KR102223534B1 - Cosmetic composition comprising centella asiatica extract and andrographolide extract - Google Patents

Abstract

The present invention relates to a cosmetic material comprising a titrated extract of Centella asiatica and an Andrographis paniculata extract at high concentrations. According to the present invention, it is possible to maintain stable emulsification ability while including one or more poorly soluble active ingredients in a complex manner using a highly concentrated fermented emulsifier. Therefore, the stability of the formulation can be greatly improved, and the cosmetic material, when applied to the skin, can maximize synergistic effects and skin improving effects due to various active ingredients, and can be easily applied to formulations requiring emulsification power.

Description

Cosmetic composition containing centella asiatica extract and Cheonsimyeon extract {COSMETIC COMPOSITION COMPRISING CENTELLA ASIATICA EXTRACT AND ANDROGRAPHOLIDE EXTRACT}

The present invention relates to a cosmetic composition comprising centrifugal centrifugal extract and cheonsimryeon extract at a high concentration.

Conventional methods of dissolving poorly soluble substances can be divided into a chemical method and a physical method. In general, the chemical method is to use a non-polar solvent, and the physical method is to increase the solubility of poorly soluble substances by mechanically producing nanoparticles with high energy. However, these methods are difficult to apply to formulations due to the use of a low concentration and often not exhibiting the functionality of the material, and due to the toxicity and low stability of the solvent.

The poorly soluble substances include most functionally excellent substances of various materials such as cosmetics, foods, and pharmaceuticals. These materials are mainly composed of an aromatic structure, have a structure having high crystallinity, are easy to precipitate and crystallize due to low formulation stability, and are difficult to dissolve in a solvent with high polarity.

For example, ingredients such as Centella asiatica extract and Cheonsimnyeon extract have excellent potential effects such as anti-inflammatory, but have low solubility for use as a cosmetic ingredient and low stability in formulation as a crystalline substance, so they are not well suited to be applied to cosmetic formulations. There is a problem in that it is inevitable to use a low concentration and it is difficult to effectively deliver the active ingredient to the skin.

Therefore, there is a need for materials and methods for effectively dispersing and dissolving poorly soluble natural substances and the like to be used effectively.

It is an object of the present invention to provide a cosmetic composition comprising centrifugal extract and Cheonsimnyeon extract using a high-purity fermentation emulsifier.

In order to solve the above problem, the present invention

High purity fermented emulsifier; And

Including centella asiatica extract, cheonsimryeon extract, or a combination thereof,

The high purity fermentation emulsifier,

Candida bombicola UA-06 (Accession No.: KCTC13855BP, Korea Research Institute of Bioscience and Biotechnology) Seed culture step of activating microorganisms;

A fermentation step of fermenting the seed cultured microorganisms and vegetable oils;

Purifying by removing moisture, fatty acids, and impurities in the fermented product obtained from the fermentation step; And

It provides a method for producing a cosmetic composition prepared by a method comprising the step of separating and recovering the supernatant by mixing ethanol with the purified fermented product.

According to one embodiment, the step of purifying comprises: adjusting the fermented product to a pH of 3 to 6, and allowing it to stand for 5 to 30 hours to separate the aqueous layer and the oil layer;

MgSO ₄ , zeolite, diatomaceous earth, activated alumina, adding 10 to 30 g/L of at least one selected from the group consisting of calcium sulfate and calcium chloride, and filtering with a filter press at 1 to 5 bar to obtain an oil layer;

Purifying with a column filled with at least one selected from the group consisting of magnesium silicate, activated carbon, white clay, acid clay, and perlite; And

It may include filtering.

According to an embodiment, in the recovering step, the purified fermented product and 70 to 95% ethanol are mixed in a volume ratio of 1:5 to 1:20 (w/w), and stirred for 3 to 10 hours to obtain the lower layer liquid and Separating the supernatant; And

After volatilizing ethanol from the supernatant, it may include the step of recovering the fermentation emulsifier.

According to an embodiment, the medium used in the seed culture step may include yeast extract, glycerin, K ₂ HPO ₄ , KH ₂ PO ₄ , NH ₄ NO ₃ and MgSO ₄ .

According to an embodiment, the medium used in the fermentation step may include yeast extract, glycerin, K ₂ HPO ₄ , KH ₂ PO ₄ , NH ₄ NO ₃ , MgSO ₄ , glucuronolactone, and vegetable oil. .

According to an embodiment, the medium used in the seed culture step is yeast extract 1 to 30 g/L, glycerin 10 to 40 g/L, K ₂ HPO ₄ 10 to 50 g/L, KH ₂ PO ₄ 3 to 15 g/L, NH ₄ NO ₃ 1 to 10 g/L and MgSO ₄ 0.5 to 5 g/L,

The medium used in the fermentation step is yeast extract 0.5 to 3 g/L, glycerin 1 to 40 g/L, K ₂ HPO ₄ 10 to 50 g/L, KH ₂ PO ₄ 3 to 15 g/L, NH ₄ NO ₃ 1 to 10 g /L, MgSO ₄ 0.5 to 5g/L, glucuronolactone 40 to 200g/L and vegetable oil 50 to 1000g/L.

