KR102735377B1 - Composition for preventing, improving or treating periodontal disease containing Spatholobi caulis exosome, Eucommia ulmoides exosome or Zanthoxylum piperitum exosome - Google Patents

Abstract

The present invention relates to a composition for preventing, improving or treating periodontal disease containing exosomes derived from Gyechuldeung, Eucommia ulmoides and Zanthoxylum piperitum as active ingredients, and more particularly, to a composition for preventing, improving or treating periodontal disease containing exosomes derived from Gyechuldeung, Eucommia ulmoides and Zanthoxylum piperitum as active ingredients, purified using freeze-thawing treatment and an aqueous two-phase system, and having excellent anti-inflammation, cell regeneration and osteocyte differentiation promotion effects.

Description

{Composition for preventing, improving or treating periodontal disease containing Spatholobi caulis exosome, Eucommia ulmoides exosome or Zanthoxylum piperitum exosome}

The present invention relates to a composition for preventing, improving or treating periodontal disease containing exosomes derived from Gyechuldeung, Eucommia ulmoides and Zanthoxylum piperitum as active ingredients, and more particularly, to a composition for preventing, improving or treating periodontal disease containing exosomes derived from Gyechuldeung, Eucommia ulmoides and Zanthoxylum piperitum as active ingredients, purified using freeze-thawing treatment and an aqueous two-phase system, and having excellent anti-inflammation, cell regeneration and osteocyte differentiation promotion effects.

Periodontitis is an inflammation of the periodontal tissue (the tissue surrounding the teeth), and it is a disease in which the periodontal tissue supporting the teeth is gradually destroyed, causing the teeth to become loose and possibly even lose the teeth. Periodontitis is caused by microorganisms that adhere to and grow on the surface of the teeth and the immune response to them. The direct cause is a bacterial film called plaque that continuously forms on the teeth. Plaque is sticky and colorless, and when it hardens, it becomes tartar. As plaque and tartar accumulate, the gums separate from the teeth, and when this gap widens, a periodontal pocket is formed. In this state, if the inflammation progresses further and the periodontal tissue is lost around the periodontal pocket, the teeth become loose.

According to what has been discovered so far, about 300 types of microorganisms exist in the human oral cavity on the surface of the teeth, between the teeth and gums at the root, on the surface of the tongue, etc. If proper oral hygiene is not performed due to the presence of these microorganisms, tooth decay, bad breath, gingivitis, or periodontal disease caused by pathogenic microorganisms can occur, and in severe cases, tooth loss can occur.

Currently, the treatment of periodontal disease has been non-surgical or surgical scaling, root planing, gingival curettage, and periodontal tissue regeneration. However, surgical methods are difficult to treat effectively because of the inconvenience of having to visit a dentist, and are limited to treatment when the disease has progressed to a certain extent rather than prevention, so most cases progress to chronic disease if left untreated. Taking antibiotics also has serious problems, as side effects and cases of resistance have been reported.

Therefore, there is an urgent need to develop a preparation that has anti-inflammatory and periodontal tissue restoration effects to complement the limitations of surgical treatment and the problems of antibiotic use and to prevent and treat periodontal disease.

It is known that two types of cells are largely involved in bone formation. One of the two cells is osteoblasts, which create bone, and the other is osteoclasts, which destroy bone. They are derived from different cells: mesenchymal stem cells differentiate into osteoblasts, myoblasts, fibroblasts, and chondrocytes, and hematopoietic stem cells differentiate into osteoclasts, macrophages, T cells, B cells, dendritic cells, eosinophils, platelets, and erythrocytes.

The activity of osteoclasts causes the absorption or destruction of old bone, which creates holes in the bone and releases a small amount of calcium into the bloodstream to be used to maintain bodily functions. Osteoblasts, which are produced from bone cells, fill the holes with collagen and cover the calcium and phosphorus deposits to create new, strong bone and rebuild the skeleton. As much bone is absorbed by osteoclasts, bone formation by osteoblasts occurs, so that a certain amount of bone tissue is always maintained. Inflammation reduces the bone-making function of osteoblasts and increases the bone-destroying or absorbing function of osteoclasts, which causes the alveolar bone to gradually decrease and be destroyed, eventually leading to tooth loss.

The present inventors completed the present invention by studying a raw material that has anti-inflammation, cell regeneration, and bone cell differentiation promotion effects without causing irritation.

In the present invention, "exosome" refers to 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 vesicles for this purpose. Exosomes are approximately 50 to 200 nm in size 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 status of the cells from which they originate, so they can be used for diagnosis and treatment. Exosomes have a double phospholipid membrane structure that easily penetrates cells, and perform various physiological and pathological functions such as immune responses and signal transduction.

Recently, research has been conducted on the various effects of plant-derived exosomes. Plant-derived exosomes are natural nanoparticles that contain physiologically active and signaling substances secreted by plant cells themselves, which help with movement and absorption between cells, and it is known that exosomes purified from plants are less toxic than mammalian exosomes.

These exosomes are materials that can be utilized in fields such as medicine, cosmetics, and food due to their various advantages and activities, but they have low dispersibility and a tendency to aggregate due to their structural characteristics consisting of a phospholipid bilayer. Therefore, in order to maintain continuous activity, it is necessary to increase the solubility and dispersibility of exosomes in aqueous solutions to increase their stability in the formulation.

The present inventors have attempted to produce plant-derived exosomes exhibiting excellent anti-inflammation, cell regeneration, and osteocyte differentiation promoting activities by applying various methods, and to use them as compositions for preventing, improving, or treating periodontal diseases. As a result, the inventors have confirmed that purified exosomes of Gyeryong-deungpo, Eucommia ulmoides, and Zanthoxylum piperitum using freeze-thawing treatment under specific conditions and an aqueous two-phase system exhibit excellent efficacy in preventing, improving, or treating periodontal diseases, thereby completing the present invention.

(0001) Republic of Korea Publication Patent No. 10-2017-0031428 (2017.03.21) (0002) Republic of Korea Publication Patent No. 10-2018-0108331 (2018.10.04) (0003) Republic of Korea Patent No. 10-2265811 (2021.06.16.)

The purpose of the present invention is to provide a composition for preventing, improving or treating periodontal disease, which contains exosomes derived from Gyehyeol-deung, Eucommia ulmoides and Chopi tree as effective ingredients and exhibits excellent anti-inflammation, cell regeneration and bone cell differentiation promotion effects.

