WO2021066431A1 - Pomegranate-derived extracellular vesicles and use thereof - Google Patents

Pomegranate-derived extracellular vesicles and use thereof Download PDF

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WO2021066431A1
WO2021066431A1 PCT/KR2020/013143 KR2020013143W WO2021066431A1 WO 2021066431 A1 WO2021066431 A1 WO 2021066431A1 KR 2020013143 W KR2020013143 W KR 2020013143W WO 2021066431 A1 WO2021066431 A1 WO 2021066431A1
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cancer
pomegranate
centrifugation
extracellular vesicles
extracellular
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PCT/KR2020/013143
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French (fr)
Korean (ko)
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백문창
조영은
허승환
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경북대학교 산학협력단
안동대학교 산학협력단
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Priority claimed from KR1020200124689A external-priority patent/KR102447448B1/en
Publication of WO2021066431A1 publication Critical patent/WO2021066431A1/en

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    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23LFOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105Plant extracts, their artificial duplicates or their derivatives
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23LFOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17Amino acids, peptides or proteins
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/33Heterocyclic compounds
    • A61K31/335Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365Lactones
    • A61K31/366Lactones having six-membered rings, e.g. delta-lactones
    • A61K31/37Coumarins, e.g. psoralen
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18Magnoliophyta (angiosperms)
    • A61K36/185Magnoliopsida (dicotyledons)
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • A61K38/16Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43Enzymes; Proenzymes; Derivatives thereof
    • A61K38/45Transferases (2)
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • A61K38/16Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46Hydrolases (3)
    • A61K38/47Hydrolases (3) acting on glycosyl compounds (3.2), e.g. cellulases, lactases
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00Antineoplastic agents

Definitions

  • the present invention relates to extracellular vesicles derived from pomegranate (Punica granatum ) and uses thereof.
  • Cancer is one of the incurable diseases that civilization must solve, and a huge amount of capital is being invested in the development to cure it worldwide.
  • Korea it is the number one cause of death for Koreans since 1983, and is about 10 per year. More than 10,000 people are diagnosed, and about 60,000 people are dying.
  • breast cancer is a cancer with the highest incidence among women, and according to statistics from the Central Cancer Registry in 2015, the number of breast cancer patients in Korea ranks fifth in the incidence rate, accounting for about 7.7% of all cancer-causing patients.
  • the risk factors for developing breast cancer are very diverse, and representatively, there are genetics, hormones, radiation, and the like, and the risk of ovarian cancer and colorectal cancer is higher.
  • Breast cancer is a difficult treatment method at the time of onset and conventional chemotherapy that is treated with anticancer drugs depending on the deterioration of the quality of life after treatment, or surgical treatment such as partial mastectomy (breast-conserving surgery) and total mastectomy, and high-energy radiation.
  • surgical treatment such as partial mastectomy (breast-conserving surgery) and total mastectomy, and high-energy radiation.
  • partial mastectomy breast-conserving surgery
  • total mastectomy total mastectomy
  • high-energy radiation high-energy radiation
  • Extracellular vesicles (Extracellular Vesicles, EVs) is also referred to as a substance appearing during cell activity, nano-vesicles with a size of 1m 10 9 minutes.
  • Extracellular vesicles are divided into three groups: exosomes, microvesicles, and apoptotic bodies.
  • the origin of exosomes is the endocytic pathway, the size of which is 30-100 nm, and can be observed in body fluids such as blood and urine of multicellular eukaryotic organisms and in the culture medium of eukaryotic cells cultured.
  • the origin of microvesicles is the plasma membrane, which is 50-1,000 nm in size.
  • the origin of apoptosis is also the plasma membrane, and the size is 500-2,000 nm.
  • Various clinical studies such as diagnosis, prognosis, and treatment of diseases are being conducted through these nanovesicles.
  • nanovesicles are secreted from food, and the secreted nanovesicles are known to be safely used as drug mediators as well as a physiologically active role.
  • the anticancer effect of nanovesicles secreted from pomegranate has not yet been reported.
  • An object of the present invention is to provide a natural product-derived extracellular vesicle.
  • Another object of the present invention is to provide an anticancer composition comprising the extracellular vesicles.
  • Another object of the present invention is to provide a method for preparing the extracellular vesicles.
  • the present invention provides an extracellular vesicle having anticancer activity derived from pomegranate (Puncha granatum).
  • the present invention provides a pharmaceutical composition for preventing or treating cancer comprising the extracellular vesicles as an active ingredient.
  • the present invention provides a health functional food composition for preventing or improving cancer comprising the extracellular vesicles as an active ingredient.
  • the present invention comprises the steps of preparing a pomegranate pulverized product; First centrifuging the prepared pomegranate crushed product at 250 to 750 g for 5 to 15 minutes; Second centrifuging the supernatant obtained by the first centrifugation at 1,000 to 3,000 g for 10 to 30 minutes; Third centrifuging the supernatant obtained by the second centrifugation at 5,000 to 15,000 g for 15 to 45 minutes; And it provides a method for producing extracellular vesicles derived from pomegranate (Punica granatum ) containing; and the step of obtaining a pellet by performing a fourth centrifugation for 1 to 3 hours at 50,000 to 150,000 g of the supernatant obtained by the third centrifugation.
  • Pomegranate-derived extracellular vesicles according to the present invention can penetrate into cancer cells and have excellent anticancer activity, such as effectively inhibiting proliferation, invasion, and migration of cancer cells. Accordingly, the pomegranate-derived extracellular vesicles may be provided as a pharmaceutical composition or a health functional food composition for preventing, improving or treating cancer diseases.
  • pomegranate-derived extracellular vesicles according to the present invention, ingestion and storage are easier, and even if a large amount of pomegranate is not consumed, there is an advantage of exhibiting an excellent anticancer effect.
  • FIG. 1 schematically shows a method of separating nanovesicles from pomegranate.
  • FIG. 2 is a graph analyzing the size of pomegranate nanovesicles according to an experimental example of the present invention.
  • FIG. 3 is a graph showing the proliferation rate of breast cancer cells according to the concentration of pomegranate nanovesicles according to an experimental example of the present invention.
  • FIG. 4 is a graph showing the effect on the invasion of breast cancer cells according to the concentration of pomegranate nanovesicles according to an experimental example of the present invention.
  • FIG. 5 shows the degree of migration of breast cancer cells according to the concentration of pomegranate nanovesicles according to an experimental example of the present invention.
  • FIG. 6 is an image showing the penetration of pomegranate nanovesicles into breast cancer cells according to an experimental example of the present invention.
  • FIG. 7 is a graph analyzing the content of ellagic acid in pomegranate nanovesicles according to an experimental example of the present invention.
  • the present inventors have completed the present invention by confirming that the nanovesicles isolated from pomegranate effectively inhibit the proliferation, invasion, and migration of breast cancer cells.
  • the present invention provides an extracellular vesicle having anticancer activity.
  • the extracellular endoplasmic reticulum may be derived from pomegranate (Punica granatum ).
  • pomegranate is a fruit of a pomegranate tree, 6-8cm in diameter, and a hard yellowish bark is wrapped around it, and there are many seeds in the flesh.
  • the skin contains tannins, and the seeds contain natural vegetable estrogens that are good for menopausal disorders, and both fruits and skins are good for preventing hypertension and arteriosclerosis, and are known to be effective against gynecological diseases and rash.
  • the pomegranate may be a pomegranate skin, flesh, seeds, or a mixed portion thereof, and preferably may be a pulp or a mixed portion thereof, but is not limited thereto.
  • extracellular vesicle refers to a small vesicle having a membrane structure secreted from various cells, and refers to vesicles released into the extracellular environment due to the fusion of the polycystic body and the plasma membrane, and nanovesicles It can also be defined as (nanovesicle).
  • the extracellular vesicles may include exosomes and microvesicles, but are not limited thereto.
  • the extracellular vesicles may be nanovesicles having an average particle diameter of 50 to 300 nm, and preferably, an average particle diameter of 100 to 200 nm, but are not limited thereto.
  • the extracellular vesicles are ultracentrifugation, density gradient centrifugation, ultrafiltration, size exclusion chromatography, ion exchange chromatography. exchange chromatography), immunoaffinity capture, microfluidics-based isolation, exosome precipitation, total exosome isolation kit, or polymer based precipitation ) May be separated from the pomegranate using a method such as, but is not limited thereto.
  • the extracellular vesicles may be obtained by centrifuging pomegranate, and more preferably, the extracellular vesicles are first centrifuged for 5 to 15 minutes at 250 to 750 g of pomegranate crushed product, and the first The supernatant obtained by centrifugation was subjected to a second centrifugation at 1,000 to 3,000 g for 10 to 30 minutes, and the supernatant obtained by the second centrifugation was subjected to a third centrifugation at 5,000 to 15,000 g for 15 to 45 minutes, and the The supernatant obtained by the third centrifugation was obtained by the fourth centrifugation at 50,000 to 150,000 g for 1 to 3 hours, and may be a pellet excluding the supernatant from the centrifuge obtained by the fourth centrifugation. It is not limited.
  • the pomegranate crushed product may be pulverized by putting pomegranate in a phosphate buffer solution (PBS), but is not limited thereto, and may be directly prepared as a pomegranate juice or extract, or commercially sold pomegranate crushed product, pomegranate
  • PBS phosphate buffer solution
  • the juice or extract may have been purchased.
  • the extracellular vesicle obtained according to the above is composed of ellagic acid, class III chitinase, lipid transfer protein, and chalcone synthase.
  • One or two or more selected from the group may be included, but the present invention is not limited thereto.
  • elagic acid is a natural vegetable phenol found in fruits or vegetables, and exists in the form of ellagitannin, a precursor, in plants. It has been reported to have antioxidant, antiviral, antimutagenic, and anticancer functions.
  • ellagic acid which is a major component of pomegranate, is contained in the pomegranate-derived extracellular vesicle, and it can be determined as a component that affects the anticancer action of the pomegranate-derived extracellular vesicle.
  • the extracellular endoplasmic reticulum may penetrate into cancer cells and may have anti-cancer activity to inhibit proliferation, migration or invasion of cancer cells.
  • the extracellular vesicles may be provided as a pharmaceutical composition or a health functional food composition for preventing or treating cancer.
  • the present invention provides a pharmaceutical composition for preventing or treating cancer comprising the pomegranate-derived extracellular vesicles as an active ingredient.
  • the composition may contain 0.001 to 50 parts by weight of the extracellular vesicles, but is not limited thereto.
  • the composition can prevent or treat cancer by penetrating into cancer cells and inhibiting proliferation, migration, or invasion of cancer cells.
  • the cancer is breast cancer, lung cancer, liver cancer, stomach cancer, colon cancer, kidney cancer, bladder cancer, acute myelogenous leukemia, acute lymphocytic leukemia, uterine cancer, ovarian cancer, laryngeal cancer, prostate cancer, thyroid cancer, head or neck cancer, brain cancer and blood cancer. It may be a disease selected from the group, but is not limited thereto.
  • the pharmaceutical composition according to the present invention can be prepared according to a conventional method in the pharmaceutical field.
  • the pharmaceutical composition may be blended with an appropriate pharmaceutically acceptable carrier according to the dosage form, and if necessary, may be prepared by further including excipients, diluents, dispersants, emulsifiers, buffers, stabilizers, binders, disintegrants, solvents, etc. have.
  • the appropriate carrier or the like is one that does not inhibit the activity and properties of the pomegranate-derived extracellular vesicles according to the present invention, and may be selected differently depending on the dosage form and formulation.
  • Carriers, excipients, diluents, etc. that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, Methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate and mineral oil.
  • diluents or excipients such as generally used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.
  • the pharmaceutical composition according to the present invention can be applied in any dosage form, and in detail, oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups and aerosols, external preparations, suppositories and sterilization according to a conventional method. It may be formulated and used in the form of a solution for injection, and preferably may be formulated and used in a unit dosage form suitable for oral administration.
  • oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups and aerosols, external preparations, suppositories and sterilization according to a conventional method. It may be formulated and used in the form of a solution for injection, and preferably may be formulated and used in a unit dosage form suitable for oral administration.
  • the solid dosage form of the oral dosage form is in the form of tablets, pills, powders, granules, capsules, etc.
  • at least one excipient such as starch, calcium carbonate, sucrose, lactose, sorbitol, mannitol. , Cellulose, gelatin, etc. can be mixed to prepare, and in addition to simple excipients, lubricants such as magnesium stearate and talc may also be included.
  • a liquid carrier such as fatty oil may be further included.
  • liquid dosage forms correspond to suspensions, liquid solutions, emulsions, syrups, etc., and various excipients, such as wetting agents, sweetening agents, fragrances, preservatives, etc., may be included in addition to water and liquid paraffin, which are commonly used simple diluents. have.
  • the parenteral formulation may include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, injections, freeze-dried preparations, suppositories, and the like.