According to an embodiment, the seed culture step is conducted under aerobic conditions of 15 to 30°C, 100 to 500 rpm, 0.5 to 2vvm,

The fermentation step may be performed at a temperature of 20 to 35° C. and a mixing condition of 50 to 200 rpm.

According to another embodiment of the present invention, there is provided a cosmetic composition comprising centrifugal extract and Cheonsimryeon extract using a high-purity fermented emulsifier prepared by the method as described above.

Other specifics of the embodiments of the present invention are included in the detailed description below.

The present invention can disperse and dissolve a poorly soluble active ingredient at a high concentration using a fermented emulsifier treated with high purity. In addition, since it is environmentally friendly to minimize toxicity and side effects to the human body, improve skin absorption of poorly soluble active ingredients, and capture high concentration of active substances, it is possible to improve formulation stability.

1 is a graph showing the amount of nitric oxide produced.
2 is a graph showing the amount of cytokine production.
3 is a graph showing NF-κB activity.
4 is a graph showing the amount of transdermal water evaporation.

Since the present invention can apply various transformations and can have various embodiments, specific embodiments are illustrated in the drawings and will be described in detail in the detailed description. However, this is not intended to limit the present invention to a specific embodiment, it should be understood to include all conversions, equivalents, and substitutes included in the spirit and scope of the present invention. In describing the present invention, when it is determined that a detailed description of a related known technology may obscure the subject matter of the present invention, a detailed description thereof will be omitted.

Emulsifiers (surfactants) are adsorbed to the interface of two different liquids to lower the interfacial tension, so that the two phases are well mixed with each other. As a structural feature, the non-polar part is composed of an alkyl group having lipophilicity, and the polar part is composed of a material having hydrophilicity.

Emulsifiers can be classified into synthetic emulsifiers and natural emulsifiers, among which, as a kind of natural emulsifier, there is a fermentation emulsifier derived from microorganisms produced by microorganisms such as yeast, mold, and bacteria. Emulsifiers produced by microorganisms are amphiphilic substances and can be used in various industries such as cosmetics, foods, pharmaceuticals, and detergents. Microbial-derived fermentation emulsifiers have low toxicity compared to conventional chemically synthesized surfactants, have high stability against extreme conditions, and are biodegradable, so they are very environmentally friendly.

On the other hand, among natural materials having excellent functional ingredients, many have disadvantages that are difficult to utilize due to poorly soluble properties. Organic solvents are sometimes used to dissolve poorly soluble substances, but their use is limited for reasons such as skin toxicity and irritation. Ethanol can be used as an organic solvent, but conventionally, even this is not preferred.

In order to more effectively utilize a poorly soluble substance having excellent functional properties, it can be easily applied to a cosmetic composition by using a biological fermentation emulsifier rather than an organic solvent. In addition, it is safe for the skin and is easily permeable to the skin, thus maximizing the efficacy of the active ingredient.

Hereinafter, according to an embodiment of the present invention, a cosmetic composition comprising centripetal centrifugal extract and Cheonsimnyeon extract will be described in more detail.

The present invention includes centripetal centrifugal extract, Cheonsimnyeon extract, or a combination thereof as a poorly soluble active ingredient.

Titrated extract of Centella asiatica (TECA) has been used for medicinal purposes, and not only for the treatment of skin lesions such as skin redness, burns, hypertrophic scars, eczema, etc. It is also widely used in diseases. The quantitative extract is obtained by quantitatively extracting the active ingredient from Centella asiatica plant, and includes asiaticoside, asiatic acid, and madecassic acid as main components. Centella asiatica extract differs from centella asiatica extract in that it is powdered by extracting and quantifying only the core components at high concentration.

Centella asiatica extract or quantitative centella asiatica extract is soluble in an organic solvent, and the organic solvent is limited for use in cosmetics. In addition, when used at a high concentration, there is a disadvantage that precipitation and stability of the formulation are extremely low. It is difficult to manufacture an aqueous dispersion, and the type of formulation is also limited for reasons of stability. In order to solve this problem, centella asiatica extract was dissolved in a high-purity fermented emulsifier to overcome the limited capacity and stability. Centella asiatica extract or quantitative centella asiatica extract dissolved using a high-purity fermentation emulsifier can increase skin improvement effect because it has high skin affinity and high permeability of active ingredients.

Andrographis paniculata extract contains diterpene lactone and flavonoids, and andrographolides as the main effective ingredients. It has been confirmed that it has antibacterial, anti-inflammatory, analgesic, antipyretic, liver protection, and immunity enhancement. Cheonsimnyeon extract dissolves well in organic solvents and does not completely dissolve in aqueous solution. As the container solvent, ethanol, dimethyl sulfoxide, dimethylformamide, etc. can be used, and an organic solvent having a polarity within the range of 5 to 7.5 can be used, but in the present invention, a high-purity fermentation emulsifier is used. use.

