KR20160068190A - Stabilization Method of Dedifferentiated Plant Protoplast Complex With Active Material Insertion - Google Patents

Abstract

The present invention relates to a method for stabilizing an active agent in a demineralized plant protoplast in order to enhance the stability and efficacy of the active material, even in an emulsified form in which the oil form and the surfactant are present in a high content will be.

Description

Technical Field [0001] The present invention relates to a method for stabilizing a structure in which an active material is introduced into a protoplast of a dedifferentiated plant,

The present invention relates to a stabilization method in which a structure in which an active material is introduced into a protoplast of a deciduous plant to maintain stability in an emulsion form in which a high amount of an oil formulation and a surfactant exist in order to enhance the stability and efficacy of the active material .

Plants present in nature contain numerous different phytochemicals, and they also have a myriad of terpenoids, flavonoids, flavonols, polyphenols, amino acids, lignans, alkaloids, vitamins and catechin compounds .

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

For example, in International Patent No. WO2013-180526, Edelweiss extract promotes skin regeneration and can be used as a cosmetic, and in International Patent No. WO2012-102456, it is mentioned that the extract of Echinacea extract is effective for improving wrinkles and can be used as various cosmetics.

In addition, Japanese Laid-Open Patent Application No. 2012-102136 discloses a cosmetic composition comprising lyophilized plant cells of a chloroplast plant, such as Criste Marine, for skin bleaching and lightening.

On the other hand, effective active substances such as retinol have useful functions in terms of physiological characteristics in the human body, but they are not stable in terms of stability, and various methods have been proposed to enhance their stability.

For example, liposomes using phospholipids or surfactants as active materials, stabilized cerasomes using ceramides, liquid crystal stabilized with liquid crystal structures, and cubic cubosomes using monoglycerides have been attempted.

In addition, studies are being conducted to apply plant cells to cosmetic compositions.

International Patent No. WO2012-173458 discloses a cosmetic composition containing plant cells in which an active substance such as gallic acid or amino acid is stabilized, wherein the plant cell is a cell wall, in which an active substance is introduced into a cell wall The stability of the active material can be enhanced.

Cell walls of plant cells contain excessive amounts of cellulose, which makes it difficult to penetrate other substances, so that removal of the active substance is necessary for removal, and when the cell wall and cell membrane are all removed, the stability of the introduced active substance is lowered .

Accordingly, the present inventors have proposed a structure in which a cell wall of a plant cell is removed through a patent application No. 10-2013-0164701, and an active material is introduced into a protoplast composed of a cell membrane and a cell membrane.

However, the structure of the above patent is disadvantageous in that it is easy to use for water-soluble products or water-phase formulations, but the stability is lowered in a product having a high content of an oil component and an oil-based product having a high content of a surfactant.

WO2013-180526 WO2012-102456 Japanese Laid-Open Patent Application No. 2012-102136 WO2012-173458

Therefore, the present inventors have conducted various studies to stabilize a structure in which an active material is introduced into a dedifferentiated plant protoplast even in an emulsified form in which oil formulations and surfactants are present in a high content. As a result, Stirring and mixing the mixture to stabilize the structure. Thus, the present invention has been completed.

Accordingly, an object of the present invention is to provide a method of stabilizing a structure in which an active material is introduced into a protoplast of a deciduous plant.

In order to achieve the above object,

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

Obtaining plant cells that have been demineralized by demineralizing the obtained plant cells with auxin;

Culturing the obtained undifferentiated plant cells in a large amount;

Removing the cell wall of the massively cultivated deciduous plant cell using an enzyme reaction to obtain a protoplast;

Introducing the active substance into the resultant deproteinized plant protoplast through a pressurized osmosis process;

A first post-treatment step;

Mixing the first post-treated structure with the first vegetable oil and stirring simultaneously with the pressurization;

A second post-treatment step; And

Mixing the second post-treated structure with the second vegetable oil

The present invention provides a method of stabilizing a structure in which an active material is introduced into a protoplast of a dedifferentiated plant.

The stabilized structure according to the method of the present invention can maintain stability even in emulsified formulations in which oil formulations and surfactants are present in high amounts.

