KR100831627B1 - Reverse-Phase Multi-Lamella Vesicle Capturing Aqueous Physiologically Active Ingredients - Google Patents

Abstract

The present invention relates to a reversed phase multilayer vesicle containing water soluble bioactive components. More specifically, the reverse-phase multilayer vesicle according to the present invention is a capsule having a hydrophilic property that can contain an aqueous solution inside and a hydrophobicity that can be applied to an anhydrous type outside, and has a physiological activity more effective than a single membrane vesicle. In addition to the high amount of encapsulation, it is very robust, better resistant to temperature and physical forces, and stable in oil, so that the reversed phase multilayer vesicles can be used to introduce water-soluble bioactive ingredients into anhydrous forms such as lipstick formulations. Encapsulated physiologically active ingredient is in a molecular state dissolved in an aqueous solution is easier to promote percutaneous absorption than the powder state.

Reversed-phase Multi-Layered Blessicle * Biologically Active Ingredients * Anhydrous Type * Lipstick

Description

Reverse-Phase Multi-Lamella Vesicle Capturing Aqueous Physiologically Active Ingredients}

1 is an electron micrograph of an inverted multilayer vesicle of Example 3 prepared by the present invention.

The present invention relates to a reversed phase multilayer vesicle containing water soluble bioactive components. More specifically, the reverse-phase multilayer vesicle according to the present invention is a capsule having a hydrophilic property that can contain an aqueous solution inside and a hydrophobicity that can be applied to an anhydrous type outside, and has a physiological activity more effective than a single membrane vesicle. In addition to the high amount of encapsulation, it is very robust, better resistant to temperature and physical forces, and stable in oil, so that the reversed phase multilayer vesicles can be used to introduce water-soluble bioactive ingredients into anhydrous forms such as lipstick formulations. Encapsulated physiologically active ingredient is in a molecular state dissolved in an aqueous solution is easier to promote percutaneous absorption than the powder state.

In general, cosmetics have been developed in various ways according to the shape and function, etc. In general, the cleanser (cleanser), such as "shampoo" for the purpose of washing the hair or body, "cream" or "lotion", Emulsion formulation cosmetics such as "Skin" supply moisture, nutrients, and moisturizing factors to the dry stratum corneum, or provide physiologically active ingredients that are excellent for anti-aging, whitening, and soothing skin. Its main function is to be able to maintain it.

In recent years, makeup products such as "powder" or "foundation" and bong cosmetics, often referred to as "lipsticks," are now a new class of products that include skin-active substances that go beyond just color and embellishment on the face. I'm trying to be born again.

However, until now, the introduction of water-soluble active substances into makeup products has been limited to emulsion products only, and it has been impossible to introduce water-soluble active substances into anhydrous formulations due to process or incompatibility with oily raw materials.

Cosmetics whose main functions are anti-aging, whitening, and skin soothing by improving skin stratum corneum by providing moisturizing effect to skin and supplying nutrients are generally o / w type (oil-in-water type) and w / o type It can be divided into (water-in-oil type). First, the type produced by emulsifying the oil phase components in the water phase components using mainly water phase components is called o / w type (water-in-oil emulsion type). On the contrary, the water phase components are mainly emulsified in the oil phase components using oil phase components. The type that is used is called w / o type (oil-in-oil type). In addition, there is a w / o / w type emulsification system using the recently developed multi-liquid crystal film emulsification technique.

For example, Korean Patent No. 10-0461458 describes a technique for stabilizing physiologically active ingredients by preparing a multi-layered liposome, but this causes a problem of sweating and poor dispersion and high temperature stability. have.

Accordingly, US Pat. No. 5843407 and Japanese Patent No. 3338057 describe hydrophobic silica, hydrophobic clay, propylene carbonate, ethyl cellulose, and N-acyl amino acids in the formulation of lipstick to prevent sweating. The addition of a gelling agent such as an Acyl Amino Acid) derivative is described to form a reversed micelle or liquid crystal to form a surfactant association structure. However, such a method has disadvantages in that an excessive amount of surfactant is used, causing skin irritation and poor dispersibility and high temperature stability of the lipstick.

