KR102523523B1 - Cosmetic composition for improving skin and hair condition containing Nepeta Cataria exosome and Spinacia Oleracea exosome - Google Patents

Abstract

The present invention relates to a cosmetic composition for improving skin and hair condition containing exosomes derived from Nepeta cataria and Spinacia oleracea as active ingredients, and specifically, to a cosmetic composition for improving skin and hair condition, which has excellent anti-inflammatory, antioxidant, anti-wrinkle effects, hair growth promotion, and hair loss prevention effects by containing, as active ingredients, the Nepeta cataria exosomes and/or Spinacia oleracea exosomes purified using coarse grinding, freeze-thaw pretreatment, and an aqueous two-phase system.

Description

Cosmetic composition for improving skin and hair condition containing catnip and spinach-derived exosomes as active ingredients

The present invention relates to a cosmetic composition for improving skin and hair conditions containing catnip and spinach-derived exosomes as active ingredients, and specifically, catnip exosomes and / Or it relates to a cosmetic composition for improving skin and hair condition, which contains spinach exosomes as an active ingredient and has anti-inflammatory, antioxidant, anti-wrinkle effects, hair growth promotion, and hair loss prevention effects.

The function of cosmetics has expanded beyond cleanliness and simple makeup to removing or suppressing causes of skin aging such as pollutants, stress, ultraviolet rays, active oxygen, and peroxide. Recently, the development of cosmetics for improving skin wrinkles and alleviating skin irritation and inflammation using safe natural materials as materials has been actively conducted. In addition, in modern society, an increasing number of people suffer from hair damage or hair loss, and to solve this problem, methods such as wearing wigs and feeding are used, or drugs for treating alopecia are used. The causes of hair loss include genetic causes, deterioration in hair cell function, excessive secretion of male hormones, and excessive secretion of sebum, and androgens, which are mainly male hormones, act as an important factor. Among the androgens, testosterone is converted to dihydrotestosterone (DHT) by 5-alpha-reductase when it reaches the hair follicle, and this hormone atrophies the hair follicle and gradually thins the hair, causing hair loss.

Although preparations containing minoxidil and finasteride are used for the treatment of such alopecia, there are many restrictions on their use due to side effects such as sexual dysfunction, possibility of birth defects, hypotension, tachycardia, and cardiovascular complications. . Therefore, studies on hair growth agents and hair-related products using natural materials without side effects are being actively conducted.

In the present invention, "Exosome" means a small membrane-structured vesicle secreted from various cells, and is defined as a type of extracellular vesicles (EVs). All cells exchange information with other cells or the external environment and secrete extracellular endoplasmic reticulum for this purpose. Exosomes have a size of about 50 to 200 nm and contain physiologically active substances such as proteins, lipids, and nucleic acids. Exosomes exist in various cells such as mammals, bacteria, and plants, and can be used for diagnosis and treatment because they reflect the state of the originating cells. Exosomes are a double-phospholipid membrane structure that is easily penetrated into cells and performs various physiological and pathological functions such as immune response and signal transduction.

Recently, studies on various effects of plant-derived exosomes have been conducted. Plant-derived exosomes are natural nanoparticles that help cell movement and absorption as they contain physiological activity and signaling substances secreted by plant cells themselves, and exosomes purified from plants are less toxic than mammalian-derived exosomes. It is known.

These exosomes are materials that can be used in the fields of medicine, cosmetics, food, etc., but have low dispersibility and tend to aggregate due to their structural characteristics consisting of a phospholipid bilayer. In addition, since it is unstable at high temperatures and can be easily broken in the process of preparing cosmetic formulations, the stability of exosomes in cosmetic formulations may be reduced and precipitation may occur. Therefore, in order to maintain continuous activity, it is necessary to increase the stability in the formulation by increasing the solubility and dispersibility of the exosomes in the aqueous solution.

The present inventors applied various methods from various plants to prepare exosomes exhibiting excellent skin and hair condition improving activity and tried to use them as cosmetics. As a result, coarse grinding under specific conditions, freeze-thaw pretreatment and aqueous two-phase The present invention was completed by confirming that catnip exosomes and/or spinach exosomes purified using the system showed excellent skin and hair condition improvement efficacy.

(0001) Republic of Korea Patent No. 10-2125567 (2020.06.16) (0002) Republic of Korea Patent No. 10-2265811 (2021.06.16.) (0003) Republic of Korea Patent Publication No. 10-2022-0122577 (2022.09.02)

The present invention contains catnip exosomes, spinach exosomes, or mixed exosomes as active ingredients and has excellent stability, anti-inflammatory, anti-oxidation, anti-wrinkle effects, hair growth promotion and hair loss prevention cosmetic composition for improving skin and hair conditions is intended to provide

Another object of the present invention is to provide a method for purifying catnip exosomes or spinach exosomes.

According to the present invention in order to achieve the above object, a cosmetic composition containing catnip exosomes, spinach exosomes, or mixed exosomes as an active ingredient is provided.

The mixed exosomes are made by mixing catnip exosomes and spinach exosomes in a weight ratio of 1 to 3:1 to 3, respectively.

Catmint exosomes and spinach exosomes as the active ingredient,

(A) coarsely grinding catnip or spinach; (B) a freeze-thaw pretreatment step of adding a solvent to coarsely ground catnip or spinach and repeating freezing and thawing; (C) extracting juice from catnip or spinach pre-treated with freeze-thaw; (D) obtaining a supernatant by centrifuging the juice at 1,000xg to 10,000xg; (E) freeze-drying the supernatant in which exosomes exist; (F) forming an aqueous two-phase system using PEG (Polyethylene glycol)/Dextran in the lyophilisate; and (G) obtaining an exosome-enriched lower layer in the aqueous two-phase system.

