KR102118751B1 - Complex having dedifferentiated plant protoplast inserted active material, preparation method thereof, and cosmetic composition comprising the same - Google Patents

Abstract

The present invention relates to a structure in which an active substance is introduced into a dedifferentiated plant protoplast, a method for manufacturing the same, and a cosmetic composition comprising the same, more specifically, a structure in which the active substance is introduced into a dedifferentiated plant protoplast, It relates to a cosmetic composition comprising a manufacturing method and this as an active ingredient.
The structure maintains the stability of the active ingredient of the existing plant cells and maintains the stability of the active substance introduced into the cell, and the cosmetic composition containing the same also improves the efficacy by the active substance to secure various efficacy Can be.

Description

Structure having an active substance introduced into a dedifferentiated plant protoplast, a manufacturing method thereof, and a cosmetic composition comprising the same{Complex having dedifferentiated plant protoplast inserted active material, preparation method thereof, and cosmetic composition comprising the same}

The present invention relates to a structure in which the active substance is introduced into a dedifferentiated plant protoplast to increase the stability and efficacy of the active substance, a manufacturing method thereof, and a cosmetic composition comprising the same as an active ingredient.

Numerous different phyto-chemicals are inherent in natural plants, and there are also numerous types of terpenoids, flavonoids, flavonols, polyphenols, amino acids, lignans, alkaloids, vitamins, and catechin compounds. consist of.

Accordingly, cosmetic compositions using only plant cells or extracting specific components have been actively proposed.

For example, International Patent WO2013-180526 mentions that Edelweiss extract promotes skin regeneration and can be used as a cosmetic, while International Patent WO2012-102456 mentions that the silkworm extract is effective in improving wrinkles and can be used as a variety of cosmetics.

In addition, the international patent WO2009-139581 is derived from the forming layer or the typical forming layer of interest, and cell lines having the following properties have antioxidant and anti-inflammatory effects, and thus can be used as a cosmetic composition as it prevents aging and suppresses melanin production. Japanese Patent Publication No. 2012-102136 discloses a cosmetic composition comprising a freeze-dried product of dedifferentiated plant cells of a inflammatory plant such as Criste Marine for skin discoloration and lightening.

On the other hand, effective active materials such as retinol, etc. have a useful function in physiological properties in the human body, but are not free from stability, and various methods have been proposed to increase their stability.

For example, many attempts have been made, such as applying liposomes using phospholipids or surfactants to active substances, stabilized cerosomes using ceramides, liquid crystalsomes stabilized with liquid crystal structures, and cubic cubosomes using monoglycerides.

In addition, research to apply plant cells to cosmetic compositions is also underway.

International patent WO2012-173458 proposes a cosmetic composition containing plant cells in which active substances such as gallic acid and amino acids are stabilized, wherein the plant cells are cell walls and draw the active substances into the cell walls. It was suggested that the stability of the active material may be increased.

The cell wall of plant cells contains excess cellulose, which makes it difficult for other substances to penetrate, so it is necessary to remove the active substance, and when removing both the cell wall and the cell membrane, the stability of the introduced active substance is rather reduced. Can be.

International patent WO2013-180526 International patent WO2012-102456 International patent WO2009-139581 International patent WO2012-173458

Accordingly, the present inventors conducted various studies to stably encapsulate the active substance in a plant cell with a high intake rate. As a result, when removing the cell wall of the plant cell and using a protoplast consisting of a cell membrane and a cell site, the living body The present invention was completed by confirming that it can be used as a cosmetic composition by increasing the penetration rate of the active material as well as the permeability to it.

Accordingly, an object of the present invention is to provide a structure capable of maximizing the efficacy of the active material, and a method for manufacturing the active material is drawn at a high withdrawal rate.

In addition, another object of the present invention is to provide a cosmetic composition comprising the structure as an active ingredient.

In order to achieve the above object, the present invention provides a structure in which an active substance is introduced into a dedifferentiated plant protoplast.

In addition, the present invention

Obtaining cells of the plant from the leaves, stems, roots, flowers, fruits and seeds of the plant;

Dedifferentiating the obtained plant cells using auxin to obtain dedifferentiated plant cells;

Culturing the obtained dedifferentiated plant cells in large quantities;

Removing the cell wall of the mass cultured dedifferentiated plant cell using an enzymatic reaction to obtain a protoplast;

Introducing the active material into the obtained dedifferentiated plant prototype by a pressure osmosis process; And

Comprising post-processing

It provides a method for producing a structure in which an active substance is introduced into a dedifferentiated plant protoplast.

In addition, the present invention provides a cosmetic composition comprising a structure in which an active substance is introduced into the dedifferentiated plant protoplast as an active ingredient.

The structure according to the present invention maintains the stability of the active ingredient of the existing plant cells and maintains the stability of the active substance introduced into the cell, and the efficacy by the active substance is also improved, so that the active ingredient composition of the cosmetic is preferable. Can be used

