KR20220109668A - Composition for oral administration comprising extract of oxya chinensis sinuosa - Google Patents

Abstract

The present invention relates to a composition for oral administration containing an extract of Oxya chinensis. More specifically, the present invention relates to a pharmaceutical composition for cancer treatment, a pharmaceutical composition for adjuvant anti-cancer, or a food composition for enhancing immunity containing an extract of Oxya chinensis.

Description

Composition for oral administration comprising rice locust extract {COMPOSITION FOR ORAL ADMINISTRATION COMPRISING EXTRACT OF OXYA CHINENSIS SINUOSA}

The present invention relates to a composition for oral administration comprising a rice locust extract, and more particularly, to a composition comprising an extract of rice locust obtained at room temperature using an aqueous solution containing an antioxidant as an extraction solvent for cancer treatment and anticancer adjuvant Or it relates to a technology used for food use for enhancing immunity.

In the rapidly changing modern society, many modern people are threatened with their health due to various lifestyle diseases such as cancer, high blood pressure, angina, diabetes, obesity, and myocardial infarction due to wrong lifestyle. According to the statistical data of the World Health Organization, cancer is the number one cause of death among lifestyle-related diseases worldwide, and it is a reality that threatens the lives of modern people.

In particular, the incidence of various lifestyle diseases is increasing due to excessive stress, lack of exercise, and changes in eating habits centered on meat among modern people. In particular, colorectal cancer is often unrecognized due to its simple initial symptoms, and is often advanced when it is discovered.

Despite the development of modern medicine and the development of various medicines, anticancer drugs that have been used until now have been limited in their use due to the formation of side effects and resistance. For example, chemotherapy is widely used for the treatment of colorectal cancer, but chemotherapy has a side effect of inhibiting immune function by inducing bone marrow damage in a patient. As an alternative to the side effects of synthetic drugs, interest in developing anticancer drugs derived from natural substances based on the pharmacological effects of natural substances is increasing. For example, since the intake of foods that can increase the patient's immunity can reduce the side effects of chemotherapy and can also increase the effectiveness of the recently applied immunotherapy, it is important to increase the patient's immunity. It is evaluated as an important factor in enhancing the therapeutic effect.

Meanwhile, edible insects are attracting attention as a future food substitute as a solution to prepare for food shortages caused by population growth and depletion of environmental resources worldwide. The amount of feed required for breeding insects is not only economical compared to the amount needed for breeding livestock, but also because the high protein content of insects is nutritionally valued, the edible insect market is expected to grow rapidly. However, studies on the use of edible insects for medicinal purposes are still lacking.

In relation to the extraction solvent for obtaining such a natural extract, for example, when extracting using hot water as disclosed in Korean Patent Application Laid-Open No. 10-2015-0097891, modification of the protein in the active ingredient may occur. , when alcohol is used as an extraction solvent, components with relatively low polarity such as polyphenols are mainly extracted and are not suitable for extracting polar protein components. If an anticancer component derived from a natural product obtained using a solvent can be provided, it is expected to be widely applied in related fields such as medicine and food.

Accordingly, one aspect of the present invention is to provide a composition for oral administration comprising a rice locust extract obtained by using an aqueous solution containing an antioxidant as an extraction solvent.

According to one aspect of the present invention, there is provided a composition for oral administration comprising an extract of rice locust (Oxya chinensis sinuosa).

The rice locust extract obtained by the present invention effectively induces the activity of CD4 T cells, CD8 T cells and dendritic cells in the spleen, and as a result can induce a reduction in tumor size, thereby providing natural product-based therapeutics and immunity for anticancer treatment. It is expected to be widely used as a functional food that can improve the health of the people.

