KR20230138306A - Composition for preventing, treating or improving cancer or immune disease comprising Calystegia dahurica (Herb.) Choisy extract as active ingredients - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for the prevention or treatment of cancer or immune diseases containing an extract of Calystegia dahurica (Herb.) Choisy as an active ingredient. Specifically, the extract of Calystegia dahurica (Herb.) Choisy is a pharmaceutical composition for preventing or treating cancer or immune diseases. Since it was confirmed that it has the effect of increasing the degranulation activity of NK cells, cytotoxicity against cancer cells, and the expression of perforin and granzyme B, the extract of Convolvulus is used as a composition for preventing, treating, or improving cancer or immune diseases. It can be useful.

Description

Composition for preventing, treating or improving cancer or immune disease comprising Calystegia dahurica (Herb.) Choisy extract as active ingredients}

The present invention relates to a composition for preventing, treating or improving cancer or immune diseases containing an extract of Calystegia dahurica (Herb.) Choisy as an active ingredient, and more specifically, to a composition for preventing, treating or improving natural killer cells by activating natural killer cells. The present invention relates to a composition for preventing, treating or improving cancer or immune diseases containing as an active ingredient a degranulation activity, cytotoxicity against cancer cells, and an extract of Convolvulus convolvulus, which increases the expression of perforin and granzyme B.

Cancer is the number one cause of death and is one of the incurable diseases that is difficult to prevent and treat because it occurs in various areas due to various triggers such as smoking, ultraviolet rays, chemicals, food, and other environmental factors.

Many of the substances currently used as cancer treatments have significant toxicity and have problems with side effects due to their inability to specifically remove cancer cells. Therefore, research on immunotherapy that can specifically eliminate cancer cells is being actively conducted. Recently, it has been known that Natural Killer cells (NK cells) can effectively eliminate cancer stem cells, which play an important role in the recurrence of cancer, in addition to preventing the development, proliferation, and metastasis of cancer cells. Treatment is receiving particular attention.

Natural killer cells are immune cells that directly attack and destroy virus-infected cells or cancer cells. They play an important role in the innate immune response to fight viruses or cancer cells and the adaptive immune response through cytokine secretion. The main mechanism used by natural killer cells to kill target cells is to secrete cytolytic granules such as perforin and granzyme into target cells through the immunological synapse. Lin creates a hole in the cell membrane of an infected cell or cancer cell, and the granzyme that enters the cell through the hole induces apoptosis of the target cell.

As such, natural killer cells play an important role in the carcinogenesis process or in the defense mechanism in the early stages of viral infection, so there is a need to discover substances that increase the activity of natural killer cells in the treatment of cancer and immune diseases.

Meanwhile, Calystegia dahurica (Herb.) Choisy is a perennial herb of the Convolvulaceae family of dicotyledonous plants that grows in the fields of the Korean Peninsula, China, eastern Siberia, and Mongolia. The young shoots are eaten as a vegetable, and the underground stems are sweet. You can eat it by steaming it. Convolvulus ( Calystegia japonica ), a close relative of Convolvulus, is known to lower blood pressure and have a diuretic effect, but its value as a material for medicines and functional foods is not well known.

Accordingly, while researching the efficacy of natural substances to develop an effective treatment for cancer or immune diseases with fewer side effects, the present inventors confirmed that the extract of Convolvulus chinensis was excellent in activating natural killer cells and thus prevented cancer or immune diseases. , the present invention was achieved by revealing that it can be used as an active ingredient in a composition for treatment or improvement.

Republic of Korea Patent Publication No. 10-2014-0131049

The purpose of the present invention is to provide a pharmaceutical composition that contains natural substances as active ingredients, has fewer side effects, and can effectively prevent or treat cancer or immune diseases by activating natural killer cells.

Another object of the present invention is to provide a food composition that contains natural substances as active ingredients, has fewer side effects, and can effectively prevent or improve cancer or immune diseases by activating natural killer cells.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating cancer or immune diseases containing Calystegia dahurica (Herb.) Choisy ) extract as an active ingredient.

Here, the columbine extract is characterized in that it is extracted with water, C1 to C2 lower alcohol, or a mixed solvent thereof.

Furthermore, the extract of Convolvulus is characterized in that it is extracted with one or more solvents selected from the group consisting of water, methanol, ethanol, and mixed solvents thereof.

In addition, the extract of Convolvulus is characterized in that it is extracted with 70% to 90% ethanol as a solvent.

In addition, the extract of Convolvulus is characterized by activating Natural Killer cells (NK cells).

In addition, the extract of Convolvulus is characterized by increasing the degranulation activity of natural killer cells.

In addition, the above-mentioned Convolvulus extract is characterized by enhancing the cytotoxicity of natural killer cells against cancer cells.

In addition, the extract of Convolvulus is characterized by increasing the expression of granzyme B or perforin in natural killer cells.