Preferably, the coarsely pulverizing process in step (A) is pulverized to a particle size of 2.0 to 5.0 mm using a pulverizing device.

In addition, in the freeze-thawing pretreatment in step (B), water was added to the coarsely ground catmint or spinach, frozen for 15 to 20 hours at a temperature of -80 to -70 ° C, and then 8 at a temperature of 40 to 50 ° C. It is characterized in that it consists of repeating the melting process 2 to 5 times for ~10 hours.

The catnip exosomes, spinach exosomes, or mixed exosomes as active ingredients are contained in an amount of 0.0001 to 30.0% (w/w) based on the total weight of the composition.

The cosmetic composition is characterized in that it is for anti-inflammatory, anti-oxidation or wrinkle improvement. In addition, the cosmetic composition is characterized in that it is for promoting hair growth or preventing hair loss.

According to the present invention in order to achieve the above other object,

(A) coarsely grinding catnip or spinach;

(B) a freeze-thaw pretreatment step of adding a solvent to coarsely ground catnip or spinach and repeating freezing and thawing;

(C) extracting juice from catnip or spinach pre-treated with freeze-thaw;

(D) obtaining a supernatant by centrifuging the juice at 1,000xg to 10,000xg;

(E) freeze-drying the supernatant in which exosomes exist;

(F) forming an aqueous two-phase system using PEG (Polyethylene glycol)/Dextran in the lyophilisate; and

(G) There is provided a method for purifying catnip or spinach exosomes comprising the step of obtaining a lower layer in which exosomes are concentrated in the aqueous two-phase system.

More preferably, the coarsely pulverizing step in step (A) is characterized in that the coarsely pulverized catnip is pulverized into a particle size of 2.0 to 5.0 mm using a grinding device, and the freeze-thaw pretreatment in step (B) is coarsely pulverized catnip. Or, add water to spinach, freeze it for 15 to 20 hours at a temperature of -80 to -70 ° C, and then repeat the process of melting for 8 to 10 hours at a temperature of 40 to 50 ° C 2 to 5 times. to be

Catmint exosomes, spinach exosomes, or mixed exosomes of the present invention purified using coarse grinding, freeze-thaw pretreatment, and an aqueous two-phase system have excellent stability, excellent anti-inflammatory effect, antioxidant effect, anti-wrinkle effect, and hair growth It can be usefully used as a cosmetic for improving skin and hair conditions because it exhibits promoting effects and hair loss prevention effects.

1 is a TEM image of catnip-derived exosomes purified according to an embodiment of the present invention.
2 is a TEM image of spinach-derived exosomes purified according to an embodiment of the present invention.
Figure 3 is a graph showing the results of NTA analysis for confirming the size distribution and particle number of purified catnip-derived exosome particles according to an embodiment of the present invention.
Figure 4 is a graph showing the NTA analysis results for confirming the size distribution and particle number of purified spinach-derived exosome particles according to an embodiment of the present invention.
5 is a graph showing the results of evaluating the cytotoxicity of exosomes derived from catnip and spinach purified according to the present invention by MTT assay.
6 is a graph showing the results obtained by evaluating the anti-inflammatory efficacy of catnip and spinach-derived exosomes purified according to the present invention in terms of IL-1 expression inhibition rate.
7 is a graph showing the results obtained by evaluating the anti-inflammatory efficacy of catnip and spinach-derived exosomes purified according to the present invention in terms of IL-6 expression inhibition rate.
8 is a graph showing the results of evaluating the anti-wrinkle efficacy of exosomes derived from catmint and spinach purified according to the present invention by COL1A1 expression.
9 is a graph showing the results of evaluating the anti-wrinkle efficacy of exosomes derived from catmint and spinach purified according to the present invention in terms of MMP-1 expression inhibition rate.
10 is a graph showing the results of evaluating the hair growth promoting effect of exosomes derived from catnip and spinach purified according to the present invention as the dermal papilla cell proliferation effect.
11 is a graph showing the results of evaluating the hair loss prevention efficacy of exosomes derived from catnip and spinach purified according to the present invention by a 5-alpha-reductase inhibitory activity test.

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

Plant-derived exosomes contain physiological activity and signaling substances secreted by plant cells themselves, and are known to be less toxic than mammalian-derived exosomes. Due to these advantages, it is a material that can be used in the fields of medicine, cosmetics, food, etc., but due to the structural feature consisting of a phospholipid double membrane, it has low dispersibility and tends to aggregate, making it difficult to maintain its activity continuously in the formulation. have a problem

The technical feature of the present invention is to separate and purify exosomes with excellent skin and hair condition improvement activity from catnip and spinach through coarse grinding and freeze-thaw pretreatment, and use them as cosmetics.

Catnip (Nepeta cataria) is a perennial plant of the Lamiaceae family and is also called catnip. It is a perennial plant that grows in the mountains or fields. The stem is square with a height of 30-100cm, has thin white hairs, and the plant body has a unique scent. The leaves are opposite, the tip is pointed, the bottom is shallow heart-shaped, and the edge is saw-toothed. Flowers bloom in June-July and are light purple in spikes at the ends of stems or branches. The calyx has spread hairs and distinct lines, and the upper part is split into 5 parts. The fruit is a small fruit, elliptical, and grows wild throughout Korea.