Figure 1 (a) shows the results of evaluating the toxicological safety of the rice locust extract, and Figure 1 (b) shows the results of evaluating the ability of the rice locust extract to induce surface molecules on dendritic cells.
Figure 2 shows the results of analysis of extracellular cytokine levels through ELISA (a) and intracellular cytokine levels through FACSverse flow cytometry analysis (b).
Figure 3 (a) shows the result of analyzing the change in antigen acquisition ability of dendritic cells through flow cytometry, and Figures 3 (b) and 3 (c) are heat-treated and non-heat-treated surface of dendritic cells of the rice locust extract. The results of comparison of expression factors and cytokine inducibility are shown.
FIG. 4 shows the results of flow cytometry analysis of the effects of dendritic cells treated with rice locust extract on the proliferation, differentiation, and activity of CD4 and CD8 T cells (a), and the results of analyzing the amount of cytokine secreted by these cells with an ELISA kit (b). ) and CTL activity evaluation results of dendritic cells treated with rice locust extract (c) are shown.
Figure 5 shows the cancer cell inhibition and dendritic cell activity evaluation results induced by the rice locust extract in CT26 tumor model mice. 5(c) shows the results of analyzing the activity of splenocyte-derived dendritic cells (splenic CD11c+MHC-II+).
6 shows the results of flow cytometry analysis of CD4 T cells by isolating the mouse spleen (a) and a bar graph of the percentages of the three groups of single, double, triple or quadruple functional CD4 T cells (b).
7 shows the results of flow cytometry analysis of CD8 + T cells by isolating the mouse spleen (a) and a histogram of the percentages of the three groups of single, double, triple or quadruple functional CD8 + T cells (b).

Hereinafter, preferred embodiments of the present invention will be described with reference to the accompanying drawings. However, the embodiments of the present invention may be modified in various other forms, and the scope of the present invention is not limited to the embodiments described below.

According to the present invention, there is provided a composition for oral administration safer to the human body, which can be used as an extract of an insect, which is a natural product, as a pharmaceutical composition for cancer treatment, a pharmaceutical composition for adjuvant anticancer or a food composition for immune enhancement.

More specifically, the composition for oral administration of the present invention includes an extract of rice locust (Oxya chinensis sinuosa), and the rice locust extract is obtained at room temperature using an aqueous solution containing an antioxidant as an extraction solvent.

In the present invention, the rice grasshopper (Oxya chinensis sinuosa) belongs to the suborder Caelifera, and is one of the insects registered as edible in the Korea Food Service.

The extract of each rice locust used in the present invention may be extracted in an aqueous solution at room temperature, wherein the room temperature is 1°C or higher to less than 40°C, for example, 4°C to 37°C, preferably 4°C to 25°C. .

As the water constituting the aqueous solution, for example, purified water may be used, and preferably, the rice locust extract of the present invention is obtained by using an aqueous solution containing 0.01 to 5% by weight of an antioxidant as an extraction solvent, preferably 0.1 to 3% by weight of an aqueous solution containing antioxidants as an extraction solvent.

At this time, the antioxidant material that can be used in the present invention is ascorbic acid (vitamin C), EDTA (disodium ethylenediaminetetracetic acid), dicalcium EDTA, tocopherol, BHT (butylated hydroxytoluene), BHA (butylated hydroxyanisole), phosphate and citric acid It may be one or more selected from the group consisting of, preferably ascorbic acid (ascorbic acid). In particular, ascorbic acid, which can also be referred to as vitamin C, is water-soluble, exists in a reduced form by glutathione, an intracellular antioxidant, plays a role in removing active oxygen, such as hydrogen peroxide, and can inhibit protein gelation. Therefore, by adding ascorbic acid to the extraction solvent, water, it is possible to prevent protein denaturation by inhibiting the oxidation of proteins and fatty acids induced through the formation of active oxygen, and it is safer to the human body by removing various active oxygen. can be applied effectively. Also, for example, a buffer to which phosphate salts are added as an extraction solvent may be used. On the other hand, when an alcohol having a relatively low polarity, such as ethanol, is used as an extraction solvent, it is not preferable to extract a polar protein.

When the aqueous solution, which is the extraction solvent of the present invention, contains less than 0.01% by weight of an antioxidant, there is a problem in that the antioxidant for preventing denaturation does not function properly.

Meanwhile, the rice locust extract may be in a dry form of the extract, for example, in a dry powder form. That is, the meaning of the extract of the present invention is to include an extract in a dry solid state.

The process of preparing the rice locust extract of the present invention includes, for example, immersing the rice locust in an aqueous solution containing an antioxidant; and obtaining an extract from an aqueous solution in which rice locusts are immersed.

