KR101627051B1 - Natural liposome comprising vitamin C, process for the preparation thereof, and cosmetic composition comprising the same - Google Patents

Abstract

The present invention relates to a natural liposome comprising vitamin c, a producing method thereof, and a cosmetic composition comprising the same. The natural liposome comprises: at least one natural emulsifier, a solvent, and a natural antiseptic as a liposome structural material; and an indian gooseberry extract as a collecting material. In addition, the natural liposome comprises 2 to 9 wt% of the natural emulsifier, 1 to 5 wt% of the natural antiseptic, 0.01 to 5 wt% of the indian gooseberry extract and the residual solvent with respect to the natural liposome total weight, and is formed by mixing and sonicating the natural emulsifier, the solvent, and the indian gooseberry extract.

Description

[0001] The present invention relates to a natural liposome containing vitamin C, a process for producing the same, and a cosmetic composition containing the same,

The present invention relates to a natural liposome containing vitamin C, a process for producing the same, and a cosmetic composition containing 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.

Skin is always exposed to oxygen and exposed to a variety of external environmental factors that directly affect the attack of oxidative stressors such as ultraviolet rays, bacteria, and pollution. Thus, skin exposed to sunlight generates active oxygen due to the effect of ultraviolet rays. Of these active oxygen species, O ₂ and OH play an important role in skin photodamage, leading to destruction of skin antioxidants, initiation of lipid peroxidation reaction, protein and DNA oxidation, chain cleavage of connective tissue components and wrinkle formation by non- , Melanin production process and accelerate skin aging.

To protect cells and tissues against oxidation, skin has built up a skin antioxidant defense network consisting of antioxidant enzymes such as SOD, catalase, and glutathione peroxidase, and non-enzymatic antioxidants such as α-tocopherol and ascorbic acid.

However, continuous exposure to ultraviolet light results in the formation of excess reactive oxygen species and their active oxygen substantially compromises the enzymatic and non-enzymatic antioxidant defense of the skin. Therefore, development of antioxidant suitable for skin antioxidant defense network is considered to be very necessary to suppress and retard skin aging.

Vitamin C (ascorbic acid) is one of the antioxidants that help skin to be antioxidant. Vitamin C is an antioxidant that is useful for the prevention and treatment of colds, various cancers and heart diseases, and has various physiological activities such as skin whitening, collagen synthesis, lipid oxidation inhibitory effect. However, vitamin C is easily dissolved in water and oxidized and denatured, and it is very weak to light or heat and has a drawback that it is easy to decompose. In addition, when applied to cosmetics, there is a problem that the stability is poor, such as a change in color or smell, and the hydrophilicity is high, and the distribution to the skin is low, so that the percutaneous absorption is difficult. As described above, vitamin C exhibits various effects, but its stability is low and its absorption into the skin is not good.

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.

Liposomes are capable of collecting aqueous solutions or oil-soluble substances and thus help to transport these materials. For example, liposomes that are sensitive and difficult to absorb into the skin can be transported to the liposomes to help them absorb the ingredients 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. 949848 discloses a nano-emulsified cosmetic composition containing a vitamin C derivative and a method for producing the same. Also, Japanese Patent Laid-Open No. 2000-0091162 discloses a cosmetic composition containing a nanoemulsion containing a vitamin C derivative and a liposome.

However, none of these prior art discloses at all about natural vitamin-containing materials with high vitamin C content, and natural liposomes containing natural preservatives and natural solvents.

Korean Patent No. 949848 Korea Patent Publication No. 2013-25988

1. V. M. Adhami, D. M. Syed, N. Khan, and F. Afaq, Phytochemicals for prevention of solar ultraviolet radiation induced damages, Photochem. Photobiol., 84 489 (2008) 2. F. Afaq and H. mukhtar, Botanical antioxidants in the prevention of photocarcinogenesis and photoaging, Exp. Dermatol., 15, 678 (2006) 3. G. T. Bowden, Prevention of non-melanoma skin cancer by targeting ultraviolet-V-light signaling, Nat. Rev. Cancer., 4, 23 (2004) 4. S. N. Park, Antioxidative Properties of Baicalein, Competet from Scutellaria baicalensis Georgi and Its Application to Cosmetics (I), J. Korean Ind. Eng. Chem., 14 (5), 657 (2003) 5. S. N. Park, Protective Effect of Isoflavone, Genistein from Soybean on Singlet Oxygen Induced Photochromolysis of Human Erythrocytes, Korean. J. Food Sci. Technol., ≪ / RTI > 35 (3) 510 (2003) 6. SH Park, DM Cho, BD Choi, YJ Choi and JH Choi, Antioxidative Effects of Skinned Mufwort (Artemisia vulgaris L.) Extracts on UV-Irradiated Hairless Mouse, J. Korea Soc Food Sci Nutr 37 20-26 (2008)