Figure 1 is a flow chart showing the manufacturing steps of the structure in which the active material is introduced into the dedifferentiated plant protoplast according to the present invention
Figure 2 (a) is a standard sample of cyclohexanediol bisethyl hexanoate, (b) GC spectrum of the structure in which cyclohexanediol bisethylhexanoate is introduced into the prototype of Example 1
Figure 3 (a) is a standard sample bisretin amido methyl pentane, (b) GC spectrum of the structure in which bisretin amido methyl pentane is introduced into the propoplast of Example 2
Figure 4 (a) is a standard sample beta-rapachon, (b) GC spectrum of the structure in which beta-rapachon is introduced into the propoplast of Example 3
Figure 5 (a) is a standard sample growth factor complex peptide, (b) MALDI-TOF mass spectrometry graph of the structure in which the growth factor complex peptide is introduced in the propoplast of Example 4
Figure 6 is a graph showing the inhibitory effect of the production of melanin according to the concentration of the structure of Example 1, cyclohexanediol bisethylhexanoate and alpha-bisabolol
7 is a graph showing collagen synthesis ability of the construct of Example 2, bisretinamido methylpentane, retinol, and adenosine.
8 is a graph showing collagen synthesis ability of the construct of Example 3, beta rapazine, retinol and adenosine.
9 is a graph showing the collagen synthesis ability of the construct, growth factor complex peptide, retinol and adenosine of Example 4
10 is an image showing epidermal regeneration before and after treatment of the structure of Example 4 for human skin tissue
11 is an image showing the degree of glycosaminoglycan increase before and after treatment of the structure of Example 4 for human skin tissue
Figure 12 is an image showing the increase in type I collagen before and after treatment of the structure of Example 4 for human skin tissue

Hereinafter, the present invention will be described in more detail.

The present invention provides a structure in which an active substance is introduced into a dedifferentiated plant protoplast.

Protoplast is a protoplast in which a cell wall of a cell is removed and a cell membrane is present, and various active substances that can be used as a cosmetic composition inside the cell membrane can be introduced. The cell wall contains an excess of cellulose, and as a result, the penetration of other materials is not easy, so removal is necessary for the introduction of the active material. When removing both the cell wall and the cell membrane, the stability of the introduced active material may be rather reduced. Therefore, a protoplast in which a cell membrane is present is preferred.

The active material may be a hydrophilic material or a hydrophobic material, and is not particularly limited in the present invention. Representatively, the active substances include organic acids, vitamins, arbutins, adenosine, niacinamide, polyphenols, flavonoids, retinol, cyclohexanediol bisethylhexanoate, bisretinamido methylpentane, beta rapazine, growth factor, growth One factor selected from the group consisting of factor complex peptides and combinations thereof is possible.

Such an active substance is used as an active ingredient of a cosmetic composition itself, but the stability was low, and thus the activity could not be sufficiently expressed. However, the active stability was increased by being introduced into the protoplast, and the biocompatibility was also greatly improved due to the protoplast. It has the advantage of being.

1 is a flow chart showing the steps of manufacturing a structure in which an active substance is introduced into a dedifferentiated plant protoplast according to the present invention, which will be described in detail at each step with reference to FIG. 1 below:

Step (a): Plant cell obtaining step

In step (a), plant cells are obtained from the leaves, stems, roots, flowers, fruits and seeds of the plants.

Plant cell yield is not particularly limited in the present invention, and a known method can be used. For example, in the embodiment of the present invention, after sterilization, the cells were immersed in an aqueous solution of sodium chlorate to which a surfactant was added, and then washed to obtain plant cells.

Plants that can be used are not particularly limited in the present invention, and all plants are possible. Representatively, curry plant ( Helichrysum) italicum ), comb ( Commiphora wightii , Commiphora myrrha ), Opuntia Ficus indica ), Peony ( Paeonia) lactiflora ), Petrification ( Adenium obesum ), Nymphaea coerulea ), Eucalyptus punctata ), Ginkgo ( Ginkgo) biloba), lilies (Lilium candidum), the olive tree (Olea europaea), Papyrus (Cyperus papyrus ), Nelumbo nucifera ), Redwood ( Sequoia sempervirens), point ka rose (Rosa gallica officinalis ), Coffea arabica ), Plumeria obtusa), gardenia (Gardenia jasminoides ), Bougainvillea spectabilis ), Jasminum sambac ), Rosa centifolia ( Rosa centifolia ), peppermint ( Mentha piperita ), Rosa Damasquena ( Rosa damascena ), Iris pallida), grapevine (Vitis vinifera ), white rose ( Rosa alba ), Cananga odorata ), almond tree ( Prunus amygdalus dulcis ), apple tree ( Malus domestica ), apricot tree ( Prunus armeniaca ), ginseng ( Panax ginseng ), blackberry ( Rubus fruticosus), gold film (Eschscholtzia californica), Centella asiatica (Centella asiatica ), Prunus cerasus ), Hibiscus , Hawaii rosa sinensis ), Juniperus communis ), cotton ( Gossypium arboreum ), date palm ( Phoenix dactylifera ), ginger ( Zingiber officinale ), Hibiscus syriacus ), Pueraria tuberosa ), pomegranate ( Punica granatum), boombox Costa lithium (Bombax costatum ), saffron ( Crocus sativus ), Salvia officinalis , Nymphaea alba ) and combinations thereof, one type selected from the group consisting of curry plant ( Helichrysum) italicum ), comb ( Commiphora wightii , Commiphora myrrha ), Opuntia Ficus indica ).

Step (b): Dedifferentiation step

In step (b), the obtained plant cells are dedifferentiated using auxin to obtain dedifferentiated plant cells.

Auxin, used for dedifferentiation, is one of the plant growth regulators. It promotes cell elongation at low concentrations but inhibits growth at high concentrations. Accordingly, as auxin, one selected from the group consisting of alpha-naphtalene acetic acid, 2,4-dichlorophenoxy acetic acid, indole-3-acetic acid, and combinations thereof It is possible, and it is possible to induce dedifferentiation of the plant cells by culturing the plant cells in a medium containing them at a concentration of 1 to 5 mg/L.

The medium is not particularly limited in the present invention, and any medium used for plant cell culture can be used.