The water in the immersion step may be an aqueous solution containing 0.01 to 5% by weight of an antioxidant as described above, wherein the step of immersing the rice locust in the aqueous solution containing the antioxidant is 1 second to 6 hours, for example It may be 10 seconds to 2 hours, preferably 1 minute to 1 hour, and the immersion step may be performed at room temperature as described above.

Subsequently, a step of obtaining a rice locust extract from the aqueous solution in which the rice locusts are immersed is performed, and the step is a step of separating the solid and obtaining a liquid extract, for example, centrifuging the aqueous solution in which the rice locusts are immersed to obtain the rice locust extract. It can be obtained as a supernatant, in which case centrifugation may be performed at a room temperature of 1 to 25 °C, preferably at a temperature of 2 to 10 °C, at 1000 to 4000 rpm for 1 minute to 20 minutes.

Furthermore, the separated supernatant is additionally filtered with a 0.2 to 0.45 μm filter, if necessary, and then again at room temperature of 1 to 25° C., preferably 5000 to 20000 g at a temperature of 2 to 10° C., for example, 1 in Separation of the supernatant by centrifugation for minutes to 20 minutes may be additionally performed. By this step, the uppermost fat layer can be removed, and the purity of the water-soluble protein extract can be increased.

Furthermore, the step of pulverizing the rice locusts may be further included before or after the step of immersing the rice locusts, and when the rice locusts are applied in powder form, the extraction efficiency may be further improved.

After the centrifugation, the rice locust extract obtained from the supernatant is additionally filtered as needed to obtain a filtrate; and/or drying the filtrate may be further included, impurities may be further removed by performing such a filtration step, and an extract in a dried form may be obtained by further drying, For example, an extract in the form of a dry powder can be obtained. A method of performing the filtration step is not particularly limited, and may be performed using a filter, filter paper, or the like.

On the other hand, the drying step is also not particularly limited in the performing method, and may be performed by room temperature drying, freeze drying, vacuum drying, hot air drying, spray drying, etc., and is preferably performed by freeze drying.

In the composition for oral administration of the present invention, the rice locust extract is included as an active ingredient, and the administration route of the composition of the present invention may be administered orally or parenterally through all general routes as long as it can reach the target tissue, but preferably is administered orally.

For example, the composition for oral administration of the present invention may include the rice locust extract in an amount of 0.01 to 100% by weight, preferably 5 to 70% by weight, based on the total weight of the total composition. will do

The preferred dosage of the composition for oral administration of the present invention is in the range of 0.001 mg/kg to 1 g/kg of rice locust extract as an active ingredient per day depending on the subject's condition, weight, sex, age, severity of disease, and route of administration, preferably For example, 1 mg/kg to 500 mg/kg, for example, 10 mg/kg to 200 mg/kg may be administered. In this case, the administration may be divided into once or several times a day. However, the dosage may be increased or decreased depending on the dosage form, disease severity, sex, weight, age, and the like.

The composition for oral administration may be a pharmaceutical composition for cancer treatment, a pharmaceutical composition for adjuvant anticancer, or a food composition for enhancing immunity.

In the present invention, "subject" refers to humans and all other animals having or not having a disease whose symptoms can be improved by administering the composition of the present invention. The composition according to the invention can be applied not only to humans (treatment, inhibition or prevention) but also to other commercially useful animals.

In the pharmaceutical composition for treating cancer and the pharmaceutical composition for adjuvant anticancer, the cancer may also be referred to as a tumor, and refers to a malignant neoplasm, and these are used interchangeably. The type of cancer is not particularly limited, but melanoma, non-small cell lung cancer, small cell lung cancer, lung cancer, liver cancer, retinoblastoma, astrocytoma, glioblastoma, gingival cancer, tongue cancer, leukemia, neuroblastoma, head cancer, neck cancer , breast cancer, pancreatic cancer, prostate cancer, kidney cancer, bone cancer, testicular cancer, ovarian cancer, mesothelioma, cervical cancer, gastrointestinal cancer, lymphoma, brain cancer, colon cancer, sarcoma, colorectal cancer, brain tumor, stomach cancer, esophageal cancer, lymphoma, fibrosarcoma, mastocytoma and/or bladder cancer.

