KR20200068416A - Cosmetic composition using unripe citrus unshiu extract containing citrus bio-gel - Google Patents

Abstract

An unripe Citrus unshiu extract containing Citrus unshiu bio-gel has a skin cell protective effect caused by hydrogen peroxide treatment and has a moisturizing effect by promoting HA production and FLG expression in HaCaT. Therefore, the unripe Citrus unshiu extract containing Citrus unshiu bio-gel can be used as a cosmetic composition for moisturizing skin or protecting skin cells.

Description

Cosmetic composition containing unripe citrus fruit extract containing citrus biogel as an active ingredient {Cosmetic composition using unripe citrus unshiu extract containing citrus bio-gel}

The present invention relates to a skin cell protection and skin moisturizing cosmetic composition containing an unripe citrus fruit extract containing citrus biogel as an active ingredient.

The skin is a part of the body that is directly exposed to the external environment. It acts as a protective film to protect vital organs of our body, as well as regulating moisture evaporation and protecting the body from other physical and chemical stimuli. However, because the skin is located on the outermost part of the body, it is exposed to environmental factors such as ultraviolet rays caused by the sun and lack of moisture due to temperature changes. It causes irritation.

The skin is largely divided into three layers of epidermis, dermis, and subcutaneous tissue in order from the outside, and the epidermis plays an important role in preventing evaporation of moisture inside the human body. The stratum corneum of the epidermis exists on the outermost side of the skin and prevents the loss of moisture and electrolyte out of the skin, thereby preventing the drying of the skin and providing an environment in which the skin can perform normal biochemical metabolism. In addition, it plays an important role in protecting the human body from external physical damage and chemicals or preventing bacteria and viruses from invading the skin. Moisture in normal skin is 70% in the body and 20-30% in the stratum corneum, supporting the elasticity and flexibility of the skin. In addition, natural moisturizing factor (NMP) and sebum are present in the stratum corneum and help maintain the moisture balance of the stratum corneum. If the amount of NMF and sebum is well balanced, the skin remains moist and soft.

The epidermis is composed of a stratum corneum, granular layer, polar layer, and base layer. In the stratum corneum of a healthy person, natural moisturing factor (NMF) is produced by using Filaggrin (FLG) protein as a precursor to maintain moisture in the skin. To help. The filaggrin (filaggrin, filament aggregating protein) is a histidine-rich cationic protein produced by the breakdown of profilagrin, which plays an important role in skin barrier by agglomerating keratin filaments in keratinocytes. In addition, the skin has a natural moisturizing factor, maintaining an appropriate moisturizing state and balancing with the surrounding environment.

The pilar green is a protein that plays an important role in the formation of the stratum corneum along with proteins such as keratin, involucrin, and locicrin. It is mainly involved in the formation of filaments by bonding with keratin, and is processed in the form of fililaggrin, filaggrin, natural moisturizing factor (NMF), etc. in the process of formation of the stratum corneum. Eventually, it forms a natural moisturizing factor, which forms 20-30% (by dry weight) of the stratum corneum, and has excellent moisture absorbing properties, thus acting as a humectant that controls moisture in the stratum corneum. Moisture content in the stratum corneum is essential for the normal functioning of various enzymes that are important for the formation of the stratum corneum. Therefore, the decrease in the amount of expression of the pilar green causes changes in the function and moisturization of the skin barrier and leads to various lesions. In particular, the formation of a normal skin barrier plays an important role in the defense against external stimuli.

In addition, hyaluronic acid, a type of glycosaminoglycan that is present in the extracellular connective tissue and makes up the skin, also acts as a barrier against moisture evaporation. Since the amino sugar constituting the hyaluronic acid is water-soluble, it effectively combines with moisture to maintain moisture in the stratum corneum to suppress the skin from drying out.

Therefore, in order to maintain the moisturization of the skin and protect the skin cells, it is necessary to protect the skin cells by suppressing the damage caused by the free radicals produced, and promote the production of keratinocyte proteins and natural moisturizing factors to maintain the moisture of the skin. Should be

Republic of Korea Patent Publication No. 10-2016-0104054

An object of the present invention is to provide a cosmetic composition for moisturizing skin or protecting skin cells comprising an unripe citrus fruit extract containing citrus biogel.

