KR101855919B1 - Active Material Stabilized in Plant Cell and Method for Manufacturing the Same - Google Patents

Abstract

The present invention relates to a composition containing plant cells containing a stabilized active substance, a method of stabilizing the active substance, and a method of producing plant cells containing the stabilized active substance, The stability of the substance can be enhanced, and when it is used as a component of functional cosmetics and external preparations for skin, stability is high and the effect of the active ingredient can be maximized.

Description

[0001] The present invention relates to an active material stabilized in plant cells,

The present invention relates to a cosmetic composition comprising an active substance stabilized in a plant cell, and a method for producing a plant cell containing the active substance stabilized after stabilizing the active substance.

There are many different kinds of active substances in natural plants and plants, and they are also made up of a myriad of flavonoids, flavonols, phenols, amino acids, lignans, alkaloids, vitamins and catechin compounds. These active substances each have different pharmacological actions, and many studies are under way. Despite the excellent effects of active ingredients having useful functions in human body physiological properties, most of the active substances are not free from the stability. Recently, in order to improve their stability and drug delivery, phospholipids or surfactants Many attempts have been made to use liposome, stabilized cerasoma using ceramide, liquid crystal stabilized with liquid crystal structure, and cubic cubosome using monoglyceride. However, these methods have problems in that the manufacturing method is complicated or the biocompatibility is deteriorated by using a compound.

The present inventors have sought to develop a method for stabilizing a simpler but more biocompatible active substance. As a result, the present inventors have completed the present invention by confirming that the stability of the active substance is enhanced when the active substance is introduced into the cell wall, particularly within the cell wall.

It is therefore an object of the present invention to provide a cosmetic composition comprising an active substance stabilized in plant cells.

It is another object of the present invention to provide a method for producing a plant cell comprising a stabilized active substance after stabilizing the active substance.

To achieve the above object, an embodiment of the present invention provides a cosmetic composition containing plant cells containing a stabilized active substance.

In one embodiment of the present invention, the active material of the cosmetic composition may be at least one selected from the group consisting of a hydrophilic substance and a hydrophobic substance.

In one embodiment of the present invention, the active material of the cosmetic composition is selected from the group consisting of gallic acid, amino acid, phytic acid, vitamin C, L-ascorbic acid, arbutin, fructose (EGCG), retinol, hesperidin, oleanolic acid, polymethylmethacrylate (EGCG), dicarboxylic acid, dibutyl phthalate, diphosphate, trisodium fructose, 1,6-diphosphate adenosine, niacinamide, genistein, flavonoids, epigallocatechin gallate Methacrylate, thymol trimethoxycinnamate, red ginseng saponin, hydrolized ginseng saponin, and ascorbyl glucoside.

 Also, in one embodiment of the present invention, the plant cell preferably uses a cell wall.

In addition, one embodiment of the present invention provides a method for preparing a plant cell comprising stabilizing an active substance comprising a stabilized active substance, comprising the steps of:

(a) introducing the active substance into plant cells under the condition of 1 - 100 MPa;

(b) causing a dehydration reaction using reverse osmosis from the plant cell into which the active substance is introduced; And

(c) obtaining a plant cell into which an active substance has been introduced

In addition, in one embodiment of the present invention, the active substance of the method for producing plant cells may be at least one selected from the group consisting of hydrophilic substances and hydrophobic substances.

In addition, in one embodiment of the present invention, the active substance of the method for producing plant cells is selected from the group consisting of gallic acid, amino acid, phytic acid, vitamin C, L-ascorbic acid, But are not limited to, arbutin, fructose diphosphate, trisodium fructose, 1,6-diphosphate adenosine, niacinamide, genistein, flavonoids, epigallocatechin gallate (EGCG), retinol, hesperidin, oleanolic acid ), Polymethyl methacrylate, thymol trimethoxycinnamate, red ginseng saponin, hydrolized ginseng saponin, and ascorbyl glucoside.

In one embodiment of the present invention, the plant cell of the method for producing the plant cell is preferably a cell wall.

The cosmetic composition containing the active substance stabilized in the plant cell of the present invention can enhance the stabilization of the active substance by using a simple method than the conventional method, and when used as a component of the functional cosmetic preparation and the external preparation for skin, The effect of the component can be maximized.

FIG. 1 is a flow chart showing a process of introducing and stabilizing an active substance into a plant cell.
FIGS. 2A to 2D are graphs showing results of comparison of each of the standard reagents as a result of analyzing the components 1 to 4, which are the hydrophilic active materials used in the present invention, by high performance liquid chromatography.
FIGS. 3A to 3C are graphs showing the results of analyzing the components 5 to 7, which are the hydrophobic active materials used in the present invention, using the HPLC chromatogram, and comparing the results with the respective standard reagents.
4A to 4D are graphs showing the results of the formulation stability after the hydrophilic active material of the present invention is introduced into plant cells.
5A to 5C are graphs showing the formulation stability results after the hydrophobic active material of the present invention is introduced into a plant cell.
6 is a photomicrograph of a gallic acid sample that has been taken in a stabilized state in a plant cell.

According to one embodiment of the present invention, there is provided a cosmetic composition comprising an active substance stabilized in a plant cell.

The plant cell in the present invention may be any kind of plant cell that can be selected in the art.

In the present invention, it is preferable that the plant cells are made into a single cell through pre-treatment with a combination of enzymes.

In the present invention, the enzyme may be any kind of enzyme that can be selected in the art.

The term " active substance " in the present invention refers to a substance that enhances or inhibits the function of a living body. According to one embodiment of the present invention, the active substance may be at least one selected from the group consisting of a hydrophilic substance and a hydrophobic substance.