Furthermore, the cancer includes, for example, mammary gland cancer, complex mammary gland cancer, mammary gland malignant mixed tumor, intraductal papillary adenocarcinoma, lung adenocarcinoma, squamous cell carcinoma, small cell carcinoma, large cell carcinoma, neuroepithelial tissue tumor glioma, ependymaloma, nerve Cellular tumor, fetal neuroectodermal tumor, schwannoma, neurofibroma, meningioma, chronic lymphocytic leukemia, lymphoma, gastrointestinal lymphoma, gastrointestinal lymphoma, small to medium cell lymphoma, appendix cancer, ascending colon cancer, descending Colon cancer, transverse colon cancer, sigmoid colon cancer, rectal cancer, ovarian epithelial cancer, germ cell tumor, stromal cell tumor, pancreatic duct cancer, invasive pancreatic duct cancer, pancreatic adenocarcinoma, acinar cell carcinoma, adenosquamous cell carcinoma, giant cell tumor, intrapancreatic papillary mucinous tumor , mucinous cystic adenocarcinoma, pancreatic blastoma, serous cystic adenocarcinoma, solid papillary carcinoma, gastrinoma, glucaconoma, insulinoma, multiple endocrine adenomatosis 1 (Wermer syndrome), nonfunctional islet cell tumor, somatostatinoma, VIP-derived tumor but is not limited to these.

In particular, cancers to which the present invention can be applied may include cancers that are classified as solid cancers or cause metastasis of cancer cells, and preferably, melanoma, brain cancer, lung cancer, stomach cancer, liver cancer, head cancer, cervical cancer, prostate cancer , pancreatic cancer, colorectal cancer, lymphoma. More preferably, it is at least one cancer selected from colorectal cancer, rectal cancer and colon cancer.

The pharmaceutical composition of the present invention may include the rice locust extract as an active ingredient in any effective amount as long as it can exhibit the ameliorating activity of cancer intended for treatment, etc. depending on the use, formulation, purpose of formulation, and the like. As used herein, the term "effective amount" means when the composition of the present invention is administered to a mammal, preferably a human subject to the application, during the administration period as suggested by a medical professional, etc. Refers to the amount of the active ingredient included in the composition of the present invention, which can exhibit a pharmacological effect.

In addition to the active ingredient, the pharmaceutical composition of the present invention may further include any compound or natural extract that has already been verified for safety and known to have anticancer activity.

More specifically, the pharmaceutical composition of the present invention can be prepared in an oral dosage form by a conventional method known in the art, including a pharmaceutically acceptable carrier in addition to the active ingredient.

Pharmaceutically acceptable carriers are well known in the art depending on the route of administration or formulation, and specifically, reference may be made to the pharmacopoeia of each country including the "Korea Pharmacopoeia".

When the pharmaceutical composition of the present invention is prepared as an oral dosage form, powder, granules, tablets, pills, dragees, capsules, liquids, gels, syrups, suspensions, wafers, etc. according to methods known in the art together with a suitable carrier It can be prepared in the form of

Examples of suitable carriers include sugars such as lactose, glucose, sucrose, dextrose, sorbitol, mannitol, and xylitol, starches such as corn starch, potato starch, wheat starch, cellulose, methylcellulose, ethylcellulose, sodium carboxymethylcellulose, Cellulose such as hydroxypropylmethylcellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate, mineral oil, malt, gelatin, talc, polyol, vegetable oil, ethanol, grease Serol etc. are mentioned. In the case of formulation activity, an appropriate binder, lubricant, disintegrant, colorant, diluent, etc. may be included as needed. Suitable binders include starch, magnesium aluminum silicate, starch ferrist, gelatin, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone, glucose, corn sweetener, sodium alginate, polyethylene glycol, wax, and the like, and oleic acid as lubricant. sodium, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, silica, talcum, stearic acid, magnesium salts and calcium salts thereof, polyethylene glycol, etc., and the disintegrant include starch, methyl cellulose , agar, bentonite, xanthan gum, starch, alginic acid or its sodium salt. Examples of the diluent include lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, glycine, and the like.

