KR102534797B1 - Preparation method of nanoparticles containing anemarrhena asphodeloides extract, polydeoxyribonucleotide and physiologically active materials and cosmetic composition comprising the same - Google Patents

Abstract

The present invention relates to a method for producing nanoparticles containing an Anemarrhena asphodeloides extract, PDRN, and water-soluble bioactive substances with antioxidant activity, and a cosmetic composition containing the nanoparticles. The nanoparticles containing the Anemarrhena asphodeloides extract, PDRN, and water-soluble bioactive substances with antioxidant activity of the present invention have a high skin absorption rate and remain on the skin for a long time, stably absorbing active substances, and accordingly, cosmetic compositions containing the active substances exhibit excellent moisturizing, antioxidant, skin inflammation relieving, skin barrier strengthening, wrinkle alleviating, and elasticity improving effects.

Description

Preparation method of nanoparticles containing anemarrhena asphodeloides extract, polydeoxyribonucleotide and physiologically active materials and cosmetic composition comprising the same}

The present invention relates to a method for preparing nanoparticles containing hair extract, PDRN, and a water-soluble physiologically active substance having antioxidant activity, and a cosmetic composition containing the nanoparticles to exhibit excellent skin improvement activity.

Cosmetics have been used for the purpose of protecting the skin and imparting beauty. However, in recent years, by emphasizing functionality, functional cosmetics with various functions such as skin wrinkles or elasticity improvement, antioxidant, and whitening beyond simple skin protection or moisturizing are in the limelight. Accordingly, components having various activities are contained, which may cause skin trouble or inflammation.

In addition, in the case of cosmetics containing these various active ingredients, it is difficult to maintain formulation stability and the skin absorption rate is low, so it is difficult to expect sufficient skin improvement activity as desired when using cosmetics. Accordingly, studies to stabilize active ingredients and improve skin absorption have been continued.

The present inventors studied the stabilization of various active ingredients and the improvement of skin absorption rate in the formulation, and produced skin-improving active ingredients such as hair extract, PDRN, and water-soluble antioxidant bioactive substances into nanoparticles by applying microfluidic chip technology. In the case of, the present invention was completed by confirming that the stability of the active ingredient is improved, the skin absorption rate is improved, and the synergistic effect shows excellent skin improvement activity.

(0001) Republic of Korea Patent Publication No. 10-2021-0060121 (2021.05.26) (0002) Republic of Korea Patent Publication No. 10-2019-0114620 (2019.10.10) (0003) Republic of Korea Patent Publication No. 10-2015-0126561 (2015.11.12)

An object of the present invention is to provide a method for producing nanoparticles containing hair extract, PDRN, and antioxidant bioactive substances.

In addition, the present invention is to provide a cosmetic composition containing the nanoparticles having high stability and excellent skin absorption as an active ingredient to exhibit excellent skin moisturizing, antioxidant, skin inflammation relief, skin barrier strengthening, wrinkle improvement and skin elasticity improvement effects. for a different purpose.

According to the present invention to achieve the above object,

(A) step of extracting juice by immersing and aging the hairy roots in water at 30 to 40 ° C;

(B) adding and mixing PDRN and a water-soluble physiologically active substance to the juice, centrifuging at 1,000xg to 10,000xg to obtain a supernatant, and freeze-drying;

(C) 2 to 5 steps of adding water to the lyophilized powder, freezing it for 15 to 20 hours at a temperature of -80 to -70 ° C, and melting it for 8 to 10 hours at a temperature of 40 to 50 ° C Repeating the steps to prepare an aqueous phase;

(D) preparing an oil phase by mixing glycerin, dipropylene glycol and plant-derived phospholipids; and

(E) homogenization by passing through a microfluidic chip using the same weight ratio of the oil phase and the aqueous phase, and then adding a water-soluble solubilizing agent and stirring to mix to form nanoparticles having a size of 20 to 200 nm Nanoparticles comprising the step of forming A manufacturing method is provided.

In step (B), at least one selected from the group consisting of low-molecular-weight hyaluronic acid, low-molecular-weight collagen, glutathione, vitamin B2, menadione, thioctic acid, and vitamin C is used as the water-soluble physiologically active substance. can

In the step (B), the hair root juice, PDRN, and physiologically active substances may be used in a weight ratio of 95 to 99: 0.5 to 2: 1 to 4, respectively.

In step (D), plant-derived phospholipids include phosphatidyl choline, sphingomyelin, phosphatidyl serine, phosphatidyl inositol, phosphatidyl ethanolamine, lecithin and at least one selected from the group consisting of polylactic acid.

In step (D), glycerin, dipropylene glycol, and plant-derived phospholipids may be used in a weight ratio of 30 to 40: 8 to 12: 4 to 7, respectively.

Preferably, the homogenization of step (E) is characterized in that it consists of repeating 1 to 3 times at 1 to 10 bar and 15 to 40 ° C conditions using a microfluidic chip having a flow channel of 5 μm to 10 μm.

According to the present invention in order to achieve the above other object, there is provided a cosmetic composition containing 0.0001 to 50.0% (w / w) of the nanoparticles prepared by the above production method based on the total weight of the composition.

The cosmetic composition is characterized in that it is for skin moisturizing, antioxidation, skin inflammation relief, skin barrier strengthening, skin elasticity improvement and skin wrinkle improvement.

The cosmetic composition is skin lotion, skin toner, pack, nutrient cream, moisture cream, essence, body cream, body lotion, body oil, shampoo, conditioner, hair conditioner, hair gel, cleansing foam, cleansing lotion, soap, patch, foundation , It is characterized in that it has a formulation such as lipstick, makeup base.