Catnip, another name for catmint, comes from cats liking the scent of this herb. Catnip contains an ingredient called nepetalactone that excites cats and has a refreshing, minty-like scent. Nepetalactone has an awakening effect on cats, but when it is absorbed by humans, it acts in the opposite way, has a calming and relaxing effect, stabilizes the mind, induces sleep, and helps relieve insomnia. Catnip contains ingredients such as tannin, iridoid, and thymol, and is rich in antioxidants such as flavonoids, caffeic acid, rosmarinic acid, coumaric acid, and phenolic acid. In particular, rosmarinic acid, which has an excellent antioxidant effect compared to vitamin E, prevents cell damage caused by free radicals. In addition, rosmarinic acid has strong anti-inflammatory properties and is known to be effective in treating gastric ulcers and asthma. In addition to this, it also contains minerals such as sodium, phosphorus, manganese and sulfur, and various vitamins such as vitamins A and B, which can help prevent and treat various diseases. In the present invention, catnip sheath is preferably used for preparing catnip exosomes as an active ingredient.

Spinach (Spinacia Oleracea) is an annual or biennial herb of the Pollack family. It is a plant cultivated as a vegetable and reaches a height of 50 cm. The main stem grows upright, is hollow, and is light green. Leaves come out from the bottom at first, but they are alternate on the main stem, and the leaves attached to the bottom are long triangular or ovate, and become smaller as they go up, becoming lanceolate spear-shaped or lanceolate. The flowers bloom in May with digahwa, and the fruit is wrapped in a calyx-like parcel, and it has two thorns, so it is similar to the fruit of water chestnut.

It is presumed to have originated from Persia and was introduced to Korea via China. Since spinach first appeared in 『Hunmongjahoe(訓蒙字會)』 compiled by Choi Se-jin in 1577 (the 10th year of King Seonjo), it is believed to have been cultivated since the early Joseon Dynasty. Currently, it is a vegetable that is widely eaten, with a cultivation area of about 5,000 hectares and 67,000 tons produced. Spinach is a food ingredient that has been recognized for its excellent efficacy enough to be selected as a world super food selected by Time magazine. It is effective for dieting and gives a feeling of fullness as it is rich in dietary fiber and water. In addition, the thylakoid component of green leaves also helps to increase satiety and suppress appetite. It also has the effect of lowering the risk of arteriosclerosis and reducing systolic blood pressure. As a result of a study published in 'Clinical Nutrition Research' in 2015, it was found that arterial stiffness and systolic blood pressure were lowered in subjects who consumed spinach soup for 7 days. Potassium, which is contained in large amounts in spinach, promotes the excretion of sodium and acts to suppress the rise in blood pressure caused by sodium. The thylakoid component, which is known to give a feeling of satiety, also has the effect of lowering blood pressure by lowering the level of fat in the blood. Spinach is an antioxidant food rich in beta-carotene that prevents aging. The content of beta-carotene, a fat-soluble vitamin, increases when boiled in water. It is also rich in various vitamins, so it is said to be effective in improving skin troubles. Vitamin A, vitamin B2, and antioxidants, which are abundant in spinach, have a good effect on hair health and prevent hair loss. In addition, various antioxidants in spinach remove active oxygen that promotes cell aging in the body, so it slows down the aging of cells. Its rich folic acid prevents brain aging and improves brain function. It also helps prevent dementia. Chlorophyll and folic acid contained in spinach are known to help prevent various cancers by stopping cancer, and according to the 'American Journal of Clinical Nutrition', people who consumed spinach daily were less likely to develop stomach cancer than those who did not. The incidence rate was reduced by 35%, colorectal and breast cancer by 40%, and pancreatic cancer by 23%. Spinach leaves are preferably used in the preparation of spinach exosomes as an active ingredient in the present invention.

Exosomes derived from catnip and spinach as active ingredients of the cosmetic composition of the present invention are purified by the following method.

(A) coarsely grinding catnip or spinach; (B) a freeze-thaw pretreatment step of adding a solvent to coarsely ground catnip or spinach and repeating freezing and thawing; (C) extracting juice from catnip or spinach pre-treated with freeze-thaw; (D) obtaining a supernatant by centrifuging the juice at 1,000xg to 10,000xg; (E) freeze-drying the supernatant in which exosomes exist; (F) forming an aqueous two-phase system using PEG (Polyethylene glycol)/Dextran in the lyophilisate; and (G) obtaining an exosome-enriched lower layer in the aqueous two-phase system.

In the present invention, in order to prepare catnip and spinach-derived exosomes having excellent skin and hair condition improving activity, the raw materials are first coarsely ground and then subjected to freeze-thaw pretreatment.

In the case of plants, the secretion characteristics of exosomes can be changed by environmental factors, and in particular, the amount of exosome secretion can be affected when the plant is stressed. Therefore, in order to confirm the optimal conditions for plants to secrete exosomes, the yield and skin improvement activity were confirmed through various pretreatments. increased, and it was confirmed that the skin and hair improvement activity increased.

According to a preferred embodiment of the present invention, the coarse grinding process in step (A) consists of grinding to a particle size of 2.0 ~ 5.0 mm using a conventional grinding device.

Subsequently, water was added to the crushed catnip and spinach, frozen for 15 to 20 hours at a temperature of -80 to -70 ° C, and then thawed for 8 to 10 hours at a temperature of 40 to 50 ° C. Repeat 5 times (step (B)).

Subsequently, the frozen catmint or spinach pre-treated is juiced (step (C)).

In step (C), it is preferable to use a screw having an agitation speed of 20 to 50 rpm.

The centrifugal separation method used in step (D) 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 the rpm to remove large contaminants, more preferably at 10,000xg to obtain a final solution for forming an aqueous two-phase system.

The freeze-drying used in the step (E) rapidly lowers the temperature of the container to freeze the material to be dried, and then the pressure inside the container is close to vacuum to sublimate the solidified solvent contained in the material into water vapor and dry it way. Freeze at -50 ~ -80 ℃ for 15 ~ 24 hours, and dry for 72 ~ 120 hours in a vacuum state in a freeze dryer. At this time, the vacuum state usually means the pressure state of the freeze dryer.