Centella asiatica and Chunsimryeon show anti-inflammatory effects through the same route, and specifically, act to inhibit the activity of NF-kappa b.

In the present invention, a fermented emulsifier treated with high purity is used to capture such a poorly soluble complex active ingredient at a high concentration.

In order to obtain a fermented emulsifier treated with high purity,

Candida bombicola UA-06 (Accession No.: KCTC13855BP, Korea Research Institute of Bioscience and Biotechnology) Seed culture step of activating microorganisms;

A fermentation step of fermenting the seed cultured microorganisms and vegetable oils;

Purifying by removing moisture, fatty acids, and impurities in the fermented product obtained from the fermentation step; And

A fermentation emulsifier may be prepared by a method comprising the step of separating and recovering the supernatant by mixing ethanol with the purified fermented product.

According to an embodiment, the ethanol may be used by adding or replacing at least one of substances having a polarity of 5 to 6, such as methanol and acetonitrile.

According to an embodiment, in the purifying step, the fermentation product is adjusted to a pH of 3 to 6, for example, pH 3.5 to 5, and for 5 to 30 hours, for example, 10 to 20 hours, for example, 15 to 20 hours. It may include the step of separating the water layer and the oil layer by standing for a period of time. In this case, the aqueous layer includes microorganisms and a medium, and the oil layer includes a fermented emulsion.

In addition, in order to remove the separated water layer and remove moisture remaining in the fermented product emulsion, _{at least one selected from the group consisting of MgSO 4} , zeolite, diatomaceous earth, activated alumina, calcium sulfate, and calcium chloride, for example, MgSO ₄ is used as 10 To 30 g/L, for example, 15 to 25 g/L may be added.

Also, _{after adding MgSO 4} , stirring at 30 to 80° C. for 1 to 3 hours at 100 to 500 rpm may be included.

Thereafter, for example, MgSO ₄ may be filtered using a filter press to obtain a transparent oil layer. The filter press can be performed at a pressure of 1 to 5 bar, for example 2 to 3 bar, for example 2 bar.

In addition, in order to remove fatty acids and impurities from the fermented emulsion, it may include the step of purifying with a column filled with at least one selected from the group consisting of magnesium silicate, activated carbon, white clay, acid clay, and pearlite, for example, magnesium silicate. have. In addition, it may include filtering with a filter, for example, a 0.45 μm filter, in order to completely remove the impurities contained in the fermented _{emulsion, for example, particles such as MgSO 4.} Filtering may include, for example, housing filtration, vacuum filtration, and the like.

According to an embodiment, in the recovering step, the purified fermented product and 70 to 95%, for example 80 to 90% ethanol, are 1:5 to 1:20 (w/w), for example 1:5 to Mixing at a volume ratio of 1:10 (w/w), stirring for 3 to 10 hours, for example, 5 to 8 hours, and then standing overnight to separate the lower layer and the supernatant. I can. In this case, the lower layer liquid contains neutral fat, and the supernatant contains an active ingredient. In addition, it may include the step of recovering the fermentation emulsifier after volatilizing ethanol from the supernatant, for example, by concentration under reduced pressure. Ethanol can be volatilized by a general method other than the above-described vacuum concentration.

According to an embodiment, the medium used in the seed culture step may include yeast extract, glycerin, K ₂ HPO ₄ , KH ₂ PO ₄ , NH ₄ NO ₃ and MgSO ₄ .

In addition, the medium used in the fermentation step may include yeast extract, glycerin, K ₂ HPO ₄ , KH ₂ PO ₄ , NH ₄ NO ₃ , MgSO ₄ , glucuronolactone and vegetable oil.

Specifically, for example, the medium used in the seed culture step is yeast extract 1 to 30 g/L, glycerin 10 to 40 g/L, K ₂ HPO ₄ 10 to 50 g/L, KH ₂ PO ₄ 3 to 15 g/L, NH ₄ NO ₃ 1 to 10 g/L and MgSO ₄ 0.5 to 5 g/L,

The medium used in the fermentation step is yeast extract 0.5 to 3 g/L, glycerin 1 to 40 g/L, K ₂ HPO ₄ 10 to 50 g/L, KH ₂ PO ₄ 3 to 15 g/L, NH ₄ NO ₃ 1 to 10 g /L, MgSO ₄ 0.5 to 5g/L, glucuronolactone 40 to 200g/L and vegetable oil 50 to 1000g/L.

In addition, according to an embodiment, the seed culture step may be performed at 15 to 30°C, for example, 18 to 25°C at 100 to 500 rpm, for example, 200 to 400 rpm, 0.5 to 2vvm, for example 0.5 to 1.5vvm aerobic conditions. Progress,

The fermentation step may be carried out in a mixing condition of 50 to 200 rpm, for example 80 to 150 rpm at 20 to 35 °C, for example 20 to 30 °C.