According to one embodiment of the present invention, the active substance may be a hydrophilic substance. The hydrophilic active material of the present invention may be selected from the group consisting of gallic acid, amino acid, Phytic acid, vitamin C, L-ascorbic acid, arbutin, fructose diphosphate, , 6-diphosphate adenosine, and niacinamide, and preferably at least one selected from the group consisting of gallic acid, amino acid, phytic acid and vitamin C, But is not limited thereto.

According to one embodiment of the present invention, the active substance may be a hydrophobic substance. The hydrophobic active material of the present invention may be selected from the group consisting of genistein, flavonoids, epigallocatechin gallate (EGCG), retinol, hesperidin, oleanolic acid, polymethylmethacrylate, thymol trimethoxycinnamate Trimethoxycinnamate), red ginseng saponin, hydrolized ginseng saponin, and ascorbyl glucoside, but the present invention is not limited thereto.

According to one embodiment of the present invention, the plant cell preferably utilizes a cell wall.

According to one embodiment of the present invention, there is provided a method of preparing a plant cell comprising a stabilized active substance after stabilizing an active substance comprising the steps of:

(a) introducing the active substance into plant cells under the condition of 1 - 100 MPa;

(b) causing a dehydration reaction using reverse osmosis from the plant cell into which the active substance is introduced; And

(c) obtaining a plant cell into which an active substance has been introduced

As used herein, the term 'soluble solvent' refers to any solvent capable of dissolving the active material, and refers to water, ethanol, ethyl acetate, butyleneglycol, methanol, propanediol, and the like.

In the step of dissolving the active material of the present invention in a solvent, the solvent is selected from the group consisting of water, ethanol, methanol, butanol, propanol, propanediol, butylene glycol, propylene glycol, pentylene glycol, hexanediol, chloroform, ethyl acetate , A solvent selected from the group consisting of dichloromethane, hexane, petroleum ether and diethyl ether, or a mixture of two or more solvents, preferably water, ethanol, methanol, more preferably water and ethanol.

In the step of mixing the active substance solution of the present invention with the plant cells, the mixing method may be performed by a method commonly used for mixing two or more substances in the art. More specifically, the active substance and the plant cell can be mixed in a container simultaneously or sequentially.

The active substance-solubilized substance formed through the dissolution process is mixed with the plant cells in an aqueous solution and equilibrated in a solution state by natural osmotic pressure by stirring. The content of plant cells according to the present invention is 0.1 to 30% by weight based on the total weight . When the content is less than 0.1% by weight, there is a problem of lowering the plant cell osmotic pressure ratio. When the content is more than 30% by weight, there is a problem of reduction in the amount of the active substance introduced into plant cells. The optimal content of the active material is preferably 0.01 to 10% by weight based on the total weight. Depending on the active substance, the optimum concentrations for different efficacies are different and exert their effect in this critical range.

In the step of mixing the active substance solution with the plant cells, the active substance is partially introduced into the plant cells. In the step of introducing the active substance into the plant cells under the high pressure condition of the present invention, The higher the rate.

The term 'high pressure condition' used in the present invention means conditions suitable for introducing an active substance into plant cells, specifically 0.01 MPa - 500 MPa, more preferably 0.1 MPa - 100 MPa, and most preferably, 0.5 MPa to 20 MPa. When the pressure is less than 0.5 MPa, the introduction of the active material is not effected effectively, and when the pressure is 20 MPa or more, the cell wall may be broken. According to a preferred embodiment of the present invention, in the method of stabilizing the active material, a step of dehydrating from the plant cell into which the active material is introduced may further include a step of raising the rate of introduction of the active material, Further comprising the step of obtaining an introduced plant cell.

The term " dehydration reaction " used in the present invention means a reaction in which water is separated in the molecule of an organic compound and a reaction in which water is separated from two molecules of the organic compound to condense. The dehydration reaction can be performed by a method commonly used in the art, such as using a hydrophilic substance or heating, but according to a preferred embodiment of the present invention, the dehydration reaction can utilize reverse osmosis.

As used herein, the term 'reverse osmosis' refers to a process in which the solute is sent to a lower concentration of solute by applying a pressure greater than osmotic pressure to the lower solute concentration (Reverse Osmosis (R / O): Selecting a Reverse Osmosis Unit By Arthur Fisher et al.). According to a preferred embodiment of the present invention, a method of reverse osmosis by adding a salt containing sodium or potassium element may be used.

In the process of reverse osmosis, dehydration occurs at a low concentration (liquid in a plant cell) and at a high concentration (mixture liquid) while leaving active substances in plant cells. The optimal soluble salt addition according to the present invention is 0.5 to 40 wt% To induce dehydration outside of the plant cell to maximize the rate of entry into the cell. In the natural state, it is normal that the water in the outside enters the cell wall (osmotic pressure), but it is reverse osmotic pressure to artificially add salt outside the cell to dehydrate the water in the cell.

The step of obtaining the plant cell into which the active substance has been introduced includes a method for obtaining cells which are conventionally used, but a method of obtaining by filtration and / or centrifugation may be used. When using a filter, 1.2 - 50 μm and 40 - 200 mesh are preferable, and when centrifuging is used, 100 - 9,000 rpm, 0 - 25 ° C. are preferable.

According to a preferred embodiment of the present invention, the plant cell in which the active material obtained through the recovery process is introduced may further include a washing step of removing impurities on the surface and active substances not introduced. In this case, it is preferable to use a solution containing 0.1 to 20% by weight of salt in distilled water, but not limited thereto.

