KR102379681B1 - Yam derived extracellular vesicles and use thereof - Google Patents

Abstract

The present invention relates to an extracellular vesicle derived from hemp ( Dioscorea japonica Thunb ) and uses thereof. It provides a composition and a health functional food composition.
The hemp-derived extracellular vesicles can penetrate into cancer cells and have excellent anticancer activity, such as effectively inhibiting proliferation, invasion, and migration of cancer cells, and thus can be provided as an effective anticancer composition.

Description

Hemp-derived extracellular vesicles and uses thereof {YAM DERIVED EXTRACELLULAR VESICLES AND USE THEREOF}

The present invention relates to extracellular vesicles derived from hemp ( Dioscorea japonica Thunb ) and uses thereof.

Cancer is one of the incurable diseases that humanity needs to solve, and huge capital is being invested in development to cure it worldwide. More than 10,000 people are being diagnosed, and more than 60,000 people are dying.

The number of cancer patients in Koreans is gradually increasing, and among them, breast cancer is reported to be the most common cancer among women. According to the 2016 national cancer registration statistics in Korea collected through the National Cancer Registration Statistics Project on December 27, 2018 by the Ministry of Health and Welfare and the Central Cancer Registry, the number of new cancer patients in 2016 was 229,180 (120 thousand males). 68 people, 109,112 females), an increase of 12,638 (5.8%) compared to the previous year (216,542). Stomach cancer was the most common cancer among men and women, followed by colorectal cancer, thyroid cancer, lung cancer, breast cancer, liver cancer, and prostate cancer. Compared with 2015, breast cancer ranks first in cancer incidence among women, and the incidence rate is continuously increasing.

Due to recent medical developments, various methods such as surgical treatment, radiation treatment, and chemotherapy have been improved, and the 5-year relative survival rate of breast cancer is 92.7%. Psychological symptoms can reduce quality of life.

Depending on the difficult treatment method at the time of onset and the deterioration of the quality of life after treatment, conventional chemotherapy, which is treated with chemotherapy, or surgical treatment such as partial mastectomy (breast-conserving surgery) and total mastectomy, high-energy radiation Unlike radiotherapy, which uses chemotherapy to treat tumors, people's interest in developing cancer prevention and treatment therapies that have fewer side effects and minimize pain on the human body has increased. Accordingly, there has been a lot of interest in plant-derived natural substances that can be easily obtained from nature, have fewer side effects, and can effectively treat cancer.

Extracellular Vesicles (EVs) are substances that come out during cell activity, and are also referred to as nano-vesicles with a size of 10 ⁹ 1m. The extracellular vesicles are divided into three groups: exosomes, microvesicles, and apoptotic bodies. The origin of exosomes is the endocytic pathway, and the size is 30-100 nm, and it can be observed in body fluids such as blood and urine of multicellular eukaryotic organisms and in the culture medium of eukaryotic cells. The origin of microvesicles is the plasma membrane, and the size is 50-1,000 nm. 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.

Recently, there is a report that nano-ERs are secreted from food, and the secreted nano-ERs are known to be used safely as drug carriers as well as for physiologically active roles. However, the anticancer effect of nano-endoplasmic reticulum secreted from hemp has not yet been reported.

Republic of Korea Patent Publication No. 10-2004-0033104 (published on April 21, 2004)

It is an object of the present invention to provide an extracellular vesicle derived from a natural product.

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 producing the extracellular vesicles.

In order to achieve the above object, the present invention provides an extracellular vesicle (extracellular vesicle) having anticancer activity, derived from hemp ( Dioscorea japonica Thunb ).

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.

In addition, the present invention comprises the steps of preparing a ground hemp; Primary centrifugation of the prepared hemp pulverized material at 250 to 750 g for 5 to 15 minutes; Second centrifugation of the supernatant obtained by the first centrifugation at 1,000 to 3,000 g for 10 to 30 minutes; Third centrifugation of the supernatant obtained by the second centrifugation at 5,000 to 15,000 g for 15 to 45 minutes; and obtaining a pellet by fourth centrifuging the supernatant obtained by the third centrifugation at 50,000 to 150,000 g for 1 to 3 hours to obtain a pellet ; .

