KR101601954B1 - Method for preparation of poor soluble materials containingnanoparticle for cosmetic composition - Google Patents

Abstract

The present invention relates to a nanoparticle having a physiologically active function but not easily dissolvable in a material such as water or oil and being easily destroyed by external stress such as heat, light or oxygen, A method for producing the nanoparticles, and a cosmetic composition containing the nanoparticles, which are capable of preventing loss of potency of an active ingredient while being easily applied to cosmetics manufacturing.
Since the nanoparticles according to the present invention are formed in a nanoscale size that contains a high amount of a poorly soluble active substance having a low solubility in a low temperature solvent and easy to be percutaneously absorbed, it is possible to improve the transdermal absorption rate The active ingredient, which is easily oxidized and destroyed by external factors such as heat, light, oxygen and the like, is protected from external factors by a coating film which surrounds the nanoparticles, thereby improving stability and content stability , Since the solvent is removed by drying, the active ingredient can be prevented from being oxidized by the dissolved oxygen dissolved in the solvent, so that loss of activity is prevented, and it is easily dispersed in water phase and excellent in dispersion stability. Lt; / RTI >

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a nanoparticle-

The present invention relates to a nanoparticle having a physiologically active function but not easily dissolvable in a material such as water or oil and being easily destroyed by external stress such as heat, light or oxygen, A method for producing the nanoparticles, and a cosmetic composition containing the nanoparticles, which are capable of preventing loss of potency of an active ingredient while being easily applied to cosmetics manufacturing.

Cosmetics are used directly for the skin to make the skin healthier and more beautiful, and are produced by mixing active substances showing activity such as skin whitening and wrinkles, solvents, additives such as surfactants, and the like.

In the cosmetic industry, oleanolic acid and ursolic acid, which are known as wrinkle-improving ingredients, are found in the natural world as triterpenoid-based substances.

Many of these substances have been reported to have an inhibitory effect on MMP-1 (Matrix metalloproteinase-1) and MMP-2 (Matrix metalloproteinas), collagen biosynthesis and anti-inflammatory effects as external preparations for skin, but the solubility in oils used in water or cosmetics is too high It is difficult to move to a targeted cell or reach a receptor in a cell.

In the case of oleanolic acid, it is almost insoluble in water at 25 ° C and dissolves in 65 times ether, 106 times 95% ethanol, 118 times chloroform, 180 times acetone and 235 times methanol, and in the case of uric acid, It is dissolved in boiling methanol, 178 times of ethanol, 140 times of ether, and 388 times of chloroform.

Thus, the low solubility for various solvents, including water, is directly related to the low skin absorption rate of the active ingredient, leading to inhibition of the physiological activity effect.

Also, in order to stabilize a poorly soluble substance, emulsions or nanoparticles of oil in water (o / w type) were prepared using a surfactant after dissolving a poorly soluble substance in a solvent to a saturated state.

The emulsion or nanoparticles thus obtained exist in the form dispersed in water, polyol or the like, and are prepared through an antibacterial filter by adding a preservative in order to prevent deterioration during the distribution period. However, in order to secure stability of the emulsion or nanoparticles, In a high content.

In Korean Patent Laid-Open Publication No. 10-2012-0054906, a mixture comprising a polyol, an oleanolic acid, a surfactant, a phospholipid and water was prepared to prepare a stabilized nanoliposome of oleanolic acid. The polyol constituting the traumatic phase of the nanoliposome was propylene Which is composed of glycerin, methylpropanediol, isopropyleneglycol, pentyleneglycol, erythritol, xylitol, sorbitol, and a mixture thereof, To 30% by weight.

Examples of the oil component for dissolving oleanolic acid include hydrocarbon oils such as hexetecane and paraffin oil, ester synthetic oils, silicone oils such as dimethicone and cyclomethicone, sunflower oil, corn oil, soybean oil, Sphingoid lipids such as sphinganoid lipids such as phytosphingosine, phytosphingosine and sphinganine, esters such as selegionolide oil, sesame oil and sesame oil, ethoxylated alkyl ether oils, propoxylated alkyl ether oils, Rosa Id cholesterol, sitosterol cholesteryl sulfate, cholesteryl sulfate, cytokines, C _{10 -40} fatty alcohol and a mixture thereof are used.

However, the above-mentioned invention can dissolve oleanolic acid in hot oil heated to dissolve oleanolic acid and prepare micelles in the form of liquid water using lecithin or the like. Therefore, solubility of oleanolic acid, which is a poorly soluble substance, It is difficult to contain it at a high content level.

In addition, in Korean Patent Registration No. 10-0715311, in order to prepare capsules containing urusholic acid, which is a poorly soluble substance, the hydroxyl groups of the uricolic acid structure are treated with an acid or base to convert them into a salt form, A capsule for promoting transdermal absorption in which ergolic acid was located was prepared.

However, the above-mentioned invention does not maintain the structure of urosolic acid, which is an effective ingredient, but converts it into a salt form treated with an acid or a base. Therefore, it is difficult to maintain the active ingredient in its original state, Can lead to problems.

Hydroxycinnamic acid, also known as a whitening agent, is furthermore known as para-coumaric acid and is known as ortho-coumaric acid, metha-coumaric acid, acid, and p-coumaric acid. Of these, p-coumaric acid is most commonly found in nature, and it is known that it has excellent effect of inhibiting melanin synthesis as an external preparation for skin.