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

One embodiment of the present invention is a natural liposome comprising an extract of Indian Gooseberry as a liposome constituent material and a captive material containing at least one natural emulsifier, a solvent and a natural preservative, wherein the natural emulsifier comprises, Wherein the natural emulsifier, the solvent, and the Indian gooseberry extract are mixed by ultrasonic treatment to form a mixture of the natural emulsifier, the solvent and the indian gooseberry extract, Liposomes are provided.

Another embodiment of the present invention provides a cosmetic composition comprising the natural liposome as an active ingredient.

In another embodiment of the present invention, there is provided a method for preparing a natural product, comprising the steps of: mixing at least one natural emulsifier with a solvent and sonicating at 50 to 80 캜; adding an Indian gooseberry extract to the product of the first step, And a third step of adding a solvent warmed to 40 to 60 캜 and a natural preservative to the product of the second step and subjecting the resulting product to ultrasonic treatment to form a liposome.

Another embodiment of the present invention provides a skin absorption promoter containing the natural liposome, and an antioxidant comprising the natural liposome as an active ingredient.

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.

1 shows a process for preparing a natural liposome according to an embodiment of the present invention.
Figs. 2A, 2B and 2C are SEM photographs (magnification of 50,000 times) of the natural liposome containing vitamin C of Example 1, the vitamin C-containing mixture of Comparative Example 1 and the encapsulated vitamin C of Comparative Example 2, respectively.
Figs. 3A, 3B, and 3C show particle size distribution graphs of the natural liposome containing vitamin C of Example 1, the vitamin C-containing mixture of Comparative Example 1, and the vitamin C-containing synthetic liposome of Comparative Example 2, respectively.
Fig. 4 is a graph showing the absorption rate of distilled water, the natural liposome containing vitamin C of Example 1, the vitamin C-containing mixture of Comparative Example 1, and the synthetic liposome containing vitamin C of Comparative Example 3.
FIG. 5A shows the vitamin C content of the natural liposome containing vitamin C of Example 1, the vitamin C-containing mixture of Comparative Example 1, and the vitamin C-containing synthetic liposome of Comparative Example 3 after 24 hours, And the vitamin C content of the skin tissue after the absorption.
6 is a graph showing the glutathione content of cells treated with distilled water, the natural liposome containing vitamin C of Example 1, the vitamin C-containing mixture of Comparative Example 1, the capsule vitamin C of Comparative Example 2, and the vitamin C-containing synthetic liposome of Comparative Example 3 .
7 shows the intracellular ATP content of cells treated with distilled water, the natural liposome containing vitamin C of Example 1, the vitamin C-containing mixture of Comparative Example 1, the capsule vitamin C of Comparative Example 2, and the vitamin C-containing synthetic liposome of Comparative Example 3 Graph.
8 is a graph showing the oxygen stability of a natural liposome containing vitamin C of Example 1. Fig.
9 is a graph showing the thermal stability of the natural vitamin C-containing liposome of Example 1. Fig.
Fig. 10 is a photograph of the external appearance of UV-damaged mice of the natural liposome containing vitamin C of Example 1, the vitamin C-containing mixture of Comparative Example 1 and the vitamin C-containing synthetic liposome of Comparative Example 3 for 4 weeks.
Fig. 11 shows the amount of hydrogen peroxide generated in the skin tissue of the UV-damaged mouse to distilled water, the natural liposome containing vitamin C of Example 1, the vitamin C-containing mixture of Comparative Example 1, and the vitamin C-containing synthetic liposome of Comparative Example 3.
Fig. 12 shows the amount of carbonyl groups in the skin tissue of UV-damaged mice to distilled water, the natural liposome containing vitamin C of Example 1, the vitamin C-containing mixture of Comparative Example 1, and the vitamin C-containing synthetic liposome of Comparative Example 3.
Fig. 13 is a graph showing the effect of UV-damaged natural liposomes of ultraviolet-damaged mice on the ultraviolet-damaged mouse skin of distilled water, the natural liposome containing vitamin C of Example 1, the vitamin C-containing mixture of Comparative Example 1 and the vitamin C- Indicates lipid peroxide content in tissues.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, a natural liposome containing vitamin C 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. Hydrogenated phosphatidylcholine is more preferable as a raw material for cosmetics which is superior in oxidation stability and has a decolorizing and deodorizing effect as compared with phosphatidylcholine containing an unsaturated fatty acid and which requires long-term stability.