In order to efficiently separate and purify exosomes, in the present invention, an aqueous two phase partition method, which is one of the separation methods using the difference in affinity between each layer by making two layers with two types of aqueous solutions that are not well dissolved in each other, is used. ) was used.

Although PEG/salts (salts such as sulfate, phosphate, citrate, etc.) can also be used in the method of forming an aqueous two-phase system, it is preferable to use PEG/Dextran to achieve the object of the present invention. Dextran is a natural polymer obtained by bacterial action and is used as a thickener, binder, and bulking agent in cosmetic formulations.

In the formation of the aqueous two-phase system in the step (F), 1 to 15% by weight, preferably 2 to 5% by weight, of PEG having a molecular weight of 10,000 to 35,000 is used, and Dextran is used in a molecular weight of 300,000 to 650,000. ~ 8% by weight, preferably 1 to 3% by weight. When the PEG and Dextran are used in a concentration ratio of 3.3% by weight: 1.7% by weight, the yield of exosomes is the highest and the stability is the best, so it is more preferable.

In order to increase the purity of exosomes, following step (G), a step of forming an aqueous two-phase system using an aqueous two-phase solution of the same concentration and obtaining a lower layer solution can be additionally performed 2-3 times. Yes (step (H)).

Catnip exosomes, spinach exosomes, and mixed exosomes prepared by this method have excellent anti-inflammatory effects compared to general catnip extract and spinach extract and compared to exosomes purified without pretreatment (Test Example 7). , Test Example 8), antioxidant effect (Test Example 9), wrinkle improvement effect (Test Example 10, Test Example 11, Test Example 12), hair growth promoting effect (Test Example 13), hair loss prevention effect (Test Example 14, Test Example 15) was shown.

Therefore, the catnip and spinach-derived exosomes can be used in a cosmetic composition for anti-inflammatory, anti-oxidation, and wrinkle improvement, and can also be used for a cosmetic composition for promoting hair growth and preventing hair loss.

At this time, catnip, spinach-derived exosomes, or mixed exosomes as the active ingredient may be contained in an amount of 0.0001 to 30.0% (w/w) based on the total weight of the cosmetic composition. The mixed exosomes are made by mixing catnip exosomes and spinach exosomes in a weight ratio of 1 to 3:1 to 3, respectively.

The cosmetic composition can be any formulation that is conventionally manufactured, and for example, skin lotion, skin toner, pack, nutrient cream, moisture cream, essence, body cream, body lotion, body oil, cleansing foam, cleansing lotion, soap , patches, foundations, lipsticks, makeup bases, lipsticks, and the like.

[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 catnip exosomes

coarse grinding

100 g of catnip was pulverized to a particle size of about 2.0 to 5.0 mm using a conventional grinding device.

Freeze Thawing Pretreatment

Purified water was added to the crushed catnip, frozen at -80 ° C for 18 hours, and then thawed at 40 ° C for 8 hours. This process was repeated a total of three times to make exosome extraction easy.

juice

Catmint subjected to freeze-thaw pretreatment was juiced with a low-speed screw at 30 rpm using a general juicer, and the obtained catnip juice was filtered through a mesh net to remove suspended matter. The collected catnip juice was stored at -80 ° C until purification.

Supernatant Recovery for Exosome Purification

Catnip juice was centrifuged at 10,000xg for 10 minutes at 4° C. because removal of large contaminants was required for exosome purification. After centrifugation, the supernatant was recovered to form an aqueous two-phase system.

Freeze-drying of the supernatant

Freeze-drying was performed for the purpose of reducing the volume of the supernatant for mass production of exosomes. It 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.

Formation of an aqueous two-phase system

Purified water was added to the lyophilized supernatant, and an aqueous two-phase system was formed using PEG (Polyethylene glycol)/Dextran. An aqueous two-phase system was formed using 3.3% by weight of PEG (purchased from Sigma Aldrich) with a molecular weight of 10,000 to 35,000 and 1.7% by weight of Dextran (purchased from Sigma Aldrich) with a molecular weight of 300,000 to 650,000.

Recovery of exosomes

After mixing the supernatant and the PEG/Dextran solution, centrifugation was performed at 1,000xg for 10 minutes at 4°C. After centrifugation, the supernatant was removed to recover exosomes.

Additional cleaning process

In order to increase the purity, an additional washing process was performed by adding the aqueous two-phase solution having the same concentration to the recovered lower layer. After repeated treatment three times, the final exosome-concentrated subnatant was recovered.

Example 2: Preparation of spinach exosomes

Spinach-derived exosomes were purified in the same manner as in Example 1, except that spinach leaves were used instead of catmint.

Example 3: Preparation of catnip and spinach mixed exosomes

Mixed exosomes were prepared by mixing catnip exosomes and spinach exosomes prepared in Examples 1 and 2 at the same weight ratio.

Examples 4-11: Preparation of catnip and spinach exosomes

In the case of plants, the secretion characteristics of exosomes may vary depending on environmental factors. Since the optimal conditions for plants to secrete exosomes are all different, the freeze-thaw pretreatment conditions of catnip and spinach were different to establish optimal conditions through comparison of exosome yields due to environmental stress, such as conditions in the freeze-thaw pretreatment step. Thus, exosomes were purified. In order to confirm the optimal conditions, only the corresponding conditions were changed and the exosomes were purified in the same manner as in Example 1, and the conditions are shown in Table 1 below. In Examples 4 and 8, catnip and spinach were directly ground and juiced without freeze-thaw pretreatment, and purified by applying an aqueous two-phase system.

raw material Number of freeze-thaw treatments Example 4 catnip 0 Example 5 catnip One Example 6 catnip 2 Example 7 catnip 4 Example 8 spinach 0 Example 9 spinach One Example 10 spinach 2 Example 11 spinach 4

Comparative Example 1: Preparation of catnip extract

10 g of dried catnip was added to 100 g of purified water and extracted at 80° C. for 3 hours. After extraction, filtration under reduced pressure was performed to obtain catnip extract, and then distillation was performed using a rotary evaporator to obtain a sample in powder form.

