KR102391636B1 - Cosmetic composition for improving skin and hair condition containing Scutellaria baicalensis exosome, Houttuynia cordata exosome - Google Patents

Abstract

The present invention relates to a cosmetic composition for improving skin and hair condition containing exosomes derived from Scutellaria baicalensis and Houttuynia cordata. Specifically, the present invention relates to a cosmetic composition for improving skin and hair condition which has excellent effects of antioxidant, anti-inflammation, anti-wrinkle, hair growth promotion, and hair loss prevention by containing Scutellaria baicalensis exosome and Houttuynia cordata exosome purified by using hot air drying pretreatment and an aqueous two-phase system as active ingredients.

Description

Golden, cosmetic composition for improving skin and hair condition containing exosomes derived from medicinal wool as an active ingredient {Cosmetic composition for improving skin and hair condition containing Scutellaria baicalensis exosome, Houttuynia cordata exosome}

The present invention relates to a cosmetic composition for improving the condition of skin and hair containing exosomes derived from gold and ryegrass as an active ingredient, and specifically, golden exosomes and/or yak-mo-mil purified using hot air drying pretreatment and an aqueous two-phase system. It relates to a cosmetic composition for improving skin and hair conditions that contains exosomes as an active ingredient and has antioxidant, anti-inflammatory, wrinkle-improving effects, hair growth promotion, and hair loss prevention effects.

The function of cosmetics has been extended beyond cleanliness and simple makeup to remove or suppress the causes of skin aging such as pollutants, stress, ultraviolet rays, free radicals, and peroxides. In recent years, the development of cosmetics that improve skin wrinkles and relieve skin irritation and inflammation by using safe natural materials as a material is being actively conducted. In addition, in modern society, more and more people are suffering from hair damage or hair loss, and various attempts are being made to treat alopecia, such as external procedures such as wigs and transplantation surgery, or drug treatment to treat alopecia. The causes of hair loss include genetic causes, decreased hair cell function, excessive secretion of male hormones, and excessive secretion of sebum, and androgens, which are male hormones, are mainly acting as important factors. Among androgens, testosterone is converted to dihydrotestosterone (DHT) by 5-alpha-reductase when it reaches the hair follicle.

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

In the present invention, "exosome" means a small vesicle with a membrane structure 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 vesicles 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 reflect the state of cells of origin, so they can be used for diagnosis and treatment. Exosomes are a double phospholipid membrane structure that facilitates intracellular penetration and performs various physiological and pathological functions such as immune response and signal transduction.

Recently, research on various efficacy of plant-derived exosomes has been conducted, and research on skin efficacy such as antioxidant and anti-inflammatory has also been started. Plant-derived exosomes are natural nanoparticles that help in cell-to-cell movement and absorption as they contain physiological activity and signal transmitters secreted by plant cells themselves. is known

These exosomes are materials that can be used in the fields of medicines, cosmetics, food, etc. due to various advantages and activities, but have low dispersibility and aggregation properties due to their structural features composed of a phospholipid bilayer. In addition, it is unstable at high temperatures and can be easily broken in the process of manufacturing a cosmetic formulation, and this property may reduce the stability of exosomes in the formulation and cause precipitation. Therefore, it is necessary to increase the stability in the formulation by increasing the solubility and dispersibility of the exosomes in the aqueous solution to maintain continuous activity.

The present inventors applied various methods to prepare plant-derived exosomes exhibiting excellent skin and hair condition improvement activity and tried to use them as cosmetics. The present invention was completed by confirming that purified golden exosomes and/or weak wheat exosomes exhibit excellent skin and hair condition improvement effects.

(0001) Republic of Korea Patent No. 10-2125567 (2020.06.16) (0002) Republic of Korea Patent Registration No. 10-2265811 (2021.06.16.) (0003) Republic of Korea Patent Publication No. 10-2021-0018735 (2021.02.18)

The present invention is a cosmetic composition for improving skin and hair conditions, which contains golden exosomes, weak wheat exosomes, or mixed exosomes as active ingredients, and has excellent antioxidant, anti-inflammatory, wrinkle-improving effects, hair growth promotion and hair loss prevention effects. is intended to provide

In addition, it is another object of the present invention to provide a method for purifying a little golden exo or yakmomil exo.

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

Golden exosomes and yakmomil exosomes as the active ingredients are,

(A) 40 ~ 60 ℃, 20 ~ 24 hours hot-air drying treatment of golden or yak buckwheat; (B) immersing the hot-air dried golden or weak wheat in water at 30 ~ 40 ℃; (C) squeezing the immersed gold or yakmomil; (D) centrifuging the juice at 1,000xg to 10,000xg to obtain a supernatant; (E) freeze-drying the supernatant in which exosomes are present; (F) forming an aqueous two-phase system using PEG (Polyethylene glycol)/Dextran in the lyophilisate; And (G) is purified by a method comprising the step of obtaining a supernatant in which the exosomes are concentrated in the aqueous two-phase system.

