KR102502664B1 - Cosmetic composition for improving skin and hair condition containing Solanum Lycopersicum(Tomato) Fruit exosome, Brassica Oleracea Italica(Broccoli) exosome - Google Patents

Abstract

The present invention relates to a cosmetic composition for improving skin and hair conditions containing tomato and broccoli exosomes as active ingredients. Specifically, the present invention relates to a cosmetic composition for improving skin and hair conditions, which contains tomato exosomes and/or broccoli exosomes purified using hot air drying, UV pretreatment, and an aqueous two-phase system as active ingredients, and thus has excellent effects on skin whitening, anti-inflammation, skin soothing, hair growth promotion, and hair loss prevention.

Description

Cosmetic composition for improving skin and hair condition containing Solanum Lycopersicum (Tomato) Fruit exosome, Brassica Oleracea Italica (Broccoli) exosome}

The present invention relates to a cosmetic composition for improving skin and hair conditions containing tomato and broccoli-derived exosomes as an active ingredient, and specifically, tomato exosomes purified using hot air drying, UV pretreatment, and an aqueous two-phase system and/or Or, it relates to a cosmetic composition for improving skin and hair conditions containing broccoli exosomes as an active ingredient and having excellent whitening, anti-inflammatory, skin soothing 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, the number of people suffering from hair damage or hair loss is increasing, and various attempts are being made to treat hair loss, such as external procedures such as wigs and transplant surgery, or drug treatment. 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. Testosterone among androgens is converted to dihydrotestosterone (DHT) by 5-alpha-reductase when it reaches the hair follicle.

For the treatment of such alopecia, preparations containing minoxidil and finasteride are representative, but there are many limitations in 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 the various effects of plant-derived exosomes have been conducted, and studies on skin effects such as whitening and anti-inflammatory have also begun. 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. due to their various advantages and activities, but have low dispersibility and tend to aggregate due to their structural characteristics consisting of a phospholipid bilayer. In addition, it is unstable at high temperatures and can be easily broken in the process of preparing cosmetic formulations, and these properties may reduce the stability of exosomes in formulations and cause precipitation. Therefore, it is necessary to increase the stability in the formulation by increasing the solubility and dispersibility of exosomes in an aqueous solution in order 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. As a result, hot air drying under specific conditions, UV pretreatment, and an aqueous two-phase system The present invention was completed by confirming that the purified tomato exosomes and/or broccoli exosomes exhibited excellent skin and hair condition improvement effects.

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

The present invention contains tomato exosomes, broccoli exosomes, or mixed exosomes as active ingredients and has excellent stability, whitening, anti-inflammatory, skin soothing 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 tomato exosomes or broccoli exosomes.

In order to achieve the above object, according to the present invention, a cosmetic composition containing tomato exosomes, broccoli exosomes, or mixed exosomes as an active ingredient is provided.

Tomato exosomes and broccoli exosomes as the active ingredient,

(A) subjecting tomatoes or broccoli to hot air drying at 40 to 60° C. for 20 to 24 hours; (B) immersing tomatoes or broccoli treated with hot air drying in water at 30 to 40 ° C. for 6 hours; (C) UV-A treatment of immersed tomatoes or broccoli for 1 to 6 hours; (D) squeezing UV-treated tomatoes or broccoli; (E) obtaining a supernatant by centrifuging the juice at 1,000xg to 10,000xg; (F) freeze-drying the supernatant in which exosomes are present; (G) forming an aqueous two-phase system using PEG (Polyethylene glycol)/Dextran in the lyophilisate; and (H) obtaining an exosome-enriched lower layer 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, and the UV treatment in step (C) is performed with UV-A at a wavelength of 365 nm for 6 hours.

The tomato exosomes, broccoli 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 whitening, anti-inflammatory or skin soothing. In addition, the cosmetic composition is characterized in that for promoting hair growth or preventing hair loss.

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

(A) subjecting tomatoes or broccoli to hot air drying at 40 to 60° C. for 20 to 24 hours;

(B) immersing tomatoes or broccoli treated with hot air drying in water at 30 to 40 ° C;

(C) UV-A treatment of immersed tomatoes or broccoli for 1 to 6 hours;

(D) squeezing UV-treated tomatoes or broccoli;

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

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

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

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

The hot air drying treatment in step (A) is performed at a temperature of 50 ° C. for 24 hours, and the UV treatment in step (C) is performed for 6 hours with UV-A at a wavelength of 365 nm.

