KR101594687B1 - Nature liposome and process for the preparation thereof - Google Patents

Abstract

Provided in the present invention is a natural liposome comprising a liposome forming component containing natural phospholipid, fatty acid, a solvent, and a natural preservative, and a natural liposome collecting material. The natural liposome is formed by a method comprising a first step of treating a mixture with ultrasonic waves at 50-80°C after mixing one or more kinds of a natural emulsifier and distilled water; and a second step of adding a collecting material to a product of the first step, treating the resultant material with ultrasonic waves, and mixing the same. The present invention has the purpose of providing a novel natural liposome easily absorbed into a body without causing a problem such as skin stimulation, and a cosmetic composition comprising the same.

Description

NATURAL LIPOSOME AND PROCESS FOR THE PREPARATION THEREOF [0002]

The present invention relates to a natural liposome and a method for producing the same.

Skin is the organ that covers the outside of the body. It consists of three parts: the outer skin, the dermis, and the subcutaneous fat layer.

The innermost subcutaneous fat layer is composed of adipocytes. The dermis on the subcutaneous fat layer is composed of matrix proteins such as collagen fibers, and there are blood vessels, nerves, and sweat glands.

The epidermis is located at the outermost part of the skin, and most of it is occupied by keratinocytes. The epidermis acts as a barrier against various external stimuli (dryness, ultraviolet light, other physicochemical stimuli). Particularly, the stratum corneum of the epidermis exists in the uppermost layer and the outermost layer of the epidermis, and functions to block ultraviolet rays, bacteria, and harmful substances. These horny layers are formed in lipid-rich form composed of keratinocytes and lamellar membranes.

The role of skin barrier in epidermis can be collapsed by active oxygen generated from ultraviolet rays or by oxidative stress caused by pollution, which promotes skin aging. Therefore, restoring these barrier functions is a way to inhibit skin aging and maintain health.

On the other hand, the skin functions as a protective film against harmful substances or harmful effects, but on the other hand it acts as a barrier against an effect substance. Therefore, in order to selectively penetrate the active substance into the skin, a special skin absorption and delivery system is required. One of the most studied ones for this purpose is a liposome.

Liposomes are vesicles formed when a phospholipid is suspended in an aqueous solution and are separated from the outer membrane by a membrane composed of a lipid bilayer, and lipids such as cholesterol glycolipids and membrane proteins can be introduced into the membrane.

The inside of the liposome contains an aqueous solution layer, and ions, low molecular substances, nucleic acids, proteins and the like can be collected in the aqueous solution layer. Therefore, liposomes help to transport these materials. For example, if the liposomes are transported and transported with ingredients that are sensitive and difficult to absorb into the skin, the ingredients can be absorbed into the skin without breaking.

Among the existing transdermal delivery systems, liposomes are composed of phospholipid, which is the main component of the biomembrane, and are biocompatible and have the advantage of collecting both water-soluble or lipid soluble components into the lipid bilayer bilayer have. Despite these advantages, liposomes, however, have problems such as unstable formulation and very low collection efficiency. In addition, cholesterol used as a raw material of liposomes has a problem that liposome lipid membrane formation becomes strong, skin permeability of a capturing material is low, and release of active substance is difficult. In addition, solvents, preservatives, and the like used in the production of liposomes may cause skin irritation. Various liposomes have been studied to solve these problems.

Korean Patent No. 603814 discloses a liposome composition comprising lecithin and ceramide. However, natural liposomes containing natural phospholipids, fatty acids and natural preservatives are not disclosed.

Korean Patent No. 603814

It is an object of the present invention to provide a novel natural liposome that can be easily absorbed into the body without causing problems such as skin irritation and the like, and a cosmetic composition containing the same.

In order to accomplish the above object, the present invention provides a method for preparing a microcapsule comprising the steps of mixing at least one natural emulsifier with distilled water and sonicating at 50 to 80 ° C, adding a capturing material to the product of the first step, And a second step, wherein the natural liposome comprises at least one natural emulsifier, a solvent, a liposome constituent material containing a natural preservative, and a natural liposome capturing material, wherein the natural preservative is a natural liposome .

Wherein the natural emulsifier comprises one or more phospholipids selected from the group consisting of phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine, soybean phosphatidylcholine, phosphatidylic acid, phosphatidylserine, phosphatidylglycerol, phosphatidylinocroline, .

The natural emulsifier may include at least one fatty acid selected from the group consisting of palmitic acid, stearic acid, oleic acid, linoleic acid, and linolenic acid (derived from soy).

The natural liposome may further include a natural emulsifier, and one or more emulsifiers selected from the group consisting of sorbitan olivate and cetearyl olivate may be used as the natural emulsifier.

As the solvent, at least one solvent selected from the group consisting of distilled water, butylene glycol, propylene glycol, glycerin and ethanol may be used. Preferably, distilled water can be used.

As the natural preservative, one or more natural extracts selected from the group consisting of a superfine fruit extract, a waxy flower extract, and an Earther extract may be used.

As the natural liposome capturing substance, at least one collecting material selected from the group consisting of adenosine, alpha-bisabolol, hyaluronic acid, cultured lactic acid bacteria, organic gooseberry extract powder (vitamin C), paprika fermentation extract and ceramide can be used.

The natural liposome can be used for promoting skin absorption.

The natural liposome may contain from 2.5 to 19 w / v% of an emulsifier comprising a natural phospholipid and a fatty acid, from 0.1 to 5 w / v% of a natural preservative, from 0.1 to 10 w / v% of a natural liposome capture agent and a residual solvent amount.

Another aspect of the present invention provides a cosmetic composition containing the natural liposome.

In another aspect of the present invention, there is provided a method for producing a microorganism, which comprises a first step of mixing at least one kind of emulsifier with distilled water and ultrasonication at 50 to 80 캜, a second step of adding a capturing material to the product of the first step, And a third step of adding a distilled water warmed to 40 to 60 ° C to the product of the second step and a natural preservative to form a liposome by ultrasonic treatment. The method may further include a fourth step of performing ultrasonic processing while gradually lowering the temperature after the third step.