Here, the cancer is selected from the group consisting of lung cancer, laryngeal cancer, liver cancer, stomach cancer, colon cancer, gallbladder cancer, pancreas cancer, bladder cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer, uterine cancer, kidney cancer, thyroid cancer, skin cancer, or blood cancer. It is characterized by

And the above blood cancers include leukemia, lymphoma, acute T-cell leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, myelodysplastic syndrome, chronic myeloid leukemia, chronic lymphocytic leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, and megaloblastic leukemia. , characterized in that it is selected from the group consisting of multiple myeloma, solitary myeloma, and erythroleukemia.

In addition, the immune disease is characterized in that it is selected from the group consisting of autoimmune disease, viral infection disease, and inflammatory disease.

Here, the autoimmune disease is characterized as being selected from the group consisting of multiple sclerosis, lupus, rheumatoid arthritis, Crohn's disease, and type 1 diabetes.

Furthermore, the viral infectious disease is characterized in that it is selected from the group consisting of acquired immunodeficiency syndrome, hepatitis B, hepatitis C, measles, herpes zoster, and papilloma virus.

And the inflammatory disease is characterized in that it is selected from the group consisting of asthma, sinusitis, atopy, periodontitis, and Behcet's disease.

In addition, the present invention provides a food composition for preventing or improving cancer or immune diseases containing an extract of Convolvulus chinensis as an active ingredient.

The extract of Calystegia dahurica (Herb.) Choisy of the present invention increases the degranulation activity of natural killer cells, cytotoxicity against cancer cells, and the expression of perforin and granzyme B through natural killer cell activation. Because it is effective, it can be usefully used as an active ingredient in pharmaceutical compositions and food compositions for preventing, treating or improving cancer or immune diseases.

Figure 1 shows CD107a (Lysosomal-associated membrane) on the surface of natural killer cells after administering Calystegia dahurica (Herb.) Choisy ) extract prepared through an example of the present invention at concentrations of 0.04, 0.2, and 0.4 mg/ml, respectively. This diagram shows the degranulation activity of natural killer cells according to the concentration of Moriflora extract through changes in protein 1; LAMP-1) expression.
Figure 2 shows the selective killing ratio of RMA-s cells to RMA cells after administering the sebum extract prepared through an example of the present invention into the mouse abdominal cavity at concentrations of 0.04, 0.2, and 0.4 mg/ml, respectively. This diagram shows the cytotoxic activity of natural killer cells against cancer cells according to the concentration.
Figure 3 is a diagram showing the mRNA expression level of perforin after administering the B. convolvulus extract prepared through an example of the present invention to natural killer cells at concentrations of 0.04, 0.2, and 0.4 mg/ml, respectively.
Figure 4 is a diagram illustrating the mRNA expression level of Granzyme B after administering the Enzyme flower extract prepared through an example of the present invention to natural killer cells at concentrations of 0.04, 0.2, and 0.4 mg/ml, respectively. .

Hereinafter, embodiments of the present invention will be described in detail so that those skilled in the art can easily implement it. Embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art. Accordingly, the embodiments of the present invention may be modified into various other forms, and the scope of the present invention is not limited to the embodiments described below.

The present invention provides a pharmaceutical composition for preventing or treating cancer or immune diseases containing Calystegia dahurica (Herb. Choisy ) extract as an active ingredient.

It is preferable that the above-mentioned Convolvulus extract is prepared by a manufacturing method comprising the following steps, but is not limited thereto:

1) Extracting by adding an extraction solvent to Convolvulus flowers;

2) filtering the Apiaceae extract of step 1);

3) concentrating the filtrate of step 2); and

4) Drying the concentrate of step 3).

In the above method, the convolvulus of step 1) can be used without limitation, whether cultivated or commercially available. The leaves, underground stems, stems, petals, flower buds, roots, or a combination of these can be used as the extract of the flowers.

In the above method, it is preferable to use water, alcohol, or a mixture thereof as the extraction solvent in step 1). It is preferable to use a C1 to C2 lower alcohol as the alcohol, and it is preferable to use ethanol or methanol as the lower alcohol. Specifically, the extract of Brimbrella columbine may be an ethanol extract of Brillion columbine. The extraction solvent is preferably 60% to 100% ethanol, more preferably 70% to 90% ethanol, but is not limited thereto. In a specific example of the present invention, the present inventors prepared an 80% ethanol extract of Convolvulus.

The extraction method is preferably, but not limited to, hot water extraction, shaking extraction, Soxhelt extraction, supercritical extraction, or reflux extraction. It is preferable to extract by adding 1 to 10 times the amount of the extraction solvent as the amount of dried Convolvulus, and more preferably to extract by adding 2 to 3 times the amount. The extraction temperature is preferably 40°C to 100°C, and most preferably 60°C to 80°C, but is not limited thereto. Additionally, the extraction time is preferably 1 to 5 hours, more preferably 2 to 4 hours, but is not limited thereto. In addition, the number of extractions is preferably 1 to 5, but is not limited thereto.

In the above method, the concentration in step 3) is preferably performed using a vacuum vacuum concentrator or a vacuum rotary evaporator, but is not limited thereto. In addition, drying is preferably performed by reduced pressure drying, vacuum drying, boiling drying, spray drying, or freeze drying, but is not limited thereto. The drying temperature is preferably 20°C to 60°C, and preferably 30°C to 50°C, but is not limited thereto. The drying time is preferably 10 to 40 hours, more preferably 20 to 30 hours, but is not limited thereto.