In the cosmetic composition of the present invention, the content of the stabilized active substance in the plant cell is preferably 0.001 to 10% by weight, more preferably 0.001 to 10% by weight, And stability.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Stabilization of hydrophilic active material

Example 1 Stabilization of Phenolic Compound Galic Acid

Galactic acid (> 99%, Sigma, USA) was added to tertiary distilled water at 10% by weight and stirred for 30 minutes. 10 wt% of gallic acid soluble matter and 10 wt% of black soybean unicell (Healthy food ingredient Co., Ltd., JPN) were added to 80 wt% of distilled water, and the mixture was mixed with a propeller mixer (BL3000, Heidon, JPN) At 800 rpm and 20 < 0 > C for 1 hour. After stirring, the plant cell mixture was pressurized at 20 MPa and 25 ° C for 1 hour using a high-pressure apparatus (TFS-2L, TOYO KOATSU CO., LTD., JPN). 2% by weight of sodium chloride (NaCl) was added to the mixed plant cell mixture under high pressure, and dehydration reaction was induced by using a propeller mixer (BL3000, Heidon, JPN) at 800 rpm and 20 ° C for 1 hour. Subsequently, high-speed centrifugation was carried out using a centrifuge (Supra 22K, Hanil, KOR) at 9,000 rpm and 15 ° C for 20 minutes to obtain plant cells into which the active material was introduced. The plant cells thus obtained were washed with 100-fold physiological saline (0.9% NaCl aqueous solution) and then centrifuged at a speed of 9,000 rpm at 15 ° C for 20 minutes using a centrifuge (Supra 22K, Hanil, KOR) A sample was obtained. The sample thus obtained was called "sample 1" and used in the following test examples.

Example 2 Stabilization of Amino Acids

Amino acid (AAS, Agilent, USA) was added to the third distilled water at 20 wt% and stirred for 30 minutes. Thereafter, 20 wt% of the amino acid solubles and 1 wt% of black soybean unicell (Healthy food ingredient Co., Ltd., JPN) were added to 79 wt% of distilled water and the mixture was blended with a propeller mixer (BL3000, Heidon, JPN) And the mixture was stirred at 800 rpm and 20 ° C for 1 hour. After stirring, the plant cell mixture was pressurized at 20 MPa and 25 ° C for 1 hour using a high-pressure apparatus (TFS-2L, TOYO KOATSU CO., LTD., JPN). 2% by weight of sodium chloride (NaCl) was added to the mixed plant cell mixture under high pressure and reverse osmosis was induced for 1 hour at 800 rpm and 20 ° C using a propeller mixer (BL3000, Heidon, JPN). Subsequently, high-speed centrifugation was carried out using a centrifuge (Supra 22K, Hanil, KOR) at 9,000 rpm and 15 ° C for 20 minutes to obtain plant cells into which the active material was introduced. The plant cells thus obtained were washed with 100-fold physiological saline (0.9% NaCl aqueous solution) and then centrifuged at a speed of 9,000 rpm at 15 ° C for 20 minutes using a centrifuge (Supra 22K, Hanil, KOR) A sample was obtained. The thus obtained sample was called "Sample 2" and used in the following test examples.

≪ Example 3 > Stabilization of organic acid phytic acid

(50% (w / w), Sigma, USA) was added to tertiary distilled water in an amount of 10% by weight and stirred for 30 minutes. Then, 10 wt% of phytic acid soluble matter and 10 wt% of black soybean unicell (Healthy food ingredient Co., Ltd., JPN) were added to 80 wt% of distilled water, and a propeller mixer (BL3000, Heidon, JPN) And the mixture was stirred at 800 rpm and 20 캜 for 1 hour. After stirring, the plant cell mixture was subjected to high pressure for 1 hour at 20 MPa and 25 ° C using a high-pressure apparatus (TFS-2L, TOYO KOATSU CO., LTD., JPN). 2% by weight of sodium chloride (NaCl) was added to the mixed plant cell mixture under high pressure and reverse osmosis was induced for 1 hour at 800 rpm and 20 ° C using a propeller mixer (BL3000, Heidon, JPN). Subsequently, high-speed centrifugation was carried out using a centrifuge (Supra 22K, Hanil, KOR) at 9,000 rpm and 15 ° C for 20 minutes to obtain plant cells into which the active material was introduced. The plant cells thus obtained were washed with 100 times physiological saline solution (0.9% NaCl solution) and centrifuged at a speed of 9,000 rpm at 15 ° C for 20 minutes using a centrifuge (Supra 22K, Hanil, KOR) To obtain a sample. The sample thus obtained was called "sample 3" and used in the following test examples.

Example 4 Stability of Water-Soluble Vitamin L-Ascorbic Acid

L-ascorbic acid (> 99%, Samchun, KOR) was added to the third distilled water in an amount of 20% by weight and stirred for 30 minutes. 10% by weight of ascorbic acid soluble substance and 10% by weight of black soybean unicell (Healthy food ingredient Co., Ltd., JPN) were added to 80% by weight of distilled water, and a propeller mixer (BL3000, Heidon, JPN) And the mixture was stirred at 800 rpm and 20 캜 for 1 hour. After stirring, the plant cell mixture was subjected to high pressure for 1 hour at 20 MPa and 25 ° C using a high-pressure apparatus (TFS-2L, TOYO KOATSU CO., LTD., JPN). 2% by weight of sodium chloride (NaCl) was added to the mixed plant cell mixture under high pressure and reverse osmosis was induced for 1 hour at 800 rpm and 20 ° C using a propeller mixer (BL3000, Heidon, JPN). Subsequently, high-speed centrifugation was carried out using a centrifuge (Supra 22K, Hanil, KOR) at 9,000 rpm and 15 ° C for 20 minutes to obtain plant cells into which the active material was introduced. The plant cells thus obtained were washed with 100-fold physiological saline (0.9% NaCl aqueous solution) and then centrifuged at a speed of 9,000 rpm at 15 ° C for 20 minutes using a centrifuge (Supra 22K, Hanil, KOR) A sample was obtained. The thus obtained sample was referred to as "sample 4" and used in the following test examples.