More preferably, the hot air drying treatment in step (A) is performed at a temperature of 50° C. for 24 hours.

As an active ingredient, the golden exosome, yakmomil exosome, or a mixed exosome thereof is 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 antioxidant, anti-inflammatory 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 to achieve the above other object,

(A) 40 ~ 60 ℃, 20 ~ 24 hours hot-air drying treatment of golden or yak buckwheat;

(B) immersing the hot-air dried golden or weak wheat in water at 30 ~ 40 ℃;

(C) squeezing the immersed gold or yakmomil;

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

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

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

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

The hot air drying treatment in step (A) is characterized in that it is performed for 24 hours at a temperature of 50 ℃.

The golden exosomes, weak wheat exosomes, or mixed exosomes of the present invention, purified using hot air drying pretreatment and an aqueous two-phase system, have excellent stability, excellent antioxidant effect, anti-inflammatory effect, anti-wrinkle effect, and hair growth promoting effect. Since it exhibits the effect of preventing hair loss, it can be usefully used as a cosmetic for improving skin and hair conditions.

1 is a TEM image of a gold-derived exosome purified according to an embodiment of the present invention.
Figure 2 is a TEM image of a yakmomil-derived exosome 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 the number of particles of the gold-derived exosome particles purified according to an embodiment of the present invention.
Figure 4 is a graph showing the results of NTA analysis for confirming the size distribution and the number of particles of exosomes derived from yakmomil purified according to an embodiment of the present invention.
5 is a graph showing the results of evaluating the cytotoxicity of gold, yakmomil-derived exosomes purified by the present invention by MTT assay.
Figure 6 is a graph showing the results of evaluation of the anti-inflammatory efficacy of the exosomes derived from gold and yakmomil purified by the present invention by the inhibition rate of nitric oxide (NO) production.
7 is a graph showing the results of evaluating the anti-wrinkle efficacy of gold-derived exosomes purified by the present invention by the collagen production ability.
Figure 8 is a graph showing the results of evaluating the hair growth promoting effect of the exosomes derived from gold and yak woolen purified by the present invention as the dermal papilla cell proliferation effect.
9 is a graph showing the results of evaluation of the hair loss prevention efficacy of gold-derived exosomes purified by 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 non-toxic compared to mammalian-derived exosomes. Due to these advantages, it is a material that can be used in pharmaceuticals, cosmetics, food, etc., but has a low dispersibility and a tendency to agglomerate due to the structural feature composed of a phospholipid bilayer, so it is difficult to continuously maintain its activity in the formulation. have a problem The present invention has a technical feature of separating and purifying exosomes from gold and yak milt, and using them as cosmetics for skin and hair.

Gold (Scutellaria baicalensis) is a herbaceous perennial plant belonging to the family Lamiaceae, and its roots are used medicinally. Gold contains ingredients such as baicalein, baicalin, wogonin, and wogonoside, and these ingredients regulate the immune system of the liver, hepatoprotective activity, inhibit cancer cell proliferation, and It helps in inducing apoptosis and inhibiting gastritis and gastric ulcer. It is also effective in allergic dermatitis such as eczema and atopic dermatitis, and has been used in oriental medicine to treat skin diseases such as boils, tumors, sores, and soles. In addition, it has excellent effects on the skin, such as used for external skin inflammatory reactions and burns, and is an effective drug for various diseases accompanied by inflammation due to its anti-inflammatory action.

Houttuynia cordata is a dicotyledonous plant, and is a perennial plant belonging to the Pepperaceae family. It is called buckwheat because it looks similar to the leaves of buckwheat. It is also called 草). The outpost of Yakmomil contains 0.0049% essential oil. Among these essential oils are Decanoyl acetaldehyde, Myrcene, Lauric aldehyde, and Capric aldehyde. Yak buckwheat has an excellent effect in removing various harmful inflammations and wastes in the body, and it has an excellent effect in discharging various harmful substances and heavy metals accumulated in the body due to fine dust. Quercitrin, which is contained in large amounts in eoseongcho, expands the capillaries of the scalp and helps to remove inflammation and wastes in the scalp, so it is effective for scalp health and is a great help in alleviating hair loss symptoms. In addition, since Quercitrin has excellent antibacterial and anti-inflammatory properties, it is effective in improving skin blemishes, acne, and various skin troubles. In addition, it helps with anticancer, blood circulation, and diuretic action, and is known as a natural product that is good for detoxification and bronchial health.

Golden, yakmomil-derived exosomes of the present invention are purified by the following method.