In the case of p-coumaric acid, a very small amount is dissolved in cold water and ethanol, and a large amount is dissolved in hot water and ethanol. In the manufacturing process, even if dissolved in hot water or ethanol, The lowered dissolved coumaric acid is recrystallized in the form of a bed, which can cause problems in consumer use.

Korean Patent Laid-Open Publication No. 10-2013-0087650 discloses a skin pharmacological composition in which a hydrophobic liquid polymer medium is impregnated with an ultraviolet screening activator. The ultraviolet screening activator includes a cupric acid and a hydrophobic liquid polymer medium such as poly Glycerin, polypropylene glycol, hydrogenated polyisobutene, hydrogenated polydodecene, etc. are used. When coumaric acid is dissolved in ethanol, glycerin, propylene glycol, butylene glycol and dipropylene glycol and the like, To prepare a stable micelle-type capsule.

However, this also has a limitation in the solubility of the coumaric acid in the alcohol or polyol used, so that it is difficult to collect a high content.

The problem to be solved by the present invention is to provide a cosmetic composition which contains a low-solubility active substance having a low solubility in a solvent in a cosmetic composition, and is characterized in that an active substance exhibiting a physiological activity effect in cosmetics is mixed with external stresses such as heat, So that the physiological activity inherent in cosmetics can be exhibited.

In order to solve the above problems, the present invention provides a nanoparticle comprising nanoparticles containing 5 to 50% by weight of an insoluble material in a wall composed of 30 to 70% by weight of an amphiphilic block copolymer and 5 to 40% by weight of lecithin, The hydrophobic block of the amphiphilic block copolymer and the lecithin is oriented to the center of the nanoparticle and the hydrophilic block is oriented out of the nanoparticle.

Also, the present invention provides a method for producing an oil-in-water emulsion, comprising heating an organic solvent to 40 to 60 ° C, dissolving the poorly soluble material and the amphiphilic block copolymer to prepare an oil phase; Dissolving lecithin in water at 0 to 10 占 폚 to prepare an aqueous phase; Mixing and homogenizing the oil phase and water phase to produce an emulsion; And drying the emulsion. The present invention also provides a method for producing nanoparticles containing a poorly soluble substance.

In this case, the mixing ratio of the poorly soluble substance, the amphiphilic block copolymer and the lecithin is 5 to 50% by weight of the poorly soluble substance, 30 to 70% by weight of the amphiphilic block copolymer and 5 to 40% by weight of the lecithin desirable.

The homogenization is preferably homogenized at 4000-6000 rpm for 5-15 minutes using a homomixer, and then homogenized one to three times at a pressure of 800-1500 bar using a high-pressure homogenizer.

It is preferable that the drying is performed by a lyophilization method.

It is preferable that the poorly soluble substance is at least one selected from the group consisting of oleanolic acid, uricolic acid, ceramide, tazotrotin, adapalene, cumaric acid and quercetin.

The organic solvent may be at least one selected from the group consisting of methanol, ethanol, acetone, tetrahydrofuran, dimethylformamide, and dimethylsulfoxide.

Also, the amphiphilic block copolymer may be selected from the group consisting of poloxamer, poly (lactide-co-glycolide), poloxarene, poly (oxyethylene) -poly (lactic acid), polyethylene glycol-polycaprolactone, methoxypolyethylene glycol- At least one selected from the group consisting of poly (ethylene oxide) -poly (propylene oxide), poly (acrylic acid) -polystyrene, poly (ethylene oxide) -polystyrene and poly (acrylic acid) -poly (butadiene) .

In addition, the lecithin is preferably hydrogenated lecithin.

The present invention also provides a cosmetic composition containing the nanoparticles as described above, wherein the cosmetic composition is selected from the group consisting of softening agents, nutritional lotion, massage cream, nutritional cream, pack, gel, skin adhesive type cosmetic, lipstick, , Shampoo, rinse, body cleanser, soap, toothpaste, mouthwash, lotion, ointment, gel, cream, patch or spray.

Since the nanoparticles according to the present invention are formed in a nanoscale size that contains a high amount of a poorly soluble active substance having a low solubility in a low temperature solvent and easy to be percutaneously absorbed, it is possible to improve the transdermal absorption rate So that the physiological activity effect of the cosmetic composition is excellent.

In addition, the active ingredient, which is easily oxidized and destroyed by external factors such as heat, light, and oxygen, is protected from external factors by a coating film surrounding the nanoparticles to improve stability and content stability, and since the solvent is removed by drying, The dissolved active oxygen can be prevented from being oxidized by the dissolved oxygen, so that the active ingredient is prevented from being lost, the active ingredient is easily dispersed in water, and the dispersion stability is excellent.

Further, the cosmetic composition of the present invention provides excellent effects on skin whitening, wrinkle improvement, skin moisturizing, anti-inflammation and acne improvement.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a diagram schematically showing the shape of nanoparticles containing a poorly soluble substance of the present invention. Fig.
FIG. 2 is a graph showing the transdermal absorption rate of nanoparticles according to the present invention.

The present invention is known to have skin whitening, wrinkle improvement, skin moisturizing, antioxidant, skin texture improvement, anti-inflammation and acne improvement effect when used as a skin external preparation in the present invention, but its solubility in water and oils used in cosmetics is too low, The poorly soluble substance, which is observed to be low in efficacy, is converted into nanoparticles to prepare a cosmetic composition.