In general, since the bioactive substance is mostly water-soluble, it is applicable only to cosmetics containing an aqueous phase, such as oil-in-water or water-in-oil formulations, such as the conventional methods described above, there is a limitation in introducing into anhydrous formulations such as lipstick, In particular, when introduced into anhydrous formulations using conventional artificial synthetic emulsifiers or polymers, there is a disadvantage that it may cause irritation problems in sensitive skin because of poor compatibility with the skin.

Accordingly, the present inventors have studied a method of introducing a water-soluble physiologically active ingredient into an anhydrous type such as a lipstick while solving the conventional problems as described above. As a result, the inside has a hydrophilicity that may include an aqueous solution and the outside is anhydrous. When preparing reversed phase multilayer vesicles with a hydrophobic capsule structure that can be applied to formulations, it has excellent skin affinity and is very mild without irritation. The present invention has been completed and found that the construction of a drug delivery system for delivering to a desired site can be realized.

Accordingly, it is an object of the present invention to provide a reversed phase multilayer vesicle in which a water-soluble physiologically active component is trapped and stabilized therein so as to be introduced into an anhydrous type.

In order to achieve the above object, the present invention provides a reverse-phase multilayer vesicle stabilized in a water-in-oil (w / o) -type multilayer by trapping the water-soluble bioactive components therein.

In the present invention, the vesicle is phosphatidylcholine (Phosphatidylcholine, PC; natural or synthetic), phosphatidic acid (Phosphatidic acid, PA), lysophosphatidylcholine (LPC), phosphatidylethanol (Phosphatidylserin, PS), phosphatidylethanolamine Phosphatidylethanolamine (PE), Sphingolipids, Phosphatidylglycerol (PG), Sphingomyelin, Cardiolipin, Glycolipids, Gangliosides, Gangrebrosides Seeds (Cerebrosides), and soybean phospholipids may include a membrane consisting of a single or two or more mixed phospholipids.

In addition, the phospholipid component includes steroid lipids including long cholesterol, long aliphatic chain amines, long aliphatic chain carboxylic acids, long aliphatic chain sulfuric acids, long aliphatic chain phosphoric acid, dicetyl phosphate, and BH, butylated hydroxytoluene (BHT), tocopherol and retinol. And a component selected from the group consisting of isoprenoid compounds, alone or in combination of two or more thereof, to form a film, and add a hydroxy group to the aliphatic chain; Branched; Has a ring structure; Aromatic derivatives; Have ether or amide bonds; Having multiple unsaturated double bonds; Halogen derivatives; Carbohydrate, Glycol, Phosphate, Phosphate, Phosphonate, Quaternary amine, Sulfate, Sulfonate, Carboxy, Amine, Attaching a hydrophilic group selected from the group consisting of sulfhydryl, and imidazole; Combinations of the above hydrophilic groups can be used to form a film by substituting or mixing the phospholipid component.

In the present invention, the water-soluble bioactive component may be selected from the group consisting of proteins, peptides, nucleic acids, synthetic compounds, natural extracts, sugars, vitamins, and minerals, the size of the vesicle is preferably 800 ~ 1500 nm. Do.

In the present invention, the vesicle may be obtained by emulsifying an aqueous phase containing a water-soluble physiologically active component on an organic solvent containing phospholipid, and then dispersing a gel paste prepared by removing the organic solvent in butter.

In another aspect, the present invention provides a cosmetic composition comprising the reversed phase multilayer vesicle. The cosmetic composition may be characterized in that it is an anhydrous type selected from the group consisting of lipstick, lip balm, lip gloss, lip treatment, stick foundation, powder foundation, deodorant stick, hair conditioner, bath oil, body stick and essence. .

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

Reversed-phase multilayer vesicles according to the present invention is characterized in that the inside is hydrophilic, which may include an aqueous solution, and the outside has a hydrophobic encapsulation structure to collect and stabilize the water-soluble physiologically active ingredients therein.