(A) 40 ~ 60 ℃, 20 ~ 24 hours hot-air drying treatment of golden or yak buckwheat;

(B) immersing the hot-air dried golden or weak wheat in water at 30 ~ 40 ℃;

(C) squeezing the immersed gold or yakmomil;

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

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

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

(G) Purified by a method comprising the step of obtaining a supernatant in which the exosomes are concentrated in the aqueous two-phase system.

In the present invention, in order to produce exosomes derived from gold and yakmomil, a hot air drying pretreatment is first performed.

In the case of plants, the amount of exosome secretion may vary depending on environmental factors. Therefore, the optimal conditions for plants to secrete exosomes may be different. It was confirmed that the secretion of exosomes differed according to the drying conditions in gold and yakmomil.

According to a preferred embodiment of the present invention, the hot-air drying pre-treatment in step (A) consists of hot-air drying treatment of golden or weak wheat at 40-60° C. for 20-24 hours using a hot-air dryer. More preferably, it consists of hot-air drying at 50° C. for 24 hours of golden or weak wheat.

Then, the hot air drying pre-treated golden and yak buckwheat is immersed in water at 30-40° C. for 3 to 6 hours and then juiced (steps (B) and (C)).

As for the screw used in the process of juicing in step (C), it is preferable to use a screw having a stirring speed of 20 to 50 rpm.

The centrifugation 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 control the rpm to remove large contaminants, and it is more preferable to carry out at 10,000xg to obtain a final solution for forming an aqueous two-phase system.

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

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

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

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

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

The golden exosomes prepared by this method, the medicinal wheat exosomes, and their mixed exosomes have excellent antioxidant effects (Test Example 8), anti-inflammatory effects (Test Example 9), and wrinkle improvement effects compared to general golden solvent extracts and medicinal wheat extracts. (Test Example 10, Test Example 11), hair growth promoting effect (Test Example 12), and hair loss prevention effect (Test Example 13, Test Example 14) were shown.

Therefore, the exosomes derived from gold and yak milt can be used in a cosmetic composition for antioxidant, anti-inflammatory, and wrinkle improvement, and can also be used in a cosmetic composition for promoting hair growth and preventing hair loss. In this case, gold, yakmomil-derived 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 cosmetic composition can be in any conventionally prepared formulation, for example, skin lotion, skin toner, pack, nourishing cream, moisture cream, essence, body cream, body lotion, body oil, cleansing foam, cleansing lotion, soap , patch, foundation, lipstick, makeup base, lipstick, and the like.

In addition, the cosmetic composition is, for example, hair tonic, hair lotion, hair oil, hair shampoo, hair conditioner, hair conditioner, hair treatment, hair cream, hair pack, hair spray, hair essence, hair mousse, hair gel, etc. can be manufactured.

[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 illustrating the present invention, and the present invention is not limited by the following examples, and can be changed to other examples with substitutions and equivalents within the scope without departing from the technical spirit of the present invention. will be apparent to those of ordinary skill in the art to which the present invention pertains.

Example 1: Preparation of golden exosomes

Golden hot air drying treatment

200 g of golden organisms were subjected to 24 hr hot air drying treatment at a temperature of 50 ° C. using a hot air dryer.

golden dipping

The hot air-dried gold was immersed in water at about 30-40 ℃ for 6 hours.

golden juice

The immersed gold was subjected to golden extraction using a general juicer with a low speed screw of 30 rpm, and the obtained golden juice was filtered through a mesh net to remove floating matter. The recovered golden juice was stored at -80°C until purification.

Supernatant recovery for exosome purification

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

Freeze-drying the supernatant

For mass production of exosomes, freeze-drying was performed for the purpose of reducing the volume of the supernatant. 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 freeze-dried supernatant, and an aqueous two-phase system was formed using PEG (Polyethylene glycol)/Dextran. An aqueous two-phase system was formed using 3.3 wt% of PEG (purchased from Sigma Aldrich) having a molecular weight of 10,000 to 35,000 and 1.7 wt% of Dextran (purchased from Sigma Aldrich) having a molecular weight of 300,000 to 650,000.

Recovery of golden 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, the aqueous two-phase solution of the same concentration was added to the recovered lower layer, and an additional washing process was performed. After repeated treatment three times, the final exosome-concentrated sublayer was recovered.

Example 2: Preparation of yakmomil exosomes

In the same manner as in Example 1, yakmomil-derived exosomes were purified.

Example 3: Preparation of golden, yakmomil mixed exosomes

Mixed exosomes were prepared by mixing the golden exosomes prepared in Examples 1 and 2 and the yakmomil exosomes in the same weight ratio.

