KR20240014859A - Pharmaceutical composition for prevention or treatment of cancer comprising Leuconostoc mesenteroides as active ingredient - Google Patents

Abstract

The present invention relates to the prevention of cancer comprising as an active ingredient a strain belonging to the genus Leuconostoc isolated from kimchi, specifically, a strain called Leuconostoc mesenteroides LB-LM1 (Leuconostoc mesenteroides LB-LM1) or a culture thereof. or to a therapeutic composition.
Since the Leuconostoc strain or its culture according to the present invention exhibits excellent anticancer activity, it can be usefully used as a composition for the treatment, prevention, or improvement of cancer in humans or animals.

Description

Pharmaceutical composition for prevention or treatment of cancer comprising Leuconostoc mesenteroides as active ingredient}

The present invention provides a method for preventing cancer comprising a strain of the genus Leuconostoc isolated from kimchi, specifically a strain of Leuconostoc mesenteroides LB-LM1 (Leuconostoc mesenteroides LB-LM1) or a culture thereof as an active ingredient. It relates to a therapeutic composition.

Cancer has a high mortality rate worldwide and is the most common cause of death in Western societies after cardiovascular disease. In particular, the consumption of high-fat diets has become common as dietary habits have become westernized, and colon cancer, breast cancer, and prostate cancer are on a continuous increase due to a rapid increase in environmental pollutants and increased drinking, and with the aging of the population, the number of smokers is increasing. Lung cancer is on the rise due to the increase in and air pollution. In this situation, there is an urgent need for the creation of anticancer substances that can contribute to the improvement of human health, healthy quality of life, and human health by enabling early prevention and treatment of cancer.

Meanwhile, lactic acid bacteria are widely distributed in the oral cavity, intestines, vagina, feces, and fermented foods such as kimchi of humans and animals, and are closely related to the health of humans and animals. Lactic acid bacteria have various health-promoting effects, including intestinal function, suppression of harmful bacteria, immune regulation, lowering of blood cholesterol, and anti-cancer.

Currently, Korean Patent Publication No. 10-2010-0013184 discloses the anticancer activity of the Leuconostoc mesenteroides strain, but only describes specific examples regarding the preventive or therapeutic effect on colon cancer and other cancer types. There are no specific experimental examples or examples described.

Korean Patent Publication No. 10-2010-0013184

The purpose of the present invention is to provide a pharmaceutical composition for preventing or treating cancer containing a strain of the genus Leuconostoc as an active ingredient.

In addition, another object of the present invention is to provide a food composition or food additive composition for preventing or improving cancer containing a strain of the genus Leuconostoc as an active ingredient.

In addition, another object of the present invention is to provide a feed composition or feed additive composition for preventing or improving cancer containing a strain of the genus Leuconostoc as an active ingredient.

Accordingly, the present inventors attempted to find lactic acid bacteria strains that exhibit cancer prevention and treatment effects from kimchi, and as a result isolated and identified novel strains of the genus Leuconostoc that have anticancer activity against various cancer types and completed the present invention. .

In order to achieve the above object, a pharmaceutical composition for preventing or treating cancer containing a strain of the genus Leuconostoc or a culture thereof as an active ingredient is provided.

In addition, the present invention provides a food composition or food additive composition for preventing or improving cancer containing a strain of the genus Leuconostoc or a culture thereof as an active ingredient.

In addition, the present invention provides a feed composition or feed additive composition for preventing or improving cancer in livestock containing a strain of the genus Leuconostoc or a culture thereof as an active ingredient.

The Leuconostoc genus strain or its culture according to the present invention exhibits excellent anticancer activity against various cancer types, and can be usefully used as a composition for the treatment, prevention, or improvement of cancer in humans or animals.