In addition, another purpose of the present invention is to provide a method for purifying exosomes derived from the above-mentioned Gyehyeol-deung, Eucommia ulmoides, and Chopi tree.

In order to achieve the above purpose, according to the present invention, a composition for preventing, improving or treating periodontal disease containing exosomes from Gyehyeol-deung, exosomes from Eucommia ulmoides or exosomes from Chopi tree as effective ingredients is provided.

Preferably, the exosomes of Gyehyeol-deung, Eucommia ulmoides exosomes, and Chopi tree exosomes as the effective ingredients are

(A) a freeze-thawing treatment step of adding a solvent to Gyechung-deung, Eucommia ulmoides, or Chopi-no-mura and repeating freezing and thawing; (B) a step of extracting the freeze-thawing-treated Gyechung-deung, Eucommia ulmoides, or Chopi-no-mura; (C) a step of centrifuging the extracted juice at 1,000xg to 10,000xg to obtain a supernatant; (D) a step of lyophilizing the supernatant in which exosomes are present; (E) a step of forming an aqueous two-phase system using PEG (Polyethylene glycol)/Dextran in the lyophilized product; and (F) a step of obtaining a lower layer in which exosomes are concentrated among the aqueous two-phase system.

Preferably, the freeze-thaw treatment in step (A) is characterized by repeating the process of freezing at a temperature of -80 to -70°C for 15 to 20 hours and then thawing at a temperature of 40 to 50°C for 8 to 10 hours 2 to 5 times.

The above composition is characterized by having anti-inflammation, cell regeneration, and bone cell differentiation promoting activities.

The above exosomes from Gyehyeol-deung, Eucommia ulmoides, or Chopi tree as effective ingredients are contained in an amount of 0.0001 to 30.0% (w/w) based on the total weight of the composition.

The above composition is characterized in that it is a pharmaceutical composition, a quasi-drug composition, a food composition or a cosmetic composition.

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

(A) A freeze-thaw treatment step of adding a solvent to a blood-stained tree, a Chinese juniper tree, or a juniper berry and repeating freezing and thawing;

(B) A step of extracting juice from freeze-thawed and thawed ginseng, Eucommia ulmoides, or Chopi tree;

(C) A step of centrifuging the above juice at 1,000xg to 10,000xg to obtain a supernatant;

(D) A step of freeze-drying the supernatant containing exosomes;

(E) a step of forming an aqueous two-phase system using PEG (Polyethylene glycol)/Dextran in the above freeze-dried product; and

(F) A method for purifying exosomes from Gyehwol-deung, Eucommia ulmoides or Chopi tree is provided, including a step of obtaining a lower layer containing concentrated exosomes from the above two-phase aqueous system.

Preferably, the freeze-thaw treatment in step (A) is characterized by repeating the process of freezing at a temperature of -80 to -70°C for 15 to 20 hours and then thawing at a temperature of 40 to 50°C for 8 to 10 hours 2 to 5 times.

The exosomes of the present invention, purified using freeze-thaw pretreatment and an aqueous two-phase system, such as those from Gyechon, Duzhong or Zanthoxylum lucidum, have excellent stability and exhibit excellent anti-inflammatory effects, cell regeneration effects and bone cell differentiation promotion effects, and thus can be usefully used as pharmaceuticals, quasi-drugs, foods or raw materials for cosmetics for preventing, improving or treating periodontal diseases.

Figure 1 is a TEM image of exosomes derived from purified Gyehwol-deung, Eucommia ulmoides, and Chopi tree according to one embodiment of the present invention.
FIG. 2 is a graph showing the results of NTA analysis to confirm the particle size distribution and particle number of purified exosomes derived from Gyehwol-deung, Eucommia ulmoides, and Chopi tree according to one embodiment of the present invention.
Figure 3 is a graph showing the results of evaluating the cytotoxicity of exosomes derived from Gyehyeol-deung, Eucommia ulmoides, and Chopi tree purified according to the present invention using an MTT assay.
Figure 4 is a graph showing the results of evaluating the anti-inflammatory efficacy of exosomes derived from Gyehwol-deung, Eucommia ulmoides, and Chopi tree purified by the present invention by suppressing TNF-α expression.
Figure 5 is a graph showing the results of evaluating the anti-inflammatory efficacy of exosomes derived from Gyehwol-deung, Eucommia ulmoides, and Chopi tree purified by the present invention by suppressing IL-8 expression.
Figure 6 is an image showing the results of evaluating the cell regeneration efficacy of exosomes derived from Gyehwol-deung, Eucommia ulmoides, and Chopi tree purified according to the present invention using a wound healing assay.
Figure 7 is a graph showing the results of evaluating the cell regeneration efficacy of exosomes derived from the plant Gyehyeol-deung and Eucommia ulmoides, purified according to the present invention, as a percentage of wound area.
Figure 8 is a graph showing the results of evaluating the effect of exosomes derived from Gyehwol-deung, Eucommia ulmoides, and Chopi tree purified according to the present invention on osteocyte differentiation through Runx2 expression.
Figure 9 is a graph showing the results of evaluating the effect of exosomes derived from Gyehwol-deung, Eucommia ulmoides, and Chopi tree purified according to the present invention on osteocyte differentiation through OSX expression.
Figure 10 is a graph showing the results of evaluating the effect of exosomes derived from Gyehwol-deung, Eucommia ulmoides, and Chopi tree purified according to the present invention on osteocyte differentiation through ALP expression.

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

Plant-derived exosomes contain physiologically active and signaling substances secreted by the plant cells themselves, and are known to be less toxic than mammalian-derived exosomes. Due to these advantages, they are materials that can be used in fields such as pharmaceuticals, cosmetics, and food. However, due to their structural characteristics consisting of a phospholipid bilayer, they have low dispersibility and a tendency to aggregate, making it difficult to continuously maintain their activity within the formulation.

The present invention is characterized in that exosomes obtained by freezing and thawing Gyechung-deung, Eucommia ulmoides, and Chopi-no-Matsutake and separating and purifying them with high purity through an aqueous two-phase system exhibit more excellent anti-inflammation, cell regeneration, and osteocyte differentiation promotion effects than extracts of Gyechung-deung, Eucommia ulmoides, and Chopi-no-Matsutake that have been used for the purpose of improving periodontal disease.