The method for producing a reversed phase multilayer vesicle according to the present invention comprises the steps of: (1) preparing an oil phase by dissolving a phospholipid component in an organic solvent; (2) dissolving a water-soluble physiologically active component in purified water to prepare an aqueous phase; (3) preparing an inverted vesicle by adding and mixing the aqueous phase of (2) to the oil phase of (1); (4) placing the vesicle of (3) in a rotary vacuum evaporator to remove the organic solvent until the vesicle emulsion is in a gel state to obtain a gel paste; And (5) adding the gel paste of (4) to a butter or anhydrous type, uniformly dispersing using a three-stage roller mixer, then cooling, and spreading to a drier to mature.

In the following, a method for manufacturing a reversed phase multilayer vesicle according to the present invention will be described in each step.

(1) dissolving the phospholipid component in an organic solvent to prepare an oil phase part.

Phospholipids are used as constituents to form the vesicle membrane in the reverse phase multilayer vesicle according to the present invention. Specifically, phosphatidylcholine (PC; natural or synthetic), phosphatidic acid (PA), and lyso Pysphatidylcholine (LPC), Phosphatidylserin (PS), Phosphatidylethanolamine (PE), Sphingolipids, Phosphatidylglycerol (PG), Sphingomyelin (Sphingomyelin) (Cardiolipin), Glycolipids, Gangliosides, Cerebrosides, mixed lipids such as soybean phospholipids, and the like, which are used alone or in combination of two or more. The phospholipid component is contained in an amount of 0.5 to 8.0% by weight based on the vesicle emulsion.

At this time, in order to give the phospholipid component an appropriate function to the vesicle membrane, steroid lipids such as cholesterol, long aliphatic chain amines, long aliphatic chain carboxylic acids, long aliphatic chain sulfuric acids, long aliphatic chain phosphoric acids, dicetyl phosphate, and BHT (BHT) , butylated hydroxytoluene), tocopherol, retinol, isoprenoid compound and the like can be used. In addition, in addition to the aliphatic chain, a hydroxyl group, a branched structure, a ring structure, an aromatic derivative, an ether or amide bond, a polyunsaturated double bond, a halogen derivative, carbohydrate, glycol, Phosphate, Organic Phosphate, Quaternary amine, Sulfate, Sulfonate, Carboxy, Amine, Sulfhydryl, Imidazole Hydrophilic groups such as imidazole) or a combination of these groups may be substituted or mixed with the phosphorus lipids.

On the other hand, the organic solvent of step (1) is an inert organic solvent that can be removed at any time, and there are aromatic, aliphatic hydrocarbons, silicones, and the like, ethers, esters, alcohols, ketones, fluorocarbons, etc., in which an aqueous phase is not dissolved. The solvents may be used alone or in a mixed solution, and the term 'inert organic solvent' refers to a solvent that does not interfere or interfere in the manufacturing step of the present invention.

The organic solvent is used in 50 to 99% by weight based on the vesicle emulsion.

The inert organic solvent used to prepare the oil phase part varies as follows depending on the conditions. For example, chloroform, tetrahydrofuran (THF), diethyl ether, and the like may be used under low temperature conditions, that is, under conditions where the solvent can be removed at a relatively low temperature, and diethyl ether is preferable. . In addition, isopropyl ether is preferred at high temperature process conditions, particularly when preparing vesicles containing phospholipids saturated with lipid components.

(2) dissolving a water-soluble physiologically active ingredient in purified water to prepare an aqueous phase.

The water-soluble physiologically active ingredient used in the present invention includes all materials that can be collected in the vesicles to enhance physiological function, and are not particularly limited. Specifically, proteins, peptides, nucleic acids, synthetic compounds, natural extracts, sugars, vitamins, minerals, and the like may be included, these may be isolated from nature, chemically synthesized or produced recombinantly. For example, water-soluble bioactive components include toxins, enzymes, hormones, neurotransmitters, immunoglobulins, polysaccharides, and the like. In another example, bioactive components include immunomodulators, antibiotics, antitumor agents, anti-inflammatory agents, antipyretics, analgesics, anti-edema agents, antitussive expectorants, sedatives, muscle relaxants, antiepileptic agents, antiulcers, antidepressants, antiallergic agents, cardiovascular agents, antiarrhythmic agents , Vasodilators, hypotensive agents, diabetes treatments, homeostatic agents, hormones, antioxidants, hair restorers, woolen agents, antibacterial agents, whitening agents, collagen synthesis promoters, wrinkle removers, emollients, skin barrier enhancers, skin moisturizers or skin cosmetics. .

