KR102556464B1 - Composition for inhibiting proliferation of tumor comprising Oenothera Radix extract as effective component - Google Patents

Abstract

The present invention relates to a composition for inhibiting tumor growth containing moonshine extract as an active ingredient, wherein the moonshine extract contains a protein (PD-L1 or CD80) present on the surface of cancer cells and a protein (PD-1) on the surface of T cells and CTLA-4) were confirmed to block the interaction (binding), and PD-1/PD-L1 of moonshine extract was confirmed using TCR/PD-1 overexpressing T cells and TCR receptor/PD-L1 overexpressing CHO cells. The interaction inhibitory effect was confirmed, and it was confirmed that the cancer cell killing effect of the moonshine extract treatment group was enhanced in the cooperative culture of colorectal cancer, breast cancer or lung cancer cells and T cells.

Description

Composition for inhibiting proliferation of tumor containing moonshine extract as an active ingredient {Composition for inhibiting proliferation of tumor comprising Oenothera Radix extract as effective component}

The present invention relates to a composition for inhibiting tumor growth containing moonshine extract as an active ingredient.

As a conventional anticancer treatment to conquer cancer, surgery to resect a tumor is known. Radiation therapy and chemotherapy are combined to reduce the size of a tumor before surgery or to kill remaining cancer cells and prevent recurrence after surgery. Radiation therapy and chemotherapy, which are referred to as first-generation anticancer drugs, are methods of inducing death of cancer cells by interfering with the process of infinite division and proliferation of cancer cells. However, radiation therapy has a problem of inducing death of normal cells as well as DNA damage of cancer cells by irradiating high-energy radiation. In addition, even in the case of chemotherapy that administers toxic chemicals that inhibit the cell division process, it does not harm cell division specifically for cancer cells, so it also inhibits the division of normal cells, causing serious side effects such as reduction of leukocytes and hair loss. do. In order to overcome this, it was expected that side effects of first-generation anticancer drugs could be reduced by developing a targeted anticancer drug, which is a second-generation anticancer drug that selectively attacks only cancer cells by distinguishing it from normal cells.

However, targeted anticancer drugs are characterized by selectively suppressing cancer cells by acting only on cancer-causing proteins to exert therapeutic effects. Depending on the type of cancer, the cancer-causing proteins and the therapeutically effective proteins are different. Appropriate anticancer drugs should be used. In addition, cancer cells have a mechanism for acquiring resistance to the target anticancer drug, and thus have the 'immune cell evasion ability' that causes mutations so that the cancer cell does not become the target of the target anticancer drug, so the target anticancer drug may not recognize the cancer cell. there is.

Therefore, a third-generation immuno-anticancer agent is being developed that reduces side effects and resistance problems caused by chemotherapy and enables immune cells to remember cancer cells and continuously attack cancer cells even after administration is stopped.

Immunotherapy is a treatment that activates the body's immune system to increase self-immunity so that immune cells attack cancer cells. Immuno-anticancer drugs focus on the function of immune cells to awaken the 'potential to attack cancer cells' of immune cells.

Immune anticancer agents can be classified into immune checkpoint inhibitors, immune cell therapy agents, therapeutic antibodies, and anticancer vaccines. Immune checkpoint inhibitors activate T cells to attack cancer cells by blocking the activity of immune checkpoint proteins involved in suppressing T cells. is an anti-cancer drug. Antibodies recognizing CTLA-4, PD-1, and PD-L1 are mainly used. Anticancer agents that enhance cellular immunity include NK cell therapy, T cell therapy, and CAR-T cell therapy, and therapeutic antibodies attack cancer cells by releasing the drug when the antibody-drug conjugate binds to cancer cells. Anticancer vaccine is an immunotherapy that activates the body's immune function and attacks cancer cells by injecting tumor-specific antigens possessed by cancer cells or protein/peptide molecules that can enhance the overall immune response in cancer patients to activate the immune system. am.