Step (c): Dedifferentiation Plant cell culture stage

In step (c), the dedifferentiated plant cells obtained in step (b) are cultured in large quantities.

Mass culture of dedifferentiated plant cells is performed in a variety of media, wherein medium as known as MS medium, B5 medium, WHITE medium, N6 medium, SH medium, Anderson medium can be used, preferably MS Use medium.

In addition, the culture conditions and period are not specifically mentioned in the present invention, and can be performed under known conditions.

Step (d): cell wall removal step through an enzymatic reaction

In step (d), the cell wall of dedifferentiated plant cells cultured in large quantities is removed using an enzymatic reaction to obtain a protoplast in which only cell membranes and organelles remain.

The enzyme consists of cellulase (EC 3.2.1.4), pectinase (EC 3.2.1.15), xylanase (EC 3.2.1.8), chitinase (EC 3.2.1.14), hemicellulase and combinations thereof. One selected from the group is possible.

These enzymes can be used in various concentrations of 0.01 to 10% by weight, preferably 0.1 to 5% by weight of cellulase, 0.01 to 2.5% by weight of pectinase, 0.1 to 5% by weight of hemicellulase, and more preferably A multicomponent enzyme mixture of 1% by weight of cellulase, 2% by weight of hemicellulase and 0.5% by weight of pectinase is used.

The enzymatic reaction is carried out by reacting at a rate of 10 to 50 rpm for 15 to 24 hours in a temperature range of 4° C. to 40° C. (more preferably 10° C. to 25° C.). Only the cell wall is removed, and the cell membrane can be stably preserved to obtain a protoplast.

Step (e): step of introducing the active substance by the pressure osmosis process

In step (e), the active material is introduced into the obtained dedifferentiated plant protoplast by a pressure osmosis process.

Specifically, when the dedifferentiated plant protoplast is dissolved in lower alcohols (C1 to C4 alcohols) and mixed with the active material and water and sodium chloride is added, the active material is introduced into the protoplast by osmosis. Incoming.

This process proceeds as a pressure osmosis process, specifically, performed at 0.01 to 10 MPa, preferably 0.05 to 1 MPa, and most preferably 0.05 to 0.5 MPa, wherein the concentration is 1% or more by addition of sodium chloride, preferably It should be 1-50% as follows. If the pressure and concentration are less than the above range, the active material is not effectively drawn in. Conversely, if it exceeds the above range, protoplast may be destroyed.

The inlet is performed so that the concentration of the active substance can be sufficiently exerted, which may vary depending on the active substance, but has an inlet ratio of 0.001 to 20% (based on weight percent). For example, in the case of cyclohexanediol bisethylhexanoate, the active material of Example 1, excellent activity at a concentration of 0.001 to 15% by weight, preferably 0.01 to 10% by weight, and more preferably 0.1 to 10% by weight Have

In addition, the active substance of Example 2, bisretin amidomethylpentane, has an excellent activity at a concentration of 0.005 to 5.0% by weight, preferably 0.01 to 2% by weight, and of the active material of Example 3, betarapachon It has excellent activity at a concentration of 0.005 to 10.0% by weight, preferably 0.01 to 5.0% by weight. In the case of the growth factor complex peptide of Example 4, the concentration may be 10 to 5000 ppm, preferably 50 to 5000 ppm, and more preferably 100 to 500 ppm.

Additionally, the inlet may further perform a dehydration reaction to increase the inlet rate.

Step (f): Post-treatment step

In step (f), a structure in which the active material is introduced into the protoplast through post-treatment is obtained through post-treatment.

This post-treatment is a conventional post-treatment process, and removes unreacted substances (active substances that are not introduced) and salts through centrifugation and washing.

The structure in which the active substance is introduced into the protoplast obtained through the above steps maintains the stability of the active ingredient existing in the plant cell, and additionally, the stability of the active substance introduced into the cell is high, and accordingly, Efficacy is also improved, and can be preferably used as an active ingredient composition of cosmetics.

Such a structure comprises 0.001 to 99.0% by weight, preferably 0.01 to 10.0% by weight, more preferably 0.1 to 3% by weight relative to the total weight of the total cosmetic composition.

In addition, the cosmetic composition can be prepared in any formulation as is known, lotion, nutrient lotion, nourishing cream, massage cream, essence, pack, paste, gel, cream, lotion, powder, soap, oil, foundation, wax or It can be formulated in the form of a spray.

In addition, in the cosmetic composition of each formulation, other components other than the above-mentioned cells may be arbitrarily selected and formulated according to other cosmetic formulations or purpose of use. In addition, the composition of each formulation may contain various bases and additives necessary and appropriate for the formulation of the formulation, and non-ionic surfactants, silicone polymers, extender pigments, fragrances, preservatives, within the scope of not impairing its effect, Fungicides, oxidation stabilizers, organic solvents, ionic or nonionic thickeners, softening agents, antioxidants, free radical disruptors, opacifying agents, stabilizers, emollients, silicones, α-hydroxy acids, antifoaming agents, moisturizing agents, It is prepared by including known compounds such as vitamins, insect repellents, fragrances, preservatives, surfactants, anti-inflammatory agents, substance P antagonists, fillers, polymers, propellants, basicizing or acidifying agents, or coloring agents.

When the formulation of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, trakant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide, etc. may be used as a carrier component. Can be.

When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder may be used as a carrier component. In particular, in the case of a spray, additionally chlorofluorohydrocarbon, propane /Propellant such as butane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, solubilizer or emulsifier is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 Fatty acid esters of ,3-butyl glycol oil, glycerol aliphatic esters, polyethylene glycol or sorbitan.