Examples 4-13: Preparation of exosomes from golden, yak-milk

In the case of plants, the amount of exosome secretion may vary depending on environmental factors. Since the optimal conditions for the secretion of exosomes from plants are all different, in order to establish the optimal drying conditions through comparison of the yield of exosomes caused by dry environmental stress, the exosomes were dried after drying the organisms by different drying conditions of gold and ryegrass. Purified. Exosomes were purified in the same manner as in Example 1 except that only the hot air drying conditions were changed, and the drying conditions are shown in Table 1 below.

raw material Drying temperature (℃) Drying time (hr) Example 4 Gold 40 6 Example 5 Gold 40 24 Example 6 Gold 50 6 Example 7 Gold 60 6 Example 8 Gold 60 24 Example 9 weak wheat 40 6 Example 10 weak wheat 40 24 Example 11 weak wheat 50 6 Example 12 weak wheat 60 6 Example 13 weak wheat 60 24

Comparative Example 1: Preparation of golden extract

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

Comparative Example 2: Preparation of Yakmomil extract

10 g of dried buckwheat wheat 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 an extract of buckwheat flour, followed by distillation using a rotary evaporator to obtain a sample in powder form.

Test Example 1: Characterization of gold-derived exosomes: TEM analysis

In order to confirm the shape of the purified gold-derived exosomes, it was analyzed by transmission electron microscopy (TEM). 1 is a TEM analysis image of a gold-derived exosome purified according to Example 1. As a result of the analysis, it was confirmed the presence of particles of about 153.9 nm in a spherical phospholipid bilayer structure.

Test Example 2: Characteristics analysis of exosomes derived from Yakmomil: TEM analysis

Transmission electron microscopy (TEM) was used to confirm the shape of the purified yakmomil-derived exosomes. Figure 2 is a TEM analysis image of the exosome derived from yakmomil purified according to Example 2 above. As a result of the analysis, it was confirmed the presence of about 160.2 nm particles having a spherical phospholipid bilayer structure.

Test Example 3: Characterization of gold-derived exosomes: NTA analysis

Nanoparticle Tracking Analysis (NTA) was used to confirm the particle size distribution and the number of particles per unit volume of the purified gold-derived exosomes.

3 is a graph showing the results of NTA analysis of gold-derived exosomes purified according to Example 1, and as a result of the analysis, the average size of particles of 153.9 nm and the concentration of 6.80 × 10 ¹⁰ per unit volume of 1 mL were confirmed.

Test Example 4: Characterization of exosomes derived from Yakmomil: NTA analysis

Nanoparticle Tracking Analysis (NTA) was used to confirm the particle size distribution and the number of particles per unit volume of the purified yakmomil-derived exosomes.

4 is a graph showing the results of NTA analysis of yakmomil-derived exosomes purified according to Example 2, and as a result of the analysis, it was confirmed that the particles had an average size of 160.2 nm and a concentration of 5.74 × 10 ¹⁰ per unit volume of 1 mL.

Test Example 5: Comparison of yields of exosomes derived from gold and yakmomil

In order to confirm the optimal conditions for hot air drying, the yields of exosomes derived from gold and yak buckwheat purified according to Examples 1, 2, and 4-13 were compared. Table 2 below is a table showing the yield comparison as a result of nanoparticle tracking analysis (NTA).

number Particle Number
(Particles/ml) Particle Size
(nm) Example 1 6.80 x 10 ¹⁰ 153.9 Example 2 5.74 x 10 ¹⁰ 160.2 Example 4 2.14 x 10 ⁹ 140.3 Example 5 3.77 x 10 ⁹ 151.9 Example 6 5.09 x 10 ⁹ 147.5 Example 7 3.11 x 10 ⁹ 150.6 Example 8 1.97 x 10 ¹⁰ 155.7 Example 9 7.11 x 10 ⁹ 161.4 Example 10 1.08 x 10 ¹⁰ 167.5 Example 11 6.19 x 10 ⁹ 166.1 Example 12 5.78 x 10 ⁹ 152.0 Example 13 1.16 x 10 ¹⁰ 152.9

As can be seen in Table 2 above, a high yield was exhibited at a temperature of 40 to 60° C. and a 24-hour hot air drying pretreatment condition (Examples 1, 2, 8, 10, 13), and among them, a hot air drying temperature of 50° C. and hot air drying The yield of exosomes was the highest in Examples 1 and 2 of the 24 hr condition. Therefore, it was confirmed that the most optimal hot air drying conditions were a temperature of 50° C. and a time of 24 hr.