  • non-aqueous solvent and suspension propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like may be used.
  • a base for suppositories witepsol, macrogol, Tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used.
  • the present invention is not limited thereto, and any suitable agent well known in the art may be used.
  • the pharmaceutical composition according to the present invention may further add an antioxidant to enhance therapeutic efficacy.
  • the antioxidants include thiamine (vitamin B1), riboflavin (vitamin B2), niacin (vitamin B3), pantothenic acid (vitamin B5), pyridoxine (vitamin B6), and cobalamin.
  • Vitamin B group compounds such as vitamin B12) and vitamin C, vitamin D, vitamin E, and the like may be used, but are not limited thereto, and all suitable agents well known in the art may be used.
  • the pharmaceutical composition according to the present invention may be administered in a pharmaceutically effective amount.
  • a pharmaceutically effective amount refers to an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment and not cause side effects.
  • the effective dosage level of the pharmaceutical composition is the purpose of use, the age, sex, weight and health condition of the patient, the type of disease, the severity, the activity of the drug, the sensitivity to the drug, the method of administration, the administration time, the administration route and rate of excretion, It can be determined differently depending on the duration, factors including the combination or co-used drugs and other factors well known in the medical field. For example, although not constant, generally 0.001 to 100 mg/kg, preferably 0.01 to 10 mg/kg may be administered once to several times a day. The above dosage does not limit the scope of the present invention in any way.
  • the pharmaceutical composition according to the present invention may be administered to any animal that may cause cancer disease, and the animal may include, for example, humans and primates as well as livestock such as cattle, pigs, horses, and dogs. .
  • the pharmaceutical composition may be administered by an appropriate route of administration according to the form of the formulation, and may be administered through various routes, such as oral or parenteral, as long as it can reach the target tissue.
  • the method of administration is not particularly limited, and may be administered by conventional methods such as, for example, oral, rectal or intravenous, intramuscular, skin application, inhalation in the respiratory tract, intrauterine dura mater or intracere-broventricular injection. have.
  • the pharmaceutical composition according to the present invention may be used alone for the prevention or treatment of cancer diseases, or may be used in combination with surgery or other drug treatment.
  • the present invention provides a health functional food composition for preventing or improving cancer comprising the pomegranate-derived extracellular vesicles as an active ingredient.
  • the composition can prevent or improve cancer by penetrating into cancer cells and inhibiting proliferation, migration, or invasion of cancer cells.
  • the cancer is breast cancer, lung cancer, liver cancer, stomach cancer, colon cancer, kidney cancer, bladder cancer, acute myelogenous leukemia, acute lymphocytic leukemia, uterine cancer, ovarian cancer, laryngeal cancer, prostate cancer, thyroid cancer, head or neck cancer, brain cancer and blood cancer. It may be a disease selected from the group, but is not limited thereto.
  • health functional food includes foods manufactured and processed using raw materials or ingredients having functions useful for the human body according to the Health Functional Food Act No. It refers to foods with high medical effects and medicines processed to efficiently exhibit biomodulatory functions such as prevention, improvement, body defense, immunity, and recovery.
  • the health functional food according to the present invention may be prepared as a powder, granule, tablet, capsule, syrup or beverage for the purpose of preventing or improving cancer.
  • the health functional food can take, and it can be formulated in the same manner as the pharmaceutical composition to be used as a functional food or added to various foods.
  • the health functional food may include all foods in a conventional sense. For example, beverages and various drinks, fruits and processed foods thereof (canned fruit, jam, etc.), fish, meat and processed foods thereof (ham, bacon, etc.), bread and noodles, cookies and snacks, dairy products (butter, cheese, etc.) ) And the like, and may include all functional foods in the usual sense. In addition, foods used as feed for animals may also be included.
  • the health functional food composition according to the present invention may be prepared by further comprising a food pharmaceutically acceptable food additive (food additive) and other appropriate auxiliary ingredients commonly used in the art.
  • a food pharmaceutically acceptable food additive food additive
  • other appropriate auxiliary ingredients commonly used in the art.
  • the suitability as a food additive may be determined according to the standards and standards for the relevant item in accordance with the general rules and general test methods of the Food Additive Code approved by the Ministry of Food and Drug Safety.
  • Examples of the items listed in the'Food Additives Code' include chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; Natural additives such as dark pigment, licorice extract, crystalline cellulose, high color pigment, and guar gum; Mixed preparations, such as a sodium L-glutamate preparation, an alkali additive for noodles, a preservative preparation, and a tar color preparation, etc. are mentioned.
  • the other auxiliary ingredients are, for example, flavoring agents, natural carbohydrates, sweetening agents, vitamins, electrolytes, colorants, pectic acids, alginic acids, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents, etc. It may further contain.
  • natural carbohydrates monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin
  • sugar alcohols such as xylitol, sorbitol, and erythritol
  • sweetener natural sweeteners such as taumatin and stevia extract, or synthetic sweeteners such as saccharin and aspartame may be used.
  • the effective dose of the extracellular vesicles contained in the health functional food according to the present invention may be appropriately adjusted according to the purpose of use, such as prevention or improvement of cancer.
  • the health functional food composition has the advantage of not having side effects that may occur during long-term use of general drugs by using food as a raw material, has excellent portability, and can be taken as an adjuvant for preventing or improving cancer.
  • the present invention provides a method for producing pomegranate-derived extracellular vesicles.
  • the manufacturing method comprises the steps of preparing a pomegranate crushed product; First centrifuging the prepared pomegranate crushed product at 250 to 750 g for 5 to 15 minutes; Second centrifuging the supernatant obtained by the first centrifugation at 1,000 to 3,000 g for 10 to 30 minutes; Third centrifuging the supernatant obtained by the second centrifugation at 5,000 to 15,000 g for 15 to 45 minutes; And obtaining a pellet by performing a fourth centrifugation for 1 to 3 hours at 50,000 to 150,000 g of the supernatant obtained by the third centrifugation.
  • the step of preparing the pomegranate pulverized product may be performed by washing the pomegranate and then pulverizing or juice by putting it in a phosphoric acid buffer solution (PBS).
  • PBS phosphoric acid buffer solution
  • the first to fourth centrifugation may be performed once or repeatedly performed two or more times, and in particular, the third centrifugation step may be performed once or repeated two to five times. However, it is not limited thereto.
  • the manufacturing method may further include one or more centrifugation steps.
  • the first centrifugation step is performed once for 10 minutes at 500 g
  • the second centrifugation step is 20 at 2,000 g of the supernatant obtained in the first centrifugation step. It is performed once for a minute
  • the third centrifugation step is performed twice for 30 minutes at 10,000 g of the supernatant obtained in the second centrifugation step
  • the fourth centrifugation step is performed in the third centrifugation step.
  • the obtained supernatant was performed once for 2 hours at 100,000 g, thereby preparing pomegranate-derived extracellular vesicles.
  • the manufacturing method includes ultrafiltration, size exclusion chromatography, ion exchange chromatography, immunoaffinity capture, microfluidic Technology separation method (microfluidics-based isolation), precipitation method (exosome precipitation), total exosome extraction kit (total exosome isolation kit), or polymer-based precipitation method (polymer based precipitation), etc. can be used, but is not limited to this technology Separation or purification methods well known in the art may be further included.
  • the pomegranate used in the experiment of the present invention was Georgia Natural Pomegranate Juice (manufactured by AROMAPRODUCT LTD), and a juice obtained by extracting 100% of pomegranate excluding pomegranate seeds was used.
  • 1 schematically shows a method of separating nanovesicles from pomegranate, referring to this, after adjusting the weight to a constant, spin once for 10 minutes at 500 g using a centrifuge, and then add a new 50 mL of the supernatant. Transferred to a conical tube and returned once for 20 minutes at 2,000 g, and the supernatant was transferred to a new 50 mL conical tube and rotated twice at 10,000 g for 30 minutes, and then the supernatant was added to a new tube and returned to 100,000 g. After returning for 2 hours, the supernatant was discarded, and the remaining nanovesicle pellet was dried, and then 1 mL of 1 ⁇ PBS was added to dissolve it.
  • Nanovesicles Extracellular Vesicles, EVs isolated from pomegranate.
  • Nanoparticle tracking analysis measured the size and concentration of specific nanovesicles in the range of 50 nm to 1000 nm in diameter in a liquid suspension.
  • Pomegranate nanovesicle protein was crushed with 1 ⁇ RIPA solution, and then the protein was quantified by bicinchoninic acid (BCA, Termo Fishers) method.
  • BCA bicinchoninic acid
  • MDA-MB-231 breast cancer cells were distributed from the ATCC cell line. MDA-MB-231 breast cancer cells were cultured in an incubator at 5% CO 2 and 37° C. by adding DMEM medium, 10% fetal bovine serum and 1% antibiotics for culture. Cells were exchanged for media every 2-3 days.
  • MDA-MB-231 breast cancer cells were placed in a 96-well plate so that the number of cells per well was 2.5 ⁇ 10 4 cells/mL, and cultured in DMEM growth medium for about 24 hours to adhere to the plate. Then, the growth medium was removed and the pomegranate nanovesicles were treated at concentrations of 0, 10, 50, and 100 ⁇ g/mL. Incubated for about 24 hours in an incubator maintained at 37°C and 5% CO 2. Then, a solution in which 2 mg/mL of MTT was dissolved in 40 ⁇ L of PBS per well was added and left in an incubator at 37° C. for 2 hours.
  • MDA-MB-231 breast cancer cells 1 mL of MDA-MB-231 breast cancer cells were added to 6 wells and cultured in an incubator at 5% CO 2 and 37°C. After the cells were wound, the healing of the cells was observed under a microscope.
  • 500 ⁇ L of a medium containing 10% FBS was treated in the side chamber to create an environment rich in nutrients that induces cell penetration in the outer chamber of the membrane.
  • MDA-MB-231 breast cancer cells were treated with vascular endothelial growth factor (VEGF), which is a positive control, at a concentration of 100 ng/mL, and pomegranate nanovesicles were treated by concentration, followed by incubation at 37°C for 24 hours. Thereafter, the cells that did not penetrate were removed, and then the cells outside the membrane that had penetrated with a cell stain solution were stained. The stained cells were counted.
  • VEGF vascular endothelial growth factor
  • MDA-MB-231 breast cancer cells were spread on an 8-well chamber slide at 1 ⁇ 10 4 cells/well and cultured in DMEM growth medium for about 24 hours. Thereafter, DID-labeled pomegranate nanovesicles were treated in each well, and then the medium was removed after 24 hours, washed with PBS, and then treated with 70% ethanol. After DAPI (4′,6-diamidino-2-phenylindol) staining was finished, it was confirmed that the pomegranate nanovesicles had penetrated into the cells under a fluorescence microscope.
  • Pomegranate nanovesicles were treated with trypsin buffer (500 ng/ ⁇ L trypsin, 50 mM ammonium bicarbonate) and reacted at 37° C. for 16 hours. After that, 5% formic acid was added to stop the action of trypsin, and then the peptide was extracted using 25 mM TEABC (triethylammonium bicarbonate) and ACN (acetonitrile). The extracted peptides were completely dried using a vacuum dryer, dissolved in A buffer (0.1% formic acid) immediately before analysis, and analyzed by mass spectrometry. Data obtained by mass spectrometry were converted into a peak list (mgf file) using Mascot Distiller. The mgf file was identified using the Mascot (Matrix Science; version 2.2.1) program.
  • Pomegranate nanovesicles were diluted 1:1 with ethanol, ultrasonically extracted for 10 minutes, centrifuged at 15,000 rpm, filtered through a 0.2 ⁇ m syringe filter, and analyzed with the Agilent 1100 Series.
  • results were obtained through three or more repeated experiments and expressed as the mean ⁇ standard deviation for each sample concentration.
  • the significance difference test for each sample concentration group was compared with the control group, and a p ⁇ 0.05 value was considered as a statistically significant result after the Student's t test.
  • NTA nanoparticle tracking analysis
  • FIG. 3 is a graph showing the proliferation rate of breast cancer cells according to the concentration of pomegranate nanovesicles according to an experimental example of the present invention.
  • pomegranate nanovesicles were respectively 0, 10, 50, and 100 ⁇ g.
  • the cell viability at each concentration was 100, 86, 80, and 77%, showing a significant decrease in cell survival from a concentration of 10 ⁇ g compared to the control group in a concentration-dependent manner.
  • pomegranate nanovesicles induce apoptosis in a concentration-dependent manner of 10 ⁇ g in MDA-MB-231 breast cancer cells, and this is the same result that pomegranate nano vesicles decrease cell viability in a concentration-dependent manner in breast cancer cells. From the results, it can be determined that the pomegranate nanovesicles have an inhibitory effect on cancer cells.
  • FIG. 4 is a graph showing the effect on the invasion of breast cancer cells according to the concentration of pomegranate nano vesicles according to an experimental example of the present invention.