When the formulation of the present invention is a suspension, liquid diluents such as water, ethanol or propylene glycol as carrier components, ethoxylated isostearyl alcohol, suspensions such as polyoxyethylene sorbitol esters and polyoxyethylene sorbitan esters, microcrystals Sex cellulose, aluminum metahydroxide, bentonite, agar or trakant, etc. can be used.

When the formulation of the present invention is a surfactant-containing cleansing agent, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide as a carrier component Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters and the like can be used.

Hereinafter, preferred embodiments are provided to help understanding of the present invention. However, the following examples are only provided to more easily understand the present invention, and the contents of the present invention are not limited by the examples.

Manufacturing example One: Dedifferentiation Plant protoplast cell production

(One) Dedifferentiation Obtaining plant cells

Curry plant ( Helichrysum) italicum ), comb ( Commiphora wightii , Commiphora myrrha ), Opuntia Ficus indica ), cut tissue sections (at least 3 cm) from stems, leaves, etc. In this operation step, all work was performed on a sterile workbench under sterile conditions.

To sterilize plant materials, tissues are immersed in 70% ethanol (Ethanol, Sigma, USA) for 60 seconds and 30% hydrogen peroxide (Hydrogen peroxide, LG Chemical, Korea) for 15 minutes to remove the solvent, after which these tissues are sterile H Washed 3 to 5 times with ₂ O, immersed in sodium hydrochlorite (Sigma, USA) with a few drops of Tween 20 for 15 minutes, and washed 3 to 5 times with sterile H ₂ O.

For tissue culture, these tissue fragments are placed in a sterile Petri dish (125 mm), cut (2-3 mm), and the bleached portion is carefully removed, and the samples thus obtained are finely incised to form a solid culture medium (Table below). I smeared it on 1) and half buried it.

Raw material Content (mg/L) KNO ₃ 2,500 (NH ₄ ) ₂ SO ₄ 134 CaCl ₂ · H ₂ O 120 NaH ₂ PO ₄ · H ₂ O 150 MgSO ₄ · H ₂ O 200 MnSO ₄ · H ₂ O 15.0 ZnSO ₄ · H ₂ O 5.0 H ₃ BO ₃ 4.000                        Kl 0.800 Na ₂ MoO ₄ · H ₂ O 0.250 CuSO ₄ · H ₂ O 0.025 CoCl ₂ · H ₂ O 0.025 FeSO ₄ · H ₂ O 32.80 Myo-inositol 200 Nicotinic acid 2.00 L-ascorbic acid 30 Citric acid 50 Biotin ((+)-Biotin) 0.01 Pyridoxine chloride (Pyridoxine-HCl) 2.00 Thiamine-HCl 10.0 Sucrose 20,000 Α-Naphtalene acetic acid 2.00 2,4-dichlorophenoxy acetic acid 0.50 Kinetin 0.50 Agar 9,000 Purified water Balance

(2) Dedifferentiation Plant cell planting ( Replanting )

After 2-3 cell clusters (1-2 cm) were collected with a spatula (Spatula), they were plated and dispersed in fresh medium. All of these procedures were performed on a sterile work surface under sterile conditions.

(3) in liquid culture medium Dedifferentiation Plant cell proliferation

The dedifferentiated plant cells were transferred to the liquid culture medium shown in Table 2, and then cultured in a rotary stirrer at 50°C to 150 rpm under dark conditions at 25°C, and their passage culture cycle was fixed every 10 days.

Raw material Content (mg/L) NH ₄ NO ₃ 400 Ca(NO ₃ ) ₂ 500 CaCl ₂ · H ₂ O 100 KH ₂ PO ₄ 200 MgSO ₄ · H ₂ O 150 MnSO ₄ · H ₂ O 20 ZnSO ₄ · H ₂ O 5.00 H ₃ BO ₃ 5.00 K ₂ SO ₄ 1,000 Na ₂ MoO ₄ · H ₂ O 0.25 CuSO ₄ · H ₂ O 0.25 FeSO ₄ · H ₂ O 32.8 Myo-inositol 200 Nicotinic acid 2.00 L-ascorbic acid 30 Citric acid 50 Biotin ((+)-Biotin) 0.01 Pyridoxine chloride (Pyridoxine-HCl) 2.00 Thiamine-HCl 10.0 Sucrose 30,000 Alpha-naphthalene acetic acid 2.00 2,4-dichlorophenoxyacetic acid 0.50 Kinetin 0.50 Purified water Balance

(4) Dedifferentiation Removed cell walls of plant cells Protoplast purchase

A multi-component enzyme mixture (cellulase 1%, hemicellulase 2% and pectinase 0.5%) was added to the obtained dedifferentiated plant cell-proliferated liquid culture medium and reacted at a rate of 50 rpm for 20 hours at a temperature range of 25°C. The cell wall was removed.

Subsequently, the protoplasts in the culture were obtained by centrifugation at 200xg for 15 minutes, and then further purified by centrifugation at 5,000xg for 15 minutes.

Example 1: Dedifferentiation  plant Protoplast Within Cyclohexanediol Bisethylhexanoate  Preparation of incoming structures

Cyclohexane diol bisethyl hexanoate (Cyclohexandiol Bis-Ethylhexanoate, RNS, Korea) was stirred in ethanol for 30 minutes to prepare a solution having a concentration of 10% by weight.

20% by weight of the obtained cyclohexanediol bisethylhexanoate solution, 20% by weight of the protoplast prepared in Preparation Example 1, and 60% by weight of distilled water, and a paddle mixer (PL-S10, Poonglim, Korea) It was stirred for 1 hour under the conditions of 500rpm and 25℃.