In order to achieve the object of the present invention as described above, the present invention provides a cosmetic composition for skin moisturization or skin cell protection, including citrus immature fruit extract containing citrus biogel.

The term "citrus biogel" of the present invention refers to a material made by culturing microorganisms in citrus juice. Citrus biogel has the advantage of being able to combine various materials and industrialize them into functional cosmetics and pharmaceutical materials because the tissues are uniform and have a lot of gaps, so they have good conditions to combine with new materials.

The microorganism is in glucon acetobacter ( Gluconacetobater sp.) gel_SEA623-2 (KACC91526P) may be a strain. The SEA623-2 strain may be pre-cultured at 25-35° C., pH 2.5-7.0, and inoculated into citrus juice medium, and more specifically, 26-30° C., pH 3.5-5.0.

The citrus fruit juice medium may have a pH in the range of 2.5 to 5.0, and specifically, a pH in the range of 2.5 to 4.0. In addition, the citrus fruit juice medium can be used that has a sugar content of 9 to 13 brix.

In addition, as the citrus fruit juice medium, not only citrus juice, but also fermentation broth obtained by treating and fermenting a glycolytic enzyme on citrus foil produced after juice of citrus can be used. It can be used, specifically those in the range of pH 3.0 ~ 4.0. In addition, the fermentation broth may be used that has a sugar content of 9 to 13 brix.

The term "citrus immature fruit" of the present invention refers to the immature fruit of Citrus unshiu , which is shipped for a month from August 15th. When it comes to citrus fruits, it usually refers to the feeling of warm jujube. The height is 3~5 m, the branches are spread and there are no thorns. The leaves are alternate, elliptical, with flat edges or wavy fine teeth. Fruits are small ball-shaped, 5-8 cm in diameter, ripening in yellowish red. It is the most commonly planted in Korea, and there are about 10 varieties of cultivar, mid-growth, and end-of-life.

The unripe citrus fruit extract of the present invention may be a solvent extract soluble extracted by adding water or an organic solvent, and the type of the extraction solvent used for extraction is not particularly limited, and any solvent known in the art may be used. You can. In the present invention, the extract can be obtained by extraction with water, C ₁ to C ₄ lower alcohol or a mixed solvent thereof. Also, non-limiting examples of the extraction solvent include water; Lower alcohols of C ₁ to C ₄ such as methanol, ethanol, propyl alcohol, and butyl alcohol; Polyhydric alcohols such as glycerin, butylene glycol, and propylene glycol; And hydrocarbon-based solvents such as methyl acetate, ethyl acetate, acetone, benzene, hexane, diethyl ether, and dichloromethane; Alternatively, a mixture of these may be used, and specifically, water, lower alcohol, 1,3-butylene glycol, and ethyl acetate may be used alone or in combination of two or more.

The citrus immature fruit extract containing the biogel of the present invention may be an extract obtained by mixing and dissolving the citrus immature fruit after extracting the solvent.

The unripe citrus fruit extract of the present invention may be prepared by a manufacturing method including the following steps, but is not limited thereto.

1) extracting by adding an extraction solvent to the unripe citrus fruits and skins and flesh;

2) filtering the extract of step 1);

3) concentrating the filtered extract of step 2) under reduced pressure and drying it to prepare an unripe citrus fruit extract; And

4) Mixing and dissolving the unripe citrus fruit extract of step 3) in citrus biogel.

In the above method, the citrus immature, peel and flesh of step 1) can be used without limitation, such as those grown or commercially available. In addition, it is not limited to step 1), but it may be to extract all of the leaves or branches other than the unripe citrus fruit and the skin and flesh.

In the above method, water, alcohol or a mixture thereof may be used as the extraction solvent of step 1). C ₁ to C ₄ lower alcohol may be used as the alcohol, and ethanol may be used as the lower alcohol. As an extraction method, shaking extraction, Soxhlet extraction, or reflux extraction may be used, but is not limited thereto. The extraction solvent may be extracted by adding 10 to 30 times the volume ratio (w/v) to the dried citrus fruits. The extraction temperature may be 20°C to 100°C, specifically 20°C to 40°C, and more specifically room temperature, but is not limited thereto. In addition, the extraction time may be 10 to 48 hours, specifically 15 to 30 hours, and more specifically, 24 hours, but is not limited thereto. In addition, the number of extractions may be 1 to 5 times, specifically, it may be repeatedly extracted 3 to 4 times, and more specifically, it may be repeatedly extracted 3 times, but is not limited thereto.