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INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a natural liposome excellent in the collection efficiency of the capturing material and the skin absorption rate without causing problems such as skin irritation and the like.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 shows a process for producing a natural liposome according to one aspect of the present invention.
Figure 2 shows a process for preparing natural liposomes according to another aspect of the present invention.
3A to 3C are SEM photographs of the liposome according to Example 1, Comparative Example 1, and Comparative Example 2, respectively.
4A to 4G are SEM photographs of natural liposomes according to Examples 2 to 8, respectively.
5A to 5F are SEM photographs of natural liposomes according to Examples 9 to 14, respectively.
6A to 6C show graphs of size distribution of the liposome according to Example 1, Comparative Example 1 and Comparative Example 2, respectively.
7A to 7G show the size distribution graphs of the natural liposomes according to Examples 2 to 8, respectively.
8A to 8F show the size distribution graphs of the natural liposomes according to Examples 9 to 14, respectively.
9A to 9H show the absorption rate graphs of the respective capturing materials of the liposome with different kinds of capturing materials.
10 is a graph comparing cell survival rates of adenosine liposomes of Example 1, Comparative Example 1 and Comparative Example 2. Fig.
Fig. 11 is a graph comparing cell survival rates of alpha-saviso rolls with alpha-safflower natural liposomes of Example 2. Fig.
12 is a graph comparing the cytotoxicity of alpha-savisrol and the alpha-safflower-free natural liposome of Example 2 at different treatment concentrations.
FIG. 13 is a graph showing cell proliferation rates of distilled water, hyaluronic acid, and hyaluronic acid natural liposome of Example 3. FIG. 14 is a photograph of each of these 100-fold magnification optical microscopes, FIG. 16 is a 500 × magnification electron microscope photograph of each of these, and FIG. 17 is a graph comparing the content of hyaluronic acid absorbed in each of these dermal cells.
18 is a SEM photograph of a 1500-fold magnification of distilled water, paprika fermentation extract, and natural liposome of paprika fermentation extract of Example 6;
19 is a graph showing the glutathione content in dermal cells of distilled water, Indian gooseberry, and Indian gooseberry natural liposome of Example 5. Fig.
20 is a graph comparing the inhibitory effects of Tyrosinase on distilled water, alpha-savisrol, alpha-safflower-based natural liposome of Example 2 and alpha-sababolol + adenosine natural liposome of Example 9;
Figure 21 shows the inhibitory effect of Tyrosinase on the liposomes of distilled water, alpha bissabolol, alpha bissabolol natural liposome of Example 2, alpha bissabolol + adenosine natural liposome of Example 9, alpha bissabolle + hyaluronic acid natural liposome of Example 10 .
22 is a photograph showing induction of cell differentiation of keratinocytes. 22 (a) shows the case immediately after the treatment with the distilled water, Fig. 22 (b) shows the case where the treatment with the distilled water is performed for 24 hours, 22 (e) shows a case immediately after treatment with hyaluronic acid natural liposome, and Fig. 22 (f) shows a case where it has been treated with hyaluronic acid natural liposome for 24 hours after 24 hours from treatment with hyaluronic acid.

Hereinafter, a natural liposome according to the present invention will be described in detail with reference to the accompanying drawings.

However, these descriptions are provided only to illustrate the present invention, and the scope of the present invention is not limited by these exemplary explanations.

1. Emulsifier

Emulsifiers consist of phospholipids and fatty acids.

Examples of the phospholipid include phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine, palmitic acid, hydrogenated phophatidylcholine (derived from soybean), phosphatidylserine, phosphatidyl At least one phospholipid selected from the group consisting of phosphatidylglycerol, phosphatidylinositol, and the like can be used.

As the fatty acid, at least one fatty acid selected from the group consisting of stearic acid, oleic acid, linoleic acid and linolenic acid (soybean-derived) may be used. The fatty acid may comprise 1 to 10 w / v%.

Other emulsifiers may be used, such as Cetearyl olivate and Sobitan olivate (from Olive).

As the emulsifier, two or more emulsifiers may be used together. For example, as the first emulsifier, phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine, palmitic acid, stearic acid, oleic acid, linoleic acid, acid and linolenic acid (derived from soybean), and hydrogenated phosphatidylcholine (derived from soybean) may be used as the second emulsifier. In this case, 1 to 5 w / v% of the first emulsifier component and 0.5 to 4 w / v% of the second emulsifier component may be included.

Further, in addition to the first emulsifier and the second emulsifier, a composition of Cetearyl olivate and sorbitan olivate (olive-derived) may be further used as the third emulsifier. In this case, 1 to 5 w / v% of the first emulsifier component, 0.5 to 4 w / v% of the second emulsifier component, and 0.5 to 2 w / v% of the third emulsifier component may be included.

2. Capable material

( Lactobacillus fermentum MieV L1106 culture medium), vitamin C (Indian gooseberry extract), hyaluronic acid (1% solution of hyaluronic acid), lactobacillus culture medium ), Paprika fermentation extract (SC complex) and ceramide (yeast-derived ceramide) can be used. Adenosine can be used as an ingredient to alleviate the wrinkles of the skin. Adenosine is 0.4 to 2 w / v%, alpha-bisabolol (whitening functional ingredients) is 1 to 10 w / v%, and Aru acid (hyaluronic acid 1% solution) is from 1 to 10 w / v%, lactic acid bacteria culture medium (Lactobacillus fermentum MieV L1106 culture medium) 0.1-5 w / v%, vitamin C (Indian gooseberry extract) 0.25-5 w / v%, paprika fermentation extract (SC complex) 1 -5 w / v%, ceramide Ceramide) may be contained in an amount of 0.1 to 1 w / v%.

3. Solvent

As the solvent, distilled water, butylene glycol, propylene glycol, glycerin, and ethanol may be used, and distilled water, glycerin, and more preferably distilled water may be used. The content of the solvent is the residual amount of the content of the emulsifier, the collectable material and the preservative.

