent is preferably extracted by adding 0.1 to 10 times the amount of the microalgal culture solution, and more preferably by adding 0.5 to 5 times, but not always limited thereto. The extraction temperature is preferably 10 to 50 ° C, more preferably 20 to 30 ° C, and most preferably at room temperature, but is not limited thereto. In addition, the extraction time is preferably 1 to 10 days, more preferably 2 to 6 days, most preferably 2 to 3 days, but is not limited thereto. In addition, the extraction number is preferably one to three times, but is not limited thereto.

In the above method, the concentration of step 5) is preferably a vacuum condenser or vacuum rotary evaporator, but is not limited thereto.

In a specific embodiment of the invention, Chlorella Kessler ( Chlorella) kessleri ), synechosis species. PCL 6803 ( Synechocystis sp . PCC 6803 ) and three kinds of microalgae of Botryococcus braunii were cultured using a photobioreactor, and the collected culture solution was centrifuged to remove cells and filtered with a filter paper to obtain a supernatant. Butanol, hexane, and chloroform were respectively added to the obtained supernatant, and the solvent layer was separated and concentrated by a vacuum evaporator. Through this, butanol extract, hexane extract, and chloroform extract were prepared from the microalgal culture.

In order to confirm the whitening effect of the obtained extract, the extract was treated with B16F10 cells and measured by comparing the change in the amount of melanin produced. As a result, in the case of the arbutin, a positive control, 67.2% of the melanin compared to the untreated drug It was confirmed that the inhibitory effect on the synthesis, the microalgal culture extract, all showed a melanin synthesis inhibitory effect regardless of the species and the type of the extracted organic solvent. Especially Chlorella Chloroform extract of kessleri ) culture solution showed an inhibitory effect of 41.9% compared to the untreated, showing a melanin synthesis inhibitory activity more superior than the arbutin positive control (see Table 1).

Therefore, it was confirmed that the microalgal culture and the microalgal culture extract had inhibitory activity of melanin synthesis.

In addition, in the embodiment of the present invention, in order to confirm the cytotoxicity of the microalgal culture extract, the extract obtained in <Example 1> was treated with B16F10 melanocytes, cell growth rate was measured, and as a result, compared with the untreated group When the extract was treated at a high concentration of 200 ug / ml, it was confirmed that the number of living cells was 90% or more, regardless of the species and the type of the extracted organic solvent except the chlorella kessler culture (Table 2). Reference).

Therefore, it was confirmed that the microalgal cultures were cytotoxic, whereas the microalgal culture extracts had little cytotoxicity.

In addition, in the embodiment of the present invention, in order to determine whether the inhibitory effect of tyrosinase, an important enzyme of melanin biosynthesis of the microalgal culture extract, in vitro ( in In vitro ) tyrosinase experiments were performed, and as a result, Chlorella Kessler ( Chlorella) kessleri ) When treated with 400 ug / ml of the culture extract, 16.2% inhibited the activity of tyrosinase (see Table 3).

Therefore, it was confirmed that the microalgal culture extract was weaker than kojic acid as a positive control, but had some tyrosinase inhibitory activity.

In addition, in the embodiment of the present invention, in order to confirm the anti-inflammatory effect of the microalgae culture extract, as a lipopolysaccharide (LPS) to Raw 264.7 cells, which is a macrophage cell line (nitrogen-producing macrophage cell line) that produces nitric oxide (NO) After inducing the production of nitric oxide (NO), the extract was treated, and compared with the measurement of the production of NO induced by LPS in cells. As a result, the better the anti-inflammatory effect, the lower the level of NO produced. Therefore, the anti-inflammatory activity was 91.9% at 1 ppm of chamomile, and Chlorella kessleri ) When treated with 200 ppm of the culture extract, it was found to be 31.9% (see Table 4). In particular, Chlorella kessleri culture extract did not show cytotoxicity at 200 ppm compared to chamomile.