The hemp-derived extracellular vesicles according to the present invention can penetrate into cancer cells and have excellent anticancer activity, such as effectively inhibiting the proliferation, invasion, and migration of cancer cells. Accordingly, the hemp-derived extracellular vesicles can be provided as a pharmaceutical composition or a health functional food composition for preventing, improving or treating cancer diseases.

In addition, by using the hemp-derived extracellular vesicles according to the present invention, ingestion and long-term storage are easy, and there is an advantage in that it can exhibit excellent anticancer effects even without ingesting a large amount of hemp.

Figure 1 schematically shows a method for isolating the nano-ER from hemp.
Figure 2 is a graph analyzing the size of the hemp nano-endoplasmic reticulum according to an experimental example of the present invention.
3 is a graph showing the proliferation rate of breast cancer cells according to the concentration of hemp nano-ER according to an experimental example of the present invention.
4 is a graph showing the effect on the infiltration of breast cancer cells according to the concentration of hemp nano-ER according to an experimental example of the present invention.
Figure 5 shows the degree of migration of breast cancer cells according to the concentration of hemp nano-ER according to an experimental example of the present invention.
6 is an image showing the penetration of hema nanovesicles into breast cancer cells according to an experimental example of the present invention.
7 is a graph showing the proliferation rate of breast cancer cells according to the concentration of hemp nano-ER and diosgenin according to an experimental example of the present invention.
8 is a graph analyzing the size of the hemp nano-ER after freezing storage according to an experimental example of the present invention.

Hereinafter, the present invention will be described in detail.

The present inventors have completed the present invention by confirming that the nano-endoplasmic reticulum isolated from hemp effectively inhibits the proliferation, invasion, migration, etc. of breast cancer cells.

The present invention provides an extracellular vesicle having anticancer activity.

The extracellular vesicles may be derived from hemp ( Dioscorea japonica Thunb ).

In the present specification, "Hemp ( Dioscorea japonica Thunb )" is a vine-like perennial plant of the Liliaceae family of monocotyledonous plants distributed in Korea, Japan, Taiwan, China, etc., which is native to the mountains or is also cultivated as a medicinal plant. In oriental medicine, the tuber root is called sanyak, and it is edible or used medicinally as a tonic, gangjeong, and antidiarrheal agent.

The hemp may be a tuber root, a vine stem, a leaf, a flower, or a mixture thereof, and preferably a tuber root portion, but is not limited thereto.

As used herein, "extracellular vesicle (EV)" refers to a small vesicle with a membrane structure secreted from various cells, and refers to a vesicle that is released into the extracellular environment due to the fusion of the polycystic body and the plasma membrane. (Nanovesicle) can also be defined.

In the present invention, the extracellular vesicles may include, but are not limited to, exosomes and microvesicles.

The extracellular vesicles may be nano-endoplasmic reticulum having an average particle diameter of 50 to 300 nm, and preferably may have an average particle diameter of 100 to 200 nm, but is not limited thereto.

In the present invention, the extracellular vesicles are ultracentrifugation (ultracentrifugation), density gradient centrifugation (density gradient centrifugation), ultrafiltration (ultrafiltration), size exclusion chromatography (size exclusion chromatography), ion exchange chromatography (ion) exchange chromatography, immunoaffinity capture, microfluidics-based isolation, exosome precipitation, total exosome isolation kit, or polymer based precipitation ) may be separated from hemp using a method such as, but is not limited thereto.

Preferably, the extracellular vesicles may be obtained by centrifuging hemp, and more preferably, the extracellular vesicles are obtained by first centrifuging the ground hemp at 250 to 750 g for 5 to 15 minutes, and the first centrifugation Second centrifugation of the supernatant obtained by separation at 1,000 to 3,000 g for 10 to 30 minutes, and third centrifugation of the supernatant obtained by the second centrifugation at 5,000 to 15,000 g for 15 to 45 minutes, the above 3 The supernatant obtained by secondary centrifugation is obtained by 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, but limited thereto it is not going to be

The pulverized hemp may be pulverized by putting hemp in a phosphate buffer solution (PBS), but is not limited thereto, and may be directly prepared from hemp juice or extract, or commercially sold hemp pulverized product, hemp juice It may be purchased with water or extract.

The extracellular vesicles obtained according to the above may contain dioscorin protein.

In the present specification, "dioscorin (dioscorin)" is a water-soluble protein contained in Yam and is known to have an antioxidant action.