In addition, the seed culture step may be terminated at the end point of the exponential growth of the bacteria. Specifically, the exponential growth end point may be 40 to 120 hours, for example, 50 to 100 hours, for example, the seed culture may be terminated at a point in time 60 hours after cultivation.

According to one embodiment, the vegetable oil is, for example, sunflower seed, grape seed, canola, rice snow, olive, soybean, argan, brown rice, perilla, sesame, almond, peanut, corn, red ginseng, avocado, macadamia, coconut, rosehip , Vitamin tree seed, shea tree fruit, oil palm, bergamot fruit, camellia seed, safflower seed, apricot seed, poppy seed, evening primrose seed, castor seed, green tea seed, meadowfoam seed, flax seed and hemp seed. It may include more than one.

According to an embodiment, bis-ethylhexyloxyphenol methoxyphenyl triazine (bis-ethylhexyloxyphenol methoxyphenyl triazine), ceramide, resveratrol (resveratrol), as a poorly soluble active ingredient that can be additionally collected according to the present invention. Phylloquinone, ubiquinone, oleanolic acid, rutin, lutein, astaxanthin, Cyclospeorine, quercetin and phytosphingosine It may contain one or more selected from the group consisting of (phytosphingosine).

According to an embodiment, the composition according to the present invention may further include one or more selected from the group consisting of, for example, conditioning agents, sunscreens, moisturizing agents, antioxidants, fat-soluble vitamins and antibacterial agents. Fat-soluble vitamins may include, for example, vitamin A, vitamin D, vitamin E, and vitamin K.

The present invention can improve the formation of reverse micelles by treating the fermentation and emulsifier with high purity by the above-described method, as well as improve the solubility of poorly soluble active ingredients. Reverse micelle means that nanometer-sized (1-10 nm) round particles are dispersed in an organic solvent by the activity of a surfactant. In addition, in the presence of a hydrophilic lysate, a polar group is oriented inward, a hydrophobic group is oriented toward an outer non-polar solvent side, and it refers to a group of molecules forming an almost spherical oriented group.

The poorly soluble active ingredient may mainly contain an aromatic structure and is difficult to dissolve in a solvent with high polarity because it has a characteristic of having a structure with high crystallinity. According to the present invention, a fermentation emulsifier can be combined and arranged between the crystal structures of the poorly soluble substance to maintain the dissolved poorly soluble substance in an amorphous state, so the stability of the poorly soluble active ingredient as a functional active substance in the formulation By improving the effect of the active ingredient can be maximized.

In addition, according to another embodiment of the present invention, there is provided a cosmetic composition comprising centripetal centrifugal extract and Cheonsimryeon extract, prepared by the method as described above.

The cosmetic composition according to the present invention may contain an active ingredient in a higher concentration in the formulation of basic products such as skins and essences, while stably maintaining the active ingredient without damage. In addition, it is easy to apply to formulations such as O/W and W/O, and the effect can be further improved by combining centrifugal extract and Cheonsimyeon extract, which are each poorly soluble substance.

According to an embodiment, indications of the composition according to the present invention may include wrinkle improvement, atopy, anti-inflammatory, damaged skin barrier recovery, and the like.

According to an embodiment, the cosmetic composition according to the present invention contains 2 to 50 times the sparingly soluble active material compared to a general fermented emulsifier that has not undergone a high-purity treatment process including the steps of purifying and recovering as described above, for example. It can be dispersed and dissolved in a high concentration of 2 to 40 times, for example 2 to 30 times, for example 2 to 20 times.

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

Manufacturing Example: Manufacture of high purity fermented emulsifier

Candida bombicola UA 06 (Accession No.: KCTC13855BP, Korea Research Institute of Bioscience and Biotechnology)

Candida bombicola UA 06 (Accession No.: KCTC13855BP, Korea Research Institute of Bioscience and Biotechnology) was used as yeast extract 1.0 g/L, glycerin 20 g/L, K ₂ HPO ₄ 25 g/L, KH ₂ PO ₄ 7 Inoculate a medium consisting of g/L, NH ₄ NO ₃ 3 g/L, MgSO ₄ 2 g/L and distilled water 933 g/L, and seed culture under aerobic conditions of 20°C, 300 rpm, 1NL/min (1vvm). Proceeded. The cultivation was terminated at 60 hours, which is the exponential growth end point of the bacteria by measuring the absorbance at 600 nm with a spectrophotometer (Epoch2, BioTek). The culture solution was centrifuged (16,000 g, 30 minutes) to precipitate the cells and washed several times with a phosphate buffer to obtain cells.

