RU2792300C2 - Pharmaceutical composition for prevention or treatment of cancer containing weissella cibaria wikim28 as an active ingredient - Google Patents

Abstract

FIELD: pharmaceuticals.

SUBSTANCE: pharmaceutical composition for prevention or treatment of colorectal cancer containing Weissella cibaria WIKIM28 (identification number KCCM11879P) or its culture as an active ingredient. Also proposed are a food composition, a food additive composition, a feed composition and a feed additive composition containing Weissella cibaria WIKIM28 (identification number KCCM11879P) or its culture as an active ingredient.

EFFECT: composition containing Weissella cibaria WIKIM28 (identification number KCCM11879P) shows excellent anti-cancer activity in animal models in which cancer cells are transplanted, and therefore can be successfully used to treat, prevent or attenuate colorectal cancer in humans or animals.

12 cl, 5 dwg

Description

[TECHNICAL FIELD]

The present invention relates to a composition for the prevention or treatment of cancer, containing Weissella cibaria WIKIM28 (identification number KCCM11879P), isolated from kimchi, as an active ingredient.

[PRIOR ART]

Cancer mortality is high worldwide, and cancer is the most common cause of death after cardiovascular disease in Western countries. In particular, as a result of the westernization of eating habits, the consumption of high-fat foods has become commonplace, and due to the rapid increase in environmental pollutants and the increase in alcohol consumption and the like, the incidence of colon cancer, breast cancer, prostate cancer, and the like continues grow and the incidence of lung cancer is on the rise due to an increase in the smoking population and air pollution in addition to an aging population. In this situation, there is an urgent need to develop anti-cancer substances that can promote human health, improve health-related quality of life and improve human health by providing early prevention and treatment of cancer.

Meanwhile, lactic acid bacteria are widely distributed in the mouth, intestines, vagina and excrement of humans and animals, as well as fermented foods such as kimchi, and are closely related to human and animal health. Lactic acid bacteria exhibit various health-promoting effects, such as gut regulation, inhibition of harmful bacteria, immunoregulation, blood cholesterol lowering, anti-cancer activity, and the like.

At present, Korean Patent Application Publication No. 10-2015-0068061 discloses the anti-cancer activity of Lactobacillus plantarum PNU (KCCM11352P) or Lactobacillus mesenteroides PNU (KCCM11353P), and Korean Patent No. 10-1287120 discloses a pharmaceutical composition for cancer treatment containing Lactobacillus plantarum DSR CK10 [identification number: KFCC-11433P] or Lactobacillus plantarum DSR M2 [identification number: KFCC-11432P] as active ingredient, but the anti-cancer activity and superiority of Weissella cibaria have not been reported.

[DESCRIPTION]

[TECHNICAL PROBLEM]

An object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of cancer containing a Weissella cibaria strain as an active ingredient.

In addition, another object of the present invention is to provide a food composition or dietary supplement composition for preventing or ameliorating cancer, containing a new lactic acid bacterium contained in kimchi, a strain of Weissella cibaria, as an active ingredient.

In addition, another object of the present invention is to provide a feed composition or a feed additive composition for preventing or alleviating cancer, containing a novel lactic acid bacterium contained in kimchi, a strain of Weissella cibaria, as an active ingredient.

[TECHNICAL SOLUTION]

Accordingly, the present inventors attempted to find a lactic acid bacteria strain which exhibits excellent effects as a probiotic and exhibits cancer prevention and treatment effects, a lactic acid bacteria strain having anticancer activity, namely Weissella cibaria WIKIM28, was isolated from kimchi and identified, thereby arriving at the present invention.

To achieve these objects, the present invention provides a pharmaceutical composition for the prevention or treatment of cancer, containing Weissella cibaria WIKIM28 (identification number KSCM11879P) or its culture as an active ingredient.

In addition, the present invention provides a nutritional composition or dietary supplement composition for preventing or ameliorating cancer, containing Weissella cibaria WIKIM28 (identification number KSCM11879P) or a culture thereof as an active ingredient.