Figure 1 is a graph showing a graph in which a breast cancer 4T1 cell line was treated with a composition according to the present invention and cell growth rate (cell viability assay) was measured.
Figure 2 is a graph showing a graph of liver cancer HepG2 cell line treated with a composition according to the present invention and cell growth rate (cell viability assay) measured.
Figure 3 is a graph showing a graph of lung cancer A549 and H1650 cell lines treated with a composition according to the present invention and cell growth rate (cell viability assay) measured.
Figure 4 is a graph showing a graph of blood cancer A20 cell line treated with a composition according to the present invention and cell growth rate (cell viability assay) measured.
Figure 5 is a graph showing a graph of normal NIH/3T3 cell line treated with a composition according to the present invention and cell growth rate (cell viability assay) measured.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

The composition containing the Leuconostoc genus strain or its culture of the present invention as an active ingredient has a cancer prevention or treatment effect and can be used as a pharmaceutical composition.

The strain of the genus Leuconostoc of the present invention is a lactic acid bacteria strain. The strains of the Leuconostoc genus are probiotics and have the general intestinal intestinal and immune-boosting effects of lactic acid bacteria. It is a well-known fact that lactic acid bacteria of the genus Leuconostoc have intestinal and immune-boosting effects.

The strains of the Leuconostoc genus include Leuconostoc mesenteroides, Leuconostoc carnosum, Leuconostoc citreum, Leuconostoc holzapfelli, or Leuconostoc It may be one of the Leuconostoc lactis species.

The Leuconostoc mesenteroides strain isolated from kimchi through an example of the present invention was found to have a nucleic acid sequence of SEQ ID NO: 1 as a result of 16S rRNA base sequence analysis for identification and classification of microorganisms. .

Therefore, the microorganism of the present invention having the 16S rRNA base sequence of SEQ ID NO: 1 was named Leuconostoc mesenteroides LB-LM1 (Leuconostoc mesenteroides LB-LM1), and was registered at the Korea Microbiological Conservation Center in 2020. It was deposited on April 24, 2018 (deposit number KCCM12701P).

The Leuconostoc strain can be used by inoculating 0.1 to 10% in MRS liquid medium and culturing at 25 to 37°C for 4 to 48 hours.

The above culturing method is preferably a stationary culture method, but is not limited thereto.

In the present invention, 'probiotics' is understood to mean 'living microorganisms that have a beneficial effect on the health of the host by improving the intestinal microbial environment of the host within the gastrointestinal tract of animals, including humans.' Probiotics are live microorganisms with probiotic activity that can have a beneficial effect on the host's intestinal flora when fed to humans or animals in the form of dried cells or fermented products in the form of single or complex bacteria.

The above cancers include bladder cancer, breast cancer, melanoma, thyroid cancer, parathyroid cancer, throat cancer, larynx cancer, esophagus cancer, pancreas cancer, stomach cancer, tongue cancer, skin cancer, brain tumor, uterine cancer, head and gallbladder cancer, oral cancer, kidney cancer, liver cancer, lung cancer, colon cancer, rectal cancer, and colon cancer. It may be any one cancer selected from the group consisting of cancer, but is not limited thereto.

Leuconostoc strains included in the composition according to the present invention may exist as live or dead cells, and may also exist in dried or freeze-dried form. Additionally, a culture of a strain of the genus Leuconostoc may be an active ingredient, and the culture may include a live cell culture medium or a dead cell supernatant. Forms and formulation methods of lactic acid bacteria suitable for inclusion in various compositions are well known to those skilled in the art.

The composition can be administered orally or parenterally. In the case of parenteral administration, it can be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, intrapulmonary administration, and intrarectal administration, and is preferably administered by intravenous injection. It can be administered, but is not limited to this.

The appropriate dosage of the composition can be prescribed in various ways depending on factors such as formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and reaction 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 pharmaceutically suitable and physiologically acceptable auxiliaries in addition to the active ingredients, and the auxiliaries include excipients and Detergents, sweeteners, binders, coating agents, leavening agents, lubricants, lubricants, or flavoring agents can be used.

For administration, the pharmaceutical composition may be preferably formulated as a pharmaceutical composition containing one or more pharmaceutically acceptable carriers in addition to the active ingredients described above.