On the other hand, Wolgyeoncho is a biennial herbaceous plant belonging to the needle family, and the name was given from the habit of flowers welcoming the moon at night. It is a naturalized plant native to South America. It grows to a height of 50 to 90 cm and has dense fine hair. The rosettes from the root spread in a rosette shape, and the leaves from the stem are alternate and wide linear, 5-15 cm long and 5-12 mm wide. The tip is pointed, the lower part directly touches the stem, there are shallow sawtooth on the edge, and the midrib is dark green with white midrib. Flowers are yellow, hanging one by one on the upper axilla, blooming in the evening and withering in the morning, with a little reddish color. The fruit is a capsule, club-shaped, 2-3 cm long, and divided into 4 pieces. The seed oil is used as a folk medicine for diabetes, and the outpost is used for antipyretic medicine.

As a technology related to Wolgyeoncho, Korean Patent Publication No. 2011-0048236 discloses a pharmaceutical composition for preventing or treating cancer containing a native plant extract as an active ingredient, and Korean Patent Publication No. 2008-0010720 discloses physiological activity Although an extract of P. japonica has been disclosed, there has been no disclosure of a composition for inhibiting tumor growth containing the extract of Moongyeoncho of the present invention as an active ingredient.

The present invention has been derived from the above needs, and the present invention provides a composition for inhibiting tumor growth containing moonshine extract as an active ingredient, and PD in which the moonshine extract, an active ingredient of the present invention, is present on the surface of cancer cells. -Binds to PD-1 on the surface of L1 or immune T cells to block the interaction of cancer cells with PD-1 on the surface of T cells, thereby activating T cells to show tumor growth inhibitory effects; with cancer, breast or lung cancer cells; In the cooperative culture of T cells, by confirming that the cancer cell killing effect of the moonshine extract administration group is enhanced, the present invention was completed.

In order to achieve the above object, the present invention provides a health functional food composition for suppressing immune checkpoints containing moonshine extract as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of immune checkpoint inhibition-related diseases containing moonshine extract as an active ingredient.

In addition, the present invention provides an antitumor pharmaceutical composition containing an anticancer active agent and an extract of moonshine as active ingredients.

In addition, the present invention provides an anticancer adjuvant comprising the moongyeoncho extract as an active ingredient.

The present invention has been derived from the above needs, and the present invention relates to a composition for inhibiting tumor growth containing moonshine extract as an active ingredient, PD in which the moonshine extract, an active ingredient of the present invention, is present on the surface of cancer cells. -By blocking the interaction between L1 and PD-1 on the surface of T cells, it activates immune T cells and has an effect of suppressing tumor growth. It has the effect of enhancing the cancer cell killing effect of the extract treatment group.