The specific formulation of pharmaceutical compositions is known in the art.

Meanwhile, according to the present invention, there is provided a food composition for improving cancer, for example, a food composition for enhancing immunity. Regarding the rice locust extract, which is an active ingredient in the food composition, the same applies to the contents described in relation to the pharmaceutical composition.

The food composition of the present invention may be prepared in any form, for example, beverages such as tea, juice, carbonated drinks, and ion drinks, processed oils such as milk and yogurt, gums, rice cakes, Korean sweets, bread, confectionery, noodles, etc. Foods, tablets, capsules, pills, granules, liquids, powders, flakes, pastes, syrups, gels, jellies, and health functional food preparations such as bars can be prepared.

In addition, the food composition of the present invention may have any product classification as long as it conforms to the enforcement regulations at the time of manufacture and distribution in legal and functional classification. For example, it is a health functional food according to the Korean 「Health Functional Food Act」, or confectionery, beans, tea, and beverages according to each food type according to the Food Ordinance of Korea 「Food Sanitation Act」 (Ministry of Food and Drug Safety Notification 「Food Standards and Specification」) , special purpose food, and the like.

The food composition of the present invention may contain food additives in addition to the active ingredients thereof. Food additives can be generally understood as substances that are added to and mixed with or infiltrated into food in manufacturing, processing or preserving food. Food additives with guaranteed safety are limited in terms of ingredients or functions in the Food Additives Ordinance according to the laws of each country that regulates the manufacture and distribution of food (“Food Sanitation Act” in Korea). In the Korean Food Additives Code (“Food Additive Standards and Specifications” notified by the Ministry of Food and Drug Safety), food additives are classified into chemically synthetic products, natural additives, and mixed preparations in terms of ingredients. It is classified into an agent, a preservative, an emulsifier, an acidulant, and a thickener.

The sweetener is used to impart appropriate sweetness to food, and both natural and synthetic ones may be used in the composition of the present invention. Preferably, a natural sweetener is used. Examples of the natural sweetener include sugar sweeteners such as corn syrup solids, honey, sucrose, fructose, lactose, and maltose.

Flavoring agents may be used to improve taste or aroma, and both natural and synthetic ones may be used. Preferably, it is a case where a natural thing is used. In the case of using a natural one, the purpose of nutritional enhancement in addition to flavor may be concurrently used. As a natural flavoring agent, it may be obtained from apple, lemon, tangerine, grape, strawberry, peach, etc., or it may be obtained from green tea leaf, dandelion, bamboo leaf, cinnamon, chrysanthemum leaf, jasmine, etc. In addition, those obtained from ginseng (red ginseng), bamboo shoots, aloe vera, ginkgo biloba, etc. can be used. The natural flavoring agent may be a liquid concentrate or a solid extract. Optionally, a synthetic flavoring agent may be used, and the synthetic flavoring agent may be an ester, an alcohol, an aldehyde, a terpene, or the like.

As a preservative, sodium calcium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, sodium benzoate, potassium benzoate, EDTA (ethylenediaminetetraacetic acid), etc. can be used, and as an emulsifier, acacia gum, carboxymethylcellulose, xanthan gum, Pectin etc. are mentioned, and acidulant, malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartaric acid, ascorbic acid, acetic acid, phosphoric acid, etc. can be used as an acidulant. The acidulant may be added so that the food composition has an appropriate acidity for the purpose of inhibiting the growth of microorganisms in addition to the purpose of enhancing the taste.

As a thickening agent, a suspending agent, a settling agent, a gel-forming agent, a bulking agent, etc. can be used.

The food composition of the present invention may contain, in addition to the food additives as described above, physiologically active substances or minerals known in the art for the purpose of supplementing and reinforcing functionality and nutrition and guaranteed stability as food additives.

In the food composition of the present invention, the food additives as described above may be included in an appropriate amount to achieve the purpose of the addition according to the product type. In relation to other food additives that may be included in the food composition of the present invention, reference may be made to the Food Ordinance of each country or the Food Additives Code.