Nanoparticles containing hair extract, PDRN, and antioxidant water-soluble bioactive substances prepared by the manufacturing method of the present invention have a high skin absorption rate and stay on the skin for a long time to stably absorb active substances, thereby providing excellent moisturizing, antioxidant, and skin inflammation. It can be usefully used as a cosmetic for improving skin because it exhibits relief, skin barrier strengthening, wrinkle improvement and elasticity improvement effects.

1 is a graph showing the results of particle size analysis of nanoparticles prepared in Examples of the present invention.

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

The present invention is a cosmetic composition that maximizes efficacy by improving stability and skin absorption by preparing hair extract, PDRN, and antioxidant water-soluble physiologically active substances as active ingredients by freeze-thawing and homogenizing them using a microfluidic chip. it's about

Nanoparticle capsules containing hair extract, PDRN, and antioxidant water-soluble bioactive substances as active ingredients according to the present invention have a liposomal double layer structure and have high skin absorption rate and stay on the skin for a long time to stably absorb active substances, thereby efficiently improving skin Its technical feature is that it can secure the effect.

Hair extract as an active ingredient included in the nanoparticles is preferably a hair root extract. The rhizome of Anemarrhena Asphodeloides contains asponin and salsapokenin, which can be used as medicinal ingredients. In oriental medicine, the rhizome is used as a medicinal material. It is used as an antipyretic and is effective for chronic bronchitis and diabetes.

Polydeoxyribonucleotide (PDRN) is known as a tissue regeneration material, and PDRN is mainly manufactured from DNA extracted from salmon semen. PDRN is used for wound healing and tissue repair caused by skin grafting. In addition, it is known as a component that stimulates the A2 receptor, a skin regeneration signal transducer, to promote the secretion of various growth factors, the formation of capillaries by VEGF (vascular endothelial cell growth factor), improvement of blood circulation, anti-inflammatory action and prevention of capillary leakage. there is. Recently, PDRN has been developed as a skin improvement cosmetic with various functions such as wound healing and normalization, as well as acne scars, accident scars, stretch marks improvement, skin atrophy, fine wrinkles improvement, acne skin improvement, melasma, and blemish removal. Effective product development is not enough.

Antioxidant water-soluble physiologically active substances as another active ingredient contained in the nanoparticles include low molecular weight hyaluronic acid, low molecular weight collagen, glutathione, vitamin B2, menadione, thioctic acid and vitamin C. At least one selected from may be used.

Glutathione reduces the melanin pigment, that is, the oxidized pigment in the skin, and releases itself out of the skin. Applying anti-oxidation technology so that glutathione is active, balancing the homeostasis and balance of glutathione, it not only has a skin whitening effect, but also Together with PDRN, it plays a role in helping cells regenerate. Glutathione is an antioxidant that maintains a thiol state in the body and plays an important role in detoxification. Reduced glutathione (GSH) is known to have a whitening effect.

Low-molecular-weight hyaluronic acid (sodium hyaluronic acid) is a kind of complex polysaccharide composed of amino sugar and uronic acid, and it is preferable to use sodium hyaluronic acid having a low molecular weight of 10,000 or less. Hyaluronic acid is widely distributed in all tissues of animals, especially interstitial tissues, and is obtained by extraction from umbilical cords or chicken combs. In particular, in the present invention, low molecular weight hyaluronic acid with improved skin permeation is used.

Low-molecular-weight collagen is obtained by enzymatically hydrolyzing collagen to have a low molecular weight, and preferably has a low molecular weight of 1,000 or less. And, although not particularly limited, the hydrolyzed collagen peptide may have a molecular weight of 150 or more. These hydrolyzed collagen peptides, for example, prepare a raw material of collagen by chopping pork skin, mix water with the raw material of collagen at a ratio of 70: 30 to 50: 50, and then adjust the pH of the raw material solution of collagen to 6.5 to 8.5, adding 0.1 to 1% by weight of proteolytic enzyme relative to the raw material of collagen, enzymatically treating it at 25 to 50 ° C., filtering the treated collagen solution to remove foreign substances, and then vacuum concentration, It can be obtained by sterilization and drying.

Riboflavin, also known as vitamin B2, is a vitamin found in foods and used as a dietary supplement. It is essential for the formation of two major coenzymes, flavin mononucleotide and flavin adenine dinucleotide. This coenzyme is involved in normal growth and development, as well as energy metabolism, cellular respiration and antibody production. Coenzymes are also required for the metabolism of niacin, vitamin B6 and folic acid.

Products containing these various ingredients have a low skin absorption rate and are difficult to maintain stability, so there is a limit to exhibiting practical effects as raw materials such as cosmetics and food.

In the present invention, a freeze-thaw method is used to prepare active ingredients contained so that these ingredients can be effectively delivered to the skin and exhibit their functions, and a microfluidic chip is used to prepare effective formulations.

A microfluidic chip is a chip configured based on microfluidics (microfluidics) technology to perform various experiments and diagnoses by flowing fluid through microchannels. Microfluidic chip technology is also being applied to emulsion production. Republic of Korea Patent Publication No. 10-2015-0126561 discloses liposomes whose uniformity and particle size are controlled by injecting two types of solutions after interposing a fine thin plate between the stacked upper and lower plates and adjusting the flow rate of each solution. A microfluidic chip and a method for preparing liposomes using the same are disclosed.