Chicken blood vine (鷄血藤) is the dried stem of Milhwadu, a legume in the Rosaceae family of the Dicotyledonous dicotyledonous plant group, and is used as a herbal medicine. The vine stem is flat and cylindrical, slightly curved, 30–40 cm long, and 3–5 cm in diameter. The outer surface is gray-brown with vertical grooves and transverse lobes, the nodes are slightly protruding, and some have marks from branches. The name Chicken blood vine (鷄血藤) comes from the fact that the sap of the vine resembles chicken blood. When a fresh stem is cut, a reddish-brown sap like chicken blood oozes out, and when this sap dries, it hardens and hardens, and when broken, it becomes flaky.

The known components of Gyehyeol include formononetin, daidzein, isoliquiritigenin, licochalcone A, (2R)-7-hydroxy-6-methoxy-flavanone, friedelin, friedelan-3β-ol, sativan, β-sistosterol, stigmasterol, campesterol, (3R,4R)-2',4'-dihydroxy-6,7-methylene-dioxyisoflavan-4-ol, genistein, medicagol, pseudobaptigenin, (3R,4R)-7',2'-dihydroxy-4'-methoxyisoflavan-4-ol, etc. Gyehyeol is being studied in various aspects such as antibacterial action, antioxidant action, anti-aging action, anti-inflammatory action, cerebral blood flow improvement action, cell cycle inhibition and apoptosis induction action, and lipid metabolism regulation action.

Eucommia ulmoides is a plant of the Eucommia family, which is extinct in the wild, but is widely cultivated because its bark is used as a herbal medicine. Like the ginkgo tree, it is a heterosexual tree, and when it is over 20 years old, it grows into a large tree over 20m tall, and small light green flowers bloom around April to May. The bark of the Eucommia tree used in oriental medicine can be harvested after 15 to 16 years of age, and the young leaves used as tea can be harvested from the second year. The bark of the Eucommia tree is gray-brown on the surface and dark gravel-brown on the inside. Eucommia is known to be surprisingly effective, and is receiving attention as a natural herbal medicine. Eucommia strengthens muscles and bones, and promotes bone formation by enhancing muscle elasticity. It contains a large amount of gum and gutta-percha, and contains resin, ash, organic acids, vitamins, and chlorogenic acid. It is characterized by a spicy and sweet taste, a warm medicinal property, and no toxicity. In the present invention, the stem, bark, and/or leaves of the Eucommia tree are used in the production of exosomes.

Zanthoxylum piperitum is a tree belonging to the Rutaceae family, and is a member of the Zanthoxylum genus, similar to the prickly ash and the Chinese pepper tree. The leaves are alternate and pinnately compound, and light yellow flowers bloom in tassel-shaped inflorescences that emerge from the leaf axils. There are no petals, but there are five sepals. The fruit is round, and when ripe, it turns from green to red, revealing black, glossy seeds. The fruit of the Zanthoxylum piperitum is used as a herbal medicine and spice. The Zanthoxylum piperitum is used in oriental medicine as a detoxifying, analgesic, anti-inflammatory, and anti-enteritis agent, and recently, it has been proven to have excellent anticancer effects, food poisoning prevention and treatment, skin whitening, and anti-aging effects, and research on this is actively being conducted. In the present invention, the fruit, root, stem, and/or leaf of the Zanthoxylum piperitum are used to produce exosomes.

The exosomes of Gyehyeol-deung, Eucommia ulmoides or Chopi tree as effective ingredients of the composition of the present invention are preferably purified by the following method.

(A) a freeze-thawing treatment step of adding a solvent to Gyechung-deung, Eucommia ulmoides, or Chopi-no-mura and repeating freezing and thawing; (B) a step of extracting the freeze-thawing-treated Gyechung-deung, Eucommia ulmoides, or Chopi-no-mura; (C) a step of centrifuging the extracted juice at 1,000xg to 10,000xg to obtain a supernatant; (D) a step of lyophilizing the supernatant in which exosomes are present; (E) a step of forming an aqueous two-phase system using PEG (Polyethylene glycol)/Dextran in the lyophilized product; and (F) a step of obtaining a lower layer in which exosomes are concentrated among the aqueous two-phase system.

In the present invention, in order to produce exosomes from Gyehwol-deung, Du-chong or Chopi tree, which have excellent anti-inflammatory effects, cell regeneration effects and bone cell differentiation promotion effects, a solvent is first added to the raw material and freeze-thaw treatment is performed.

In the case of plants, the secretion characteristics of exosomes can vary depending on environmental factors, and in particular, when the plant is stressed, the amount of exosomes secreted can be affected. Therefore, in order to identify the optimal conditions for plants to secrete exosomes, the yield and activity were confirmed through various pretreatments, and it was confirmed that when freeze-thawing treatment was performed as a pretreatment for Gyeryongdeungpo, Eucommia ulmoides, and Zinnia serrata, the secretion of exosomes increased and the activity increased.

According to a preferred specific example of the present invention, the freeze-thaw treatment in step (A) is performed by repeating the process of freezing at a temperature of -80 to -70°C for 15 to 20 hours and then thawing at a temperature of 40 to 50°C for 8 to 10 hours 2 to 5 times.

It is preferable that the screw used in the juicing process in the above step (B) has a stirring speed of 20 to 50 rpm.

The centrifugation method used in the above step (C) refers to a method of separating particles of a solution by using centrifugal force according to size, shape, density, viscosity, and rotor speed. It is necessary to remove large contaminants by sequentially adjusting the rpm, and it is more preferable to perform it at 10,000xg to obtain a final solution for forming an aqueous two-phase system.

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

In order to efficiently separate and purify exosomes, the present invention used the aqueous two-phase partition method, which is a separation method that utilizes the difference in affinity between two layers formed by two types of aqueous solutions that do not dissolve well in each other.

In general, PEG/salt (salts such as sulfate, phosphate, and citrate) can be used in the method of forming a two-phase aqueous system, but in order to achieve the purpose of the present invention, it is preferable to use PEG/Dextran. Dextran is a natural polymer obtained by bacterial action and is used as a thickening agent, binder, and bulking agent in cosmetic formulations.