Comparative Example 2: Preparation of spinach extract

10 g of dried spinach was added to 100 g of purified water and extracted at 80° C. for 3 hours. After extraction, vacuum filtration was performed to obtain a spinach extract, and then distillation was performed using a rotary evaporator to obtain a sample in powder form.

Test Example 1: Characteristic analysis of catnip-derived exosomes: TEM analysis

In order to confirm the shape of the purified catnip-derived exosomes, they were analyzed by transmission electron microscopy (TEM). 1 is a TEM analysis image of catnip-derived exosomes purified according to Example 1 above. As a result of the analysis, the existence of about 150 nm particles having a spherical phospholipid bilayer structure was confirmed.

Test Example 2: Characterization of spinach-derived exosomes: TEM analysis

In order to confirm the shape of the purified spinach-derived exosomes, they were analyzed by transmission electron microscopy (TEM). 2 is a TEM analysis image of spinach-derived exosomes purified according to Example 2 above. As a result of the analysis, the existence of about 150 nm particles having a spherical phospholipid bilayer structure was confirmed.

Test Example 3: Characteristic analysis of catnip-derived exosomes: NTA analysis

In order to confirm the particle size distribution and the number of particles per unit volume of the purified catnip-derived exosomes, nanoparticle tracking analysis (NTA) was analyzed.

3 is a graph showing the results of NTA analysis of catmint-derived exosomes purified according to Example 1. As a result of the analysis, the average particle size of 150.7 nm and the concentration of 5.30 × 10 ¹⁰ particles per unit volume of 1 mL were confirmed.

Test Example 4: Characterization of spinach-derived exosomes: NTA analysis

In order to confirm the particle size distribution and the number of particles per unit volume of the purified spinach-derived exosomes, nanoparticle tracking analysis (NTA) was analyzed.

4 is a graph showing the results of NTA analysis of spinach-derived exosomes purified according to Example 2. As a result of the analysis, the average particle size of 152.1 nm and the concentration of 4.60 × 10 ¹⁰ particles per unit volume of 1 mL were confirmed.

Test Example 5: Comparison of the yield of catnip and spinach-derived exosomes

In order to confirm the optimal conditions for freezing and thawing, the yields of catnip and spinach-derived exosomes purified according to Examples 1, 2, and 4 to 11 were compared. Table 2 below is a table showing yield comparison as a result of nanoparticle tracking analysis (NTA).

number Particle Number
(Particles/ml) Particle Size
(nm) Example 1 5.30 x 10 ¹⁰ 150.7 Example 2 4.60 x 10 ¹⁰ 152.1 Example 4 3.50 x 10 ⁸ 149.7 Example 5 2.80 x 10 ⁹ 151.4 Example 6 6.70 x 10 ⁹ 151.0 Example 7 9.40 x 10 ⁹ 154.6 Example 8 1.70 x 10 ⁸ 157.3 Example 9 1.90 x 10 ⁹ 155.0 Example 10 5.60 x 10 ⁹ 154.1 Example 11 8.70 x 10 ⁹ 154.6

As confirmed in Table 2, when the freeze-thaw pre-treatment was performed (Examples 1, 2, 5, 6, 7, 9, 10, and 11), high yields were exhibited, and among them, conditions in which the treatment was repeated three times In Examples 1 and 2, the yield of exosomes was the highest. From the results of Example 4 and Example 8, the yield of exosomes not subjected to pre-freezing and thawing was the lowest. This indicates that the pretreatment conditions greatly affect the yield of exosomes, and it was confirmed that the most optimal condition was to repeat the freeze-thaw process three times.

Test Example 6: Evaluation of cytotoxicity

Catmint exosome (Example 1, Example 4), spinach exosome (Example 2, Example 8) and mixed exosome (Example 3) and catmint extract (Comparative Example 1), spinach extract (Comparative Example 2 ), MTT assay was evaluated to confirm the cytotoxicity. Human fibroblast cell line (human dermal fibroblast, HDFa) was 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 then catnip exosomes, spinach exosomes, the mixed exosomes, catnip extract, and spinach extract were administered to a new medium according to 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.

5 is a graph showing the results of evaluating the cytotoxicity of the samples by MTT assay. As a result of the test, when the exosomes of catnip, exosomes of spinach, mixed exosomes of catnip and spinach, catnip extract, and spinach extract were treated, no cytotoxicity was observed at all concentrations.

Test Example 7: Evaluation of anti-inflammatory efficacy of catnip and spinach exosomes (inhibition of IL-1 expression)

In order to confirm the anti-inflammatory efficacy of catnip and spinach-derived exosomes, an IL-1 mRNA expression inhibition test was performed in comparison with the extracts. Human Keratinocyte (HaCaT) cells were inoculated at a concentration of 6×10 ⁵ cells/well, cultured for 24 hours at 37°C and 5% CO ₂ until they reached 70-80% growth, and then the medium in each well was removed. and replaced with fresh serum-free DMEM. Pretreatment was performed for 4 hours by administering catnip exosomes, spinach exosomes, catnip extract, and spinach extract by concentration to each well. Each well plate was irradiated with 10 mJ/cm ² UVB by a UVB irradiator (Vilber Loumet, France). Samples were further cultured for 24 hours after treatment with diluted serum-free DMEM culture medium. At this time, PBS was treated as a negative control group, and 0.01% Allantoin was treated as a positive control group. Ribo Ex TM Total RNA Isolation Solution (GeneAll Biotechnology, Korea) and scraper were used to dissolve the cells after cell culture, and then 0.2mL chloroform (Sigma-Aldrich, USA) was added and centrifuged (12,000rpm, 4℃, 30 minutes). )do. Separate the supernatant with RNA, add isopropanol (Merck-Millipore, Germany) in the same amount as the supernatant, invert, and centrifuge (12,000 rpm, 4 ℃, 30 minutes). 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 of 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, and the StepOnePlus Real-Time PCR System ( PCR was performed using Applied Biosystems, USA).