Therefore, it was confirmed that the microalgal culture extract showed anti-inflammatory activity.

Although the microalgal culture and the extract of the culture of the present invention exhibited a whitening effect, the microalgal culture was found to have a skin cytotoxic effect, which is inappropriate for use as a cosmetic composition, whereas the microalgal culture extract is a cell. Since there is little toxicity, microalgal culture extract can be usefully used for the development of skin cosmetic composition.

In addition, the color of chlorophyll in the development of cosmetics using plant extracts may be a problem in the aesthetics in the development of cosmetics, and the research to remove the color by selectively removing only chlorophyll without changing the composition and function of cells. There is a situation. However, the present invention relates to a composition using a culture supernatant extract in which microalgae are cultured and cells are removed through centrifugation, and the culture supernatants in which cells are removed are yellowish green when the microalgae are cultured and cells are not removed. It has a yellow color, and thus does not require a color removal process compared to other plant extracts, and thus can be used more efficiently when developing a skin cosmetic composition.

In the composition of the present invention, the microalgal culture extract is preferably contained in an amount of 0.005 to 90% by weight based on the total weight of the composition. have.

The composition of the present invention may further contain at least one active ingredient exhibiting the same or similar function in addition to the microalgal culture extract.

When the microalgal culture extract of the present invention is used as an external preparation for skin, it is further used as a fatty substance, an organic solvent, a dissolving agent, a thickening agent and a gelling agent, an emollient, an antioxidant, a suspending agent, a stabilizer, a foaming agent, a fragrance, Surfactants, water, ionic or nonionic emulsifiers, fillers, metal ion sequestrants and chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles or external preparations for skin It may contain adjuvants commonly used in the field of dermatology, such as any other ingredients commonly used in the art. In addition, the ingredients may be introduced in amounts generally used in the field of dermatology.

In addition, as a specific formulation of the cosmetic composition of the present invention, skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisturizing lotion, nutrition lotion, massage cream, nutrition cream, moisture cream, hand cream, essence, nutrition essence, Packs, soaps, shampoos, cleansing foams, cleansing lotions, cleansing creams, body lotions, body cleansers, emulsions, press powders, loose powders, eye shadows and the like.

In addition, the cosmetic composition is in addition to the microalgal culture extract of the present invention, fatty substances, organic solvents, solubilizers, thickening and gelling agents, emollients, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, interfaces Common to active agents, water, ionic or nonionic emulsifiers, fillers, metal ion sequestrants and chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles or cosmetics It may contain auxiliaries commonly used in the cosmetic field, such as any other ingredients used as.

The present invention also provides a pharmaceutical composition for skin whitening containing the microalgal culture extract as an active ingredient.

The microalgal culture extract is preferably prepared by a manufacturing method comprising the following steps, but not always limited thereto:

1) culturing the microalgae;

2) separating the supernatant from the culture solution of step 1);

3) filtering the supernatant of step 2);

4) extracting by adding an extraction solvent to the filtered supernatant of step 3); And

5) concentrating the extract of step 4).

The microalgae are Chlorella kessleri , cinecosistis species. PCL 6803 ( Synechocystis sp. PCC 6803) , and Botryococcus brownies braunii )} is preferably any one selected from the group consisting of, but is not limited thereto.

In the above method, the microalgal culture of step 1) is preferably cultured using a photobioreactor, but is not limited thereto, and general microalgal culture methods may be used.

In the above method, the microalgal culture of step 1) is preferably an N-8 medium, but any medium used for microalgal culture is possible, and it is preferable to incubate at 20 to 30 ° C, but at 22 to 25 ° C. It is more preferable to incubate, and most preferably, but not limited thereto, at 24 ° C. Oxygen conditions are preferably from 0.05 to 0.5 vvm, more preferably from 0.1 to 0.3 vvm, most preferably incubated under 0.2 vvm oxygen conditions, but is not limited thereto, preferably incubated for 1 to 15 days, 5 More preferably, the culture is carried out for 12 to 12 days, most preferably for 10 days, but is not limited thereto. The amount of light is preferably 50 to 200 μE / m 2 / s, more preferably 90 to 150 μE / m 2 / s, and most preferably irradiated at 100 μE / m 2 / s, but is not limited thereto.