In the step (E) above, in the formation of the aqueous two-phase system, PEG having a molecular weight of 10,000 to 35,000 is used in an amount of 1 to 15 wt%, preferably 2 to 5 wt%, and Dextran having a molecular weight of 300,000 to 650,000 is used in an amount of 1 to 8 wt%, preferably 1 to 3 wt%. When PEG and Dextran are used in a concentration ratio of 3.3 wt%: 1.7 wt%, the yield of exosomes is the highest and the stability is the most excellent, which is more preferable.

To increase the purity of exosomes, a process 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 to 3 times following the step (F).

The exosomes of Gyechul-deung, Eucommia ulmoides, and Zanthoxylum piperitum produced by the above method exhibited better anti-inflammatory effects (Test Examples 4 and 5), cell regeneration effects (Test Example 6), and bone cell differentiation promotion effects (Test Examples 7, 8, and 9) compared to the general Gyechul-deung extract, Eucommia ulmoides extract, and Zanthoxylum piperitum extract.

Therefore, the exosomes derived from the above-mentioned Gyehyeol-deung, Eucommia ulmoides, and Chopi tree can be usefully used as a composition for preventing, improving, or treating periodontal disease.

The above composition may be a pharmaceutical composition, a quasi-drug composition, a food composition or a cosmetic composition.

At this time, the exosomes derived from Gyehyeol-deung, Eucommia ulmoides or Chopi tree as the effective ingredients may be contained in an amount of 0.0001 to 30.0% (w/w) based on the total weight of the composition.

The above pharmaceutical composition may contain an acceptable carrier, excipient or diluent, and the pharmaceutically acceptable carrier, excipient or diluent may be saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, sorbitol, mannitol, starch, alginate, gelatin, cellulose, methyl cellulose, methylhydroxy benzoate, talc, glycerol, ethanol, and a mixture of one or more of these components. If necessary, other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added. In addition, a diluent, a dispersant, a surfactant, a binder and a lubricant may be additionally added to formulate the composition into a dosage form such as an aqueous solution, a suspension, an emulsion, a powder, a tablet, a capsule, a pill, a granule or an injection.

The pharmaceutical composition of the present invention can be formulated into tablets, capsules, powders, granules, suspensions, emulsions, syrups, and other liquid preparations by conventional methods. Specifically, the pharmaceutical composition of the present invention is formulated into dosage forms for oral administration, for example, tablets, troches, lozenges, aqueous or concentrated suspensions, prepared powders or granules, emulsions, hard or soft capsules, syrups, or elixirs. In order to formulate into dosage forms such as tablets and capsules, the composition contains a binder such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose, or gelatin, an excipient such as dicalcium phosphate, a disintegrant such as corn starch or sweet potato starch, and a lubricant such as magnesium stearate, calcium stearate, sodium stearyl fumarate, or polyethylene glycol wax. In the case of capsule formulations, in addition to the substances mentioned above, a liquid carrier such as fatty oil is contained.

In addition, the pharmaceutical composition of the present invention can be administered orally or parenterally, and when administered parenterally, it is preferable to select a subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection method. The pharmaceutical composition according to the present invention can vary depending on various factors including the activity of the active ingredient, age, body weight, general health, sex, administration time, and administration route, and the dosage of the pharmaceutical composition is appropriately selected depending on the absorption rate, inactivation rate, and excretion rate of the active ingredient in the body, the age, sex and condition of the patient, administration route, administration time, and the severity of the disease to be treated, but can be appropriately selected by a person skilled in the art. Generally, it can be administered to adults in an amount of 0.0001 to 500 mg per 1 kg of body weight per day, and can be administered once or several times a day, but it is preferable to administer in an amount of 0.001 to 100 mg.

The above-mentioned pharmaceutical composition is not particularly limited in its formulation and may have a conventional formulation, specifically, may have a formulation such as toothpaste, mouthwash, or oral hygiene agent. The oral composition provided by the present invention may contain a base and additives necessary for formulation according to its formulation, and the types and amounts of these ingredients may be easily selected by those skilled in the art. For example, it may be manufactured by adding an abrasive, a wetting agent, a foaming agent, a sweetener, a binder, a preservative, a pH regulator, a flavoring agent, a pigment, etc.

The above food composition may contain various flavoring agents or natural carbohydrates as additional ingredients. Natural carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. As a sweetener, natural sweeteners such as thaumatin and stevia, synthetic sweeteners such as saccharin and aspartame, etc. can be used. In addition, the food composition of the present invention may contain various nutrients, vitamins, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, protective colloid thickeners, stabilizer glycerin, alcohol, carbonating agents, etc. The type of the above food composition is not particularly limited. Examples of foods to which the above substances can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and include all health foods in the conventional sense.

The above cosmetic composition can be manufactured in any formulation that is commonly manufactured, and can be manufactured as, for example, a skin lotion, a skin toner, a pack, a nourishing cream, a moisturizing cream, an essence, a body cream, a body lotion, a body oil, a cleansing foam, a cleansing lotion, a soap, a patch, a foundation, a lipstick, a makeup base, a lipstick, etc.

[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 to the following examples, and it will be apparent to a person having ordinary skill in the art to which the present invention pertains that the present invention may be changed into other equivalent examples and substituted within the scope that does not depart from the technical spirit of the present invention.

Example 1: Preparation of exosomes from blood vessels

Pretreatment of blood vessels for freezing and thawing

Purified water was added to 100 g of blood plasma, frozen at -80°C for 18 hours, and then thawed at 40°C for 8 hours. This process was repeated a total of 3 times to create a condition suitable for exosome extraction.

Bloodletting and juice extraction

Freeze-thawed pretreated scutellaria baicalensis was juiced using a general juicer with a low-speed screw of 30 rpm, and the obtained scutellaria baicalensis juice was filtered through a mesh screen to remove floating substances. The recovered scutellaria baicalensis juice was stored at -80℃ until purification.

Supernatant recovery for exosome purification

Because the blood extract required the removal of large contaminants for exosome purification, centrifugation was performed at 10,000 × g for 10 minutes at 4°C. After centrifugation, the supernatant was collected to form an aqueous two-phase system.

Freeze drying of supernatant

To reduce the volume of the supernatant for mass production of exosomes, freeze-drying was performed. Freezing was performed at -80°C for 20 hours, and drying was performed in a freeze dryer under vacuum for 100 hours. Here, the vacuum state usually refers to the pressure state of the freeze dryer, and the freezing and drying times may vary depending on the volume of the solution.