According to the present invention, (A) immersing and aging the hairy roots in water at 30 to 40 ° C. to extract juice;

(B) adding and mixing PDRN and a water-soluble physiologically active substance to the juice, centrifuging at 1,000xg to 10,000xg to obtain a supernatant, and freeze-drying;

(C) 2 to 5 steps of adding water to the lyophilized powder, freezing it for 15 to 20 hours at a temperature of -80 to -70 ° C, and melting it for 8 to 10 hours at a temperature of 40 to 50 ° C Repeating the steps to prepare an aqueous phase;

(D) preparing an oil phase by mixing glycerin, dipropylene glycol and plant-derived phospholipids; and

(E) homogenization by passing through a microfluidic chip using the same weight ratio of the oil phase and the aqueous phase, and then adding a water-soluble solubilizing agent and stirring to mix to form nanoparticles having a size of 20 to 200 nm Nanoparticles comprising the step of forming A manufacturing method is provided.

First, the hairy roots are immersed in water to retain the active ingredients, aged for 8 to 24 hours, and then juiced with a screw stirring speed of 20 to 50 rpm.

According to one embodiment of the present invention, in step (B), PDRN and water-soluble physiologically active substances are added in a weight ratio of 95 to 99: 0.5 to 2: 1 to 4, respectively, to the juice of the hairy root, and 1 to 6 hours After stirring and mixing, centrifugation is performed. Centrifugal separation refers to a method of separating particles of a solution using centrifugal force according to size, shape, density, viscosity, and rotor speed. It is necessary to sequentially adjust rpm to remove polymers and large contaminants, and it is more preferable to perform at 3,000xg to obtain a solution for preparing an aqueous phase of nanoparticle capsules for nanoparticle formation.

As the water-soluble physiologically active substance, at least one selected from the group consisting of low molecular weight hyaluronic acid, low molecular weight collagen, glutathione, vitamin B2, menadione, thioctic acid and vitamin C may be used.

The supernatant obtained by centrifugation is lyophilized.

In step (C), the freeze-dried product of the supernatant containing the hair root extract, PDRN, and water-soluble physiologically active substances is separated and extracted using a freeze-thaw method. The freeze-thawing method is a method for efficiently extracting active ingredients by repeating freezing and thawing. Preferably, in the step (C), water is added to the lyophilized powder and 15 After freezing for ~20 hours, it consists of repeating a process of melting for 8 to 10 hours at a temperature condition of 40 to 50 ° C. 2 to 5 times, more preferably 3 times. By not using heat treatment, it has the advantage of being able to prevent destruction of active ingredients by heat or denaturation of PDRN and physiologically active substances. In addition, it was confirmed that the nanocapsules containing the active ingredient prepared by applying the freeze-thaw method had better stability.

According to one embodiment of the present invention, in step (D), an oil phase portion is prepared by using glycerin, dipropylene glycol, and plant-derived phospholipids in a weight ratio of 30 to 40: 8 to 12: 4 to 7, respectively.

The plant-derived phospholipids include phosphatidyl choline, sphingomyelin, phosphatidyl serine, phosphatidyl inositol, phosphatidyl ethanolamine derived from sunflower, soybean, shea butter, ceramide, etc. ethanolamine), at least one selected from the group consisting of lecithin and polylactic acid may be used.

Subsequently, the prepared oil phase and aqueous phase solution are homogenized by applying a ratio of 1:1 to a microfluidic chip having a flow channel of 5 μm to 10 μm or less at 1 to 10 bar. It is preferably homogenized by repeating 1 to 3 times. Subsequently, a water-soluble solubilizing agent is added and mixed and stirred to prepare W/O/W nanoparticle capsules having a size of 20 to 200 nm. The water-soluble solubilizing agent may be used in an amount of 3 to 5% by weight based on the total weight of the oil phase and the aqueous phase.

In addition, solubilizers, stabilizers, fragrances, preservatives, pH adjusters, and the like may be further included within a range that does not impair the object of the present invention.

The nanoparticles containing hair extract, PDRN, and water-soluble bioactive substances stabilized by the manufacturing method of the present invention have moisturizing effect (Test Example 4, Test Example 13), antioxidant effect (Test Example 5), and active stability maintenance effect (Test Example 6). , Test Example 7), MMP-1 production inhibitory effect (Test Example 8), collagen synthesis enhancing effect (Test Example 9), anti-inflammatory effect (Test Example 10), skin barrier strengthening (Test Example 11), skin wrinkle improvement effect ( Test Example 12) and skin elasticity improvement effect (Test Example 14) were shown.

The nanoparticles as an active ingredient are contained in an amount of 0.0001 to 50.0% (w/w) based on the total weight of the cosmetic composition.

The cosmetic composition is characterized in that it is for moisturizing the skin, for antioxidant, for relieving skin inflammation, for strengthening the skin barrier, for improving skin wrinkles, or for enhancing skin elasticity.

The cosmetic composition is skin lotion, skin toner, pack, nutrient cream, moisture cream, essence, body cream, body lotion, body oil, shampoo, conditioner, hair conditioner, hair gel, cleansing foam, cleansing lotion, soap, patch, foundation , It can be prepared into formulations such as lipsticks and makeup bases.

[Example]

Hereinafter, the present invention will be described in more detail based on the following examples and test examples. However, the following examples are only for exemplifying the present invention, and the present invention is not limited by the following examples, and may be replaced with other equivalent examples without departing from the technical spirit of the present invention. will be clear to those skilled in the art to which the present invention pertains.