In the above method, the separation of the supernatant of step 2) is preferably carried out by centrifugation after culturing, but not limited thereto, and general cell separation methods may be used.

In the above method, the filtration of step 3) is preferably filtered with a filter paper, but is not limited thereto.

In the above method, it is preferable to use water, a lower alcohol of C ₁ to C ₄ or a mixed solvent thereof, a solvent selected from butanol, hexane and chloroform as the extraction solvent of step 4), and butanol, hexane and chloroform It is more preferable to use. As the extraction method, it is preferable to use shaking extraction or reflux extraction, but not always limited thereto. The extraction solvent is preferably extracted by adding 0.1 to 10 times the amount of the microalgal culture solution, and more preferably by adding 0.5 to 5 times, but not always limited thereto. Extraction temperature is preferably 50 to 100 ℃ but is not limited thereto. In addition, the extraction time is preferably 1 to 10 days, more preferably 2 to 5 days, but is not limited thereto. In addition, the extraction number is preferably one to three times, but is not limited thereto.

In the above method, the concentration of step 5) is preferably a vacuum condenser or vacuum rotary evaporator, but is not limited thereto.

The microalgal culture extract of the present invention has little cytotoxicity, has anti-inflammatory activity, and has a remarkable effect on inhibiting melanin synthesis, and thus can be usefully used as an effective ingredient of an effective pharmaceutical composition for skin whitening.

The pharmaceutical composition of the present invention may further include conventionally used excipients, disintegrants, sweeteners, lubricants, flavoring agents, etc., tablets, capsules, powders, granules, suspensions, emulsions, It can be formulated into syrups, other liquids.

In particular, the pharmaceutical compositions of the present invention may be formulated for oral administration such as, for example, stagnation, troches, lozenges, aqueous or dominant suspensions, prepared powders or granules, emulsions, hard or soft capsules, syrups or It is formulated with elixirs. Formulations such as tablets and capsules for lactose, saccharose, sorbitol, mannitol, starch, amylopectin, binders such as cellulose or gelatin, excipients such as dicalcium phosphate, disintegrants such as corn starch or sweet potato starch, stearic acid Lubricants such as magnesium, calcium stearate, sodium stearyl fumarate or polyethylene glycol wax. In the case of a capsule formulation, in addition to the above-mentioned substances, a liquid carrier such as fatty oil is contained.

In addition, the pharmaceutical composition of the present invention may be administered orally or parenterally, and it is preferable to select subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection injection method. To formulate into a parenteral formulation, the microalgal culture extract of the present invention is mixed in water with a stabilizer or buffer to prepare a suspension, which is formulated in unit dosage forms of ampoules or vials.

The dosage of the active ingredient according to the present invention is appropriately selected according to the absorption rate, inactivation rate and excretion rate of the active ingredient in the body, the age, sex and condition of the patient, the severity of the disease to be treated, but in the case of oral administration in general Adults can be administered once to several times in an amount of 0.0001 to 500 mg of the extract of the present invention per kg of body weight per day, it is preferable to administer in an amount of 0.001 to 100 mg.

The present invention also provides a health food for skin whitening containing the microalgal culture extract as an active ingredient.

The microalgal culture extract is preferably prepared by a manufacturing method comprising the following steps, but not always limited thereto:

1) culturing the microalgae;

2) separating the supernatant from the culture solution of step 1);

3) filtering the supernatant of step 2);

4) extracting by adding an extraction solvent to the filtered supernatant of step 3); And

5) concentrating the extract of step 4).