Hereinafter, the present invention will be described in more detail through specific examples. The following examples are only examples to help the understanding of the present invention, and the scope of the present invention is not limited thereto.

Example

1. Preparation of rice locust extract

Rice grasshopper (Oxya chinensis sinuosa) was obtained from S-WORM (S-WORM, Cheonan, Korea), dried and ground to prepare a powder.

600 mL of 0.16% ascorbic acid in water was mixed with 100 g of the pulverized rice locust powder, and the mixture was stored at 4° C. for 1 hour. Thereafter, the mixture was centrifuged at 4° C. and 3,000 rpm for 5 minutes to separate the supernatant. The separated supernatant was filtered with filter paper (filter paper, No. 4), and then centrifuged again at 4° C. and 10,000 g for 10 minutes to separate the supernatant. The separated supernatant was further filtered with a 0.45 μm filter and then freeze-dried to obtain a dry powder of the extract.

2. Changes in surface expression factors of dendritic cells

After dissolving 10 mg of the freeze-dried extract powder obtained in 1. in 1 mL of PBS and performing a 0.45 μm filter, the degree of contamination with LPS (lipopolysccharide) to exclude immune activity caused by contamination with gram negative bacteria As a result of performing LAL (Limulus Amebocyte Lysate) analysis that can measure

Differentiation of dendritic cells using bone marrow-derived cells from mice was performed through GM-CSF/IL-4 (dendritic cell growth factor). In more detail, femoral bone marrow was collected from C57BL/6 mice by injection for bone marrow collection, and after washing the collected bone marrow, red blood cells were removed using ammonium chloride. The isolated cells were treated with RPMI 1640 (10% FBS, 100 U/ml penicillin/streptomycin, 20 ng/ml GM-CSF, 0.5 ng/ml IL-4) in a 6-well plate for 8 days. cultured.

Before evaluating the immune activity by the rice locust extract, intracellular toxicological safety was evaluated at various concentrations prepared as described above. After diluting 10 mg/mL of the rice locust extract in PBS to 100 μg or 1 mg/mL (OcsP), the dendritic cells were treated so that the final concentration in the cell culture solution was 1, 10, and 50 100 μg/mL, respectively. As a result, it was confirmed that toxicity was not induced at the concentrations of 1, 10, 50, and 100 μg/mL (FIG. 1(a)). At this time, the control (control, con) is the PBS-only treatment group.

Next, the activity of dendritic cells was evaluated based on the concentration showing such toxicological safety. More specifically, the surface expression factors CD80, CD86, MHC-I and MHC-II are known to be important factors for the activation and differentiation of T cells. and high expression of these factors can promote the activation and differentiation of T cells.

Based on these contents, the rice locust extract was treated with the rice locust extract at concentrations of 1, 10, 50, and 100 μg/mL for 20 hours in dendritic cells cultured for 8 days. After 20 hours of treatment, the treated dendritic cells were stained with anti-CD11c-V450, anti-CD80-FITC, anti-CD86-PE, anti-MHC-I-APC, and anti-MHC-II-PE-Cy7 antibodies. It was then analyzed with a flow cytometer FACSverse.

As a result, it was confirmed that the expression of CD80, CD86, MHC-I and MHC-II of dendritic cells was strongly induced in the group treated with the rice locust extract, as shown in FIG. 1(b). At this time, 100 ng/ml of LPS was used as a positive control for the maturation of dendritic cells.

3. Cytokine inducing ability and antigen acquisition ability evaluation

(1) Cytokine inducibility evaluation

TNF-alpha, an inflammatory cytokine induced upon activation of dendritic cells, and IL-12p70, which are important for Th1 polarization of T cells, are known as important index factors for differentiation of Th1-type cells. On the other hand, IL-10 has the ability to suppress such immunity, so it can suppress anticancer immunity. Therefore, analysis of the increase pattern of these index factors can be an important data to predict whether various diseases such as cancer, tuberculosis, and non-tuberculous mycobacteria can be controlled.

Based on this, dendritic cells were treated with samples of rice locust extract 1, 10, 50, and 100 μg/mL concentrations for 20 hours. Meanwhile, 100 ng/ml of LPS was used as a positive control for the maturation of dendritic cells. After 20 hours of treatment, TNF-alpha, IL-12p70 and IL-10 in the culture medium were analyzed by ELISA.