  • pomegranate nano vesicles are treated with 0, 10, 50, and 100 ⁇ g, respectively, for 24 hours. When doing so, it was found that the invasion of MDA-MB-231 breast cancer cells decreased in a concentration-dependent manner.
  • the pomegranate nanovesicles inhibit cell invasion in a concentration-dependent manner on MDA-MB-231 breast cancer cells, and through these results, it can be determined that the pomegranate nanovesicles exhibit an inhibitory effect on cancer cells.
  • FIG. 5 shows the degree of migration of breast cancer cells according to the concentration of pomegranate nanovesicles according to an experimental example of the present invention. Referring to this, when pomegranate nanovesicles were treated at 0, 10, and 100 ⁇ g for 24 hours, respectively, concentration-dependent As a result, the migration of MDA-MB-231 breast cancer cells was decreased.
  • the pomegranate nanovesicles inhibit cell migration in a concentration-dependent manner on MDA-MB-231 breast cancer cells, and through these results, it can be determined that the pomegranate nanovesicles exhibit an inhibitory effect on cancer cells.
  • FIG. 6 is an image showing that pomegranate nanovesicles infiltrate into breast cancer cells according to an experimental example of the present invention. Referring to this, when treated for 24 hours after DID labeling of pomegranate nano vesicles, MIA-MB-231 breast cancer cells It was confirmed to migrate to the cytoplasm.
  • the pomegranate nanovesicles have an inhibitory effect on the cancer cells through the penetration of the pomegranate nanovesicles into the MBA-MD-231 breast cancer cells.
  • chitinase protein is an enzyme that breaks down the ⁇ -1,4-glycosidic bond of chitin, and provides metal storage capacity in pomegranate. It is known to provide. It was confirmed that this enzyme is present in nanovesicles, and also a lipid transfer protein that transfers fat proteins was identified. Chalcone synthase exists in many plants and is known to play a role in flavonoid biosynthesis. These proteins were found to be present in pomegranate nanovesicles.
  • FIG. 7 is a graph analyzing the content of ellagic acid in the pomegranate nanovesicles according to an experimental example of the present invention.
  • the acid was contained, and it could be confirmed that the three pomegranate nanovesicle samples contained 61.5502, 62.05329 and 61.61564 mg/L of ellagic acid, respectively. It was found that ellagic acid, a major component of pomegranate, is also contained in nanovesicles, and it is considered a very important discovery.

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Abstract

The present invention relates to extracellular vesicles derived from pomegranate (Punica granatum) and a use thereof, and provides: extracellular vesicles which are derived from pomegranate and have anticancer activity; a pharmaceutical composition which is for preventing or treating cancer and contains the extracellular vesicles as an active ingredient; and a health functional food composition. The pomegranate-derived extracellular vesicles can penetrate into cancer cells, have excellent anticancer activity, such as that of effectively suppressing the proliferation, infiltration, migration, etc. of cancer cells, and thus may be provided as an effective anticancer composition.

Description

석류 유래 세포외 소포체 및 이의 용도Pomegranate-derived extracellular vesicles and uses thereof
본 발명은 석류(Punica granatum)에서 유래된 세포외 소포체 및 이의 용도에 관한 것이다.The present invention relates to extracellular vesicles derived from pomegranate (Punica granatum ) and uses thereof.
암은 인류가 해결해야 할 난치병 중의 하나로, 전 세계적으로 이를 치유하기 위한 개발에 막대한 자본이 투자되고 있는 실정이며, 우리나라의 경우, 1983년 이후로 한국인의 사망원인 중 1위의 질병으로 연간 약 10만명 이상이 진단되고 있으며, 약 6만 명 이상이 사망하고 있다.Cancer is one of the incurable diseases that humanity must solve, and a huge amount of capital is being invested in the development to cure it worldwide. In the case of Korea, it is the number one cause of death for Koreans since 1983, and is about 10 per year. More than 10,000 people are diagnosed, and about 60,000 people are dying.
특히, 유방암은 여성에게 발병률이 가장 높은 암으로, 2015년 중앙암등록본부의 통계에 의하면 국내 유방암 환자 수는 전체 암 발생 환자의 약 7.7%로 발병률 5위를 차지한다. 유방암을 발생시키는 위험 요인은 매우 다양하며, 대표적으로 유전, 호르몬, 방사선 등이 있고 난소암 대장암 경험자일수록 위험성이 높아진다.In particular, breast cancer is a cancer with the highest incidence among women, and according to statistics from the Central Cancer Registry in 2015, the number of breast cancer patients in Korea ranks fifth in the incidence rate, accounting for about 7.7% of all cancer-causing patients. The risk factors for developing breast cancer are very diverse, and representatively, there are genetics, hormones, radiation, and the like, and the risk of ovarian cancer and colorectal cancer is higher.
최근 의학적 발달로 수술적 치료, 방사선 치료, 항암 치료 등 방법이 다양하게 향상되어 유방암의 5년 상대 생존율이 92.7%로 생존율이 높음에도 불구하고 환자들은 재발에 대한 두려움과 치료과정에서 겪는 신체적ㆍ사회 심리적 증상들로 인해 삶의 질이 저하될 수 있다.With recent medical developments, various methods such as surgical treatment, radiation therapy, and chemotherapy have improved, and the 5-year relative survival rate of breast cancer is 92.7%. Despite the high survival rate, patients are afraid of recurrence and the physical and social experiences of the treatment process. Psychological symptoms can reduce your quality of life.
유방암은 발병했을 때의 힘든 치료방법과 치료 후의 삶의 질의 저하 따라 항암제를 통해 치료하는 기존의 화학요법과 또는 유방부분절제술(유방보존술)과 유방전절제술과 같은 수술적 치료, 고 에너지의 방사선을 이용하여 종양을 치료하는 방서선 치료와 달리 부작용이 적으면서 인체에 미치는 고통을 최소화하는 암 예방 및 치료 요법을 개발하는 데 사람들의 관심이 증가되었다. 그에 따라 자연에서 쉽게 얻을 수 있고 부작용은 적고 암을 효율적으로 치료할 수 있는, 식물 유래의 천연 물질들에 많은 관심을 가지게 되었다. Breast cancer is a difficult treatment method at the time of onset and conventional chemotherapy that is treated with anticancer drugs depending on the deterioration of the quality of life after treatment, or surgical treatment such as partial mastectomy (breast-conserving surgery) and total mastectomy, and high-energy radiation. Unlike Bangseosun therapy, which treats tumors by using a method, people's interest has increased in developing a cancer prevention and treatment regimen that minimizes pain to the human body with fewer side effects. Accordingly, a lot of attention has been paid to plant-derived natural substances that can be easily obtained from nature, have few side effects, and can effectively treat cancer.
세포외 소포체(Extracellular Vesicles, EVs)는 세포 활동 중에 나오는 물질로, 109분의 1m 크기를 가진 나노소포체라고도 한다. 세포외 소포체는 엑소좀(exosomes), 미세소포(microvesicles), 세포 자살체(apoptotic bodies)로 3가지 군으로 구분된다. 엑소좀의 기원은 내포작용 경로(endocytic pathway)로, 크기는 30-100 nm이며, 다세포 진핵 생명체의 혈액, 오줌 등 체액과 진핵세포를 배양한 배양액에서 관찰할 수 있다. 미세소포의 기원은 원형질막(plasma membrane)으로, 크기는 50-1,000 nm이다. 세포 자살체의 기원도 원형질막이며, 크기는 500-2,000 nm 크기이다. 이러한 나노소포체를 통해 질병의 진단, 예후, 치료 등 다양한 임상 연구가 진행되고 있다.The extracellular vesicles (Extracellular Vesicles, EVs) is also referred to as a substance appearing during cell activity, nano-vesicles with a size of 1m 10 9 minutes. Extracellular vesicles are divided into three groups: exosomes, microvesicles, and apoptotic bodies. The origin of exosomes is the endocytic pathway, the size of which is 30-100 nm, and can be observed in body fluids such as blood and urine of multicellular eukaryotic organisms and in the culture medium of eukaryotic cells cultured. The origin of microvesicles is the plasma membrane, which is 50-1,000 nm in size. The origin of apoptosis is also the plasma membrane, and the size is 500-2,000 nm. Various clinical studies such as diagnosis, prognosis, and treatment of diseases are being conducted through these nanovesicles.
최근에는 식품에서 나노소포체들이 분비된다는 보고가 있고, 이때 분비되는 나노소포체는 생리 활성 역할뿐만 아니라 약물 매개체로 안전하게 사용된다고 알려져 있다. 하지만, 석류에서 분비되는 나노소포체의 항암 효과에 대해서는 아직 보고된 바 없다.Recently, there has been a report that nanovesicles are secreted from food, and the secreted nanovesicles are known to be safely used as drug mediators as well as a physiologically active role. However, the anticancer effect of nanovesicles secreted from pomegranate has not yet been reported.
본 발명의 목적은 천연물 유래 세포외 소포체를 제공하는 데에 있다.An object of the present invention is to provide a natural product-derived extracellular vesicle.
본 발명의 다른 목적은 상기 세포외 소포체를 포함하는 항암용 조성물을 제공하는 데에 있다.Another object of the present invention is to provide an anticancer composition comprising the extracellular vesicles.
본 발명의 또 다른 목적은 상기 세포외 소포체의 제조방법을 제공하는 데에 있다.Another object of the present invention is to provide a method for preparing the extracellular vesicles.
상기의 목적을 달성하기 위하여, 본 발명은 석류(Punica granatum)로부터 유래된, 항암 활성을 가지는 세포외 소포체(extracellular vesicle)를 제공한다.In order to achieve the above object, the present invention provides an extracellular vesicle having anticancer activity derived from pomegranate (Puncha granatum).
본 발명은 상기의 세포외 소포체를 유효성분으로 포함하는 암 예방 또는 치료용 약학 조성물을 제공한다.The present invention provides a pharmaceutical composition for preventing or treating cancer comprising the extracellular vesicles as an active ingredient.
본 발명은 상기의 세포외 소포체를 유효성분으로 포함하는 암 예방 또는 개선용 건강기능식품 조성물을 제공한다.The present invention provides a health functional food composition for preventing or improving cancer comprising the extracellular vesicles as an active ingredient.
또한, 본 발명은 석류 분쇄물을 제조하는 단계; 상기 제조된 석류 분쇄물을 250 내지 750 g에서 5 내지 15분간 1차 원심분리하는 단계; 상기 1차 원심분리하여 수득한 상층액을 1,000 내지 3,000 g에서 10 내지 30분간 2차 원심분리하는 단계; 상기 2차 원심분리하여 수득한 상층액을 5,000 내지 15,000 g에서 15 내지 45분간 3차 원심분리하는 단계; 및 상기 3차 원심분리하여 수득한 상층액을 50,000 내지 150,000 g에서 1 내지 3시간 4차 원심분리하여 펠렛을 수득하는 단계;를 포함하는 석류(Punica granatum) 유래 세포외 소포체 제조방법을 제공한다.In addition, the present invention comprises the steps of preparing a pomegranate pulverized product; First centrifuging the prepared pomegranate crushed product at 250 to 750 g for 5 to 15 minutes; Second centrifuging the supernatant obtained by the first centrifugation at 1,000 to 3,000 g for 10 to 30 minutes; Third centrifuging the supernatant obtained by the second centrifugation at 5,000 to 15,000 g for 15 to 45 minutes; And it provides a method for producing extracellular vesicles derived from pomegranate (Punica granatum ) containing; and the step of obtaining a pellet by performing a fourth centrifugation for 1 to 3 hours at 50,000 to 150,000 g of the supernatant obtained by the third centrifugation.
본 발명에 따른 석류 유래 세포외 소포체는 암 세포 내로 침투가 가능하고 암 세포의 증식, 침윤, 이동 등을 효과적으로 억제하는 등 우수한 항암 활성을 가진다. 이에, 상기 석류 유래 세포외 소포체를 암 질환 예방, 개선 또는 치료를 위한 약학 조성물 또는 건강기능식품 조성물로 제공할 수 있다.Pomegranate-derived extracellular vesicles according to the present invention can penetrate into cancer cells and have excellent anticancer activity, such as effectively inhibiting proliferation, invasion, and migration of cancer cells. Accordingly, the pomegranate-derived extracellular vesicles may be provided as a pharmaceutical composition or a health functional food composition for preventing, improving or treating cancer diseases.
또한, 본 발명에 따른 석류 유래 세포외 소포체를 이용함으로써, 섭취 및 보관이 보다 용이하며, 많은 양의 석류를 섭취하지 않더라도 우수한 항암 효과를 나타낼 수 있는 장점이 있다.In addition, by using the pomegranate-derived extracellular vesicles according to the present invention, ingestion and storage are easier, and even if a large amount of pomegranate is not consumed, there is an advantage of exhibiting an excellent anticancer effect.