For example, for formulation in the form of tablets or capsules, the active ingredient may be combined with an oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, etc. Additionally, if desired or necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included in the mixture. Suitable binders include, but are not limited to, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tracacance or sodium oleate, sodium stearate, magnesium stearate, sodium benzoate. Includes sodium acetate, sodium chloride, etc. Disintegrants include, but are not limited to, starch, methylcellulose, agar, bentonite, xanthan gum, etc. Acceptable pharmaceutical carriers for compositions formulated as liquid solutions include those that are sterile and biocompatible, such as saline solution, sterile water, Ringer's solution, buffered saline solution, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and One or more of these ingredients can be mixed and used, and other common additives such as antioxidants, buffers, and bacteriostatic agents can be added as needed. In addition, diluents, dispersants, surfactants, binders, and lubricants can be additionally added to formulate injectable formulations such as aqueous solutions, suspensions, emulsions, etc., pills, capsules, granules, or tablets.

Furthermore, it can be preferably formulated according to each disease or ingredient using a method disclosed by Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA, as an appropriate method in the field.

The composition containing the Leuconostoc genus strain or its culture of the present invention as an active ingredient can be used as a food composition or food additive composition for preventing or improving cancer.

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

The above “health functional food” refers to food manufactured and processed using raw materials or ingredients with functionality useful to the human body in accordance with the Act on Health Functional Food (Article 3, Paragraph 1), and “functional” refers to It means adjusting nutrients to the structure and function of the human body or obtaining useful effects for health purposes such as physiological effects (Article No. 2).

The above food composition may additionally contain food additives, and its suitability as a “food additive” is determined by the specifications for the item in accordance with the general provisions and general test methods of the food additive code approved by the Ministry of Food and Drug Safety, unless otherwise specified. and standards.

Items listed in the "Food Additive Code" include, for example, chemical compounds such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamic acid, natural additives such as subchromic pigment, licorice extract, crystalline cellulose, and guar gum, L- These include mixed preparations such as sodium glutamate preparations, noodle additive alkaline preparations, preservative preparations, and tar coloring preparations.

Foods containing the active ingredient of the present invention include confectionery such as bread, rice cake, dried fruit, candy, chocolate, chewing gum, and jam, ice cream products such as ice cream, frozen confectionery, and ice cream powder, milk, low-fat milk, lactose-digested milk, Dairy products such as processed milk, goat milk, fermented milk, butter milk, concentrated milk, milk cream, butter oil, natural cheese, processed cheese, powdered milk, and whey Processed meat products, processed egg products, meat products such as hamburgers Fish cakes, ham, Fish products such as processed fish products such as sausage and bacon, noodles such as ramen, dried noodles, fresh noodles, fried noodles, deluxe dried noodles, modified overnight noodles, frozen noodles, pasta, fruit drinks, vegetable drinks, carbonated drinks, soy milk, Lactobacillus beverages such as yogurt, beverages such as mixed drinks, seasonings such as soy sauce, soybean paste, red pepper paste, chunjang, cheonggukjang, mixed soybean paste, vinegar, sauces, tomato ketchup, curry, and dressing, but limited to margarine, shortening, and pizza. It doesn't work.

In addition to the above, the 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 adjusters, stabilizers, preservatives, glycerin, alcohol, It may include carbonating agents used in carbonated beverages. Additionally, the composition of the present invention may include pulp for the production of natural fruit juice, fruit juice beverages, and vegetable beverages. These ingredients can be used independently or in combination.

The beverage composition containing the active ingredient of the present invention has no particular limitations on other ingredients and may contain various flavoring agents or natural carbohydrates as additional ingredients like a conventional beverage. Examples of the above-mentioned natural carbohydrates include monosaccharides, (e.g., glucose, fructose, etc.); Disaccharides, (e.g. maltose, sucrose, etc.); and polysaccharides, common sugars such as dextrin, cyclodextrin, etc., and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. .

In addition, the composition containing the Leuconostoc genus strain or its culture of the present invention as an active ingredient can be used as a feed composition or feed additive composition for preventing or improving cancer in livestock.