The microalgae are Chlorella kessleri , cinecosistis species. PCL 6803 ( Synechocystis sp. PCC 6803) , and Botryococcus brownies braunii )} is preferably any one selected from the group consisting of, but is not limited thereto.

In the above method, the microalgal culture of step 1) is preferably cultured using a photobioreactor, but is not limited thereto, and general microalgal culture methods may be used.

In the above method, the microalgal culture of step 1) is preferably an N-8 medium, but any medium used for microalgal culture is possible, and it is preferable to incubate at 20 to 30 ° C, but at 22 to 25 ° C. It is more preferable to incubate, and most preferably, but not limited thereto, at 24 ° C. Oxygen conditions are preferably from 0.05 to 0.5 vvm, more preferably from 0.1 to 0.3 vvm, most preferably incubated under 0.2 vvm oxygen conditions, but is not limited thereto, preferably incubated for 1 to 15 days, 5 More preferably, the culture is carried out for 12 to 12 days, most preferably for 10 days, but is not limited thereto. The amount of light is preferably 50 to 200 μE / m 2 / s, more preferably 90 to 150 μE / m 2 / s, and most preferably irradiated at 100 μE / m 2 / s, but is not limited thereto.

In the above method, the separation of the supernatant of step 2) is preferably carried out by centrifugation after culturing, but not limited thereto, and general cell separation methods may be used.

In the above method, the filtration of step 3) is preferably filtered with a filter paper, but is not limited thereto.

In the above method, it is preferable to use water, a lower alcohol of C ₁ to C ₄ or a mixed solvent thereof, a solvent selected from butanol, hexane and chloroform as the extraction solvent of step 4), and butanol, hexane and chloroform It is more preferable to use. As the extraction method, it is preferable to use shaking extraction or reflux extraction, but not always limited thereto. The extraction solvent is preferably extracted by adding 0.1 to 10 times the amount of the microalgal culture solution, and more preferably by adding 0.5 to 5 times, but not always limited thereto. Extraction temperature is preferably 50 to 100 ℃ but is not limited thereto. In addition, the extraction time is preferably 1 to 10 days, more preferably 2 to 5 days, but is not limited thereto. In addition, the extraction number is preferably one to three times, but is not limited thereto.

In the above method, the concentration of step 5) is preferably a vacuum condenser or vacuum rotary evaporator, but is not limited thereto.

The microalgal culture extract of the present invention has little cytotoxicity, has anti-inflammatory activity, and has a remarkable effect in inhibiting melanin synthesis, and thus can be usefully used as an effective health food for skin whitening.

The health food of the present invention may be added to the active fraction separated from the microalgal culture extract as it is, or used with other foods or food ingredients, and may be appropriately used according to conventional methods.

There is no particular limitation on the kind of food. Examples of the food to which the active fraction separated from the microalgal culture extract can be added include meat, sausage, bread, chocolate, candy, snacks, confectionary, pizza, ramen, other noodles, gums, dairy products including ice cream, various Soups, beverages, tea, drinks, alcoholic beverages and vitamin complexes, etc., and includes all the health food in the usual sense.

The health beverage composition of the present invention may contain various flavors, natural carbohydrates, and the like as additional components, as in a conventional beverage. Such natural carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like. The proportion of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 ml of the composition of the present invention.

In addition to the above, the active fractions isolated from the microalgal culture extract of the present invention are various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners, pH regulators, stable It may contain a topical agent, a preservative, glycerin, alcohol, carbonation agent used in the carbonated beverage and the like. In addition, the active fraction isolated from the microalgal culture extract of the present invention may contain a flesh for the production of natural fruit juice, fruit juice beverage and vegetable beverage. These components may be used independently or in combination. The proportion of such additives is not critical, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

Hereinafter, the present invention will be described in detail with reference to Examples, Experimental Examples and Preparation Examples.