Tomato exosomes, broccoli exosomes, or mixed exosomes of the present invention purified using hot air drying, UV pretreatment, and an aqueous two-phase system have excellent stability, excellent whitening effect, anti-inflammatory effect, skin soothing effect, and hair growth promotion It can be usefully used as a cosmetic for improving skin and hair conditions because it shows the effect of preventing hair loss and hair loss.

1 is a TEM image of tomato-derived exosomes purified according to an embodiment of the present invention.
2 is a TEM image of purified broccoli-derived exosomes according to an embodiment of the present invention.
Figure 3 is a graph showing the NTA analysis results for confirming the size distribution and particle number of purified tomato-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 broccoli-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 tomato and broccoli purified according to the present invention by MTT assay.
6 is a graph showing the results obtained by evaluating the whitening efficacy of tomato and broccoli-derived exosomes purified according to the present invention in terms of tyrosinase activity inhibition rate.
7 is a graph showing the results obtained by evaluating the whitening efficacy of tomato and broccoli-derived exosomes purified according to the present invention in terms of melanin production inhibition rate.
8 is a graph showing the results obtained by evaluating the anti-inflammatory efficacy of tomato and broccoli-derived exosomes purified according to the present invention in terms of IL-1 expression inhibition rate.
9 is a graph showing the results obtained by evaluating the anti-inflammatory efficacy of tomato and broccoli-derived exosomes purified according to the present invention in terms of IL-6 expression inhibition rate.
10 is a graph showing the results of evaluating the hair growth promoting effect of exosomes derived from tomato and broccoli purified according to the present invention as the dermal papilla cell proliferation effect.
11 is a graph showing the results obtained by evaluating the anti-hair loss efficacy of tomato and broccoli-derived exosomes 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 from tomatoes and broccoli with high purity and use them as cosmetics for skin and hair.

Tomato ( Solanum lyclpersicum ) is a plant belonging to the solanaceae family, and its fruits are edible. It is an annual plant native to Latin America, 1-3m tall with yellow flowers. The fruit is red due to lycopene. As a vine plant, it originally crawls on the ground and grows roots from the stem, but when grown, it is grown standing upright. It should be noted that solanine, a component of the seeds of species belonging to the Solanaceae family, is contained in very small amounts. Tomatoes were selected as one of the world's top 10 superfoods by Time magazine, and they are also called the fruit of heaven in the West because of their excellent taste and nutrition. There are about 500 varieties in addition to the well-known varieties such as Charlal Tomato, Cod Tomato, Stevia Tomato, Cherry Tomato, Date Tomato, and Black Tomato. 95% of tomato ingredients are water, and it is an alkaline food rich in vitamins and minerals. In particular, vitamins are rich enough to consume most of the recommended amount by eating just two tomatoes. It also contains beta-carotene (450μg), citric acid, malic acid, succinic acid, and lutein. It is especially rich in lycopene, which has a strong anti-cancer effect, helps prevent freckles and blemishes caused by melanin pigment transformation, and helps keep skin tone bright. give In addition, it can protect the skin from ultraviolet rays, which is helpful for skin beauty. In addition, it contains a lot of various antioxidants, so it is effective in preventing hair damage and hair loss symptoms.

Broccoli ( Brassica Oleracea Italica ) is a kind of vegetable belonging to Brassica oleracea, a type of cabbage. Broccoli is a cultivated plant of the same system as cauliflower, which originates from cabbage, and the bud is used as a vegetable. The word broccoli is the plural of the Italian word broccolo, meaning the end of a flower. Like other green leafy vegetables, it is rich in nutrients. Vitamin E content per 100g is excellent enough to be in the upper class, and beta-carotene and vitamin A are also quite high, except for carrots, spinach, and lettuce. Broccoli contains high amounts of glucoraphanin, a pigment that is converted to an antioxidant called sulforaphane. Sulforaphane is known to have a strong antioxidant effect, and has recently been reported to inhibit the growth of Helicobacter pylori, known as the causative agent of gastritis, and to have an effect of preventing gastric cancer. It is also rich in lutein and zeaxanthin, which help reduce oxidative stress and cell damage in the eyes. In addition, broccoli contains zinc, calcium, and folic acid, which prevent hair loss and enhance hair growth. Recently, a paper has been published that NMN (nicotinamide mononucleotide) in broccoli is called a longevity substance and suppresses various diseases and aging phenomena. NMN, called 'dream substance', exists in the body and plays a role in activating genes involved in longevity called sirtuin, but decreases with age. In particular, because broccoli naturally contains these NMN ingredients, it is famous as one of the top 10 cancer prevention foods and a representative antioxidant food selected by the National Cancer Institute in the United States.