Example 1: Preparation of nanocapsules containing hair extract, PDRN and physiologically active substances

Immersion of hairy roots, juice extraction

100 g of dried hair root was immersed at a temperature of 35 ° C. for 8 hr. The immersed hairy roots were squeezed with a low-speed screw at 30 rpm using a general juicer, and the obtained juice from the hairy roots was filtered through a mesh net to remove suspended matter.

Water phase and oil phase manufacturing using freeze-thaw method

In 96.7 g of the hair root juice, 1 g of PDRN, 0.2 g of low molecular weight hyaluronic acid as a physiologically active substance, 0.02 g of low molecular weight collagen, 0.02 g of glutathione, 0.02 g of vitamin B2 (Riboflavin), 0.02 g of menadione, thioctic acid After mixing and stirring 0.02 g of Thioctic Acid and 2 g of vitamin C, the supernatant was obtained by centrifugation at 3,000xg for 10 minutes at 4° C. to remove polymers and large contaminants. The supernatant was frozen at -80 °C for 20 hours and dried for 100 hours in a vacuum in a freeze dryer. At this time, the vacuum state usually means the pressure state of the freeze dryer, and the freezing and drying time may vary depending on the volume of the solution.

Purified water was added to the lyophilized powder, frozen at -80 °C for 18 hours, and then thawed at 40 °C for 8 hours. This process was repeated three times to separate and extract the physiologically active substances so that they could be purified with high purity to prepare 50 g of the aqueous phase.

An oil phase was prepared by mixing and stirring 5 g of Phosphatidyl choline with 35 g of glycerin and 10 g of dipropylene glycol.

Formation of nanoparticle encapsulation

Nanoparticles were prepared by passing the prepared water phase part and oil phase part through a microfluidic chip.

A microfluidic chip with a flow channel of 5 μm was prepared and used. Using 48% by weight of the aqueous phase and 48% by weight of the oil phase, a microfluidic chip with a flow channel of 5 μm was passed through at room temperature at 5 bar to homogenize. The process was repeated a total of three times to increase the degree of homogenization through self-assembly.

4% by weight of Polyglyceryl-10 Laurate, a water-soluble solubilizing agent, was added thereto, and mixing was stirred to prepare W/O/W nanoparticle capsules having a size of 20 to 200 nm.

Comparative Example 1: Preparation of nanocapsules

100 g of dried hair root was added to 1,000 g of purified water and extracted at 80° C. for 3 hours. 96.7 g of the hot water extract of the hairy root, 1 g of PDRN, 0.2 g of low molecular weight hyaluronic acid as a physiologically active substance, 0.02 g of low molecular weight collagen, 0.02 g of glutathione, 0.02 g of vitamin B2 (Riboflavin), 0.02 g of menadione, thioctic acid An aqueous phase was prepared by mixing 0.02 g of (Thioctic Acid) and 2 g of vitamin C. The rest of the method was the same as in Example 1 to obtain nanocapsules.

Comparative Example 2: Preparation of a mixture of hair extract, PDRN and bioactive substances

Put 100 g of dried hair root in 1,000 g of purified water and extract at 80 ° C for 3 hours. After extraction, filtration under reduced pressure was performed to prepare an extract of the hairy root. 98.35 g of the hair root extract, 0.5 g of PDRN, 0.1 g of low molecular weight hyaluronic acid as a physiologically active substance, 0.01 g of low molecular weight collagen, 0.01 g of glutathione, 0.01 g of vitamin B2 (Riboflavin), 0.01 g of menadione, thioctic acid A mixture was prepared by mixing 0.01 g of (Thioctic Acid) and 1 g of vitamin C.

Test Example 1: Particle size analysis of nanoparticles

In order to confirm the particle size of the nanoparticles prepared in Example 1, it was analyzed with a particle size analyzer.

1 is a graph showing the analysis results of the particles according to Example 1, and as a result of the analysis, the average size of the particles was confirmed to be 143 nm.

Test Example 2: Analysis of zeta potential of nanoparticles

Colloids, which are particles dispersed in a solution, are charged by dissociation of surface polar groups on the surface of particles and adsorption of ions when present in an aqueous solution. The degree of repulsive force between particles is used as an important criterion for evaluating the stability of a colloid. That is, if the value of negative (-) or positive (+) zeta potential is large, it can be seen that the repulsive force between the particles is high and the dispersion stability is good, and if it is small, the attractive force between the particles is large and the cohesive force is high. Therefore, in order to confirm dispersion and aggregation in the solution in which the nanoparticles were formed, zeta potential analysis was performed through a Zetasizer. Table 1 below shows the zeta potential values of the nanoparticles containing the hair extract, PDRN, and water-soluble bioactive substances prepared in Example 1 and the zeta potentials of the nanoparticles prepared in Comparative Example 1. potential) value.

Example 1 Comparative Example 1 Zeta potential (mV) -40.9 -5.3

As a result of zeta potential analysis, it can be seen that the dispersion stability of the nanoparticles prepared by applying the freeze-thaw method according to the examples of the present invention is better, as confirmed in Table 1 above.

Test Example 3: Evaluation of cytotoxicity

MTT assay was evaluated to confirm the cytotoxicity of the nanoparticle capsules containing the hair root extract, PDRN, and water-soluble bioactive substances. Human keratinocytes (HaCaT) were inoculated in a 96-well plate at a concentration of 1×10 ⁵ cells/mL, and then incubated at 37° C. for 18 hours under 5% CO ₂ . After culturing, the medium was removed, washed with PBS buffer, and fresh medium and samples corresponding to Example 1 and Comparative Examples 1 and 2 were administered by concentration and cultured again for 24 hours. After adding MTT solution (5mg/mL) to measure cell viability, formazan formed for 4 hours was dissolved in dimethyl sulfoxide (DMSO), and absorbance was measured at 570 nm using an ELISA reader.