According to an experimental example of the present invention, as it was confirmed for the first time that the dioscorin protein is included in the nano-ER isolated from the hemp, the protein can be determined as a component exhibiting the anticancer activity of the hemp-derived nano-ER.

In the present invention, the extracellular vesicles may penetrate into cancer cells, and may have anticancer activity to inhibit the proliferation, migration or invasion of cancer cells.

According to an experimental example of the present invention, it was confirmed that the extracellular vesicles penetrate into the cells in breast cancer cells or lung cancer cells, and it was confirmed that the proliferation, invasion and migration of the cancer cells were significantly inhibited.

Accordingly, 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 hemp-derived extracellular vesicles as an active ingredient.

The composition may include 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, colorectal 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 consisting of It may be a disease selected from the group, but is not limited thereto.

The pharmaceutical composition according to the present invention may be prepared according to a conventional method in the pharmaceutical field. The pharmaceutical composition may be combined with a suitable pharmaceutically acceptable carrier according to the formulation, and if necessary, excipients, diluents, dispersants, emulsifiers, buffers, stabilizers, binders, disintegrants, solvents, etc. may be prepared further comprising there is. The appropriate carrier and the like do not inhibit the activity and properties of the hemp-derived extracellular vesicle 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, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxy benzoate, propyl hydroxy benzoate, talc, magnesium stearate and mineral oil. When formulating the composition, it can be prepared using a diluent or excipient such as a filler, extender, binder, wetting agent, disintegrant, surfactant, etc. commonly used.

The pharmaceutical composition according to the present invention can be applied in any dosage form, and specifically, oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterilization according to conventional methods It may be formulated and used in the form of an injection solution, and preferably may be formulated into a unit dosage form suitable for oral administration.

More specifically, the solid dosage form among the oral dosage forms is in the form of tablets, pills, powders, granules, capsules, etc., and at least one or more excipients, for example, starch, calcium carbonate, sucrose, lactose, sorbitol, mannitol , cellulose, gelatin, etc. can be mixed and prepared, and lubricants such as magnesium stearate and talc in addition to simple excipients may be included. In addition, the capsule formulation may further include a liquid carrier such as fatty oil in addition to the above-mentioned substances.

Among the oral dosage forms, liquid formulations include suspensions, solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients, for example, wetting agents, sweeteners, fragrances, preservatives, etc. may be included. there is.

The parenteral formulation may include a sterile aqueous solution, a non-aqueous solution, a suspension, an emulsion, an injection, a freeze-dried formulation, a suppository, and the like. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, Tween 61, cacao butter, laurin fat, glycerogelatin, and the like can be used. Without being limited thereto, any suitable formulation well known in the art may be used.

In addition, the pharmaceutical composition according to the present invention may further add an antioxidant to enhance therapeutic efficacy. The antioxidant includes thiamin (vitamin B1), riboflavin (vitamin B2), niacin (vitamin B3), pantothenic acid (vitamin B5), pyridoxine (vitamin B6) and cobalamin (cobalamin, Vitamin B group compounds such as vitamin B12) and vitamin C, vitamin D, vitamin E, etc. may be used, but are not limited thereto, and all suitable formulations well known in the art may be used.

The pharmaceutical composition according to the present invention may be administered in a pharmaceutically effective amount.

As used herein, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment and not to cause side effects.

The effective dose level of the pharmaceutical composition may be determined according to the purpose of use, the age, sex, weight and health status of the patient, the type of disease, the severity, the activity of the drug, the sensitivity to the drug, the administration method, the administration time, the administration route and the excretion rate, the treatment The duration, formulation or concomitant use may be determined differently depending on factors including 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 capable of developing a cancer disease, and the animal may include, for example, not only humans and primates, but also livestock such as cattle, pigs, horses, and dogs. .

The pharmaceutical composition may be administered by an appropriate administration route according to the form of the formulation, and may be administered through various routes, either oral or parenteral, as long as it can reach the target tissue. The administration method is not particularly limited, and for example, oral, rectal or intravenous, muscle, skin application, respiratory inhalation, intrauterine dural or intracerebroventricular injection, etc. can be administered in a conventional manner. there is.

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 hemp-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, colorectal 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 consisting of It may be a disease selected from the group, but is not limited thereto.