FIG. 1 is a flowchart showing a method of stabilizing a structure in which an active material is introduced into a protoplast of a deconditioned plant according to the present invention.
FIG. 2 is a graph showing the results of measuring the stability of vegetable oil in a mixture of a structure obtained by the stabilization method according to the present invention and a glycerin-dispersed structure.
FIG. 3 is a graph showing the results of measuring the stability of emulsion formulations containing a 5 wt% surfactant of a mixture of the structure obtained by the stabilization method according to the present invention and the glycerin-dispersed structure.
Figure 4 is a graph showing the results of measuring the stability of an emulsion formulation comprising a 15 wt% surfactant in a mixture of the structure obtained by the stabilization method according to the present invention and the glycerin dispersed structure.

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

The present invention proposes a method of stabilizing a structure in which a structure in which an active material is introduced into a protoplast of a decidased plant maintains stability even in an emulsion form in which a high amount of an oil formulation and a surfactant are present.

A protoplast is a protoplast in which a cell wall of a cell is removed and a cell membrane is present, and various active materials which can be used as a cosmetic composition can be introduced into the cell membrane. The cell wall contains an excess amount of cellulose, which makes it difficult to penetrate other substances. Therefore, it is necessary to remove the active substance for the introduction of the active substance. If the cell wall and the cell membrane are all removed, the stability of the introduced active substance may be lowered Therefore, a protoplast in which a cell membrane is present is preferable.

The active substance may be a hydrophilic substance or a hydrophobic substance, and is not particularly limited in the present invention. Representative examples of the active substance include organic acids, vitamins, arbutin, adenosine, niacinamide, polyphenol, flavonoid, retinol, cyclohexanediol bisethylhexanoate, bisretenamidomethylpentane, Factor complex peptide, and combinations thereof.

Such an active material is used as an effective ingredient of cosmetic composition itself, but its stability is low and its activity can not be fully manifested. However, the active substance is introduced into the prooctlast to increase its activity stability, and the protoplast also greatly improves biocompatibility .

FIG. 1 is a flow chart showing a method of stabilizing a structure in which an active material is introduced into a protoplast of a dehulling plant according to the present invention.

Step (a): step of obtaining plant cells

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

The production of plant cells is not particularly limited in the present invention, and known methods can be used. For example, in the example of the present invention, after sterilization, the plant cells were obtained by immersing in an aqueous sodium chlorate solution to which a surfactant was added, followed by washing.

The plants which can be used are not particularly limited in the present invention, and all plants are possible. Typical examples are curry plant ( Helichrysum italicum ), Commiphora wightii (Commiphora myrrha ), Opuntia Ficus indica , Paeonia lactiflora , Adenium obesum , Nymphaea coerulea), eucalyptus Tooth (eucalyptus punctata), ginkgo (ginkgo biloba), lilies (Lilium candidum), olive trees (Olea europaea), papyrus (Cyperus papyrus), lotus (Nelumbo nucifera), redwood (Sequoia sempervirens), point But are not limited to, Rosa gallica officinalis , Coffea arabica , Plumeria obtusa , Gardenia jasminoides , Bougainvillea spectabilis , Jasminum sambac, Rosa centifolia , It has been found that it can be used in a variety of applications such as peppermint ( Mentha piperita ), Rosa damascena , Iris pallida , Vitis vinifera , Rosa alba , Cananga odorata ,

Prunus amygdalus dulcis , apple tree ( Malus domestica ), apricot tree ( Prunus amygdalus dulcis )

armeniaca), ginseng (Panax ginseng), blackberry (Rubus fruticosus), gold film (Eschscholtzia californica), Centella asiatica (Centella asiatica), Xinyang Cherry (P runus

cerasus), Hawaii Hibiscus (Hibiscus rosa sinensis), juniper berry (Juniperus communis), cotton (Gossypium arboreum), date palm (Phoenix dactylifera), ginger (Zingiber officinale), Rose of Sharon (Hibiscus syriacus), Pueraria investment Vero four (Pueraria tuberosa ), Pomegranate ( Punica granatum ), Bombax costatum , Crocus sativus , Salvia officinalis , Nymphaea alba , and combinations thereof. Preferably, curry plant ( Helichrysum italicum ), Commiphora wightii ( Commiphora myrrha ), Opuntia ficus indica , etc. are used.