4. Preservatives

As a preservative, one or more natural extracts selected from the group consisting of a superfine fruit extract, a flower leaf extract, and an Earther extract may be used. It is distinguished from benzoic acid, a paraoxybenzoic acid ester, a mixture of methylchloroisothiazolinone, phenoxyethanol and the like, which is a commonly used preservative in that it is a natural extract. Preservatives may generally comprise from 0.1 to 5 w / v%.

5. Preparation of liposome

The liposome according to the present invention can be manufactured using ultrasonic waves or using emulsification equipment.

end. How to use ultrasonic waves

One or more emulsifiers are mixed with a part of distilled water and ultrasonicated at 50 to 80 ° C using an ultrasonic liquid processor. Ultrasonic fracturing is an operation to homogenize the mixture. When the emulsifier is homogenized, it is mixed with ultrasonic wave after addition of the capturing material. When the emulsifier and the capturing material are completely homogenized, the remaining distilled water and the natural extract (preservative) warmed to 40 to 60 ° C are added and mixed by ultrasonic disruption to form liposome type particles. Thereafter, the temperature is gradually lowered and ultrasonication is finally performed for 5 minutes to make the particles smaller and uniform so that a natural liposome is finally produced. The manufacturing process is shown in Fig.

I. How to use oil painting equipment

Mix at least one kind of emulsifier with a part of distilled water and mix by using homomixer at 1300 ~ 1500rpm and 50 ~ 80 ℃ for 10 ~ 30 minutes. After the emulsifier is homogenized, the emulsifier is added to the emulsifier and mixed with the emulsifier. When the mixing of the emulsifier and the capturing material is finished, the remaining distilled water and the natural extract are added to prepare the natural liposome. Then the particles are made small with an ultrasonic disintegrator to produce the final natural liposome. The manufacturing process is shown in Fig.

The liposome according to the present invention can increase the skin permeability and improve the absorption rate, so that the active ingredient can effectively act on the skin. Therefore, the liposome according to the present invention can be used in a cosmetic composition. For example, softening longevity, convergent lotion, nutritional lotion, nutritional cream, massage cream, essence, eye cream, eye essence, cleansing cream, cleansing foam, cleansing water, pack, powder, body lotion, body cream, body oil, body Cosmetics, essences, makeup base, foundation, hair dye, shampoo, rinse, body cleanser, toothpaste, mask sheet and oral care form composition.

≪ Examples 1 to 6 >

The liposomes were prepared according to the composition shown in Table 1 below.

Raw material Content (unit (w / v)%) Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 Example 7 Example 8 Emulsifier Emulsifier 1 4.5 4.5 4.5 4.5 4.5 4.5 4.5 4.5 Emulsifier 2 4 4 4 4 4 4 4 4 Emulsifier 3 - - - - - - fatty acid Evening Primrose Oil - - - - - - antiseptic 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 menstruum Distilled water (D. I. WATER) Balance Balance Balance Balance Balance Balance Balance Balance Capture material

AD 0.4 - - - - - 5 - alpha -bis - 2 - - - - - - HA - - 3 - - - - - L1106 - - - 5 - - - - VC - - - - 4 - - - SC - - - - - 5 - - CG - - - - - - 5 - LC - - - - - - - 2

Emulsifier 1 Emulsifier 1 Emulsifier 1 A mixture of 67 wt% of phosphatidylcholine, 8 wt% of lysophosphatidylcholine, 8 wt% of phosphatidyl ethanolamine, 3 wt% of palmitic acid, 1 wt% of stearic acid, 3 wt% of oleic acid, 7 wt%, linolenic acid 3 wt%

Emulsifier 2: Hydrogenated phophatidylcholine (derived from soy)

Emulsifier 3: 50% by weight of cetearyl olivate and 50% by weight of sorbitan olivate (derived from olive), based on 100%

Preservative: Pine nuts fruit extract: Waxy pine extract: Earth nere extract is included in weight ratio of 1: 1: 1

* AD: adenosine * α-bisa: alpha-bisabolol (whitening functional ingredient)

* L1106: Culture medium of Lactobacillus fermentum MieV L1106

* HA: Hyaluronic acid (1% solution) * VC: Vitamin C (Indian gooseberry extract powder)

* SC: paprika fermented extract * CPS: natural liposome

* CG: collagen (from acacia plant) * LC: lidocaine

≪ Examples 7 to 12 >

Natural liposomes were prepared according to the compositions shown in Table 2 below.

Raw material Content (unit (w / v)%) Example 9 Example 10 Example 11 Example 12 Example 13 Example 14 Emulsifier Emulsifier 1 4.5 4.5 4.5 4.5 4.5 4.5 Emulsifier 2 4 4 4 4 4 4 Emulsifier 3 - - - - One One fatty acid Evening Primrose Oil - - - - 3 3 antiseptic 0.1 0.1 0.1 0.1 0.1 0.1 menstruum D. I. WATER Balance Balance Balance Balance Balance Balance Capture material AD 0.4 - 0.4 - - - alpha -bis 2 2 - - - - L1106 - - 5 5 - - HA - 3 - 3 3 3 SC - - - - - 5 CER - - - - One One

* CER: Ceramide (yeast-derived ceramide)

Other components are the same as in Examples 1 to 8

≪ Comparative Example 1 &

As the liposome of Comparative Example 1, Bounsphere AD (sold by GFC Co., LTD) was used. Bounsphere AD contains water, glycerin, hydrogenated lecithin, adenosine, dipotassium glycyrrhizate (synthetic), ceramide 3, phenoxyethanol (synthetic preservative), and triethanolamine (pH regulator).

≪ Comparative Example 2 &

As the liposome of Comparative Example 2, NLT adenosphere 2.0 (Biospectrum) was used. The NLT adenosphere contains water, butylene glycol, lecithin, and adenosine as constituents.