도 1은 석류에서 나노소포체를 분리하는 방법을 개략적으로 나타낸 것이다. 1 schematically shows a method of separating nanovesicles from pomegranate.
도 2는 본 발명의 일 실험예에 따른 석류 나노소포체의 크기를 분석한 그래프이다. 2 is a graph analyzing the size of pomegranate nanovesicles according to an experimental example of the present invention.
도 3은 본 발명의 일 실험예에 따른 석류 나노소포체의 농도에 따른 유방암 세포의 증식률을 나타낸 그래프이다. 3 is a graph showing the proliferation rate of breast cancer cells according to the concentration of pomegranate nanovesicles according to an experimental example of the present invention.
도 4는 본 발명의 일 실험예에 따른 석류 나노소포체의 농도에 따른 유방암 세포의 침윤에 미치는 영향을 나타낸 그래프이다. 4 is a graph showing the effect on the invasion of breast cancer cells according to the concentration of pomegranate nanovesicles according to an experimental example of the present invention.
도 5는 본 발명의 일 실험예에 따른 석류 나노소포체의 농도에 따른 유방암 세포의 이동 정도를 나타낸 것이다.5 shows the degree of migration of breast cancer cells according to the concentration of pomegranate nanovesicles according to an experimental example of the present invention.
도 6은 본 발명의 일 실험예에 따른 석류 나노소포체가 유방암 세포 내에 침투함을 나타낸 이미지이다.6 is an image showing the penetration of pomegranate nanovesicles into breast cancer cells according to an experimental example of the present invention.
도 7은 본 발명의 일 실험예에 따른 석류 나노소포체 내 엘라그산(ellagic acid)의 함량을 분석한 그래프이다.7 is a graph analyzing the content of ellagic acid in pomegranate nanovesicles according to an experimental example of the present invention.
이하, 본 발명을 상세하게 설명하기로 한다.Hereinafter, the present invention will be described in detail.
본 발명자는 석류로부터 분리한 나노소포체가 유방암 세포의 증식, 침윤, 이동 등을 효과적으로 억제함을 확인함으로써, 본 발명을 완성하였다.The present inventors have completed the present invention by confirming that the nanovesicles isolated from pomegranate effectively inhibit the proliferation, invasion, and migration of breast cancer cells.
본 발명은 항암 활성을 가지는 세포외 소포체(extracellular vesicle)를 제공한다.The present invention provides an extracellular vesicle having anticancer activity.
상기 세포외 소포체는 석류(Punica granatum)로부터 유래된 것일 수 있다. The extracellular endoplasmic reticulum may be derived from pomegranate (Punica granatum ).
본 명세서에서, "석류(Punica granatum)"는 석류나무의 열매로, 지름 6~8cm에 단단하고 노르스름한 껍질이 감싸고 있으며, 과육 속에는 많은 종자가 있다. 껍질에는 타닌, 종자에는 갱년기 장애에 좋은 천연식물성 에스트로겐이 들어있고, 열매와 껍질 모두 고혈압·동맥경화 예방에 좋으며, 부인병·부스럼에 효과가 있다고 알려져 있다. In the present specification, "pomegranate ( Puncha granatum )" is a fruit of a pomegranate tree, 6-8cm in diameter, and a hard yellowish bark is wrapped around it, and there are many seeds in the flesh. The skin contains tannins, and the seeds contain natural vegetable estrogens that are good for menopausal disorders, and both fruits and skins are good for preventing hypertension and arteriosclerosis, and are known to be effective against gynecological diseases and rash.
상기 석류는 석류 껍질, 과육, 종자 또는 이의 혼합 부위일 수 있고, 바람직하게는 과육 또는 이의 혼합 부위일 수 있으나, 이에 제한되는 것은 아니다.The pomegranate may be a pomegranate skin, flesh, seeds, or a mixed portion thereof, and preferably may be a pulp or a mixed portion thereof, but is not limited thereto.
본 명세서에서, "세포외 소포체(extracellular vesicle, EV)"는 다양한 세포들로부터 분비되는 막 구조의 작은 소낭으로, 다낭체와 원형질막의 융합이 일어나 세포 밖 환경으로 방출되는 소낭을 의미하며, 나노소포체(nanovesicle)로도 정의될 수 있다. In the present specification, "extracellular vesicle (EV)" refers to a small vesicle having a membrane structure secreted from various cells, and refers to vesicles released into the extracellular environment due to the fusion of the polycystic body and the plasma membrane, and nanovesicles It can also be defined as (nanovesicle).
본 발명에 있어서, 상기 세포외 소포체는 엑소좀(exosomes), 미세소포(microvesicles)를 포함할 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the extracellular vesicles may include exosomes and microvesicles, but are not limited thereto.
상기 세포외 소포체는 평균 입경이 50 내지 300 nm인 나노소포체일 수 있으며, 바람직하게는 평균 입경이 100 내지 200 nm 일 수 있으나, 이에 제한되는 것은 아니다.The extracellular vesicles may be nanovesicles having an average particle diameter of 50 to 300 nm, and preferably, an average particle diameter of 100 to 200 nm, but are not limited thereto.
본 발명에 있어서, 상기 세포외 소포체는 초원심분리(ultracentrifugation), 밀도구배원심분리(density gradient centrifugation), 초미세여과(ultrafiltration), 사이즈 배제 크로마토그래피(size exclusion chromatography), 이온 교환 크로마토그래피(ion exchange chromatography), 면역친화성 분리(immunoaffinity capture), 미세유체기술 분리(microfluidics-based isolation), 침전(exosome precipitation), 총 엑소좀 추출 키트(total exosome isolation kit), 또는 폴리머 기반 침전(polymer based precipitation) 등의 방법을 사용하여 석류로부터 분리된 것일 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the extracellular vesicles are ultracentrifugation, density gradient centrifugation, ultrafiltration, size exclusion chromatography, ion exchange chromatography. exchange chromatography), immunoaffinity capture, microfluidics-based isolation, exosome precipitation, total exosome isolation kit, or polymer based precipitation ) May be separated from the pomegranate using a method such as, but is not limited thereto.
바람직하게는 상기 세포외 소포체는 석류를 원심분리하여 얻은 것일 수 있으며, 보다 바람직하게는, 상기 세포외 소포체는 석류 분쇄물을 250 내지 750 g에서 5 내지 15분간 1차 원심분리하고, 상기 1차 원심분리로 수득한 상층액을 1,000 내지 3,000 g에서 10 내지 30분간 2차 원심분리하며, 상기 2차 원심분리로 수득한 상층액을 5,000 내지 15,000 g에서 15 내지 45분간 3차 원심분리하고, 상기 3차 원심분리로 수득한 상층액을 50,000 내지 150,000 g에서 1 내지 3시간 4차 원심분리하여 수득한 것으로, 상기 4차 원심분리하여 수득한 원심분리물 중 상층액을 제외한 펠렛일 수 있으나, 이에 제한되는 것은 아니다.Preferably, the extracellular vesicles may be obtained by centrifuging pomegranate, and more preferably, the extracellular vesicles are first centrifuged for 5 to 15 minutes at 250 to 750 g of pomegranate crushed product, and the first The supernatant obtained by centrifugation was subjected to a second centrifugation at 1,000 to 3,000 g for 10 to 30 minutes, and the supernatant obtained by the second centrifugation was subjected to a third centrifugation at 5,000 to 15,000 g for 15 to 45 minutes, and the The supernatant obtained by the third centrifugation was obtained by the fourth centrifugation at 50,000 to 150,000 g for 1 to 3 hours, and may be a pellet excluding the supernatant from the centrifuge obtained by the fourth centrifugation. It is not limited.
상기 석류 분쇄물은 석류를 인산완충용액(PBS)에 넣어 분쇄한 것일 수 있으나, 이에 제한되지 않고, 석류 착즙물, 추출물 등으로 직접 제조한 것일 수 있으며, 또는 상업적으로 판매하는 석류 분쇄물, 석류 착즙물 또는 추출물 등을 구매한 것일 수 있다.The pomegranate crushed product may be pulverized by putting pomegranate in a phosphate buffer solution (PBS), but is not limited thereto, and may be directly prepared as a pomegranate juice or extract, or commercially sold pomegranate crushed product, pomegranate The juice or extract may have been purchased.
본 발명에 있어서, 상기에 따라 수득된 세포외 소포체는 엘라그산(ellagic acid), 클래스 III 키티나아제(class III chitinase), 지질 전달 단백질(lipid transfer protein) 및 칼콘 합성효소(chalcone synthase)로 이루어진 군에서 선택되는 하나 또는 둘 이상을 포함할 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the extracellular vesicle obtained according to the above is composed of ellagic acid, class III chitinase, lipid transfer protein, and chalcone synthase. One or two or more selected from the group may be included, but the present invention is not limited thereto.
본 명세서에서, "엘라그산(ellagic acid)"은 과일 또는 채소에서 발견되는 천연 식물성 페놀로, 식물체 내에서는 전구물질인 엘라기탄닌(ellagitannin) 형태 존재한다. 항산화, 항바이러스, 항돌연변이, 항암 기능을 가지는 것으로 보고되어 있다. In the present specification, "elagic acid" is a natural vegetable phenol found in fruits or vegetables, and exists in the form of ellagitannin, a precursor, in plants. It has been reported to have antioxidant, antiviral, antimutagenic, and anticancer functions.
본 발명의 일 실험예에 따르면, 석류 유래 세포외 소포체에서 석류의 주요 성분인 엘라그산이 포함되어 있음을 확인하였고, 상기 석류 유래 세포외 소포체의 항암 작용에 영향을 주는 성분으로 판단될 수 있다. According to an experimental example of the present invention, it was confirmed that ellagic acid, which is a major component of pomegranate, is contained in the pomegranate-derived extracellular vesicle, and it can be determined as a component that affects the anticancer action of the pomegranate-derived extracellular vesicle.
본 발명에 있어서, 상기 세포외 소포체는 암 세포 내로 침투할 수 있고, 암 세포의 증식, 이동 또는 침윤을 억제하는 항암 활성을 가질 수 있다.In the present invention, the extracellular endoplasmic reticulum may penetrate into cancer cells and may have anti-cancer activity to inhibit proliferation, migration or invasion of cancer cells.
본 발명의 일 실험예에 따르면, 상기 세포외 소포체가 유방암 세포 또는 폐암 세포에서 세포 내로 침투함을 확인하였고, 상기 암 세포의 증식, 침윤 및 이동을 유의하게 억제함을 확인할 수 있었다.According to an experimental example of the present invention, it was confirmed that the extracellular vesicles penetrated into the cells from breast cancer cells or lung cancer cells, and it was confirmed that the proliferation, invasion and migration of the cancer cells were significantly inhibited.
이에, 상기 세포외 소포체는 암 예방 또는 치료용 약학 조성물 또는 건강기능식품 조성물로 제공될 수 있다.Thus, the extracellular vesicles may be provided as a pharmaceutical composition or a health functional food composition for preventing or treating cancer.
본 발명은 상기 석류 유래 세포외 소포체를 유효성분으로 포함하는 암 예방 또는 치료용 약학 조성물을 제공한다.The present invention provides a pharmaceutical composition for preventing or treating cancer comprising the pomegranate-derived extracellular vesicles as an active ingredient.
상기 조성물은 상기 세포외 소포체가 0.001 내지 50 중량부 포함될 수 있으나, 이에 제한되는 것은 아니다.The composition may contain 0.001 to 50 parts by weight of the extracellular vesicles, but is not limited thereto.
상기 조성물은 암 세포 내로 침투하여 암 세포의 증식, 이동 또는 침윤을 억제함으로써 암을 예방 또는 치료할 수 있다.The composition can prevent or treat cancer by penetrating into cancer cells and inhibiting proliferation, migration, or invasion of cancer cells.
상기 암은 유방암, 폐암, 간암, 위암, 대장암, 신장암, 방광암, 급성 골수성 백혈병, 급성 림프구성 백혈병, 자궁암, 난소암, 후두암, 전립선암, 갑상선암, 두부 또는 경부암, 뇌암 및 혈액암으로 이루어진 군에서 선택되는 질환일 수 있으나, 이에 제한되는 것은 아니다.The cancer is breast cancer, lung cancer, liver cancer, stomach cancer, colon cancer, kidney cancer, bladder cancer, acute myelogenous leukemia, acute lymphocytic leukemia, uterine cancer, ovarian cancer, laryngeal cancer, prostate cancer, thyroid cancer, head or neck cancer, brain cancer and blood cancer. It may be a disease selected from the group, but is not limited thereto.