However, the following Examples, Experimental Examples and Preparation Examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following Examples, Experimental Examples, and Preparation Examples.

< Example  1> Preparation of microalgal culture extract

Three types of microalgae used in the present invention are Chlorella kessleri , University of Texas), Synecosistis species. PCL 6803 ( Synechocystis sp . PCC 6803 , Pasteur Bird Bank), and Botryococcus braunii (University of Texas), the microalgae were incubated for 10 days using a photobioreactor in N-8 medium. Culture conditions were 24 ℃, oxygen was supplied at 0.2 vvm, the light amount was irradiated at 100 μE / m2 / s intensity.

The microalgae were cultured and collected, and the culture medium was removed by centrifugation, and the supernatant was taken by filtration with filter paper. The obtained supernatant was extracted for 3 days by adding 50% of butanol, hexane and chloroform, and the solvent layer was separated and concentrated using a vacuum evaporator. , Extracted for 2 to 3 days at room temperature, the solvent layer was separated and concentrated using a vacuum evaporator, it was confirmed that the yield of the chloroform extract of Chlorella Kessler is 0.0083%.

< Example  2> Cultivation of B16F10 melanocytes

B16F10 cells are melanin-producing cell lines, which are generally used for screening of whitening substances, and were cultured by the Suwon Forestry Research Institute. The cells were incubated in a 37 ° C., 5% CO ₂ environment incubator in sterile DMEM medium with 10% FBS and 1% penicillin streptomycin. Subsequently, subcultures were performed when the grown cells occupy about 90% of the 100 mm dish.

< Experimental Example  1> Confirmation of melanin inhibitory activity of microalgal culture extract

In order to confirm the whitening effect of the microalgal culture extract, the extract obtained in Example 1 was treated with B16F10 melanocytes, and the change in the amount of melanin produced was measured. Specifically, the cells were seeded in 6-well plates, treated with the extract in an amount of 200 ug / ml and incubated for two days, and then the cells were recovered. 1 N sodium chloride (NaOH) containing 10% DMSO was added to the recovered cells and reacted at 90 ° C. for one hour to fully dissolve the melanin. Then, ELISA was performed to measure the amount of melanin through measurement of absorbance. . As a positive control, arbutin, which is known as a general whitening substance, was treated at the same concentration, and the relative absorbance was analyzed by setting the absorbance of the non-treated microalgae extract as 100%.

As a result, it was confirmed that Arbutin, a positive control, exhibited 67.2% of melanin synthesis inhibition effect compared to the untreated drug, and microalgal culture extract inhibited melanin synthesis regardless of the species and the type of the extracted organic solvent. It showed the effect, particularly chlorella Kessler Li (chlorella Chloroform extract of kessleri culture broth showed 41.9% inhibitory activity compared to untreated, showing melanin synthesis inhibitory activity superior to that of arbutin, a positive control (Table 1).

Sample Name Melanin amount (%) Untreated 100.00 Arbutin 67.21 Hexane Extract of Chlorella Kessler Culture 57.71 Chloroform Extract of Chlorella Kessler Culture 41.94 Butanol Extract of Chlorella Kessler Culture 78.16 Chlorella Kessler Culture 52.89 Cynecosistis species. Hexane Extract in PCL 6803 Culture 72.14 Cynecosistis species. Chloroform Extract of PCL 6803 Culture 56.20 Cynecosistis species. Butanol Extract of PCL 6803 Culture 78.35 Chloroform Extract of Boturiococcus Brownie Culture 53.28

Therefore, it was confirmed that the microalgal culture and the microalgal culture extract had inhibitory activity of melanin synthesis.

< Experimental Example  2> Confirmation of Cytotoxicity of Microalgae Extract

In order to confirm the cytotoxicity of the microalgal culture extract, the extract obtained in Example 1 was treated with B16F10 melanocytes, and cell growth rate was measured. Specifically, the cells were seeded in 96 well plates, and the extract was treated and cultured in an amount of 200 ug / ml to measure the number of living cells. As a positive control, arbutin, which is known as a general whitening substance, was treated at the same concentration, and the number of living cells in the non-treated microalgae extract was 100% as the number of living cells.