Mercury binary system formation

Purified water was added to the freeze-dried supernatant, and an aqueous two-phase system was formed using PEG (polyethylene glycol)/Dextran. PEG (purchased from Sigma Aldrich) with a molecular weight of 10,000–35,000 was used in an amount of 3.3 wt%, and Dextran (purchased from Sigma Aldrich) with a molecular weight of 300,000–650,000 was used in an amount of 1.7 wt% to form an aqueous two-phase system.

Recovery of exosomes from blood vessels

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

Additional washing process

To increase the purity, an additional washing process was performed by adding the same concentration of the aqueous two-phase solution to the recovered supernatant. After three repeated treatments, the final supernatant containing the exosomes was recovered.

Example 2: Preparation of Eucommia exosomes

Exosomes derived from Eucommia ulmoides were purified in the same manner as in Example 1, except that Eucommia ulmoides bark was used instead of the cinquefoil in Example 1.

Example 3: Preparation of Coptis chinensis exosomes

Exosomes derived from the C. japonica tree were purified in the same manner as in Example 1, except that the C. japonica tree stem was used instead of the blood lamp in Example 1.

Examples 4-6: Preparation of exosomes from Gyehyeol-deung, Eucommia ulmoides, and Chopi tree

In the case of plants, the secretion characteristics of exosomes can vary depending on environmental factors. Since the optimal conditions for plants to secrete exosomes are all different, the yield of exosomes was compared due to environmental stresses such as freeze-thaw treatment to establish the optimal conditions, and exosomes were purified by varying the freeze-thaw treatment conditions of Gyechung, Eucommia, and Zinnia. In order to confirm the optimal conditions, exosomes were purified in the same manner as in Example 1 except that the conditions were varied, and the conditions are shown in Table 1 below.

Original Freeze-thaw treatment Example 4 Bloodletting lamp X Example 5 Eucommia X Example 6 Chopi tree X

Comparative Example 1: Preparation of extracts of Gyehyeol-deung

10g of dried Gye-hyeol-deung was added to 100g of purified water and extracted at 80℃ for 3 hours. After extraction, reduced pressure filtration was performed to obtain Gye-hyeol-deung extract, which was then distilled using a rotary evaporator to obtain a sample in powder form.

Comparative Example 2: Preparation of Eucommia extract

10g of dried Eucommia bark was added to 100g of purified water and extracted at 80℃ for 3 hours. After extraction, vacuum filtration was performed to obtain the Eucommia extract, which was then distilled using a rotary evaporator to obtain a sample in powder form.

Comparative Example 3: Preparation of Coptis chinensis extract

10g of dried branches of the Chinese juniper tree were added to 100g of purified water and extracted at 80℃ for 3 hours. After extraction, reduced pressure filtration was performed to obtain a Chinese juniper tree extract, which was then distilled using a rotary evaporator to obtain a sample in powder form.

Experimental Example 1: Characterization of Exosomes: TEM Analysis

To confirm the shape of the purified exosomes derived from Gyechuldeung, Eucommia ulmoides, and Chopi tree, transmission electron microscopy (TEM) was used. Figure 1 is a TEM analysis image of the purified exosomes derived from Gyechuldeung, Eucommia ulmoides, and Chopi tree, derived from the above Examples 1 to 3. The analysis results confirmed the presence of particles of approximately 150 nm in a spherical phospholipid bilayer structure.

Test Example 2: Characterization of exosomes: NTA analysis

To confirm the particle size distribution and the number of particles per unit volume of purified exosomes from Ginseng, Eucommia ulmoides, and Zanthoxylum bark, nanoparticle tracking analysis (NTA) was used.

Figure 2 shows the NTA graph results of the purified exosomes derived from Gyehwol-deung, Eucommia-derived exosomes, and Zinnia-derived exosomes according to the above Examples 1 to 3, and the analysis results are shown in Table 2 below.

Particle Number
(Particles/ml) Particle Size
(nm) Example 1 5.8 x 10 ¹⁰ 149.6 Example 2 7.1 x 10 ¹⁰ 150.3 Example 3 3.7 x 10 ¹⁰ 178.4 Example 4 2.1 x 10 ⁹ 150.1 Example 5 1.8 x 10 ⁹ 137.4 Example 6 4.7 x 10 ⁹ 169.7

As confirmed in Table 2 above, the yield of exosomes was higher in Examples 1, 2, and 3, which were conditions for freeze-thaw pretreatment. It was confirmed that the pretreatment conditions had a significant effect on the yield of exosomes.

Test Example 3: Cytotoxicity Evaluation

To confirm the cytotoxicity of Gyechuldeung exosomes (Example 1), Eucommia exosomes (Example 2), and Zinc oxide exosomes (Example 3) and Gyechuldeung extracts (Comparative Example 1), Eucommia extracts (Comparative Example 2), and Zinc oxide extracts (Comparative Example 3), an MTT assay was performed. Human keratinocyte (HaCaT) cells were inoculated into 96-well plates at a concentration of 1Х10 ⁵ cells/mL and cultured at 37°C for 18 hours under 5% CO _2. After culture, the medium was removed, washed with PBS buffer, and Gyechuldeung exosomes, Eucommia exosomes, Zinc oxide exosomes, and Gyechuldeung extracts, Eucommia extracts, and Zinc oxide extracts were added at different concentrations to new media, and cultured again for 24 hours. To measure cell viability, MTT solution (5 mg/mL) was added, and the formazan formed over 4 hours was dissolved with dimethyl sulfoxide (DMSO) and the absorbance was measured at 570 nm using an ELISA reader.

Figure 3 is a graph showing the results of evaluating the cytotoxicity of the above samples using an MTT assay. As a result of the test, when treated with exosomes from Gyechuldung, Eucommia ulmoides, and Zinnia pipa exosomes, no cytotoxicity was observed at any concentration. However, when treated with 20% of Gyechuldung extract, Eucommia ulmoides, and Zinnia pipa extract, cytotoxicity was observed, so the treatment concentration was set to 10% or less.