본 발명에 따른 약학 조성물은 약학적 분야의 통상적인 방법에 따라 제조될 수 있다. 상기 약학 조성물은 제형에 따라 약학적으로 허용가능한 적절한 담체와 배합될 수 있고, 필요에 따라, 부형제, 희석제, 분산제, 유화제, 완충제, 안정제, 결합제, 붕해제, 용제 등을 더 포함하여 제조될 수 있다. 상기 적절한 담체 등은 본 발명에 따른 석류 유래 세포외 소포체의 활성 및 특성을 저해하지 않는 것으로, 투여 형태 및 제형에 따라 달리 선택될 수 있다.The pharmaceutical composition according to the present invention can be prepared according to a conventional method in the pharmaceutical field. The pharmaceutical composition may be blended with an appropriate pharmaceutically acceptable carrier according to the dosage form, and if necessary, may be prepared by further including excipients, diluents, dispersants, emulsifiers, buffers, stabilizers, binders, disintegrants, solvents, etc. have. The appropriate carrier or the like is one that does not inhibit the activity and properties of the pomegranate-derived extracellular vesicles according to the present invention, and may be selected differently depending on the dosage form and formulation.
상기 약학 조성물에 포함될 수 있는 담체, 부형제, 희석제 등으로는 락토오스, 덱스트로오스, 수크로오스, 소르비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로오스, 메틸 셀룰로오스, 미정질 셀룰로오스, 폴리비닐 피롤리돈, 물, 메틸히드록시 벤조에이트, 프로필히드록시 벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유 등이 있다. 상기 조성물을 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제될 수 있다.Carriers, excipients, diluents, etc. that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, Methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate and mineral oil. When formulating the composition, it may be prepared using diluents or excipients such as generally used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.
본 발명에 따른 약학 조성물은 어떠한 제형으로도 적용될 수 있고, 상세하게는 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 및 멸균 주사용액의 형태로 제형화하여 사용될 수 있으며, 바람직하게는 경구 투여에 적합한 단위투여형의 제제로 제형화시켜 사용될 수 있다.The pharmaceutical composition according to the present invention can be applied in any dosage form, and in detail, oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups and aerosols, external preparations, suppositories and sterilization according to a conventional method. It may be formulated and used in the form of a solution for injection, and preferably may be formulated and used in a unit dosage form suitable for oral administration.
보다 상세하게는, 상기 경구형 제형 중 고형 제형은 정제, 환제, 산제, 과립제, 캡슐제 등의 형태로, 적어도 하나 이상의 부형제, 예를 들면, 전분, 칼슘카보네이트, 수크로스, 락토오스, 솔비톨, 만니톨, 셀룰로오스, 젤라틴 등을 섞어 조제할 수 있고, 단순한 부형제 이외에 마그네슘 스테아레이트, 탈크 같은 윤활제들도 포함될 수 있다. 또한, 캡술제형의 경우 상기 언급한 물질 외에도 지방유와 같은 액체 담체를 더 포함할 수 있다.More specifically, the solid dosage form of the oral dosage form is in the form of tablets, pills, powders, granules, capsules, etc., at least one excipient, such as starch, calcium carbonate, sucrose, lactose, sorbitol, mannitol. , Cellulose, gelatin, etc. can be mixed to prepare, and in addition to simple excipients, lubricants such as magnesium stearate and talc may also be included. In addition, in the case of the capsul formulation, in addition to the above-mentioned substances, a liquid carrier such as fatty oil may be further included.
상기 경구형 제형 중 액상 제형은 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순 희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다.Among the oral dosage forms, liquid dosage forms correspond to suspensions, liquid solutions, emulsions, syrups, etc., and various excipients, such as wetting agents, sweetening agents, fragrances, preservatives, etc., may be included in addition to water and liquid paraffin, which are commonly used simple diluents. have.
상기 비경구 제형은 멸균된 수용액, 비수성용제, 현탁제, 유제, 주사제, 동결건조 제제, 좌제 등이 포함될 수 있다. 비수성용제, 현탁제로는 프로필렌글리콜, 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈 61, 카카오지, 라우린지, 글리세로제라틴 등이 사용될 수 있다. 이에 제한되지 않고, 당업계에 널리 공지된 적합한 제제를 모두 사용 가능하다.The parenteral formulation may include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, injections, freeze-dried preparations, suppositories, and the like. As the non-aqueous solvent and suspension, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like may be used. As a base for suppositories, witepsol, macrogol, Tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used. The present invention is not limited thereto, and any suitable agent well known in the art may be used.
또한, 본 발명에 따른 약학 조성물은 치료 효능의 증진을 위해 항산화제를 더 첨가할 수 있다. 상기 항산화제로는 티아민(thiamin, 비타민 B1), 리보플라빈(riboflavin, 비타민 B2), 나이아신(niacin, 비타민 B3), 판토펜산(pantothenic acid, 비타민 B5), 피리독신(pyridoxine, 비타민 B6) 및 코발라민(cobalamin, 비타민 B12) 등의 비타민 B군의 화합물과 비타민 C, 비타민 D, 비타민 E 등이 사용될 수 있으나, 이에 제한되지 않고, 당업계에 널리 공지된 적합한 제제를 모두 사용 가능하다.In addition, the pharmaceutical composition according to the present invention may further add an antioxidant to enhance therapeutic efficacy. The antioxidants include thiamine (vitamin B1), riboflavin (vitamin B2), niacin (vitamin B3), pantothenic acid (vitamin B5), pyridoxine (vitamin B6), and cobalamin. Vitamin B group compounds such as vitamin B12) and vitamin C, vitamin D, vitamin E, and the like may be used, but are not limited thereto, and all suitable agents well known in the art may be used.
본 발명에 따른 약학 조성물은 약학적으로 유효한 양으로 투여될 수 있다. The pharmaceutical composition according to the present invention may be administered in a pharmaceutically effective amount.
본 명세서에서, "약학적으로 유효한 양"이란, 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분하며 부작용을 일으키지 않을 정도의 양을 의미한다.As used herein, "a pharmaceutically effective amount" refers to an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment and not cause side effects.
상기 약학 조성물의 유효 용량 수준은 사용 목적, 환자의 연령, 성별, 체중 및 건강 상태, 질환의 종류, 중증도, 약물의 활성, 약물에 대한 민감도, 투여 방법, 투여 시간, 투여 경로 및 배출 비율, 치료기간, 배합 또는 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 달리 결정될 수 있다. 예를 들어, 일정하지는 않지만 일반적으로 0.001 내지 100mg/kg으로, 바람직하게는 0.01 내지 10mg/kg을 일일 1회 내지 수회 투여될 수 있다. 상기 투여량은 어떠한 면으로든 본 발명의 범위를 한정하는 것은 아니다.The effective dosage level of the pharmaceutical composition is the purpose of use, the age, sex, weight and health condition of the patient, the type of disease, the severity, the activity of the drug, the sensitivity to the drug, the method of administration, the administration time, the administration route and rate of excretion, It can be determined differently depending on the duration, factors including the combination or co-used drugs and other factors well known in the medical field. For example, although not constant, generally 0.001 to 100 mg/kg, preferably 0.01 to 10 mg/kg may be administered once to several times a day. The above dosage does not limit the scope of the present invention in any way.
본 발명에 따른 약학 조성물은 암 질환이 발생할 수 있는 임의의 동물에 투여될 수 있고, 상기 동물은 예를 들어, 인간 및 영장류뿐만 아니라 소, 돼지, 말, 개 등의 가축 등을 포함할 수 있다.The pharmaceutical composition according to the present invention may be administered to any animal that may cause cancer disease, and the animal may include, for example, humans and primates as well as livestock such as cattle, pigs, horses, and dogs. .
상기 약학 조성물은 제제 형태에 따른 적당한 투여 경로로 투여될 수 있고, 목적 조직에 도달할 수 있는 한 경구 또는 비경구의 다양한 경로를 통하여 투여될 수 있다. 투여 방법은 특히 한정할 필요 없이, 예를 들면, 경구, 직장 또는 정맥, 근육, 피부 도포, 호흡기내 흡입, 자궁내 경막 또는 뇌혈관내(intracere-broventricular) 주사 등의 통상적인 방법으로 투여될 수 있다.The pharmaceutical composition may be administered by an appropriate route of administration according to the form of the formulation, and may be administered through various routes, such as oral or parenteral, as long as it can reach the target tissue. The method of administration is not particularly limited, and may be administered by conventional methods such as, for example, oral, rectal or intravenous, intramuscular, skin application, inhalation in the respiratory tract, intrauterine dura mater or intracere-broventricular injection. have.
본 발명에 따른 약학 조성물은 암 질환의 예방 또는 치료를 위하여 단독으로 사용될 수 있고, 수술 또는 다른 약물 치료 등과 병용하여 사용될 수 있다.The pharmaceutical composition according to the present invention may be used alone for the prevention or treatment of cancer diseases, or may be used in combination with surgery or other drug treatment.
본 발명은 상기 석류 유래 세포외 소포체를 유효성분으로 포함하는 암 예방 또는 개선용 건강기능식품 조성물을 제공한다.The present invention provides a health functional food composition for preventing or improving cancer comprising the pomegranate-derived extracellular vesicles as an active ingredient.
상기 조성물은 암 세포 내로 침투하여 암 세포의 증식, 이동 또는 침윤을 억제함으로써 암을 예방 또는 개선할 수 있다.The composition can prevent or improve cancer by penetrating into cancer cells and inhibiting proliferation, migration, or invasion of cancer cells.
상기 암은 유방암, 폐암, 간암, 위암, 대장암, 신장암, 방광암, 급성 골수성 백혈병, 급성 림프구성 백혈병, 자궁암, 난소암, 후두암, 전립선암, 갑상선암, 두부 또는 경부암, 뇌암 및 혈액암으로 이루어진 군에서 선택되는 질환일 수 있으나, 이에 제한되는 것은 아니다.The cancer is breast cancer, lung cancer, liver cancer, stomach cancer, colon cancer, kidney cancer, bladder cancer, acute myelogenous leukemia, acute lymphocytic leukemia, uterine cancer, ovarian cancer, laryngeal cancer, prostate cancer, thyroid cancer, head or neck cancer, brain cancer and blood cancer. It may be a disease selected from the group, but is not limited thereto.
이에 상응하는 특징들은 상술된 부분에서 대신할 수 있다.Corresponding features can be substituted in the above-described section.
본 명세서에서, "건강기능식품"이란, 건강기능식품에 관한 법률 제6727호에 따른 인체에 유용한 기능성을 가진 원료나 성분을 사용하여 제조 및 가공한 식품을 포함하며, 영양 공급 외에도 본 발명의 목적상 암의 예방, 개선, 생체 방어, 면역, 회복 등의 생체 조절 기능이 효율적으로 나타나도록 가공된 의학, 의료 효과가 높은 식품을 의미한다.In the present specification, the term "health functional food" includes foods manufactured and processed using raw materials or ingredients having functions useful for the human body according to the Health Functional Food Act No. It refers to foods with high medical effects and medicines processed to efficiently exhibit biomodulatory functions such as prevention, improvement, body defense, immunity, and recovery.
본 발명에 따른 건강기능식품은 암의 예방 또는 개선 목적으로, 분말, 과립, 정제, 캡슐, 시럽 또는 음료 등으로 제조될 수 있다. 상기 건강기능식품이 취할 수 있는 형태에는 제한이 없으며, 상기 약학 조성물과 동일한 방식으로 제제화되어 기능성 식품으로 이용하거나, 각종 식품에 첨가될 수 있다. The health functional food according to the present invention may be prepared as a powder, granule, tablet, capsule, syrup or beverage for the purpose of preventing or improving cancer. There is no limitation on the form that the health functional food can take, and it can be formulated in the same manner as the pharmaceutical composition to be used as a functional food or added to various foods.
상기 건강기능식품은 통상적인 의미의 식품을 모두 포함할 수 있다. 예를 들어, 음료 및 각종 드링크, 과실 및 그의 가공식품(과일통조림, 잼 등), 어류, 육류 및 그 가공식품(햄, 베이컨 등), 빵류 및 면류, 쿠키 및 스낵류, 유제품(버터, 치즈 등) 등이 가능하며, 통상적인 의미에서의 기능성 식품을 모두 포함할 수 있다. 또한, 동물을 위한 사료로 이용되는 식품도 포함할 수 있다.The health functional food may include all foods in a conventional sense. For example, beverages and various drinks, fruits and processed foods thereof (canned fruit, jam, etc.), fish, meat and processed foods thereof (ham, bacon, etc.), bread and noodles, cookies and snacks, dairy products (butter, cheese, etc.) ) And the like, and may include all functional foods in the usual sense. In addition, foods used as feed for animals may also be included.