Corresponding features may be substituted for the above-mentioned parts.

As used herein, the term "health functional food" includes food manufactured and processed using raw materials or ingredients useful for the human body according to Act No. 6727 on Health Functional Foods, and in addition to nutrition, the purpose of the present invention It refers to foods with high medical and medical effects that are processed to efficiently exhibit bioregulatory functions such as prevention, improvement, bio-defense, immunity, and recovery from cancer.

The health functional food according to the present invention may be prepared in the form of powder, granules, tablets, capsules, syrups or beverages for the purpose of preventing or improving cancer. There is no limitation in the form that the health functional food can take, and it can be formulated in the same manner as the pharmaceutical composition and used as a functional food or added to various foods.

The health functional food may include any food in a conventional sense. For example, beverages and various drinks, fruits and their processed foods (canned fruit, jam, etc.), fish, meat and their processed foods (ham, bacon, etc.), breads and noodles, cookies and snacks, dairy products (butter, cheese, etc.) ), etc. are possible, and may include all functional foods in a conventional sense. It may also include food used as feed for animals.

The health functional food composition according to the present invention may be prepared by further including pharmaceutically acceptable food additives (food additives) and other suitable auxiliary ingredients commonly used in the art. Whether or not it is suitable as a food additive can be judged according to the standards and standards for the relevant item in accordance with the general rules and general test methods of the Food Additives Code approved by the Ministry of Food and Drug Safety, unless otherwise specified. The items listed in the 'Food Additives Codex' include, for example, chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; natural additives such as dark pigment, licorice extract, crystalline cellulose, high pigment, and guar gum; Mixed preparations, such as a sodium L-glutamate preparation, a noodle-added alkali agent, a preservative preparation, and a tar color preparation, etc. are mentioned.

The other auxiliary ingredients include, for example, flavoring agents, natural carbohydrates, sweeteners, vitamins, electrolytes, coloring agents, pectic acid, alginic acid, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonation agents, etc. may further contain. In particular, as the natural carbohydrate, monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol can be used. , As the sweetener, natural sweeteners such as taumatine and stevia extract, or synthetic sweeteners such as saccharin and aspartame can 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 its use, such as prevention or improvement of cancer.

The health functional food composition has the advantage that there are no side effects that may occur during long-term administration of general drugs by using food as a raw material, and has excellent portability, and can be taken as an adjuvant for the prevention or improvement of cancer.

In addition, the present invention provides a method for producing hemp-derived extracellular vesicles.

The manufacturing method comprises the steps of preparing a ground hemp; Primary centrifugation of the prepared hemp pulverized material at 250 to 750 g for 5 to 15 minutes; Second centrifugation of the supernatant obtained by the first centrifugation at 1,000 to 3,000 g for 10 to 30 minutes; Third centrifugation of 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 of the supernatant obtained by the third centrifugation at 50,000 to 150,000 g for 1 to 3 hours.

In the above manufacturing method, the step of preparing the ground hemp may be performed by washing the hemp and then pulverizing or juicing the hemp by putting it in a phosphate buffer solution (PBS).

In the above manufacturing method, the first to fourth centrifugation may be performed once or repeated two or more times, and in particular, the third centrifugation step is performed once or repeated two to five times. may be performed, but is not limited thereto.

The manufacturing method may further include a first or more centrifugation step.

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 performed by removing the supernatant obtained in the first centrifugation step at 2,000 g at 20 It is performed once for 1 minute, 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 By carrying out once for 2 hours at 100,000 g of the obtained supernatant, hemp-derived extracellular vesicles were prepared.

Through the manufacturing method as described above, it is possible to effectively obtain high-purity hemp-derived extracellular vesicles having excellent anticancer activity and uniform particle size.

In addition, the preparation method is, in addition to the centrifugation method, ultrafiltration, size exclusion chromatography, ion exchange chromatography, immunoaffinity capture, microfluid Microfluidics-based isolation, exosome precipitation, total exosome isolation kit, or polymer based precipitation may be used, but the present invention is not limited thereto Separation or purification methods well known in the art may be further included.

Hereinafter, examples will be described in detail to help the understanding of the present invention. However, the following examples are merely illustrative of the content 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 explain the present invention to those of ordinary skill in the art.