Subsequently, 2% by weight of sodium chloride (NaCl, Sigma, USA) was added to the mixed solution of the active material solution, protoplast and distilled water, and reverse osmosis was performed at 0.5 MPa and 25° C. for 1 hour using a high-pressure reactor (Miniclave, Buchi AG, Switzerland). Permeation, dehydration and induction reaction of cyclohexanediol bisethylhexanoate were induced.

Thereafter, centrifugation was performed at 5,000×g for 20 minutes using a centrifuge (Supra 22K, Hanil, Korea) to obtain a structure in which cyclohexanediol bisethylhexanoate was introduced into the protoplast.

In order to preserve the thus obtained structure, 80% by weight of glycerin was added to 20% by weight of the structure to obtain a cell composition.

Example 2: Dedifferentiation  plant Protoplast  Within Bisretin amido Methyl pentane  Preparation of incoming structures

Performed in the same manner as in Example 1, using bisretinamido methylpentane (DermaLab, Korea) instead of cyclohexanediol bisethylhexanoate as the active material, bisretinami in the protoplast Also obtained was a structure in which methylpentane was introduced (structure/glycerin, 20/80% by weight).

Example 3: Dedifferentiation  plant Protoplast  Within Beta La Pachon Preparation of incoming structures

The same structure as in Example 1, using beta-rapachon (β-Lapachone, Sigma, USA) instead of cyclohexanediol bisethylhexanoate as an active material, a structure in which beta-rapachon was introduced into the protoplast Obtained (Structure/Glycerin, 20/80% by weight).

Example 4: Dedifferentiation plant Protoplast Growth factor complex peptide ( growth factor mimic Preparation of peptide-incorporated constructs

Performed in the same manner as in Example 1, using a growth factor complex peptide composition of the following composition instead of cyclohexanediol bisethylhexanoate as an active material, to obtain a structure in which a growth factor complex peptide was introduced into the protoplast (structure /Glycerin, 20/80% by weight).

Growth factor complex peptide composition is oligopeptide-34 (Oligopeptide-34, Caregen, Korea), oligopeptide-24 (Oligopeptide-24, Caregen, Korea), decapeptide-4 (Decapeptide-4, Caregen, Korea), acetyl deca Peptide-3 (Acetyl Decapeptide-3, Caregen, Korea) and rh-polypeptide-4 (rh-Polypeptide-4, Bio-FD&C, Korea) were added to tertiary distilled water at 4,000 ppm (total 20,000 ppm) for 30 minutes each. Stirred ones were used.

Experimental Example  1: Component analysis of the structure in which the active substance is introduced

In this experiment, it was confirmed through the GC (gas chromatography, gas chromatography) whether the active material was well introduced into the protoplast in the structures prepared in Examples 1 to 4, wherein the standard reagents of each active material were compared for comparison. Used.

Figure 2 (a) is a standard sample of cyclohexanediol bisethyl hexanoate, (b) is a GC spectrum of the cyclohexanediol bisethyl hexanoate in the protoplast of Example 1 is introduced, It can be seen that the active substance cyclohexanediol bisethylhexanoate is introduced into the cell.

It was confirmed that these results were the same in Examples 2 and 3.

Figure 3 (a) is a standard sample of bisretin amido methyl pentane, (b) is a GC spectrum of the structure in which bisretin amido methyl pentane is introduced into the propoflast of Example 2, the active substance in the cell bis Retinami also shows that methylpentane is introduced.

In addition, Figure 4 (a) is a standard sample beta-rapachon, (b) is a GC spectrum of the structure in which beta-rapachon is introduced into the propoplast of Example 3, beta-rapachon, an active substance in the cell It can be seen that.

Cells into which the growth factor complex peptide of Example 4 was introduced were made through a MALDI-TOF mass spectrometer (Matrix assisted laser desorption/ionization time-of-flight Mass Spectrometry).

Figure 5 (a) is a standard sample growth factor complex peptide, (b) is a MALDI-TOF mass spectrometry graph of the structure in which the growth factor complex peptide is introduced in the propoflast of Example 4, the active material growth in the cell It can be seen that the factor complex peptide is introduced.

Efficacy analysis

The active substances obtained in Examples 1 to 4 were carried out as follows to confirm the effect and efficacy of the inclusion cells as cosmetic.

Experimental Example  2: Analysis of the whitening effect of the structure of Example 1

Cyclohexanediol bisethylhexanoate has a whitening effect and was tested using melanin-producing cells to compare the whitening effect with the construct prepared in Example 1.

This experiment was conducted in vitro using melanin-producing cell line and melanin-stimulating hormone. By establishing an assay system, the skin whitening effect was confirmed by measuring the effect of inhibiting melanin production and the effect of inhibiting tyrosinase activity by cyclohexanediol bisethylhexanoate.

(1) Cell line and cell culture

The rodent melanoma cell line B16 cell line (ATCC CRL-6323) was 10% FBS (Gibco Co), 100 μg/mL streptomycin, 100 U/mL penicillin at 37° C. and 5% CO ₂ conditions. The cells were cultured using DMEM (Dulbecco's modified Eagle medium) (Incubator; Thermo-scientific, USA).

B16 cells are 100 cm ² After sufficiently proliferating in the flask (Corning, USA), the surface of the cultured cells was washed with PBS at intervals of 3 days after cultivation, and then the 0.25% trypsin-EDTA solution was added and treated in the incubator for 3 minutes, then the trypsin-EDTA solution was discarded and the mixture was discarded at 37°C. Cells were detached by storing for a minute. The detached cells were transferred to 10 ml of DMEM containing 10% FBS in a new culture container, and cultured at 37°C and 5% CO ₂ under a 1:5 split ratio.