본 발명에 따른 건강기능식품 조성물은 당업계에서 통상적으로 사용되는 식품학적으로 허용 가능한 식품 첨가제(식품 첨가물) 및 적절한 기타 보조 성분을 더 포함하여 제조될 수 있다. 식품 첨가물로서의 적합 여부는 다른 규정이 없는 한, 식품의약품안전처에 승인된 식품첨가물공전의 총칙 및 일반시험법 등에 따라 해당 품목에 관한 규격 및 기준에 의하여 판정할 수 있다. 상기 '식품첨가물공전'에 수재된 품목으로는 예를 들어, 케톤류, 글리신, 구연산칼슘, 니코틴산, 계피산 등의 화학적 합성물; 감색소, 감초 추출물, 결정셀룰로오스, 고량색소, 구아검 등의 천연첨가물; L-글루타민산나트륨 제제, 면류첨가알칼리제, 보존료 제제, 타르색소제제 등의 혼합 제제류 등을 들 수 있다. The health functional food composition according to the present invention may be prepared by further comprising a food pharmaceutically acceptable food additive (food additive) and other appropriate auxiliary ingredients commonly used in the art. Unless otherwise specified, the suitability as a food additive may be determined according to the standards and standards for the relevant item in accordance with the general rules and general test methods of the Food Additive Code approved by the Ministry of Food and Drug Safety. Examples of the items listed in the'Food Additives Code' include chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; Natural additives such as dark pigment, licorice extract, crystalline cellulose, high color pigment, and guar gum; Mixed preparations, such as a sodium L-glutamate preparation, an alkali additive for noodles, a preservative preparation, and a tar color preparation, etc. are mentioned.
상기 기타 보조 성분은 예를 들어, 향미제, 천연 탄수화물, 감미제, 비타민, 전해질, 착색제, 펙트산, 알긴산, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알콜, 탄산화제 등을 추가로 함유할 수 있다. 특히, 상기 천연 탄수화물로는 포도당, 과당과 같은 모노사카라이드, 말토스, 수크로오스와 같은 디사카라이드, 및 덱스트린, 사이클로덱스트린과 같은 폴리사카라이드, 자일리톨, 소르비톨, 에리트리톨 등의 당알콜을 사용할 수 있으며, 감미제로서는 타우마틴, 스테비아 추출물과 같은 천연 감미제나 사카린, 아스파르탐과 같은 합성 감미제 등을 사용할 수 있다.The other auxiliary ingredients are, for example, flavoring agents, natural carbohydrates, sweetening agents, vitamins, electrolytes, colorants, pectic acids, alginic acids, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents, etc. It may further contain. In particular, as the natural carbohydrates, monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, sugar alcohols such as xylitol, sorbitol, and erythritol may be used. , As the sweetener, natural sweeteners such as taumatin and stevia extract, or synthetic sweeteners such as saccharin and aspartame may be used.
본 발명에 따른 건강기능식품에 함유된 상기 세포외 소포체의 유효 용량은 암의 예방 또는 개선 등 그 사용 목적에 따라 적절하게 조절될 수 있다. The effective dose of the extracellular vesicles contained in the health functional food according to the present invention may be appropriately adjusted according to the purpose of use, such as prevention or improvement of cancer.
상기 건강기능식품 조성물은 식품을 원료로 하여 일반 약품의 장기 복용 시 발생할 수 있는 부작용 등이 없는 장점이 있고, 휴대성이 뛰어나, 암의 예방 또는 개선을 위한 보조제로 섭취될 수 있다.The health functional food composition has the advantage of not having side effects that may occur during long-term use of general drugs by using food as a raw material, has excellent portability, and can be taken as an adjuvant for preventing or improving cancer.
또한, 본 발명은 석류 유래 세포외 소포체의 제조방법을 제공한다.In addition, the present invention provides a method for producing pomegranate-derived extracellular vesicles.
상기 제조방법은 석류 분쇄물을 제조하는 단계; 상기 제조된 석류 분쇄물을 250 내지 750 g에서 5 내지 15분간 1차 원심분리하는 단계; 상기 1차 원심분리하여 수득한 상층액을 1,000 내지 3,000 g에서 10 내지 30분간 2차 원심분리하는 단계; 상기 2차 원심분리하여 수득한 상층액을 5,000 내지 15,000 g에서 15 내지 45분간 3차 원심분리하는 단계; 및 상기 3차 원심분리하여 수득한 상층액을 50,000 내지 150,000 g에서 1 내지 3시간 4차 원심분리하여 펠렛을 수득하는 단계;를 포함할 수 있다.The manufacturing method comprises the steps of preparing a pomegranate crushed product; First centrifuging the prepared pomegranate crushed product at 250 to 750 g for 5 to 15 minutes; Second centrifuging the supernatant obtained by the first centrifugation at 1,000 to 3,000 g for 10 to 30 minutes; Third centrifuging the supernatant obtained by the second centrifugation at 5,000 to 15,000 g for 15 to 45 minutes; And obtaining a pellet by performing a fourth centrifugation for 1 to 3 hours at 50,000 to 150,000 g of the supernatant obtained by the third centrifugation.
상기 제조방법에 있어서, 석류 분쇄물을 제조하는 단계는 석류를 세척한 후 인산완충용액(PBS)에 넣어 분쇄하거나 착즙함으로써, 수행될 수 있다.In the above manufacturing method, the step of preparing the pomegranate pulverized product may be performed by washing the pomegranate and then pulverizing or juice by putting it in a phosphoric acid buffer solution (PBS).
상기 제조방법에 있어서, 상기 1차 내지 4차의 원심분리는 1회 수행되거나 또는 2회 이상 반복하여 수행될 수 있으며, 특히 상기 3차 원심분리 단계는 1회 수행되거나 또는 2회 내지 5회 반복하여 수행될 수 있으나, 이에 제한되는 것은 아니다.In the above manufacturing method, the first to fourth centrifugation may be performed once or repeatedly performed two or more times, and in particular, the third centrifugation step may be performed once or repeated two to five times. However, it is not limited thereto.
상기 제조방법은 1차 이상의 원심분리 단계를 더 포함할 수 있다.The manufacturing method may further include one or more centrifugation steps.
본 발명의 일 실험예에 따르면, 상기 1차 원심분리 단계는 500 g 에서 10분 동안 1회 수행되고, 상기 2차 원심분리 단계는 상기 1차 원심분리 단계에서 수득한 상층액을 2,000 g에서 20분 동안 1회 수행되며, 상기 3차 원심분리 단계는 상기 2차 원심분리 단계에서 수득한 상층액을 10,000 g에서 30분씩 2회 수행되고, 상기 4차 원심분리 단계는 상기 3차 원심분리 단계에서 수득한 상층액을 100,000 g로 2시간 동안 1회 수행됨으로써, 석류 유래 세포외 소포체를 제조하였다.According to an experimental example of the present invention, the first centrifugation step is performed once for 10 minutes at 500 g, and the second centrifugation step is 20 at 2,000 g of the supernatant obtained in the first centrifugation step. It is performed once for a minute, and the third centrifugation step is performed twice for 30 minutes at 10,000 g of the supernatant obtained in the second centrifugation step, and the fourth centrifugation step is performed in the third centrifugation step. The obtained supernatant was performed once for 2 hours at 100,000 g, thereby preparing pomegranate-derived extracellular vesicles.
상기와 같은 제조방법을 통해 항암 활성이 우수하며, 입자 크기가 균일한 고순도의 석류 유래 세포외 소포체를 효과적으로 수득할 수 있다.Through the manufacturing method as described above, it is possible to effectively obtain high-purity pomegranate-derived extracellular vesicles having excellent anticancer activity and uniform particle size.
또한, 상기 제조방법은 상기 원심분리 방법 외에도, 초미세여과법(ultrafiltration), 사이즈 배제 크로마토그래피(size exclusion chromatography), 이온 교환 크로마토그래피(ion exchange chromatography), 면역친화성 분리법(immunoaffinity capture), 미세유체기술 분리법(microfluidics-based isolation), 침전법(exosome precipitation), 총 엑소좀 추출 키트(total exosome isolation kit), 또는 폴리머 기반 침전법(polymer based precipitation) 등을 이용할 수 있으나, 이에 제한되지 않고 당 기술분야에 널리 알려진 분리 또는 정제 방법을 추가로 더 포함하여 진행할 수 있다.In addition, in addition to the centrifugal separation method, the manufacturing method includes ultrafiltration, size exclusion chromatography, ion exchange chromatography, immunoaffinity capture, microfluidic Technology separation method (microfluidics-based isolation), precipitation method (exosome precipitation), total exosome extraction kit (total exosome isolation kit), or polymer-based precipitation method (polymer based precipitation), etc. can be used, but is not limited to this technology Separation or purification methods well known in the art may be further included.
이하, 본 발명의 이해를 돕기 위하여 실시예를 들어 상세하게 설명하기로 한다. 다만 하기의 실시예는 본 발명의 내용을 예시하는 것일 뿐 본 발명의 범위가 하기 실시예에 한정되는 것은 아니다. 본 발명의 실시예는 당업계에서 평균적인 지식을 가진 자에게 본 발명을 보다 완전하게 설명하기 위해 제공되는 것이다.Hereinafter, examples will be described in detail to aid understanding of the present invention. However, the following examples are merely illustrative of the contents of the present invention, and the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely describe the present invention to those of ordinary skill in the art.
<실험예 1> 석류에서 분리된 나노소포체의 특성 분석<Experimental Example 1> Characterization of nanovesicles isolated from pomegranate
<실험 방법><Experiment method>
1. 석류의 나노소포체 분리1. Separation of Pomegranate Nanovesicles
본 발명의 실험에 사용된 석류는 조지아스내추럴 석류주스(AROMAPRODUCT LTD 제조)로, 석류 씨앗을 제외한 석류 100%를 그대로 착즙한 주스를 사용하였다. The pomegranate used in the experiment of the present invention was Georgia Natural Pomegranate Juice (manufactured by AROMAPRODUCT LTD), and a juice obtained by extracting 100% of pomegranate excluding pomegranate seeds was used.
도 1은 석류에서 나노소포체를 분리하는 방법을 개략적으로 나타낸 것으로, 이를 참조하면, 무게를 일정하게 맞춘 후 원심분리기를 이용하여 500 g에서 10분 동안 1회 돌려준 후, 상층액을 새로운 50 mL 코니컬 튜브로 옮겨 2,000 g에서 20분 동안 1회 돌려주었고, 다시 상층액을 새로운 50 mL 코니컬 튜브에 옮겨 담아 10,000 g에서 30분씩 2회 돌린 후, 다시 새 튜브에 상층액을 담고 100,000 g로 2시간 돌려준 후 상층액을 버리고 남은 나노소포체 펠렛(pellet)은 건조시킨 뒤, 1 mL의 1× PBS를 넣어서 녹여주었다.1 schematically shows a method of separating nanovesicles from pomegranate, referring to this, after adjusting the weight to a constant, spin once for 10 minutes at 500 g using a centrifuge, and then add a new 50 mL of the supernatant. Transferred to a conical tube and returned once for 20 minutes at 2,000 g, and the supernatant was transferred to a new 50 mL conical tube and rotated twice at 10,000 g for 30 minutes, and then the supernatant was added to a new tube and returned to 100,000 g. After returning for 2 hours, the supernatant was discarded, and the remaining nanovesicle pellet was dried, and then 1 mL of 1×PBS was added to dissolve it.
2. 나노 입자 추적 분석 (NTA; Nano particle Tracking Analysis)2. Nano particle Tracking Analysis (NTA)
Malvern의 NanoSight를 이용하여 석류에서 분리한 나노소포체(Extracellular Vesicles, EVs)를 분석하였다. 나노 입자 추적 분석(NTA)은 현탁액(liquid suspension) 내 직경 50 nm ~ 1000 nm 범위의 특정의 나노소포체의 크기 및 농도를 측정하였다.Malvern's NanoSight was used to analyze nanovesicles (Extracellular Vesicles, EVs) isolated from pomegranate. Nanoparticle tracking analysis (NTA) measured the size and concentration of specific nanovesicles in the range of 50 nm to 1000 nm in diameter in a liquid suspension.
3. 석류 나노소포체 단백질 정량3. Quantification of pomegranate nanovesicle protein
석류의 나노소포체 단백질은 1× RIPA 용액을 넣어서 파쇄한 후에 비신코닌산(bicinchoninic acid, BCA, Termo Fishers)법으로 단백질을 정량하였다.Pomegranate nanovesicle protein was crushed with 1× RIPA solution, and then the protein was quantified by bicinchoninic acid (BCA, Termo Fishers) method.
4. 유방암 세포 배양 4. Breast cancer cell culture
MDA-MB-231 유방암 세포는 ATCC 세포주에서 분양 받았다. MDA-MB-231 유방암 세포는 배양을 위해 DMEM 배지, 10% 소 태아 혈청(fetal bovine serum)과 1% 항생제를 첨가하여 5% CO2 및 37℃의 인큐베이터에서 배양을 하였다. 세포는 미디어를 2-3일마다 교환하였다.MDA-MB-231 breast cancer cells were distributed from the ATCC cell line. MDA-MB-231 breast cancer cells were cultured in an incubator at 5% CO 2 and 37° C. by adding DMEM medium, 10% fetal bovine serum and 1% antibiotics for culture. Cells were exchanged for media every 2-3 days.