≪ Preparation Example 1 &

In Examples 1 to 12, emulsifier 1 and emulsifier 2 were mixed with 1/3 of distilled water and sonicated at 65 ° C using an ultrasonic liquid processor (QSONICA, USA). When the emulsifier was homogenized, the supernatant was added, followed by ultrasonic disruption. When the emulsifier and the capturing material were completely homogenized, the remaining 2/3 of distilled water heated to 50 ° C and the natural extract (preservative) were added and mixed by ultrasonic disruption to form liposome type particles. Thereafter, the temperature was gradually lowered, and ultrasonic disruption was performed for 5 minutes to make the particles smaller and uniform so that a natural liposome was finally prepared (see FIG. 1). The natural liposomes of Examples 1-12 were prepared in this manner.

≪ Preparation Example 2 &

In Examples 13 and 14, Emulsifier 1, Emulsifier 2, and Emulsifier 3 were mixed with 1/3 of distilled water and ultrasonicated at 65 ° C using an ultrasonic liquid processor (QSONICA, USA). When the emulsifier was homogenized, the capturing material and evening primrose oil were added, followed by ultrasonic disruption and mixing. When the emulsifier, the capturing material and evening primrose oil were completely homogenized, the remaining 2/3 of distilled water heated to 50 ° C and the natural extract (preservative) were added and mixed by ultrasonic disruption to form liposome type particles. Thereafter, the temperature was gradually lowered, and ultrasonication was performed for 5 minutes at the end to make the particles smaller and uniform, thereby finally producing a natural liposome.

<Test Example 1> Analysis of morphology and size of liposome by electron microscope

The shape and size of the liposome particles prepared in Example 1 and Comparative Examples 1 and 2 were analyzed using a scanning electron microscope (S-1700, hitachi). The measurement was performed after lyophilization of the prepared sample, followed by thin coating and coating with a PdPt catalyst once.

Particle size was measured using a laser particle size analyzer (Electrophoretic Light Scattering Spectrophotometer ELS-8000, Otsuka).

&Lt; Test Example 2 > Measurement of collection rate

An aliquot of the finished liposomal suspension was taken and the unadsorbed material in the liposomes was removed using a 1.2 μm syringe filter. Then, ethanol was used to destroy the liposome membrane. In the case of the functional material, the content of the substance collected by HPLC was quantitatively determined, and the absorbance of the extracted extract, such as fermentation extract, was measured and converted into the following formula.

[Functional Substance]

Charging efficiency (%) = Concentration of functional material through filter / Concentration of first functional material * 100

[extract]

Charge efficiency (%) = Absorbance of the extract through the filter / Absorbance of the first extract * 100

&Lt; Test Example 3 > - Confirmation of skin absorbency

To examine the effects of the natural liposomes on the skin permeation enhancement, a skin absorption test was conducted using a Franz diffusion cell. In the skin absorption test, the degree of absorption was confirmed using a skin oil film (Strat-M membrane Millipore). The membranes were fixed between the donor and receptor phases, and 1 ml of the natural liposome was added to the prepared Franz diffusion cell and the temperature was maintained at about 32.5 ° C using a constant temperature bath. 0.25-0.5 ml of receptor phase was collected with a Pasteur pipette through a sampling port at intervals of a certain time and stored in a tube.

&Lt; Test Example 4 >

Fibroblast cells (1 × 10 ⁴ ) were dispensed into 48 wells, and after 24 hours, the cells were replaced with fresh medium and treated with a predetermined concentration of natural liposome material for 24 hours. Then, 3-4,5-dimethylthiazol-2-yl) -2,5-diphenyltetra zolium bromide (0.1 mg / ml) was added to each well and incubated for 3 hours. Insoluble formazan formed was dissolved in 0.04 N HCl / Absorbance was measured at 570 nm through an ELISA reader.

<Test Results>

1. Form and Size Analysis of Natural Liposome Particles

The natural liposome of Example 1, the liposome of Comparative Example 1, and the liposome of Comparative Example 2, which captured adenosine, were confirmed by scanning electron microscopy and then SEM photographs were taken. The results are shown in FIG. FIG. 3A is an SEM photograph of a natural liposome according to Example 1, FIG. 3B is an SEM photograph of a liposome according to Comparative Example 1, and FIG. 3C is an SEM photograph of a liposome according to Comparative Example 2.

As shown, the natural liposomes according to Example 1 are spherical and relatively uniformly distributed (Figure 3a), whereas the liposomes according to Comparative Example 1 are very large and irregularly elliptical (Figure 3b) The liposome according to Comparative Example 2 has a large particle size and a rough surface (Fig. 3C). As described above, it was confirmed that the particle size of the liposome according to Example 1 was smaller than that of the adenosine liposomes of Comparative Examples 1 and 2 by comparing 10 ⁵ magnification liposome photographs.

The particle shape of the natural liposome according to the entrapping material is shown in Fig. The natural liposome of Example 2 and the natural liposome of Example 6 using the paprika fermentation extract (SC complex), which used alpha-bisabolol as a trapping material, exhibited flexion of the surface (Figs. 4A and 4E, respectively) .

On the other hand, the natural liposome of Example 3 using hyaluronic acid as a capturing material and the natural liposome of Example 5 using vitamin C (Indian gooseberry extract) as a capturing material were formed into elliptical shapes (FIGS. 4B and 4D, respectively).

The natural liposome of Example 4 using lactic acid bacteria culture ( Lactobacillus fermentum MieV L1106 culture medium) as a capturing material was confirmed to have a hexagonal shape (Fig. 4C).

The natural liposome of Example 7 showed a circular shape with sharp protrusions, and the natural liposome of Example 8 showed a shape with a curved surface (FIGS. 4F and 4G).

On the other hand, the natural liposomes of Examples 9 to 14 using two or more substances as a capturing material showed relatively spherical shape. This can be confirmed in FIGS. 5A to 5F, respectively.

2. Size analysis of natural liposome particles

Using Orsuka ELS-Z series, the size of natural liposome particles was measured by light scattering and the particle size distribution was analyzed. The average particle size is shown in Table 3, and the particle size distribution is shown in Fig.