Fermentation of oil

For oil fermentation, yeast extract 1.0 g/L, glycerin 20 g/L, K ₂ HPO ₄ 25 g/L, KH ₂ PO ₄ 7 g/L, NH ₄ NO ₃ 3 g/L , MgSO ₄ 2 g/L, 100 g/L of glucuronolactone, 500 g/L of vegetable oil, and 333 g/L of distilled water are mixed with fermentation broth at 25°C and 100 rpm in a 5 L fermentor Was prepared. When the temperature of the fermentation broth was 25° C., 2.5 g/L of cells were added, and fermentation was performed for 3 days. Sunflower seed oil was used as the vegetable oil.

refine

After 3 days of fermentation, the remaining enzymatic activity of the fermented product was lost to a high temperature of 90°C, and the pH was adjusted to 4 with 1N HCl, and the fermented product was allowed to stand for 18 hours, and the aqueous layer (microorganisms and medium) and the oil layer (fermented emulsion) ) To remove the aqueous layer. To remove moisture remaining in the fermented emulsion _{, 20 g/L of MgSO 4} was added, and a transparent oil layer (fermented emulsion) was obtained by filtering at 2 bar to 3 bar using a filter press. In addition, the fatty acids and impurities of the fermented emulsion were purified by a column filled with magnesium silicate, and then filtered through a 0.45 µm filter to completely remove _{remaining small particles of MgSO 4 to obtain a fermented emulsion.}

extraction

The obtained fermented product was extracted with 80% ethanol. Specifically, the ratio of the fermented product and the ethanol solvent was added in a ratio of 1:9 (w/w) and stirred for 6 hours, and the neutral fat and the active ingredient (natural surfactant) contained in the fermented product were added overnight. ) After standing, the lower layer (neutral fat) and the supernatant (active ingredient) were separated. The lower layer (triglyceride) layer was removed, and the supernatant was recovered and concentrated under reduced pressure to volatilize ethanol, and a high-purity fermentation surfactant part was obtained.

Examples 1 to 3: Preparation of cosmetic composition

A cosmetic composition was prepared in the composition shown in Table 1. The units in Table 1 are weight percent.

division Example One 2 3 Centella asiatica extract (TECA) 15.0 - 7.5 Cheonsimnyeon extract - 15.0 7.5 Manufacturing example (high purity fermentation emulsifier) 50.0 50.0 50.0 Butanediol (2,3-butanediol) 25.0 25.0 25.0 Caprylic/Capric Triglyceride 5.0 5.0 5.0 Purified water 5.0 5.0 5.0 Sum 100.0 100.0 100.0

Examples 4 to 6: Essence formulation

According to the composition of Table 3, a composition of the essence formulation including Examples 1 to 3 was prepared. Specifically, raw materials 2 to 5 were sequentially added to raw materials 1 in Table 3, stirred, and heated and dissolved at 70°C. Then, the raw materials 6 to 10 were put in a separate container and heated to 70°C to dissolve. Materials 11 to 13 were placed in a separate container and heated to 70°C to dissolve. Thereafter, the raw material reaction vessels 1 to 5 were sequentially introduced and emulsified. Then, it was dissolved and mixed uniformly and cooled to 30°C. The essence formulation was completed with a total concentration of 1.0% of the active ingredient. The unit of content in Table 2 is% by weight.

delete

Examples 7 to 10: Skin formulation

According to the composition of Table 3, a composition of a skin formulation including Examples 1 to 3 was prepared. Specifically, raw materials 2 to 5 were sequentially added to raw materials 1 in Table 3, stirred, and heated and dissolved at 50°C. Then, the raw materials 6 to 8 were put in a separate container, heated to 50°C, and dissolved. Thereafter, the raw material reaction vessels 1 to 5 were sequentially introduced and emulsified. Then, it was dissolved and mixed uniformly and cooled to 30°C. A skin formulation with a total concentration of 0.6% of the active ingredient was completed. The unit of content in Table 3 is% by weight.

division Raw material Example 7 8 9 One Purified water 80.4 80.4 80.4 2 Carbomer 0.1 0.1 0.1 3 Propanediol 8.0 8.0 8.0 4 1,2 hexanediol 1.5 1.5 1.5 5 Xanthan gum 0.1 0.1 0.1 6 Example 1 4.0 - - Example 2 - 4.0 - Example 3 - - 4.0 7 Dipropylene glycol 4.0 4.0 4.0 8 Ceteares-20 0.8 0.8 0.8 9 Tromethamine 0.1 0.1 0.1 10 Hyaluronic Acid (1%) 1.0 1.0 1.0 Sum 100.0 100.0 100.0

Experimental Example 1: Nitric Oxide Measurement

In order to evaluate the anti-inflammatory efficacy of the compositions according to Examples 1 to 3, the ability to inhibit nitric oxide production was measured.

Nitric oxide is known as a chemical molecule that is produced by immune cells and regulates the immune system against external or internal harmful substances.