5. 세포 증식 분석 (MTT assay)5. Cell proliferation assay (MTT assay)
MDA-MB-231 유방암 세포를 96 웰 플레이트의 각 웰당 세포수가 2.5×104 cells/mL가 되도록 넣고 플레이트에 붙을 수 있도록 DMEM 성장 배지에서 약 24시간 동안 배양했다. 그러고 나서 그 성장 배지는 제거하고 석류 나노소포체를 0, 10, 50, 100μg/mL 농도 조건으로 처리하였다. 37℃, 5% CO2가 유지되는 배양기 내에서 약 24시간 동안 배양하였다. 그 후 각 웰당 40μL의 PBS에 2 mg/mL의 MTT를 녹인 용액을 넣고 37℃ 배양기에 2시간 동안 방치하였다. 다음으로 MTT 용액을 제거하고 웰당 100μL의 DMSO(dimethyl sulfoxide)를 첨가하여 형성된 포르마잔(formazan)을 녹였다. 완전히 용해된 포르마잔의 흡광도를 ELISA-reader (Bio-Rad Lab- oratories Inc., Hercules, CA, USA)로 595 nm에서 측정하였다. MDA-MB-231 breast cancer cells were placed in a 96-well plate so that the number of cells per well was 2.5×10 4 cells/mL, and cultured in DMEM growth medium for about 24 hours to adhere to the plate. Then, the growth medium was removed and the pomegranate nanovesicles were treated at concentrations of 0, 10, 50, and 100 μg/mL. Incubated for about 24 hours in an incubator maintained at 37°C and 5% CO 2. Then, a solution in which 2 mg/mL of MTT was dissolved in 40 μL of PBS per well was added and left in an incubator at 37° C. for 2 hours. Next, the MTT solution was removed, and formazan formed by adding 100 μL of dimethyl sulfoxide (DMSO) per well was dissolved. The absorbance of completely dissolved formazan was measured at 595 nm with an ELISA-reader (Bio-Rad Lab-oratories Inc., Hercules, CA, USA).
6. 세포 이동 분석 (Migration assay)6. Cell migration assay
MDA-MB-231 유방암 세포를 6 웰에 1 mL을 넣고 5% CO2 및 37℃인 인큐베이터에서 배양하였다. 세포를 상처(wound) 낸 다음 세포의 치유(healing)를 현미경으로 관찰하였다.1 mL of MDA-MB-231 breast cancer cells were added to 6 wells and cultured in an incubator at 5% CO 2 and 37°C. After the cells were wound, the healing of the cells was observed under a microscope.
7. 세포 침윤 분석 (Invasion assay)7. Invasion assay
재수화(Rehydration)된 8μm 포어(pore) 크기의 폴리카보네이트 막(polycarbonate membrane)이 도포된 인서트(insert)에 무 혈청 배지(serum free medium)에 부유된 MDA-MB-231 유방암 세포를 300μL 넣고 바깥쪽 챔버(chamber)에 10% FBS를 함유한 배지를 500μL 처리하여 막의 바깥 챔버에는 세포의 침투를 유도하는 영양분이 풍부한 환경을 조성하였다. MDA-MB-231 유방암 세포에 양성대조군인 혈관내피성장인자(vascular endothelial growth factor, VEGF)를 100 ng/mL의 농도로 처리하고 석류 나노소포체를 농도별로 처리한 뒤 37℃에서 24시간 배양하였다. 그 후 침투되지 않은 세포를 제거한 뒤, 세포 염색 용액(cell stain solution)으로 침투된 막 바깥쪽 세포를 염색하였다. 염색된 세포를 수를 측정하였다.Insert 300 μL of MDA-MB-231 breast cancer cells suspended in a serum free medium into an insert coated with a rehydrated 8 μm pore-sized polycarbonate membrane. 500 μL of a medium containing 10% FBS was treated in the side chamber to create an environment rich in nutrients that induces cell penetration in the outer chamber of the membrane. MDA-MB-231 breast cancer cells were treated with vascular endothelial growth factor (VEGF), which is a positive control, at a concentration of 100 ng/mL, and pomegranate nanovesicles were treated by concentration, followed by incubation at 37°C for 24 hours. Thereafter, the cells that did not penetrate were removed, and then the cells outside the membrane that had penetrated with a cell stain solution were stained. The stained cells were counted.
8. MMP-9 (Matrix metallopeptidase 9) ELISA 분석8. MMP-9 (Matrix metallopeptidase 9) ELISA analysis
MMP-9 ELISA (Enzyme-Linked ImmunoSorbent Assay, 효소결합면역흡착검사, R&D system) 실험은 항체(또는 항원)에 효소를 표지하고 효소 활성을 업체의 프로토콜을 이용해서 실험하였다.In the MMP-9 ELISA (Enzyme-Linked ImmunoSorbent Assay, Enzyme-Linked Immunosorbent Assay, R&D system) experiment, an antibody (or antigen) was labeled with an enzyme and the enzyme activity was tested using the manufacturer's protocol.
9. DID 라벨링(labelling)9. DID labeling
MDA-MB-231 유방암 세포를 8 웰 챔버 슬라이드에 1×104 cells/well로 깔고 DMEM 성장배지에서 약 24시간 동안 배양하였다. 그 후 각 웰에 DID 라벨링된 석류 나노소포체를 처리한 다음, 24시간 뒤에 배지를 제거하고 PBS로 세척한 뒤 70% 에탄올 처리를 하였다. DAPI(4′,6-diamidino-2-phenylindol) 염색을 마치고 현광 현미경으로 석류 나노소포체가 세포로 침투되었는지 확인하였다.MDA-MB-231 breast cancer cells were spread on an 8-well chamber slide at 1×10 4 cells/well and cultured in DMEM growth medium for about 24 hours. Thereafter, DID-labeled pomegranate nanovesicles were treated in each well, and then the medium was removed after 24 hours, washed with PBS, and then treated with 70% ethanol. After DAPI (4′,6-diamidino-2-phenylindol) staining was finished, it was confirmed that the pomegranate nanovesicles had penetrated into the cells under a fluorescence microscope.
10. 프로테오믹스(proteomics) 분석10. Proteomics analysis
석류 나노소포체를 트립신 완충액 (500 ng/μL trypsin, 50mM ammonium bicarbonate)을 처리하여 37℃에서 16시간 동안 반응시켰다. 이 후 5% 포름산(formic acid)을 첨가하여 트립신의 작용을 중지시킨 후, 25 mM TEABC(triethylammonium bicarbonate)와 ACN(acetonitrile)을 이용하여 펩타이드를 추출하였다. 추출된 펩타이드들은 감압건조기를 이용하여 완전히 건조시켰고, 분석 직전에 A 완충액 (0.1% formic acid)에 녹여 질량분석기로 분석하였다. 질량분석기로 얻은 데이터는 Mascot Distiller를 이용하여 피크 리스트(mgf 파일)로 변환하였다. mgf 파일을 Mascot (Matrix Science; version 2.2.1) 프로그램을 이용하여 단백질을 동정하였다. Pomegranate nanovesicles were treated with trypsin buffer (500 ng/μL trypsin, 50 mM ammonium bicarbonate) and reacted at 37° C. for 16 hours. After that, 5% formic acid was added to stop the action of trypsin, and then the peptide was extracted using 25 mM TEABC (triethylammonium bicarbonate) and ACN (acetonitrile). The extracted peptides were completely dried using a vacuum dryer, dissolved in A buffer (0.1% formic acid) immediately before analysis, and analyzed by mass spectrometry. Data obtained by mass spectrometry were converted into a peak list (mgf file) using Mascot Distiller. The mgf file was identified using the Mascot (Matrix Science; version 2.2.1) program.
11. HPLC 분석11. HPLC analysis
석류 나노소포체는 에탄올을 1:1로 희석하여 10분간 초음파 추출한 후, 15,000 rpm으로 원심분리하여, 0.2μm syringe filter로 여과한 뒤, Agilent 1100 Series로 분석하였다.Pomegranate nanovesicles were diluted 1:1 with ethanol, ultrasonically extracted for 10 minutes, centrifuged at 15,000 rpm, filtered through a 0.2 μm syringe filter, and analyzed with the Agilent 1100 Series.
12. 통계처리12. Statistical processing
3 가지 이상 반복실험을 통하여 그 결과를 얻어 각각의 시료 농도에 대해 평균±표준편차로 나타내었다. 각 시료 농도군에 대한 유의차 검정은 대조군과 비교하여 Student’s t test 한 후 p<0.05 값을 통계적으로 유의성 있는 결과로 간주하였다. The results were obtained through three or more repeated experiments and expressed as the mean ± standard deviation for each sample concentration. The significance difference test for each sample concentration group was compared with the control group, and a p<0.05 value was considered as a statistically significant result after the Student's t test.
<실험 결과><Experiment result>
1. 석류 나노소포체 크기 확인1. Check the size of pomegranate nanovesicles
도 2는 본 발명의 일 실험예에 따른 석류 나노소포체의 크기를 분석한 그래프로, 이를 참조하면, 상기 도 1에 따라 분리한 석류의 나노소포체의 나노입자추적분석(NTA)한 결과, 직경 173nm의 나노소포체의 크기를 확인할 수 있었다.2 is a graph analyzing the size of the pomegranate nano vesicles according to an experimental example of the present invention. Referring to this, as a result of nanoparticle tracking analysis (NTA) of the pomegranate nano vesicles separated according to FIG. 1, a diameter of 173 nm The size of the nanovesicles could be confirmed.
2. 석류 나노소포체의 유방암 세포 생존 억제 효과 확인2. Confirmation of the effect of pomegranate nanovesicles on the inhibition of breast cancer cell survival
도 3은 본 발명의 일 실험예에 따른 석류 나노소포체의 농도에 따른 유방암 세포의 증식률을 나타낸 그래프이다. 3 is a graph showing the proliferation rate of breast cancer cells according to the concentration of pomegranate nanovesicles according to an experimental example of the present invention.
석류 나노소포체가 MDA-MB-231 유방암 세포 생존에 미치는 영향을 확인하기 위하여 농도별로 처리를 하여 MTT assay을 통해 관찰한 결과, 도 3과 같이, 석류 나노소포체를 각각 0, 10, 50, 100μg으로 24시간 처리하였을 때, 각각 농도별 세포 생존율은 100, 86, 80, 77%로 대조군에 비해 10μg의 농도에서부터 농도 의존적으로 세포 생존에 대하여 유의적인 감소를 나타내었다. In order to confirm the effect of pomegranate nanovesicles on the survival of MDA-MB-231 breast cancer cells, the results were observed through MTT assay by treatment at each concentration. As shown in FIG. 3, pomegranate nanovesicles were respectively 0, 10, 50, and 100 μg. When treated for 24 hours, the cell viability at each concentration was 100, 86, 80, and 77%, showing a significant decrease in cell survival from a concentration of 10 μg compared to the control group in a concentration-dependent manner.
따라서 석류 나노소포체가 MDA-MB-231 유방암 세포에 10μg의 농도 의존적으로 세포 사멸을 유도함을 확인할 수 있고, 이는 석류 나노소포체가 유방암 세포에 농도 의존적으로 세포 생존율을 감소시킨 것과 같은 결과로 나타나, 이러한 결과를 통해 석류 나노소포체가 암 세포에 억제 효과를 보이는 것으로 판단할 수 있다.Therefore, it can be confirmed that pomegranate nanovesicles induce apoptosis in a concentration-dependent manner of 10 μg in MDA-MB-231 breast cancer cells, and this is the same result that pomegranate nano vesicles decrease cell viability in a concentration-dependent manner in breast cancer cells. From the results, it can be determined that the pomegranate nanovesicles have an inhibitory effect on cancer cells.
3. 석류 나노소포체의 유방암 세포 침윤 억제 효과 확인3. Confirmation of the effect of pomegranate nanovesicles to inhibit breast cancer cell invasion
도 4는 본 발명의 일 실험예에 따른 석류 나노소포체의 농도에 따른 유방암 세포의 침윤에 미치는 영향을 나타낸 그래프로, 이를 참조하면, 석류 나노소포체를 각각 0, 10, 50, 100μg으로 24시간 처리하였을 때, 농도 의존적으로 MDA-MB-231 유방암 세포의 침윤이 감소하는 것으로 나타났다. 4 is a graph showing the effect on the invasion of breast cancer cells according to the concentration of pomegranate nano vesicles according to an experimental example of the present invention. Referring to this, pomegranate nano vesicles are treated with 0, 10, 50, and 100 μg, respectively, for 24 hours. When doing so, it was found that the invasion of MDA-MB-231 breast cancer cells decreased in a concentration-dependent manner.