Capture material Average particle size
(Unit: nm) Example 1 AD 136.5 Comparative Example 1 AD 153.8 Comparative Example 2 AD 3634.4 Example 2 alpha -bis 243.9 Example 3 HA 148.3 Example 4 L1106 81.6 Example 5 VC 135.6 Example 6 SC 79.7 Example 7 CG 81.4 Example 8 LC 121.4 Example 9 alpha -bis + AD 298.7 Example 10 α-bisa + HA 254.1 Example 11 L1106 + AD 134.4 Example 12 L1106 + HA 126.2 Example 13 CER + HA 174.4 Example 14 SC + CER + HA 196.7

As shown in Table 3, the average particle size of the adenosine natural liposome of Example 1 was 136.5 nm, which was smaller than that of the liposomes of Comparative Examples 1 and 2. Adenosine of Example 1 showed stable particle distribution (Fig. 6A).

The liposome of Comparative Example 1 had an average particle size of 153.8 nm, which was not large, but the particle distribution was unstable (Fig. 6b), and the liposome of Comparative Example 2 had an average particle size of 3 탆 or more, (Fig. 6C). As a result, it was found that the particles were not stably formed.

The natural liposome of Example 2, which captured the alpha-bisabolol, had an average particle size of 200 nm or more. The natural liposome of Example 3 in which hyaluronic acid was captured had an average particle size of 140 nm or more. Examples 4 to 7 are all natural liposomes in which a liquid sample is used as a capture material. As shown in Table 3, it can be seen that when the natural liposome is made into a liquid sample, the particles are small.

7A to 7G show the particle distributions of the natural liposomes according to Examples 2 to 8, respectively. As shown, it was confirmed that the natural liposome capturing a single substance has a relatively constant particle size due to a narrow particle distribution range (FIGS. 7A to 7G).

The liposomes of Example 9 prepared by mixing alpha-bisabolol and adenosine, the liposomes of Example 10 prepared by mixing alpha-bisabolol and hurulonic acid, showed narrow particle size distribution (FIGS. 8A, 8b), the liposome of Example 11 prepared by mixing the culture of lactic acid bacteria and adenosine, and the liposome of Example 12 prepared by mixing lactic acid bacteria culture with hyruronic acid had a relatively wide particle size distribution and showed various particle sizes (Fig. 8C and Fig. 8D, respectively). The liposome of Example 13 prepared by mixing ceramide and hyaluronic acid and the liposome of Example 14 prepared by mixing ceramide, paprika extract and hyruronic acid showed narrow particle size distribution (Figs. 8E and 8F, respectively).

3. Measurement of collection rate

The total amount of adenosine adsorbed on adenosine liposomes was determined using HPLC to determine the entrapment rate of the liposomes of Example 1 and Comparative Examples 1 and 2. As a result of the measurement, the natural liposome of Example 1 showed a high collection rate of 99%. On the other hand, the liposomes of Comparative Example 1 and Comparative Example 2 had a collection rate of 60 to 75%. As a result, it was confirmed that the entrapment ratio of the natural liposome of Example 1 was high (Table 4 below).

As shown in Table 4 below, the collection rate was different depending on the collected material. As shown in Table 4, the entrapment rate of natural liposomes obtained by collecting a single substance was 80% or more, which was higher than that of Comparative Example 1 and Comparative Example 2. In addition, the capture rate of natural liposomes using 2 or 3 substances as a capture material was 70% or higher.

Capture material Collection rate (%) Example 1 AD 99.0 Comparative Example 1 AD 75.0 Comparative Example 2 AD 60.5 Example 2 alpha -bis 92.0 Example 3 HA 91.0 Example 4 L1106 87.3 Example 5 VC 98.2 Example 6 SC 86.2 Example 7 CG 91.3 Example 8 LC 80.1 Example 9 alpha -bis + AD alpha -bis: 94.4, AD: 97.2 Example 10 α-bisa + HA alpha -bisa: 98.5, HA: 98.8 Example 11 L1106 + AD L1106: 72.7, AD: 72.6 Example 12 L1106 + HA L1106: 87.2, HA: 86.5 Example 13 CER + HA CER: 95.1, HA: 93.7 Example 14 SC + CER + HA SC: 80.2, CER: 80.1, HA: 86.8

4. Measurement of skin absorption

(1) adenosine natural liposome

Skin permeation experiments were carried out using Franz diffusion cell to determine the effect of adenosine-captured natural liposome (Example 1) on skin permeation enhancement. To exclude animal experiments, a membrane (size 25 mm Strat-M ^™ Membrane, Millipore, USA), the most similar to the skin, was used in the experiment. Adenosine concentration was equally diluted and supplied onto the membrane, and the absorbance of samples taken at certain time intervals was compared to determine the amount of absorbed material.

Experiments were conducted on adenosine, adenosine natural liposome (Example 1) and adenosine liposome (Comparative Example 2) of the prior art, and as a result, the absorption capacity of adenosine natural liposome of Example 1 was increased over time Adenosine absorption did not change much (Fig. 9A). The adenosine natural liposomes of Example 1 were found to be faster than the adenosine liposomes of the prior art (Fig. 9A).

(2) Hyaluronic acid natural liposome

The absorption rate of hyaluronic acid having a large molecular weight was measured. As a result of comparing absorption capacities of hyaluronic acid and natural hyaluronic acid liposome (Example 2), the absorption rate of natural hyaluronic acid liposome (Example 2) was increased from 6 hours (FIG. 9B).

(3) Other

In the same manner as in (1) and (2) above, cultures of alpha-secretase, alpha-secretase + adenosine, alpha-secretase + hyaluronic acid, lactic acid bacteria (L1106) + hyaluronic acid, lactic acid bacteria (L1106) + adenosine and lidocaine The absorption rate was measured and the results are shown in Figs. 9c to 9h.

5. Cytotoxicity measurement

The safety of the liposome was confirmed by comparing the cytotoxicity of the natural liposome of Example 1 that captured adenosine with the adenosine liposome of the prior art (Comparative Examples 1 and 2). Cell survival rates were compared by treating cells with NLT (Comparative Example 2), Bounsphere (Comparative Example 1), and adenosine natural liposome (Example 1) 1%. As a result, the survival rate of adenosine natural liposome of Example 1 (Fig. 10).