1 shows that PD-1/PD-L1 binding is inhibited in a concentration-dependent manner when PD-L1 is bound to PD-L1 after treatment with 0.001 to 10 μg/ml of anti-PD-L1 on a PD-L1-coated plate. is the result of *, **, *** indicate statistically significant PD-1/PD-L1 binding when treated with 0.001 to 10 μg/ml of anti-PD-L1 compared to those not treated with anti-PD-L1. That is, * is p <0.05, ** is p <0.01, *** is p <0.001.
Figure 2 confirms that PD-1 / PD-L1 binding is inhibited in a concentration-dependent manner when PD-1 is bound to PD-L1-coated plates after treatment with 5 to 500 μg / ml of moonshine extract This is the result. *, *** indicate that the binding of PD-1/PD-L1 was statistically significantly reduced when 50-500 μg/ml of moonshine extract was treated compared to untreated moonshine extract, * indicates p<0.05 and *** indicates p<0.001.
FIG. 3 shows the results of concentration-dependent inhibition of CD80/CTLA-4 binding when CD80 was bound to CTLA-4-coated plates after treatment with 0.01 to 10 μg/ml of anti-CTLA-4. **, *** indicate that CD80/CTLA-4 binding was statistically significantly reduced when treated with 0.01 to 10 μg/ml of anti-CTLA-4 compared to not treated with anti-CTLA-4. As such, ** is p<0.01, and *** is p<0.001.
Figure 4 is a result confirming that CTLA-4 / CD80 binding is inhibited in a concentration-dependent manner when CD80 is bound after treatment with 10 to 500 μg / ml of moonshine extract on a plate coated with CTLA-4. *** indicates that the binding of CD80/CTLA-4 was statistically significantly reduced when 10 to 500 μg/ml of moonshine extract was treated compared to untreated moonshine extract, p<0.001 .
Figure 5 is the result of confirming the PD-1 / PD-L1 interaction inhibitory effect of the moonshine extract using TCR / PD-1 overexpressing T cells and TCR receptor / PD-L1 overexpressing CHO cells. *, *** means that luminescence was statistically significantly enhanced in the group treated with moonshine extract or PD-1 compared to those not treated with moonshine extract or PD-1, * is p<0.05, *** indicates p<0.001. Fold induction means the ratio of the relative light emission level based on the control group (CON) that has not been treated with anything.
Figure 6 is a result of confirming the cancer cell viability according to the co-cultivation of human colon cancer cells (HCT116) and T cells, Moongyeoncho extract treatment. (A) is a culture of colon cancer cells (HCT116) without T cells, and (B) is a co-culture of colon cancer cells (HCT116) and T cells at a ratio of 1:5. **, *** means that the cancer cell survival rate of the moonshine extract treatment group was statistically significantly decreased compared to the control group not treated with moonshine extract, ** is p <0.01 and *** is p <0.001 .
Figure 7 is a result of confirming the cancer cell viability according to the co-cultivation of breast cancer cells (MCF-7) and T cells, Moongyeoncho extract treatment. (A) cultured breast cancer cells (MCF-7) without T cells, (B) was co-cultured breast cancer cells (MCF-7) and T cells at a ratio of 1:5. **, *** means that the cancer cell survival rate of the moonshine extract-treated group compared to the control group not treated with moonshine extract was statistically significantly reduced, ** is p <0.01, and *** is p <0.001.
Figure 8 is the result of confirming the cancer cell survival rate according to the treatment of the moongyeoncho extract during the co-cultivation of lung cancer (A549) and T cells. (A) is cultured lung cancer cells (A549) without T cells, (B) is air-cultured lung cancer cells (A549) and T cells at a ratio of 1:5. *, *** means that the survival rate of cancer cells in the moonshine extract treatment group was statistically significantly reduced compared to the control group that was not treated with anything, * is p <0.05, and *** is p <0.001.
Figure 9 confirms the decrease in body weight (B) and tumor size (C) according to the passage of time (A) after administration of the moongyeoncho extract of the present invention in a colorectal cancer animal model. Vehicle (PBS) is a colorectal cancer induction group that does not eat moonshine extract, αPD-1 (5mpk) is a group administered with 5mg/kg of αPD-1, and WGC (50mpk) is a group administered with 50mg/kg moonshine extract was administered, WGC (100mpk) was a group administered with 100mg/kg of moonshine extract, WGC (200mpk) was a group administered with 200mg/kg of moonshine extract, and αPD-1 (2.5mpk)+ WGC (200mpk) was a group administered with 2.5 mg/kg of αPD-1 and 200 mg/kg of moonshine extract. **, *** means that the tumor size of the group administered with the lunar weed extract of the present invention or the positive control group was statistically significantly reduced compared to the colorectal cancer induction group (Vehicle), ** is p<0.01, *** is p<0.001.

The present invention relates to a health functional food composition for inhibiting immune checkpoints containing an extract of moonshine as an active ingredient.

The moongyeoncho extract of the present invention may be extracted from the outpost, leaf, root or seed of moongyeoncho, but is preferably extracted from the root of moongyeoncho, but is not limited thereto.

The moongyeoncho extract may be prepared by a method comprising the following steps, but is not limited thereto:

(1) wolgyeoncho ( Oenothera Radix ) Step of extracting by adding an extraction solvent;

(2) filtering the extract of step (1); and

(3) preparing an extract by concentrating the filtered extract of step (2) under reduced pressure and drying.

In the step (1), the extraction solvent is preferably selected from water, C ₁ ~ C ₄ lower alcohol or mixtures thereof, more preferably C ₁ ~ C ₄ lower alcohol, and still more preferably 70% (v/v) ethanol, but is not limited thereto.