Moreover, the present invention provides a feed composition or a feed additive composition for preventing or ameliorating livestock cancer (livestock) containing Weissella cibaria WIKIM28 (identification number KSCM11879P) or a culture thereof as an active ingredient.

[EFFECTS PROVIDING BENEFITS]

Weissella cibaria WIKIM28 according to the present invention shows excellent anti-cancer activity in standard animal models and animal models transplanted with human cancer cells, and therefore it can be successfully used as a composition for use in treating, preventing or attenuating cancer in humans or animals.

[BRIEF DESCRIPTION OF THE DRAWINGS]

FIG. 1 is a drawing showing photographs of a tumor part of a mouse to observe how the size of tumor cells changes over time after intravenous injection of a composition of the present invention into mice transplanted with CT26 cells.

FIG. 2 is a drawing showing a plot of volume change over time following intravenous injection of a composition of the present invention into mice transplanted with CT26 cells.

FIG. 3 is a drawing showing photographs of a tumor part of a mouse to observe how tumor cell size changes over time after intravenous injection of a composition of the present invention into mice transplanted with MC38 cells.

FIG. 4 is a drawing showing a plot of volume change over time following intravenous injection of a composition of the present invention into mice transplanted with MC38 cells.

FIG. 5 is a drawing showing a graph of tumor cell volume growth rate over time after intravenous injection of a composition of the present invention into mice transplanted with MC38 cells.

[IMPLEMENTATION OF THE INVENTION]

Hereinafter, the present invention will be described in more detail using examples. These examples are intended solely to more specifically illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples in accordance with the gist of the present invention.

The composition containing Weissella cibaria WIKIM28 (identification number KSCM11879P) or its culture as an active ingredient according to the present invention has the effect of preventing or treating cancer and can be used as a pharmaceutical composition.

Weissella cibaria WIKIM28 is a kimchi-derived lactic acid bacterium. Although Weissella cibaria WIKIM28 according to the present invention is isolated and identified from kimchi, production methods are not limited to this method.

Sequencing of 16S rDNA to identify and classify Weissella cibaria WIKIM28 showed that it has the nucleotide sequence of SEQ ID NO: 1.

Accordingly, the microorganism of the present invention, which has the 16S rDNA sequence of SEQ ID NO: 1, is named Weissella cibaria WIKIM28 and deposited with the Korean Microorganism Culture Center on October 8, 2015 (identification number KCCM11879P).

Weissella cibaria WIKIM28 according to the present invention has a general intestinal regulation effect and an immunostimulating effect of lactic acid bacteria as a probiotic. It is well known that lactic acid bacteria Lactobacillus sp. have the effect of regulating the intestines and immunostimulating effect.

As used herein, the term "probiotics" is intended to mean live microorganisms that have a positive effect on the health of the host by improving the intestinal microbial environment in the gastrointestinal tract of animals, including humans. Probiotics are live microorganisms with probiotic activity and are in the form of single or mixed strains, and when consumed in the form of dried cells or fermented products by humans or animals, they can have a positive effect on the intestinal flora of the host.

The cancer may be any cancer selected from the group consisting of bladder cancer, breast cancer, melanoma, thyroid cancer, parathyroid cancer, rectal cancer, throat cancer, larynx cancer, esophageal cancer, pancreatic cancer, gastric cancer, tongue cancer, skin cancer, brain tumor, uterine cancer, gallbladder cancer, oral cancer, colon cancer, anal cancer, liver cancer, lung cancer and colorectal cancer, and preferably it may be colorectal cancer, but is not limited to them.

The Weissella cibaria WIKIM28 contained in the composition of the present invention may be present as a live cell or dead cell, and in addition it may be present in a dried or lyophilized form. Forms and methods of obtaining lactic acid bacteria suitable for inclusion in various compositions are well known to specialists in this field of technology.

Said composition may be administered orally or parenterally. In the case of parenteral administration, it may be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration and rectal administration and the like, and it may preferably be administered by intravenous injection, but is not limited to this. .