Raw264.7 cells, a mouse-derived macrophage line, were cultured in a 12 well culture plate at a ^{concentration of 5x10 4} cells/well for 24 hours, and each sample was treated with different concentrations (0~1.0%) for 2 hours, and then Lipopolysaccharide (0.1 μg/ml) and cultured for 24 hours. A culture solution was obtained, centrifuged (3,000 rpm, 5 minutes), and 100 μl of the supernatant was mixed with 100 μl of a griess reagent (a mixture of 1% sulfanilamide and 0.1% N-(1-naphthyl) ethylene diamine dihydrochloride) to block light. It was reacted for 15 minutes. The resulting reaction product was measured by absorbance at 450 nm with a microplate reader. In addition, as samples to be treated with cells, a concentrate obtained by extracting Examples 1 to 3 with 70% ethanol was prepared. The concentrate was dissolved in DMSO (dimethylsulfoxide) and diluted with a medium to be used. The anti-inflammatory effect was confirmed by measuring the nitric oxide inhibition rate when the cells were treated, and shown in FIG. 1. However, the experiment was conducted at a concentration without toxicity.

As shown in FIG. 1, it can be seen that the amount of nitric oxide produced was inhibited according to Examples 1 to 3. When the nitric oxide inhibition rate was 100% of nitric oxide generated during LPS treatment, the nitric oxide produced of the sample was calculated as the inhibition rate.

As a result, at the same concentration, it was confirmed that the Chunsimnyeon extract is superior to the Nitric Oxide inhibitory rate compared to the Centella asiatica extract, and in particular, a sample treated with Centella asiatica extract or Cheonsimyeon extract alone at all concentrations (0.01~1%) ( Compared to Examples 1 and 2), the sample (Example 3) obtained by complexly treating Centella asiatica extract and Cheonsimyeon extract showed more excellent results in inhibiting nitric oxide production.

Experimental Example 2: Cytokine measurement

In order to evaluate the anti-inflammatory effect of the compositions according to Examples 1 to 3, the rate of inhibition of cytokine production was measured. Raw264.7 cells, a mouse-derived macrophage line, were cultured in a 12 well culture plate at a ^{concentration of 5x10 4} cells/well for 24 hours, and each sample was treated with different concentrations (0~1.0%) for 2 hours, and then Lipopolysaccharide (0.1 μg/ml) and cultured for 24 hours. A culture solution was obtained, centrifuged (3,000 rpm, 5 minutes), and the amount of TNF-α secreted in the supernatant was analyzed using an ELISA Kit (Thermo Scientific, IL, USA).

Samples to be treated on the cells were prepared as a concentrate obtained by extracting Examples 1 to 3 with 70% ethanol. The concentrate was dissolved in DMSO (dimethylsulfoxide) and diluted with a medium to be used. The cells were treated to confirm the TNF-α inhibition rate, and the anti-inflammatory effects were compared, and the results are shown in FIG. 2. However, the experiment was conducted at a concentration without toxicity.

As shown in Figure 2, it can be confirmed that the amount of TNF-α production was inhibited according to Examples 1 to 3. In particular, in the group treated with 1% concentration of Example Centellar Centella asiatica extract or Cheonsimnyeon extract alone (Examples 1 and 2), a sample treated with Centellar Centella asiatica extract and Cheonsimnyeon extract in combination (Example 3 ), the amount of TNF-α production was markedly reduced to about 88%.

Experimental Example 3: NF-κB activity inhibition rate measurement

In order to evaluate the anti-inflammatory of the compositions according to Examples 1 to 3, the inhibition of NF-κB activity was measured using a luciferase reporter gene assay. Raw264.7 cells, a mouse-derived macrophage line, were cultured in a 12 well culture plate at a ^{concentration of 5x10 4} cells/well for 24 hours, and each sample was treated with different concentrations (0~1.0%) for 2 hours, and then Lipopolysaccharide (0.1 μg/ml) and cultured for 24 hours. The culture solution was obtained and centrifuged (3,000 rpm, 5 minutes), and the cell pellet was collected and the NF-κB content was performed using a dual-luciferase reporter assay (Promega, Madison, WI, USA).

Samples to be treated on the cells were prepared as a concentrate obtained by extracting Examples 1 to 3 with 70% ethanol. The concentrate was dissolved in DMSO (dimethylsulfoxide) and diluted with a medium to be used. The anti-inflammatory effect was compared by measuring the NF-κB activity when the cells were treated. A reporter gene analysis was performed by transfection of pNF-kB-Luc plasmid into Raw 264.7 cells, and the results are shown in FIG. 3. However, the experiment was conducted at a concentration without toxicity.

As shown in Figure 3, compared to the negative control (pNF-kB transfected conrol) into which pNF-kB was introduced, in the case of the positive control treated with LPS, Luciferase reporter acivity increased 5 times.

It can be seen that NF-κB activity was inhibited according to Examples 1 to 3. In particular, in the group treated with 1% concentration of Example Centella asiatica extract or samples treated with Cheonsimyeon extract alone (Examples 1 and 2), a sample treated with Centella asiatica extract and Cheonsimyeon extract in combination (Example 3 ), the NF-κB activity was markedly reduced to about 59%.