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.

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, the amount of the first emulsifier component may be 1 to 5 wt%, and the amount of the second emulsifier component may be 0.5 to 2 wt%.

Natural fatty acids may also be used together with these. Evening primrose oil may be used as a natural fatty acid. Preferably, the natural fatty acid may be contained in an amount of 1 to 10% by weight.

2. Collecting material

A material containing vitamin C is used as a capturing substance that can be captured inside the phospholipid layer of the liposome. Vitamin C is also made by synthesis, but it is also found in many natural plants. Specifically, it is preferable to use an extract of Indian gooseberry as a capturing substance.

The Indian gooseberry is a health nutrient found in the traditional Indian medicine Ayubeda, which is rich in vitamin C (10 times lemon) and polyphenol (30 times red). Indian gooseberry is Phyllanthus ( Phyllanthus) It is also known as emblica, amla, amalika and contains more than 20 times more vitamin C than orange. The Indian gooseberry has anti aging, moisturizing protection, collagen protection, inflammation and allergy relief It is also resistant to heat and prevents the decomposition of chemical components, which can be used as a natural product to compensate for the shortcomings of vitamin C.

The vitamin C containing Indian gooseberry extract may be included in an amount of 0.01 to 5% by weight based on the whole liposome.

The vitamin C content of the Indian gooseberry extract is, for example, 50%.

The Indian gooseberry extract contains 50% vitamin C. It is known to have anti-aging, moisturizing protection, collagen protection, inflammation and alleviation of allergic effects by the effect on skin health. It is also resistant to heat and prevents the decomposition of chemical components, so it can be used as a natural product to overcome the shortcomings of vitamin C.

3. Solvent

As the solvent, distilled water, naturally occurring butylene glycol, propanediol, glycerin, fermented alcohol can be used, and distilled water can be preferably used. The content of the solvent is the residual amount of the emulsifier, the trapping agent, 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. The preservative may generally be included in an amount of 1 to 5% by weight.

5. Preparation of liposome

The liposome according to the present invention can be produced using ultrasonic waves.

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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.

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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, in the form of a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, cleansing oil, foundation, More specifically, 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 essence , A makeup base, a foundation, a hair dye, a shampoo, a rinse, a body cleansing agent, and a mask sheet. Indian gooseberry extract can also be used as a natural hair nutrient in hair care products such as hair oils and hair conditioners.

<Examples>

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

content Raw material name Example 1
(weight%) Comparative Example 1
(weight%) Emulsifier Emulsifier 1 2.5 0.0 Emulsifier 2 1.0 0.0 antiseptic 1.0 1.0 menstruum Distilled water (D. I. WATER) Balance Balance Capture material Vitamin C 0.02 0.02

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)

Preservative: Pine nuts fruit extract: Rhizome extract: Earth nere extract in a ratio of 1: 1: 1

Vitamin C: Indian gooseberry extract powder

The specific manufacturing process is as follows.

Emulsifier 1 and emulsifier 2 were mixed with 1/3 of distilled water and sonicated at 65 ° C using an ultrasonic disrupter (Ultrasonic Liquid Processor, QSONICA, USA). When the emulsifier was homogenized, the Indian gooseberry extract powder was added, followed by ultrasonic disruption. When the emulsifier and the Indian gooseberry extract powder were completely homogenized, distilled water (2/3) heated to 50 ° C and 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 to make the particles smaller and uniform. Finally, vitamin C-containing natural liposomes were prepared. The manufacturing process is shown in Fig.

&Lt; Comparative Example 1 &

An orange gooseberry mixture, i.e., a vitamin C-containing mixture, which was not liposomized, was prepared by mixing the extract of the Indian gooseberry extract powder with the same concentration as that of Example 1, preservative, and remaining amount of distilled water.

&Lt; Comparative Example 2 &

Commercially available encapsulated vitamin C ((SYAY-C 50, KS CHEM) was used as Comparative Example 2.