(2) Measurement of melanin production inhibitory effect

To a 96-well plate, 1 to 10 ⁵ cells per well were inoculated using medium DMEM, and the structure prepared in Example 1 and cyclohexanediolbisethylhexanoate and alpha-bisabolol, each of which had a known whitening effect, were each concentrated. 2 ml of sample solution was processed by making a star. Thereafter, 1 ppm of α-Melanocyte stimulating hormone (α-MSH) was treated and cultured for 72 hours. Next, after washing the medium with phosphate buffered saline (PBS), melanin extraction solution (1N NaOH+50% DMSO) was added at 100 µl per well to lyse the cells at 80° C. and then using an ELISA reader. The absorbance of melanin was measured at 492 nm.

[Equation 1]

Melanin production inhibition rate (%) = [(Mm -Ms)/(Mm -Mc)]×100

Mc: Miss melanin from the control group

Mm: amount of melanin in the α-MSH treated group

MS: amount of melanin in the group treated with the sample and α-MSH

(3) Analysis of results

6 is a graph showing the inhibitory effect of melanin production according to each concentration of the structure of Example 1, cyclohexanediol bisethylhexanoate, and alpha-bisabolol.

Referring to FIG. 6, in the case of a structure in which cyclohexanediol bisethylhexanoate was introduced into the protoplast prepared in Example 1 according to the present invention, the effect of inhibiting melamine was cyclohexanediol bisethylhexanoate and alpha- It was superior to bisabolol, and the higher the concentration, the greater the inhibitory effect of melanin production.

Experimental Example 3: Analysis of collagen biosynthesis effect of the construct of Example 2

Bisretinamido methylpentane has a skin regeneration effect, and the collagen biosynthetic effect was compared with the construct prepared in Example 2, and retinol and adenosine were additionally used as comparative examples.

(1) Cell line and cell culture

Human dermal fibroblasts (Human dermal fibroblast, Gibco, USA) were inoculated to each well of a 96-well microplate to be 1×10 ⁴ cells, 100 U/ml penicillin, 100 μg/ml Cultured in 10% DMEM (Dulbecco's Modified Eagle's Medium) medium supplemented with streptomycin. The medium was maintained until 80% confluence of the attached cells and passage culture was performed at 80% confluence. Medium was changed every 2 days until confluence. In the passage culture, the flask from which the culture solution was removed was washed with phosphate buffer saline (PBS), the cells were dropped with 0.25% trypsin-EDTA (Gibco BRL, Grand Island, NY, USA), and the cell suspension was centrifuged. After that, the number of cells was measured, and cultured again 3 times in the culture medium.

(2) collagen biosynthesis effect

Human normal fibroblasts were inoculated at a concentration of 1x10 ⁵ cells/well into a 48-well microplate containing DMEM medium containing 10% FBS, and a CO ₂ incubator at a concentration of 5% (CO ₂ ) Incubated at 37°C for 24 hours.

After incubation, the construct of Example 2, bisretinamido methylpentane, retinol, and adenosine were replaced with serum-free DMEM medium adjusted for each concentration, and then cultured for 48 hours. Collagen synthesis is promoted by adding 50 µg/ml of ascorbic acid 24 hours before the last incubation. After incubation, each well was washed and replaced with DMEM medium without serum, followed by further incubation for 24 hours.

After cultivation, the supernatant of each well was collected, and the amount of collagen newly synthesized using the kit (Kit, Takara Shuzo Co., Ltd, Japan) was used as the amount of procollagen type IC-peptide (PICP). The amount of PICP was measured, and the amount of PICP was converted to ng/2×10 ⁴ cells to measure the synthesis effect of collagen.

(3) Analysis of results

7 is a graph showing the collagen synthesis ability of the construct of Example 2, bisretinamido methylpentane, retinol, and adenosine, and the collagen synthesis ability of the active substance-derived cell of Example 2 according to the present invention is very excellent, and the concentration is It shows that the higher the higher the synthesis ability.

Experimental Example 4: Analysis of collagen biosynthesis effect of the construct of Example 3

Perform the same procedure as in Experimental Example 3, but after culturing, replace the structure, beta rapazine, retinol, and adenosine prepared in Example 3 with a serum-free DM medium adjusted for each concentration, and then culture and analyze the collagen synthesis effect. Did.

8 is a graph showing the collagen synthesis ability of the construct of Example 3, beta-rapachon, retinol, and adenosine, and the collagen synthesis ability of the active substance-derived cell of Example 3 according to the present invention is very excellent, and the higher the concentration, the It shows that the synthesis ability is further improved.

Experimental Example  5: Analysis of the effect of the structure of Example 4

(One) in - vitro Experiment

The same procedure as in Experimental Example 4, but after culture, the structure, growth factor complex peptide, retinol, and adenosine prepared in Example 4 were replaced with serum-free DMM medium adjusted for each concentration, and then cultured to induce collagen synthesis effect. Analysis.

Figure 9 is a graph showing the collagen synthesis ability of the construct, growth factor complex peptide, retinol and adenosine of Example 4, the collagen synthesis ability of the active substance inlet cell of Example 3 according to the present invention is very excellent, the higher the concentration It shows that the synthesis ability is further improved.

(2) ex - vivo Experiment

In order to confirm the epidermal regeneration effect and the increase effect of glycosaminoglycan and the collagen increase effect in the human skin tissue of the structure prepared in Example 4, it was performed as follows, and the results are shown below.