Tomato and broccoli-derived exosomes of the present invention are purified by the following method.

(A) subjecting tomatoes or broccoli to hot air drying at 40 to 60° C. for 20 to 24 hours;

(B) immersing tomatoes or broccoli treated with hot air drying in water at 30 to 40 ° C for 6 hours;

(C) UV-A treatment of immersed tomatoes or broccoli for 1 to 6 hours;

(D) squeezing UV-treated tomatoes or broccoli;

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

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

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

(H) obtaining an exosome-enriched lower layer in the aqueous two-phase system.

In the present invention, in order to prepare tomato and broccoli-derived exosomes, pretreatment with hot air drying is performed first, followed by UV-A treatment.

In the case of plants, the amount of exosome secretion can vary depending on environmental factors, and in particular, the amount of exosome secretion can be affected when the plant is under stress. Therefore, in order to confirm the optimal conditions for plants to secrete exosomes, the yield was confirmed through various pretreatments, and it was confirmed that the secretion of exosomes increased in tomato and broccoli under hot air drying and UV-A pretreatment conditions. In addition, it was confirmed that the purified tomato and broccoli exosomes after hot air drying and UV-A pretreatment exhibited better skin and hair improvement effects.

According to a preferred embodiment of the present invention, the hot air drying pre-treatment in step (A) consists of hot air drying treatment for 24 hours at 50 ° C. using a hot air dryer for tomatoes or broccoli.

Subsequently, tomatoes and broccoli pretreated with hot air drying are immersed in water at 30 to 40 ° C. for 6 hours (steps (A) and (B)).

Tomatoes or broccoli immersed in step (C) are exposed to UV-A at a wavelength of 365 nm for 1 to 6 hours.

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

The centrifugal separation method used in step (E) 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, and it is more preferable to carry out at 10,000 x g to obtain a final solution for forming an aqueous two-phase system.

The freeze-drying used in step (F) 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 step (G), PEG is used in an amount of 1 to 15% by weight, preferably 2 to 5% by weight, with a molecular weight of 10,000 to 35,000, and Dextran is used in an amount of 1 to 8 with a molecular weight of 300,000 to 650,000 It is characterized by using 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 the step (H), 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. there is.

Tomato exosomes, broccoli exosomes, and mixed exosomes prepared by this method have excellent whitening effect compared to general tomato extract and broccoli extract and compared to exosomes purified without pretreatment (Test Example 7 , Test Example 8), anti-inflammatory effect (Test Example 9, Test Example 10), skin soothing effect (Test Example 11), hair growth promoting effect (Test Example 12), hair loss prevention effect (Test Example 13, Test Example 14) showed up

Therefore, the tomato and broccoli-derived exosomes can be used in cosmetic compositions for whitening, anti-inflammatory, and skin soothing, and can also be used in cosmetic compositions for promoting hair growth and preventing hair loss. At this time, tomato and broccoli-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 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.

In addition, the cosmetic composition may be, for example, hair tonic, hair lotion, hair oil, hair shampoo, hair rinse, 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 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 Tomato Exosomes

Tomato hot air drying treatment

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

tomato dipping

Tomatoes treated with hot air drying were immersed in water at about 30 to 40 ° C. for 6 hours.

Tomato UV-A treatment

The immersed tomatoes were treated with UV-A at a wavelength of 365 nm for 6 hours.

tomato juice

UV-treated tomatoes were subjected to tomato juice with a low-speed screw at 30 rpm using a general juicer, and the obtained tomato juice was filtered through a mesh net to remove suspended matter. The recovered tomato juice was stored at -80 ° C until purification.

Supernatant Recovery for Exosome Purification

Tomato juice was centrifuged at 4° C. for 10 minutes at 10,000xg 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 tomato 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 broccoli exosomes

Broccoli-derived exosomes were purified in the same manner as in Example 1.