When the composition is prepared as a feed additive, the composition may be a 20 to 90% highly concentrated solution or may be prepared in the form of powder or granules. The feed additives include organic acids such as citric acid, malic acid, adipic acid, lactic acid, and malic acid, phosphates such as sodium phosphate, potassium phosphate, acid pyrophosphate, and polyphosphate (polymerized phosphate), polyphenol, catechin, alpha-tocopherol, and rosemary. It may additionally contain one or more of natural antioxidants such as extract, vitamin C, green tea extract, licorice extract, chitosan, tannic acid, and phytic acid. When manufactured as feed, the composition may be formulated in the form of a normal feed and may include common feed ingredients.

The feed and feed additives include grains such as ground or broken wheat, oats, barley, corn and rice; Vegetable protein feeds, such as those based on rape, soybean, and sunflower; Animal protein feeds such as blood meal, meat meal, bone meal and fish meal; It may further include dry ingredients such as sugar and dairy products, such as various powdered milk and whey powder, and may further include nutritional supplements, digestion and absorption enhancers, growth promoters, etc.

The feed additive may be administered to animals alone or in combination with other feed additives in an edible carrier. Additionally, the feed additives can be easily administered to animals as top dressing, by mixing them directly into animal feed, or in an oral dosage form separate from the feed. When the feed additive is administered separately from animal feed, it can be prepared into an immediate-release or sustained-release formulation by combining it with a pharmaceutically acceptable edible carrier, as is well known in the art. These edible carriers may be solid or liquid, such as corn starch, lactose, sucrose, soy flakes, peanut oil, olive oil, sesame oil and propylene glycol. If a solid carrier is used, the feed additive may be a tablet, capsule, powder, troche or sugar-containing tablet or a top dressing in microdisperse form. If a liquid carrier is used, the feed additive may be in the form of gelatin soft capsules, or in the form of syrup, suspension, emulsion, or solution.

Additionally, the feed and feed additives may contain auxiliaries such as preservatives, stabilizers, wetting or emulsifying agents, solution accelerators, etc. The feed additive can be used by adding it to animal feed by injecting, spraying, or mixing it.

The feed or feed additive of the present invention can be applied to a number of animal diets, including mammals, poultry and fish.

It can be used on mammals such as pigs, cows, horses, sheep, rabbits, goats, rodents, and laboratory rodents such as rats, hamsters, and guinea pigs, as well as pets (e.g., dogs, cats), and as poultry, chicken, It can also be used for turkey, duck, goose, pheasant, and quail, and can be used for carp, carp, and trout as the fish, but is not limited to this.

Example 1. Isolation and identification of strains

After crushing various types of kimchi samples, the kimchi solution was serially diluted (10 ³ to 10 ⁸ ) with PBS (phosphate buffered saline), plated on MRS, the primary lactic acid bacteria selection medium, and incubated at 30°C or 37°C for 24 hours, respectively. Cultivate. The formed colonies were separated into single colonies, plated on Phenylethyl alcohol sucrose agar, a leuconostoc selection medium, and cultured at 20°C for 24 hours. After culturing, the formed single colonies were collected and cultured in MRS liquid medium. Genomic DNA was extracted from the culture medium incubated for 24 hours. To confirm the 16S rDNA base sequence, the obtained genomic DNA product was submitted to Macrogen for analysis. The identified base sequences were analyzed for similarity using the Basic Local Alignment Search Tool (BLAST) of the National Center for Biotechnology information (NCBI, www.ncbi.nlm.nih.gov), and the results were ultimately used to determine Leuconostoc mesenteroides. The strain was named LB-LM1 (Leuconostoc mesenteroides LB-LM1) and deposited at the Korea Microbiological Conservation Center on April 24, 2020.

Example 2. Cultivation of strains

The isolated Leuconostoc mesenteroides LB-LM1 (Leuconostoc mesenteroides LB-LM1) strain was inoculated at 1% in 30 ml of MRS liquid medium and cultured for 6 hours at 37°C. After culturing, the culture medium was removed by centrifugation at 3500 rpm for 10 minutes, and the cells were washed three times with PBS (phosphate buffered saline) solution to remove remaining medium components.