The water-soluble bioactive component is contained in an amount of 0.001 to 10.00% by weight based on the vesicle emulsion.

The water phase part has a water-soluble physiologically active ingredient to be contained in the vesicle capsule prepared by the present invention in a dissolved or dispersed form, preferably the water phase part pH 3 to maintain the stability of the physiologically active ingredient into the vesicle capsule Buffer to ~ 8. Much of the physiologically active ingredients such as drugs, compounds, and macromolecules dissolved in the aqueous buffer solution are collected in vesicle capsules.The type of phospholipid used in the manufacturing process, the type of inert organic solvent, the ratio of the organic solvent award to the aqueous phase, and the aqueous phase Depending on the volume, the amount of phospholipids, etc., it is collected up to at least about 60%.

The optimum ratios of the phospholipids, the organic solvent and the aqueous phase are different, and are determined in some cases.

(3) forming an inverted vesicle by adding and mixing the water phase of (2) to the oil phase of (1).

The water phase part of said (2) was thrown into the oil phase part of said (1), and it is 1-30 minutes, Preferably it is 3-10 minutes, More preferably, it is 500-9000 rpm, Preferably 2000-4000 rpm, More preferably, 3000 rpm Preferably for 4-6 minutes.

This emulsification method is a method commonly used to make an oil-in-water or water-in-oil type emulsion in the art, so no special skill acquisition is required. The stirring emulsification uses various dispersion emulsifiers widely used in the art such as propeller mixers, dispersers, homo mixers, homogenizers, colloid mills, ultrasonic emulsifiers, and the like. Among the above, the homomixer is preferable, and the homomixer is a stirring device generally used for homogeneous mixing in medicines, cosmetics, and foods, and can adjust the vesicle of the appropriate size by controlling the stirring speed and the stirring time.

In step (3), the vesicle according to the present invention is a water-in-oil emulsification which is a reverse of oil-in-water emulsification prepared by a reverse phase evaporation method (REV). It is produced in a form, which is formed by adding a small amount of an aqueous solution to a large amount of organic solvent and mixing it strongly to break the water phase into fine and numerous spherical shapes in the organic solvent phase. At this time, the phospholipids dissolved in the organic solvent form a reverse phase single film at the interface between the aqueous solution spheres and the surrounding organic solvent phase to stabilize the aqueous solution spheres. This is a reversed phase single membrane vesicle.

When the vesicle obtained in step (3) is to be manufactured in nano size, it may further include a secondary processing process. Specifically, a more uniform and smaller nano vesicle can be obtained by passing the prepared vesicle at high pressure using a high pressure homogenizer or a microfluidizer. The microfluidizer is a machine for performing emulsification using high pressure, and emulsification by cavitation and impact force caused by pressure fluctuations generated when the oil is discharged at atmospheric pressure 1 atm using a high pressure of 200 to 2000 atm. Use the principle of making particles fine.

On the other hand, the emulsification can be carried out in a variety of temperature range, for example -10 ~ 50 ℃, preferably at 0 ~ 20 ℃. In addition, the optimum conditions of the emulsification is determined according to the volume, phospholipid, solvent, etc. of the aqueous phase used in the production method.

(4) putting the vesicle of (3) into a rotary depressurization evaporator to remove the organic solvent until the vesicle emulsion is in a gel state to obtain a gel paste.