Stabilization of hydrophobic active material

Example 5 Stabilization of Isoflavonan Genistein

5% by weight of genistein (> 99%, Sigma, USA) was added to 99.5% ethyl alcohol (Ethanol) and stirred for 30 minutes. 10 wt% of genistein solubles and 10 wt% of black soybean unicell (Healthy food ingredient Co., Ltd., JPN) were added to 80 wt% of distilled water, and the mixture was treated with a propeller mixer (BL3000, Heidon, JPN) And the mixture was stirred at 800 rpm and 20 ° C for 1 hour. After stirring, the plant cell mixture was subjected to high pressure for 1 hour at 20 MPa and 25 ° C using a high-pressure apparatus (TFS-2L, TOYO KOATSU CO., LTD., JPN). 2% by weight of sodium chloride (NaCl) was added to the mixed plant cell mixture under high pressure and reverse osmosis was induced for 1 hour at 800 rpm and 20 ° C using a propeller mixer (BL3000, Heidon, JPN). Subsequently, high-speed centrifugation was carried out using a centrifuge (Supra 22K, Hanil, KOR) at 9,000 rpm and 15 ° C for 20 minutes to obtain plant cells into which the active material was introduced. The plant cells thus obtained were washed with 100-fold physiological saline (0.9% NaCl aqueous solution) and then centrifuged at a speed of 9,000 rpm at 15 ° C for 20 minutes using a centrifuge (Supra 22K, Hanil, KOR) A sample was obtained. The sample thus obtained was referred to as "sample 5" and used in the following test examples.

Example 6 Stabilization of a flavonoid epigallocatechin gallate (EGCG)

Epigallocatechin gallate (> 99%, Sigma, USA) was added to an aqueous solution of 70% ethyl alcohol (Ethyl alcohol, Ethanol) in an amount of 5% by weight and stirred for 30 minutes. Then, 10 wt% of epigallocatechin gallate (EGCG) solubles and 10 wt% of black soybean unicell (Healthy food ingredient Co., Ltd., JPN) were added to 80 wt% of distilled water, BL3000, Heidon, JPN) at 800 rpm and 20 ° C for 1 hour. After stirring, the plant cell mixture was pressurized at 20 MPa and 25 ° C for 1 hour using a high-pressure apparatus (TFS-2L, TOYO KOATSU CO., LTD., JPN). 2% by weight of sodium chloride (NaCl) was added to the mixed plant cell mixture under high pressure and reverse osmosis was induced for 1 hour at 800 rpm and 20 ° C using a propeller mixer (BL3000, Heidon, JPN). Subsequently, high-speed centrifugation was carried out using a centrifuge (Supra 22K, Hanil, KOR) at 9,000 rpm and 15 ° C for 20 minutes to obtain plant cells into which the active material was introduced. The plant cells thus obtained were washed with 100-fold physiological saline (0.9% NaCl aqueous solution) and centrifuged at 9,000 rpm and 15 ° C for 20 minutes using a centrifuge (Supra 22K, Hanil, KOR) A sample was obtained. The sample thus obtained was referred to as "sample 6" and used in the following test examples.

<Example 7> Stabilization of fat-soluble vitamin retinol

5% by weight of retinol (> 99%, Sigma, USA) was added to an aqueous solution of 99.5% ethyl alcohol (Ethanol) and stirred for 30 minutes. Then, 10 wt% of retinol-soluble matter and 10 wt% of black soybean unicell (Healthy food ingredient Co., Ltd., JPN) were added to 80 wt% of distilled water and the mixture was blended with a propeller mixer (BL3000, Heidon, JPN) And the mixture was stirred at 800 rpm and 20 ° C for 1 hour. After stirring, the plant cell mixture was subjected to high pressure for 1 hour at 20 MPa and 25 ° C using a high-pressure apparatus (TFS-2L, TOYO KOATSU CO., LTD., JPN). 2% by weight of sodium chloride (NaCl) was added to the mixed plant cell mixture under high pressure, and dehydration reaction was induced by using a propeller mixer (BL3000, Heidon, JPN) at 800 rpm and 20 ° C for 1 hour. Subsequently, high-speed centrifugation was carried out using a centrifuge (Supra 22K, Hanil, KOR) at 9,000 rpm and 15 ° C for 20 minutes to obtain plant cells into which the active material was introduced. The plant cells thus obtained were washed with 100-fold physiological saline (0.9% NaCl aqueous solution) and then centrifuged at a speed of 9,000 rpm at 15 ° C for 20 minutes using a centrifuge (Supra 22K, Hanil, KOR) A sample was obtained. The sample thus obtained was called "sample 7" and used in the following test examples.

[Test Example 1] High-performance liquid chromatography (HPLC) component analysis

In order to analyze the efficiency and components of the active substance stabilized in plant cells of the present invention, analysis was conducted as follows.

[Hydrophilic active substance]

(1) Phenolic Compound Glaric Acid

The analysis was carried out using high performance liquid chromatography (HPLC) for the gallic acid inclusion efficiency and component analysis in the plant cells of the present invention. Samples were prepared at appropriate concentrations with each solvent and filtered into a 0.2 μm disk filter, pre-treated and injected into the instrument. The instrument was analyzed in the UV 254 to 340 nm region using high performance liquid chromatography (Agilent Technologies 1200 Series HPLC, Agilent, USA) and analyzed using a C18 column (Zorbax XDB-C18 4.6 x 250 mm, Agilent, USA) Acetic acid solvent and acetonitrile (ACN) solvent. The analytical solvents used were HPLC grade reagents and component analysis was performed using gallic acid (> 99%, Sigma, USA) as a standard. The analysis results are shown in [Figure 2a] and [Table 1].