As a result, as can be seen in FIG. 2(a), TNF-alpha and IL-12p70 were induced higher than that of the control group when the rice locust extract was treated.

As shown in Fig. 2(b), after 8 hours of treatment with rice locust extract and GolgiPlug, anti-CD11c-V450, anti-TNF-alpha-APC, anti-IL-12p70-PE and anti-IL-10-FITC antibodies were After staining using a flow cytometer, it was confirmed again with FACSverse.

(2) Evaluation of antigen acquisition ability

Dendritic cells have a characteristic that their ability to acquire antigens decreases during maturation. Based on this, dendritic cells were treated with samples of 1, 10, 50, and 100 μg/mL of rice locust extract for 20 hours, and these cells were treated with 1 mg/mL dextran for 20 minutes and then antigen acquisition ability Changes were analyzed by flow cytometry.

As a result, as can be seen in FIG. 3(a), it was confirmed that the dextran uptake ability was significantly reduced in the dendritic cells treated with the rice locust extract sample. Based on this, it was confirmed that the treatment of dendritic cells with the rice locust extract could effectively induce dendritic cell maturation.

(3) Immune activity evaluation

When a protein is heat-treated, it causes denaturation of the protein and loses its intrinsic properties. Based on this, rice locust extract (100 μg/mL) was heated at 100 °C for 30 minutes on dendritic cells, and the non-heated sample (Ocs-P) and the heated sample (Boiled Ocs-P) were treated on the dendritic cells. did. As a result, it was confirmed that the heat-treated rice locust protein significantly reduced the surface expression factors of dendritic cells (Fig. Based on these results, it could be confirmed that the rice locust extract contains protein as an active ingredient, and the unique properties of the rice locust extract protein induce immune activity.

4. Characterization of dendritic cells treated with rice locust extract

(1) T cell activity, proliferation and differentiation capacity

The most important feature of mature dendritic cells in vivo is the induction of T lymphocyte activation. An experiment was performed to determine whether dendritic cells treated with rice locust extract could enhance cytotoxic T lymphocyte (CTL) response and induce differentiation into Th1-type cells. In order to carry out the experiment by the MLR (Allogenic mixed lymphocyte reaction) method, CD4 ⁺ and CD8 ⁺ T cells isolated from the spleen of BALB/C mice were cultured with C57BL/6-derived locust extract-treated dendritic cells, followed by T cells. The degree of proliferation was measured. As a result, as can be seen from the flow cytometry results in FIG. 4(a) , the dendritic cells treated with the rice locust extract effectively induced the proliferation of naive CD4 and CD8 T cells.

In addition, the ELISA kit was applied to analyze the amount of cytokines (IFN-g, IL-2, IL-5) secreted by CD4 and CD8 T cells. It was confirmed that it can induce (Fig. 4(b)).

(2) CTL activation ability

CTL cells can directly kill antigen-specific target cells. Therefore, PBS only, immature dendritic cells (iDC), OVA _257-264 treated dendritic cells (OVA-DC), rice locust extract and 1 ug/mL OVA _257-264 treated dendritic cells (Ocs-P/OVA-DC) were immunized 3 times (on day 1, 3, 5) with IV in mice of each group. Five days after the last immunization, splenic T cells from naive mice were isolated, and T cells treated with OVA _257-264 were treated with 5 mM CPD (Cell proliferation dye) (OVA-treated T cells), and OVA _257-264 . Untreated T cells were stained with 0.5 mM (OVA-untreated T cells), respectively, and these T cells were mixed 1:1 and administered IV to dendritic cell immunized mice 6 hours later, OVA-treated T cells (5 mM CPD staining) ) to evaluate the CTL activity of dendritic cells treated with rice locust extract.

As a result, as can be seen in FIG. 4(c), it was confirmed that the dendritic cell immunized mice treated with the rice locust extract induced a rapid decrease in OVA-treated T cells (5 mM CPD staining).