따라서 석류 나노소포체가 MDA-MB-231 유방암 세포에 농도 의존적으로 세포 침윤을 억제함을 확인할 수 있고, 이러한 결과를 통해 석류 나노소포체가 암 세포에 억제 효과를 보이는 것으로 판단할 수 있다.Therefore, it can be confirmed that the pomegranate nanovesicles inhibit cell invasion in a concentration-dependent manner on MDA-MB-231 breast cancer cells, and through these results, it can be determined that the pomegranate nanovesicles exhibit an inhibitory effect on cancer cells.
4. 석류 나노소포체의 유방암 세포 이동 억제 효과 확인4. Confirmation of the effect of pomegranate nanovesicles on the inhibition of breast cancer cell migration
도 5는 본 발명의 일 실험예에 따른 석류 나노소포체의 농도에 따른 유방암 세포의 이동 정도를 나타낸 것으로, 이를 참조하면, 석류 나노소포체를 각각 0, 10, 100μg으로 24시간 처리하였을 때, 농도 의존적으로 MDA-MB-231 유방암 세포의 이동이 감소하는 것으로 나타났다. 5 shows the degree of migration of breast cancer cells according to the concentration of pomegranate nanovesicles according to an experimental example of the present invention. Referring to this, when pomegranate nanovesicles were treated at 0, 10, and 100 μg for 24 hours, respectively, concentration-dependent As a result, the migration of MDA-MB-231 breast cancer cells was decreased.
따라서 석류 나노소포체가 MDA-MB-231 유방암 세포에 농도 의존적으로 세포 이동을 억제함을 확인할 수 있고, 이러한 결과를 통해 석류 나노소포체가 암 세포에 억제 효과를 보이는 것으로 판단할 수 있다.Therefore, it can be confirmed that the pomegranate nanovesicles inhibit cell migration in a concentration-dependent manner on MDA-MB-231 breast cancer cells, and through these results, it can be determined that the pomegranate nanovesicles exhibit an inhibitory effect on cancer cells.
5. 석류 나노소포체의 유방암 세포 내재화(internalization) 효과 확인5. Confirmation of the effect of pomegranate nanovesicles on breast cancer cell internalization
도 6은 본 발명의 일 실험예에 따른 석류 나노소포체가 유방암 세포 내에 침투함을 나타낸 이미지로, 이를 참조하면, 석류 나노소포체를 DID 라벨링한 후 24시간 처리하였을 때 MIA-MB-231 유방암 세포의 세포질로 이동하는 것이 확인되었다.6 is an image showing that pomegranate nanovesicles infiltrate into breast cancer cells according to an experimental example of the present invention. Referring to this, when treated for 24 hours after DID labeling of pomegranate nano vesicles, MIA-MB-231 breast cancer cells It was confirmed to migrate to the cytoplasm.
따라서 석류 나노소포체가 MBA-MD-231 유방암 세포에 침투하는 것을 통해, 석류 나노소포체가 암 세포에 억제 효과를 보이는 것으로 판단할 수 있다.Therefore, it can be determined that the pomegranate nanovesicles have an inhibitory effect on the cancer cells through the penetration of the pomegranate nanovesicles into the MBA-MD-231 breast cancer cells.
6. 석류 나노소포체의 단백질 동정(identification)6. Protein identification of pomegranate nanovesicles
석류 나노소포체를 트립신 소화(trypsin digestion)를 통해서 펩타이드를 만든 후에 프로테오믹스 분석을 하였다. 그 결과, 하기 표 1과 같이 15개의 단백질들을 석류 나노소포체에서 동정을 하였다. 클래스 III 키티나아제(class III chitinase) 단백질은 키틴의 β-1,4-클리코시드 결합(β-1,4-glycosidic bond)을 분해하는 효소로 석류에 금속 저장 용량(metal storage capacity)을 제공하는 것으로 알려져 있다. 이 효소가 나노소포체에 존재하는 것을 확인할 수 있었으며, 또한 지방 단백질을 이동하는 지질 전달 단백질(lipid transfer protein)도 동정하였다. 칼콘 합성효소(chalcone synthase)는 식물에 많이 존재하며, 플라보노이드 생합성(flavonoid biosynthesis) 역할을 하는 것으로 알려져 있다. 이러한 단백질들이 석류 나노소포체에 존재하는 것을 알 수 있었다.After making a peptide through trypsin digestion of pomegranate nanovesicles, proteomic analysis was performed. As a result, as shown in Table 1 below, 15 proteins were identified in pomegranate nanovesicles. Class III chitinase protein is an enzyme that breaks down the β-1,4-glycosidic bond of chitin, and provides metal storage capacity in pomegranate. It is known to provide. It was confirmed that this enzyme is present in nanovesicles, and also a lipid transfer protein that transfers fat proteins was identified. Chalcone synthase exists in many plants and is known to play a role in flavonoid biosynthesis. These proteins were found to be present in pomegranate nanovesicles.
Figure PCTKR2020013143-appb-T000001
Figure PCTKR2020013143-appb-T000001
7. 석류 나노소포체에서 엘라그산(ellagic acid) 함량 확인7. Confirmation of ellagic acid content in pomegranate nanovesicles
도 7은 본 발명의 일 실험예에 따른 석류 나노소포체 내 엘라그산의 함량을 분석한 그래프로, HPLC 분석을 통해서 석류 나노소포체 내 엘라그산을 분석한 결과, 도 7과 같이, 석류 나노소포체는 엘라그산을 함유하고 있었으며, 3개의 석류 나노소포체 시료에서 각각 61. 5502, 62.05329 및 61.61564 mg/L의 엘라그산을 포함하고 있음을 확인할 수 있었다. 석류의 주요 성분인 엘라그산이 나노소포체에도 포함하고 있음을 알 수 있었으며, 아주 중요한 발견이라고 사료된다.7 is a graph analyzing the content of ellagic acid in the pomegranate nanovesicles according to an experimental example of the present invention. As a result of analyzing the ellagic acid in the pomegranate nanovesicles through HPLC analysis, as shown in FIG. The acid was contained, and it could be confirmed that the three pomegranate nanovesicle samples contained 61.5502, 62.05329 and 61.61564 mg/L of ellagic acid, respectively. It was found that ellagic acid, a major component of pomegranate, is also contained in nanovesicles, and it is considered a very important discovery.
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 즉, 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다.As described above, a specific part of the present invention has been described in detail, and for those of ordinary skill in the art, it is obvious that this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby. Do. That is, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims (12)

  1. 석류(Punica granatum)로부터 유래된 항암 활성을 가지는 세포외 소포체(extracellular vesicle).Pomegranate ( Punica granatum ) derived from extracellular vesicles having anticancer activity.
  2. 제 1 항에 있어서,The method of claim 1,
    상기 세포외 소포체는,The extracellular vesicles,
    석류 분쇄물을 250 내지 750 g에서 5 내지 15분간 1차 원심분리하고, 상기 1차 원심분리로 수득한 상층액을 1,000 내지 3,000 g에서 10 내지 30분간 2차 원심분리하며, 상기 2차 원심분리로 수득한 상층액을 5,000 내지 15,000 g에서 15 내지 45분간 3차 원심분리하고, 상기 3차 원심분리로 수득한 상층액을 50,000 내지 150,000 g에서 1 내지 3시간 4차 원심분리하여 수득한 것을 특징으로 하는, 세포외 소포체. The pomegranate crushed product was first centrifuged at 250 to 750 g for 5 to 15 minutes, and the supernatant obtained by the first centrifugation was secondarily centrifuged at 1,000 to 3,000 g for 10 to 30 minutes, and the second centrifugation The supernatant obtained by 3rd centrifugation at 5,000 to 15,000 g for 15 to 45 minutes, and the supernatant obtained by the 3rd centrifugation at 50,000 to 150,000 g for 1 to 3 hours obtained by 4th centrifugation. As an extracellular vesicle.
  3. 제 1 항에 있어서,The method of claim 1,
    상기 세포외 소포체는,The extracellular vesicles,
    평균 입경이 50 내지 300 nm인 나노소포체인 것을 특징으로 하는, 세포외 소포체. Extracellular vesicles, characterized in that the nanovesicles having an average particle diameter of 50 to 300 nm.
  4. 제 1 항에 있어서,The method of claim 1,
    상기 세포외 소포체는,The extracellular vesicles,
    암 세포 내로 침투하는 것을 특징으로 하는, 세포외 소포체. Extracellular endoplasmic reticulum, characterized in that it penetrates into cancer cells.
  5. 제 1 항에 있어서,The method of claim 1,
    상기 세포외 소포체는,The extracellular vesicles,
    암 세포의 증식, 이동 또는 침윤을 억제하는 것을 특징으로 하는, 세포외 소포체. Extracellular endoplasmic reticulum, characterized in that it inhibits the proliferation, migration or invasion of cancer cells.
  6. 제 1 항에 있어서,The method of claim 1,
    상기 세포외 소포체는,The extracellular vesicles,
    엘라그산(ellagic acid), 클래스 III 키티나아제(class III chitinase), 지질 전달 단백질(lipid transfer protein) 및 칼콘 합성효소(chalcone synthase)로 이루어진 군에서 선택되는 하나 또는 둘 이상을 포함하는 것을 특징으로 하는, 세포외 소포체. Characterized in that it comprises one or two or more selected from the group consisting of ellagic acid, class III chitinase, lipid transfer protein, and chalcone synthase. Which, extracellular endoplasmic reticulum.
  7. 제 1 항 내지 제 6 항 중 어느 한 항에 따른 세포외 소포체를 유효성분으로 포함하는 암 예방 또는 치료용 약학 조성물.A pharmaceutical composition for preventing or treating cancer comprising the extracellular vesicle according to any one of claims 1 to 6 as an active ingredient.
  8. 제 7 항에 있어서,The method of claim 7,
    상기 조성물은,The composition,
    암 세포 내로 침투하여 암 세포의 증식, 이동 또는 침윤을 억제하는 것을 특징으로 하는, 암 예방 또는 치료용 약학 조성물. A pharmaceutical composition for preventing or treating cancer, characterized in that it penetrates into cancer cells to inhibit proliferation, migration or invasion of cancer cells.
  9. 제 7 항에 있어서,The method of claim 7,
    상기 암은,The cancer,
    유방암, 폐암, 간암, 위암, 대장암, 신장암, 방광암, 급성 골수성 백혈병, 급성 림프구성 백혈병, 자궁암, 난소암, 후두암, 전립선암, 갑상선암, 두부 또는 경부암, 뇌암 및 혈액암으로 이루어진 군에서 선택되는 질환인 것을 특징으로 하는, 암 예방 또는 치료용 약학 조성물. Select from the group consisting of breast cancer, lung cancer, liver cancer, gastric cancer, colon cancer, kidney cancer, bladder cancer, acute myeloid leukemia, acute lymphocytic leukemia, uterine cancer, ovarian cancer, laryngeal cancer, prostate cancer, thyroid cancer, head or neck cancer, brain cancer and blood cancer. A pharmaceutical composition for preventing or treating cancer, characterized in that it is a disease.
  10. 제 1 항 내지 제 6 항 중 어느 한 항에 따른 세포외 소포체를 유효성분으로 포함하는 암 예방 또는 개선용 건강기능식품 조성물.A health functional food composition for preventing or improving cancer comprising the extracellular vesicles according to any one of claims 1 to 6 as an active ingredient.
  11. 석류 분쇄물을 제조하는 단계;Preparing a pomegranate crushed product;
    상기 제조된 석류 분쇄물을 250 내지 750 g에서 5 내지 15분간 1차 원심분리하는 단계;First centrifuging the prepared pomegranate crushed product at 250 to 750 g for 5 to 15 minutes;
    상기 1차 원심분리하여 수득한 상층액을 1,000 내지 3,000 g에서 10 내지 30분간 2차 원심분리하는 단계;Second centrifuging the supernatant obtained by the first centrifugation at 1,000 to 3,000 g for 10 to 30 minutes;
    상기 2차 원심분리하여 수득한 상층액을 5,000 내지 15,000 g에서 15 내지 45분간 3차 원심분리하는 단계; 및Third centrifuging the supernatant obtained by the second centrifugation at 5,000 to 15,000 g for 15 to 45 minutes; And
    상기 3차 원심분리하여 수득한 상층액을 50,000 내지 150,000 g에서 1 내지 3시간 4차 원심분리하여 펠렛을 수득하는 단계;를 포함하는 석류(Punica granatum) 유래 세포외 소포체 제조방법. Pomegranate (Punica granatum )-derived extracellular vesicle production method comprising; the step of obtaining a pellet by performing the fourth centrifugation for 1 to 3 hours at 50,000 to 150,000 g of the supernatant obtained by the third centrifugation.
  12. 제 11 항에 있어서,The method of claim 11,
    상기 원심분리 단계는,The centrifugation step,
    1회 수행되거나 또는 2회 내지 5회 반복하여 수행되는 것을 특징으로 하는, 세포외 소포체 제조방법.A method for producing extracellular vesicles, characterized in that it is performed once or repeatedly performed 2 to 5 times.
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