<Experimental Example 1> Characterization of nano-vesicles isolated from hemp

<Experiment method>

1. Hemp nano-vesicle separation

The hemp used in the experimental example of the present invention was harvested in Andong, Gyeongsangbuk-do, purchased from the Nonghyup. The purchased hemp was washed with clean water so that there was no dirt. After washing, the skin of the hemp was removed, and 150 g of hemp and 500 mL of 1×PBS (phosphate buffer saline) were put in a blender and ground for 5 minutes. 50 mL of the previously ground hemp was placed in a 50 mL conical tube.

Figure 1 schematically shows a method of isolating the nanovesicles from hemp, referring to this, after adjusting the weight to a constant, and returning it once at 500 g for 10 minutes using a centrifuge, a new 50 mL of the supernatant Transfer to a conical tube and return it once for 20 minutes at 2,000 g, then transfer the supernatant to a new 50 mL conical tube and spin it twice at 10,000 g for 30 minutes each, then put the supernatant in a new tube again and wash it with 100,000 g. After returning for 2 hours, the supernatant was discarded and the remaining nano-endoplasmic reticulum pellet was dried, and then dissolved by adding 1 mL of 1× PBS.

2. Nano particle tracking analysis (NTA)

Malvern's NanoSight was used to analyze extracellular vesicles (EVs) isolated from horses. 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. Quantification of Hema Nanovesicle Proteins

Hemp nano-ER protein was quantified by the bicinchoninic acid (BCA, Termo Fishers) method after crushing by adding 1× RIPA solution.

4. Breast Cancer Cell Culture

MDA-MB-231 breast cancer cells were harvested from the ATCC cell line. MDA-MB-231 breast cancer cells were cultured in RPMI-1640 medium, 5% fetal bovine serum, and 1% antibiotics added for culture in an incubator at 5% CO ₂ and 37°C. Cells were replaced with media every 2-3 days.

5. Cell proliferation assay (MTT assay)

MDA-MB-231 breast cancer cells were placed in each well of a 96-well plate so that the number of cells was 2.5 × 10 ⁴ cells/mL, and incubated in RPMI 1640 growth medium for about 24 hours to attach to the plate. Then, the growth medium was removed and the hemp nanovesicles were treated with concentrations of 0, 10, 50, and 100 μg/mL. 37° C., 5% CO ₂ Incubated in an incubator maintained for about 24 hours. Thereafter, a solution of 2 mg/mL MTT 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. Cell migration assay (Migration assay)

MDA-MB-231 breast cancer cells were placed in 1 mL of 6 wells and cultured in an incubator at 5% CO ₂ and 37 °C. After the cells were wounded, the healing of the cells was observed under a microscope.

7. Cell invasion assay (Invasion assay)

300 μL of MDA-MB-231 breast cancer cells suspended in serum free medium was placed in an insert coated with a rehydrated 8 μm pore size polycarbonate membrane, and the outside 500 μL of medium containing 10% FBS was treated in the side chamber to create a nutrient-rich environment that induces cell infiltration in the outer chamber of the membrane. MDA-MB-231 breast cancer cells were treated with vascular endothelial growth factor (VEGF), a positive control, at a concentration of 100 ng/mL, treated with hemp nano-ERs by concentration, and then cultured at 37°C for 24 hours. After removing the non-infiltrating cells, cells outside the infiltrated membrane were stained with a cell stain solution. The number of stained cells was counted.

8. MMP-9 (Matrix metallopeptidase 9) ELISA assay

In the MMP-9 ELISA (Enzyme-Linked ImmunoSorbent Assay, enzyme-linked immunosorbent assay, R&D system) experiment, an enzyme was labeled with an antibody (or antigen) and the enzyme activity was tested using the company's protocol.

9. DID labeling

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. After that, each well was treated with DID-labeled hemp nanovesicles, and after 24 hours, the medium was removed, washed with PBS, and then treated with 70% ethanol. After DAPI (4',6-diamidino-2-phenylindol) staining, it was checked whether hema nanovesicles had penetrated into the cells under a fluorescence microscope.