(2) Amino acid

In order to analyze the efficiency and components of the stabilized amino acid in the plant cells of the present invention, a sample was prepared at a proper concentration, filtered through a 0.2 μm disk filter, pre-treated with o-phthalaldehyde (OPA Agilent) Fluorescence detector (FLD) using high performance liquid chromatography (Agilent Technologies 1200 Series HPLC, Agilent, USA) after forming fluorescence isoindole by using 9-fluorenylmethylchloroformate (FOC) , Agilent Technologies, USA). Amino acid analysis was performed using 250 pM standard amino acid analysis standard (AAS, Agilent, USA) as a reference material. The analysis results are shown in [Figure 2b] and [Table 1].

(3) Organic acid phytic acid

In order to analyze the filling efficiency and components of the stabilized phytic acid in the plant cells of the present invention, the samples were prepared at appropriate concentrations with each solvent, filtered and pre-treated on a 0.2 μm disk filter, and then subjected to high performance liquid chromatography (Agilent Technologies 1200 Series HPLC analysis was carried out using an evaporative light scattering detector (ELSD 3300, Alltech, USA) using HPLC, Agilent, USA. The eluent was tertiary distilled water, the column temperature was 25 ° C, the flow rate was 0.5 ml / min , The gas flow rate was fixed at 1.4 L / min and the gain was set at 1, and a calibration curve was prepared using a standard substance (Sigma, USA), and component analysis was performed. The results of the analysis are shown in [Figure 2c] and [Table 1].

(4) Water-soluble vitamin Vitamin C

For the analysis of the efficiency and components of the stabilized vitamin C in the plant cells of the present invention, samples were filtered with a 0.2 μm disk filter and purified by high performance liquid chromatography (Agilent Technologies 1200 Series HPLC, Agilent, The mobile phase was fixed with 0.05M KH ₂ PO ₄ : ACN = 60:40 (v / v) and the concentration of 1.0 ml / min was measured using a diode array detector (DAD; Flow rate. Calibration curves were prepared using L-ascorbic acid (> 99%, Samchun, KOR) as a reference material and component analysis was performed. The results of the analysis are shown in [Figure 2d] and [Table 1]. The content of the stabilized sample in the plant cell of the hydrophilic active substance using the high performance liquid chromatography (Agilent Technologies 1200 Series HPLC, Agilent, USA) is shown in Table 1 below and the filling efficiency was calculated by the following formula (1).

[Equation 1]

※

sample Active substance type content (%) Sealing efficiency  (%) Remarks Sample 1 Garlic Mountain 2.80 28.0 Sample 2 amino acid 0.03 37.5 Sample 3 Pete Mountain 0.54 10.8 Sample 4 Vitamin C 3.11 15.6

[Hydrophobic active material]

(5) Isoflavones Genistein

Analysis was carried out by means of high performance liquid chromatography (HPLC) in order to analyze the encapsulation efficiency and components of stabilized genistein in plant cells of the present invention. Samples were prepared at appropriate concentrations with each solvent, filtered through a 0.2 um distill filter, and pretreated and injected into the instrument. The instrument was analyzed in the UV 254 to 340 nm region using high performance liquid chromatography (Agilent Technologies 1200 Series HPLC, Agilent, USA) and analyzed using a C18 column (Zorbax XDB-C18 4.6 x 250 mm, Agilent, USA) Acetic acid solvent and acetonitrile (ACN) solvent. The solvent used for the analysis was high-performance liquid chromatography reagent, and component analysis was performed using genistein (> 99%, Sigma, USA) as a reference material. The analysis results are shown in [Figure 3a] and [Table 2].

(6) Flavonoids Epigallocatechin gallate (EGCG)

Analysis was performed using high performance liquid chromatography (HPLC) for the analysis of the encapsulation efficiency and composition of epigallocatechin gallate (EGCG) stabilized in plant cells of the present invention. Samples were prepared at appropriate concentrations with each solvent and filtered into a 0.2 μm disk filter, pre-treated and injected into the instrument. The instrument was analyzed in the UV 254 to 340 nm region using high performance liquid chromatography (Agilent Technologies 1200 Series HPLC, Agilent, USA) and analyzed using a C18 column (Zorbax XDB-C18 4.6 x 250 mm, Agilent, USA) Acetic acid solvent and acetonitrile (ACN) solvent. The solvent used for the analysis was a high-performance liquid chromatography reagent, and the component analysis was performed using epigallocatechin gallate (> 99%, Sigma, USA) as a reference material. The analysis results are shown in [Figure 3b] and [Table 2].

(7) fat soluble vitamin retinol

Analysis was carried out using high performance liquid chromatography (HPLC) for the purpose of analyzing the retention efficiency and components of stabilized retinol in plant cells of the present invention. Samples were prepared at appropriate concentrations with each solvent and filtered into a 0.2 μm disk filter, pre-treated and injected into the instrument. The instrument was analyzed in the UV 325 nm region using high performance liquid chromatography (Agilent Technologies 1200 Series HPLC, Agilent, USA) and analyzed on a C18 column (Zorbax XDB-C18 4.6 x 150 mm, Agilent, USA) using a 90% methanol (MeOH) aqueous solution. The solvent used for the analysis was high-performance liquid chromatography reagent and component analysis was performed using retinol (> 99%, Sigma, USA) as a reference material. The analysis results are shown in [Figure 3c] and [Table 2]. The content of the stabilized sample in the plant cell of the hydrophobic active material using the high-performance liquid chromatography (Agilent Technologies 1200 Series HPLC, Agilent, USA) is shown in Table 2 below and the filling efficiency was calculated according to the formula (1).