Meanwhile, in a specific example of the present invention, the present inventors confirmed the degranulation activity by comparing the expression level of CD107a on the surface of natural killer cells administered with the Curbweed extract, and as a result, as the concentration of the Curbweed extract increased, the concentration-dependent It was confirmed that the expression of CD107a on the surface of natural killer cells significantly increased, and that the degranulation activity of natural killer cells increased in a concentration-dependent manner as the concentration of Convolvulus extract increased (see Figure 1).

In addition, in a specific example of the present invention, the present inventors confirmed the cytotoxicity of cancer cells by comparing the cancer cell killing ability of natural killer cells administered with the above-mentioned Convolvulus extract, and as a result, as the concentration of the Convolvulus extract increased, RMA cells It was confirmed that the cytotoxicity of natural killer cells to cancer cells increases in a concentration-dependent manner as the concentration of the A. columbine extract increases, as the selective killing rate of RMA-s cells increases (see Figure 2).

In addition, in a specific example of the present invention, the present inventors confirmed the change in the mRNA expression level of perforin and granzyme B in natural killer cells administered with the extract of Convolvulus convolvulus, and as a result, the concentration of the extract of Convolvulus convolvulus was confirmed. It was confirmed that as the increases, the expression of perforin and granzyme B in natural killer cells increases in a concentration-dependent manner (see Figures 3 and 4).

Accordingly, the inventors of the present invention have found that the extract of Convolvulus chinensis of the present invention has the effect of increasing the degranulation activity of natural killer cells, cytotoxicity against cancer cells, and the expression of perforin and granzyme B through natural killer cell activation. As confirmed, the extract of Convolvulus chinensis of the present invention can be usefully used as a pharmaceutical composition for preventing or treating cancer or immune diseases.

The cancer of the present invention is selected from the group consisting of lung cancer, larynx cancer, liver cancer, stomach cancer, colon cancer, gallbladder cancer, pancreas cancer, bladder cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer, uterine cancer, kidney cancer, thyroid cancer, skin cancer, and blood cancer. It may be possible, but it is not limited to this. Here, the blood cancer includes leukemia, lymphoma, acute T-cell leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, myelodysplastic syndrome, chronic myeloid leukemia, chronic lymphocytic leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, and megaloblastic It may be selected from the group consisting of leukemia, multiple myeloma, solitary myeloma, and erythroleukemia, but is not limited thereto.

Additionally, the immune disease of the present invention may be selected from the group consisting of autoimmune disease, viral infection disease, and inflammatory disease. More specifically, the autoimmune disease may be selected from the group consisting of multiple sclerosis, lupus, rheumatoid arthritis, Crohn's disease, and type 1 diabetes, and the viral infectious disease may be an acquired disease. It may be selected from the group consisting of acquired immune deficiency syndrome (AIDS), hepatitis B, hepatitis C, measles, shingles, and papilloma virus, but is not limited thereto. The inflammatory disease may be selected from the group consisting of asthma, sinusitis, atopic dermatitis, periodontitis, and Behcet's disease, but is not limited thereto.

The pharmaceutical composition for preventing or treating cancer or immune diseases containing the extract of Convolvulus convolvulus of the present invention as an active ingredient may contain one or more active ingredients exhibiting the same or similar functions to the extract of Convolvulus convolvulus of the present invention.

The composition of the present invention may further include pharmaceutically acceptable additives, wherein the pharmaceutically acceptable additives include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, and lactose. , mannitol, taffy, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, lead carbauba, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate. , white sugar, dextrose, sorbitol, and talc can be used. The pharmaceutically acceptable additive according to the present invention is preferably contained in an amount of 0.1 to 90 parts by weight based on the composition, but is not limited thereto.

The composition of the present invention can be formulated according to a conventional method and used as a preparation. The preparation may be formulated as a preparation selected from the group consisting of tablets, capsules, injections, troches, powders, granules, solutions, suspensions, oral solutions, emulsions, syrups, suppositories, vaginal tablets and pills. It is not limited.

The composition can be formulated in various ways depending on the route of administration using a pharmaceutically acceptable carrier using methods known in the art. The carrier may be selected from one or two or more of diluents, lubricants, binders, disintegrants, sweeteners, stabilizers, and preservatives.

When formulating the composition, it can be prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, troches, etc. These solid preparations contain one or more excipients, such as starch, calcium carbonate, sucrose, and lactose, in the composition of the present invention. , or mixed with gelatin, etc. Additionally, in addition to simple excipients, lubricants such as magnesium styrate talc are also used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, or syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, they contain various excipients such as wetting agents, sweeteners, fragrances, and preservatives. You can. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, etc. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, wethepsol, macrogol, Tween 61, cacao, laurin, glycerogeratin, etc. can be used.

The composition of the present invention can be administered orally or parenterally according to the desired method. When administered parenterally, it is administered externally under the skin or intraperitoneally, intrarectally, subcutaneously, intravenously, intramuscularly, or intrathoracically. It is desirable to select . The dosage range varies depending on the patient's weight, age, gender, health condition, diet, administration time, administration method, excretion rate, and severity of the disease.