In the preparation method, all conventional methods known in the art such as filtration, hot water extraction, immersion extraction, reflux cooling extraction and ultrasonic extraction may be used as the extraction method. The extraction solvent is preferably extracted by adding 1 to 20 times the weight of dried moonshine, and more preferably 5 to 15 times. The extraction temperature is preferably 4 to 50 ° C, but is not limited thereto. In addition, the extraction time is preferably 0.5 to 10 hours, more preferably 0.5 to 5 hours, but is not limited thereto. In the above method, the vacuum 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 reduced pressure drying, vacuum drying, boiling drying, spray drying or freeze drying, but is not limited thereto.

The moonshine extract targets PD-L1 or CD80 of cancer cells; It is characterized by targeting PD-1 or CTLA-4 of T cells, and the cancer cells are liver cancer, lung cancer, breast cancer, melanoma, stomach cancer, colorectal cancer, colon cancer, skin cancer, bladder cancer, prostate cancer, ovarian cancer, cervical cancer, It is preferably a cancer cell derived from any one cancer selected from thyroid cancer, renal cancer, fibrosarcoma and hematological cancer, but is not limited thereto.

The health functional food composition of the present invention may be added as it is or used together with other foods or food ingredients, and may be appropriately used according to conventional methods. There is no particular limitation on the type of health functional food composition. Examples of foods to which the moonshine extract can be added include meat, sausages, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, There are drinks, alcoholic beverages, and vitamin complexes, and includes all health functional foods in a conventional sense. A health drink containing the composition of the present invention may contain various flavoring agents or natural carbohydrates as additional components like conventional drinks. The aforementioned natural carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As the sweetener, natural sweeteners such as thaumatin and stevia extract, or synthetic sweeteners such as saccharin and aspartame may be used. The proportion of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 g of the composition of the present invention.

In addition to the above active ingredients, the health functional food composition of the present invention contains various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH regulators, stabilizers, and preservatives. , glycerin, alcohol, a carbonating agent used in carbonated beverages, and the like may be further contained. In addition, it may further contain fruit flesh for the manufacture of fruit juice or vegetable beverages. These components may be used independently or in combination. The ratio of these additives is not very important, but is generally selected in the range of 0.01 to 2 parts by weight based on 100 parts by weight of the composition of the present invention.

In addition, the present invention relates to a pharmaceutical composition for preventing or treating diseases related to immune checkpoint inhibition, containing an extract of moonshine as an active ingredient.

The immune checkpoint inhibition-related disease is preferably cancer, infection, sepsis or immunosenescence, more preferably cancer, but is not limited thereto. The cancer is as described above.

In addition, the present invention relates to an antitumor pharmaceutical composition containing an anticancer active agent and an extract of moonshine as active ingredients.

The anticancer activator is preferably, but not limited to, an anticancer agent or an immune checkpoint inhibitor, and the anticancer agent is actinomycin D, bleomycin sulfate, daunomycin, daunorubicin ), doxorubicin, epirubicin, idarubicin, mitomycin, mitomycin-C, mitramycin, irinotecan, campto camptothecin, novobiocin, epirubicin, dactinomycin, amsacrine, teniposide, etoposide, cisplatin, It is preferably at least one selected from carboplatin, oxaliplatin, paclitaxel, docetaxel, gefitinib, erlotinib, and afatinib. The immune checkpoint inhibitor is preferably, but not limited to, an anti-PD-L1 antibody, an anti-PD-1 antibody, an anti-CD80 antibody, or an anti-CTLA-4 antibody.

The pharmaceutical composition of the present invention may be in various oral or parenteral dosage forms. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient, for example, starch, calcium carbonate, sucrose or lactose ( lactose) and gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid formulations for oral administration include suspensions, internal solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included. there is. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending solvents. As the base of the suppository, witepsol, macrogol, tween) 61, cacao butter, laurin paper, glycerogelatin, etc. may be used.