&Lt; Comparative Example 3 &

(Vitamin C-liposome-Konzentrat dermaviduals®) containing vitamin C in the form of a solution containing purified water, pentyleneglycol, alcohol, glycerin, sorbitol, sodium ascorbyl phosphate, lecithin, citric acid, Was used as Comparative Example 3. &lt; tb &gt;&lt; TABLE &gt;

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

The particle shape and size of the vitamin C-containing natural liposome of Example 1 were analyzed using a scanning electron microscope (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.

The natural liposome of Example 1, the vitamin C mixture of Comparative Example 1, and the capsule vitamin C of Comparative Example 2, which had captured the vitamin C, were confirmed by a scanning electron microscope (SEM), and then SEM photographs were taken. The results are shown in FIGS. , And FIG. 2C (magnification of 50,000 times).

As shown in the figure, the vitamin C-containing natural liposome according to Example 1 has a spherical shape and the particles are relatively uniformly distributed (FIG. 2A), whereas the extract of the Indian gooseberry according to Comparative Example 1 has a rugged shape (FIG. 2B), and the capsule vitamin C according to the comparative example 2 had a square particle shape and a particle size of about 1 μm (FIG. 2C). It was confirmed that the particle size of the liposome according to Example 1 was smaller than that of Comparative Examples 1 and 2 by comparing 5x10 ⁴ magnification liposome photographs.

<Test Example 2> Size analysis of liposome particles

The particle size of the sample was measured by using a laser particle size analyzer (Electrophoretic Light Scattering Spectrophotometer ELS-8000, Otsuka) and the particle size distribution was analyzed. The average particle size is shown in Table 2, and the particle size distribution is shown in Fig.

Average particle size (nm) Example 1 135.6 Comparative Example 1 2058.3 Comparative Example 2 6390.8

As shown in Table 2 and FIG. 3, the average particle size of the vitamin C-containing natural liposome of Example 1 was 135.6 nm, which was smaller than the vitamin C mixture of Comparative Example 1 and the capsule vitamin C of Comparative Example 2. The vitamin C-containing natural liposome of Example 1 (Fig. 3a) was more uniform and stable than the vitamin C mixture of Comparative Example 1 (Fig. 3b) and Comparative Example 2 of capsule vitamin C (Fig. 3c).

&Lt; Test Example 3 > Measurement of collection rate

A certain amount of the completed suspension of vitamin C-containing natural liposomes was taken and a non-captured substance in the liposome was removed using a 0.45 μm syringe filter. Ethanol was then added to the suspension through the filter to destroy the liposome membrane. The content of collected vitamin C was quantitatively analyzed. The collection rate was calculated by the following formula.

(%) = Concentration of the functional substance passing through the filter / Concentration of the first functional substance * 100

The collection rates are shown in Table 3 below.

Collection rate (%) Example 1 98.2 Comparative Example 3 54.4

As a result of measurement, the vitamin C-containing natural liposome of Example 1 showed a high capture ratio of 98.2%, and the synthetic vitamin C liposome of Comparative Example 3 had a capture ratio of 54.4%, which was lower than that of Example 1. In Comparative Examples 1 and 2, the charging efficiency was not measured since it was not the collected raw material.

&Lt; Test Example 4 > - In vitro skin absorption test using black mouse skin tissue

Skin permeation experiments were conducted using a Franz diffusion cell to determine the effect of vitamin C-containing natural liposome (Example 1) on Indian gooseberry on skin permeation enhancement. In the experiment, black mouse skin tissue (C57BL / 60) was used to remove hair. Black mouse skin tissue was fixed between the donor and receptor phases. 1 ml of sample 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. The absorbance of a sample collected at predetermined time intervals at 235 nm, which is the maximum absorption wavelength of Indian gooseberry, which was previously measured by absorbance scanning, was measured and the results are shown in FIG.

Absorbance measurement result

As shown in FIG. 4, the natural liposome containing vitamin C of Example 1 is higher in absorbance than the distilled water, the Indian gooseberry of Comparative Example 1 and the vitamin C-containing synthetic liposome of Comparative Example 3, that is, And the absorbency was better even after the lapse of time.

pH measurement result

The pH of the lower layer in which vitamin C was absorbed was measured with a pH meter and is shown in Table 4 below.

sample pH Distilled water 7.27 Comparative Example 1 7.09 Example 1 6.77

The pH was compared between the samples taken from the Franz diffusion cell receptor phase after absorption of distilled water, the natural gooseberry of Example 1, and the natural liposome containing vitamin C for 24 hours. As a result, distilled water and the Indian gooseberry of Comparative Example 1 showed neutral , But it was confirmed that the vitamin C-containing natural liposome of Example 1 exhibits weak acidity. This indirectly confirmed that the natural liposome containing vitamin C of Example 1 was more absorbed than the mixture containing distilled water and vitamin C of Comparative Example 1.