A dosage unit may contain, for example, 1, 2, 3 or 4 times the individual dosage or 1/2, 1/3 or 1/4 times the amount. The individual dosage specifically contains the amount of the active drug administered at one time, which usually corresponds to all, 1/2, 1/3 or 1/4 times the daily dose. The effective dose of the composition of the present invention may be 0.0001 to 10 g/kg, specifically 0.001 to 1 g/kg, and may be administered 1 to 6 times a day. However, since it may increase or decrease depending on the route of administration, severity of disease, gender, weight, age, etc., the above dosage does not limit the scope of the present invention in any way.

In addition, the present invention provides a food composition for preventing or improving cancer or immune diseases containing an extract of Convolvulus chinensis as an active ingredient.

The above-mentioned Morphospermia extract, cancer, and immune diseases are the same as the description of the pharmaceutical composition for preventing or treating cancer or immune diseases containing the Morphospermia extract as an active ingredient, and the above content is used for the specific description, and hereinafter, food Only the components specific to the composition will be described.

Meanwhile, in a specific embodiment of the present invention, in order to confirm the effect of the natural killer cell activation effect of the bovine columbine extract, the present inventors compared the expression level of CD107a on the surface of the natural killer cells administered with the bovine columbine extract to determine degranulation activity. As a result, the expression of CD107a on the surface of natural killer cells increased significantly in a concentration-dependent manner as the concentration of the Amaranthecus extract increased, and as the concentration of the B. convolvulus extract increased, the degranulation activity of natural killer cells increased in a concentration-dependent manner. was confirmed (see Figure 1).

In addition, in a specific example of the present invention, the present inventors confirmed the cytotoxicity of cancer cells by comparing the cancer cell killing ability of natural killer cells administered with the above-mentioned Convolvulus extract, and as a result, as the concentration of the Convolvulus extract increased, RMA cells It was confirmed that the cytotoxicity of natural killer cells to cancer cells increases in a concentration-dependent manner as the concentration of the A. columbine extract increases, as the selective killing rate of RMA-s cells increases (see Figure 2).

In addition, in a specific example of the present invention, the present inventors confirmed the change in the mRNA expression level of perforin and granzyme B in natural killer cells administered with the extract of Convolvulus convolvulus, and as a result, the concentration of the extract of Convolvulus convolvulus was confirmed. It was confirmed that the expression of perforin and granzyme B in natural killer cells increased in a concentration-dependent manner as increased (see Figures 3 and 4).

Accordingly, the inventors of the present invention have found that the extract of Convolvulus chinensis of the present invention has the effect of increasing the degranulation activity of natural killer cells, cytotoxicity against cancer cells, and the expression of perforin and granzyme B through natural killer cell activation. As confirmed, the extract of Convolvulus chinensis of the present invention can be usefully used as a food composition for preventing or improving cancer or immune diseases.

The food composition according to the present invention refers to a natural product or processed product containing one or more nutrients. For example, it may mean that it has undergone a certain degree of processing and is ready to be eaten directly, and is usually used as a food, Includes food additives, functional foods and beverages.

The food composition for preventing or improving cancer or immune disease containing the extract of Convolvulus chinensis as an active ingredient can be formulated in the same manner as the pharmaceutical composition and used as a functional food or added to various foods.

Examples of foods to which the extract of Convolvulus chinensis of the present invention can be added include meat, bread, chocolate, candy, pizza, gum, various soups, tea, drinks, alcoholic beverages and vitamin complexes, health supplements, functional foods, etc. . Additionally, in the present invention, foods include special nutritional foods (e.g., infant formula, infant food, etc.), processed meat products, fish products, tofu, jelly, noodles (e.g., ramen and noodles, etc.), and health supplements. , seasoned foods (e.g., soy sauce, soybean paste, red pepper paste, and mixed paste, etc.), sauces, confectionery (e.g., snacks), dairy products (e.g., fermented milk and cheese, etc.), other processed foods, kimchi, and pickled foods. It includes (e.g., various kimchi, pickled vegetables, etc.), beverages (e.g., fruit, vegetable drinks, soy milk, fermented beverages, etc.) and natural seasonings (e.g., ramen soup, etc.), in the usual sense. Includes all foods. The food, beverage or food additive can be manufactured by conventional manufacturing methods.

In the present invention, functional food refers to a food group or food composition in which added value is added to the food to function and express the function of the food for a specific purpose using physical, biochemical, biotechnological methods, etc., regulating biological defense rhythm, disease prevention and recovery. It refers to foods that have been designed and processed to sufficiently express body regulatory functions related to the body to the living body. The functional food may include food auxiliary additives that are foodologically acceptable, and may further include appropriate carriers, excipients, and diluents commonly used in the production of functional foods.

The food composition of the present invention contains various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavors, colorants and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts. , organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the food composition of the present invention may contain pulp for the production of natural fruit juice, fruit juice drinks, and vegetable drinks. The food composition of the present invention can use these ingredients independently or in combination. The proportion of these additives is not critical, but may be selected in the range of 0 parts by weight to 20 parts by weight per 100 parts by weight of the composition of the present invention.