Cell viability
(% of control) Example 1 Comparative Example 1 Comparative Example 2 Control 100 100 100 0.01% 125.72 114.50 105.65 0.1% 120.64 108.82 103.62 One% 107.44 106.15 100.15 5% 104.35 102.45 90.40

As confirmed in Table 2, cytotoxicity was not observed in all samples of Example 1 and Comparative Examples 1 and 2.

Test Example 4: Moisturizing efficacy evaluation (AQP3 expression)

To confirm the moisturizing effect of nanoparticle capsules containing hair extract, PDRN, and water-soluble bioactive substances, the effect on AQP3 expression was evaluated. After inoculation of human dermal fibroblast (HDFa), 100 IU/mL penicillin and 100 μg/mL streptomycin were added in Fibroblast Basal Medium (Medium 106) culture medium at 37°C for 24 hours under 5% CO ₂ cultured. The medium in each well was removed and replaced with a new serum-free medium. Samples corresponding to Example 1 and Comparative Examples 1 and 2 were administered to each well according to concentration, and pretreatment was performed for 4 hours. Each well plate was irradiated with 15 mJ/cm ² UVB by a UVB irradiator (Vilber Loumet, France). After processing the diluted serum-free medium, the samples were additionally cultured for 24 hours. At this time, PBS was treated as a negative control group, and Hyaluronic acid 0.005% was treated as a positive control group. Cell cultured cells were lysed using Ribo Ex TM Total RNA Isolation Solution (GeneAll Biotechnology, Korea) and a scraper, and then 0.2mL chloroform (Sigma-Aldrich, USA) was added and centrifuged (12,000 rpm, 4℃, 30 minutes). ) was done. The supernatant with RNA was separated, and the same amount of isopropanol (Merck-Millipore, Germany) was added to the supernatant, followed by inverting and centrifugation (12000 rpm, 4° C., 30 minutes). The RNA was precipitated and the supernatant except for the precipitate was discarded, and the remaining precipitate was washed with 70% ethanol (Merck-Millipore, Germany) and centrifuged (12,000 rpm, 4° C., 10 minutes). After removing ethanol and drying at room temperature, total RNA was extracted by dissolving in Nuclease-Free Water (Affymetrix, USA). After measuring the purity and concentration of RNA at the A260/A280 wavelength using a MaestroNano ^® Micro-volume Spectrophotometer (MN-913, Maestrogen, USA), it was confirmed that the ratio between 260 nm and 280 nm was in the range of 2.0-2.2. For cDNA, 1 μg RNA, Oligo dT (Bionics, Korea), dNTP (Takara, Korea), and nuclease-free water were prepared in a total of 13 μL in a PCR tube, reacted at 65 ° C for 5 minutes, and M-MLV Reverse Transcriptase (Invitrogen, Thermo Fisher Scientific, Waltham, Massachusetts, USA) was synthesized by reacting at 37 ° C. for 50 minutes. qRT-PCR was performed to quantitatively analyze gene expression patterns occurring within each cell by the sample. For qRT-PCR, a total of 20 μL of primer, cDNA, 2X SYBR green PCR Master Mix (Applied Biosystems, USA), and HPLC (J. T baker, USA) were mixed in a PCR tube to make a reaction solution, which was then run on the StepOnePlus Real-Time PCR System ( PCR was performed using Applied Biosystems, USA).

The PCR primer sequence of the AQP3 gene is shown in Table 3 below, and the results of evaluating the moisturizing efficacy of the hair extract, PDRN, and nanoparticle capsules containing water-soluble bioactive substances prepared according to the above example by AQP3 mRNA expression rate are shown in Table 4 below. showed up

gene Forward primer (5'→3') Reverse primer (5'→3') AQP3 5'-CCT TTG GCT TTG CTG CAC TC-3' 5'-ACG GGG TTG TTG TAA GGG TCA-3' GAPDH 5'-GTC TCC TCT GAC TTC AAC AGC G-3' 5'-ACC ACC CTG TTG CTG TAG CCA A-3'

Relative AQP3 expression
(% of control) Hyaluronic acid
(0.005%) Example 1 Comparative Example 1 Comparative Example 2 Control 125.64% 145.64 0.01% 105.67 101.42 109.35 0.1% 120.16 105.94 112.36 One% 135.62 119.63 118.60 5% 145.55 126.07 125.41

As a result of the test, when the sample of Example 1 was treated, AQP3 mRNA, which is a moisturizing factor involved in cell water movement and plays an important role in skin moisturizing, increased in a concentration-dependent manner, compared to samples corresponding to Comparative Examples 1 and 2. It was confirmed that the expression rate increased even more in the treatment concentration range of Example 1. At this time, when treated with hyaluronic acid 0.005% as a positive control, the expression rate was 125.64%.