The nanoparticles according to the present invention are prepared by dissolving a poorly soluble material exhibiting activity in cosmetics in an organic solvent heated to 40 to 60 DEG C together with an amphiphilic block copolymer as a first wall to form an oil phase, Dissolving lecithin in a cooled water at 0 to 10 ° C to prepare a water phase, mixing the oil phase and water phase, homogenizing the mixture to prepare nanoparticles of fine particles, and drying the nanoparticles, And removing the organic solvent and water present on the outside to form nanoparticles containing a poorly soluble substance therein.

At this time, the mixing ratio of the materials to be mixed is 5 to 50% by weight of the poorly soluble substance, 30 to 70% by weight of the amphiphilic block copolymer and 5 to 40% by weight of the lecithin, The content of the amphiphilic block copolymer and lecithin is sufficient to form a coating film while the remaining organic solvent and water are removed during the production of the nanoparticles, And to the extent that lecithin is sufficiently soluble.

The poorly soluble substance in the composition may be used alone or in admixture of two or more selected from the group consisting of oleanolic acid, erosolic acid, ceramide, tazarotene, adapalene, coumaric acid, and quercetin Lt; / RTI >

The organic solvent for dissolving the poorly soluble material in the composition may be selected from the group consisting of methanol, ethanol, acetone, tetrahydrofuran, dimethylformamide, and dimethyl It is preferable to select one or a mixture of two or more thereof in the group consisting of dimethyl sulfoxide.

In addition, the amphiphilic block copolymer used as the first wall in the composition has hydrophilic blocks and hydrophobic blocks in the copolymer to form micelles in water or oil phase. As such amphiphilic block copolymers, poly (lactide-co-glycolide), poloxalene, poly (oxyethylene) -poly (oxyethylene) -poly lactic acid), polyethylene glycol-polycaprolactone, methoxy polyethylene glycol-polycaprolactone, poly (ethylene oxide) -poly (propylene oxide) {(poly (ethylene oxide) -poly (propylene oxide)}, poly (acrylic acid) -polystyrene, poly (ethylene oxide) -polystyrene, and poly ) - poly (butadiene) {poly (ac rylic acid) -poly (butadiene)}, or a mixture of two or more thereof.

In addition, lecithin, which is used as a second wall in the composition, is one of phospholipids present in large amounts in egg yolk, soybean oil, liver, brain, etc., and has a lipophilic group having a strong lipophilic property on one side and a phosphoric acid and choline part Which can serve as an emulsifier to stabilize a mixture of water and oil, and can be hydrogenated lecithin or conventional lecithin.

The homogenization of the oil phase and the water phase is preferably homogenized using a high-pressure homogenizer, homogenized at 4000 to 6000 rpm for 5 to 15 minutes using a homomixer, and then homogenized using a high-pressure homogenizer at 800 to 1500 It is more preferred to homogenize one to three times at the pressure of bar.

In addition, it is preferable to remove the organic solvent and water through the lyophilization method, not the heat drying or the vacuum distillation method, from the viewpoint of preventing the breakage of the heat-sensitive active material.

Since the active substance exhibiting the physiological activity effect in cosmetics is easily destroyed by external stress such as heat, light, oxygen and the like in the air and thus loses its original physiological activity, there is a problem in using the active substance in cosmetics as it is in pure form. It is necessary to block the active substance from external stress and dissolved oxygen dissolved in the solvent.

For this purpose, in the present invention, the poorly soluble material is dried in a state in which the amphiphilic block copolymer and the lecithin are dissolved in the solvent, so that the water-soluble organic solvent and the aqueous phase are removed and the amphiphilic block copolymer and lecithin The nanoparticulate material is coated on the surface of the poorly soluble material, wherein the hydrophobic block of the amphiphilic block copolymer and lecithin is oriented to the center and the hydrophilic block is oriented to the outside to serve as a wall surrounding the poorly soluble material.

That is, nanoparticles in which an insoluble physiologically active substance is a core substance and an external solvent in which an organic solvent or water is removed while forming a coating film with an amphiphilic block copolymer and lecithin are formed, , Oxygen and the like, and the organic solvent or water is removed from the nanoparticles. Therefore, it is possible to prevent the oxidation mechanism of the insoluble matter due to oxygen by blocking the dissolved oxygen from the dissolved oxygen, and the nanoparticles have a hydrophilic block And is excellent in dispersibility in an aqueous phase and is easily dissolved in water heated to 40 ° C or higher.

As shown in FIG. 1, the nanoparticles containing the insoluble active substance prepared as described above are contained in a wall composed of 30 to 70% by weight of the amphiphilic block copolymer (B) and 5 to 40% by weight of lecithin (C) The hydrophobic block is formed in a form containing 5 to 50% by weight of the poorly soluble substance (A), the hydrophobic block of the amphiphilic block copolymer and the lecithin is oriented to the center of the nanoparticle and the hydrophilic block is oriented to the outside of the nanoparticle .

The nanoparticles prepared as described above can collect an insoluble substance, which is an effective ingredient, in an amount exceeding a conventionally known amount, and can eliminate the sterilization process while reducing the volume, thereby providing excellent effects in terms of economy.

The nanoparticles containing the poorly soluble substance prepared by the above method can be contained in an amount of 0.1 to 20.0% by weight based on the total weight of the entire cosmetic composition and can be used for skin whitening, wrinkle improvement, skin moisturizing, antioxidation, It can be used for improvement and it can be used for improvement purpose and it can be used for softening longevity, nutritional lotion, massage cream, nutritional cream, pack, gel, skin adhesive type cosmetic, lipstick, makeup base, foundation, shampoo, rinse, body cleanser, Lotions, ointments, gels, creams, patches or sprays.