sample Active substance type content(%) Sealing efficiency (%) Remarks Sample 5 Genistein 0.35 7.0 Sample 6 Epigallocatechin gallate 1.21 24.2 Sample 7 Retinol 0.41 8.2

[Formulation Example 1 and Formulation Comparative Example 1]

The composition of the nutritional cream containing galactic acid stabilized in plant cells according to Example 1 was prepared as shown in Table 3 below.

number ingredient Formulation Example  One Formulation Comparative Example  One One Pro-type glycerin monostearate 2.0 2.0 2 Stearic acid 1.5 1.5 3 Cetearyl alcohol 2.2 2.2 4 Wax 1.0 1.0 5 Squalane 3.0 3.0 6 Hardened vegetable oil 1.0 1.0 7 Sorbitan stearate 0.6 0.6 8 Mineral oil 5.0 5.0 9 Polysorbate 60 1.5 1.5 10 Dimethicone 1.0 1.0 11 Trioctanoin 5.0 5.0 12 Betaine 3.0 3.0 13 Triethanolamine 1.0 1.0 14 glycerin 5.0 5.0 15 Sodium hyaruronate 3.0 3.0 16 Sample 1 2.0 - 17 Garlic Mountain - 2.0 18 Distilled water Balance Balance 19 Preservative, fragrance, pigment a very small amount a very small amount

First, the aqueous components of the raw materials 12 to 18 were completely dissolved by heating at 80 DEG C among the components constituted by the composition of Table 3, and then the raw materials 1 to 11 were heated to 80 DEG C and added to the solutions of the raw materials 12 to 18, (Homo Mixer Mark II, Primix, JPN) at 3000 rpm for 15 minutes. Thereafter, the raw material 19 was added, stirred for 5 minutes, and then cooled to room temperature.

[Formulation Example 2 and Formulation Comparative Example 2]

The composition of the nutritional cream containing the amino acid stabilized in the plant cell according to Example 2 was prepared as shown in Table 4 below.

number ingredient Formulation Example  One Formulation Comparative Example  One One Pro-type glycerin monostearate 2.0 2.0 2 Stearic acid 1.5 1.5 3 Cetearyl alcohol 2.2 2.2 4 Wax 1.0 1.0 5 Squalane 3.0 3.0 6 Hardened vegetable oil 1.0 1.0 7 Sorbitan stearate 0.6 0.6 8 Mineral oil 5.0 5.0 9 Polysorbate 60 1.5 1.5 10 Dimethicone 1.0 1.0 11 Trioctanoin 5.0 5.0 12 Betaine 3.0 3.0 13 Triethanolamine 1.0 1.0 14 glycerin 5.0 5.0 15 Sodium hyaruronate 3.0 3.0 16 Sample 2 2.0 - 17 Amino acid standard - 2.0 18 Distilled water Balance Balance 19 Preservative, fragrance, pigment a very small amount a very small amount

First, the aqueous components of raw materials 12 to 18 were completely dissolved by heating at 80 DEG C among the components constituted by the composition of Table 4, and then the raw materials 1 to 11 were heated to 80 DEG C and added to the solutions of raw materials 12 to 18, (Homo Mixer Mark II, Primix, JPN) at 3000 rpm for 15 minutes. Thereafter, the raw material 19 was added, stirred for 5 minutes, and then cooled to room temperature.

[Formulation Example 3 and Formulation Comparative Example 3]

The composition of the nutritional cream containing the phytic acid stabilized in the plant cell according to Example 3 was constructed as shown in Table 5 below.

number ingredient Formulation Example  One Formulation Comparative Example  One One Pro-type glycerin monostearate 2.0 2.0 2 Stearic acid 1.5 1.5 3 Cetearyl alcohol 2.2 2.2 4 Wax 1.0 1.0 5 Squalane 3.0 3.0 6 Hardened vegetable oil 1.0 1.0 7 Sorbitan stearate 0.6 0.6 8 Mineral oil 5.0 5.0 9 Polysorbate 60 1.5 1.5 10 Dimethicone 1.0 1.0 11 Trioctanoin 5.0 5.0 12 Betaine 3.0 3.0 13 Triethanolamine 1.0 1.0 14 glycerin 5.0 5.0 15 Sodium hyaruronate 3.0 3.0 16 Sample 3 2.0 - 17 Pete Mountain - 2.0 18 Distilled water Balance Balance 19 Preservative, fragrance, pigment a very small amount a very small amount

First, the aqueous components of the raw materials 12 to 18 were completely dissolved by heating at 80 ° C among the components constituted by the composition of Table 5, and then the raw materials 1 to 11 were heated to 80 ° C and added to the solutions of the raw materials 12 to 18, (Homo Mixer Mark II, Primix, JPN) at 3000 rpm for 15 minutes. Thereafter, the raw material 19 was added, stirred for 5 minutes, and then cooled to room temperature.

[Formulation Example 4 and Formulation Comparative Example 4]

The composition of the nutritional cream containing vitamin C stabilized in plant cells according to Example 4 was prepared as shown in Table 6 below.

number ingredient Formulation Example  One Formulation Comparative Example  One One Pro-type glycerin monostearate 2.0 2.0 2 Stearic acid 1.5 1.5 3 Cetearyl alcohol 2.2 2.2 4 Wax 1.0 1.0 5 Squalane 3.0 3.0 6 Hardened vegetable oil 1.0 1.0 7 Sorbitan stearate 0.6 0.6 8 Mineral oil 5.0 5.0 9 Polysorbate 60 1.5 1.5 10 Dimethicone 1.0 1.0 11 Trioctanoin 5.0 5.0 12 Betaine 3.0 3.0 13 Triethanolamine 1.0 1.0 14 glycerin 5.0 5.0 15 Sodium hyaruronate 3.0 3.0 16 Sample 4 2.0 - 17 Vitamin C - 2.0 18 Distilled water Balance Balance 19 Preservative, fragrance, pigment a very small amount a very small amount

First, the aqueous components of raw materials 12 to 18 were completely dissolved by heating at 80 DEG C among the components constituted by the composition of Table 6, and then the raw materials 1 to 11 were heated to 80 DEG C to put them into the solutions of raw materials 12 to 18, (Homo Mixer Mark II, Primix, JPN) at 3000 rpm for 15 minutes. Thereafter, the raw material 19 was added, stirred for 5 minutes, and then cooled to room temperature.