The content of absorbed vitamin C was measured

The receptor phase and mouse tissue were recovered to confirm the amount of vitamin C absorbed and the results are shown in FIGS. 5A and 5B. It was also confirmed that the natural liposome containing vitamin C of Example 1 was more absorbed than the vitamin C-containing mixture of Comparative Example 1 and the vitamin C-containing synthetic liposome of Comparative Example 3 through this.

&Lt; Test Example 5 > The glutathione e content

6 × 10 ⁴ fibroblast cells were dispensed and replaced with fresh medium after 24 hours. Then, distilled water, the Indian gooseberry mixture of Comparative Example 1, and the natural vitamin C-containing liposome of Example 1 were mixed at a concentration of 1% for 48 hours Respectively. After 48 hours of treatment, the cells were removed from the culture medium. Cells were recovered and disrupted by sonication. Cell lysate was obtained by salicylic acid treatment. The intracellular glutathione (GSH) content was confirmed using a glutathione assay kit (Sigma-aldrich).

The extract of Indian gooseberry powder of Comparative Example 1, the encapsulated vitamin C of Comparative Example 2, the natural liposome containing vitamin C of Example 1, and the prior art synthetic vitamin C liposome of Comparative Example 3 were treated with the antioxidant (Glutathione), and the results are shown in Fig.

As shown in FIG. 6, the content of glutathione in the cells treated with Indian gooseberry of Comparative Example 1 was similar to that of the distilled water treatment group. However, the glutathione content of cells treated with vitamin C-containing natural liposome of Example 1 was 1.5 times higher than that of the Indian gooseberry-treated cells, and compared with the capsule vitamin C of Comparative Example 2 and the vitamin C synthetic liposome of Comparative Example 3 , Respectively.

&Lt; Test Example 6 > Confirmation test for intracellular ATP content

In order to measure the cellular activity of vitamin C - containing natural liposomes, the content of ATP, which plays an important role in energy metabolism, was measured by releasing free energy of living things. After 5 hours, fibroblast cells (5 × 10 ⁴ ) were dispensed into 6 wells. After 24 hours, the cells were replaced with fresh medium. Distilled water, 0.1% concentration of Indian gooseberry, Comparative Example 1, Vitamin C containing natural liposome, Of the encapsulated vitamin C and the vitamin C-containing synthetic liposome of Comparative Example 3 were treated for 48 hours. After removing the culture medium for 48 hours, only the cells were recovered, disrupted by ultrasonication, centrifuged, and the supernatant was recovered. The intracellular ATP content was confirmed using ATP Assay Kit (Colorimetric / Fluorometric) (abcam) and the results are shown in Fig.

As shown in FIG. 7, the ATP content of the extract of the Indian gooseberry extract of Comparative Example 1, the encapsulated vitamin C of Comparative Example 2, and the synthetic vitamin C liposome of the prior art of Comparative Example 3 showed no significant difference from the distilled water . However, it was confirmed that the vitamin C-containing natural liposome of Example 1 significantly increased the content of ATP.

&Lt; Test Example 7 > Oxidation and thermal stability test

In order to examine the stability of vitamin C-containing natural liposome of Example 1 against the external environment, oxidation and heat stability experiments were conducted.

1) Oxidation stability test

0.003% and 0.03% hydrogen peroxide solution (30% (w / w)) was added to each of the natural liposome containing vitamin C of Example 1, the encapsulated vitamin C liposome (stay-c 50, DSM) of Comparative Example 2, and the Indian gooseberry of Comparative Example 1, (SIGMA, USA) 0.05 ml of 85% phosphoric acid (MATSUNOEN CHEMICALS LTD., JAPAN) and 8% 2,2'-Bipyridyl (SIGMA, USA) ml, and 3% ferric chloride hexahydrate (JUNSEI, JAPAN) were added to the wells, and the mixture was allowed to stand at room temperature for 1 hour to produce ferrous dipyridyl chromophore, and the absorbance was measured at 525 nm. The additives and the addition amounts are shown in Table 5 below.