As a result, despite the high concentration of the extract of 200 ug / ml compared to the untreated group, except for the chlorella Kessler culture, the number of living cells, regardless of the species and the type of organic solvent extracted, more than 90% It was confirmed that (Table 2).

Sample Name Cell amount (%) Untreated 100.00 Arbutin 105.80 Hexane Extract of Chlorella Kessler Culture 95.10 Chloroform Extract of Chlorella Kessler Culture 102.73 Butanol Extract of Chlorella Kessler Culture 95.97 Chlorella culture 52.47 Cynecosistis species. Hexane Extract in PCL 6803 Culture 103.86 Cynecosistis species. Chloroform Extract of PCL 6803 Culture 100.94 Cynecosistis species. Butanol Extract of PCL 6803 Culture 90.45 Chloroform Extract of Boturiococcus Brownie Culture 91.14

Therefore, it was confirmed that the microalgal cultures were cytotoxic, whereas the microalgal culture extracts had little cytotoxicity.

< Experimental Example  3> In vitro of microalgal culture extract ( In in vitro ) Tyrosinase ( tyrosinase ) Confirmation of Inhibitory Effect

In order to determine whether the inhibitory effect of tyrosinase (tyrosinase) in the microalgae key enzyme in melanin biosynthesis of the culture extract of ties, in vitro (in In vitro ) tyrosinase experiments were performed. Specifically, L-3,4-dihydroxyphenylalanine (L-DOPA) was used as a substrate for measuring tyrosinase activity, and L-DOPA was phosphate buffered at 1 mg / ml. The solution was completely dissolved in 0.1 M, pH 6.8, and tyrosinase was prepared at a concentration of 1250 units / ml. Chlorella Kessler ( Chlorella) prepared in Example 1 kessleri ) culture extract was prepared at a concentration of 400 ug / ml, and after the addition of tyrosinase or phosphate buffer solution, and finally L-DOPA was added, the reaction was reacted for 10 minutes at 37 ℃ and then 475 nm Absorbance was measured at. As a positive control, kojic acid, known as a tyrosinase inhibitor, was treated at 100 ug / ml, and as a negative control, an equivalent amount of phosphate buffer solution (0.1 M, pH 6.8) was used instead of tyrosinase. Tyrosinase inhibitory activity was expressed as a decrease in absorbance of the extract treated group and the untreated group, and the relative absorbance was compared with the absorbance of the untreated group as 100%.

As a result, in the case of kojic acid, when treated at a concentration of 100ug / ml, it inhibited the tyrosinase activity by 87.5%, Chlorella kessleri ) culture extract, when treated at 400 ug / ml inhibited the activity of tyrosinase by 16.2% (Table 3).

Sample Name Kojic acid
Chlorella Kessler
( Chlorella kessleri ) culture extract Tyrosinase Inhibition Rate (%) 87.5% 16.2%

Therefore, it was confirmed that the microalgal culture extract was weaker than Kojisan, a positive control group, but had some tyrosinase inhibitory activity.

< Experimental Example  4> Confirmation of anti-inflammatory effect of microalgal culture extract

In order to confirm the anti-inflammatory effect of the microalgal culture extract, Raw 264.7 cells were used as a macrophage. Specifically, nitric oxide (NO) production is induced by lipopolysaccharide (LPS) in Raw 264.7 cells, which are nitric oxide (NO) -producing macrophage cell lines, and then prepared in <Example 1>. The extracts were treated with 200 ppm and the level of NO production induced by LPS in the cells was measured. Positive controls were treated with chamomile, known as an anti-inflammatory.