Example 3: Preparation of Tomato and Broccoli Mixed Exosomes

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

Examples 4 to 15: Preparation of tomato and broccoli exosomes

In the case of plants, the amount of exosome secretion may vary depending on environmental factors. Since the optimum conditions for plant secretion of exosomes are all different, exosomes were purified by varying the UV treatment conditions of tomatoes and broccoli in order to establish optimal drying conditions through comparison of exosome yields by UV treatment stress. Exosomes were purified in the same manner as in Example 1 except for Example 4 and Example 10, except for the UV treatment conditions, and the UV treatment conditions are shown in Table 1 below. In Examples 4 and 10, tomato and broccoli organisms were directly extracted without hot air drying and UV pretreatment, and purified by applying an aqueous two-phase system.

raw material Hot air drying UV treatment conditions (hr) Example 4 tomato X X Example 5 tomato O 3 Example 6 tomato O One Example 7 tomato X 6 Example 8 tomato X 3 Example 9 tomato X One Example 10 broccoli X X Example 11 broccoli O 3 Example 12 broccoli O One Example 13 broccoli X 6 Example 14 broccoli X 3 Example 15 broccoli X One

Comparative Example 1: Preparation of tomato extract

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

Comparative Example 2: Preparation of broccoli extract

10 g of dried broccoli 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 broccoli extract, and then distillation was performed using a rotary evaporator to obtain a sample in powder form.

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

In order to confirm the shape of the purified tomato-derived exosomes, they were analyzed by transmission electron microscopy (TEM). 1 is a TEM analysis image of tomato-derived exosomes purified according to Example 1 above. As a result of the analysis, the existence of about 160 nm particles having a spherical phospholipid bilayer structure was confirmed.

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

In order to confirm the shape of the purified broccoli-derived exosomes, they were analyzed by transmission electron microscopy (TEM). 2 is a TEM analysis image of broccoli-derived exosomes purified according to Example 2 above. As a result of the analysis, the existence of about 160 nm particles having a spherical phospholipid bilayer structure was confirmed.

Experimental Example 3: Characterization of tomato-derived exosomes: NTA analysis

The purified tomato-derived exosomes were analyzed by Nanoparticle Tracking Analysis (NTA) to confirm the particle size distribution and number of particles per unit volume.

3 is a graph showing the results of NTA analysis of tomato-derived exosomes purified according to Example 1. As a result of the analysis, the average particle size of 159.7 nm and the concentration of 6.40 x 10 ¹⁰ particles per unit volume of 1 mL were confirmed.

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

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

4 is a graph showing the results of NTA analysis of broccoli-derived exosomes purified according to Example 2. As a result of the analysis, it was confirmed that the particles had an average size of 167.3 nm and a concentration of 5.90 x 10 ¹⁰ particles per unit volume of 1 mL.

Test Example 5: Comparison of the yield of tomato and broccoli-derived exosomes

In order to confirm the optimal conditions for hot air drying and UV treatment, the yields of tomato and broccoli-derived exosomes purified according to Examples 1, 2 and 4 to 15 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 6.40 x 10 ¹⁰ 159.7 Example 2 5.90 x 10 ¹⁰ 167.3 Example 4 3.40 x 10 ⁸ 151.6 Example 5 1.10 x 10 ¹⁰ 142.7 Example 6 1.70 x 10 ¹⁰ 160.3 Example 7 6.70 x 10 ⁹ 167.5 Example 8 3.10 x 10 ⁹ 170.5 Example 9 6.20 x 10 ⁹ 166.8 Example 10 1.80 x 10 ⁸ 150.9 Example 11 9.00 x 10 ⁹ 159.4 Example 12 2.70 x 10 ¹⁰ 161.7 Example 13 5.60 x 10 ⁹ 171.6 Example 14 4.70 x 10 ⁹ 150.6 Example 15 7.10 x 10 ⁹ 159.7

As confirmed in Table 2, when UV treatment was performed after hot air drying treatment (Examples 5, 6, 11, and 12), high yields were exhibited, and among them, Examples 1 and 2 under the condition of UV treatment for 6 hours The yield of exosomes was the highest in . Judging from the results of Example 4 and Example 10, exosomes not subjected to hot air drying and UV pretreatment showed the lowest yield. This was confirmed to have a significant effect on the yield of exosomes under pretreatment conditions, and therefore, it was confirmed that the most optimal conditions were pretreatment with hot air drying followed by UV-A treatment for 6 hours.