10. Proteomics analysis

Hemp nanovesicles were treated with trypsin buffer (500 ng/μL trypsin, 50 mM ammonium bicarbonate) and reacted at 37° C. for 16 hours. After stopping the action of trypsin by adding 5% formic acid, the peptide was extracted using 25 mM triethylammonium bicarbonate (TEABC) and acetonitrile (ACN). The extracted peptides were completely dried using a vacuum dryer, and immediately before analysis, they were dissolved in A buffer (0.1% formic acid) and analyzed by mass spectrometry. The data obtained by mass spectrometry was 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. Statistical processing

The results were obtained through three or more repeated experiments and expressed as the mean ± standard deviation for each sample concentration. For each sample concentration group, a value of p<0.05 was considered as a statistically significant result after Student's t test compared to the control group.

<Experiment result>

1. Check the size of hemp nanovesicles

Figure 2 is a graph analyzing the size of the hemp nano-ER according to an experimental example of the present invention. Referring to this, as a result of nanoparticle tracking analysis (NTA) of the hemp nano-ER separated according to FIG. 1, a diameter of 168 nm was able to confirm the size of the nano-ER.

2. Confirmation of breast cancer cell survival inhibitory effect of hemp nano-endoplasmic reticulum

3 is a graph showing the proliferation rate of breast cancer cells according to the concentration of hemp nano-ER according to an experimental example of the present invention.

In order to confirm the effect of hema nanovesicles on the survival of MDA-MB-231 breast cancer cells, each concentration was treated and observed through MTT assay, as shown in FIG. When treated for 24 hours, the cell viability for each concentration was 100, 92, 85, and 78%, indicating a significant decrease in cell viability in a concentration-dependent manner from a concentration of 10 μg compared to the control.

Therefore, it can be confirmed that hema nanovesicles induce apoptosis in a concentration-dependent manner in MDA-MB-231 breast cancer cells at a concentration of 10 μg or more, which is the same result as the hema nanovesicles reduced the cell viability in a concentration-dependent manner in breast cancer cells. , Through these results, it can be judged that hemp nanovesicles have an inhibitory effect on cancer cells.

3. Confirmation of the inhibitory effect of hema nanovesicles on breast cancer cell invasion

Figure 4 is a graph showing the effect on the infiltration of breast cancer cells according to the concentration of hemp nano-ER according to an experimental example of the present invention. It was shown that the infiltration of MDA-MB-231 breast cancer cells was decreased in a concentration-dependent manner.

Therefore, it can be confirmed that hema nanovesicles inhibit cell invasion in a concentration-dependent manner in MDA-MB-231 breast cancer cells, and through these results, it can be determined that hemp nanovesicles have an inhibitory effect on cancer cells.

4. Confirmation of the inhibitory effect of hema nanovesicles on breast cancer cell migration

Figure 5 shows the degree of movement of breast cancer cells according to the concentration of hemp nanovesicles according to an experimental example of the present invention. It was found that the migration of MDA-MB-231 breast cancer cells was decreased in a concentration-dependent manner.

Therefore, it can be confirmed that hema 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 hemp nanovesicles have an inhibitory effect on cancer cells.

5. Confirmation of breast cancer cell internalization effect of hemp nanoendoplasmic reticulum

6 is an image showing the penetration of hemp nanovesicles into breast cancer cells according to an experimental example of the present invention. A sample in which DID was removed after labling and 1:200 DID reagent was added to the concentration of 10 μg/mL of hemp nanovesicles. After treatment with MDA-MB-231 breast cancer cells for 24 hours, the presence in the cytoplasm was confirmed with a fluorescence microscope. As a result, it was confirmed that hema nanovesicles penetrated into MIA-MB-231 breast cancer cells.

Therefore, it can be determined that hema nanovesicles have an inhibitory effect on cancer cells by penetrating the MBA-MD-231 breast cancer cells into cells.

6. Protein identification of hemp nanoER

Proteomics analysis was performed after making a peptide through trypsin digestion of hemp nano-ER. As a result, as shown in Table 1 below, eight proteins were identified in the hemp nanoER. Several types of dioscorin proteins have been identified. Dioscorin is known as a hemp storage protein and is known to have various physiologically active functions such as antioxidant and anti-inflammatory. However, it was confirmed for the first time that the nano-ER-dioscorin exists in the form, so it is thought that the dioscorin present in the nano-ER has an anticancer effect.

7. Confirmation of breast cancer cell survival inhibitory effect of hemp nanoendoplasmic reticulum and diosgenin

7 is a graph showing the proliferation rate of breast cancer cells according to the concentration of hemp nano-ER and diosgenin according to an experimental example of the present invention.