In the above method, the reduced pressure concentration of step 3) may use a vacuum decompressor or a vacuum rotary evaporator, but is not limited thereto. In addition, drying may be performed under reduced pressure, vacuum drying, boiling drying, spray drying or freeze drying, but is not limited thereto.

In the above method, the citrus biogel of step 4) may be added to the dried unripe fruit extract in a weight ratio (w/w) of 5 to 20 times, but is not limited thereto.

In the present invention, the citrus immature fruit extract containing the citrus biogel may exhibit skin cell protective activity through an action of inhibiting damage and inhibiting toxicity of cells exposed to hydrogen peroxide.

In addition, the unripe citrus fruit extract containing the citrus biogel has a moisturizing effect by forming a skin barrier, preventing evaporation of water, and increasing the production of pilargreen and hyaluronic acid involved in moisture retention.

The cosmetic composition of the present invention may include an unripe citrus fruit extract containing 0.001 to 100% by weight of the citrus biogel based on the total composition weight, specifically, 0.01 to 50% by weight of the citrus biogel Citrus fruits may include, but are not limited to.

In addition, the cosmetic composition of the present invention may include, without limitation, ingredients that are normally allowed in addition to the active ingredient, for example, antioxidants, stabilizers, solubilizers, vitamins, pigments, fragrances, and common adjuvants, and carriers. can do. The cosmetic composition of the present invention may be prepared in various forms, for example, the cosmetic composition of the present invention may be prepared in any formulation conventionally prepared in the art, for example, solutions, suspensions, emulsions, pastes, It may be formulated as a gel, cream, lotion, powder, soap, cleansing, oil, powder foundation, emulsion foundation, wax foundation, and spray, but is not limited thereto. In addition, specifically, skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nutrition lotion, massage cream, nutrition cream, moisture cream, hand cream, essence, nutrition essence, pack, soap, shampoo, It may have a formulation selected from the group consisting of cleansing foam, cleansing lotion, cleansing cream, body lotion, body cleanser, emulsion, lipstick, makeup base, foundation, press powder and rust powder, but is not limited thereto.

The citrus immature fruit extract containing citrus biogel has a skin cell protective effect caused by hydrogen peroxide treatment and has a moisturizing effect through promoting expression of FLG and HA production in HaCaT. Therefore, it is possible to use the unripe citrus fruit extract containing the citrus biogel as a cosmetic composition for moisturizing skin or protecting skin cells.

1 is a graph showing the cell viability confirmed through MTT assay in a HaCaT cell culture system.
2 is a graph showing the cell protective effect of the citrus immature fruit extract containing citrus biogel against damage induced by hydrogen peroxide.
3 is a graph showing the cell viability confirmed through WST-1 assay in the HaCaT cell culture system.
Figure 4 is a graph showing the results of measuring the expression level of FLG, a keratinocyte protein according to the concentration of immature citrus fruit extract containing citrus biogel.
Figure 5 is a graph showing the results of measuring the production amount of natural moisturizing factor HA according to the concentration of the unripe citrus fruit extract containing citrus biogel.

Hereinafter, the configuration and effects of the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Example 1 Preparation of citrus immature fruit extract containing citrus biogel

Example 1-1. Citrus biogel production

Citrus biogels are of the genus Gluconacetobater sp. It was produced using the gel_SEA623-2 (KACC91526P) strain. In order to produce the citrus biogel, 1×10 ⁵ cells of the strain SEA623-2 in the glucon acetobacter were cultured in a medium containing a warm nectar juice (10% (v/v)) with a sugar content of 10 brix and a pH of 3.5. It was inoculated at /ml, and was incubated at 25°C for 20 days under aerobic conditions. As a result, the initial gel generation rate of the SEA623-2 strain was very excellent, and a gel film began to be formed from the 2nd day of culture, and the citrus biogel produced after 20 days was collected and used.