Step (b): Decomination step

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

Auxin, used for dedifferentiation, is one of the plant growth regulators, which promotes cell growth at low concentrations but inhibits growth at high concentrations. As the auxin, there may be mentioned one species selected from the group consisting of alpha -naphthalene acetic acid, 2,4-dichlorophenoxyacetic acid, indole-3-acetic acid, , And plant cells may be cultured in a medium containing 1 to 5 mg / L of the cells to induce dedifferentiation of the plant cells.

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

Step (c): the step of culturing the dedifferentiated plant cells

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

Bulk cultivation of dedifferentiated plant cells is carried out in various media. As the medium, known media such as MS medium, B5 medium, WHITE medium, N6 medium, SH medium and Anderson medium can be used, preferably MS Use a medium.

In addition, the culture conditions and duration are not specifically mentioned in the present invention, and can be carried out under known conditions.

Step (d): Removal of cell wall through enzyme reaction

In step (d), the cell wall of the dehydrated plant cells cultivated in large quantities is removed using an enzyme reaction to obtain a protoplast free from the cell membrane and the cell micro-organ.

The enzyme is composed of a 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 species selected from the group is possible.

These enzymes may be used in various concentrations of 0.01 to 10 wt%, preferably 0.1 to 5 wt% of a cellulase, 0.01 to 2.5 wt% of a pectinase and 0.1 to 5 wt% of a hemicellulase, 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 at a temperature of 4 ° C to 40 ° C (more preferably 10 ° C to 25 ° C) for 15 hours to 24 hours at a rate of 10 to 50 rpm. By this enzymatic reaction, Only the cell wall is removed and the cell membrane is stably preserved.

Step (e): Activated substance introduction step by pressure osmosis process

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

Specifically, when the depleted plant protoplasts are dissolved in a lower alcohol (C1 to C4 alcohol), mixed with the active substance and water, and then sodium chloride is added, the active substance is introduced into the protoplast by osmosis do.

This process is carried out in a pressurized osmosis process, specifically at a pressure of 0.01 to 10 MPa, preferably 0.05 to 1 MPa, and most preferably 0.05 to 0.5 MPa, wherein the concentration is increased to 1 wt% or more by the addition of sodium chloride By weight to 1% by weight to 50% by weight. If the pressure and the concentration are less than the above ranges, the introduction of the active material will not occur effectively. On the contrary, if the above range is exceeded, the protoplast may be destroyed.

The introduction is carried out in such a concentration that the efficacy of the active substance can be sufficiently exhibited, which may vary depending on the active substance, but has an incorporation rate (based on% by weight) of 0.001 to 20%. In addition, the inlet may further perform a dehydration reaction to increase the inlet rate.

Step (f): the first post-treatment step

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

This primary post-treatment is a conventional post-treatment process, which removes unreacted materials (unreacted active material) and salts through centrifugation and washing.

The structure in which the active material is introduced into the protoplast obtained through the above steps has the stability of the active ingredient of the plant cell as it is and the stability of the active ingredient introduced into the cell is high, The effect is also improved and can be preferably used as the active ingredient composition of the cosmetic.

Step (g): mixing with the first vegetable oil followed by pressure stirring

In the step (g), the structure obtained through the first post-treatment is mixed with the first vegetable oil and then stabilized by stirring simultaneously with the pressurization.

Through the pressure agitation process, the outer wall of the protoplast structure, which is mainly composed of phospholipids, ceramides, cholesterol, etc., is impregnated into the first vegetable oil, and the solubility change is induced and stabilized.

The first vegetable oil may be olive oil, sunflower seed oil, meadowfoam seed oil or argan oil. Preferably, refined olive oil or extra virgin olive oil is used as the olive oil.

At this time, the structure and the first vegetable oil are preferably mixed at a weight ratio of 1: 1.

This pressurizing stirring process is concretely carried out at a pressure of 0.01 to 10 MPa, preferably 0.05 to 1 MPa, and most preferably 0.05 to 0.5 MPa. If the pressure is less than the above range, the effect of stabilizing the structure is insufficient. Conversely, if the pressure exceeds the above range, the protoplast may be destroyed.

Step (h): Second post-treatment step

In step (h), the second vegetable oil is pressurized and stirred together with the first vegetable oil to obtain a stabilized structure.

This second post-treatment removes the first vegetable oil through centrifugation and washing.