Experimental Example 4: Evaluation of skin efficacy of the compositions according to Examples 7 to 9

In order to evaluate the skin efficacy of the compositions according to Examples 7 to 9, an experiment was conducted on the skin of the human body.

Specifically, after starting to form wrinkles or being divided into a test group and a control group by 10 adult men and women who have already been generated, the test groups applied Examples 7 to 9 each morning and evening twice for 4 weeks. The skin condition was precisely measured with normal light, polarized light, and ultraviolet light using a facial analyzer (Janus). Normal light can analyze skin texture, pores, and wrinkles, and polarized light can analyze pigmentation and inflammatory expressions such as acne, and ultraviolet light can analyze sebum, pigmentation, and dead skin cells. This was comprehensively evaluated to derive the effect of the skin, and the results are shown in Tables 4 and 5.

Sample/evaluation Skin tone and pigmentation improvement Great usually No change Example 4 3 2 - Example 5 2 6 One Example 6 8 2 -

Sample/evaluation Wrinkle and skin texture improvement Great usually No change Example 4 2 7 One Example 5 - 5 5 Example 6 9 One -

As shown in Tables 4 and 5, as a result of evaluating the degree of improvement in skin tone, pigmentation, wrinkles, and skin texture, Centella asiatica extract and Cheonsimryeon than in Examples 7 and 8 containing Centellarella extract or Cheonsimryeon extract alone. It was confirmed that the skin formulation according to Example 9 containing the extract exhibited excellent results in skin effect.

It can be confirmed that the two poorly soluble active ingredients of Centella asiatica extract and Cheonsimyeon extract are stably incorporated into skin formulations and are easily applied to skin formulations, thereby showing excellent effects on skin aging and skin tone improvement due to excellent skin transmission power. It is the result.

Experimental Example 5: Skin barrier damage recovery evaluation

The damaged skin barrier restoration test was a tape-striping test for 10 volunteers in each test group.The stratum corneum was repeated 15 to 20 times by attaching and detaching it to the skin with a dedicated tape on the entire cut area. Damaged. Thereafter, the essence formulations of Examples 4 to 6 were applied, and as a comparative group, an essence formulation having the same composition as Examples 4 to 6 was used, except that Examples 1 to 3 were not added. Comparative group and Examples 4 to 6 were applied to the skin in the same amount, respectively.

After application to the skin, after damage (before application) for each test group, the value of the transdermal moisture evaporation amount according to the lapse of 3 days, 4 days, and 5 days of application was measured, and the resilience of the damaged skin stratum corneum was confirmed and shown in FIG. . As a measuring instrument, Teawameter TM300 (Courage & Khazaka, Germany) was used.

As shown in FIG. 4, when the skin barrier was damaged by taping, the amount of transdermal moisture evaporation increased, and the loss of transdermal moisture in the test group coated with Example 6 compared to the test group coated with Examples 4 and 5 Since it appears to be the most excellent recovery, it was determined that the formulation according to the present invention exhibited a synergistic effect of the two types of composites to quickly restore the skin barrier.

Experimental Example 6: Stability evaluation by long-term storage temperature

In order to evaluate the stability according to the storage temperature conditions, the stability of each condition was confirmed for the compositions of Examples 1 to 3. Each composition was stored at room temperature, 25°C for 1 month, low temperature (-5°C), high temperature (50°C), and circulation (-5 to 40°C/12 hr) for 3 months to observe its properties. The evaluation results are shown in Tables 6 and 7.

division Example One 2 3 Room temperature Appearance No change No change No change Precipitation No change No change No change 25℃ Appearance No change No change No change Precipitation No change No change No change

division Example One 2 3 -5 ℃ Appearance No change No change No change Precipitation No change No change No change 50 ℃ Appearance No change No change No change Precipitation No change There is change No change cycle
(-5~40℃)/12hr Appearance No change No change No change Precipitation No change No change No change

From the results of Tables 6 and 7, it was confirmed that Examples 1 to 3 have excellent stability depending on the change of properties and precipitation according to the storage conditions. This is a result of confirming that the compositions of Examples 1 to 3 maintain a stably dissolved property even in a high temperature and long-term environment.

Experimental Example 7: Stability evaluation by long-term storage temperature by formulation

In order to evaluate the stability according to the storage conditions for each formulation, the stability for each condition was confirmed for the compositions of Examples 4 to 9. Each composition was stored at room temperature, 25°C for 1 month, low temperature (-5°C), high temperature (50°C), and circulation (-5 to 40°C/12 hr) for 3 months to observe its properties. The evaluation results are shown in Tables 8 and 9.

division Example 4 5 6 7 8 9 Room temperature Appearance No change No change No change No change No change No change Precipitation No change No change No change No change No change No change 25 ℃ Appearance No change No change No change No change No change No change Precipitation No change No change No change No change No change No change

division Example 4 5 6 7 8 9 -5 ℃ Appearance No change No change No change No change No change No change Precipitation No change No change No change No change No change No change 50 ℃ Appearance No change No change No change No change No change No change Precipitation No change There is change No change No change There is change No change cycle
(-5 ~40℃)/12hr Appearance No change No change No change No change No change No change Precipitation No change No change No change No change No change No change

From the results of Tables 8 and 9, it was confirmed that all of Examples 4 to 9 had excellent stability depending on the change of properties and precipitation according to the storage conditions. These results are the result of confirming that the compositions of Examples 4 to 9 including the compositions of Examples 1 to 3 maintain a stably dissolved property even in a high temperature and long-term environment.