Test Example 6: Evaluation of cytotoxicity

Tomato exosome (Example 1, Example 4), broccoli exosome (Example 2, Example 10) and its mixed exosome (Example 3) and tomato extract (Comparative Example 1), broccoli 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 tomato exosomes, broccoli exosomes, the mixed exosomes, tomato extract, and broccoli extract were administered to a new medium by concentration and cultured again for 24 hours. To measure cell viability, MTT solution (5mg/mL) was added, 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 tomato exosome, broccoli exosome, tomato and broccoli mixed exosome, tomato extract, and broccoli extract were treated, no cytotoxicity was observed at all concentrations.

Test Example 7: Evaluation of whitening efficacy of tomato and broccoli exosomes (tyrosinase activity inhibition)

As tyrosine is oxidized to dopaquinone by tyrosinase, pigmentation occurs in the skin. In order to confirm the whitening efficacy of the exosomes, a tyrosinase enzyme activity inhibition test was conducted to darken the skin compared to the extract. Mushroom-derived tyrosinase and tyrosine were purchased and used from Sigma Chemical. For tyrosinase activity, 0.1M phosphate buffer (pH 6.5) 150μl and mushroom tyrosinase (2100unit/ml, 0.05M phosphate buffer, pH 6.5) 8μl, 1.5 Tomato exosomes, broccoli exosomes, their mixed exosomes, tomato extract, and broccoli extract were treated with 36 μl of L-tyrosine at mM concentration for each concentration. Tyrosinase inhibitory activity was confirmed by measuring the absorbance at 490 nm after the sample was reacted at 37° C. for 15 minutes. At this time, PBS was treated as a negative control, and Niacinamide 0.01% was treated as a positive control.

6 is a graph showing the results obtained by evaluating the whitening efficacy of tomato and broccoli-derived exosomes purified according to the above example in terms of tyrosinase activity inhibition rate. As a result of the test, when tomato exosomes, broccoli exosomes, tomato extract, and broccoli extract were treated, the tyrosinase activity inhibition rate increased in a concentration-dependent manner, and when tomato and broccoli-derived exosomes were treated, the activity inhibition rate was further increased confirmed. In addition, it was confirmed that the highest activity inhibition rate was obtained when the tomato and broccoli mixed exosomes were treated. At this time, when Niacinamide 0.01%, a positive control, was treated, 40.2% inhibition rate was shown.

Test Example 8: Evaluation of whitening efficacy of tomato and broccoli exosomes (inhibition of melanin production)

In order to confirm the whitening efficacy of tomato and broccoli-derived exosomes, a melanin production inhibition test was conducted in comparison with the extracts. B16F10 cell line (melanin-secreting cells) derived from mice was inoculated and cultured in Dulbecco Modified Eagle Medium (DMEM) culture medium supplemented with 10% FBS at 37° C. for 24 hours under 5% CO ₂ . After culturing, the medium was discarded and replaced with a new medium, and then the tomato exosomes, the broccoli exosomes, the mixed exosomes, the tomato extract, and the broccoli extract were treated by concentration and cultured for 72 hours. After culturing, the cells were recovered by centrifugation after treatment with Trypsin-EDTA. The recovered cells were washed, treated with 500 μL of 1N NaOH, and reacted at 100° C. for 10 minutes to dissolve melanin. The amount of melanin was confirmed by measuring absorbance at 405 nm. At this time, PBS was treated as a negative control, and arbutin, a synthetic material known as a whitening agent, was used as a standard sample, and the result was converted into %.

7 is a graph showing the results obtained by evaluating the whitening efficacy of tomato and broccoli-derived exosomes purified according to the above example in terms of melanin production inhibition rate. As a result of the test, when tomato exosomes, broccoli exosomes, tomato extract, and broccoli extract were treated, the melanin production inhibition rate increased in a concentration-dependent manner. did In addition, it was confirmed that the highest melanin production inhibition rate was obtained when the tomato and broccoli mixed exosomes were treated.