As a result of comparing the toxicity of α-bisabolol, which is a whitening functional substance, with that of α-bisabolol and α-bisabolol obtained by liposomizing it (Example 2), the cell survival rate was similar to that of 0.01% , But the cell viability was significantly different at the 0.05% concentration, and it was confirmed that the toxicity was decreased when the liposome was prepared as a natural liposome (FIGS. 11 and 12).

6. Cell experiment

end. Cell proliferation of cells treated with hyaluronic acid liposome (Example 3)

(1) Experimental method

Fibroblast cells (5 × 10 ⁴ ) were dispensed into 6 wells. After 24 hours, the cells were replaced with fresh medium and treated with 0.05% concentration of distilled water, hyaluronic acid, and hyaluronic acid liposome (Example 3) for 48 hours. After the sample was treated, the cell proliferation process of dermal cells of each sample was observed with an optical microscope (100 times, 200 times) and a scanning electron microscope (500 times) every 24 hours. The cells were treated with 0.1% concentration in the same manner to observe the cell proliferation process.

(2) Experimental results

- cell proliferation rate

As a result of the experiment, when treated with 0.05% and 0.1%, the cell proliferation rate of the sample treated with the hyaluronic acid liposome of Example 3 was high (see FIG. 13).

- Observation of optical microscope (100 times) (when treated with 0.05% concentration)

Cells treated with the hyaluronic acid liposome of Example 3 showed increased cell density than cells treated with distilled water and hyaluronic acid. After 48 hours of the sample treatment, cells treated with distilled water and hyaluronic acid had a space between the cells, but cells treated with the hyaluronic acid liposome of Example 3 showed little space between the cells (see FIG. 14).

- Observation of optical microscope (200 times) (when treated with 0.05% concentration)

Dermal cells treated with distilled water, hyaluronic acid, and natural liposomes of hyaluronic acid were magnified 200-fold and observed with an optical microscope (Figs. 15A, 15B and 15C, respectively). The dermal cells treated with hyaluronic acid showed no significant difference from the cells treated with distilled water. However, the cells treated with the natural liposomes of hyaluronic acid showed a substance which is presumed to be hyaluronic acid inside the cells, and the cell size was enlarged.

- Scanning electron microscope (500 times) Observation result (when treated with 0.05% concentration)

The size and curvature of the cells were observed using a scanning transfer microscope, and it was confirmed that the volume became larger in the order of distilled water <hyaluronic acid <hyaluronic acid natural liposomes. (Figs. 16A, 16B and 16C, respectively)

I. The absorbed content of cells treated with hyaluronic acid liposomes (Example 3)

(1) Experimental method

Fibroblast cells (5 × 10 ⁴ ) were dispensed into 6 wells, and after 24 hours, they were replaced with fresh medium and treated with 15% concentration of distilled water, hyaluronic acid, and hyaluronic acid liposome (Example 3) for 15 hours. After the sample was treated, the medium of the treated cells was removed and only the cells were recovered and then disrupted by ultrasound to obtain cell lysate. The content of hyaluronic acid was determined by using a hyaluronan enzyme-linked immunosorbent assay kit (echelon) to determine the amount of hyaluronic acid contained in the cell lysate.

(2) Experimental results

Dermal cells were treated with distilled water, hyaluronic acid, and hyaluronic acid natural liposomes, and the content of the recovered hyaluronic acid in the cells was measured. The content of hyaluronic acid in the cells was 1.85 mg / g in the hyaluronic acid-treated group and 5.6 mg / g in the hyaluronic acid natural liposome-treated group. Cells treated with hyaluronic acid showed similar hyaluronic acid content to those treated with distilled water, indicating that the amount of hyaluronic acid absorbed into the cells was almost zero. It was confirmed that the cells treated with the natural liposome of hyaluronic acid absorbed hyaluronic acid into the cells and increased in number compared to the group treated with distilled water (FIG. 17).

All. Cell proliferation of cells treated with paprika fermented extract liposome (Example 6)

(1) Experimental method

^Four 5 × 10 ⁴ keratinocyte cells were dispensed in 6 wells, and after 24 hours, they were replaced with fresh medium and treated with 1% concentration of distilled water, paprika fermentation extract, and paprika fermented extract natural liposome (Example 6) for 48 hours. After the sample was treated with distilled water, the cells were removed by freeze-drying, and the size and surface curvature of the cells were observed with a scanning electron microscope (SEM, 1500 ×). The results are shown in Fig. 18A (distilled water treatment), Fig. 18B (paprika fermentation extract treatment) and Fig. 18C (paprika fermentation extract natural liposome treatment).

(2) Experimental results

As a result, the cells treated with the paprika fermented extract did not show a significant increase in volume compared with the cells treated with distilled water. However, in the case of cells treated with the paprika fermented extract liposome, the central part of the cell was rounded and the cell internal space was increased. (Fig. 18)

la. Cell proliferation of cells treated with Indian gooseberry liposome (Example 5)

(1) Experimental method

Fibroblast cells in 6 well dispense the 5 × 10 ⁴ dogs, and a new culture medium after 24 hours of 0.1% concentration and then replace the Indian gooseberry natural Liposomes in the control group (distilled water, Indian gooseberry) as in Example 5 was treated for 48 hours. After 48 hours of cell culture, the cells were harvested, disrupted by sonication, and treated with salicylic acid to remove cell lysate. The intracellular glutathione (GSH) content was confirmed using the Glutathione assay kit (Sigma-aldrich).

(2) Experimental results

The Indian gooseberry used in this experiment contains a large amount of antioxidants such as vitamin C. Indocyanine gooseberry natural liposomes were prepared using the Indian gooseberry, and the amounts of the antioxidants (glutathione) contained in the cells were determined when the natural liposomes were treated with the cells. The content of glutathione in the cells treated with Indian gooseberry was similar to that of distilled water. However, the glutathione content of cells treated with Indian gooseberry natural liposomes was 1.5 times higher than that of the Indian gooseberry treated group (FIG. 19).

hemp. Cell proliferation of cells treated with alpha liposorbrol natural liposomes (Example 2)

(1) Experimental method

6 well in Melanocyte Cells 5 × 10 ⁴ dogs dispensed, and the control group in an amount of 0.01% concentration and then replaced with fresh medium after 24 hours (distilled, alpha bisabolol), Example 2 alpha-bisabolol natural liposomes, Example 9 Of &lt; / RTI &gt; alpha bisabolol + adenosine natural liposomes for 48 hours. Cells treated for 48 hours were removed and cells were recovered and disrupted by sonication to obtain cell lysate. Tyrosinase (tyrosinase from mushroom-sigma-aldrich) was inhibited by cell lysate.