Test Example 5: Evaluation of antioxidant efficacy

DPPH radical scavenging activity test was conducted to confirm the antioxidant efficacy of nanoparticle capsules containing hair extract, PDRN and water-soluble bioactive substances. The DPPH radical scavenging activity was measured by the reducing power of the sample for the DPPH radical. A 0.4 mM solution was prepared by dissolving DPPH (1,1-Diphenyl-2-picrylhydrazyl) reagent in ethanol, and samples corresponding to Examples 1 and Comparative Examples 1 and 2 or positive control BHT (Butylated Butylated hydroxyl toluene or 2,6-Di-tert-butyl-p-cresol) in ethanol, and 100 μl of a 0.4 mM DPPH solution were added and mixed. At this time, ethanol was treated as a negative control, and after mixing, the mixture was reacted at 37° C. for 30 minutes. The absorbance was measured at 517 nm using an ELISA reader, and the DPPH radical scavenging ability (%) of the sample or positive control, BHT, was calculated using the formula below.

DPPH radical scavenging ability (%) = {1-(absorbance of positive control or sample/absorbance of control)} × 100

Table 5 is a table showing the results of evaluating the antioxidant effect of each sample by DPPH radical scavenging ability.

Concentration (%) DPPH radical scavenging activity (%) Example 1 Comparative Example 1 Comparative Example 2 0.01 25.01 20.27 18.55 0.1 58.45 32.40 35.40 1.0 75.49 53.08 40.19 5.0 85.79 64.78 54.05 BHT (0.01%) 64.84

As a result of the test, when the samples corresponding to Example 1 and Comparative Examples 1 and 2 were treated, DPPH radical scavenging activity increased in a concentration-dependent manner, and it was confirmed that the highest activity was exhibited when the nanoparticles of Example 1 were treated.

Test Example 6: Antioxidant activity stability test

Antioxidant activity retention rates of the samples prepared in Example 1 and Comparative Examples were evaluated. The antioxidant activity test was performed in the same manner as in Test Example 5, and antioxidant retention rates of Examples and Comparative Examples 1 and 2 were compared based on a reference concentration of 0.1%. Antioxidant activity was evaluated for each week at a high temperature (45 ℃) based on the antioxidant result measured at week 0 as a 100% standard. The results are shown in Table 6 below.

by parking Example 1 Comparative Example 1 Comparative Example 2 Week 0 100 100 100 Week 1 95 91 85 Week 2 92 84 59 Week 4 87 79 55 week 8 85 60 42 week 12 80 52 29

As shown in Table 6, it can be seen that the ability to maintain antioxidant activity of the hair extract, PDRN, and nanoparticles containing water-soluble bioactive substances prepared in Example 1 of the present invention is far superior to those of Comparative Examples. Compared to the comparative examples, the retention rate of antioxidant activity was far superior.

Test Example 7: Content stability evaluation

In order to confirm the stability of the physiologically active ingredients prepared in Example 1 and Comparative Examples, changes in vitamin C content were observed. Based on the content result measured at week 0 as a 100% standard, the content was evaluated for each week at a high temperature (45 ° C).

by parking Example 1 Comparative Example 1 Comparative Example 2 Week 0 100 100 100 Week 1 92.5 91.4 89.1 Week 2 91.2 85.7 75.2 Week 4 90.4 80.2 62.5 week 8 87.0 71.5 55.2 week 12 81.3 65.7 52.8

As shown in Table 7, it can be confirmed that the vitamin C content retention rate of the nanoparticles prepared according to Example 1 of the present invention is far superior to those of Comparative Examples.

Test Example 8: MMP-1 production inhibitory effect

In order to determine whether there is an effect of inhibiting MMP-1 production, TGF-β (10 ng / ml, Roche), a substance known to have an effect of inhibiting MMP-1 production, was used as a control group, and MMP-1 The effect of inhibiting the production of was measured.

Fibroblasts (Korea Cell Line Bank, Korea), which are human normal skin cells, were inoculated into 48-well microplates (Nunc, Denmark) to be 1×10 ⁶ cells per well, and cultured in DMEM medium (Sigma) and 24 °C at 37 °C. After culturing for 48 hours, the cells were further cultured in serum-free DMEM medium to which a final concentration of 0.1% of Example 1 was added.

After culturing, the supernatant of each well was collected and the amount (ng/ml) of newly synthesized MMP-1 was measured using an MMP-1 assay kit (Amersham), and the inhibition rate (%) of MMP-1 production was determined according to the following formula. was calculated, and the results are shown in Table 8.

MMP-1 production inhibition rate (%) = [1-amount of MMP-1 in the experimental group / amount of MMP-1 in the control group] × 100

sample name MMP-1 production inhibition rate (%) Example 1 (0.1%) 67.8 Comparative Example 1 (0.1%) 58.2 Comparative Example 2 (0.1%) 47.5 Positive control TGF-β (10 ng/ml) 72.9

As shown in the results of Table 8, the nanoparticle capsule of Example 1 exhibited an inhibition rate of 67.8% when the MMP-1 production inhibition rate was measured at a concentration of 0.1%.

Test Example 9: Collagen synthesis enhancing effect

Fibroblasts, which are human normal epithelial cells, were inoculated into 1×10 ⁶ cells per well in a 48-well microplate, and cultured in DMEM medium for 24 hours. The nanoparticle capsules of Example 1 were additionally cultured for 48 hours in a serum-free DMEM medium added at a final concentration of 0.1%. After culturing, the supernatant of each well was collected and the amount of procollagen type I C-peptide (PICP) was measured using a collagen kit (Takara, Japan) and converted to ng/ml. The amount of collagen synthesized thereby measured.

As a comparison group in this experiment, TGF-β (10ng/ml, Roche), a substance known to have a collagen synthesis enhancing effect, was used as a comparison group, and the collagen biosynthesis increase rate (%) was calculated according to the following formula. is shown in Table 9.