A suitable dose according to the present invention may be administered in various ways depending on factors such as preparation method, patient age, body weight, sex, disease state, food, administration time, administration route, excretion rate, and response sensitivity.

When the composition of the present invention is used as a pharmaceutical composition, the pharmaceutical composition of the present invention can be prepared using a pharmaceutically suitable and physiologically acceptable adjuvant along with the active ingredient, and as an adjuvant, an excipient, a disintegrating agent, a sweetener, a binding agent, a coating material, an expanding agent, a lubricating agent, a glidant or flavoring agent, or the like.

Said pharmaceutical composition may preferably be formulated into a pharmaceutical composition by additionally including one or more kinds of pharmaceutically acceptable carriers along with the described active ingredient for administration.

For example, for preparation in the form of a tablet or capsule, said active ingredient may be combined with an oral and non-toxic pharmaceutically acceptable inactive carrier such as ethanol, glycerol, water, and the like. In addition, if desired or necessary, a suitable binding agent, lubricating agent, loosening agent and coloring agent can also be included in this mixture. Suitable binding agent includes, but is not limited to, starch, gelatin, natural sugars such as glucose or beta-lactose, natural and synthetic gums such as corn sweetener, gum arabic, sodium tragacanth or oleate, sodium stearate, magnesium stearate, sodium benzoate , sodium acetate, sodium chloride, and the like. The disintegrating agent includes, but is not limited to, starch, methylcellulose, agar, bentonite, xanthan gum, and the like. Saline solution, sterile water, Ringer's solution, buffered saline solution, albumin injection solution, dextrose solution, maltodextrin solution, glycerin, ethanol and a mixture of one or more components selected from them, and other conventional additives such as an antioxidant, a buffer solution, a bacteriostatic agent, and the like can be added if necessary. In addition, it can be formulated into an injection formulation such as an aqueous solution, suspension, emulsion, etc., pill, capsule, granule, or tablet by further adding a diluent, a dispersing agent, a surfactant, a binder, and lubricant agent.

In addition, it can preferably be prepared depending on each disease or component using the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA, as a suitable method in the art.

A composition containing Weissella cibaria WIKIM28 (identification number KSCM11879P) or a culture thereof as an active ingredient according to the present invention can be used as a food composition or dietary supplement composition for preventing or attenuating cancer.

Said food composition may be in the form of a health functional food product.

The term “health functional food” means a food product that is produced and processed using raw materials or ingredients that have beneficial functions for the human body in accordance with the Health Functional Food Act (Article 3, No. 1), and “functionality " means obtaining beneficial effects for health applications such as nutrient regulation or physiological effects and the like. based on the structure and functions of the human body (the same article, No. 2).

Said food composition may additionally contain a food additive, and, unless otherwise indicated, suitability as a "food additive" must be determined in accordance with the standards and criteria for the relevant name under the General Rules and General Test Methods of the Food Additive Code (Food Additive Code) approved by the Ministry of Food and Drug Safety.

The name given in the Food Additives Code may include, for example, chemical synthetic products such as ketones, glycine, potassium citrate, nicotinic acid, cinnamic acid, etc., natural additives such as persimmon dye (Persimmon color), licorice extract, crystalline cellulose, guar gum, etc., and mixed formulations such as monosodium L-glutamine formulations, noodle alkaline agents, preservative formulations, tar color (tar color) formulations, etc.

The food product containing the active ingredient of the present invention may include, but is not limited to, confectionery products such as bread, rice cakes, dried cakes, candy, chocolate, chewing gum and jam, ice cream related products such as ice cream, ice and ice cream powder, dairy products such as milk, reduced fat milk, lactose split milk, processed milk, goat milk, cultured milk product, buttermilk, condensed milk, milk cream, buttermilk, natural cheese, processed cheese, milk powder and whey, meat products such as processed meat products, processed egg products and hamburgers, fish meat products such as fish cakes, ham, sausage, bacon, etc. noodles such as ramen, dried noodles, fresh noodles, instant fried noodles, gelatinized dried noodles, boiled l improved cooked noodles, frozen noodles and pasta, beverages such as fruit juice beverages, vegetable beverages, soy milk, lactobacillus-containing beverages such as yogurt, etc., and mixed beverages, sauces such as soy sauce, soy paste, red pepper paste, black soybean paste, mixed paste and vinegar, food condiments such as tomato ketchup, curry and dressing, margarine, confectionery fat and pizza.