As a result, the better the anti-inflammatory effect, the lower the level of NO produced. Therefore, the anti-inflammatory activity was 91.9% at 1 ppm of chamomile, and when treated with 200 ppm of Chlorella kessleri culture extract, 31.9 It was expressed in% (Table 4). In particular, Chlorella kessleri culture extract did not show cytotoxicity at 200 ppm compared to chamomile.

Sample Name Chamomile
(1 ppm) Chlorella Kessler Chlorella kessleri ) culture extract (200 ppm) Anti-inflammatory activity 91.9% 31.9%

Therefore, it was confirmed that the microalgal culture extract showed anti-inflammatory activity.

The preparation examples for the compositions of the present invention are illustrated below.

< Manufacturing example  1> Skin whitening Cosmetic  Produce

<1-1> preparation of flexible lotion

The flexible purified water containing the microalgae culture extract of the present invention as an active ingredient was prepared as shown in [Table 5].

Raw material Content (parts by weight) Microalgal Culture Extract 10.00 1,3-butylene glycol 1.00 Disodium ID 0.05 Allantoin 0.10 Dipotassium glycylizate 0.05 Citrix Acid 0.01 Sodium citrate 0.02 Glyceres-26 1.00 Arbutin 2.00 Hydrogenated Castor Oil 1.00 ethanol 30.00 Preservative a very small amount coloring agent a very small amount Flavoring agent a very small amount Purified water Balance

<1-2> Preparation of nourishing cream

Nutritional cream containing the microalgal culture extract active ingredient of the present invention was prepared as shown in the following [Table 6].

Raw material Content (parts by weight) Microalgal Culture Extract 10.0 1,3-butylene glycol 7.0 glycerin 1.0 D-panthenol 0.1 Plant extracts 3.2 Magnesium aluminum silicate 0.3 PEG-40 Stearate 1.2 Stearic acid 2.0 Polysorbate 60 1.5 Chin type glyceryl stearate 2.0 Sorbitan sesquioleate 1.5 Cetearyl Alcohol 3.0 Mineral oil 4.0 Squalane 3.8 Carlylic / Capric Triglycerides 2.8 vegetable oil 1.8 Dimethicone 0.4 Dipotassium glycylizate a very small amount Allantoin a very small amount Sodium hyaruronate a very small amount Tocopheryl acetate Quantity Triethanolamine Quantity Preservative Quantity Flavoring agent Quantity Purified water Balance

< Manufacturing example  2> Preparation of skin whitening agent

<2-1> Preparation of Syrup

Syrup containing the microalgal culture extract of the present invention as an active ingredient was prepared as in the composition of the following [Table 7].

Ingredient Content (parts by weight) Microalgal Culture Extract 2 saccharin 0.8 Party 25.4 glycerin 8 Spices 0.04 ethanol 4 Sorbic acid 0.4 Distilled water 60

<2-2> Preparation of Tablet

Tablets containing the microalgal culture extract of the present invention as an active ingredient were prepared as shown in Table 8 below.

Ingredient Content (parts by weight) Microalgal Culture Extract 250 Lactose 175.9 Potato starch 180 Colonde silicic acid 32 10% gelatin solution 5 Potato starch 160 talc 50 Magnesium stearate 5

The microalgal culture extract of the present invention was mixed with 250 parts by weight, lactose 175.9 parts, potato starch 180 parts by weight and colloidal silicic acid 32 parts by weight. 10% gelatin solution was added to the mixture, which was then ground and passed through a 14 mesh sieve. This was dried and the mixture obtained by adding 160 parts by weight of potato starch, 50 parts by weight of talc and 5 parts by weight of magnesium stearate was prepared as a tablet.

< Manufacturing example  3> Preparation of health food for skin whitening

Foods containing the microalgal culture extract of the present invention as an active ingredient were prepared as follows.