5. Effect of reducing colon cancer tumor size Confirm

(1) experimental animals

Seven-week-old female BALB/c mice were purchased from Orient Bio and acclimatized for 1 week, and then used in the experiment. The temperature of 23±2℃, humidity of 55±5%, and light-dark cycle were acclimatized for one week in an experimental animal kennel maintained at 12-hour intervals, and solid feed and water were freely supplied. Animal experiments for this study were performed under the approval of the Animal Experiment Ethics Committee of the Advanced Radiation Research Institute, Korea Atomic Energy Research Institute.

(2) Evaluation of cancer cell suppression ability

To construct a BALB/C mouse-derived colorectal cancer model, 1Х10 ⁵ /mouse of CT26 cells was subcutaneously injected into BALB/C mice. Six days after the injection, the rice locust protein was orally administered 5 times at 3-day intervals (FIG. 5(a)).

As a result, as can be seen in FIG. 5(b), it was confirmed that cancer growth was significantly slowed when the rice locust extract was administered. In particular, there was no change in tumor volume in the untreated group, but it was confirmed that the tumor volume decreased significantly from the 15th day of tumor administration in the group treated with the rice locust extract.

(3) Evaluation of the activity of dendritic cells

Additionally, as a result of analyzing the activity of splenocyte-derived dendritic cells (splenic CD11c+MHC-II+), as can be seen in FIG. MHC-I) was remarkably decreased, and importantly, it was confirmed that this decrease was recovered when the rice locust extract was administered.

6. Confirmation of Inducing Effect of Multifunctional T Cells

In order to analyze whether the reduction in tumor size caused by oral administration of the rice locust extract in 5. above is due to the immune activation effect of the rice locust extract, CT26 cells were subcutaneously injected into BALB/C mice 6 days later, and the rice locust extract was orally administered for 3 days. Changes in T cells induced when administered 5 times at an interval were analyzed in splenocytes.

The mouse spleen was isolated, and CD4 T cells (FIG. 6) and CD8 T cells (FIG. 7) were analyzed. As a result, flow cytometry analysis of FIGS. 6 (a) and 7 (b) and single, double, triple or quadruple functionalities were performed. As can be seen from the histogram showing the percentages of the three groups of CD8 + T cells, multifunctional CD4 T cells that simultaneously induce CD107a, IFN-gamma, TNF-alpha and IL-2, which can induce strong anticancer immunity, and It was confirmed that CD8 T cells were significantly increased. It is known that these multifunctional cells have significantly higher effector functions than cells that induce cytokines alone.

Although the embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and various modifications and variations are possible within the scope without departing from the technical spirit of the present invention described in the claims. It will be apparent to those of ordinary skill in the art.

Claims (9)

A composition for oral administration comprising an extract of rice locust (Oxya chinensis sinuosa).
The composition for oral administration according to claim 1, wherein the extract is obtained at room temperature using an aqueous solution containing an antioxidant as an extraction solvent.
The composition for oral administration according to claim 1, wherein the antioxidant is contained in an amount of 0.01 to 5% by weight based on the total weight of the aqueous solution.
4. The method of claim 3, wherein the antioxidant is selected from the group consisting of ascorbic acid, disodium ethylenediaminetetracetic acid (EDTA), dicalcium EDTA, tocopherol, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), phosphate and citric acid. One or more kinds of compositions for oral administration.
The composition for oral administration according to claim 1, wherein the composition for oral administration contains the rice locust extract in an amount of 0.01 to 100% by weight based on the total weight of the total composition.
The composition for oral administration according to claim 1, wherein the composition for oral administration has an oral dosage in the range of 0.001 mg/kg to 1 g/kg per day.
The composition for oral administration according to claim 1, wherein the composition for oral administration is a pharmaceutical composition for cancer treatment, a pharmaceutical composition for adjuvant anticancer, or a food composition for enhancing immunity.
The composition for oral administration according to claim 7, wherein in the pharmaceutical composition for cancer treatment and the pharmaceutical composition for adjuvant anticancer, the cancer is at least one cancer selected from colorectal cancer, rectal cancer and colon cancer.
The composition for oral administration according to claim 1, wherein the composition for oral administration is in the form of tablets, pills, powders, granules, powders, solutions, sprays or capsules.

Images