division Contents A The Indian gooseberry of Comparative Example 1 A-1 The addition of 0.003% of the Indian gooseberry-hydrogen peroxide of Comparative Example 1 A-2 0.0 &gt;% &lt; / RTI &gt; of the Indian gooseberry-hydrogen peroxide of Comparative Example 1 B The vitamin C-containing natural liposome of Example 1 B-1 The natural liposome containing vitamin C in Example 1-added with 0.003% hydrogen peroxide B-2 0.03% addition of natural liposome-hydrogen peroxide containing vitamin C of Example 1 C The encapsulated vitamin C of Comparative Example 2 C-1 The encapsulated vitamin C of Comparative Example 2-added with 0.003% hydrogen peroxide C-2 0.03% of encapsulated vitamin C-hydrogen peroxide of Comparative Example 2

As a result of measurement of absorbance, as shown in FIG. 8, in the case of the Indian gooseberry of Comparative Example 1, when the hydrogen peroxide was added at 0.03%, all the vitamins were oxidized, but the vitamin C-containing natural liposome of Example 1 was oxidized It was confirmed that the oxidation stability was higher than that of Comparative Example 1 and Comparative Example 2.

2) Thermal Stability Test

Vitamin C In order to confirm the thermal stability of the natural liposome, the natural liposome containing vitamin C of Example 1, the Indian gooseberry of Comparative Example 1 and the encapsulated vitamin C (stay-c 50, DSM) of Comparative Example 2 were diluted 110 Lt; 0 &gt; C for 1 hour and then cooled naturally. After completion of the cooling, the DPPH assay was used to confirm the antioxidant activity. For the DPPH assay, DPPH (Diphenyl-1-picrylhydrazyl, sigma) was used. For comparison, 0.05 wt% of the synthetic antioxidant 2,6-ditert-butyl-4-methylphenol (BHT) was tested in the same manner. 50 μl of each sample was added to 96 wells, and 250 μl of DPPH reagent was added. After 30 minutes of reaction, absorbance was measured at 528 nm using an ELISA reader. The additives and the addition amounts are shown in Table 6 below.

division Contents BHT Synthetic antioxidants (comparison group) A (before heating) The Indian gooseberry of Comparative Example 1 - before heating A (after heating) Indian gooseberry of Comparative Example 1 - after heating B (before heating) The Indian gooseberry natural liposome of Example 1 - preheated B (after heating) After the Indian gooseberry natural liposome-heating of Example 1 C (before heating) The encapsulated vitamin C of Comparative Example 2 - before heating C (after heating) The encapsulated vitamin C of Comparative Example 2 - after heating

As shown in FIG. 9, the antioxidative capacity of the Indian gooseberry of Comparative Example 1 was slightly reduced compared to that before heating, whereas the vitamin C-containing natural liposome of Example 1 showed a slight increase in antioxidant activity after heating appear. As a result, it can be seen that the vitamin C-containing natural liposome of Example 1 is more thermally stable than the Indian gooseberry of Comparative Example 1. The encapsulated vitamin C of Comparative Example 2 lost the antioxidant ability after heating. As a result, it was confirmed that the natural liposome containing vitamin C of Example 1 was excellent in heat stability (Fig. 9).

&Lt; Test Example 8 > Effect of vitamin C-containing natural liposome on UV-damaged skin

In order to confirm the skin damaging activity of the vitamin C-containing natural liposome of Example 1, animal experiments were conducted using damaged skin after irradiation with ultraviolet rays.

1) Experimental animals and experimental procedures

Five week old male C57BL / 60 mice (22-24 g) were adapted for one week and used in animal studies. During the test period, feed and water were freely consumed, and the temperature of the breeding room (22 ± 2 ℃), relative humidity (55 ± 5%) and intensity were maintained for 12 hours.

Three rabbits were used for each group. The UV-irradiated mice were removed with an animal clipper (THRIVE S-138A), and then the hair cream (Veet Creme Depilatoire) was applied. After 1 minute, the hair was wiped with alcohol cotton Respectively. UVB was irradiated to the rest of the groups except for the normal group at a certain time of day. Light irradiation dose was 60 mJ corresponding to 1 MED for the first week, followed by 100 mJ for 3 weeks from 2 weeks to 4 weeks For 6 weeks, for a total of 4 weeks. Experiments were carried out in the same manner as in Example 1, except that the UV and experimental groups were not treated, the UV treated, and the samples were applied to the distilled water group, the Indian gooseberry containing mixture treated group of Comparative Example 1, the vitamin C containing natural liposome treated group of Example 1, And then irradiated with 0.3 ml of the irradiated vitamin C. The results are shown in Table 8 below. The results are shown in Table 8 below.