<3-1> Preparation of Flour Food

0.5 to 5.0 parts by weight of the microalgal culture extract of the present invention was added to the flour, and bread, cake, cookies, crackers and noodles were prepared using the mixture.

<3-2> soup  And juicy ( gravies Manufacturing

0.1 to 5.0 parts by weight of the microalgal culture extract of the present invention was added to soups and broths to prepare meat products for health promotion, soups and noodles for noodles.

<3-3> ground Beef  Produce

10 parts by weight of the microalgal culture extract of the present invention was added to the ground beef to prepare ground beef for health promotion.

<3-4> Dairy Products ( dairy products Manufacturing

5 to 10 parts by weight of the microalgal culture extract of the present invention was added to milk, and various dairy products such as butter and ice cream were prepared using the milk.

<3-5> Solar  Produce

Brown rice, barley, glutinous rice, and yulmu were dried by a known method and dried, and the mixture was granulated to a powder having a particle size of 60 mesh.

Black soybeans, black sesame seeds, and perilla seeds were steamed and dried by a conventional method, and then they were prepared into powder having a particle size of 60 mesh by a pulverizer.

The microalgal culture extract of the present invention was concentrated under reduced pressure in a vacuum concentrator, dried by spraying and drying with a hot air dryer, and ground to a particle size of 60 mesh using a grinder to obtain a dry powder.

The grains, seeds and the microalgal culture extract of the present invention prepared above were formulated in the following ratios:

Cereals (30 parts by weight brown rice, 15 parts by weight brittle, 20 parts by weight of barley);

Seeds (7 parts by weight perilla, 8 parts by weight black beans, 7 parts by weight black sesame seeds);

Microalgal culture extract of the present invention (3 parts by weight);

Ganoderma (0.5 parts by weight); And

Sulfur (0.5 part by weight).

< Manufacturing example  4> Preparation of skin whitening drink

<4-1> Health drink  Produce

Instant sterilization by homogeneously mixing 5 g of microalgal culture extract of the present invention with subsidiary materials such as liquid fructose (0.5%), oligosaccharide (2%), sugar (2%), salt (0.5%) and water (75%) After it was prepared by packaging in a small packaging container such as glass bottles, plastic bottles.

<4-2> Preparation of Vegetable Juice

5 g of the microalgal culture extract of the present invention was added to 1,000 ml of tomato or carrot juice to prepare vegetable juice.

<4-3> Preparation of Fruit Juice

1 g of the microalgal culture extract of the present invention was added to 1,000 ml of apple or grape juice to prepare a fruit juice.

As described above, the microalgal culture extract of the present invention has little cytotoxicity, shows a remarkable effect on the anti-inflammatory activity and the inhibition of melanin synthesis, and has an excellent effect on skin whitening, cosmetics, skin external preparations, drugs or It can be usefully used for the development of functional foods and production of products.

Claims (6)

Cosmetic composition for skin whitening containing the extract of the microalgal culture as an active ingredient.
The method of claim 1, wherein the microalgae are Chlorella Kessler ( Chlorella) kessleri ), synechosis species. PCL 6803 ( Synechocystis sp . PCC 6803 ) and Botryococcus braunii ( Botryococcus braunii ) is a cosmetic composition for skin whitening, characterized in that any one selected from the group consisting of.
The method of claim 1, wherein the culture medium
1) culturing for 10 days at 24 ℃, 0.2 vvm oxygen conditions, 100 μE / m2 / s light conditions to obtain a culture solution;
2) centrifuging the obtained culture to remove cells; And
3) A cosmetic composition for skin whitening, characterized in that it is prepared by the method comprising the step of separating the supernatant by filtration of the culture medium from which cells have been removed.
The cosmetic composition for skin whitening according to claim 1, wherein the extract is extracted with hexane, chloroform or butanol.
A pharmaceutical composition for skin whitening containing the extract of the microalgal culture as an active ingredient.
Health food for skin whitening containing extract of microalgae culture as an active ingredient.