Step (i): mixing with the second vegetable oil

In step (i), the stabilized structure obtained through the second post-treatment is again mixed with the second vegetable oil to obtain a mixture.

The second vegetable oil may be at least one selected from the group consisting of avocado oil, wheat germ oil, rosehip oil, almond oil, castor oil, camellia oil, corn oil, sunflower seed oil, It can be used in a variety of applications such as oil, safflower oil, soybean oil, rapeseed oil, macanadia nut oil, jojoba oil, palm oil, palm oil, coconut oil, mango butter oil, shea butter oil, cocoa seed oil, It is possible to use four-flower oil or peach seed oil.

The stabilized structure obtained through the second post-treatment and the second vegetable oil are preferably mixed in a weight ratio of 1: 9 to 5: 5.

The mixture of the structure obtained by mixing with the second vegetable oil includes 0.001 to 99.0% by weight, preferably 0.01 to 10.0% by weight, more preferably 0.1 to 3% by weight based on the total weight of the entire cosmetic composition.

In addition, the cosmetic composition may be prepared by any known formulation and may be in the form of lotion, nutritional lotion, nutritional cream, massage cream, essence, pack, paste, gel, cream, lotion, powder, soap, oil, foundation, It can be formulated in spray form.

In addition, in the cosmetic composition of each formulation, other components other than the above-mentioned cells can be arbitrarily selected and formulated according to the formulation of the other cosmetics, the purpose of use, and the like. In addition, the compositions of each formulation may contain various bases and additives necessary for formulation of the formulation, and may contain non-ionic surfactants, silicone polymers, extender pigments, flavorings, preservatives, But are not limited to, disinfectants, oxidative stabilizers, organic solvents, ionic or nonionic thickeners, plasticizers, antioxidants, free radical scavengers, opacifiers, stabilizers, emollients, silicones, A preservative, a surfactant, an anti-inflammatory agent, a substance P antagonist, a filler, a polymer, a propellant, a basicizing or acidifying agent, or a coloring agent.

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

In the case where 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. Especially, in the case of a spray, a chlorofluorohydrocarbon, / Propane or dimethyl ether.

When the formulation of the present invention is a solution or an emulsion, a solvent, a dissolving agent or an emulsifying agent is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, , 3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan fatty acid esters.

In the case where the formulation of the present invention is a suspension, a carrier such as water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Cellulose, aluminum metahydroxide, bentonite, agar or tracant, etc. may be used.

When the formulation of the present invention is an interfacial active agent-containing cleansing, the carrier component is selected from aliphatic alcohol sulfates, aliphatic alcohol ether sulfates, sulfosuccinic acid monoesters, isethionates, imidazolinium derivatives, methyltaurate, sarcosinate, fatty acid amides Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided to further understand the present invention, and the present invention is not limited by the examples.

Manufacturing example  One: Depletion plant Protoplast cell Produce

(1) Obtaining of the plant cells of dedifferentiation

Tissue sections (at least 3 cm) were cut from the stem, leaf, etc. of the curry plant ( Helichrysum italicum ), Commiphora wightii , and Opuntia Ficus indica .

In this operating step, all work was carried out in aseptic workbenches under sterile conditions.

To sterilize the plant material, the tissue was immersed in 70% ethanol (Ethanol, Sigma, USA) for 60 seconds, 30% hydrogen peroxide (LG Chemical, Korea) for 15 minutes, (Sodium hydrochlorite, Sigma, USA) for 15 minutes and washed three to five times with sterile H2O.

For tissue culture, these tissue fragments were inserted into an aseptic Petri dish (125 mm), cut (2-3 mm) and carefully removed from the bleached sections, and the thus obtained samples were finely cut into solid culture medium 1) and half buried.