In addition, in the food composition for preventing or improving cancer or immune diseases containing the extract of Convolvulus chinensis as an active ingredient, the content of the active ingredient is 0.0001% by weight to 99.99% by weight or 0.001% by weight to 99.99% by weight of the total food weight. % or 0.01% by weight to 90% by weight or 0.1% by weight to 50% by weight.

In addition, the intake amount of the food composition for preventing or improving cancer or immune diseases containing the extract of Convolvulus chinensis as an active ingredient varies depending on the condition and weight of the individual to be consumed, the degree of disease, drug type, administration route and period, It can be appropriately selected by a person skilled in the art. The administration method can be administered once a day or divided into several times, and the above dosage and administration method do not limit the scope of the present invention in any way.

Hereinafter, the present invention will be described in detail through examples, experimental examples, and production examples.

However, the following Examples, Experimental Examples, and Preparation Examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following Examples, Experimental Examples, and Preparation Examples.

<Example 1> Preparation of Convolvulus extract

An 80% ethanol solution (Sigma-Aldrich, St. Louis, Missouri) was added to the dried and ground bindweed flowers and extracted at 70°C for 3 hours. The obtained extract was filtered, concentrated under reduced pressure, and dried at 40° C. for 24 hours to prepare an extract of Convolvulus. The extract of Convolvulus used in the experiment was prepared by diluting it to 0.04, 0.2, and 0.4 mg/ml using physiological saline (Sigma-Aldrich, St. Louis, Missouri).

cell culture

Human natural killer cells were obtained from healthy donors after informed consent according to a protocol approved by the Institutional Review Board (IRB) of Catholic University of Pusan and the Declaration of Helsinki, and peripheral blood mononuclear cells from healthy donors were obtained. Mononuclear cells (PBMC) were separated using lymphocyte separation medium (LSM, MP Biomedicals, Santa Ana, CA, USA).

K562 cell line (chronic myeloid leukemia cell line) was purchased from ATCC (American Type Culture Collection, USA), and K562 cells were cultured in 10% Fetal Bovine Serum (FBS, Sigma) and 2 mM L-glutamine. The cells were cultured in supplemented IMDM (Iscove's modified Dulbecco's medium) at 37°C and 5% CO ₂ conditions.

RMA cells and RMA-s cells were cultured in RPMI1640 (GIBCO Life Technologies, Grand island, NY, USA) supplemented with 5% fetal bovine serum (FBS) and 2 mM L-glutamine at 37°C and 5% CO ₂ conditions. .

<Experimental Example 1> Confirmation of increase in degranulation of natural killer cells by columbine extract

In order to confirm the effect of increasing the activity of natural killer cells by the extract of Convolvulus convolvulus, degranulation analysis of natural killer cells was performed by measuring the expression level of CD107a on the surface of natural killer cells after administering the extract of Convolvulus convolvulus prepared in <Example 1> above. (Degranulation assay) was performed.

Specifically, the mononuclear cells (PBMC) isolated from the lymphocyte separation medium (LSM) were washed twice with DPBS (Dulbecco's Phosphate-Buffered Saline), and then washed with 1% penicillin/streptomycin and 10% bovine 4 × 10 ⁵ cells/200 per well in a 96-well plate using IMDM medium supplemented with fetal serum (FBS), 1% glutamine, and 400 U/ml interleukin-2 (IL-2). It was dispensed in ㎕ volume and cultured for 24 hours at 37°C and 5% CO ₂ conditions.

The cultured mononuclear cells were dispensed into a 96-well plate at 2×10 ⁵ cells/100 ㎕ per well, and then pretreated with the Convolvulus extract at concentrations of 0.04, 0.2, and 0.4 mg/ml for 1 hour, respectively.

Afterwards, monensin, anti-CD107a-FITC antibody (FITC-conjugated anti-CD107a antibody, BD Biosciences), and K562, a cell line sensitive to natural killer cells, were added to the mononuclear cells (PBMC). The same concentrations were mixed and cultured for 2 hours at 37°C and 5% CO ₂ conditions. A positive control group in which only K562 cells were administered without administration of the Convolvulus flower extract, and a control group (untreated group) in which neither the extract nor K562 cells were administered was used as a control group (untreated group).

After incubation, the sample was treated with anti-CD56-PE antibody (PE-conjugated anti-CD56 antibody, BD Biosciences) and anti-CD3-PerCP antibody (PerCP-conjugated anti-CD3 antibody, BD Biosciences) at 4°C for 30 minutes. . The reaction product was washed twice with Phosphate Buffer Saline (PBS) and the fluorescence intensity was measured with Accuri C6 (BD Biosciences), and data analysis was performed using the FlowJo 7.6.5 program (FlowJo. LLC, Ashland, OR, USA). was used.

As a result, as shown in FIG. 1, it was confirmed that the expression level of CD107a on the surface of natural killer cells administered with the bovine bindweed extract was significantly increased compared to the untreated group and the positive control group. In addition, it was confirmed that the amount of CD107a expression increased in cells administered 0.2 mg/ml of the bovine bindweed extract compared to cells administered 0.04 mg/ml, and in cells administered 0.4 mg/ml compared to cells administered 0.2 mg/ml. It was confirmed that the degranulation activity of natural killer cells increased in a concentration-dependent manner of the Convolvulus extract.