The PCR primer sequence of the IL-1 gene is shown in Table 3 below, and FIG. 6 is a graph showing the results of evaluating the anti-inflammatory efficacy of exosomes derived from catnip and spinach purified according to the above example in terms of IL-1 mRNA expression inhibition rate. . As a result of the test, it was confirmed that the treatment of catnip exosomes, spinach exosomes and catnip extract, and spinach extract reduced IL-1 mRNA, an inflammatory factor, in a concentration-dependent manner, and the inhibition rate increased significantly in the exosome treatment concentration range compared to the extract. At this time, when Allantoin 0.01%, a positive control, was treated, 41.1% inhibition rate was shown.

gene Forward primer (5'→3') Reverse primer (5'→3') IL-1 5′-AAT CTG TAC CTG TCC TGC GTG TT-3′ 5'-TGG GTA ATT TTT GGG ATC TAC ACT CT-3' GAPDH 5'-GTC TCC TCT GAC TTC AAC AGC G-3' 5'-ACC ACC CTG TTG CTG TAG CCA A-3'

Test Example 8: Evaluation of anti-inflammatory efficacy of catnip and spinach exosomes (inhibition of IL-6 expression)

In order to confirm the anti-inflammatory efficacy of catnip and spinach-derived exosomes, an IL-6 mRNA expression inhibition test was performed in comparison with the extracts. The experimental method is the same as in Test Example 7 above, and the PCR primer sequence of the IL-6 gene is shown in Table 4 below. 7 is a graph showing the results obtained by evaluating the anti-inflammatory efficacy of catnip and spinach-derived exosomes purified according to the above example in terms of IL-6 mRNA expression inhibition rate. As a result of the test, it was confirmed that the treatment of catnip exosomes, spinach exosomes and catnip extract, and spinach extract reduced IL-6 mRNA, an inflammatory factor, in a concentration-dependent manner, and the inhibition rate increased significantly in the exosome treatment concentration range compared to the extract. At this time, when Allantoin 0.01%, a positive control, was treated, 47.6% inhibition rate was shown.

gene Forward primer (5'→3') Reverse primer (5'→3') IL-6 5'-AAC CTG AAC CTT CCA AAG ATG G-3' 5'-TCT GGC TTG TTC CTC ACT ACT-3' GAPDH 5'-GTC TCC TCT GAC TTC AAC AGC G-3' 5'-ACC ACC CTG TTG CTG TAG CCA A-3'

Test Example 9: Antioxidant efficacy evaluation of catnip and spinach-derived exosomes (DPPH radical scavenging activity)

In order to confirm the antioxidant efficacy of catnip-derived exosomes, spinach-derived exosomes, and mixed exosomes, a DPPH radical scavenging activity test was conducted in comparison with each extract. The DPPH radical scavenging activity was measured by the reducing power of the sample for the DPPH radical. DPPH (1,1-Diphenyl-2-picrylhydrazyl) reagent was dissolved in ethanol to prepare a 0.4 mM solution, and samples (exosomes, extracts) prepared by concentration in a 96 well plate or BHT (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 Example 2 Example 3 Example 4 Example 8 Comparative Example 1 Comparative Example 2 0.01 19.4 22.7 34.8 10.7 9.8 6.7 10.1 0.1 33.8 37.6 50.3 27.4 25.6 17.8 20.3 One 52.6 55.3 67.5 44.6 42.7 35.4 37.9 BHT (0.01%) 66.3

As a result of the test, the DPPH radical scavenging activity increased in a concentration-dependent manner when treated with catnip-derived exosomes, spinach-derived exosomes, catnip extract, and spinach extract. confirmed to increase. In addition, it was confirmed that the highest activity was exhibited when the mixed exosomes of catnip and spinach were treated.

Test Example 10: Evaluation of anti-wrinkle efficacy of catnip and spinach-derived exosomes (COL1A1 expression)

To confirm the anti-wrinkle efficacy of catnip and spinach-derived exosomes, the effect on COL1A1 expression was evaluated in comparison with the extracts. 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 in 5% CO ₂ cultured under. The medium in each well was removed and replaced with a new serum-free medium. Pretreatment was performed for 4 hours by administering catnip exosomes, spinach exosomes, catnip extract, and spinach extract by concentration to each well. 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 TGF-β 5ng/mL 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). )do. Separate the supernatant with RNA, add isopropanol (Merck-Millipore, Germany) in the same amount as the supernatant, invert, and centrifuge (12,000 rpm, 4°C, 30 minutes). 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, and the StepOnePlus Real-Time PCR System ( PCR was performed using Applied Biosystems, USA).