In addition, the composition according to the present invention may contain various nutrients, vitamins, electrolytes, flavors, coloring agents, pectin acid and its salts, alginic acid and its salts, organic acids, thickeners, which are protective colloids, pH adjusting agents, stabilizers, preservatives, glycerol, alcohols, carbonizing agents used in carbonated drinks, and the like. Moreover, the composition according to the present invention may contain pulp for the production of natural fruit juices, juice-containing fruit drinks and vegetable drinks. These ingredients may be used independently or in combination.

The beverage composition containing the active ingredient of the present invention is not particularly limited with respect to other components, and, like conventional beverages, may contain an additional component such as various flavors or natural carbohydrates or the like. An example of the natural carbohydrates described above are conventional sugars such as monosaccharides (eg glucose, fructose, etc.); disaccharides (eg, maltose, sucrose, etc.) and polysaccharides (eg, dextrin, cyclodextrin, etc.), and sugar alcohols such as xylitol, sorbitol, erythritol, and the like. As a flavor other than the flavors described above, natural flavors (thaumatin, stevia extracts (eg, rebaudioside A, glycyrrhizin, etc.)) and synthetic flavors (saccharin, aspartame, etc.) can be successfully used.

In addition, the composition containing Weissella cibaria WIKIM28 (identification number KCCM11879P) or its culture as an active ingredient according to the present invention can be used as a feed composition or feed additive composition for preventing or ameliorating livestock cancer.

When said composition is obtained as a feed additive, said composition can be produced in the form of a strong (20-90%) concentrate or in the form of a powder or granules. Said feed additive may further contain any or one or more of organic acids such as citric acid, fumaric acid, adipic acid, lactic acid, malic acid, etc., or phosphates such as sodium phosphate, potassium phosphate, acid pyrophosphate. , polyphosphate (polyphosphate), etc., or natural antioxidants such as polyphenol, catechin, alpha-tocopherol, rosemary extract, vitamin C, green tea extract, liquorice extract, keto acid, tannic acid, phytic acid, etc. When obtained as a feed, said composition may be prepared in the usual form of feed and may contain conventional feed components.

The feed and feed additive may additionally contain grains, such as powdered or crushed wheat, oats, barley, corn and rice; vegetable protein food, such as food containing rapeseed, beans (bean) and sunflower as the main component; food with animal protein, such as powdered blood, meat meal, bone dust and fish meal; sugars and dairy products, such as dried components consisting of various types of milk powder and whey powder, and the like, and in addition may further contain a nutritional supplement, a digestion and absorption enhancer, a growth promoting agent, etc. P.

Said feed additive may be administered to the animals alone or in combination with another feed additive in an edible carrier. In addition, said feed additive can be mixed in the form of top dressing or directly with animal feed or easily administered to animals as an oral formulation separate from the feed. When said feed additive is administered separately from the animal feed, as is well known in the art, it may be formulated as an immediate or sustained release formulation in combination with a pharmaceutically acceptable edible carrier. Said edible carrier may be solid or liquid, such as corn starch, lactose, sucrose, bean flakes, peanut butter, olive oil, sesame oil, and propylene glycol. When a solid carrier is used, said feed additive may be in the form of a tablet, capsule, powder, troche, lozenge, or non-dispersed form of supplement. When a liquid carrier is used, said feed additive may be a soft gelatin capsule formulation or a syrup or suspension, emulsion or solution.

In addition, the feed and feed additive may contain an additive such as a preservative, stabilizer, wetting agent or emulsifier, dissolution accelerator or the like. The specified feed additive can be applied by adding to animal feed by dissolving (salivating), spraying or mixing.