The pharmaceutical composition of the present invention may be administered orally or parenterally, and in the case of parenteral administration, it is preferable to select an external skin or intraperitoneal, rectal, intravenous, intramuscular, subcutaneous, intrauterine epidural or intracerebrovascular injection method.

The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level is based on the type, severity, and activity of the drug in the patient. , sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, factors including drugs used concurrently, and other factors well known in the medical field. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple times. Considering all of the above factors, it is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by those skilled in the art.

The dosage of the composition of the present invention varies in its range depending on the patient's body weight, age, sex, health condition, diet, administration time, administration method, excretion rate and severity of the disease, and the daily dosage is the amount of moonshine extract 0.01 to 2,000 mg/kg as a standard, preferably 30 to 500 mg/kg, and more preferably 50 to 300 mg/kg, and may be administered 1 to 6 times a day. The pharmaceutical composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers.

In addition, the present invention relates to an anticancer adjuvant comprising moonshine extract as an active ingredient.

The anticancer adjuvant may contain at least one active ingredient exhibiting the same or similar function in addition to the moongyeoncho extract. The anticancer adjuvant can be administered orally or parenterally during clinical administration, and in the case of parenteral administration, intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, intramuscular injection, intrauterine intrathecal injection, intracerebrovascular injection or It can be administered by intrathoracic injection, and can be used in the form of a general pharmaceutical preparation.

The anticancer adjuvant may be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers. The daily dose of the anti-cancer adjuvant is about 0.0001 to 100 mg/kg, preferably 0.001 to 10 mg/kg, and it is preferable to administer once or several times a day in divided doses, but the patient's weight, age, sex, health condition, The range varies according to diet, administration time, administration method, excretion rate, and severity of disease. The anticancer adjuvant of the present invention can be administered in various parenteral formulations during actual clinical administration. When formulated, diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants are used. It is prepared by Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base for the suppository, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogeratin and the like may be used.

Hereinafter, the present invention will be described in more detail using examples. These examples are only for explaining the present invention in more detail, and it is obvious to those skilled in the art that the scope of the present invention is not limited thereto.

Example 1. Preparation of Moongyeoncho extract

10g of wolgyeoncho ( Oenothera Radix ) After drying the roots, 100㎖ of 70% (v / v) ethanol was added as a solvent, extracted under reflux three times for 2 hours, and then concentrated under reduced pressure to prepare an extract of wolgyeoncho.

Example 2. Competitive ELISA analysis of the interaction between ethanol extract of moonshine and PD-L1

Using competitive ELISA analysis, it was confirmed that the ethanol extract of Moongyeoncho binds to PD-L1 in a concentration-dependent manner and blocks the interaction with PD-1.

A competitive ELISA assay was performed using a PD-1/PD-L1 inhibitor ELISA screening kit according to the manufacturer's instructions. 100 μl of 1 μg/ml of the combined human PD-L1 in PBS was coated on 96-well plates overnight. The 96-well plate was washed with PBS containing 0.1% Tween (PBS-T), blocked with PBS containing 2% (w/v) BSA for 1 hour at room temperature, and washed again. Then, 50 μl of 0.5 μg/ml biotinylated hPD-1 was added to the wells and the plate was incubated at room temperature for 2 hours. After washing 3 times in PBS-T, 50 μl of 0.2 μg/ml HRP-conjugated streptavidin was added to each well and the plate was incubated for 1 hour. After incubation, plates were washed three times with 0.1% PBS-T and relative chemiluminescence was measured using a SpectraMax L luminometer.

As a result, it was confirmed that the binding of PD-1/PD-L1 was inhibited in a concentration-dependent manner (FIG. 1), and the moonshine extract statistically significantly reduced the binding of PD-1/PD-L1 in a concentration-dependent manner ( Fig. 2).

Example 3. Competitive ELISA analysis of the interaction of moonshine ethanol extract and CTLA-4

Using competitive ELISA analysis, it was confirmed that the ethanol extract of Moongyeoncho binds to CTLA-4 in a concentration-dependent manner and blocks the interaction with CD80.