(2) Experimental results

The inhibitory effect of cell lysate on tyrosinase was examined in order to examine the whitening effect of the whitening substance (alpha - bisabolol) adsorbed into the cell when treated with natural liposome using melanin - forming cells using alpha - sabolol and alpha - sabolol. The tyrosinase inhibitory activity of the cells treated with alpha bisabolol was about 17%, whereas the activity of the alpha liposorbyl natural liposome-treated cells was more than 3 times inhibitory. A natural liposome (Example 9) in which alpha-bisabolol and adenosine were mixed showed an increase in activity by 13% as compared to the group treated with alpha-bisabolol (Example 2) (Fig. 20)

bar. Cell proliferation of cells treated with alpha liposomes

(1) Experimental method

6 well in melanocyte cells 5 × 10 ⁴ dogs dispensed, and the control group in an amount of 0.01% concentration and then replaced with fresh medium after 24 hours (distilled, alpha bisabolol), Example 2 alpha-bisabolol natural liposomes, Example 9 Adenosine natural liposome, alpha bissabolol + hyaluronic acid natural liposome of Example 10 were treated for 48 hours. After the sample was treated, the degree of melanin formation in the cells was observed with an optical microscope (200 times). The results of the observation are shown in Fig. 21 (treated with distilled water), Fig. 21 (treated with alpha-isobore), Fig. 21 (treated with alpha liposorbyl natural liposome) (Alpha bsobrol + hyaluronic acid natural liposome treatment).

(2) Experimental results

After melanin-forming cells were treated with natural liposomes using alpha-saborol and alpha-saborol, the melanin formed in the cells was confirmed with a microscope. The cells treated with distilled water had melanin throughout the cells and dark black (FIG. 21A). The cells treated with alpha bissabolol (Fig. 21B) had less melanin production than the cells treated with distilled water (Fig. 21A). On the other hand, cells treated with alpha liposorbyl natural liposomes (FIG. 21c), alpha bisabolol + adenosine natural liposomes (FIG. 21d), and alpha bisabolol + hyaluronic acid natural liposomes (FIG. 21e) The degree of melanin formation was smaller than that of treated cells and tended to decrease from the center of the cell to the outside (FIG. 21).

four. Induction of keratinocyte cell differentiation

(1) Experimental method

Human epidermal keratinocyte cell division well in 6 to 5 × 10 ⁴ dogs, and a new culture medium after 24 hours of 0.1% concentration and then replace the control group (distilled water and hyaluronic acid), and hyaluronic acid, natural liposomes of Example 3 for 24 hours. The sample was treated for 24 hours and then magnified 200-fold by an optical microscope to observe the differentiation state of the cells.

22 is a photograph showing induction of cell differentiation of keratinocytes. 22 (a) shows the case immediately after the treatment with the distilled water, Fig. 22 (b) shows the case where the treatment with the distilled water is performed for 24 hours, 22 (e) shows a case immediately after treatment with hyaluronic acid natural liposome, and Fig. 22 (f) shows a case where it has been treated with hyaluronic acid natural liposome for 24 hours after 24 hours from treatment with hyaluronic acid.

(2) Experimental results

The keratinocytes differentiate cells to function as a skin barrier. The keratinocytes are differentiated to increase ceramide production and reduce the transepidermal water loss, ultimately enhancing the skin barrier. Cell differentiation changes the cell shape to form a cornified cell envelope (CE). The keratinocytes were maintained in a form capable of proliferating when well divided. When the distilled water and the hyaluronic acid were treated for 24 hours, no change in the shape was observed. On the other hand, keratinocytes treated with hyaluronic acid natural liposome of Example 3 induced keratinocyte formation and induced differentiation.

Ah. Measurement of absorption of natural liposome

(1) Experimental method

In order to further increase the skin permeability of the natural liposome, a skin permeation enhancer was added to the skin absorbency test.

The raw material used in the experiment was adenosine, and the experiment was carried out under the same conditions as in <Test Example 3>. To determine adenosine uptake over time, samples were taken with a Pasteur pipette by time slot through a sampling port and stored in tubes. Adenosine natural liposomes added with a skin penetration enhancer used for absorption power were prepared as follows. Emulsifier 1 and Emulsifier 2 were mixed in glycerin, which acts as a skin penetration promoter component, not only in daily use instead of purified water, which is a solvent for dissolving an emulsifier, and then heated to 65 ° C before ultrasonication. Next, adenosine was added, sonicated, and distilled water, a natural preservative, and capric acid (skin penetration enhancer) were added. Thereafter, the adenosine natural liposome was prepared by adding ultrasound to the skin permeation enhancer through a final ultrasonic treatment step while lowering the temperature. The procedure is shown in FIG. 23, and the amounts used are shown in Table 5 below.

Raw material name Content (unit (w / v)%) Emulsifier Emulsifier 1 4.5 Emulsifier 2 4 antiseptic 0.1 menstruum Distilled water (D. I. WATER) Balance Capture material AD 4 Skin permeation accelerator glycerin 30 Capric acid 0.1

Adenosine natural liposomes were prepared by the above-mentioned method, and the skin absorption test was conducted using a Franz diffusion cell. The absorbance of the sample was measured at 287 nm using an Elisa leader.