The PCR primer sequences of the COL1A1 gene are shown in Table 6 below, and FIG. 8 is a graph showing the results of evaluating the anti-wrinkle efficacy of exosomes derived from catmint and spinach purified according to the above example by COL1A1 mRNA expression rate. As a result of the test, it was confirmed that when the catnip exosomes, spinach exosomes and catnip extract, and spinach extract were treated, the collagen production factor, COLA1A mRNA, increased in a concentration-dependent manner, and the expression rate increased significantly in the exosome treatment concentration range compared to the extract. At this time, when treated with TGF-β 5ng/mL as a positive control group, the expression rate was 143.7%.

gene Forward primer (5'→3') Reverse primer (5'→3') COL1A1 5'-GAG AGC ATG ACC GAT GGA TT-3' 5'-CCT TCT TGA GGT TGC CAG TC-3' GAPDH 5'-GTC TCC TCT GAC TTC AAC AGC G-3' 5'-ACC ACC CTG TTG CTG TAG CCA A-3'

Test Example 11: Evaluation of anti-wrinkle efficacy of catnip and spinach exosomes (inhibition of MMP-1 expression)

In order to confirm the anti-wrinkle efficacy of catnip and spinach-derived exosomes, the effect on MMP-1 expression was confirmed by comparing with the extracts. The experimental method is the same as in Test Example 10 above, and the PCR primer sequence of the MMP-1 gene is shown in Table 7 below. 9 is a graph showing the results obtained by evaluating the anti-wrinkle efficacy of exosomes derived from catmint and spinach purified according to the above example in terms of inhibition of MMP-1 mRNA expression. As a result of the test, when the catnip exosomes, spinach exosomes and catnip extract, and spinach extract were treated, the collagenase, MMP-1 mRNA, decreased in a concentration-dependent manner, and the inhibition rate was significantly increased in the concentration range of exosome treatment compared to the extract. did At this time, when treated with 5 ng/mL of TGF-β as a positive control group, the expression rate was 54.6%.

gene Forward primer (5'→3') Reverse primer (5'→3') MMP-1 5′-GGG CTT GAA GCT GCT TAC GA-3′ ACA GCC CAG TAC TTA TTC CCT TTG-3' GAPDH 5'-GTC TCC TCT GAC TTC AAC AGC G-3' 5'-ACC ACC CTG TTG CTG TAG CCA A-3'

Formulation Examples 1 to 3: Preparation of Cream

With the composition shown in Table 8 below, a cream containing catnip exosomes and spinach exosomes purified according to the above example was prepared by a conventional method. The cream containing the catmint extract of Comparative Example 1 was used as Comparative Formulation Example 1, and the cream containing the spinach extract of Comparative Example 2 was used as Comparative Formulation Example 2.

ingredient Formulation example 1 Formulation Example 2 Formulation Example 3 Comparative Formulation Example 1 Comparative Formulation Example 2 Content (% by weight) Catnip exosomes (Example 1) 5 - - - - Spinach exosomes (Example 2) - 5 - - - Catmint + spinach exosomes (Example 3) - - 5 - - Catmint extract (Comparative Example 1) - - - 5 - Spinach extract (Comparative Example 2) - - - - 5 glycerin 10 10 10 10 10 Butylene Glycol 5 5 5 5 5 glyceryl oleate 1.8 1.8 1.8 1.8 1.8 Cetearyl Olivate 0.5 0.5 0.5 0.5 0.5 Sorbitan Olivate 0.5 0.5 0.5 0.5 0.5 Caprylic/Capric Triglyceride 5.0 5.0 5.0 5.0 5.0 Cetylethylhexanoate 1.0 1.0 1.0 1.0 1.0 beeswax 0.5 0.5 0.5 0.5 0.5 squalane 0.2 0.2 0.2 0.2 0.2 1,2-Hexanediol 0.2 0.2 0.2 0.2 0.2 Cholesteryl/Behenyl/Octyldodecyllauroylglutamate 1.0 1.0 1.0 1.0 1.0 dimethicone 0.5 0.5 0.5 0.5 0.5 Cyclopentasilonesac/Cyclohexasiloxane 2.0 2.0 2.0 2.0 2.0 Cetialyl Alcohol 1.0 1.0 1.0 1.0 1.0 mineral oil 2.5 2.5 2.5 2.5 2.5 Disodium EDT 0.02 0.02 0.02 0.02 0.02 BHT 0.05 0.05 0.05 0.05 0.05 Tocopheryl Acetate 0.3 0.3 0.3 0.3 0.3 panthenol 0.2 0.2 0.2 0.2 0.2 Ethylhexylmethoxycinnamate 0.2 0.2 0.2 0.2 0.2 incense 0.01 0.01 0.01 0.01 0.01 Purified water balance balance balance balance balance

Test Example 12: Evaluation of catnip and spinach-derived exosomes for wrinkle improvement

In order to confirm the anti-wrinkle effect of catnip exosomes (Example 1), spinach exosomes (Example 2) and their mixed exosomes (Exosome 3), 20 adult women in their 30s to 50s were tested in the formulation example above. The prepared cream was applied to both sides of the face, and 633 nm red light was irradiated with an LED light source device for 24 hours for a cumulative period of 6 weeks, and then the wrinkle improvement effect was evaluated through an actual use test.

Table 9 is a table showing the results of evaluation of the anti-wrinkle effect of catnip and spinach-derived exosomes purified according to the above example through actual use tests.

Wrinkle improvement effect Great a little doesn't exist Formulation Example 1 6 11 3 Formulation Example 2 5 10 5 Formulation Example 3 12 7 One Comparative Formulation Example 1 One 3 16 Comparative Formulation Example 2 0 5 15

As can be seen from the results of Table 9, the cream containing the exosomes of catnip and spinach and the mixed exosomes of catnip and spinach of the present invention showed an excellent skin wrinkle improvement effect.