Raw material Content (mg / L) KNO ₃ 2,500 (NH _{4) 2} SO ₄ 134 CaCl ₂ .2H ₂ O 120 NaH ₂ PO ₄ .2H ₂ O 150 MgSO ₄ .7H ₂ O 200 MnSO ₄ · 4H ₂ O 15 ZnSO ₄ .7H ₂ O 5 H ₃ BO ₃ 4 KI 0.8 Na ₂ MoO ₄ .2H ₂ O 0.25 CuSO ₄ · 5H ₂ O 0.025 CoCl ₂ .6H ₂ O 0.025 FeSO ₄ .7H ₂ O 32.8 Myo-inositol 200 Nicotinic acid 2 L-ascorbic acid 30 Citric acid 50 Biotin ((+) - Biotin) 0.01 Pyridoxine-HCl 2 Thiamine-HCl 10 Sucrose 20,000 Alpha-Naphthalene acetic acid < RTI ID = 0.0 > 2 2,4-dichlorophenoxy acetic acid (2,4-dichlorophenoxy acetic acid
acid) 0.5 Kinetin 0.5 Agar 9,000 Purified water Balance

(2) Replanting of deciduous plant cells

Two to three cell clusters (1-2 cm) were collected with a Spatula and then spread on a new medium and dispersed. All of these procedures were performed on aseptic workbenches under sterile conditions.

(3) proliferation of deciduous plant cells in a liquid culture medium

The above demineralized plant cells were transferred to the liquid culture medium of Table 2, and then cultured in a rotary shaker at 50 to 150 rpm under a dark condition at 25 캜, wherein the subculture period was fixed to 10 days.

Raw material Content (mg / L) NH ₄ NO ₃ 400 Ca (NO _{3) 2} 500 CaCl ₂ .2H ₂ O 100 KH ₂ PO ₄ 200 MgSO ₄ .7H ₂ O 150 MnSO ₄ · 4H ₂ O 20 ZnSO ₄ .7H ₂ O 5 H ₃ BO ₃ 5 K ₂ SO ₄ 1,000 Na ₂ MoO ₄ .2H ₂ O 0.25 CuSO ₄ · 5H ₂ O 0.25 FeSO ₄ .7H ₂ O 32.8 Myo-inositol 200 Nicotinic acid 2 L-ascorbic acid 30 Citric acid 50 Biotin ((+) - Biotin) 0.01 Pyridoxine-HCl 2 Thiamine-HCl 10 Sucrose 30,000 Alpha-Naphthalene acetic acid < RTI ID = 0.0 > 2 2,4-dichlorophenoxy acetic acid (2,4-dichlorophenoxy acetic acid
acid) 0.5 Kinetin 0.5 Purified water Balance

(4) Obtain a protoplast free of the cell walls of the dedifferentiated plant cells

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

Then, the protoplast in the culture broth was obtained by centrifugation at 200 x g for 15 minutes, followed by centrifugation at 5,000 x g for 15 minutes for further purification.

(5) Production of a structure in which growth factor mimic peptides are introduced into a dedifferentiated plant protoplast

Oligopeptide-24 (Caregen, Korea), Decapeptide-4 (Caregen, Korea), Acetyl Decapeptide-3 (Oligopeptide- 3, Caregen, Korea) and rh-Polypeptide-4 (Bio-FD & C, Korea) were added to the third distilled water at 4,000 ppm (total 20,000 ppm) and stirred for 30 minutes.

20% by weight of the protoplast prepared in Preparation Example 1 and 60% by weight of distilled water were added to 20% by weight of the obtained peptide mixture solution, and the mixture was stirred at 500 rpm and 25 占 폚 using a paddle mixer (PL-S10, Poonglim, Korea) ≪ / RTI > for 1 hour.

Then, 2% by weight of sodium chloride (NaCl, Sigma, USA) was added to the mixture of the peptide mixture solution, protoplast and distilled water, and the mixture was filtered through a high pressure reactor (Miniclave, Buchi AG, Switzerland) , Dehydration and the induction reaction of the peptide. Thereafter, the mixture was centrifuged at 5,000 × g for 20 minutes using a centrifuge (Supra 22K, Hanil, Korea) to obtain a structure in which peptides were introduced into the protoplast.

Example  One : Structures stabilized in vegetable oils mixture Produce

The structure in which the growth factor complex peptide was introduced into the dedifferentiated plant protoplast obtained in Preparation Example 1 was mixed with extra virgin olive oil at a weight ratio of 1: 1. The mixture was stirred for 24 hours while being pressurized to 0.5 MPa.

Thereafter, the mixture was centrifuged at 5,000xg for 20 minutes using a centrifugal separator (Supra 22K, Hanil, Korea) to obtain a stabilized structure in vegetable oil. This was stabilized in a vegetable oil, and the structure: extra virgin olive oil in a ratio of 2: 8 Redispersed again to have a weight ratio. The thus obtained structure mixture has a concentration of about 5,000,000 structures / mL.