(2) Experimental results

As a result, it was confirmed that the group to which the skin permeation enhancer was added showed a higher absorption capacity than that of the adenosine group and the natural adipocin group. The results are shown in Fig.

character. Evaluation of the stability of natural liposome raw materials

The stability of the natural liposomes of Examples 1 to 14 over time was measured. The pH, color, flavor, and sinking degree were measured at room temperature (25 ° C) for 30 days, and the stability was evaluated. The evaluation results are shown in Table 6 below.

pH (25 캜) color incense Degree of sinking 0 days 30 days 0 days 30 days 0 days 30 days 0 days 30 days Example 1 6.63 6.62 Opaque yellow Opaque yellow none none slightly slightly Example 2 6.32 6.51 Opaque milk color Opaque milk color none none X X Example 3 5.99 6.12 Opaque yellow Opaque yellow none none slightly slightly Example 4 4.63 4.72 Opaque ocher Opaque ocher none none X X Example 5 4.80 4.22 Brown Brown slightly slightly slightly slightly Example 6 4.35 4.15 Transparent brown Transparent brown slightly slightly X X Example 7 5.22 5.22 Transparent yellow Transparent yellow slightly slightly X X Example 8 8.92 8.96 Brown Brown slightly slightly slightly slightly Example 9 6.84 6.60 cream color cream color slightly slightly X X Example 10 6.58 6.43 cream color cream color slightly slightly X X Example 11 4.75 4.75 Brown Brown slightly slightly slightly slightly Example 12 4.75 4.62 Brown Brown slightly slightly slightly slightly Example 13 6.35 6.54 cream color cream color slightly slightly slightly slightly Example 14 4.64 4.52 cream color cream color slightly slightly slightly slightly

car. Identification of variable form of raw material of natural liposome

The degree of penetration through the artificial permeation barrier was measured with the natural liposome samples of Examples 1 to 14 to confirm the variable formation of the natural liposome membrane before and after skin permeation.

Specifically, the amounts and particle sizes of the natural liposomes passed through the polycarbonate membrane (having a pore size of 0.08 μm) when the pressure of 0.2 MPa was applied to the natural liposome samples of Examples 1 to 14 for 1 minute were measured with MINI-EXTRUDER Avanti polar lipids, inc., USA) and a syringe pump (KDS330 Revodix, Korea). The results of measurement of the variable shaping index are shown in Table 7 below.

The particle size of the formulation was measured using a laser particle size analyzer (Electrophoretic Light Scattering Spectrophotometer ELS-8000, Otsuka). The variable formation of the natural liposome membrane is proportional to the following equation

Variable index = J _flux x (rv / rp) ²

(Where J _flux = amount of natural liposome extruded for 1 min

rv = particle size of natural liposome after extrusion

rp = pore size of filter membrane)

No. Raw material Size before extrusion (nm) Size after extrusion (nm) Extradability (%) Variable Formation Index Example 1 AD 131 129.5 85 7.597318 Example 2 alpha -bis 255.3 241 94 28.43549 Example 3 HA 159.9 148.3 95 12.65084 Example 4 L1106 80.9 79.9 94 3.20423 Example 5 VC 127 124.5 91 7.346473 Example 6 SC 92.4 91.8 93 4.081944 Example 7 CG 113.7 107.3 94 5.636715 Example 8 LC 134 132.7 88 8.070925 Example 9 alpha -bis + AD 274.8 230.3 91 25.13784 Example 10 α-bisa + HA 282.8 235.8 94 27.22164 Example 11 L1106 + AD 102.5 104 94 5.295333 Example 12 L1106 + HA 108.5 107.7 92 5.678819 Example 13 CER + HA 255 259.8 91 31.99031 Example 14 SC + CER + HA 222 202.1 85 18.08216

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention. The present invention is not limited to the drawings.

Claims (16)

A first step of mixing at least one natural emulsifier with distilled water and ultrasonic treatment at 50 to 80 ° C, a second step of adding a capturing material to the product of the first step, A natural liposome formed,
A liposome constituent material containing at least one natural emulsifier, a solvent and a natural preservative, and
Containing a natural liposome capturing material,
The natural preservative is a natural liposome, which is a natural-derived extract. The method according to claim 1,
Wherein the natural emulsifier comprises at least one phospholipid selected from the group consisting of phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine, soybean phosphatidylcholine, phosphatidylic acid, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, Liposome. The method according to claim 1,
Wherein the natural emulsifier comprises at least one fatty acid selected from the group consisting of palmitic acid, stearic acid, oleic acid, linoleic acid, and linolenic acid (derived from soy). The method according to claim 1,
Wherein the natural emulsifier comprises a first natural emulsifier containing at least one fatty acid and at least one phospholipid and a second natural emulsifier containing at least one phospholipid. 5. The method of claim 4,
Wherein the second natural emulsifier is at least one emulsifier selected from the group consisting of sorbitan olivate and cetearyl olivate. The method according to claim 1,
Wherein the solvent is at least one solvent selected from the group consisting of distilled water, butylene glycol, propylene glycol, glycerin and ethanol. The method according to claim 1,
The solvent is distilled water. The method according to claim 1,
Wherein the natural preservative is at least one natural extract of natural liposomes selected from the group consisting of an extract of Acerola japonica, an extract of Acerola sp. The method according to claim 1,
Wherein the natural liposome capturing material is at least one capturing material selected from the group consisting of adenosine, alpha-bisabolol, hyaluronic acid, cultured lactic acid bacteria, organic gooseberry extract powder (vitamin C), paprika fermentation extract and ceramide. The method according to claim 1,
Natural liposome for promoting skin absorption. The method according to claim 1,
2.5 to 19 w / v% of a natural emulsifier,
0.1 to 5 w / v% of a natural preservative,
0.1 to 10 w / v% of natural liposome capture material and
Solvent residue
&Lt; / RTI &gt; A cosmetic composition comprising the natural liposome of claim 1. A first step of mixing at least one natural emulsifier with distilled water and sonicating at 50 to 80 ° C,
A second step of adding the natural liposome capturing material to the product of the first step,
Adding a distilled water warmed at 40 to 60 ° C to the product of the second step and a natural preservative and sonicating to form a liposome;
12. The method according to any one of claims 1 to 11, 14. The method of claim 13,
The fourth step of performing ultrasonic processing while gradually lowering the temperature after the third step
&Lt; / RTI &gt; delete delete

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