1. 15 tissues are removed from the skin of a woman before and after age 40. Preparation of skin tissue cultured alive.

2. These skin tissues are tested under the condition of 3 sets 1 set as follows.

Batch division Organization quantity sampling D0 Control 3 D0 T An untreated organization 6 D6 and D9 P Example treatment tissue 6 D6 and D9

3. 0.1g of sample is applied to skin tissue at D0 (sample treatment is performed in the same way for D2, D5, D6 and D8)-Sample concentration: 0.2%

4. D0, 3 control batch skin tissue samples, D6 and D9, 3 skin tissue samples respectively.

5. Fixed to paraffin. Section to 5μm thickness

6. Check the degree of epidermal regeneration: After staining paraffin fixation tissue with Masson® trichrome-Goldner variant, Masson-Goldner trichrome staining kit, MERCK, USA, using Cell ^D Olympus software (Olympus, Japan) Image analysis.

7. Confirmation of Glycosaminoglycan Increase: After increasing the amount of glycosaminoglycan after staining the paraffin fixation tissue with Alcian blue PAS, Alcian Blue/PAS Kit, Polyscience, Inc., USA, LEICA Check using Q Win and analyze using Kodak in vivo image analyzer (Eastern Kodak, Rochester, USA).

8. Confirmation of increase in collagen: frozen paraffin fixation tissue is labeled with FITC-labeled type 1 collagen antibody, rabbit polyclonal antibody (Poab Rabbit anti Collagen I affinity purified Cat#PS047, Monosan, Netherlands) 1 After staining at room temperature for a period of time, staining the nucleus with Propidium Iodid (Sigma-Aldrich, USA), the amount of collagen was checked using LEICA Q Win and Kodak in vivo image analyzer (Eastern Kodak, Rochester, USA).

10 is an image showing epidermal regeneration before and after treatment of the structure of Example 4 with respect to human skin tissue, and it can be seen that the regenerating effect is very excellent.

These results were the same in the increase effect of glycosaminoglycan and in the collagen increase experiment.

11 is an image showing the degree of increase in glycosaminoglycan before and after treatment of the structure of Example 4 with respect to human skin tissue, and FIG. 12 is an image showing the degree of increase in type I collagen. Accordingly, it can be seen that the above effect is excellent when a growth factor complex peptide is introduced into the protoplast.

Hereinafter, a preferred formulation example is presented to aid the understanding of the present invention. However, the following formulation examples are provided only for easier understanding of the present invention, and the contents of the present invention are not limited by the formulation examples.

Formulation example  1: Preparation of nutrition cream

A nutritional cream was prepared using a known method to have the composition shown in Table 4 below.

ingredient Content (% by weight) Structure of Example 4 0.5 Lipophilic glycerin monostearate 2.0 Cetearyl alcohol 2.0 Stearic acid 1.5 Polysorbate 60 1.5 Sorbitan stearate 0.6 Hydrogenated polyisobutene 1.0 Squalane 3.0 Mineral oil 5.0 Cyclomethicone 5.0 Dimethicone 1.0 Tocopherol acetate 0.5 glycerin 5.0 Betaine 3.0 Triethanolamine 1.0 Shotgun 0.05 incense Proper antiseptic Proper Coloring Proper Distilled water Balance Sum 100.00

Formulation example 2: Preparation of flexible lotion (skin lotion)

A flexible lotion was prepared using a known method to have the composition shown in Table 5 below.

ingredient Content (% by weight) Structure of Example 4 0.5 glycerin 5.0 1,3-butylene glycol 3.0 Betaine 1.0 Allantoin 0.1 DL-panthenol 0.3 EDTA-2Na 0.02 Sodium hyaluronate powder 0.05 ethanol 5.0 Octyldodeseth-16 0.2 Polyoxyethylene cured castor oil 0.2 incense Proper antiseptic Proper Coloring Proper Purified water Balance Sum 100.00

Formulation example  3: Nutrition lotion

Nutritional lotion was prepared using a known method to have the composition shown in Table 6 below.

ingredient Content (% by weight) Structure of Example 3 0.5 Glyceryl stearate SE 1.5 Cetearyl alcohol 1.0 Shea butter 1.5 Polysorbate 60 1.3 Sorbitan stearate 0.5 Hydrogenated vegetable oil 1.0 Mineral oil 5.0 Squalane 3.0 Cyclomethicone 2.0 Dimethicone 0.8 Tocopherol acetate 0.5 Carbomer 0.12 glycerin 5.0 1,3-butylene glycol 3.0 Sodium hyaluronate powder 0.05 Triethanolamine 0.12 incense Proper antiseptic Proper Coloring Proper Distilled water Balance Sum 100.00

Formulation example  4 : Massage  cream

Massage cream was prepared using a known method to have the composition shown in Table 7 below.

ingredient Content (% by weight) Structure of Example 3 0.5 Lipophilic monostearate glycerin 1.5 Cetearyl alcohol 1.5 Stearic acid 1.0 Polysorbate 60 1.5 Sorbitan stearate 0.6 Isostearyl Isosterate 5.0 Squalane 5.0 Mineral oil 35 Dimethicone 0.5 Hydroxyethyl cellulose 0.12 glycerin 6.0 1,3-butylene glycol 3.0 Triethanolamine 0.3 incense Proper antiseptic Proper Coloring Proper Distilled water Balance Sum 100.00

Formulation example 4: Essence

Essence was prepared using a known method to have the composition shown in Table 8 below.