Hereinafter, the present invention will be described in more detail with reference to the following examples and test examples.

It is to be understood, however, that the invention is not to be construed as being limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. Will be apparent to those skilled in the art to which the present invention pertains.

Example 1: Preparation of nanoparticles containing coumaric acid

100 g of ethanol was added to a 300 ml beaker, and 10 g of coumaric acid and 60 g of methoxypolyethylene glycol-polycaprolactone were gradually dissolved while being warmed to 50 占 폚.

One liter of water was added to another 1.5 L beaker, and 30 g of lecithin was dissolved. The mixture was cooled to 0 ° C and homogenized by using a homomixer (TK HOMOMIXER MARK II Model 2.5) at 5000 rpm for 10 minutes Homogenized.

The mixture was treated twice at 1200 bar with a high-pressure homogenizer (Microfludizer, Medel: M-110F), allowed to stand in a freezer at -85 ° C for 2 days, and lyophilized using a freeze dryer (Ilshin BioBase Model FD8508) Nanoparticles containing coumaric acid were prepared.

Example 2: Preparation of nanoparticles containing oleanolic acid

In Example 1, except that tetrahydrofuran was used as a solvent instead of ethanol and oleanolic acid was used instead of coumaric acid as an insoluble active ingredient, nanoparticles containing an oleanolic acid .

Example 3: Preparation of nanoparticles containing ceramide

100 g of ethanol was added to a 300 ml beaker and heated to 50 캜 while slowly dissolving 25 g of ceramide and 20 g of poloxamer slowly.

One liter of water was added to another 1.5 L beaker, and 5 g of lecithin was dissolved. The mixture was cooled to 0 DEG C and homogenized at 5000 rpm for 10 minutes using a homomixer while slowly adding the ethanol mixture.

The mixture was treated with a high-pressure homogenizer three times at 1000 bar, allowed to stand in a -85 ° C freezer for 2 days, and then lyophilized using a freeze dryer to prepare ceramide-containing nanoparticles.

Example 4: Preparation of nanoparticles containing tazatorin

Methanol (100 g) was added to a 300 ml beaker and heated to 50 캜 while dissolving 15 g of tazotroin and 25 g of poloxamer slowly.

One liter of water was added to another 1.5 L beaker, and 10 g of lecithin was dissolved. The mixture was cooled to 0 占 폚 and homogenized at 5000 rpm for 10 minutes using a homomixer while slowly adding the methanol mixture.

The mixture was treated with a high pressure homogenizer three times at 1000 bar, allowed to stand in a freezer at -85 ° C for 2 days, and then lyophilized using a freeze dryer to prepare nanoparticles containing tazadotin.

Example 5 Preparation of Quercetin-Containing Nanoparticles

100 g of acetone was added to a 300 ml beaker, and 10 g of quercetin and 30 g of poloxamer were slowly added to dissolve while heating to 40 ° C.

One liter of water was added to another 1.5 L beaker, and 10 g of lecithin was dissolved. The mixture was cooled to 0 ° C and homogenized for 10 minutes at 5000 rpm using a homomixer while slowly adding an acetone mixture.

The mixture was treated with a high-pressure homogenizer three times at 1000 bar, allowed to stand in a -85 ° C freezer for 2 days, and then lyophilized using a freeze dryer to prepare ceramide-containing nanoparticles.

≪ Test Example 1 > Evaluation of appearance stability of nanoparticles containing a poorly soluble substance

In order to confirm the stability of the nanoparticles prepared in Examples 1 to 5, the prepared nanoparticles were placed in 20 ml glass vials and stored at 5 ° C, 25 ° C, 45 ° C and daylight, And the change in external appearance of the drying property were observed. The results are shown in Table 1 below.

Appearance stability evaluation result division Immediately after manufacture After 1 week after 2 weeks After 3 weeks After 4 weeks Example 1 5 ℃ ○ ○ ○ ○ ○ 25 ℃ ○ ○ ○ ○ ○ 45 ° C ○ ○ ○ ○ ◑ daylight ○ ○ ○ ○ ○ Example 2 5 ℃ ○ ○ ○ ○ ○ 25 ℃ ○ ○ ○ ○ ○ 45 ° C ○ ○ ○ ○ ○ daylight ○ ○ ○ ○ ○ Example 3 5 ℃ ○ ○ ○ ○ ○ 25 ℃ ○ ○ ○ ○ ○ 45 ° C ○ ○ ○ ○ ○ daylight ○ ○ ○ ○ ○ Example 4 5 ℃ ○ ○ ○ ○ ○ 25 ℃ ○ ○ ○ ○ ○ 45 ° C ○ ○ ○ ○ ◑ daylight ○ ○ ○ ○ ○ Example 5 5 ℃ ○ ○ ○ ○ ○ 25 ℃ ○ ○ ○ ○ ○ 45 ° C ○ ○ ○ ○ ○ daylight ○ ○ ○ ○ ◑  ○: No change, ◑: Weak discoloration, ▣: Weak discoloration, ★: Completely discolored and discolored

As shown in Table 1, the nanoparticles were stored under various conditions and observed changes with time. As a result, quartic acid and tazarotene discolored to weak yellow at 45 ° C after 4 weeks, and quercetin was observed under daylight It was discolored to a slight darkness, but this level is at a level that can be overcome in cosmetic formulations.