Through the above results, it was confirmed that the extract of the present invention increases the degranulation activity of natural killer cells, and thus the extract of the flowers of the present invention can be usefully used as a composition for preventing, treating or improving cancer or immune diseases.

<Experimental Example 2> Confirmation of the effect of increasing cytotoxicity of natural killer cells on cancer cells by extract of Convolvulus columbine

In order to confirm the effect of increasing the cytotoxicity of natural killer cells against cancer cells by the extract of Wild Convolvulus, the extract of Wild Convolvulus prepared in <Example 1> was administered to RMA cells according to the concentration of the Natural Killer cells. The selective death rate of RMA-s cells was confirmed.

For the experiment, C57BL/6 mice were used to deplete natural killer cells by injecting 10 μM rabbit anti-asialo-GM1 (Cedarlane Laboratories Ltd, Burlington, Ontario, Canada) for 3 consecutive days. .

Specifically, the mice were divided into four groups, and the control group (untreated group) was not administered the bovine convolvulus extract, and the remaining three groups were intraperitoneally administered the bovine convolvulus extract at doses of 0.04, 0.2, and 0.4 mg/ml, respectively. . Next, natural killer cell-resistant RMA cells and natural killer cell-sensitive RMA-s cells were each grown in PBA (Potato Bamboo Extract Agar) containing 0.5% bovine serum albumin (BSA) at 10 ⁷ cells/ml. After resuspension, 5 mM CFSE (Carboxylfluorescein succinimidyl ester) was administered to final concentrations of 1 μM and 4 μM, left at 37°C for 10 minutes, and cultured with culture medium containing 5% fetal bovine serum (FBS). Washed twice. Afterwards, the RMA cells and RMA-s cells labeled with EFSE were mixed at a ratio of 1:1 and injected into the mouse's abdominal cavity along with 500 μl phosphate-buffered saline (PBS). At this time, the number of cells administered into the mouse abdominal cavity is 2×10 ⁵ cell/mouse.

Afterwards, the mouse abdominal cavity was washed with 100 ml of low-temperature culture medium containing 5% fetal bovine serum (FBS), and the peritoneal fluid was collected and stored in a low temperature, dark place until use. The peritoneal fluid was centrifuged at a speed of 300 /[CFSE low/CFSE high]output)}×100%.

As a result, as shown in Figure 2, the number of RMA-s cells was significantly reduced compared to the RMA cells in all experimental groups and control groups, confirming the selective killing effect of natural killer cells on RMA-s cells, and the extract of B. columbine It was confirmed that the selective killing ratio of the RMA-s to the RMA cells increased in the experimental group administered compared to the control group. In addition, as the concentration of the lineage extract increases, the selective killing rate of RMA-s cells relative to RMA cells increases, and as the concentration of the lineage extract increases, the cytotoxicity of natural killer cells to cancer cells increases in a concentration-dependent manner. An increase was confirmed.

Through the above results, it was confirmed that the extract of the present invention increases the cytotoxicity of natural killer cells to cancer cells, and therefore, the extract of the convolvulus of the present invention can be usefully used as a composition for preventing, treating or improving cancer or immune diseases. there is.

<Experimental Example 3> Confirmation of the effect of increasing the expression level of granzyme B and perforin by columbine extract

In order to confirm the effect of increasing the expression of granzyme B and perforin in natural killer cells by the extract of Convolvulus convolvulus, the extract prepared in <Example 1> was administered to the natural killer cells, and then the natural killer cells were treated using RT-PCR. Perforin and granzyme B mRNA expression levels in killer cells were confirmed.

Specifically, the mononuclear cells (PBMC) isolated from the lymphocyte separation medium (LSM) were washed twice with DPBS (Dulbecco's Phosphate-Buffered Saline), and then washed with 1% penicillin/streptomycin and 10% bovine 4 × 10 ⁵ cells/200 per well in a 96-well plate using IMDM medium supplemented with fetal serum (FBS), 1% glutamine, and 400 U/ml interleukin-2 (IL-2). It was dispensed in ㎕ volume and cultured for 24 hours at 37°C and 5% CO ₂ conditions.

The cultured mononuclear cells were dispensed into a 96-well plate at 2×10 ⁵ cells/100 ㎕ per well, and then pretreated with the Convolvulus extract at concentrations of 0.04, 0.2, and 0.4 mg/ml for 1 hour, respectively.