As can be seen from the above results, according to the present invention, by stabilizing crystalline substances or unstable substances in a cosmetic formulation using a fermented emulsifier treated with high purity as in the present invention, O/W (oil in water), etc. The formulation of can be stably prepared. In addition, since a product with improved emulsifying power can be manufactured while containing an active ingredient in combination, the synergistic effect of the active ingredients in the skin can be maximized.

The above description is merely illustrative of the technical idea of the present invention, and those of ordinary skill in the technical field to which the present invention pertains can make various modifications and variations without departing from the essential characteristics of the present invention. In addition, the embodiments disclosed in the present invention are not intended to limit the technical idea of the present invention, but to describe it, and the scope of the technical idea of the present invention is not limited by these embodiments. The scope of protection of the present invention should be interpreted by the following claims, and all technical ideas within the scope equivalent thereto should be construed as being included in the scope of the present invention.

Claims (8)

Fermentation emulsifier; And
Including centella asiatica extract, cheonsimryeon extract, or a combination thereof,
The fermentation emulsifier,
Candida bombicola UA-06 (Accession No.: KCTC13855BP, Korea Research Institute of Bioscience and Biotechnology) Seed culture step of activating microorganisms;
A fermentation step of fermenting the seed cultured microorganisms and vegetable oils;
Purifying by removing moisture, fatty acids, and impurities in the fermented product obtained from the fermentation step; And
Including the step of separating and recovering the supernatant by mixing ethanol with the purified fermentation,
The recovering step is a step of mixing the purified fermented product and 70 to 95% ethanol in a volume ratio of 1:5 to 1:20 (w/w) and stirring for 3 to 10 hours to separate the lower layer and the supernatant. ; And
After volatilizing ethanol from the supernatant, it is prepared by a method comprising the step of recovering the fermentation emulsifier. The method of claim 1,
In the purifying step, the fermented product is adjusted to a pH of 3 to 6 and allowed to stand for 5 to 30 hours to separate the aqueous layer and the oil layer;
MgSO ₄ , zeolite, diatomaceous earth, activated alumina, adding 10 to 30 g/L of at least one selected from the group consisting of calcium sulfate and calcium chloride, and filtering with a filter press at 1 to 5 bar to obtain an oil layer;
Purifying with a column filled with at least one selected from the group consisting of magnesium silicate, activated carbon, white clay, acid clay, and perlite; And
The method for producing a cosmetic composition comprising the step of filtering. delete The method of claim 1,
The medium used in the seed culture step is to contain yeast extract, glycerin, K ₂ HPO ₄ , KH ₂ PO ₄ , NH ₄ NO ₃ and MgSO ₄ , a method for producing a cosmetic composition. The method of claim 1,
The method for producing a cosmetic composition, wherein the medium used in the fermentation step contains yeast extract, glycerin, K ₂ HPO ₄ , KH ₂ PO ₄ , NH ₄ NO ₃ , MgSO _{4, glucuronolactone and vegetable oil} . The method of claim 1,
The medium used in the seed culture step is yeast extract 1 to 30 g/L, glycerin 10 to 40 g/L, K ₂ HPO ₄ 10 to 50 g/L, KH ₂ PO ₄ 3 to 15 g/L, NH ₄ NO ₃ 1 to Including 10g/L and 0.5 to 5g/L of _{MgSO 4,}
The medium used in the fermentation step is yeast extract 0.5 to 3 g/L, glycerin 1 to 40 g/L, K ₂ HPO ₄ 10 to 50 g/L, KH ₂ PO ₄ 3 to 15 g/L, NH ₄ NO ₃ 1 to 10 g /L, MgSO ₄ 0.5 to 5g / L, glucuronolactone 40 to 200g / L and vegetable oil 50 to 1000g / L containing that, a method for producing a cosmetic composition. The method of claim 1,
The seed cultivation step is carried out in 15 to 30 ℃, 100 to 500rpm, 0.5 to 2vvm, aerobic conditions,
The fermentation step is to proceed at a temperature of 20 to 35 ℃ and mixing conditions of 50 to 200rpm, the method for producing a cosmetic composition. A cosmetic composition comprising centella asiatica extract and centella asiatica extract prepared by the method according to any one of claims 1, 2 and 4 to 7.

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