[Formulation Example 5 and Formulation Comparative Example 5]

The composition of the nutritional cream containing the stabilized genistein in the plant cell according to Example 5 was prepared as shown in Table 7 below.

number ingredient Formulation Example  One Formulation Comparative Example  One One Pro-type glycerin monostearate 2.0 2.0 2 Stearic acid 1.5 1.5 3 Cetearyl alcohol 2.2 2.2 4 Wax 1.0 1.0 5 Squalane 3.0 3.0 6 Hardened vegetable oil 1.0 1.0 7 Sorbitan stearate 0.6 0.6 8 Mineral oil 5.0 5.0 9 Polysorbate 60 1.5 1.5 10 Dimethicone 1.0 1.0 11 Trioctanoin 5.0 5.0 12 Betaine 3.0 3.0 13 Triethanolamine 1.0 1.0 14 glycerin 5.0 5.0 15 Sodium hyaruronate 3.0 3.0 16 Sample 5 2.0 - 17 Genistein - 2.0 18 Distilled water Balance Balance 19 Preservative, fragrance, pigment a very small amount a very small amount

The aqueous components of the raw materials 12 to 16 and 18 were completely dissolved by heating to 80 ° C among components constituted by the composition of Table 7, and then the raw materials 1 to 11 and 17 were heated to 80 ° C to prepare the raw materials 12 to 16 and 18 (Homo Mixer Mark II, Primix, JPN) and emulsified at 3000 rpm for 15 minutes. Thereafter, the raw material 19 was added, stirred for 5 minutes, and then cooled to room temperature.

[Formulation Example 6 and Formulation Comparative Example 6]

The composition of the nutritional cream containing epigallocatechin gallate (EGCG) stabilized in the plant cell according to Example 6 was prepared as shown in Table 8 below.

number ingredient Formulation Example  One Formulation Comparative Example  One One Pro-type glycerin monostearate 2.0 2.0 2 Stearic acid 1.5 1.5 3 Cetearyl alcohol 2.2 2.2 4 Wax 1.0 1.0 5 Squalane 3.0 3.0 6 Hardened vegetable oil 1.0 1.0 7 Sorbitan stearate 0.6 0.6 8 Mineral oil 5.0 5.0 9 Polysorbate 60 1.5 1.5 10 Dimethicone 1.0 1.0 11 Trioctanoin 5.0 5.0 12 Betaine 3.0 3.0 13 Triethanolamine 1.0 1.0 14 glycerin 5.0 5.0 15 Sodium hyaruronate 3.0 3.0 16 Sample 6 2.0 - 17 EGCG - 2.0 18 Distilled water Balance Balance 19 Preservative, fragrance, pigment a very small amount a very small amount

First, the aqueous components of the raw materials 12 to 18 were completely dissolved by heating to 80 DEG C among the components constituted by the composition of Table 8, and then the raw materials 1 to 11 were heated to 80 DEG C and added to the solutions of the raw materials 12 to 18, (Homo Mixer Mark II, Primix, JPN) at 3000 rpm for 15 minutes. Thereafter, the raw material 19 was added, stirred for 5 minutes, and then cooled to room temperature.

[Formulation Example 7 and Formulation Comparative Example 7]

The composition of the nutritional cream containing retinol stabilized in the plant cell according to Example 7 was prepared as shown in Table 9 below.

number ingredient Formulation Example  One Formulation Comparative Example  One One Pro-type glycerin monostearate 2.0 2.0 2 Stearic acid 1.5 1.5 3 Cetearyl alcohol 2.2 2.2 4 Wax 1.0 1.0 5 Squalane 3.0 3.0 6 Hardened vegetable oil 1.0 1.0 7 Sorbitan stearate 0.6 0.6 8 Mineral oil 5.0 5.0 9 Polysorbate 60 1.5 1.5 10 Dimethicone 1.0 1.0 11 Trioctanoin 5.0 5.0 12 Betaine 3.0 3.0 13 Triethanolamine 1.0 1.0 14 glycerin 5.0 5.0 15 Sodium hyaruronate 3.0 3.0 16 Sample 7 2.0 - 17 Retinol - 2.0 18 Distilled water Balance Balance 19 Preservative, fragrance, pigment a very small amount a very small amount

The aqueous components of the raw materials 12 to 16 and 18 were completely dissolved by heating to 80 ° C among the components constituted by the composition of Table 9, and then the raw materials 1 to 11 and 17 were heated to 80 ° C to prepare the raw materials 12 to 16 and 18 (Homo Mixer Mark II, Primix, JPN) and emulsified at 3000 rpm for 15 minutes. Thereafter, the raw material 19 was added, stirred for 5 minutes, and then cooled to room temperature.

[Test Example 2] Formulation stability experiment

For the stability test in the formulation of the active substance stabilized in the plant cell of the present invention, each of the formulation examples and the formulation comparative examples prepared as described above were sampled under the harsh condition at 90 캜 under the harsh conditions and analyzed according to the time course after high performance liquid chromatography analysis And showed stability by decreasing the arbitrary unit (Arbitary unit. AU) of the active ingredient (see Figs. 4A to 4D and Figs. 5A to 5C).