Afterwards, total RNA was isolated from the natural killer cells using the RNeasy kit (Qiagen, Hilden, Germany), and then the mRNA was isolated using the ReverTra Ace qPCR RT kit (Toyobo, Osaka, Japan) according to the manufacturer's protocol. cDNA was synthesized from 1 μM. Afterwards, RT-PCR was performed using SYBR Green Real-Time PCRMaster Mix (Toyobo, Osaka, Japan) and LightCycler 480 Real-Time PCR system (Roche Diagnostics). b-actin mRNA level was used as a normalization control. Primer sequences used in RT-PCR are shown in Table 1 below.

primer F/R order
PRF1 F 5'-CGCCTACCTCAGGGCTTATCTC-3' (SEQ ID NO: 1) R 5'-CCTCGACAGTCAGGCAGTC-3' (SEQ ID NO: 2)
GzmB F 5'-CCCTGGGAAAAACACTCACACA-3' (SEQ ID NO: 3) R 5'-CACAACTCAATGGTACTGTCGT-3' (SEQ ID NO: 4)
IGF-1 F 5'-AGGAAGTACATTTGAAGAACGCAAGT-3' (SEQ ID NO: 5) R 5'-CCTGCGGTGGCATGTCA-3' (SEQ ID NO: 6)
ACTB F 5'-ACTCCATCATGAAGTGTGACG-3' (SEQ ID NO: 7) R 5'-CATACTCCTGCTTGCTGATCC-3' (SEQ ID NO: 8)

As a result, as shown in Figures 3 and 4, it was confirmed that the mRNA expression levels of granzyme B and perforin were significantly increased in natural killer cells treated with the extract of Convolvulus. In addition, the levels of perforin and granzyme B were higher in cells administered 0.2 mg/ml of the bovine bindweed extract than in cells administered 0.04 mg/ml, and in cells administered 0.4 mg/ml of the extract, compared to cells administered 0.2 mg/ml. By confirming that the expression level increased, it was confirmed that the expression level of perforin and granzyme B increased in a concentration-dependent manner of the Convolvulus extract.

Through the above results, it was confirmed that the extract of the present invention increases the expression of perforin and granzyme B in natural killer cells, and thus the extract of the convolvulus of the present invention is useful as a composition for preventing, treating or improving cancer or immune diseases. It can be used effectively.

The following illustrates preparation examples for the composition of the present invention.

<Preparation Example 1> Preparation of pharmaceutical preparation

<1-1> Production of powder

Convolvulus extract of the present invention 10mg

lactose 1 g

The above ingredients were mixed and filled into an airtight bubble to prepare a powder.

<1-2> Preparation of tablets

Convolvulus extract of the present invention 0.1mg

all the corn 100 mg

lactose 100 mg

Magnesium Stearate 2mg

After mixing the above ingredients, tablets were manufactured by tableting according to a conventional tablet manufacturing method.

<1-3> Manufacturing of capsules

Convolvulus extract of the present invention 0.1mg

corn starch 100 mg

lactose 100 mg

Magnesium Stearate 2mg

After mixing the above ingredients, a capsule was prepared by filling a gelatin capsule according to a typical capsule manufacturing method.

<1-4> Manufacturing of pills

Convolvulus extract of the present invention 1 mg

lactose 1.5g

glycerin 1 g

xylitol 0.5g

After mixing the above ingredients, it was prepared in an amount of 4 g per ring according to a conventional method.

<1-5> Preparation of granules

Convolvulus extract of the present invention 0.15mg

soybean extract 50mg

glucose 200mg

starch 600mg

After mixing the above ingredients, 100 mg of 30% ethanol was added and dried at 60°C to form granules, which were then filled into bags.

<Manufacture Example 2> Manufacture of health food

Foods containing the convolvulus extract of the present invention as an active ingredient were prepared as follows.

<2-1> Manufacturing of wheat flour food

0.5 to 5.0 parts by weight of the convolvulus extract of the present invention was added to flour, and bread, cakes, cookies, crackers, and noodles were prepared using this mixture.

<2-2> Preparation of soups and gravies

Health-enhancing meat products and noodle soup and gravy were prepared by adding 0.1 to 5.0 parts by weight of the Convolvulus extract of the present invention to soup and gravy.

<2-3> Production of ground beef

Ground beef for health promotion was prepared by adding 10 parts by weight of the convolvulus extract of the present invention to ground beef.

<2-4> Manufacturing of dairy products

5 to 10 parts by weight of the columbine extract of the present invention was added to milk, and various dairy products such as butter and ice cream were manufactured using the milk.

<2-5> Manufacturing of Seonsik

Brown rice, barley, glutinous rice, and coix seed were gelatinized and dried using a known method, roasted, and then made into powder with a particle size of 60 mesh using a grinder.

Black beans, black sesame seeds, and perilla seeds were steamed and dried using a known method, roasted, and then made into powder with a particle size of 60 mesh using a grinder.

The extract of Convolvulus chinensis of the present invention was concentrated under reduced pressure in a vacuum concentrator, dried with a spray and hot air dryer, and the dried product was pulverized with a grinder to a particle size of 60 mesh to obtain a dry powder.

The grains, seeds, and complex extract of the present invention prepared above were mixed in the following ratio.

Grains (30 parts by weight of brown rice, 15 parts by weight of coix radish, 20 parts by weight of barley),

Seeds (7 parts by weight perilla seeds, 8 parts by weight black beans, 7 parts by weight black sesame seeds),

Convolvulus extract of the present invention (3 parts by weight)

Reishi (0.5 parts by weight)

Rehmannia glutinosa (0.5 parts by weight)

<Manufacture Example 3> Manufacture of health drink

<3-1> Manufacturing of health drinks

Instant sterilization is achieved by homogeneously mixing 0.5 g of the convolvulus extract of the present invention with auxiliary ingredients such as high fructose corn syrup (0.5%), oligosaccharide (2%), sugar (2%), table salt (0.5%), and water (75%). It was then manufactured by packaging it in small containers such as glass bottles and plastic bottles.