Test Example 6: Stability analysis by temperature

The stability of the exosomes derived from gold and yakmomil purified according to Examples 1 and 2 of the present invention at each temperature was confirmed. Table 3 below shows the size and concentration of the particles through nanoparticle tracking analysis (NTA) after storing the exosomes derived from gold and yak buckwheat purified according to Examples 1 and 2 at 4°C, 25°C, and 45°C for 12 weeks. table shown.

golden exosome
(Storage at 4℃) golden exosome
(Stored at 25℃) golden exosome
(Storage at 45℃) Particle Number
(Particles/ml) Particle Size(nm) Particle Number
(Particles/ml) Particle Size(nm) Particle Number
(Particles/ml) Particle Size(nm) Early 6.80 x 10 ¹⁰ 153.9 6.80 x 10 ¹⁰ 153.9 6.80 x 10 ¹⁰ 153.9 2nd week 6.67 x 10 ¹⁰ 149.1 6.59 x 10 ¹⁰ 151.7 6.71 x 10 ¹⁰ 153.4 4 weeks 6.53 x 10 ¹⁰ 151.6 6.54 x 10 ¹⁰ 152.6 6.68 x 10 ¹⁰ 152.9 Week 8 6.55 x 10 ¹⁰ 152.7 6.63 x 10 ¹⁰ 148.9 6.70 x 10 ¹⁰ 155.4 Week 12 6.57 x 10 ¹⁰ 155.7 6.57 x 10 ¹⁰ 151.8 6.69 x 10 ¹⁰ 155.1 Mildew wheat exosome
(Storage at 4℃) Mildew wheat exosome
(Stored at 25℃) Mildew wheat exosome
(Storage at 45℃) Particle Number
(Particles/ml) Particle Size(nm) Particle Number
(Particles/ml) Particle Size(nm) Particle Number
(Particles/ml) Particle Size(nm) Early 5.74 x 10 ¹⁰ 160.2 5.74 x 10 ¹⁰ 160.2 5.74 x 10 ¹⁰ 160.2 2nd week 5.74 x 10 ¹⁰ 161.8 5.71 x 10 ¹⁰ 158.0 5.69 x 10 ¹⁰ 157.4 4 weeks 5.68 x 10 ¹⁰ 159.4 5.69 x 10 ¹⁰ 157.1 5.70 x 10 ¹⁰ 155.7 Week 8 5.62 x 10 ¹⁰ 159.1 5.70 x 10 ¹⁰ 159.7 5.75 x 10 ¹⁰ 156.7 Week 12 5.69 x 10 ¹⁰ 158.6 5.75 x 10 ¹⁰ 158.4 5.79 x 10 ¹⁰ 159.0

As a result of analyzing the stability for 12 weeks by temperature, it was confirmed that the size and concentration changes of the exosomes derived from gold and yakmomil corresponding to Examples were all stable at 4 ℃, 25 ℃, and 45 ℃ conditions.

Test Example 7: Cytotoxicity evaluation

To confirm the cytotoxicity of golden exosomes (Example 1), yakmomil exosomes (Example 2) and their mixed exosomes (Example 3) and golden extracts (Comparative Example 1), and yakmomil extracts (Comparative Example 2) MTT assay was evaluated. Human dermal fibroblast (HDFa) was inoculated into a 96-well plate at a concentration of 1×10 ⁵ cells/mL and then cultured at 37° C. for 18 hours under 5% CO ₂ .

After incubation, the medium was removed and washed with PBS buffer, and then golden exosomes, yakmomil exosomes, their mixed exosomes, golden extracts, and yakmomil extracts were administered to a new medium by concentration, and cultured again for 24 hours. To measure the cell viability, formazan formed for 4 hours after addition of MTT solution (5 mg/mL) 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, no cytotoxicity was observed at all concentrations when treated with golden exosome, yakmomil exosome, golden and yakmomil mixed exosome, golden extract, and yakmomil extract.

Test Example 8: Antioxidant efficacy evaluation

In order to confirm the antioxidant efficacy of gold-derived exosomes, yakmomil-derived exosomes, and their 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 to the DPPH radical. A 0.4 mM solution was prepared by dissolving DPPH (1,1-Diphenyl-2-picrylhydrazyl) reagent in ethanol, 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 was mixed with 100 μl of a solution and 0.4 mM DPPH solution 100 μl. At this time, ethanol was treated as a negative control, and after mixing, it was reacted at 37° C. for 30 minutes. 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 4 is a table showing the results of evaluating the antioxidant effect of each sample by DPPH radical scavenging ability.

Concentration (%) DPPH radical scavenging activity (%) golden exosome
(Example 1) Mildew wheat exosome
(Example 2) gold + wheat
mixed exosomes
(Example 3) golden extract
(Comparative Example 1) Mildew extract
(Comparative Example 2) Stock 6.80 x 10 ¹⁰ Particles/mL 5.74 x 10 ¹⁰ Particles/mL 6.27 x 10 ¹⁰ Particles/mL 100mg/mL 100mg/mL 0.001 8.07 6.87 11.18 0.84 1.27 0.01 18.44 16.08 22.96 4.81 4.51 0.1 52.55 51.40 54.19 27.49 25.87 One 78.47 77.18 82.59 54.17 52.77 BHT (0.01%) 71.49

As a result of the test, DPPH radical scavenging activity was increased in a concentration-dependent manner when gold-derived exosomes, yak-milk-derived exosomes, golden extract, and yak-mo-mil extract were treated. was confirmed. In addition, it was confirmed that the highest activity was shown when the golden and yakmomil mixed exosomes were treated.