Test Example 9: Evaluation of anti-inflammatory efficacy of tomato and broccoli exosomes (inhibition of IL-1 expression)

In order to confirm the anti-inflammatory efficacy of tomato and broccoli-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 6x10 ⁵ cells/well and cultured for 24 hours at 37℃ and 5% CO ₂ until they reached 70-80% growth. It was replaced with free DMEM. Tomato exosomes, broccoli exosomes, tomato extract, and broccoli extract were administered to each well at different concentrations, and pretreatment was performed for 4 hours. 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 cells after cell culture, and then centrifugation (12,000 rpm, 4℃ for 30 minutes) by adding 0.2mL chloroform (Sigma-Aldrich, USA) 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℃ for 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 for 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 PCR tube in a total of 13 μL, reacted at 65 ° C for 5 minutes, and then M-MLV Reverse Transcriptase (Invitrogen, Thermo Fisher Scientific, Waltham, Massachusetts, USA) was synthesized by reacting at 37 ° C. for 50 minutes. qRT-PCR was performed to quantitatively analyze gene expression patterns occurring within each cell by the sample. For qRT-PCR, a total of 20 μL of primer, cDNA, 2X SYBR green PCR Master Mix (Applied Biosystems, USA), and HPLC (J. T baker, USA) were mixed in a PCR tube to make a reaction solution, which was then run on a StepOnePlus Real-Time PCR System ( PCR was performed using Applied Biosystems, USA).

The PCR primer sequences of the IL-1 gene are shown in Table 3 below, and FIG. 8 is a graph showing the results of evaluating the anti-inflammatory efficacy of exosomes derived from tomato and broccoli 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 when the tomato exosomes, broccoli exosomes, tomato extract, and broccoli extract were treated, IL-1 mRNA, an inflammatory factor, was reduced in a concentration-dependent manner, and the inhibition rate was significantly increased in the exosome treatment concentration range compared to the extract. At this time, when Allantoin 0.01%, a positive control, was treated, 43.1% 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 10: Evaluation of anti-inflammatory efficacy of tomato and broccoli exosomes (inhibition of IL-6 expression)

In order to confirm the anti-inflammatory efficacy of tomato and broccoli-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 9 above, and the PCR primer sequence of the IL-6 gene is shown in Table 4 below. 9 is a graph showing the results obtained by evaluating the anti-inflammatory efficacy of tomato and broccoli-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 when tomato exosomes, broccoli exosomes, tomato extract, and broccoli extract were treated, IL-6 mRNA, an inflammatory factor, was reduced in a concentration-dependent manner, and the inhibition rate was significantly increased in the concentration range of exosome treatment compared to the extract. At this time, when Allantoin 0.01%, a positive control, was treated, 46.8% 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'

Formulation Example 1: Preparation of Cream

With the composition shown in Table 5 below, a cream containing purified tomato exosomes and broccoli exosomes according to the above example was prepared by a conventional method. The cream containing the tomato extract of Comparative Example 1 was used as Comparative Formulation Example 1, and the cream containing the broccoli 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) Tomato exosomes (Example 1) 5 - - - - Broccoli exosomes (Example 2) - 5 - - - Tomato + Broccoli Exosomes (Example 3) - - 5 - - Tomato extract (Comparative Example 1) - - - 5 - Broccoli 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 11: Evaluation of skin soothing efficacy of tomato and broccoli exosomes

Twenty healthy adult men and women without skin allergy symptoms were divided into three groups and the skin soothing effect was evaluated through the tape stripping method (a method of stimulating the skin after attaching the tape). After inducing skin irritation and erythema on the inside of the habak using a tape, a certain amount of the cream prepared in the above formulation was applied to confirm the degree to which the irritation subsided after 3 hours. The results are shown in Table 6 below.

skin soothing effect - + ++ +++ ++++ Formulation example 1 11 8 One 0 0 Formulation Example 2 10 8 2 0 0 Formulation Example 3 14 5 One 0 0 Comparative Formulation Example 1 5 8 4 3 0 Comparative Formulation Example 2 2 5 9 3 One

(-; no erythema, +; very mild erythema, ++; faint erythema, +++; marked erythema, ++++; severe erythema)

As can be seen from the results of Table 6, the cream containing the tomato exosome, broccoli exosome, and tomato/broccoli mixed exosome of the present invention showed excellent skin soothing effect.