Comparative Example  One : Structure dispersed in glycerin mixture Produce

80% by weight of glycerin was added to 20% by weight of the structure for preservation of the structure in which the growth factor complex peptide was introduced into the dedifferentiated plant protoplast obtained in Preparation Example 1, followed by dispersion.

Experimental Example  One: Of vegetable oils Stability measurement

A mixture of the stabilized structure (Example 1) and the dispersed glycerin-containing structure (Comparative Example 1) in vegetable oil was prepared as shown in Table 3 below And uniformly dispersed in olive oil at a concentration of 500,000 structures / mL.

ingredient Content (% by weight) Experiment 1 Comparison group 1 Olive oil 90.00 90.00 The structure mixture of Example 1 10.00 - The structure mixture of Comparative Example 1 - 10.00 Sum 100.00 100.00

The cells were stored at 45 ° C and 5% CO ₂ and sampled at 1 week, 2 weeks, 3 weeks, and 4 weeks to determine the number of plant protoplast cells not destroyed by the Cell Counter (TC20 Automated Cell Counter, BMS) Were measured. The results are shown in Fig.

Referring to FIG. 2, it was confirmed that the structure stabilized in vegetable oil was stable even under harsh conditions, whereas the structure redispersed in glycerin was not stable under harsh conditions.

Experimental Example  2: 5 wt% Surfactant Containing emulsion  Stability measurement

An emulsion formulation containing 5 wt% surfactant was prepared as shown in Table 4 below using a mixture of a stabilized structure (Example 1) and a glycerin-dispersed structure (Comparative Example 1) in vegetable oil.

ingredient Content (% by weight) Experiment 2 Control group 2 Pro-type glycerin monostearate 1.80 1.80 PEG-100 stearate 1.80 1.80 Polysorbate 60 1.00 1.00 Sorbitan stearate 0.40 0.40 Cetearyl alcohol 1.50 1.50 Squalane 10.00 10.00 Isostearyl isoserate 5.00 5.00 Dimethicone 0.50 0.50 glycerin 6.00 6.00 1,3-butylene glycol 3.00 3.00 Carbomer 0.20 0.20 Triethanolamine 0.20 0.20 Example 1 Mixture 10.00 - Comparative Example 1 [ - 10.00 incense Suitable amount Suitable amount antiseptic Suitable amount Suitable amount Pigment Suitable amount Suitable amount Distilled water Balance Balance Sum 100.00 100.00

The cells were stored at 45 ° C and 5% CO ₂ and sampled at 1 week, 2 weeks, 3 weeks, and 4 weeks to determine the number of plant protoplast cells not destroyed by the Cell Counter (TC20 Automated Cell Counter, BMS) Were measured. The results are shown in Fig.

3, the number of protoplast structures dispersed in glycerin decreased with time. On the other hand, in the case of the structure stabilized by the method of the present invention, in the emulsion containing 5% by weight of surfactant, There was almost no change.

Experimental Example  2: One 5 wt% Surfactant Containing emulsion  Stability measurement

An emulsion formulation containing 15 wt% surfactant was prepared as shown in Table 5 below using a mixture of a stabilized structure (Example 1) in vegetable oil and a structure mixture dispersed in glycerin (Comparative Example 1).

ingredient Content (% by weight) Experiment group 3 Control group 3 Pro-type glycerin monostearate 5.00 5.00 PEG-100 stearate 5.00 5.00 Polysorbate 60 3.50 3.50 Sorbitan stearate 1.50 1.50 Cetearyl alcohol 2.00 2.00 Squalane 15.00 15.00 Isostearyl isoserate 5.00 5.00 Dimethicone 1.00 1.00 glycerin 6.00 6.00 1,3-butylene glycol 3.00 3.00 Carbomer 0.40 0.40 Triethanolamine 0.40 0.40 Example 1 Mixture 10.00 - Comparative Example 1 [ - 10.00 incense Suitable amount Suitable amount antiseptic Suitable amount Suitable amount Pigment Suitable amount Suitable amount Distilled water Balance Balance Sum 100.00 100.00

The cells were stored at 45 ° C and 5% CO ₂ and sampled at 1 week, 2 weeks, 3 weeks, and 4 weeks to determine the number of plant protoplast cells not destroyed by the Cell Counter (TC20 Automated Cell Counter, BMS) Were measured. The results are shown in Fig.