Example 1-2. Preparation of citrus immature fruit extracts containing citrus biogels

The unripe citrus fruits, rinds, and flesh were prepared by the Jeju Special Self-Governing Province's Citrus Research Institute. 100 g of dried and crushed citrus fruits, peels, and flesh were added to 2 L of 70% (v/v) ethanol (EtOH) and stirred at room temperature for 24 hours. The leached sample was collected only by the filtrate using a vacuum filtration device, and the residue separated in this way was repeated twice more under the same conditions. Thereafter, the filtrate obtained by filtration was concentrated on a rotary evaporator in a water bath at 40° C. or lower, and dried to obtain 38.6 g of 70% ethanol extract. 1 g of the obtained 70% ethanol extract was dissolved in 10 g of citrus biogel and used in the experiment.

Example 2. Cell culture

HaCaT cells, which are immortalized human keratinocyte cell lines, were distributed from Korean Cell Line Bank (KCLB) and used in the experiment. Using dulbecco's modified eagle's medium (DMEM) medium containing 1% penicillin/streptomycin (100 U/mL penicillin, 100 μg/mL streptomycin) and 10% fetal bovine serum (FBS) at 37°C and 5% CO ₂ conditions The culture was carried out, and passage culture was performed at intervals of 3 days.

Example 3. Measurement of the effect of inhibiting skin damage induced by hydrogen peroxide

In order to confirm the effect of inhibiting skin cell damage caused by hydrogen peroxide of the citrus immature fruit extract containing citrus biogel obtained in Example 1, hydrogen peroxide was treated with the hydrogen peroxide cultured in Example 2 to induce skin cell damage and samples To confirm the cytoprotective effect of the sample, the sample was treated, and then MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay was performed to evaluate cytotoxicity.

Example 3-1. Cytotoxicity evaluation of unripe citrus fruits and extracts, including citrus biogels

In order to determine the concentration of the sample to be used in the experiment, the cytotoxicity of the unripe citrus fruit extract including citrus biogel was confirmed for HaCaT cells. Cells were dispensed into a 96-well plate at 3.0×10 ⁴ cells/well and cultured for 24 hours in a cell incubator at 37° C. and 5% CO ₂ conditions. After processing the samples for each concentration in a medium not containing FBS to exchange the cell culture supernatant, incubate for 24 hours, add MTT solution at a concentration of 500 μg/mL, and in a cell incubator at 37°C and 5% CO ₂ conditions 3 Time reaction. DMSO was dissolved in the formazan precipitate formed by reacting with living cells and absorbance was measured at 570 nm using a microplate reader. When the sample was not added, the untreated group and the citrus immature fruit extract containing citrus biogel samples were compared with the experimental group to measure the cytotoxicity by concentration of the citrus immature fruit extract containing citrus biogel (FIG. 1). .

As a result, as shown in FIG. 1, it was confirmed that the citrus immature fruit extract containing citrus biogel had little cytotoxicity in keratinocytes (HaCaT cells) at a concentration of 0.0125 to 0.05% (w/w).

Example 3-2. Measurement of cell protection effect against damage of hydrogen peroxide induced HaCaT cells

HaCaT cells were dispensed into a 96-well plate at 3.0×10 ⁴ cells/well and cultured for 24 hours in a cell incubator at 37° C. and 5% CO ₂ conditions. After removing the cell culture supernatant, hydrogen peroxide was treated in order to damage the cells and cultured for 30 minutes in a cell incubator. The cultured cells were washed twice with DPBS, and the samples were treated in a medium not containing FBS at a concentration in a range where cytotoxicity did not appear, followed by incubation for 24 hours, followed by MTT assay. The case where no hydrogen peroxide was added was set as the untreated group and the case where no sample was added as a control group. It was confirmed that the cell protection effect by concentration of the sample was compared to the control group, and the citrus fruit extract containing citrus biogels as an experimental group (Fig. 2).

As a result, as shown in Figure 2, it was confirmed that the reduced cell viability by hydrogen peroxide treatment is recovered by the treatment with the citrus immature fruit extract containing citrus biogel. Therefore, it was confirmed that the citrus immature fruit extract containing citrus biogel had a skin cell protective effect by inhibiting skin cell damage induced by hydrogen peroxide.