Test Example 4: Evaluation of anti-inflammatory efficacy of exosomes from Gyehyeol-deung, Du-chong, and Chopi-namul (inhibition of TNF-α expression)

To confirm the anti-inflammatory efficacy of exosomes derived from Gyehyeoldeng and Eucommia ulmoides, their effects on TNF-α expression were evaluated compared to the extracts. Human gingival fibroblasts (HGF-1) were inoculated and cultured in DMEM culture medium containing 10% FBS for 24 hours at 37℃, 5% _CO2 . The medium in each well was removed and replaced with new serum-free medium. Gyehyeoldeng exosomes, Eucommia ulmoides exosomes, Zanthoxylum piperitum exosomes, and Gyehyeoldeng extracts, Eucommia ulmoides extracts, and Zanthoxylum piperitum extracts were administered to each well at different concentrations, and pretreatment was performed for 4 hours. Each well plate was irradiated with 20 mJ/ ^cm2 UVB using a UVB irradiation device (vilber loumet, France). After treating the diluted serum-free medium, the samples were additionally cultured for 24 hours. At this time, PBS was treated as a negative control and 0.01% Allantoin was treated as a positive control. After lysing the cells after cell culture using Ribo Ex TM Total RNA Isolation Solution (GeneAll Biotechnology, Korea) and a scraper, 0.2 mL of chloroform (Sigma-Aldrich, USA) was added and centrifuged (12,000 rpm, 4℃, 30 min). The supernatant containing RNA was separated, and isopropanol (Merck-Millipore, Germany) was added in the same amount as the supernatant, inverted, and centrifuged (12,000 rpm, 4℃, 30 min). The RNA was precipitated, and the supernatant except for the precipitate was discarded. The remaining precipitate was washed by adding 70% ethanol (Merck-Millipore, Germany) to it and centrifuging (12,000 rpm, 4℃, 10 min). After removing the ethanol and drying at room temperature, total RNA was extracted by dissolving in Nuclease-Free Water (Affymetrix, USA). The purity and concentration of RNA were measured at A260/A280 wavelengths using a MaestroNano ^® Micro-volume Spectrophotometer (MN-913, Maestrogen, USA), and the ratio of 260 nm to 280 nm was confirmed to be in the range of 2.0-2.2. cDNA was prepared by adding 1 μg RNA, Oligo dT (Bionics, Korea), dNTP (Takara, Korea), and nuclease free water to a total of 13 μL in a PCR tube, incubating for 5 minutes at 65°C, and then reacting for 50 minutes at 37°C with M-MLV Reverse Transcriptase (Invitrogen, Thermo Fisher Scientific, Waltham, Massachusetts, USA). qRT-PCR was performed to quantitatively analyze the gene expression pattern occurring in each cell by the sample. qRT-PCR was performed by mixing primers, cDNA, 2X SYBR green PCR Master Mix (Applied Biosystems, USA), and HPLC (J. T baker, USA) in a PCR tube (total 20 μL) to create a reaction solution, and PCR was performed using the StepOnePlus Real-Time PCR System (Applied Biosystems, USA).

The PCR primer sequences of the TNF-α gene are shown in Table 3 below, and Figure 4 is a graph showing the results of evaluating the anti-inflammatory efficacy of exosomes derived from Gyehyeoldeung, Eucommia ulmoides, and Chopi tree purified according to the above example by inhibition of TNF-α mRNA expression.

As a result of the test, when treated with Gyehyeol-deung, exosomes, Eucommia exosomes, Zanthoxylum exosomes, and Gyehyeol-deung extracts, Eucommia extracts, and Zanthoxylum extracts, the inflammatory factor TNF-α mRNA decreased in a concentration-dependent manner, and it was confirmed that the inhibition rate increased significantly in the range of exosome treatment concentrations compared to the extract. At this time, when Allantoin 0.01%, which is a positive control, was treated, the fold change value was confirmed to be 1.934.

gene Forward primer (5'→3') Reverse primer (5'→3') TNF-α 5'-CAG GGA CCT CTC TCT AAT CA-3' 5'-AGC TGG TTA TCT CTC AGC TC-3' GAPDH 5'-GTC TCC TCT GAC TTC AAC AGC G-3' 5'-ACC ACC CTG TTG CTG TAG CCA A-3'

Test Example 5: Evaluation of anti-inflammatory efficacy of exosomes from Gyehyeol-deung, Du-chong, and Chopi-namul (inhibition of IL-8 expression)

In order to confirm the anti-inflammatory efficacy of exosomes derived from Gyehyeoldeung, Eucommia ulmoides, and Chopi tree, the effect on IL-8 expression was confirmed by comparing them with the extracts. The experimental method is the same as Test Example 4 above, and the PCR primer sequences of the IL-8 gene are shown in Table 4 below. Figure 5 is a graph showing the results of evaluating the anti-inflammatory efficacy of exosomes derived from Gyehyeoldeung, Eucommia ulmoides, and Chopi tree purified according to the above example by inhibition of IL-8 mRNA expression.

As a result of the test, when treated with Gyehyeol-deung, exosomes, Eucommia exosomes, Zanthoxylum exosomes, and Gyehyeol-deung extracts, Eucommia extracts, and Zanthoxylum extracts, the level of IL-8 mRNA, an inflammatory factor, was decreased in a concentration-dependent manner, and it was confirmed that the inhibition rate was significantly increased in the range of exosome treatment concentrations compared to the extract. At this time, when Allantoin 0.01%, which is a positive control, was treated, the fold change value was confirmed to be 2.645.

gene Forward primer (5'→3') Reverse primer (5'→3') IL-8 5'-GTG AAG GTG CAG TTT TGC CA-3' 5'-TCT CCA CAA CCC TCT GCA C-3' GAPDH 5'-GTC TCC TCT GAC TTC AAC AGC G-3' 5'-ACC ACC CTG TTG CTG TAG CCA A-3'

Test Example 6: Evaluation of cell regeneration efficacy of exosomes derived from Gyehyeol-deung, Eucommia ulmoides, and Chopi tree (Wound healing assay)

In order to confirm the cell regeneration efficacy of exosomes derived from Gyechuldeng, Eucommia ulmoides, and Zanthoxylum pipi, a wound healing assay was performed. When cells are damaged by chemical and physical stimuli, they secrete various chemokines and cytokines and simultaneously migrate to the damaged skin area to induce cell proliferation. Therefore, the cell regeneration induction effect by the sample was confirmed through a wound healing assay that tests the growth and migration of skin cells. Gingival fibroblasts (HGF-1) were seeded in 6-well plates and cultured at 37°C for 24 hours under 5% _CO2 . After culture, the cells were scraped using a 200 ㎕ pipette tip. After scraping, the detached cells were washed repeatedly with PBS buffer and replaced with DMEM medium containing 1.5% bovine serum, and then treated with Gyechuldeng exosomes, Eucommia ulmoides exosomes, Zanthoxylum pipi exosomes, and Gyechuldeng extracts, Eucommia extracts, and Zanthoxylum pipi extracts. Afterwards, the cells were cultured for 18 hours, and the same locations were photographed under a microscope at 0 and 18 hours after the wound was created. The area reduced over the 18 hours was measured using ImageJ (National Institutes of Health, USA), and the average value was calculated.