A competitive ELISA assay was performed using the CTLA-4/CD80 inhibitor ELISA screening kit according to the manufacturer's instructions. 100 μl of recombinant human CTLA-4 at 1 μg/ml in PBS was coated on a 96-well plate overnight. The 96-well plate was washed with PBS containing 0.1% Tween (PBS-T), blocked with PBS containing 2% (w/v) BSA for 1 hour at room temperature, and washed again. Then, 50 μl of 1.25 μg/ml biotinylated hCD80 was added to the wells and the plate was incubated for 2 hours at room temperature. After washing 3 times in PBS-T, 50 μl of 0.2 μg/ml HRP-conjugated streptavidin was added to each well and the plate was incubated for 1 hour. After incubation, plates were washed three times with 0.1% PBS-T and relative chemiluminescence was measured using a SpectraMax L luminometer.

As a result, it was confirmed that the binding of CTLA-4/CD80 was inhibited in a concentration-dependent manner (FIG. 3), and the moonshine extract statistically significantly reduced the binding of CTLA-4/CD80 in a concentration-dependent manner (FIG. 4).

Example 4. Confirmation of PD-1/PD-L1 interaction inhibitory effect of moonshine extract using TCR/PD-1 overexpressing T cells and TCR receptor/PD-L1 overexpressing CHO cells

To confirm the efficacy of moonshine extract for inhibiting the PD-1/PD-L1 interaction, a cell-based PD-1/PD-L1 blocking assay was performed. The cell-based PD-1/PD-L1 blocking assay uses the principle that luciferase emission is reduced when TCR binds to the TCR receptor by PD-1/PD-L1 interaction, and the extract and It is possible to confirm whether the PD-L1 blocking antibody blocks PD-1/PD-L1 binding on the cell surface.

To this end, TCR/PD-1 overexpressing T cells and TCR receptor/PD-L1 overexpressing CHO cells were used. 5×10 ⁴ PD-L1 aAPC/CHO-K1 cells were seeded in a 96-well plate and cultured in DMEM medium containing 10% FBS. On the day of performing the cell-based PD-1/PD-L1 blocking assay, the medium was removed, PD-L1 blocking antibody or moonshine extract was added at each concentration, and then 1×10 ⁵ PD-1 overexpressing T cells was added. Then, after a certain period of time, Bio-Glo ™ reagent (Promega) was mixed and luminescence was measured.

As a result, as shown in Figure 5, when treated with moonshine extract and PD-1 blocking antibody (PD-1 100ng, PD-1 50ng), the binding of PD-1 / PD-L1 was inhibited in a concentration-dependent manner It was confirmed that the degree of luminescence of luciferase increased.

Example 5. Confirmation of colorectal cancer, breast cancer and lung cancer apoptosis enhancement according to moonshine extract treatment and immune T cell cooperative culture

(1) Confirmation of colorectal cancer cell death effect

Cancer cell apoptosis was confirmed by co-culture of colorectal cancer cells (HCT116) and T cells and treatment with moonshine extract. Co-culture of the colon cancer cells (HCT116) and T cells was cultured at a ratio of 1:5.

Colorectal cancer cells (HCT116) treated with 10 ng/ml of interferon gamma were spread on a 96-well plate in an amount of 5×10 ⁴ cells/well, cultured for 18 hours, and adhered to the bottom. Thereafter, the PD-1 overexpressed T cells were dispensed in an amount of 2.5×10 ⁵ cells/well in a 96-well plate to which colon cancer cells were attached, and a moonshine extract (12.5, 25 or 50 μg/ml) was added. After 72 hours of air conditioning culture. Thereafter, 10 μl of the CCK solution was added, incubated at 37° C. for 2 hours, and absorbance at 450 nm of the microplate was measured.

As a result, as shown in Figure 6, when the co-cultivated by treating the moongyeoncho extract, it was confirmed that the colorectal cancer cell viability was significantly reduced.

(2) Confirmation of breast cancer cell death effect

Cancer cell apoptosis was confirmed by co-culture of breast cancer cells (MCF-7) and T cells and by treatment with the extract of moonshine. Co-culture of the breast cancer cells (MCF-7) and T cells was cultured at a ratio of 1:5.