The food or feed additive according to the present invention can be used with many animal feeds, including mammals, poultry and fish.

It can be applied to pigs, cows, sheep, goats, experimental rodents and domestic animals (eg dogs, cats) along with experimental rodents and the like. as mammals, can be applied to chickens, turkeys, ducks, geese, pheasants and quails, etc. as poultry and can be used for trout, etc. as a fish, but not limited to.

In addition, the composition containing Weissella cibaria WIKIM28 (identification number KSCM11879P) or its culture as an active ingredient according to the present invention can be used as a lactic acid bacteria starter for fermentation.

Example 1 Isolation and cultivation of a strain

Weissella cibaria WIKIM28 (identification number KSCM11879P) was provided from the Microorganism and Gene Bank of the World Kimchi Institute to carry out the experiment.

A single colony of the provided Weissella cibaria WIKIM28 was subcultured in an agar medium containing Mann, Cattail and Sharp (MRS) Lactobacillus Culture Agar at 30° C. for 48 hours, and then the single culture colony was cultured at 37° C. for 24 hours at 200 rpm after inoculation into 10 ml liquid medium containing MRS agar. After cultivation, the cells of the strain were centrifuged at 8000 rpm for 5 minutes to remove the culture solution and washed three times with PBS (phosphate-buffered saline) to remove the remaining medium components.

Example 2. Conditions for keeping experimental animals (mice)

As experimental animals used for the experiment, male BALB/c mice (Orient Bio, Korea) and C57BL/6 at the age of 5 weeks were provided, which were reared during the period of the experiment after a stabilization period of 1 week in the animal breeding room in a pathogen-free environment, where room temperature was maintained at 20±2°C and humidity 55±15%. A general sample of pellets without antibiotic was provided as a sample, and water was available for frequent ingestion. During the experiment, all animals were reared, subjected to experimental procedures and euthanasia in accordance with the protocol approved by the Ethical Committee for Animal Research of the World Kimchi Institute. Tumor size change was monitored by measuring the volume (mm ³ ) of the tumor using the equation 3.14×(length×height×area)/6.

Example 3 Generation of Animal Models with Transplanted CT26 and MC38 Mouse Colorectal Cancer Cells

3-1. Cell culture

CT26 mouse colorectal cancer cells (Korea Bank of Cell Lines, Korea) were purchased and used, and MC38 mouse colorectal cancer cells (Chonnam National University, College of Medicine) were provided and used. These CT26 and MC38 mouse colorectal cancer cells were cultured at 5% CO ₂ and 37°C in DMEM medium (Hyclone, USA) containing 10% fetal bovine serum and 1% penicillin-streptomycin.

3-2. Obtaining an animal model of cancer with transplanted cells

6 week old BALB/c mice (18-21 g) were used for the CT26 cell transplant animal model, and 6 week old C57BL/6 mice (18-21 g) were used for the MC38 cell transplant animal model. G). Cultured mouse colorectal cancer cells CT26 and MC38 were administered by subcutaneous injection into the right thigh of mice after harvesting 1×10 ⁵ cells, respectively, and resuspended in 50 μl of PBS.

Example 4 Analysis of the antitumor effect of Weissella cibaria WIKIM28

4-1. Analysis of the antitumor effect in animal models with transplanted CT26 cells

Weissella cibaria WIKIM28 was administered by tail intravenous injection to mice transplanted with CT26 cells that developed a tumor of about 80~100 mm ³ . Weissella cibaria WIKIM28 was prepared by providing a bacterial count of 1 x 10 ¹⁰ cfu/ml using PBS, and 0.1 ml (1 x 10 ⁹ cfu) was administered by intravenous injection to the experimental animal, and the negative control group was injected with PBS. A result confirming the change in colorectal cancer tumor size as determined by the naked eye over time in mice transplanted with CT26 cells is shown in FIG. 1.