First week Week Two The third week Week Four UVB 1MED (60mJ) (daily)
300 μ sample treatment (daily) UVB 100 mJ (6 days)
300μ sample treatment (daily)

2) External appearance of experimental animals

To observe the symptoms of UV rays damage, the external appearance of mouse skin was photographed using a digital camera (Canon IXUS 100 IS). Fig. 10 shows a photograph of the appearance of dermatitis-induced mice. There was no significant difference between normal group and 4th week, but skin damage was observed in the group treated with ultraviolet light. In the UV distilled water test group, the improvement of the skin damage was slower from 1 to 4 weeks. The vitamin C of Comparative Example 1 showed similar pattern to that of distilled water until the fourth week. It was confirmed that the UV damage was gradually alleviated in the case of the vitamin C-containing natural liposome of Example 1, and it was confirmed that the vitamin C-containing natural liposome recovered faster than the encapsulated vitamin C of Comparative Example 2.

3) Preparation of skin homogenate

To evaluate the effect of vitamin C-containing natural liposomes on UV damage, the skin of the dorsal skin was recovered by lightly anesthetizing with ether after the last 12 hours of the experiment. The skin tissue was washed with a buffer solution (1.15% KCl / 10 mM phosphate buffer containing 5 mM EDTA) at pH 4 at 4 ° C and subcutaneous fat was removed to make a skin homogenate. In other words, the skin tissue was homogenized by applying a buffer solution corresponding to 10 times of the skin tissue, using an ultrasonic sonicator, and then centrifuged at 3,000 xg for 15 minutes to obtain a skin tissue homogenate.

4) Measurement of protein content

The total protein content of the dermal tissue homogenate was measured using the BCA method. 80 μL of BCA solution (Copper Ⅱ sulfate: Bicinchoninicacid = 1: 50) was added to 10 μL of the sample and allowed to stand at 37 ° C. for 30 minutes. Absorbance was measured at 562 nm using an ELISA reader. The standard curve was bovine serum albumin (Sigma) Were used to calculate the content of the calibration curve. And the protein content of each sample was adjusted to 200 μg / μl. Dilution was performed using a buffer solution (1.15% KCl / 10 mM phosphate buffer containing 5 mM EDTA, pH 7.4) and the same diluted dermal tissue homogenate was used in the experiment.

5) Confirmation of production of hydrogen peroxide (H ₂ O ₂ )

The production of active oxygen species was confirmed by confirming the amount of hydrogen peroxide directly involved in the oxidation of the tissue. Based on the ferrithiocyanate complex of red color produced by the skin tissue, 400 μL of 400 mM phosphate buffer (pH 7.4), 200 μL of 200 mM nicotinamide, 200 μL of 100 mM MgCl 2, 200 μL of 50 mM NaN ₃ , 64.1 μL of dermal tissue homogenate, 735.9 μL was added and mixed, and then 200 μL of 60 mM NADPH was added. After incubating for 30 minutes in a constant temperature water bath at 37 ° C, 1 mL of 1.2 M trichloroacetic acid (TCA) solution was added to stop the reaction and centrifuged (3000 xg, 10 min) to take the supernatant. To 1 mL of the supernatant, 200 μL of ferrous ammonium and 100 μL of 2.5 M potassium thiocyanate were added, mixed, and reacted at room temperature for 10 minutes. The absorbance of the reaction solution was measured at 480 nm and the content of hydrogen peroxide (nM / mg-protein / min) was measured using a standard curve prepared with hydrogen peroxide.

Oxidative stress was induced in the dorsal skin of mice using UV and the amount of hydrogen peroxide produced in the skin tissue was compared.

As shown in FIG. 11, the amount of hydrogen peroxide generated in the group treated with distilled water after UV treatment was increased by 1.5 times or more than the amount of hydrogen peroxide produced in the untreated group. 164.5% of the indian gooseberry treated group of Comparative Example 1 and 158.1% of the encapsulated vitamin C treated group of Comparative Example 2, respectively. In contrast, in the case of the natural vitamin C-containing natural liposome of Example 1, 90.3% A similar amount of hydrogen peroxide production could be confirmed.