In the water-in-oil emulsification method of step (3), the vesicle is prepared in two steps. First, the membrane is formed in reverse phase, and then the outer surface is formed. The next step is to concentrate the solvent under reduced pressure. To remove it. This can be easily carried out under a vacuum condition (10 ~ 450 mmHg) with a rotary evaporator. The temperature under which the reduced pressure evaporation is determined depends on the stability of the captured bioactive components and the boiling point of the organic solvent used for emulsification. Under reduced pressure evaporation, the emulsion initially turns into a viscous gel type as an intermediate. At this time, if the volume of the aqueous phase is small or the concentration of the phospholipid in the organic solvent is high, the remaining phospholipid used to form the film is moved around the organic solvent, and the excess phospholipids in the organic solvent are initially reversed as the organic solvent is removed. Single membranes are enclosed to naturally form a multilayered phospholipid membrane. At this stage, the phospholipid monolayers enclosing each water particle are arranged in opposite directions so that a double layer is formed, and a plurality of double layers are formed on the double layer to serve as a rigid capsule to protect each water particle. . There are more than 10 double membranes thus formed, called reversed phase multilayer vesicles. The gel paste thus produced is stable and well preserved even at high temperatures.

(5) After the gel paste of (4) is added to a butter or anhydrous type, uniformly dispersed using a three-stage roller mixer, cooled, and spread to a dryer to mature.

The prepared vesicles are fixed and dispensed into butter for ease of use in anhydrous formulations.

The butters include Astrocaryum Murumuru Butter, Bassia Butyracea Seed Butter, Red Leaf Bassia Latifolia Seed Butter, Butylospermum Paki (Shea Butter) Butyrospermum Parkii (Shea Butter), Garcinia Indica Seed Butter, Goat Butter, Hydrogenated Shea Butter, Persea Gratissima (Avocado) Butter ), Shorea Robusta Seed Butter, Theobroma Cacao (Cocoa) Seed Butter, Theobroma Grandiflorum Seed Butter, Bacteria Indica Seed Butter (Vateria Indica Seed Butter).

The multi-layer reversed phase vesicle gel prepared in step (4) was added to the fully melted butter at 60-70 ° C. and stirred while maintaining the temperature. The mixed solution was evenly filtered through 200mesh to remove foreign substances, and then put into a three-stage roller mixer at about 40 ~ 50 ℃ to disperse three times. After dispersing, cool rapidly from room to room temperature. Chop it to a flat drying container so that it is about 5 ~ 10cm, and then mature it in a cool, dry place.

In addition, it is also possible to directly add to the anhydrous type to be applied in the step (5) to disperse, then cooled and extended by aging in a drier to use.

The reversed phase multilayer vesicles prepared in the steps (1) to (5) according to the present invention can be stably introduced into an anhydrous cosmetic. Examples of the anhydrous cosmetics include lipstick, lip balm, lip gloss, lip treatment, stick foundation, powder foundation, deodorant stick, hair conditioner, bath oil, body stick, essence and the like.

Hereinafter, although an Example and a test example are given and this invention is demonstrated in detail, this invention is not limited only to these examples.

[Examples 1-3]

The reverse phase multilayer vesicles of Examples 1 to 3 containing adenosine as a water-soluble physiologically active ingredient in the composition of Table 1 were prepared in the following steps.

(1) Components such as phosphatidylcholine, cholesterol, and phosphatidic acid were added to diethyl ether and completely dissolved.

(2) After raising the purified water at a temperature of about 50 ℃, adenosine was added and mixed with a paddle mixer to completely dissolve.

(3) The water phase part of (2) was put into the oil phase part dissolution tank of said (1), and it mixed for 5 minutes with the homo mixer of 3000 rpm of stirring speed, and the reverse phase vesicle was prepared first.

(4) The first formed reverse phase vesicle was placed in a high pressure homogenizer, followed by repeated treatment three times at a pressure of 600 bar to obtain a nanosized reverse phase vesicle.

(5) The resulting vesicle emulsion was placed in a rotary evaporator, and the inert organic solvent was removed until the vesicle emulsion became a gel under 37 ° C. and vacuum conditions (about 450 mmHg).

(6) The vesicle gel of (5) was added to Butyrospermum Parkii (Shea Butter) melted at 60 ° C., and then uniformly dispersed using a three-stage roller mixer.