As shown in FIG. 1, it was found that the number of tumor cells decreased to such an extent that it could be seen with the naked eye, after 12 days in the case of the introduction of Weissella cibaria WIKIM28 by intravenous injection compared with the negative control group (PBS).

In addition, in FIG. 2 shows the result of determining the change in tumor volume over time in mice transplanted with CT26 cells.

As shown in FIG. 2, it was confirmed that the anti-tumor effects on tumor size in colorectal cancer mice transplanted with CT26 cells when injected with Weissella cibaria WIKIM28 after 16 days were about 2.4 times better than the negative control group.

Example 5 Biodistribution study after injection of Weissella cibaria WIKIM28

Weissella cibaria WIKIM28 was administered by tail intravenous injection to mice transplanted with CT26 cells that developed a tumor of about 80~100 mm ³ . Weissella cibaria WIKIM28 was prepared by providing a bacterial count of 1 x 10 ¹⁰ cfu/ml using PBS, and 0.1 ml (1 x 10 ⁹ cfu) was injected intravenously into the tail of the experimental animal, and the negative control group was injected with PBS. A result confirming change in colorectal cancer tumor size as determined by the naked eye over time in MC38 transplanted mice is shown in FIG. 3.

As shown in FIG. 3, it could be observed that the number of tumor cells was reduced to such an extent that it could be seen with the naked eye, in the case of the introduction of Weissella cibaria WIKIM28 by intravenous injection compared with the negative control group (PBS). It was confirmed that after 15 days, the tumor was reduced to such an extent that it could not be seen with the naked eye.

In addition, in FIG. 4 and FIG. 5 shows the result of determining the change in tumor volume and volume growth rate over time in mice transplanted with MC38 cells.

As shown in FIG. 4 and FIG. 5, it can be confirmed that colorectal cancer tumor volume and growth rate in MC38 transplanted mice rapidly decreased when Weissella cibaria WIKIM28 was injected compared with the negative control group, and it can be determined that after 12 days, antitumor effects were about 16.3 times better.

Based on the experimental results, the present inventors confirmed that the antitumor effects of Weissella cibaria WIKIM28 are excellent, and therefore the value of the invention disclosed in the present application as a composition for preventing, alleviating and treating cancer is high.

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Sequence listing

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<120> Pharmaceutical composition for the prevention or treatment of cancer,

containing Weissella cibaria WIKIM28 as an active ingredient

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<---

Claims (19)

1. Pharmaceutical composition for the prevention or treatment of colorectal cancer, containing Weissella cibaria WIKIM28 (identification number KCCM11879P) or its culture as an active ingredient. 2. Pharmaceutical composition according to claim 1, where Weissella cibaria WIKIM28 has the nucleotide sequence of SEQ ID NO: 1. 3. Pharmaceutical composition according to claim 1, wherein said composition may be administered by oral or parenteral route. 4. Pharmaceutical composition according to claim 3, where the specified method of parenteral administration is an introduction by intravenous injection. 5. Pharmaceutical composition according to claim 1, wherein said composition is in the form of a living cell. 6. Food composition containing Weissella cibaria WIKIM28 (identification number KCCM11879P) or its culture as an active ingredient. 7. Food composition according to claim 6, wherein said food product is a health functional food product. 8. Food composition according to claim 6, wherein said food product is any food product selected from the group consisting of kimchi, soy sauce, soy paste, red pepper paste, beverages and a fermented milk product. 9. Food supplement composition containing Weissella cibaria WIKIM28 (identification number KCCM11879P) or its culture as an active ingredient. 10. Feed composition containing Weissella cibaria WIKIM28 (identification number KCCM11879P) or its culture as an active ingredient. 11. Feed composition according to claim 10, where said livestock is any livestock selected from the group consisting of pig, cow, horse, sheep, rabbit, goat, mouse, hamster, guinea pig, dog, cat, chicken, turkey, duck, goose, pheasant, quail , carp, golden carp and trout. 12. Feed additive composition containing Weissella cibaria WIKIM28 (identification number KCCM11879P) or its culture as an active ingredient.

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