ingredient Content (% by weight) Structure of Example 2 0.5 glycerin 6.0 Betaine 5.0 PEG 1500 2.0 Allantoin 0.1 DL-panthenol 0.3 EDTA-2Na 0.02 Hydrogenated lecithin 0.6 Hydroxyethyl cellulose 0.1 Sodium hyaluronate powder 0.08 Carboxyvinyl polymer 0.2 Triethanolamine 0.2 Ceramide 0.2 Octyldodecanol 3.0 Squalane 3.0 Polysorbate 60 0.4 Glyceryl stearate SE 1.5 incense Proper antiseptic Proper Coloring Proper Distilled water Balance Sum 100.00

Formulation example  5: Pack

A pack was prepared using a known method to have the composition shown in Table 9 below.

ingredient Content (% by weight) Structure of Example 1 0.5 Polyvinyl alcohol 15 Cellulose gum 0.15 glycerin 3.0 PEG 1500 2.0 Betaine 2.0 DL-panthenol 0.4 Allantoin 0.1 Triethanolamine 0.2 Nicotinamide 0.5 ethanol 6.0 PEG 40 hardened castor oil 0.3 incense Proper antiseptic Proper Coloring Proper Distilled water Balance Sum 100.00

Claims (13)

A structure in which an active substance is introduced into a dedifferentiated plant protoplast. The method of claim 1, wherein the plant is a curry plant (curry plant, Helichrysum italicum ), gulgal ( Commiphora wightii , Commiphora myrrha), prickly pear (Opuntia Ficus indica), peony (Paeonia lactiflora ), Petrification ( Adenium obesum ), Nymphaea coerulea ), Eucalyptus punctata ), Ginkgo biloba , Lilium candidum , olive tree ( Olea) europaea), Papyrus (Cyperus papyrus ), Nelumbo nucifera ), Redwood ( Sequoia sempervirens), point ka rose (Rosa gallica officinalis ), Coffea arabica ), Plumeria obtusa), gardenia (Gardenia jasminoides ), Bougainvillea spectabilis ), Jasminum sambac ), Rosa centifolia ( Rosa centifolia ), peppermint ( Mentha piperita ), Rosa Damasquena ( Rosa damascena ), Iris pallida), grapevine (Vitis vinifera ), white rose ( Rosa alba ), Cananga odorata ), almond tree ( Prunus amygdalus dulcis ), apple tree ( Malus domestica ), apricot tree ( Prunus armeniaca ), ginseng ( Panax ginseng ), blackberry ( Rubus fruticosus ), gold film ( Eschscholtzia californica ), Centella asiatica ), Prunus cerasus , Hibiscus , Hawaii rosa sinensis ), Juniperus communis , Cotton ( Gossypium arboreum ), date palm ( Phoenix dactylifera ), ginger ( Zingiber officinale ), Hibiscus syriacus ), Pueraria tuberosa ), pomegranate ( Punica granatum), boombox Costa lithium (Bombax costatum ), saffron ( Crocus sativus ), salvia officinalis ), Nymphaea alba ) and combinations thereof. The structure according to claim 1, wherein the active material is one selected from the group consisting of hydrophilic materials and hydrophobic materials. The method according to claim 1, wherein the active substance is organic acid, vitamin, arbutin, adenosine, niacinamide, polyphenol, flavonoid, retinol, cyclohexanediol bisethylhexanoate, bisretinamido methylpentane, betarapachon, growth Structure characterized in that it is one member selected from the group consisting of factor, growth factor complex peptide, and combinations thereof. The structure according to claim 1, wherein the active material has a draw rate of 0.001 to 20% by weight. Obtaining cells of the plant from the leaves, stems, roots, flowers, fruits and seeds of the plant;
Dedifferentiating the obtained plant cells using auxin to obtain dedifferentiated plant cells;
Culturing the obtained dedifferentiated plant cells in large quantities;
Removing the cell wall of the mass cultured dedifferentiated plant cell using an enzymatic reaction to obtain a protoplast;
Introducing the active material into the obtained dedifferentiated plant prototype by a pressure osmosis process; And
Comprising post-processing
Method for manufacturing a structure in which an active substance is introduced into a dedifferentiated plant prototype. The method of claim 6, wherein the auxin is alpha-naphtalene acetic acid (α-Naphtalene acetic acid), 2,4-dichlorophenoxy acetic acid, indole-3-acetic acid (Indole-3-acetic acid), and a combination thereof Method of manufacturing a structure characterized in that it is used at a concentration of 1 to 5 mg/ℓ as one selected from the group. The method according to claim 6, wherein the enzymatic reaction is cellulase (EC 3.2.1.4), pectinase (EC 3.2.1.15), xylanase (EC 3.2.1.8), chitinase (EC 3.2.1.14), hemi. A method for producing a structure, characterized in that it is reacted for 15 hours to 24 hours at a temperature of 4°C to 40°C by mixing with one of the enzymes selected from the group consisting of cellulase and combinations thereof. The method of claim 6, wherein the pressure osmosis process is a condition of adding 1 to 50% concentration by adding sodium chloride under a pressure of 0.01 to 10 MPa. The method of claim 6, wherein the dehydration reaction is further performed before the post-treatment step after the active material is introduced. The method of claim 6, wherein the post-treatment is centrifugation and washing. A cosmetic composition comprising the structure in which the active substance is introduced into the dedifferentiated plant protoplast of claim 1 as an active ingredient. The cosmetic composition of claim 12, wherein the cosmetic composition is a formulation of lotion, nutrient lotion, nutrient cream, massage cream, essence, pack, paste, gel, cream, lotion, powder, soap, oil, foundation, wax or spray. Cosmetic composition to be said.

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