(7) After cooling to room temperature, it was extended to mature in a drier.

division Ingredient Name Content (% by weight) Example 1 Example 2 Example 3 The upper part Phosphatidylcholine 5.00 2.50 2.00 cholesterol _ 2.50 2.50 Force Party Dick Acid _ _ 0.50 Diethyl ether to 80 to 80 to 80 Award Adenosine 2.00 2.00 2.00 Purified water to 20 to 20 to 20

[Test Example 1]

The size of the vesicles prepared in Examples 1 to 3 was determined using a particle size analyzer (Particle size analyser, Model 370 Nicomp, USA). After measuring the size of the emulsified particles three times each with the particle counter and the average value of the results and the results observed under a microscope of 600X magnification are shown in Table 2 below. In addition, the Examples 1 to 3 were observed with an electron microscope, of which the electron micrograph of Example 3 is shown in FIG. 1.

Measurement of vesicle size by particle analyzer Item Example 1 Example 2 Example 3 Particle Size Distribution 150-350 1000-15000 800-1500 Average particle size (nm) 250 4000 1200 Constellation Multilayer vesicle Multilayer vesicle Multilayer vesicle

From the results of Table 2, the distribution of particles in Examples 1 to 3 was found to be 150 ~ 350nm, 1000 ~ 15000nm, 800 ~ 1500nm, respectively, and the average size was about 250, 4000, 1200 nm respectively. In addition, all of the electron microscopic readings showed that they were multilayer liposomes, and the number of layers was found to range from 10 to 20 or more.

[Test Example 2]

In order to confirm the stability of the vesicles prepared in Examples 1 to 3, the relative humidity of 70 ± 5%, stored in a thermostat adjusted to a temperature of 25 ℃ two weeks later, after one month, three months later using a particle counter The particle size was measured. The results are shown in Table 3 below.

Vesicle stability Item Example 1 Example 2 Example 3 Right after manufacturing 250 nm 4000 nm 1200 nm after 2 weeks 280 nm 4020 nm 1200 nm 1 month later 410 nm 4200 nm 1213 nm 3 months later 1100 nm 6020 nm 1197 nm

From the results of Table 3, the 250-nm nanoscale vesicles prepared in Example 1 are thermodynamically stable and show little change in their size for two weeks, but only about one month and three months in size. It was confirmed that the fold increased. In addition, the multilayer vesicle of Example 2 was maintained in size from about 4000 nm to 6000 nm. In addition, it was confirmed that the vesicle obtained in Example 3 did not change the liposome size significantly with time, and from this, the vesicle of Example 3 showed the most excellent degree of stabilization.

Formulation Example 1 Preparation of Lipstick Added with Reversed Phase Blessicle Containing Adenosine

(1) In the composition of Example 3, the components of phosphatidylcholine, cholesterol, and phosphatidic acid were added to diethyl ether to completely dissolve them.

(2) After raising the purified water by the composition of Example 3 to about 50 ° C., adenosine was added and mixed and stirred with a paddle mixer to completely dissolve it.

(3) The water phase part of (2) was put into the oil phase part dissolution tank of said (1), and it mixed for 5 minutes with the homo mixer of 3000 rpm of stirring speed, and the reverse phase vesicle was prepared first.

(4) The resulting vesicle emulsion was placed in a rotary evaporator, and the inert organic solvent was removed until the vesicle emulsion became a gel under 37 ° C. and vacuum conditions (about 450 mmHg).

(5) In order to prepare a lipstick formulation for measuring skin penetrability, stability and safety of adenosine, the vesicle gel of (4) was added 0.04% by weight after the vesicle gel of (4) was melted at 90 ° C. After the addition, the mixture was uniformly dispersed using a three-stage roller mixer.

(6) After cooling to room temperature, it was matured by spreading widely in a dryer.

ingredient Content (% by weight) Castor oil 24.5 Caprylic / capric triglycerides 16 Octyldodecanol 17 Lanolin oil 5 Acetylated lanolin alcohol 5 Microcrystalline wax 2 Ozokerite 5 Candelilla wax 7 Carnauba wax 3 Color 15

[ Comparative formulation example  One] Reverse Vesicle  Preparation of Lipstick Containing Adenosine Without Use

(1) Into the lipstick composition of Table 4, which was melted at 90 ° C, 0.04 wt% of adenosine powder and 0.46 wt% of castor oil were added, and then uniformly dispersed using a three-stage roller mixer.