[Test Example 3] Observation of microscopic cells by staining method

In order to observe cells of the active substance stabilized in plant cells of the present invention, microscopic observation was carried out as follows. Microscopic observations were performed using staining. Sample 1 was diluted with 100 times of third distilled water, vortexed for 30 seconds using a laboratory stirrer, and then stained with methylene blue (Sigma, USA). After staining, the cells were observed at a magnification of 100x using an optical microscope (Eclipse E600, Nikon, JPN), and images were captured (FIG. 6).

Examples of formulations of the present invention are a soft lotion, a convergent lotion, a nutritional lotion, a massage cream, an essence and a pack, but the formulation of the cosmetic composition of the present invention should not be construed as being limited thereto. Lt; / RTI &gt; is possible.

Table 10 shows an example of a softening longevity formulation [Formulation Example 1].

number ingredient content(%) One glycerin 5.00 2 1,3-butylene glycol 3.00 3 PEG 1500 1.00 4 Allantoin 0.10 5 DL-Panthenol 0.30 6 EDTA-2Na 0.02 7 Benzophenone-9 0.04 8 ethanol 10.00 9 Octidodeces-16 0.20 10 Polysorbate 20 0.20 11 Active substance stabilized in plant cells 5.0 12 Preservative, fragrance, pigment a very small amount 13 Distilled water Balance

Table 11 shows a formulation example of convergent lotion [Formulation Example 2].

number ingredient content(%) One glycerin 2.00 2 1,3-butylene glycol 2.00 3 Allantoin 0.10 4 DL-Panthenol 0.30 5 EDTA-2Na 0.02 6 Benzophenone-9 0.04 7 ethanol 15.00 8 Polysorbate 20 0.20 9 Active substance stabilized in plant cells 3.0 10 Citric acid a very small amount 11 Preservative, fragrance, pigment a very small amount 12 Distilled water Balance

Table 12 shows a formulation example of a nutritional lotion [Formulation Example 3].

number ingredient content(%) One Cetearyl alcohol 1.00 2 Glyceryl stearate 0.50 3 Polysorbate 60 1.00 4 Sorbitan sesquioleate 0.30 5 Cetyl octanoate 6.00 6 Squalane 4.00 7 Shapower Oil 4.00 8 Butylene glycol 4.00 9 glycerin 4.00 10 Carbomer 0.10 11 Triethanolamine 0.10 12 Active substance stabilized in plant cells 1.00 13 Preservative, fragrance, pigment a very small amount 14 Distilled water Balance

Table 13 shows a formulation example of essence [Formulation Example 4].

number ingredient content(%) One glycerin 10.00 2 Betaine 5.00 3 PEG 1500 2.00 4 Allantoin 0.10 5 DL-Panthenol 0.30 6 EDTA-2Na 0.02 7 Benzophenone-9 0.04 8 Hydroxyethylcellulose 0.10 9 Carboxyvinyl polymer 0.20 10 Triethanolamine 0.18 11 Octyldodecanol 0.30 12 Octyldodec-16 0.40 13 ethanol 6.00 14 Active substance stabilized in plant cells 5.00 15 Preservative, fragrance, pigment a very small amount 16 Distilled water Balance

Table 14 shows a formulation example of a pack [Formulation Example 5].

number ingredient content(%) One Polyvinyl alcohol 15.00 2 Cellulose sword 0.15 3 glycerin 3.00 4 PEG 1500 2.00 5 Sheik Destrin 0.15 6 DL-Panthenol 0.30 7 Allantoin 0.10 8 Monoammonium glycyrrhizin acid 0.20 9 Nicotinamide 0.40 10 ethanol 5.00 11 PEG 40 hardened castor oil 0.30 12 Active substance stabilized in plant cells 1.00 13 Preservative, fragrance, pigment a very small amount 14 Distilled water Balance

Claims (8)

A cosmetic composition comprising plant cells in which an active substance is introduced into a cell wall.
The method according to claim 1,
Wherein the active material is at least one selected from the group consisting of a hydrophilic substance and a hydrophobic substance.
The method according to claim 1,
The active substance may be selected from the group consisting of gallic acid, amino acid, Phytic acid, L-Ascorbic acid, arbutin, fructose diphosphate, trisodium fructose, 1,6- , Niacinamide, genistein, flavonoids, epigallocatechin gallate (EGCG), retinol, hesperidin, oleanolic acid, polymethylmethacrylate, thymol trimethoxycinnamate, Wherein the cosmetic composition is at least one selected from the group consisting of red ginseng saponin, hydrolized ginseng saponin, and ascorbyl glucoside.
delete A method for producing a plant cell comprising an active substance through the following manufacturing steps.
(a) introducing an active substance into plant cells under conditions of 1-100 MPa;
(b) causing a dehydration reaction using reverse osmosis from the plant cell into which the active substance is introduced; And
(c) obtaining a plant cell into which the active substance has been introduced.
6. The method of claim 5,
Wherein the active material is at least one selected from the group consisting of hydrophilic and hydrophobic.
6. The method of claim 5,
The active substance may be selected from the group consisting of gallic acid, amino acid, Phytic acid, L-Ascorbic acid, arbutin, fructose diphosphate, trisodium fructose, 1,6- , Niacinamide, genistein, flavonoids, epigallocatechin gallate (EGCG), retinol, hesperidin, oleanolic acid, polymethylmethacrylate, thymol trimethoxycinnamate, Wherein the plant cell is at least one selected from the group consisting of red ginseng saponin, hydrolized ginseng saponin, and ascorbyl glucoside. delete

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