Test Example 13: Evaluation of hair growth promoting efficacy (hair papilla cell proliferation)

In order to confirm the hair growth promoting effect of catnip and spinach-derived exosomes, their effects on dermal papilla cell proliferation were compared with those of catnip and spinach extracts by concentration. HGDPC cell line (Human follicle dermal papilla cell), which is a dermal papilla cell constituting hair follicles, was divided into 1 × 10 ⁴ cells / mL in a 96 well plate and cultured, and then exosomes of catnip, exosomes of spinach, mixed exosomes and catnip extract, Spinach extract and DHT (5α-dihydrotestosterone) were treated to be 10 μM and cultured for 48 hours. After incubation, the cells were treated with 10x BrdU solution and incubated for 6 hours, then the solution was removed, treated with Anti-BrdU Antibody, and allowed to react at room temperature for 1 hour. Secondary Antibody HRP Conjugate was added and reacted, washed with PBS, reacted with substrate solution, and the reaction was terminated using stop solution, and absorbance was measured at 450 nm.

10 is a graph showing the results of evaluating the hair growth promoting effect of each sample as the dermal papilla cell proliferation effect. As a result of the test, it was confirmed that treatment with catnip and spinach-derived exosomes and catnip and spinach extracts increased cell proliferation in a concentration-dependent manner, and further increased proliferation when treated with catnip and spinach-derived exosomes compared to the extracts. Also, the highest cell proliferation rate was shown when the mixed exosomes of catnip and spinach were treated, and at this time, when the positive control, Minoxidil 10Μm, was treated, the proliferation rate was 88.8%.

Test Example 14: Evaluation of anti-hair loss efficacy (inhibition of 5α-reductase activity)

In order to confirm the anti-hair loss efficacy of catnip and spinach-derived exosomes, the effects on 5α-reductase inhibitory activity were compared with those of catnip and spinach extracts for each concentration. HGDPC cell line (Human follicle dermal papilla cell), which is a dermal papilla cell constituting hair follicles, was dispensed in a 24 well plate at 4×10 ⁵ cells/mL and cultured for 24 hours. time incubated. After culturing, testosterone was treated, and the supernatant was separated to obtain a cell pellet. The obtained pellet was measured for the concentration of dihydrotestosterone (DHT) using enzyme immunoassay. DHT ElISA Kit strip-well was dispensed with 100 μl of the cell pellet and 25 μl of DHT antiserum, and reacted at 4° C. for about 18 hours. After the reaction, 100 μl of HRP-Streptavidin Conjugate was dispensed into each well, reacted at room temperature for 30 minutes, and washed. After washing, 90 μl of the TMB substrate was dispensed and reacted at room temperature within 20 minutes. Then, 50 μl of the stop solution was treated, and the absorbance was measured at 450 nm immediately after the treatment to quantify the concentration of DHT.

Figure 11 is a graph showing the results of evaluating the hair loss prevention efficacy of each sample by alpha-reductase (5α-reductase) inhibitory activity test. Based on the negative control group, which was not treated with the sample, the 5 alpha-reductase inhibition rate was calculated. As a result of the test, the inhibitory activity increased in a concentration-dependent manner when exosomes derived from catnip and spinach were treated. The best inhibitory activity was shown when catnip and spinach mixed exosomes were treated.

Test Example 15: Evaluation of hair loss prevention efficacy

To confirm the anti-hair loss efficacy of catnip exosomes (Example 1, Example 4), spinach exosomes (Example 2, Example 8), and mixed exosomes (Example 3), catnip extract by concentration, spinach extract and In comparison, an experiment was conducted for 12 weeks on 30 adults with early hair loss. Catnip-derived exosomes, spinach-derived exosomes, and mixed exosomes purified according to the above example were prescribed at a concentration of 5% and applied once a day, and hair was washed after about 20 minutes. After 12 weeks, the fallen hairs were collected and counted, and the results are shown in Table 10 below. As a negative control group, fallen hairs were collected and compared without any treatment.

Treatment concentration (%) Average number of fallen hairs (pcs) negative control - 70 Catnip exosomes (Example 1) 5 50 Spinach exosomes (Example 2) 5 56 Catmint + spinach exosomes (Example 3) 5 44 Catmint extract (Comparative Example 1) 5 69 Spinach extract (Comparative Example 2) 5 73

As a result of the experiment, it was confirmed that the number of exfoliated hairs significantly decreased when treated with exosomes compared to catnip extract and spinach extract.

Claims (11)

In the cosmetic composition containing catnip exosomes, spinach exosomes, or mixed exosomes as an active ingredient,
Catmint exosomes and spinach exosomes as the active ingredient,
(A) coarsely grinding catnip or spinach; (B) a freeze-thaw pretreatment step of adding a solvent to coarsely ground catnip or spinach and repeating freezing and thawing; (C) extracting juice from catnip or spinach pre-treated with freeze-thaw; (D) obtaining a supernatant by centrifuging the juice at 1,000xg to 10,000xg; (E) freeze-drying the supernatant in which exosomes exist; (F) forming an aqueous two-phase system using PEG (Polyethylene glycol)/Dextran in the lyophilisate; and (G) obtaining a lower layer in which exosomes are concentrated in the aqueous two-phase system. delete The method of claim 1, wherein the freeze-thawing pretreatment in step (B) is performed by adding water to the coarsely ground catmint or spinach, freezing for 15 to 20 hours at a temperature of -80 to -70 ° C, and then to 40 to 50 ° C A cosmetic composition characterized in that the melting process is repeated 2 to 5 times for 8 to 10 hours at a temperature condition of. The cosmetic composition according to claim 1, wherein the catnip exosome, spinach exosome, or a mixed exosome is contained in an amount of 0.0001 to 30.0% (w/w) based on the total weight of the composition. The cosmetic composition according to claim 1, wherein the cosmetic composition is for anti-inflammatory use. The cosmetic composition according to claim 1, wherein the cosmetic composition is for antioxidation. The cosmetic composition according to claim 1, wherein the cosmetic composition is for wrinkle improvement. The cosmetic composition according to claim 1, wherein the cosmetic composition is for promoting hair growth. The cosmetic composition according to claim 1, wherein the cosmetic composition is for preventing hair loss. delete delete

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