In order to confirm the effect of hemp nanoER and diosgenin on the survival of MDA-MB-231 breast cancer cells, each concentration was treated and observed through MTT assay. As shown in FIG. 7, hemp nanoER (EVs) 0, 5 , 10, and 20 μg concentration for 24 hours, the cell viability for each concentration showed a significant decrease with respect to cell viability from the concentration of 5 μg compared to the control.

It was found that even when treated with diosgenin of Formula 1, a type of saponin contained in hemp, the survival rate of MDA-MB-231 breast cancer cells was reduced at concentrations of 10 and 20 μg. It was confirmed that the hema nano-ER also had an inhibitory effect on cancer cells at a similar concentration, and this is the first report on the inhibitory effect of hemp nano-ER on cancer cell viability.

<Formula 1>

In addition, it was found that hemp nanovesicles (EVs) had a greater inhibitory effect on cancer cell viability than those of hemp microvesicles (MVs), which were larger in size than nanovesicles.

8. Confirmation of stability of cryopreservation of hemp nanovesicles

Figure 8 is a graph analyzing the size of the hemp nano-ER after cryopreservation according to an experimental example of the present invention, to confirm the safety of the frozen storage (-80 degree) of the hemp nano-ER, the size and concentration of the hemp nano-ER As a result of nanoparticle tracking analysis (NTA), it was confirmed that nano-vesicles with a diameter of 152 nm in the liquid suspension were confirmed, and had a size and concentration similar to that of 168 nm measured 1 year ago.

As described above in detail a specific part of the content of the present invention, for those of ordinary skill in the art, it is clear 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)

Derived from hemp ( Dioscorea japonica Thunb ), containing a dioscorin protein, characterized in that it has anticancer activity, the average particle diameter of the extracellular vesicles (extracellular vesicle) of 50 to 300 nm. The method of claim 1,
The extracellular vesicles,
The ground hemp is first centrifuged at 250 to 750 g for 5 to 15 minutes, the supernatant obtained by the first centrifugation is secondarily centrifuged at 1,000 to 3,000 g for 10 to 30 minutes, and the second centrifugation 3rd centrifugation of the supernatant obtained by 5,000 to 15,000 g for 15 to 45 minutes, and the fourth centrifugation of the supernatant obtained by the third centrifugation at 50,000 to 150,000 g for 1 to 3 hours to the extracellular endoplasmic reticulum. delete The method of claim 1,
The extracellular vesicles,
An extracellular endoplasmic reticulum, characterized in that it penetrates into cancer cells. The method of claim 1,
The extracellular vesicles,
An extracellular vesicle, characterized in that it inhibits the proliferation, migration or invasion of cancer cells. delete A pharmaceutical composition for preventing or treating cancer comprising the extracellular vesicles according to any one of claims 1, 2, 4 and 5 as an active ingredient. 8. The method of claim 7,
The composition is
A pharmaceutical composition for preventing or treating cancer, characterized in that it penetrates into cancer cells and inhibits the proliferation, migration or invasion of cancer cells. 8. The method of claim 7,
The cancer is
Select from the group consisting of breast cancer, lung cancer, liver cancer, stomach cancer, colorectal cancer, kidney cancer, bladder cancer, acute myeloid leukemia, acute lymphoblastic 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. A health functional food composition for preventing or improving cancer comprising the extracellular vesicles according to any one of claims 1, 2, 4 and 5 as an active ingredient. preparing a hemp pulverized product;
Primary centrifugation of the prepared hemp pulverized material at 250 to 750 g for 5 to 15 minutes;
Second centrifugation of the supernatant obtained by the first centrifugation at 1,000 to 3,000 g for 10 to 30 minutes;
Third centrifugation of the supernatant obtained by the second centrifugation at 5,000 to 15,000 g for 15 to 45 minutes; and
Hemp ( Dioscorea japonica Thunb )-derived extracellular vesicle production method comprising a; the supernatant obtained by the third centrifugation to obtain a pellet by a fourth centrifugation at 50,000 to 150,000 g for 1 to 3 hours. 12. The method of claim 11,
The centrifugation step is
Method for producing extracellular vesicles, characterized in that performed once or repeated 2 to 5 times.

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