6) Comparison of carbonyl group production

The production of oxidized protein was measured by the amount of carbonyl group which is the decomposition product of peroxides or monosaccharides. Add 0.5 mL of 30% TCA to 0.1 mL of sample, mix well and centrifuge (800 × g, 10 minutes) to remove supernatant. Add 0.5 mL of 10 mM dinitrophenyl hydrazine (DNPH) (800 x g, 10 min) after allowing to stand at room temperature for a period of time. To the residue was added 3 mL of ethanol / ethyl acetate (1: 1, v / v), mixed well, left at room temperature for 10 minutes, centrifuged (800 × g, 10 minutes) and 6 M guanidine / 20 1 mM potassium phosphate (pH 2.3) solution was added thereto. The carbonyl production of the supernatant obtained by incubation in a constant temperature water bath at 37 ° C for 30 min and centrifugation (800 × g, 10 min) was measured at 360 nm and compared.

As shown in FIG. 12, the groups of UV treated skin treated with 100% of the untreated skin were 110.46%, respectively, and the amount of carbonyl produced was higher In the case of the Indian gooseberry group of Example 1, the amount of carbonyl group was 103.05%. On the other hand, the vitamin C-containing natural liposome of Example 1 showed 92.42% lower than the untreated group, indicating that the amount of carbonyl group was the lowest. That is, it appeared that the carbonyl group, which is a decomposition product of peroxide, appeared to be less. Also, the value was lower than that of the synthetic liposome of Comparative Example 3, and it was predicted that the UV damage was alleviated through the natural liposome of Example 1.

7) Confirm the content of lipid peroxide

We measured the lipid peroxide concentration, which is used as an index of oxidative stress in skin tissue, to determine how much skin damage is caused by UV - induced oxidative stress. The lipid peroxidation content of the homogenate fraction of the skin tissue was determined by adding 2 ml of thiobarburic acid (TBA) reagent to 1 ml of the sample to be measured, reacting in boiling water for 30 minutes, cooling the mixture at room temperature, centrifuging at 3,000 rpm for 10 minutes, Absorbance was measured at 535 nm. The content of lipid peroxide was expressed as nmol / g of malondialdehyde.

As shown in FIG. 13, the lipid peroxidation in the skin tissue was lowest at 61.41 nmol / ml in the untreated group and 66.30 nmol / ml in the group treated with distilled water after UV treatment, which was higher than that of the untreated group I could. In addition, the content of 68.38 nmol / ml was higher in the Indian gooseberry-treated group of Comparative Example 1 than in the untreated group and did not show a significant effect on lipid peroxidation by UV treatment. On the other hand, in the case of the natural vitamin-C-containing liposome of Example 1, the lipid content was 62.76 nmol / ml, and the lipid peroxidation content of the liposome of Comparative Example 3 was 63.80 nmol / ml But higher than the vitamin C-containing natural liposome of Example 1.

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 (13)

1. A natural liposome comprising an extract of Indian gooseberry as a liposome constituent material and a capturing material containing at least one natural emulsifier, a solvent and a natural preservative,
2 to 9% by weight of the natural emulsifier, 1 to 5% by weight of the natural preservative, 0.01 to 5% by weight of the extract of the Indian gooseberry, and the residual solvent amount based on the total weight of the natural liposome,
Wherein the natural emulsifier, the solvent, and the Indian gooseberry extract are ultrasonicated and mixed. The method according to claim 1,
Wherein said natural emulsifier comprises at least one phospholipid selected from the group consisting of phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine, hydratidylated phosphatidylcholine, phosphatidylic acid, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, and their hydrogenation products Lt; / RTI &gt; 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 solvent is at least one solvent selected from the group consisting of distilled water, natural-derived butylene glycol, propanediol, glycerin and fermented liquor. The method according to claim 1,
Wherein the preservative is at least one natural-derived extract selected from the group consisting of a superfine fruit extract, a honeycomb extract, and an Earther extract. The method according to claim 1,
The solvent is distilled water. The method according to claim 1,
Wherein said 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. The method according to claim 1,
Natural liposomes for antioxidants. A cosmetic composition comprising the natural liposome of claim 1 as an active ingredient. A first step in which one or more natural emulsifiers are mixed with a solvent and then subjected to ultrasonic treatment at 50 to 80 ° C,
A second step of adding an Indian gooseberry extract to the product of the first step,
A third step of adding a solvent warmed to 40 to 60 캜 and a natural preservative to the product of the second step and forming a liposome by ultrasonication
9. A method for producing a natural liposome according to any one of claims 1 to 8, 11. The method of claim 10,
The fourth step of performing ultrasonic processing while gradually lowering the temperature after the third step
&Lt; / RTI &gt; A skin absorption promoter comprising the natural liposome of claim 1. An antioxidant comprising the natural liposome of claim 1 as an active ingredient.

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