Example 4. Measurement of skin moisturizing effect

In order to confirm the skin moisturizing effect of the citrus immature fruit extract containing the citrus biogel obtained in Example 1, the cytotoxicity of the sample for the HaCaT cells cultured in Example 2 was confirmed, and the cultured cells and the cell culture supernatant were used. Using the expression amount of Filaggrin (FLG, Filaggrin) and the amount of production of hyaluronic acid (HA) was confirmed.

Example 4-1. Cytotoxicity evaluation by WST-1 method

HaCaT cells were divided into 1.0×10 ⁵ cells/well in a 24-well plate and cultured for 24 hours in a cell incubator at 37° C. and 5% CO ₂ conditions. After replacing the cell culture supernatant with a medium treated with samples for each concentration, the cells were cultured for 24 hours and the WST-1 solution was added to 10% per well (v/v) to react for 1 hour in a cell incubator. The absorbance of soluble formazan precipitates generated by reaction with living cells was measured at 440 nm using a microplate reader. Control the case where the citrus immature fruit extract containing citrus biogel is not added, compare the case where the citrus immature fruit extract containing citrus biogel is added to the experimental group, and measure the cytotoxicity by concentration of the citrus immature fruit extract containing citrus biogel. (Fig. 3).

As a result, as shown in FIG. 3, it was confirmed that there was little cytotoxicity in keratinocytes (HaCaT cells) at a concentration of 0.25 to 1% (w/w) of citrus immature fruits including citrus biogels.

Example 4-2. Measurement of FLG expression in HaCaT cells

In Example 4-1, the culture solution remaining in the well plate treated with the WST-1 solution was removed, washed twice with phosphate buffered saline (PBS), and then treated with 0.5% EDTA. After incubation at 37°C for 10 minutes, the cells were removed and centrifuged. Thereafter, PBS was treated with the obtained pellet, and the cells were frozen at -80°C and room temperature and thawed repeatedly to break the cells. Subsequently, the amount of pilar green (FLG) produced was performed using a human filaggrin ELISA kit (Cusabio biotech co., LTD.), and absorbance was measured and quantified at 520 nm using a microplate reader. Control the case where the untreated citrus fruit extract containing citrus biogel and untreated citrus fruit extract containing citrus biogel are compared with the experimental group, and measure the expression level of Filaggrin (FLG) according to the concentration of the sample. (Fig. 4).

As a result, as shown in FIG. 4, it was confirmed that the expression of keratinocyte protein, Filaggrin (FLG, Filaggrin) is increased by the treatment of the unripe citrus fruit extract containing citrus biogel, thereby increasing the water retention effect of the skin. Could know.

Example 4-3. Measurement of HA production in HaCaT cells

In Example 4-1, after the cell culture supernatant was collected before processing the WST-1 solution, the amount of HA was generated using a human hyaluronic ELISA kit (Cusabio biotech co., LTD.), and 520 using a microplate reader. Absorbance at nm was measured and quantified. Controlling the case where the tangerine immature fruit extract containing citrus biogel was not treated, and comparing the case where the tangerine immature fruit extract containing citrus biogel was treated with the experimental group, the amount of HA produced by the concentration of the sample was measured (FIG. 5).

As a result, as shown in FIG. 5, it was confirmed that the production of natural moisturizing factor hyaluronic acid (HA, Hyaluronic acid) was increased by the treatment of unripe citrus fruits and citrus fruits containing citrus biogels, thereby having a skin moisturizing effect by the extract. I could see that.

Claims (5)

A cosmetic composition for moisturizing skin or protecting skin cells comprising citrus immature fruit extract containing citrus biogel. According to claim 1,
The extract is extracted with water, alcohol having 1 to 4 carbon atoms or a mixed solvent thereof, cosmetic composition. According to claim 1,
The extract is extracted with ethanol, cosmetic composition. According to claim 1,
Citrus biogel is a blend of 5 to 20 times the weight ratio (w/w) of dried citrus fruits and unripe citrus extracts, cosmetic composition. The method of claim 1, wherein the cosmetic composition is a skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nutrition lotion, massage cream, nutrition cream, moisture cream, hand cream, essence, nutrition essence, pack , Soap, shampoo, cleansing foam, cleansing lotion, cleansing cream, body lotion, body cleanser, emulsion, lipstick, makeup base, foundation, press powder, and has a formulation selected from the group consisting of pulsing, cosmetic composition.

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