(2) After cooling to room temperature, it was matured by spreading widely in a dryer.

Test Example 3 Percutaneous Absorption Test

Percutaneous absorption was tested using Formulation Example 1 and Comparative Formulation Example 1. Percutaneous absorption was measured by using PermeGear, Inc. on skinned guinea pigs (Jackson Laboratories). Immediately before the test, the skin of the abdomen of the guinea pig, from which hair was removed, was cut and cut into an area of 1 cm 2, which was then mounted in a transmission cell having a diameter of 0.9 cm and fixed with a clamp. One side of the skin (donor container) was added 0.5 ml of the vesicles of Formulation Example 1 and Comparative Formulation Example 1. The opposite side (receptor vessel) was in contact with the solvent mixed with purified water and ethanol in a 4: 1 weight ratio, the temperature was maintained at 32 ℃ the actual skin temperature during the test. After the start of the test, a portion of the solvent was collected at regular time intervals, and then the amount of adenosine passed through the skin was measured and expressed as the amount of skin absorption (μg / cm 2 / wt%) per application concentration, and the results are shown in Table 5 below. Indicated. At this time, quantitative analysis of adenosine was performed by a gas chromatography method and a high performance liquid chromatography method, and the conditions are as follows.

[Gas Chromatography Method]

Injector: Split Ratio 1:50

Detector: FID (Flame Ionization Detector)

Column: 30m DBWAX 0.25mm LD

Column pressure: 10psi

Injector Temperature: 250 ℃

Detector temperature: 250 ℃

Oven temperature program

Start: 200 ℃

Heating rate: 4 ℃ / min, up to 250 ℃

[High speed liquid chromatography method]

Instrument: Dionex P530 pump, ASI-100 automated sample injector

Column: Phenomenex Luna 5u (C18 (2)), 150x 4.6 mm

Mobile phase: 10 mM KH2PO4: water = 92: 8 (0-6.5 min) -40: 60 (7.5-12 min)-92: 8 (12.5-15 min) gradient system

Flow rate: 0.6 ml / min

Detector: UV / Vis Detector UVD 340S 260 nm

Injection volume: 10 ul

Uptime: 25 min

Test Example 4 Human Safety Test

As a stimulus test for humans, a patch test was performed to determine whether there was a stimulus and to check if there was a stimulus and whether it could be alleviated with other sedative components. The trial was conducted by DERMAPRO Co., Ltd., a clinical institution. At least 30 people participated in the trial. The patch site was closed patch appropriate for evaluating human use tests such as the upper part of the human being (except for the midline) or forearm. In principle, the patch was removed 48 hours after the patch, waited for the loss of transient erythema by removal, and then observed and judged. The judgment was made under the responsibility of a dermatologist for more than five years.

Specifically, the patch test was conducted according to the following method. Thirty subjects were subjected to a patch test using a Fin chamber (diameter 5 mm) in normal human skin (back or forearm). A small amount of Formulation Example 1 and Comparative Formulation Example 1 were added to the pin chamber as a sample, and then attached to the skin using a scanpor tape. After the patch was applied and attached for 2 days, the patch was removed and 30 minutes later, the skin condition of the patch was read. Two days later the same site was read again. The reading standard was based on the reading standard of the patch test results recommended by the International Contact Dermatitis Research Committee of Table 6 below. Subjects were prohibited from taking any antihistamines three days before the patch test until the reading was complete (one week total). Patch tests conducted on 30 subjects were read to determine whether IR responses (irritants) and allergic reactions were generated. The results are shown in Table 7 below.