Test Example 9: Anti-inflammatory efficacy evaluation (NF-κB expression)

Inflammatory mediators expressed at the site of inflammation, that is, cytokines, chemokines, reactive oxygen species intermediates, cyclooxygenase-2 (COX-2), 5-lipoxygenase (5- Lipoxygenase, 5-LOX) and matrix metalloproteinase (MMP) play an important role in the generation and maintenance of an inflammatory response, and the expression of these inflammatory mediators is a transcription factor, nuclear factor κB ), STAT3 (signal transducer and activator of transcription 3), AP-1 (activator protein 1), HIF-1a (hypoxia-inducible factor 1a), etc. are known to be regulated.

Therefore, the NF-κB gene expression test was performed in comparison with the extract to confirm the anti-inflammatory efficacy of exosomes derived from gold and yakmomil. After inoculation of a human epidermal keratinocyte line (HEKa), cultured in Dulbecco Modified Eagle Medium (DMEM) culture medium supplemented with 100 IU/mL penicillin and 100 μg/mL streptomycin at 37°C for 24 hours under 5% CO ₂ did After culturing, the medium was discarded and UVB 20 mJ/cm ² was irradiated to induce cell damage, followed by treatment with golden exosomes, yakmomil exosomes, their mixed exosomes, golden extracts, and yakmomil extracts, and cultured for 48 hours. After that, RNA was extracted from the cultured cells using TransZol reagent, followed by RT-PCR (real-time gene polymerase chain reaction), and the PCR product was electrophoresed on 1% agarose gel and confirmed with the Gel Documentation system . At this time, PBS was treated as a negative control and 0.01% of Allantoin was treated as a positive control.

6 is a graph showing the results of evaluating the anti-inflammatory efficacy of the samples by NF-κB expression. As a result of the test, NF-κB expression was decreased in a concentration-dependent manner when gold-derived exosomes and yak-momil-derived exosomes were treated, and a much higher reduction rate was confirmed than when gold extracts and yak-milk extracts were treated. In addition, a higher reduction rate was confirmed when the golden and yakmomil mixed exosomes were treated. At this time, when 0.01% of Allantoin, a positive control, was treated, the expression rate was 50.66%.

Test Example 10: Wrinkle improvement efficacy evaluation

In order to confirm the anti-wrinkle efficacy of exosomes derived from gold and yakmomil, the effect on COL1A1 expression was confirmed compared with the extract. 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 under 5% CO ₂ for 24 hours. cultured. After culturing, the medium was discarded and treated with golden exosomes, yakmomil exosomes and their mixed exosomes, golden extracts, and yakmomil extracts, respectively, and cultured for 48 hours. After culturing, RNA was extracted using TransZol reagent, and then RT-PCR (real-time gene polymerase chain reaction) was performed to measure the amount of change in mRNA of collagen that maintains the skin connective tissue. The product generated by PCR was electrophoresed on 1% agarose gel and confirmed with the Gel Documentation system. At this time, PBS was treated as a negative control and 50 μM of Retinyl palmitate was treated as a positive control.

7 is a graph showing the results of evaluating the wrinkle-improving efficacy of the exosomes derived from gold and yakmomil purified according to Examples 1 and 2 and the mixed exosomes by COL1A1 expression. As a result of the test, it was confirmed that the expression of COL1A1 was increased in a concentration-dependent manner when the gold-derived exosomes and the medicinal wheat-derived exosomes were treated, and there was a higher wrinkle improvement effect than when the golden extracts and the medicinal woolly extracts were treated. It was confirmed that the wrinkle improvement effect was excellent even when the golden and yakmomil mixed exosomes were treated. In this case, when the positive control group, Retinyl palmitate 50M, was treated, the expression rate was 161.33%.

Formulation Examples 1-3: Preparation of cream

With the composition shown in Table 5 below, a cream containing golden exosomes and yakmomil exosomes purified according to the above example was prepared by a conventional method. The cream containing the golden extract of Comparative Example 1 was used as Comparative Formulation Example 1, and the cream containing the Yakmomil extract of Comparative Example 2 was used as Comparative Formulation Example 2.