Breast cancer cells (MCF-7) treated with 10 ng/ml of interferon-gamma were spread on a 96-well plate in an amount of 5×10 ⁴ cells/well, cultured for 18 hours, and adhered to the bottom. Thereafter, PD-1 overexpressed T cells were dispensed in an amount of 2.5×10 ⁵ cells/well in a 96-well plate to which breast cancer cells were attached, and after the addition of moonshine extract (6.25, 12.5 or 25 μg/ml) It was cultured in air for 72 hours. Thereafter, 10 μl of the CCK solution was added, incubated at 37° C. for 2 hours, and absorbance at 450 nm of the microplate was measured.

As a result, as shown in Figure 7, when the co-cultivation by treatment with the moongyeoncho extract, it was confirmed that the breast cancer cell viability was significantly reduced.

(3) Confirmation of lung cancer cell death effect

Cancer cell apoptosis was confirmed by co-culture of lung cancer cells (A549) and T cells and by treatment with moonshine extract. Co-culture of the lung cancer cells (A549) and T cells was cultured at a ratio of 1:5.

Lung cancer cells (A549) treated with 10 ng/ml of interferon-gamma were spread on a 96-well plate in an amount of 5×10 ⁴ cells/well, cultured for 18 hours, and adhered to the bottom. Thereafter, PD-1 overexpressed T cells were dispensed in an amount of 2.5×10 ⁵ cells/well into a 96-well plate to which lung cancer cells were attached, and after the addition of moonshine extract (12.5, 25 or 50 μg/ml) It was cultured in air for 72 hours. Thereafter, 10 μl of the CCK solution was added, incubated at 37° C. for 2 hours, and absorbance at 450 nm of the microplate was measured.

As a result, it was confirmed that the survival rate of lung cancer cells was significantly reduced when the coculture was performed by treating the moongyeoncho extract as shown in FIG. 8 .

Example 6. Confirmation of tumor size reduction effect according to administration of moonshine extract using colorectal cancer animal model

Using six mice (C57BL) in which mPD-1 was removed and replaced with hPD-1, MC-38 mice in which mouse PD-L1 (mPD-L1) was removed and replaced with human PD-1 (hPD-L1) were used. (mouse colon cancer cells) cells were injected, and αPD-1 (positive control) and moonshine extract (50, 100, and 200 mg/Kg) were administered at intervals as shown in FIG. 7 after 14 days had elapsed, Body weight and tumor size were measured.

As a result, there was no significant change in the body weight of the mice, but it was confirmed that the size of the tumor was significantly reduced in the group administered with the moonshine extract of the present invention and the positive control group compared to the colorectal cancer induction group (vehicle) (FIG. 9).

[Statistical Processing]

The present invention was repeated 3 times and expressed as mean ± standard deviation, and statistical processing was performed by Tukey's post-hoc test.

Claims (12)

A health functional food composition for preventing or improving colorectal cancer or lung cancer, characterized in that it contains moonshine root ethanol extract as an active ingredient and has an immune checkpoint inhibitory effect. delete The method of claim 1, wherein the ethanol extract of moonshine root targets PD-L1 or CD80 of cancer cells; A health functional food composition for preventing or improving colorectal cancer or lung cancer, characterized in that it targets PD-1 or CTLA-4 of T cells. delete A pharmaceutical composition for preventing or treating colorectal cancer or lung cancer, comprising an ethanol extract of moonshine root as an active ingredient and having an immune checkpoint inhibitory effect. delete delete A pharmaceutical composition for preventing or treating colorectal cancer, comprising moonshine root extract and an immune checkpoint inhibitor as active ingredients and having an immune checkpoint inhibitory effect. delete delete The pharmaceutical composition for preventing or treating colorectal cancer according to claim 8, wherein the immune checkpoint inhibitor is an anti-PD-L1 antibody, an anti-PD-1 antibody, an anti-CD80 antibody, or an anti-CTLA-4 antibody. An anti-cancer adjuvant for colorectal cancer comprising moonshine root extract and an immune checkpoint inhibitor as active ingredients and having an immune checkpoint inhibitory effect.