Fig. 6 is an image showing the results of evaluating the skin regeneration efficacy of purified Gyechuldeung, Eucommia ulmoides, and Chopi tree-derived exosomes according to the above examples using a wound healing assay, and shows the results when Gyechuldeung, Eucommia ulmoides, and Chopi tree exosomes were treated at 1.0E+08 particles/mL and when Gyechuldeung, Eucommia ulmoides, and Chopi tree extracts were treated at 20%. Fig. 7 shows the graph results for the wound area ratio. As a result of the test, when Gyechuldeung-derived exosomes, Eucommia ulmoides-derived exosomes, and Chopi tree-derived exosomes were treated, the wound area was reduced more significantly. Through this, it was confirmed that cell growth and movement increased, which was effective in cell regeneration.

Test Example 7: Evaluation of osteocyte differentiation efficacy of exosomes from Gyehyeoldeung, Eucommia ulmoides, and Chopi tree (increased RUNX2 expression)

To confirm the effect of exosomes derived from Gyechung, Eucommia ulmoides, and Zanthoxylum lucidum on osteocyte differentiation, the expression of Runx2 (runt-related transcription factor2), the most essential transcription factor for osteogenesis, was evaluated by comparing it with the extracts.

Osteoblastic MC3T3-E1 cells were cultured in alpha-MEM (high glucose) medium containing 10% FBS at 37℃ in a 5% CO ₂ incubator. After stabilization for 24 hours, the cells were treated with Gyechuldung exosomes, Eucommia ulmoides exosomes, Zanthoxylum piperitum exosomes, and Gyechuldung extracts, Eucommia ulmoides extracts, and Zanthoxylum piperitum extracts, and 10 mM β-glycerophosphate and 50 ug/ml ascorbic acid were added to induce osteogenic differentiation for 4 days. At this time, 10 mM β-glycerophosphate and 50 ug/ml ascorbic acid were added every time the medium was replaced. PBS was treated as a negative control and 100 nM dexamethasone was treated as a positive control. After lysing the cells after cell culture using Ribo Ex TM Total RNA Isolation Solution (GeneAll Biotechnology, Korea) and a scraper, 0.2 mL of chloroform (Sigma-Aldrich, USA) is added and centrifuged (12,000 rpm, 4℃, 30 min). The supernatant containing RNA is separated, and isopropanol (Merck-Millipore, Germany) is added in the same amount as the supernatant, inverted, and centrifuged (12,000 rpm, 4℃, 30 min). The RNA is precipitated, and the supernatant except for the precipitate is discarded. The remaining precipitate is washed by adding 70% ethanol (Merck-Millipore, Germany) to it and centrifuging (12,000 rpm, 4℃, 10 min). After removing the ethanol and drying at room temperature, total RNA was extracted by dissolving in Nuclease-Free Water (Affymetrix, USA). The purity and concentration of RNA were measured at A260/A280 wavelengths using a MaestroNano ^® Micro-volume Spectrophotometer (MN-913, Maestrogen, USA), and the ratio of 260 nm to 280 nm was confirmed to be in the range of 2.0-2.2. cDNA was prepared by adding 1 μg RNA, Oligo dT (Bionics, Korea), dNTP (Takara, Korea), and nuclease free water to a total of 13 μL in a PCR tube, incubating for 5 minutes at 65°C, and then reacting for 50 minutes at 37°C with M-MLV Reverse Transcriptase (Invitrogen, Thermo Fisher Scientific, Waltham, Massachusetts, USA). qRT-PCR was performed to quantitatively analyze the gene expression pattern occurring in each cell by the sample. qRT-PCR was performed by mixing primers, cDNA, 2X SYBR green PCR Master Mix (Applied Biosystems, USA), and HPLC (J. T baker, USA) in a PCR tube (total 20 μL) to create a reaction solution, and PCR was performed using the StepOnePlus Real-Time PCR System (Applied Biosystems, USA).

The PCR primer sequences of the Runx2 gene are shown in Table 5 below, and Fig. 8 is a graph showing the results of evaluating the effect of purified Gyechuldeung, Eucommia ulmoides, and Zanthoxylum piperitum-derived exosomes on osteocyte differentiation according to the above examples, as measured by the increase in Runx2 mRNA expression. As a result of the test, when Gyechuldeung, exosomes, Eucommia ulmoides exosomes, Zanthoxylum piperitum exosomes, and Gyechuldeung extracts, Eucommia ulmoides extracts, and Zanthoxylum piperitum extracts were treated, Runx2 mRNA increased in a concentration-dependent manner, and it was confirmed that the expression rate increased much more in the range of exosome treatment concentrations compared to the extracts. At this time, when 100 nM dexamethasone, which is a positive control, was treated, the fold change value was confirmed to be 3.877.

gene Forward primer (5'→3') Reverse primer (5'→3') Runx2 5'- TAC AAA CCA TAC CCA GTC CCT GTT T-3' 5'-AGT GCT CTA ACC ACA GTC CAT GCA-3' GAPDH 5'-GTC TCC TCT GAC TTC AAC AGC G-3' 5'-ACC ACC CTG TTG CTG TAG CCA A-3'

Test Example 8: Evaluation of osteocyte differentiation efficacy of exosomes from Gyehyeol-deung, Eucommia ulmoides, and Chopi tree (increased OSX expression)

To confirm the effect of exosomes derived from Gyechung, Eucommia ulmoides, and Zinnia japonica on osteoblast differentiation, the expression of OSX (Osterix), a transcriptional regulatory gene in osteoblast differentiation, was evaluated by comparing it with the extracts. The experimental method was the same as in Test Example 7 above, and the PCR primer sequences of the OSX gene are shown in Table 6 below.