Criteria for reading test results recommended by the International Committee on Contact Dermatitis ± Medicinal 1+ Weak Positive (Non bullous) 2+ Strong positive (bullous) 3+ Super strong positive 4+ pepper

Test substance 48 hours after kill 72 hours after kill Reactivity (n = 30) ± 1+ 2+ 3+ 4+ ± 1+ 2+ 3+ 4+ 48 hours 72 hours Average Formulation Example 1 - - - - - - - - - - 0 0 0 Comparative Formulation Example 1 2 - - - - - - - - - 0.8 0 0.4

From the results of Table 7, it was confirmed that Formulation Example 1 according to the present invention has excellent safety that does not cause skin irritation even after 72 hours.

As described above, the reversed phase multilayer vesicles provided by the present invention have a larger amount of effective bioactive components and structurally enclosed effective bioactive components than the general vesicles, and are not emulsion type lipsticks. It can be conveniently applied to anhydrous formulations such as, so that the water-soluble physiologically active ingredients are collected inside, so that there is no impediment to the functionalization of the anhydrous formulation. In addition, since the reversed phase multilayer vesicle according to the present invention is protected by 10 or more membranes, it is possible to reduce the concern about instability when applying the formulation compared to the single membrane vesicle, and to penetrate into the dermis layer, thereby promoting percutaneous absorption of the bioactive active ingredient. It can be used to make it.

Claims (9)

In a vesicle having a water-soluble bioactive component as an inner phase, (1) dissolving a phospholipid component in an organic solvent to prepare an oil phase part; (2) dissolving a water-soluble physiologically active component in purified water to prepare an aqueous phase; (3) preparing an inverted vesicle by adding and mixing the aqueous phase of (2) to the oil phase of (1); (4) placing the vesicle of (3) in a rotary vacuum evaporator to remove the organic solvent until the vesicle emulsion is in a gel state to obtain a gel paste; And (5) adding the gel paste of (4) to a butter or anhydrous type, and then uniformly dispersing using a three-stage roller mixer, then cooling and spreading in a drier to mature; Reversed-phase multilayer vesicle stabilized with a water-in-oil (w / o) type of multilayer prepared by the method of. The method of claim 1, wherein the vesicle is phosphatidylcholine (PC; natural or synthetic), phosphatidic acid (Phosphatidic acid, PA), lysophosphatidylcholine (LPC), phosphatidylethanol (Phosphatidylserin (PS), phosphatidylethanol) Phosphhatidylethanolamine (PE), Sphingolipids, Phosphatidylglycerol (PG), Sphingomyelin, Cardiolipin, Glycolipids, Gangliosides (Ganglioside) A vesicle comprising a membrane made of cerebrosides and sole or phospholipids selected from the group consisting of soybean phospholipids. 3. The phospholipid component according to claim 2, wherein the phospholipid component includes cholesterol, aliphatic chain amine having 10 to 24 carbon atoms, aliphatic chain carboxylic acid having 10 to 24 carbon atoms, aliphatic chain sulfuric acid having 10 to 24 carbon atoms, and carbon number 10 And a membrane comprising a mixture of two or more components selected from the group consisting of aliphatic chain phosphoric acid, dicetyl phosphate, butylated hydroxytoluene (BHT), tocopherol, retinol, and isoprenoid compounds of ˜24; Vesicles. 3. The method of claim 2, wherein the vesicles comprise: adding a hydroxy group to the aliphatic chain; Branched; Has a ring structure; Aromatic derivatives; Have ether or amide bonds; Having multiple unsaturated double bonds; Halogen derivatives; Carbohydrate, Glycol, Phosphate, Phosphate, Phosphonate, Quaternary amine, Sulfate, Sulfonate, Carboxy, Amine, Attaching a hydrophilic group selected from the group consisting of sulfhydryl, and imidazole; And a combination of the above hydrophilic groups in a phospholipid component or mixed with the vesicle. delete The vesicle according to claim 1, wherein the size of the vesicle is 800 to 1500 nm. delete A cosmetic composition comprising the reversed phase multilayer vesicle of any one of claims 1 to 4 and 6. The cosmetic composition of claim 8 wherein the cosmetic composition is selected from the group consisting of lipstick, lip balm, lip gloss, lip treatment, stick foundation, powder foundation, deodorant stick, hair conditioner, bath oil, body stick and essence A composition characterized in that.

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