As shown in FIG. 4, the protoflast structure dispersed in glycerin decreased in number with time, while the structure stabilized by the method of the present invention showed that the number of cells in the emulsion containing 15% by weight of surfactant was 4 weeks There was little change until the time.

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

Formulation Example 1: Preparation of nutritional cream

Nutritive creams were prepared using known methods such as those shown in Table 6 below.

ingredient Content (% by weight) The structure mixture of Example 1 0.5 Pro-type 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 Tocopheryl acetate 0.5 glycerin 5.0 Betaine 3.0 Triethanolamine 1.0 Xanthan gum 0.05 incense Suitable amount antiseptic Suitable amount Pigment Suitable amount Distilled water Balance Sum 100.00

Formulation Example 2: Preparation of Flexible Lotion (Skin Lotion)

A flexible lotion was prepared by using a known method so as to have a composition as shown in Table 7 below.

ingredient Content (% by weight) The structure mixture of Example 1 0.3 glycerin 5.0 1,3-butylene glycol 3.0 Betaine 1.0 Allantoin 0.1 DL-Panthenol 0.3 EDTA-2Na 0.02 Sodium hyaruronate powder 0.05 ethanol 5.0 Octyldodec-16 0.2 Polyoxyethylene hardened castor oil 0.2 incense Suitable amount antiseptic Suitable amount Pigment Suitable amount Purified water Balance Sum 100.00

Formulation Example 3: Nutritional lotion

Nutritional lotion was prepared using known methods to have the composition shown in Table 8 below.

ingredient Content (% by weight) The structure mixture of Example 1 0.5 Glyceryl stearate SE 1.5 Cetearyl alcohol 1.0 Sheer butter 1.5 Polysorbate 60 1.3 Sorbitan stearate 0.5 Hardened vegetable oil 1.0 Mineral oil 5.0 Squalane 3.0 Cyclomethicone 2.0 Dimethicone 0.8 Tocopheryl acetate 0.5 Carbomer 0.12 glycerin 5.0 1,3-butylene glycol 3.0 Sodium hyaruronate powder 0.05 Triethanolamine 0.12 incense Suitable amount antiseptic Suitable amount Pigment Suitable amount Distilled water Balance Sum 100.00

Formulation Example 4: Massage Cream

Massage cream was prepared using known methods to have the composition shown in Table 9 below.

ingredient Content (% by weight) The structure mixture of Example 1 0.5 Pro-type glycerin monostearate 1.5 Cetearyl alcohol 1.5 Stearic acid 1.0 Polysorbate 60 1.5 Sorbitan stearate 0.6 Isostearyl isoserate 5.0 Squalane 5.0 Mineral oil 35 Dimethicone 0.5 Hydroxyethylcellulose 0.12 glycerin 6.0 1,3-butylene glycol 3.0 Triethanolamine 0.3 incense Suitable amount antiseptic Suitable amount Pigment Suitable amount Distilled water Balance Sum 100.00

Formulation Example 4: Essence

Essences were prepared using known methods so as to have the composition shown in Table 10 below.

ingredient Content (% by weight) The structure mixture of Example 1 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 Hydroxyethylcellulose 0.1 Sodium hyaruronate 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 Suitable amount antiseptic Suitable amount Pigment Suitable amount Distilled water Balance Sum 100.00

Formulation Example 5: Pack

Packs were prepared using known methods so as to have the composition shown in Table 11 below.

ingredient Content (% by weight) The structure mixture of Example 1 0.5 Polyvinyl alcohol 15 Cellulose sword 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 Suitable amount antiseptic Suitable amount Pigment Suitable amount Distilled water Balance Sum 100.00

Claims (16)