Figure 9 is a graph showing the results of evaluating the effect of purified Gyechuldeung, Eucommia ulmoides, and Zanthoxylum piperitum-derived exosomes on osteocyte differentiation according to the above examples, as measured by the OSX mRNA expression rate. As a result of the test, when Gyechuldeung, exosomes, Eucommia ulmoides exosomes, Zanthoxylum piperitum exosomes, and Gyechuldeung extracts, Eucommia ulmoides extracts, and Zanthoxylum piperitum extracts were treated, OSX mRNA increased in a concentration-dependent manner, and it was confirmed that the expression rate increased much more in the range of exosome treatment concentrations compared to the extracts. At this time, when 100 nM dexamethasone, which is a positive control, was treated, the fold change value was confirmed to be 3.312.

gene Forward primer (5'→3') Reverse primer (5'→3') OSX 5'- TGC TTG AGG AGG AAG TTC AC -3' 5'-AGG TCA CTG CCC ACA GAG TA-3' GAPDH 5'-GTC TCC TCT GAC TTC AAC AGC G-3' 5'-ACC ACC CTG TTG CTG TAG CCA A-3'

Test Example 9: Evaluation of osteocyte differentiation efficacy of exosomes from Gyehyeol-deung, Eucommia ulmoides, and Chopi tree (increased ALP expression)

In order to confirm the effect of exosomes derived from Gyehwol-deung, Eucommia ulmoides, and Zanthoxylum piperitum on osteocyte differentiation, the expression of ALP (Alkaline phosphatase), a bone formation marker, was evaluated by comparing it with the extracts. The experimental method was the same as in Test Example 7 above, and the PCR primer sequences of the ALP gene are shown in Table 7 below.

Figure 10 is a graph showing the results of evaluating the effect of purified exosomes from Gyechuldeung, Eucommia ulmoides, and Zanthoxylum piperitum on osteocyte differentiation according to the above examples, as measured by the ALP mRNA expression rate. As a result of the test, when Gyechuldeung, exosomes, Eucommia ulmoides exosomes, Zanthoxylum piperitum exosomes, and Gyechuldeung extracts, Eucommia ulmoides extracts, and Zanthoxylum piperitum extracts were treated, ALP mRNA increased in a concentration-dependent manner, and it was confirmed that the expression rate increased much more in the range of exosome treatment concentrations compared to the extracts. At this time, when 100 nM dexamethasone, which is a positive control, was treated, the fold change value was confirmed to be 3.319.

gene Forward primer (5'→3') Reverse primer (5'→3') ALP 5'-ACG TGG CTA AGA ATG TCA TC-3' 5'- CTG GTA GGC GAT GTC CTT A-3' GAPDH 5'-GTC TCC TCT GAC TTC AAC AGC G-3' 5'-ACC ACC CTG TTG CTG TAG CCA A-3'

Claims (11)

In a composition for preventing or improving periodontal disease containing exosomes from Gyehyeol-deung, exosomes from Duzhong, or exosomes from Chopi tree as active ingredients,
The exosomes of Gyechul-deung, Eucommia ulmoides, and Zinnia rhizome as the effective ingredients are purified by a method including: (A) a freeze-thawing treatment step of adding a solvent to Gyechul-deung, Eucommia ulmoides, or Zinnia rhizome and repeating freezing and thawing; (B) a step of extracting the juice of Gyechul-deung, Eucommia ulmoides, or Zinnia rhizome that has been freeze-thawing treated; (C) a step of centrifuging the extracted juice at 1,000xg to 10,000xg to obtain a supernatant; (D) a step of lyophilizing the supernatant in which exosomes are present; (E) a step of forming an aqueous two-phase system using PEG (Polyethylene glycol)/Dextran in the lyophilized product; and (F) a step of obtaining a lower layer in which exosomes are concentrated among the aqueous two-phase system.
A composition for preventing or improving periodontal disease, characterized in that the composition has anti-inflammation, cell regeneration, and bone cell differentiation promoting activities. In a composition for treating periodontal disease containing exosomes from Gyehyeol-deung, Eucommia ulmoides exosomes or Zanthoxylum lucidum exosomes as active ingredients,
The exosomes of Gyechul-deung, Eucommia ulmoides, and Zinnia rhizome as the effective ingredients are purified by a method including: (A) a freeze-thawing treatment step of adding a solvent to Gyechul-deung, Eucommia ulmoides, or Zinnia rhizome and repeating freezing and thawing; (B) a step of extracting the juice of Gyechul-deung, Eucommia ulmoides, or Zinnia rhizome that has been freeze-thawing treated; (C) a step of centrifuging the extracted juice at 1,000xg to 10,000xg to obtain a supernatant; (D) a step of lyophilizing the supernatant in which exosomes are present; (E) a step of forming an aqueous two-phase system using PEG (Polyethylene glycol)/Dextran in the lyophilized product; and (F) a step of obtaining a lower layer in which exosomes are concentrated among the aqueous two-phase system.
A composition for treating periodontal disease, characterized in that the composition has anti-inflammation, cell regeneration, and bone cell differentiation promoting activities. A composition for preventing or improving periodontal disease, characterized in that in the first paragraph, the freezing and thawing treatment in step (A) is a process of freezing at a temperature of -80 to -70°C for 15 to 20 hours, and then thawing at a temperature of 40 to 50°C for 8 to 10 hours, repeated 2 to 5 times. delete A composition for preventing or improving periodontal disease, characterized in that in claim 1, the exosomes of Gyehyeol-deung, Eucommia ulmoides, or Chopi tree are contained in an amount of 0.0001 to 30.0% (w/w) based on the total weight of the composition. A composition for treating periodontal disease, characterized in that the composition in claim 2 is a pharmaceutical composition. A composition for preventing or improving periodontal disease, characterized in that in claim 1, the composition is an over-the-counter drug composition. A composition for preventing or improving periodontal disease, characterized in that the composition in claim 1 is a food composition. A composition for preventing or improving periodontal disease, characterized in that the composition in claim 1 is a cosmetic composition. delete delete