ingredient Formulation Example 1 Formulation Example 2 Formulation Example 3 comparison
Formulation Example 1 comparison
Formulation Example 2 Content (wt %) Golden exosomes (Example 1) 5 - - - - Yakmomil exosome (Example 2) - 5 - - - Golden + Mildew wheat exosome (Example 3) - - 5 - - Golden Extract (Comparative Example 1) - - - 5 - Yak buckwheat 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 Oliveate 0.5 0.5 0.5 0.5 0.5 Caprylic/Capric Triglycerides 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 Cyclopentasiloxane/Cyclohexasiloxane 2.0 2.0 2.0 2.0 2.0 cetiaryl alcohol 1.0 1.0 1.0 1.0 1.0 mineral oil 2.5 2.5 2.5 2.5 2.5 Disodium EDIT 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 remaining amount remaining amount remaining amount remaining amount remaining amount

Test Example 11: Wrinkle improvement evaluation of exosomes derived from gold and ryegrass wheat

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

Table 6 is a table showing the results of evaluating the wrinkle-improving effect of the exosomes derived from gold and yakmomil purified according to the above example through an actual use test.

Wrinkle improvement effect Great slightly doesn't exist Formulation Example 1 12 3 5 Formulation Example 2 12 7 One Formulation Example 3 14 4 2 Comparative Formulation Example 1 3 3 14 Comparative Formulation Example 2 4 5 11

As can be seen from the results of Table 6, the cream containing the golden exosomes of the present invention, the yakmomil exosomes and the golden, yakmomil mixed exosomes exhibited an excellent skin wrinkle improvement effect.

Test Example 12: Hair growth promoting effect test

In order to confirm the hair growth promoting effect of exosomes derived from gold and ryegrass, the effect on the proliferation of dermal papilla cells was confirmed by comparing with the extracts of gold and ryegrass by concentration. Human follicle dermal papilla cells (HGDPC), which are dermal papilla cells constituting hair follicles, were dispensed and cultured at 1×10 ⁴ cells/mL in a 96 well plate, followed by golden exosomes, weak hairy wheat exosomes, mixed exosomes and golden extracts; Yakmomil extract and DHT (5α-dihydrotestosterone) were treated to 10 μM and cultured for 48 hours. After incubation, 10x BrdU solution was treated and incubated for 6 hours, the solution was removed, and Anti-BrdU antibody was treated and reacted for 1 hour at room temperature. Secondary Antibody HRP Conjugate was added and reacted, washed with PBS, reacted by adding a substrate solution, and the reaction was terminated using a stop solution, and absorbance was measured at 450 nm.

8 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 the cell proliferation was increased in a concentration-dependent manner when the gold- and yak-milk-derived exosomes and the golden-haired wheat extract were treated. Again, the highest cell proliferation rate was shown when treated with golden and yakmomil mixed exosomes, and at this time, when Minoxidil 10Mm, a positive control, was treated, the growth rate was 93.14%.

Test Example 13: 5 alpha-reductase (5α-reductase) inhibitory activity test

In order to confirm the anti-hair loss efficacy of exosomes derived from gold and buckwheat, the effect on the 5 alpha-reductase inhibitory activity was confirmed by comparing the extracts of gold and yak buckwheat for each concentration. Human follicle dermal papilla cells (HGDPC), which are dermal papilla cells constituting hair follicles, were dispensed in a 24 well plate at 4×10 ⁵ cells/mL and cultured for 24 hours. time incubation. After incubation, testosterone was treated, and the supernatant was separated to obtain a cell pellet. The obtained pellet was measured for the concentration of dihydrotestosterone (DHT, dihydrotestosterone) using an enzyme immunoassay. 100 μl of cell pellet was dispensed in each DHT ElISA Kit strip-well, and 25 μl of DHT antiserum was dispensed 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 then washed. After washing, 90 μl of TMB substrate was dispensed and reacted at room temperature within 20 minutes, treated with 50 μl of stop solution, and absorbance was measured at 450 nm immediately after treatment to quantify the concentration of DHT.

9 is a graph showing the results of evaluating the hair loss prevention efficacy of each sample by a 5-alpha-reductase (5α-reductase) inhibitory activity test. The inhibition rate of 5 alpha-reductase was calculated based on the negative control group that was not treated with the sample. As a result of the test, the inhibitory activity was increased in a concentration-dependent manner when the exosomes derived from gold and yakmomil were treated. The best inhibitory activity was exhibited when treated with golden and yakmomil mixed exosomes.

Test Example 14: Hair Loss Prevention Effect Test

In order to confirm the hair loss prevention efficacy of the golden exosomes (Example 1), the weak wheat exosomes (Example 2), and the mixed exosomes (Example 3), adults with initial hair loss in comparison with the golden extracts and yakmomil extracts by concentration The experiment was conducted on 30 subjects for 12 weeks. Gold-derived exosomes purified according to the above example, yakmomil-derived exosomes and their mixed exosomes were prescribed at a concentration of 5%, applied once a day, and the hair was washed after about 20 minutes. After 12 weeks, the deciduous hairs were collected and counted, and the results are shown in Table 7 below. As a negative control, deciduous hairs were collected and compared in an untreated state.