<3-2> Production of vegetable juice

Vegetable juice was prepared by adding 0.5 g of the Convolvulus extract of the present invention to 1,000 ml of tomato or carrot juice.

<3-3> Production of fruit juice

Fruit juice was prepared by adding 0.1 g of the Convolvulus extract of the present invention to 1,000 ml of apple or grape juice.

<110> Industry-University Cooperation Foundation Catholic University of Pusan <120> Composition for preventing, treating or improving cancer or immune disease comprising Calystegia dahurica (Herb.) Choisy extract as active ingredients <130>P22-0052 <160> 8 <170> KoPatentIn 3.0 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> PRF1 primer F <400> 1 cgcctacctc aggcttatct c 21 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> PRF1 primer R <400> 2 cctcgacagt caggcagtc 19 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> GzmB primer F <400> 3 ccctgggaaa acactcacac a 21 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> GzmB primer R <400> 4 cacaactcaa tggtactgtc gt 22 <210> 5 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> IGF-1 primer F <400> 5 aggaagtaca tttgaagaac gcaagt 26 <210> 6 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> IGF-1 primer R <400> 6 cctgcggtgg catgtca 17 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> ACTB primer F <400> 7 actccatcat gaagtgtgac g 21 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> ACTB primer R <400> 8 catactcctg cttgctgatc c 21

Claims (15)

A pharmaceutical composition for preventing or treating cancer or immune diseases containing Calystegia dahurica (Herb.) Choisy ) extract as an active ingredient. According to paragraph 1,
A pharmaceutical composition for the prevention or treatment of cancer or immune diseases, wherein the extract of Convolvulus is extracted with water, C1 to C2 lower alcohol, or a mixed solvent thereof. According to paragraph 2,
A pharmaceutical composition for the prevention or treatment of cancer or immune diseases, characterized in that the extract of Convolvulus is extracted with one or more solvents selected from the group consisting of water, methanol, ethanol, and mixed solvents thereof. According to paragraph 3,
A pharmaceutical composition for the prevention or treatment of cancer or immune diseases, characterized in that the extract of Convolvulus chinensis is extracted with 70% to 90% ethanol as a solvent. According to paragraph 1,
A pharmaceutical composition for the prevention or treatment of cancer or immune disease, characterized in that the extract of Convolvulus convolvulus activates Natural Killer cells (NK cells). According to clause 5,
A pharmaceutical composition for the prevention or treatment of cancer or immune diseases, wherein the extract of Convolvulus convolvulus increases the degranulation activity of Natural Killer cells (NK cells). According to clause 5,
A pharmaceutical composition for the prevention or treatment of cancer or immune diseases, wherein the extract of Convolvulus lily increases the cytotoxicity of Natural Killer cells (NK cells) to cancer cells. According to clause 5,
A pharmaceutical composition for the prevention or treatment of cancer or immune diseases, wherein the extract of Convolvulus is characterized by increasing the expression of Granzyme B or Perforin in Natural Killer cells (NK cells). According to paragraph 1,
The cancer is selected from the group consisting of lung cancer, larynx cancer, liver cancer, stomach cancer, colon cancer, gallbladder cancer, pancreas cancer, bladder cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer, uterine cancer, kidney cancer, thyroid cancer, skin cancer, and blood cancer. A pharmaceutical composition for preventing or treating cancer or immune diseases. The method of claim 9, wherein the blood cancer is leukemia, lymphoma, acute T-cell leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, myelodysplastic syndrome, chronic myelogenous leukemia, chronic lymphocytic leukemia, Hodgkin's lymphoma, and non-Hodgkin's lymphoma. , a pharmaceutical composition for the prevention or treatment of cancer or immune disease, characterized in that it is selected from the group consisting of megaloblastic leukemia, multiple myeloma, solitary myeloma, and erythroleukemia. According to paragraph 1,
A pharmaceutical composition for preventing or treating cancer or immune diseases, wherein the immune diseases are selected from the group consisting of autoimmune diseases, viral infectious diseases, and inflammatory diseases. According to clause 11,
A pharmaceutical composition for preventing or treating cancer or an immune disease, wherein the autoimmune disease is selected from the group consisting of multiple sclerosis, lupus, rheumatoid arthritis, Crohn's disease, and type 1 diabetes. According to clause 11,
A pharmaceutical composition for preventing or treating cancer or immune diseases, wherein the viral infectious disease is selected from the group consisting of acquired immunodeficiency syndrome, hepatitis B, hepatitis C, measles, shingles, and papilloma virus. According to clause 11,
A pharmaceutical composition for preventing or treating cancer or immune diseases, wherein the inflammatory disease is selected from the group consisting of asthma, sinusitis, atopy, periodontitis, and Behcet's disease. A food composition for preventing or improving cancer or immune diseases, containing extract of Convolvulus chinensis as an active ingredient.