Other than that, it showed no discoloration, detachment and dampness.

≪ Test Example 2 > Evaluation of content stability of active ingredient

In order to confirm the stability of the active ingredient contained in the nanoparticles of the present invention over time, the produced nanoparticles were analyzed for constituents of the poorly soluble substance using high performance liquid chromatography (HPLC).

The analytical samples used in the above Test Example 1 were used, and the analytical conditions of HPLC for each sample were as follows.

(1) Analysis conditions of cumaric acid

1) Detector: UV-Vis Detector (310 nm)

2) Column: Mightysil RP-18 GP 250-4.6 (5 占 퐉)

3) Column oven: 30 ° C

4) Mobile phase: 1% Acetic acid / ACN (gradient)

5) Flow rate: 1.0 ml / min

6) Inject volume: 20 μl

(2) Oleanolic acid

1) Detector: UV-Vis Detector (203 nm)

2) Column: Mightysil RP-18 GP 250-4.6 (5 占 퐉)

3) Column oven: 30 ° C

4) Mobile phase: 0.1% Phosphoric acid / ACN = 10/90

5) Flow rate: 1.0 ml / min

6) Inject volume: 20 μl

(3) ceramide

1) Detector: ELS-Detector (Gain: 5, 40 psi, 60 캜)

2) Column: SunFire pre-silica C18 4.6 X 150 mm, 5 μm

3) Column oven: 30 ° C

4) Mobile phase: Chloroform / MeOH = 95/5

5) Flow rate: 1.0 ml / min

6) Inject volume: 20 μl

(4) Tazarotin

1) Detector: UV-Vis detector (233 nm)

2) Column: Mightysil RP-18 GP 250-4.6 mm (5 占 퐉)

3) Column oven: 30 ° C

4) Mobile phase: AN / 0.1% Phosporic aicd = 9/1

5) Flow rate: 1.0 ml / min

6) Inject volume: 20 μl

(5) Quercetin

1) Detector: UV-Vis detector (363 nm)

2) Column: Mightysil RP-18 GP 250-4.6 mm (5 占 퐉)

3) Column oven: 30 ° C

4) Mobile phase: MeOH / 2.5% Acetic aicd = 5/5

5) Flow rate: 1.0 ml / min

6) Inject volume: 20 μl

Results of evaluation of content stability of active ingredient Results of stability test of content of active ingredient division Immediately after manufacture After 1 week after 2 weeks After 3 weeks After 4 weeks Example 1 5 ℃ 100.0 100.0 100.0 100.0 100.0 25 ℃ 100.0 100.0 100.0 100.0 100.0 45 ° C 100.0 99.8 ± 0.1 100.0 98.2 ± 0.4 97.8 ± 0.6 daylight 100.0 100.0 99.2 ± 0.3 99.4 ± 0.4 98.5 ± 0.7 Example 2 5 ℃ 100.0 100.0 100.0 100.0 100.0 25 ℃ 100.0 100.0 100.0 100.0 99.2 ± 0.3 45 ° C 100.0 100.0 99.9 ± 0.2 99.6 ± 0.2 99.1 ± 0.1 daylight 100.0 100.0 98.8 ± 0.7 99.3 ± 0.8 99.0 + - 0.8 Example 3 5 ℃ 100.0 100.0 100.0 100.0 100.0 25 ℃ 100.0 99.3 ± 0.6 100.0 100.0 99.4 ± 0.4 45 ° C 100.0 99.9 ± 0.1 99.3 ± 0.7 98.7 ± 0.6 98.3 ± 0.5 daylight 100.0 100.0 99.4 ± 0.2 99.1 ± 0.2 98.5 ± 0.7 Example 4 5 ℃ 100.0 100.0 100.0 100.0 100.0 25 ℃ 100.0 100.0 100.0 100.0 98.4 ± 0.6 45 ° C 100.0 99.2 ± 0.3 99.1 ± 0.2 97.9 ± 0.6 97.6 ± 0.5 daylight 100.0 100.0 100.0 100.0 99.4 ± 0.5 Example 5 5 ℃ 100.0 100.0 100.0 100.0 100.0 25 ℃ 100.0 100.0 100.0 100.0 99.4 ± 0.7 45 ° C 100.0 100.0 99.4 ± 0.8 98.7 ± 0.1 97.9 ± 0.1 daylight 100.0 100.0 100.0 100.0 98.6 ± 0.7

As shown in Table 2, after storing the nanoparticles under various conditions, the content of the poorly soluble active ingredient was measured using HPLC. As a result, the stability was 97% or more until the fourth week. As a result, It can be effectively encapsulated and protected from external stresses such as air, light, and heat, thus maintaining stability even in cosmetic formulations.

≪ Test Example 3 > Measurement of average particle size distribution and zeta potential of nanoparticles using a particle size analyzer

The nanoparticles have a phenomenon in which the stability of dispersion is degraded at a constant temperature over time. Particularly, the particle migration is classified into particle migration and particle size variation.

Particle migration is a phenomenon in which the dispersed particles move to the upper or lower layer and is called a creaming phenomenon when moving to the upper layer and is a sedimentation phenomenon when moving to the lower layer. Flocculation phenomenon and coalescence phenomenon in which small particles aggregate to form one large particle.

In order to observe the change of the nanoparticles, the particle size distribution was measured. Samples for measuring the particle size distribution were prepared by diluting the nanoparticles of Examples 1 to 5 with 0.5% (w / w) spectrophotometer cuvette PS, ratiolab®).