Collagen biosynthesis increase rate (%) = (amount of collagen in the experimental group / amount of collagen in the control group - 1) × 100

sample name Collagen biosynthesis increase rate (%) Example 1 (0.1%) 75.8 Comparative Example 1 (0.1%) 52.5 Comparative Example 2 (0.1%) 25.4 Positive control TGF-β (10 ng/ml) 67.4

As confirmed in Table 9, it can be seen that the hair extract, PDRN, and nanoparticle capsules containing water-soluble bioactive substances (Example 1) exhibit a biosynthesis rate of 75.8% when the collagen biosynthesis rate is measured at a concentration of 0.1%. .

Test Example 10: Evaluation of anti-inflammatory efficacy

In order to confirm the anti-inflammatory effect, NO (Nitric oxide) production inhibition rate was evaluated. Raw264.7 macrophages were divided into 8×10 ⁴ cells/well in a 96 well plate and cultured for 24 hours, and then the samples of Example 1 and Comparative Examples 1 and 2 were treated for 1 hour. After treatment, LPS was treated at a concentration of 100 ng/ml and cultured for 24 hours to induce inflammation. Thereafter, 100 μL of cell culture supernatant and Griess reagent were mixed in equal amounts and reacted in the dark at room temperature for 10 minutes, and then absorbance was measured at 540 nm using an ELISA reader. Table 10 is a table showing the results of evaluating the anti-inflammatory efficacy in terms of nitric oxide (NO) production inhibition rate. As a result of the test, it was confirmed that the amount of NO production decreased in a concentration-dependent manner in all samples, and the NO production inhibitory activity was the best in the sample of Example 1.

Concentration (%) NO Production (% of control) Example 1 Comparative Example 1 Comparative Example 2 Control 100 100 100 0.01 89.06 92.27 88.10 0.1 82.62 85.63 84.50 1.0 65.11 81.08 82.62 5.0 59.54 72.70 77.57

Test Example 11: Evaluation of skin barrier strengthening efficacy (filaggrin expression)

Skin barrier protein is an important component of skin structure and plays an important role in blocking external antigens from entering the body. Types of skin barrier proteins include filaggrin, involucrin, and loricrin, among which filaggrin plays an important role in the skin barrier by coagulating keratin filaments within keratinocytes. Therefore, in order to confirm the skin barrier strengthening effect of Example 1 nanoparticle capsules containing hair extract, PDRN and water-soluble bioactive substances, a filaggrin gene expression test was conducted in comparison with Comparative Examples 1 and 2.

After inoculation of human keratinocyte (HEKa) cell line, 100 IU/mL penicillin and 100 μg/mL streptomycin were added to Dulbecco's Modified Eagle Medium (DMEM) culture medium at 37℃ for 24 hours under 5% CO ₂ culture. did After culturing, the medium was discarded, and the samples of Example 1 and Comparative Examples 1 and 2 were treated and cultured for 48 hours. Thereafter, RNA was extracted from the cultured cells using TransZol reagent, followed by RT-PCR (real-time gene polymerase chain reaction), and the products generated by PCR were electrophoresed on 1% agarose gel and confirmed with a Gel Documentation system. . At this time, PBS was treated as a negative control group, and CaCl ₂ 20 mM was treated as a positive control group.

Concentration (%) Relative FLG expression (% of control) Example 1 Comparative Example 1 Comparative Example 2 Control 100 100 100 0.01 105.90 104.69 102.18 0.1 115.73 110.43 103.61 1.0 121.06 115.16 107.76 5.0 130.46 121.33 113.96

Table 11 is a table showing the results of evaluating the skin barrier strengthening efficacy of the samples by filaggrin expression. As a result of the test, when the nanoparticle capsules of Example 1 were treated, the expression of filaggrin increased in a concentration-dependent manner, and showed a much higher increase rate than when the samples of Comparative Example were treated.

Example 2: Preparation of Lotion

A lotion containing the nanoparticle capsules of Example 1 according to the composition shown in Table 12 was prepared in a conventional manner.

Ingredients (% by weight) Content (% by weight) Hair extract, nanoparticles containing PDRN and water-soluble bioactive substances
(Example 1) 3.00 glycerin 10.00 Butylene Glycol 5.00 glyceryl oleate 1.80 Cetearyl Olivate 0.50 Sorbitan Olivate 0.50 Caprylic/Capric Triglyceride 5.00 Cetylethylhexanoate 1.00 beeswax 0.50 squalane 0.20 1,2-Hexanediol 2.00 Cholesteryl/Behenyl/Octyldodecyllauroylglutamate 1.00 dimethicone 0.50 Cyclopentasilonesac/Cyclohexasiloxane 2.00 Cetialyl Alcohol 1.00 mineral oil 2.50 Disodium EDT 0.02 BHT 0.05 Tocopheryl Acetate 0.30 panthenol 0.20 Ethylhexylmethoxycinnamate 0.20 incense 0.01 Purified water balance

Comparative Examples 3 to 5: Preparation of lotion

A lotion was prepared in the same manner as in Example 2 by adding Comparative Example 1 instead of the nanoparticles of Example 1 (Comparative Example 3). In addition, a lotion containing 0.04% of adenosine, which is known to have an anti-wrinkle effect, and a lotion containing 3.00% of hyaluronic acid, which is known to have a moisturizing effect, were prepared and used as Comparative Example 4 and Comparative Example 5, respectively.