Obtaining cells of a plant from the leaves, stems, roots, flowers, fruits and seeds of plants;
Obtaining plant cells that have been demineralized by demineralizing the obtained plant cells with auxin;
Culturing the obtained undifferentiated plant cells in a large amount;
Removing the cell wall of the massively cultivated deciduous plant cell using an enzyme reaction to obtain a protoplast;
Introducing the active substance into the protoplast through a pressurized osmosis process;
A first post-treatment step;
Mixing the first post-treated structure with the first vegetable oil and stirring simultaneously with the pressurization;
A second post-treatment step; And
Mixing the second post-treated structure with the second vegetable oil
≪ / RTI > wherein the active material is entrapped in a protoplast of a dehulled plant comprising the active ingredient. 2. The plant according to claim 1,
Curry plant (curry plant, Helichrysum italicum), dry mouth (Commiphora wightii, Commiphora myrrha), prickly pear (Opuntia Ficus indica), peony (Paeonia lactiflora), petrochemical (Adenium obesum), s Pie Oh Oh Dora Other (Nymphaea coerulea), Eucalyptus punctata , Ginkgo biloba , Lilium candidum , Olea europaea , Papyrus ( Cyperus papyrus ), Nelumbo nucifera , Redwood ( Sequoia sempervirens ), Garica rose Rasa gallica officinalis , Coffea arabica , Plumeria obtusa , Gardenia jasminoides , Bougainvillea spectabilis , Jasminum sambac , Rosa centifolia , Peppermint Mentha piperita, Rosa damascena , Iris pallida , Vitis vinifera , Rosa alba , Cananga odorata , Almond trees ( Prunus amygdalus dulcis ), Apple trees ( Malus domestica), apricot (Prunus armeniaca), ginseng (Panax ginseng), blackberry (Rubus fruticosus), gold film (Eschscholtzia californica), Centella asiatica (Centella asiatica), Xinyang Cherry (Prunus cerasus), Hawaii Hibiscus (Hibiscus rosa sinensis) Juniperus communis , cotton ( Gossypium arboreum ), Phoenix dactylifera , ginger ( Zingiber officinale ), Hibiscus syriacus , Pueraria tuberosa , Punica granatum, Wherein the composition is one selected from the group consisting of Bombax costatum, Crocus sativus , Salvia officinalis , Nymphaea alba , and combinations thereof. The stabilization method according to claim 1, wherein the active material is one selected from the group consisting of a hydrophilic substance and a hydrophobic substance. The method of claim 1, wherein the active material is selected from the group consisting of organic acids, vitamins, arbutin, adenosine, niacinamide, polyphenols, flavonoids, retinol, cyclohexanediol bisethylhexanoate, bisretenamidomethylpentane, Factor, growth factor complex peptide, and combinations thereof. The stabilization method according to claim 1, wherein the penetration rate of the active material is 0.001 to 20% by weight. The method of claim 1, wherein the auxin is selected from the group consisting of alpha -naphthalene acetic acid, 2,4-dichlorophenoxyacetic acid, indole-3-acetic acid, , And the concentration is 1 to 5 mg / l. The method according to claim 1, wherein the enzyme reaction is selected from the group consisting 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) Cellulase
And a combination thereof, and the mixture is heated at a temperature of 4 ° C to 40 ° C
At a temperature of from 15 to 24 hours. The stabilization method according to claim 1, wherein the pressurized osmosis process is a condition of adding 1 to 50 wt% of sodium chloride under a pressure of 0.01 to 10 MPa. The stabilization method according to claim 1, further comprising performing a dehydration reaction before the first post-treatment step after the introduction of the active material. The stabilization method according to claim 1, wherein the first post-treatment is centrifugal separation and washing. The stabilization method according to claim 1, wherein the secondary post-treatment is centrifugal separation. The method according to claim 1, wherein the first vegetable oil
Olive oil, sunflower seed oil, meadowfoam seed oil or argan oil. The method of claim 1, wherein the second vegetable oil is selected from the group consisting of olive oil, sunflower seed oil, meadowfoam seed oil or argan oil, avocado oil, wheat germ oil, rosehip oil, almond oil, castor oil, camellia oil, corn oil, Oil, soybean oil, rapeseed oil, macanadia nut oil, jojoba oil, palm oil, palm oil, coconut oil, mango butter oil, shea butter oil, cocoa seed beet oil, refined grape seed oil, rose hip oil, Or peach seed oil. The method of claim 1, wherein the first post-treated structure and the first vegetable oil are mixed at a weight ratio of 1: 1. The method according to claim 1, wherein the pressure is applied at a pressure of 0.01 to 10 MPa. The method according to claim 1, wherein the second post-treated structure and the second vegetable oil are mixed at a weight ratio of 1: 9 to 5: 5.

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