The particles dispersed in the solution are electrically or negatively charged by the dissociation of the surface polar group and the adsorption of the ions. In the vicinity of the particles, ions having an opposite sign existing in excess to neutralize the interface charge and a small amount Ions with the same charge are distributed densely, which is called an eletric double layer.

The zeta potential is calculated in consideration of the strength of the electric field and the hydrodynamic effect of moving the colloid particle in the opposite direction when an external electric field is applied.

The zeta potential was measured using a Malvern Zetasizer Nano-ZS nanoseries (Zetasizer). For the measurement of the zeta potential, the nanoparticles of Examples 1 to 5 were dissolved in 0.1% (w / w) And then placed in 0.7 ml of capillary folded cells (Malvern®).

Average particle size distribution and zeta potential measurement results division Average particle size distribution (nm) Zeta potential (㎷) Example 1 113.4 -37.4 Example 2 107.3 -35.9 Example 3 172.4 -31.4 Example 4 109.9 -35.5 Example 5 125.6 -35.8

In Table 3, all the nanoparticles showed a size between 100 and 200 nm. In Example 3, the content of the poorly soluble substance was higher than that of the other examples, and the average particle size distribution of the particles was relatively large .

When the zeta potential of the nanoparticles is more than +30 또는 or less than -30 ㎷, the dispersion stability is considered to be good. When the zeta potential is less than +30 또는 or more than -30 ㎷, the dispersion stability is not good.

All the zeta potentials of Examples 1 to 5 were measured to be -30 ㎷ or less, confirming excellent dispersion stability of the nanoparticles according to the present invention.

≪ Test Example 4 > Measurement of physiological activity before and after preparation of nanoparticles for poorly soluble substances

(1) Measurement of whitening activity of coumaric acid

The whitening physiological activity was measured by comparing the degree of decrease of melanin pigment during the artificial culture of cells.

The B16F10 melanin-forming cells used in this test were used from ATCC (American Type Culture Collection), and the inhibitory effect on melanin biosynthesis was measured as follows.

B16F10 melanin-forming cells were divided into 6 well plates at a concentration of 2x10 < ⁶ > cells per well. After adhering the cells, pure kumaric acid at a concentration not causing toxicity and nanomaterials containing the coumaric acid of Example 1 And cultured for 72 hours.

After culturing, the cells were detached with trypsin-EDTA, and the number of cells was measured, followed by centrifugation to recover the cells.

The cell pellet was washed once with phosphate buffered saline (PBS), and then homogenized. The cell pellet was washed twice with phosphate buffered saline (PBS) 1 ml of buffer solution (50 mM sodium phosphate, pH 6.8, 1% Triton X-100, 2 mM PMSF) was added and vortexed for 5 minutes to disrupt the cells.

After centrifugation (3000 rpm, 10 min), 1N NaOH (10% DMSO) was added to the cell filtrate to dissolve the extracted melanin, and the absorbance of melanin was measured at 405 nm using a microplate reader. The inhibition rate (%) of melanin formation was measured.

Melanin formation inhibition rate (%) of B16F1 melanin-forming cells was calculated by the following formula, and the results are shown in Table 4. [

Melanin formation inhibition rate (%) = (A-B) / A x 100

A: Amount of melanin in wells to which no sample was added

B: Amount of melanin in the well to which the sample was added

Results of Melanin Inhibition Analysis Name of sample Treatment concentration (%, w / v) Melanin inhibition rate (%) Pure Kumaric acid 0.1 1.83 Example 1 0.1 1.91

The results of Table 4 indicate that the nanoparticles containing the coumaric acid of the present invention significantly inhibit melanin formation in the B16F10 melanin-forming cells. The nanoparticles prepared in Example 1 were found to contain melanin using pure coumaric acid It was confirmed that the cosmetic composition containing the nanoparticles containing the coumaric acid had an excellent effect for inhibiting melanin production.

(2) Measurement of wrinkle-improving physiological activity of oleanolic acid

Fibroblasts were inoculated into IMDM (Isocove's Modified Dulbecco's Medium) supplemented with 10% fetal bovine serum (FBS) at a cell concentration of 5 × 10 ⁵ cells and cultured in a 5% CO ₂ incubator at 37 ° C. for 24 hours Respectively.

The diluted solution prepared by diluting the pure oleanolic acid with the dimethyl sulfoxide so that the final concentration of the nanoparticles prepared in Example 2 was 50, 100, 500 쨉 g / ml was added to the cultured fibroblasts, (UV) light for 1 hour after incubation under the same conditions.

After recovering the fibroblasts using Trizol reagent (Invitrogen, USA) and extracting mRNA, cDNA was synthesized through a series of steps. The gene of PROCOLLAGEN TYPE I gene was amplified from the synthesized cDNA and the amount of gene expression was confirmed by electrophoresis Respectively.

Gene amplification was carried out using 10x taq polymerase buffer, 10 mM dNTP, 10 pmol primer (F: AGC CAG CAG ATG GAG AAC AT, R: TCT TGT CCT) using a thermal cycler (GenePro, Hangzhou bioer tech. TGG GGT TCT TG) and taq polymerase were mixed and distilled water was added to adjust to 50 μl. The mixture was amplified at 95 ° C for 5 minutes, 1 cycle at 95 ° C for 1 minute, 51 ° C for 2 minutes, 72 ° C for 1 minute at 28 ° C, Reaction conditions.