Test Example 12: Evaluation of hair growth promoting efficacy (hair papilla cell proliferation)

In order to confirm the hair growth promoting effect of tomato and broccoli-derived exosomes, the effect on dermal papilla cell proliferation was confirmed by comparing with tomato and broccoli extracts at each 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 tomato exosomes, broccoli exosomes, the mixed exosomes and tomato extract, Broccoli 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 the cell proliferation was increased in a concentration-dependent manner when the tomato and broccoli-derived exosomes were treated with the tomato and broccoli extracts, and the proliferation was further increased when the tomato and broccoli-derived exosomes were treated compared to the extracts. Also, the highest cell proliferation rate was shown when the tomato and broccoli mixed exosomes were treated, and at this time, when the positive control, Minoxidil 10Μm, was treated, the proliferation rate was 90.7%.

Test Example 13: Evaluation of anti-hair loss efficacy (inhibition of 5α-reductase activity)

In order to confirm the anti-hair loss efficacy of tomato and broccoli-derived exosomes, the effect on 5α-reductase inhibitory activity was compared with tomato and broccoli 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 the tomato and broccoli-derived exosomes were treated. The best inhibitory activity was shown when tomato and broccoli mixed exosomes were treated.

Test Example 14: Evaluation of hair loss prevention efficacy

Tomato extract, broccoli extract and In comparison, an experiment was conducted for 12 weeks on 30 adults with early hair loss. Tomato-derived exosomes, broccoli-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 7 below. As a negative control group, fallen hairs were collected and compared in the state of not being treated with anything.

Treatment concentration (%) Average number of fallen hairs (pcs) negative control - 74 Tomato exosomes (Example 1) 5 52 Broccoli exosomes (Example 2) 5 57 Tomato + Broccoli Exosomes (Example 3) 5 49 Tomato extract (Comparative Example 1) 5 70 Broccoli extract (Comparative Example 2) 5 69

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

Claims (9)

In the cosmetic composition containing tomato exosomes, broccoli exosomes or mixed exosomes as an active ingredient,
Tomato exosomes and broccoli exosomes as the active ingredient,
(A) subjecting tomatoes or broccoli to hot air drying at 40 to 60° C. for 20 to 24 hours;
(B) immersing tomatoes or broccoli treated with hot air drying in water at 30 to 40 ° C for 6 hours;
(C) UV-A treatment of immersed tomatoes or broccoli for 1 to 6 hours;
(D) squeezing UV-treated tomatoes or broccoli;
(E) obtaining a supernatant by centrifuging the juice at 1,000xg to 10,000xg;
(F) freeze-drying the supernatant in which exosomes are present;
(G) forming an aqueous two-phase system using PEG (Polyethylene glycol)/Dextran in the lyophilisate; and
(H) A cosmetic composition characterized in that it is purified by a method comprising obtaining a lower layer in which 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 UV-A treatment in step (C) is performed for 6 hours with UV-A of 365 nm wavelength. The cosmetic composition according to claim 1, wherein the tomato exosome, broccoli 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, characterized in that the cosmetic composition is for whitening, anti-inflammatory or skin soothing. The cosmetic composition according to claim 1, wherein the cosmetic composition is for promoting hair growth or preventing hair loss. (A) subjecting tomatoes or broccoli to hot air drying at 40 to 60° C. for 20 to 24 hours;
(B) immersing tomatoes or broccoli treated with hot air drying in water at 30 to 40 ° C for 6 hours;
(C) UV-A treatment of immersed tomatoes or broccoli for 1 to 6 hours;
(D) squeezing UV-treated tomatoes or broccoli;
(E) obtaining a supernatant by centrifuging the juice at 1,000xg to 10,000xg;
(F) freeze-drying the supernatant in which exosomes are present;
(G) forming an aqueous two-phase system using PEG (Polyethylene glycol)/Dextran in the lyophilisate; and
(H) A method for purifying tomato or broccoli exosomes comprising the step of obtaining a lower layer in which exosomes are concentrated in the aqueous two-phase system. The method of claim 8, wherein the hot air drying treatment in step (A) is performed at a temperature of 50 ° C. for 24 hours, and the UV treatment in step (C) is performed for 6 hours with UV-A of 365 nm wavelength. A method for purifying tomato or broccoli exosomes, characterized in that to do.

Images