Treatment concentration (%) Average number of deciduous hairs (pieces) negative control - 77 Golden exosomes (Example 1) 5 51 Yakmomil exosome (Example 2) 5 54 Golden + Mildew wheat exosome (Example 3) 5 55 Golden Extract (Comparative Example 1) 5 73 Yakmomil extract (Comparative Example 2) 5 72

As a result of the experiment, it was confirmed that the number of deciduous hairs was significantly reduced when the exosomes were treated compared to the golden extract and yakmomil extract.

Formulation Example 4: Preparation of hair tonic

Hair tonic was prepared according to a conventional method with the composition shown in Table 8 below.

number Raw material content (% by weight) One castor oil 5.00 2 Golden exosomes (Example 1) 3.00 3 Yakmomil exosome (Example 2) 3.00 4 panthenol 0.20 5 resorcinol 0.10 6 salicylic acid 0.10 7 tocopheryl acetate 0.10 8 Pyridoxine HCL 0.10 9 menthol 0.05 10 pigment appropriate amount 11 combination spice appropriate amount 12 ethanol appropriate amount 13 Purified water To 100

Formulation Example 5: Preparation of hair shampoo

A hair shampoo was prepared according to a conventional method with the composition shown in Table 9 below.

number Raw material content (% by weight) One Ammonium Lauryl Sulfate 20.00 2 Ammonium Laureth Sulfate 20.00 3 cocamide propyl betaine 6.00 4 Golden exosomes (Example 1) 3.00 5 Yakmomil exosome (Example 2) 3.00 6 Dimethicone (50%) 2.00 7 Dimethicone (70%) 2.00 8 Disodium EDIT 0.03 9 Methylchloroisothiazolinone Methylisothiazolinone Mixture 0.03 10 cocamide mipa appropriate amount 11 Carbomer appropriate amount 12 ethanol appropriate amount 13 DL-Panthenol appropriate amount 14 tocopheryl acetate appropriate amount 15 triethanolamine appropriate amount 16 combination spice appropriate amount 17 menthol appropriate amount 18 Purified water To 100

Formulation Example 6: Preparation of hair conditioner

A hair conditioner was prepared according to a conventional method with the composition shown in Table 10 below.

number Raw material content (% by weight) One Stearylmethylbenzyl ammonium chloride (25%) 5.00 2 cetanol 3.50 3 Golden exosomes (Example 1) 3.00 4 Yakmomil exosome (Example 2) 3.00 5 propylene glycol 2.50 6 Self-emulsifying glycerin monostearate 1.50 7 methyl paraoxybenzoate 0.30 8 pigment appropriate amount 9 combination spice appropriate amount 10 Purified water To 100

Claims (8)

In a cosmetic composition containing golden exosome, yakmomil exosome or a mixed exosome thereof as an active ingredient,
Golden exosomes and yakmomil exosomes as the active ingredients are,
(A) hot-air drying treatment of golden or weak wheat at 40 to 60° C. for 20 to 24 hours;
(B) immersing the hot air-dried golden or weak wheat in water at 30-40 ℃;
(C) squeezing the immersed gold or yakmomil;
(D) centrifuging the juice at 1,000xg to 10,000xg to obtain a supernatant;
(E) freeze-drying the supernatant in which exosomes are present;
(F) forming an aqueous two-phase system using PEG (Polyethylene glycol)/Dextran in the lyophilisate; and
(G) A cosmetic composition, characterized in that it is purified by a method comprising the step of obtaining a lower layer solution in which the exosomes are concentrated in the aqueous two-phase system. delete The cosmetic composition according to claim 1, wherein the hot air drying treatment in step (A) is performed at a temperature of 50° C. for 24 hours. The cosmetic composition according to claim 1, wherein the golden exosome, yakmomil exosome or a mixture thereof 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 antioxidant, anti-inflammatory, or wrinkle improvement. The cosmetic composition according to claim 1, wherein the cosmetic composition is for promoting hair growth or preventing hair loss. (A) hot-air drying treatment of golden or weak wheat at 40 to 60° C. for 20 to 24 hours;
(B) immersing the hot air-dried golden or weak wheat in water at 30-40 ℃;
(C) squeezing the immersed gold or yakmomil;
(D) centrifuging the juice at 1,000xg to 10,000xg to obtain a supernatant;
(E) freeze-drying the supernatant in which exosomes are present;
(F) forming an aqueous two-phase system using PEG (Polyethylene glycol)/Dextran in the lyophilisate; and
(G) A method of purifying golden or weak wheat exosomes comprising the step of obtaining a lower layer in which the exosomes are concentrated in the aqueous two-phase system. The method of claim 7, wherein the hot air drying treatment in step (A) is performed at a temperature of 50° C. for 24 hours.

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