The expression level of the produced procollagen was calculated according to the following equation. As a control, normal fibroblasts without irradiation with ultraviolet light were used.

Procollagen Expression Rate (%) = B / A x 100

A: The amount of procollagen expression in the control group

B: The amount of procollagen expression of the sample treated and irradiated fibroblasts

As a result of the test, the results shown in Table 5 were obtained.

Analysis of prolactin expression rate (%) Sample concentration (/ / ml) Pure oleanolic acid Example 2 0 2.62 2.62 50 46.54 44.46 100 98.34 103.45 500 131.82 159.16

As shown in the results of Table 5, in Example 2 in which oleanolic acid was converted into nanoparticles, the effect of promoting collagen biosynthesis related to skin wrinkles was higher than that in the case of using pure oleanolic acid, and more excellent synergistic effect was shown.

(3) Measurement of moisture activity of ceramide

The keratinocytes were inoculated in DMEM (Dulbecco's modified Eagle's medium) supplemented with 10% fetal bovine serum at a cell concentration of 1 × 10 ⁶ cells and cultured in a 5% CO ₂ incubator at 37 ° C. for 24 hours.

The diluted solution prepared by diluting the pure ceramide and the nanoparticles containing 50% ceramides prepared in Example 3 in a DMEM medium to a final concentration of 0.5% was added to the cultured keratinocytes and incubated for 18 hours under the same conditions Lt; / RTI >

After that, the keratinocyte was recovered by using Trizol reagent, and the mRNA was extracted, and the cDNA was synthesized through a series of steps. The HAS-3 (hyaluronan synthase-3) gene region was amplified from the synthesized cDNA, And the gene amplification method is the same as that of Test Example 1 above.

10x taq polymerase buffer, 10 mM dNTP, 10 pmol primer (F: GAG GAC TGG TAC CAT CAG AA, R: TTT GTT GAT GGT AGC) and taq polymerase were mixed and distilled water was added to adjust the volume to 50 μl. 5 minutes 1 cycle / 94 degrees 30 seconds / 50 degrees 30 seconds / 72 degrees 1 minute 28 cycles amplification and reaction at 72 degrees for 5 minutes.

The expression level of HAS-3 produced was calculated according to the following equation and normal keratinocytes not treated with the sample were used as a control.

Expression rate (%) of HAS-3 = B / A × 100

A: Expression of HAS-3 in the control

B: HAS-3 expression level of the sample-treated cells

The test results are shown in Table 6 below.

HAS-3 expression rate (%) analysis result Sample concentration (/ / ml) Pure ceramide Example 3 500 27.46 35.97

As shown in the results of Table 6 above, the test results of the nanoparticles of Example 3 containing 50% ceramides showed a higher effect of promoting the expression of HAS-3 associated with moisturizing than the pure ceramides Able to know.

(4) Measurement of anti-inflammatory physiological activity of tazarotine

Human normal fibroblasts were cultured in a 12-well plate incubator at a concentration of 1 × 10 ⁵ , treated with LPS (lipopolysaccharide), and then treated with 1 ppm of the nanoparticles of Example 4 containing 30% of the pure haploctin and tazarotin, 10 ppm, and 20 ppm, respectively.

Cells were harvested on the second day of culture and the amounts of tumor necrosis factor alpha (TNF-alpha) and cyclooxygenase-2 (TNF-alpha) induced by Western blotting were quantified. And the untreated group which was cultured without being treated was counted as 100, and a comparison value with the measured value was calculated.

Analysis of TNF-α and COX-2 biosynthesis rates division TNF-α biosynthesis rate (%) COX-2 biosynthesis rate (%) Control 100 100 Pure hottest lotin 1 ppm 84 91 10 ppm 72 76 20 ppm 43 66 Example 4 1 ppm 79 83 10 ppm 67 78 20 ppm 38 69

As shown in Table 7, nanoparticles containing 30% of tyrosin compared to pure haploxin resulted in inhibition of the inflammatory reaction by reducing the biosynthesis of TNF-α and COX-2 in a concentration-dependent manner. As a result, It was confirmed that the cosmetic composition is also excellent in relieving skin troubles.

(5) Antioxidant measurement of quercetin

In order to evaluate the antioxidant activity, the DPPH free radical scavenging effect assay (DPPH) of 1,1-diphenyl-2-picrylhydrazyl radical of Example 5 nanoparticles containing pure quercetin and quercetin 20% ) Was performed in the following manner.

The DPPH radical scavenging activity was measured by the reducing power of DPPH radicals. DPPH was dissolved in ethanol to prepare a 0.4 mM solution, and BHT (butylated hydroxyl toluene or 2,6-Di- tert-butyl-p-cresol) was prepared by dissolving in ethanol.

100 .mu.l of BHT and 100 .mu.l of 0.4 mM DPPH solution were added to the 96 well plate, and the mixture was reacted at 37.degree. C. for 30 minutes and the absorbance was measured at 517 nm using an ELISA reader.

The DPPH free radical scavenging activity (%) of the sample or the reference material (BHT) was calculated using distilled water instead of the sample as a control.

DPPH free radical scavenging ability (%) = {1- (absorbance of sample or reference material / absorbance of control)} x 100

DPPH free radical scavenging ability (%) analysis result Concentration (ppm) Pure quercetin Example 5 10 0.00 0.00 50 13.41 16.78 100 44.29 49.46 500 84.57 89.84 BHT * (100 ppm) 78.12

As shown in Table 8, it can be seen that all of the nanoparticles of Example 5 containing quercetin are more excellent in DPPH free radical scavenging ability depending on the concentration than pure quercetin.