Test Example 12: Skin wrinkle improvement effect test

The anti-wrinkle effect of the prepared lotions of Example 2, Comparative Example 3 and Comparative Example 4 was evaluated through actual use tests. The degree of wrinkle improvement was confirmed by visually evaluating the wrinkle improvement effect after 6 weeks using both sides of the face for 60 women. The wrinkle improvement effect results based on this evaluation are shown in Table 13 below.

sample name Wrinkle improvement effect Effectiveness (%) Great a little doesn't exist Example 2 15 3 2 49 Comparative Example 3 8 9 3 25 Comparative Example 4 14 4 2 38

As confirmed in the results of Table 13, the lotion of the formulation of Example 2 containing the hair extract of the present invention, PDRN and nanoparticles containing water-soluble bioactive substances as active ingredients showed a higher anti-wrinkle effect than Comparative Example 3. and showed an excellent skin wrinkle improvement effect similar to that of the lotion of Comparative Example 4 containing adenosine.

Test Example 13: Skin Moisture Improvement Effect

In order to find out the effect of improving skin moisturizing power, the following experiment was conducted. 60 people in their 20s and 40s without skin diseases were divided into 3 groups of 20 people per group, and Example 2 and Comparative Example 3 prepared with the composition of Table 12 for each group and substances known to have a moisturizing effect A lotion containing hyaluronic acid (Comparative Example 5) was applied to the face and forearm twice daily for one month.

Before starting application in advance, skin conductivity was measured using a corneometer (CM820 courage Khazaka electronic GmbH) under constant temperature and humidity conditions (24 degrees, 40% humidity), and the skin conductivity was set as the basic value, and skin conductivity after 1 week, 2 weeks, and 4 weeks was measured to evaluate the rate of increase. The results are shown in Table 14 below.

sample name 1 week later 2 weeks later 4 weeks later Example 2 35 43 49 Comparative Example 3 23 22 25 Comparative Example 5 45 43 45

As shown in the results of Table 14, the lotion of the formulation of Example 2 containing the hair extract of the present invention, PDRN, and nanoparticle capsules containing water-soluble bioactive substances was very superior in skin conductivity increase rate compared to Comparative Example 3. In addition, compared to Comparative Example 5, which is a cosmetic containing hyaluronic acid known to have a moisturizing effect, it showed a similar skin conductivity increase rate.

In general, since skin conductivity is proportional to the amount of skin moisture, it can be confirmed from the above results that the cosmetics contained in the present invention maintain a higher skin moisture content than cosmetics containing purified water.

Test Example 14: Skin elasticity improvement effect

The skin elasticity improvement effect of Example 2 and Comparative Examples 3 and 5 was measured on 30 healthy women (average age 33.7 years old) in a constant temperature and humidity room at 22 ° C. and 45% relative humidity.

The formulation was applied to the eye area for 4 weeks by attaching it once every night, and the skin elasticity of each group was measured using a skin elasticity measuring device (Cutometer SEM 575, C+KElectronic Co., Germany). The change values are shown in Table 15 below.

division Example 2 Comparative Example 3 Comparative Example 5 skin elasticity level 48.5 26.2 32.5

Through the results of Table 15, when using the lotion of Example 2, it was confirmed that the skin elasticity improvement was higher than Comparative Examples 3 and 5, and the skin elasticity improvement effect was excellent.

Claims (11)

(A) step of extracting juice by immersing and aging the hairy roots in water at 30 to 40 ° C;
(B) adding and mixing PDRN and a water-soluble physiologically active substance to the juice, centrifuging at 1,000xg to 10,000xg to obtain a supernatant, and freeze-drying;
(C) 2 to 5 steps of adding water to the lyophilized powder, freezing it for 15 to 20 hours at a temperature of -80 to -70 ° C, and melting it for 8 to 10 hours at a temperature of 40 to 50 ° C Repeating the steps to prepare an aqueous phase;
(D) preparing an oil phase by mixing glycerin, dipropylene glycol and plant-derived phospholipids; and
(E) Using the oil phase and the water phase in the same weight ratio, passing through a microfluidic chip with a flow channel of 5 μm to 10 μm under conditions of 1 to 10 bar and 15 to 40 ° C. is repeated 1 to 3 times to homogenize, and then dissolved in water. A method for producing nanoparticles comprising the step of adding an agent and mixing and stirring to form nanoparticles having a size of 20 to 200 nm. The method of claim 1, wherein the water-soluble physiologically active substance is at least one selected from the group consisting of low molecular weight hyaluronic acid, low molecular weight collagen, glutathione, vitamin B2, menadione, thioctic acid and vitamin C. Method for producing nanoparticles, characterized in that. The method of claim 1, wherein the plant-derived phospholipids are phosphatidyl choline, sphingomyelin, phosphatidyl serine, phosphatidyl inositol, phosphatidyl ethanolamine, and lecithin. ) And a method for producing nanoparticles, characterized in that at least one selected from the group consisting of polylactic acid (Polylactic Acid). delete A cosmetic composition containing 0.0001 to 50.0% (w/w) of the nanoparticles prepared by the method of any one of claims 1 to 3 based on the total weight of the composition. The cosmetic composition according to claim 5, characterized in that the cosmetic composition is for antioxidation. The cosmetic composition according to claim 5, wherein the cosmetic composition is for improving skin wrinkles. The cosmetic composition according to claim 5, wherein the cosmetic composition is for improving skin elasticity. The cosmetic composition according to claim 5, wherein the cosmetic composition is for moisturizing the skin. The cosmetic composition according to claim 5, wherein the cosmetic composition is for relieving skin inflammation. The cosmetic composition according to claim 5, characterized in that the cosmetic composition is for strengthening the skin barrier.

Images