<Test Example 5> Percutaneous absorption rate measurement

The in vitro skin absorption test method is a method for measuring the amount of a test substance that has passed through the skin and moved to a solution reservoir. The skin of the human skin or other species can be used and repeated measurement is possible for the test substance.

It also has the advantage of being able to study the relationship between skin damage and skin absorption that can be used for a wide range of test substances and which can not be assessed by in vivo testing for ethical reasons, And can be used as a useful model for evaluating the risk due to skin absorption in the human body.

For the measurement of the percutaneous absorption rate, a cosmetic material containing nanoparticles containing a poorly soluble substance was prepared as shown in Table 9 (Examples 6 to 10), and the transdermal absorption rate of a poorly soluble substance with time was measured using HPLC Respectively.

The thigh surface of the donated white female was placed in a Franz-type diffusion cell (Lab fine instruments, Korea), and 50 mM phosphate buffer (pH 7.4, 0.1 M NaCl) was added to the receptor container (10 ml) 37 ° C and 600 rpm, and 1 ml of each of the cosmetic compositions of Examples 6 to 10 was placed in a donor container.

2 ml of the stabilized whitening derivative absorbed in the vessel was collected once every 2, 4, 6, 8, 10, 12 and 24 hours after the start of the absorption for 24 hours according to the defined time, The area of skin was 1.28 ㎠.

After the absorption of the whitening derivative is completed, the emulsion remaining on the skin that has not been absorbed is washed with dried Kimwipes paper or 10 ml of phosphate buffer, and the skin with whitening derivative absorbed and diffused using a forcep , The emulsion absorbed into the skin was extracted with 4 ml of dichloromethane.

Thereafter, the extract was filtered through a 0.45 탆 nylon membrane filtration membrane and quantified by HPLC conditions as in Test Example 2. The results are shown in FIG.

Preparation of cosmetic composition containing nanoparticles containing poorly soluble substance (% by weight) ingredient Example 6 Example 7 Example 8 Example 9 Example 10 Example 1 2.0 0.0 0.0 0.0 0.0 Example 2 0.0 2.0 0.0 0.0 0.0 Example 3 0.0 0.0 2.0 0.0 0.0 Example 4 0.0 0.0 0.0 2.0 0.0 Example 5 0.0 0.0 0.0 0.0 2.0 Pro-type glycerin monostearate 2.0 2.0 2.0 2.0 2.0 Cetearyl alcohol 2.2 2.2 2.2 2.2 2.2 Stearic acid 1.5 1.5 1.5 1.5 1.5 Wax 1.0 1.0 1.0 1.0 1.0 Polysorbate 60 1.5 1.5 1.5 1.5 1.5 Sorbitan stearate 0.6 0.6 0.6 0.6 0.6 Hardened vegetable oil 1.0 1.0 1.0 1.0 1.0 Squalane 3.0 3.0 3.0 3.0 3.0 Mineral oil 5.0 5.0 5.0 5.0 5.0 Trioctanoin 5.0 5.0 5.0 5.0 5.0 Dimethicone 1.0 1.0 1.0 1.0 1.0 Sodium magnesium silicate 0.1 0.1 0.1 0.1 0.1 glycerin 5.0 5.0 5.0 5.0 5.0 Betaine 3.0 3.0 3.0 3.0 3.0 Triethanolamine 1.0 1.0 1.0 1.0 1.0 Sodium hyaruronate 4.0 4.0 4.0 4.0 4.0 Preservative, fragrance, pigment a very small amount a very small amount a very small amount a very small amount a very small amount Purified water Balance Balance Balance Balance Balance Sum 100 100 100 100 100

As shown in FIG. 2, it was confirmed by HPLC test that the nanoparticles containing the poorly soluble substance prepared according to Examples 6 to 10 according to the passage of time actually permeated through human skin.

A: poorly soluble substance, B: amphiphilic block copolymer, C: lecithin

Claims (15)

delete delete delete delete delete At least one organic solvent selected from the group consisting of methanol, ethanol, acetone and tetrahydrofuran is heated to 40 to 60 DEG C and then at least one selected from the group consisting of oleanolic acid, ceramide, tazurocin, coumaric acid and quercetin Preparing an oil phase by dissolving at least one ampholytic block copolymer selected from the group consisting of flushamer and methoxypolyethylene glycol-polycaprolactone in the organic solvent;
Dissolving the hydrogenated lecithin in water at 0 to 10 占 폚 to prepare an aqueous phase;
Mixing and homogenizing the oil phase and water phase to produce an emulsion; And
And lyophilizing the emulsion. The method for producing a nanoparticle containing a poorly soluble substance for a cosmetic composition according to claim 1, The method of claim 6,
The mixing ratio of the poorly soluble substance, the amphiphilic block copolymer and the hydrogenated lecithin is 5 to 50% by weight of the poorly soluble substance, 30 to 70% by weight of the amphiphilic block copolymer and 5 to 40% by weight of the hydrogenated lecithin Wherein the nanoparticles are mixed with the nanoparticles. delete delete delete delete The method of claim 6,
Wherein the homogenization is homogenized using a homomixer at 4000-6000 rpm for 5-15 minutes and then homogenized at a pressure of 800-1500 bar using a high pressure homogenizer for 1-3 times. Containing nanoparticles. delete delete delete

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