Example 3. In vitro anticancer activity efficacy analysis

3-1. Preparation of cell lines

To observe the anticancer activity effect of the isolated Leuconostoc mesenteroides LB-LM1 strain, cell viability assay was measured using Cell counting Kit-8. The cells used in the anticancer activity efficacy test were the breast cancer 4T1 cell line, the liver cancer HepG2 cell line, the lung cancer A549 and H1650 cell lines, the blood cancer A20 cell line, and the normal cell line NIH/3T3.

To measure cell growth rate, 2x10 ⁵ cells of each cancer cell were plated in a 6 well plate and cultured in a cell incubator at 37°C with 5% CO ₂ for 24 hours.

3-2. Anti-cancer activity results

Afterwards, the Leuconostoc mesenteroides LB-LM1 strain was treated for 48 hours, and the cell culture medium and cells were recovered and washed three times with PBS. 100 ul of PI solution was added to the cells, the cells were stained for 15 minutes at room temperature, washed twice with PBS, and the ratio of PI positive cells was measured using a flow cytometer. The results are shown in Figures 1 to 4.

As shown in Figures 1 to 4, it was confirmed that cancer cell death increased in a LB-LM1 concentration-dependent manner in the group treated with the Leuconostoc mesenteroides LB-LM1 strain compared to the group untreated.

Specifically, apoptosis increased 2.5-fold in 4T1, a breast cancer cell line (see Figure 1), 2.6-fold in HepG2 cells, a liver cancer cell line (see Figure 2), and 1.7-fold in H1650 and A549 lung cancer cell lines (see Figure 3). It was confirmed that 3.7 times more cell death (see Figure 4) occurred at the high concentration of LB-LM1 in A20, a blood cancer cell line.

In light of the above results, it was confirmed that the Leuconostoc mesenteroides LB-LM1 strain can exhibit anticancer activity by effectively inhibiting the growth of blood cancer cells as well as major solid cancers.

In contrast to the above experiment, the results of measuring cell growth rate by treating the NIH/3T3 normal cell line with the Leuconostoc mesenteroides LB-LM1 strain are shown in Figure 5.

As shown in Figure 5, it can be confirmed that the Leuconostoc mesenteroides LB-LM1 strain does not show toxicity to the NIH/3T3 normal cell line.

Korea Microbiological Conservation Center (Overseas) KCCM12701P 20200424

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Claims (10)

A pharmaceutical composition for preventing or treating breast cancer, liver cancer, lung cancer, or blood cancer, comprising a strain of the genus Leuconostoc or a culture thereof as an active ingredient. According to paragraph 1,
The Leuconostoc mesenteroides strain is Leuconostoc mesenteroides LB-LM1 (Accession number KCCM 12701P). A pharmaceutical composition for preventing or treating breast cancer, liver cancer, lung cancer, or blood cancer. . According to paragraph 1,
A pharmaceutical composition for the prevention or treatment of breast cancer, liver cancer, lung cancer, or blood cancer, characterized in that the composition can be administered by oral or parenteral administration. According to paragraph 3,
The method of parenteral administration is intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, intrapulmonary administration, or intrarectal administration, for the prevention of breast cancer, liver cancer, lung cancer, or blood cancer. or therapeutic pharmaceutical compositions. A food composition for preventing or improving breast cancer, liver cancer, lung cancer, or blood cancer, comprising Leuconostoc mesenteroides LB-LM1 (Accession No. KCCM 12701P) strain or a culture thereof as an active ingredient. According to clause 5,
The food is a health functional food, a food composition for preventing or improving breast cancer, liver cancer, lung cancer, or blood cancer. According to clause 5,
The above foods include bread, rice cake, candy, chocolate, gum, ice cream, milk, cheese, processed meat products, processed fish products, kimchi, soy sauce, soybean paste, red pepper paste, chunjang, cheonggukjang, vinegar, ketchup, curry, dressing, beverages, and fermented milk. A food composition for preventing or improving breast cancer, liver cancer, lung cancer, or blood cancer, which is any one food selected from the group. A food additive composition for preventing or improving breast cancer, liver cancer, lung cancer, or blood cancer, comprising Leuconostoc mesenteroides LB-LM1 (Accession No. KCCM 12701P) strain or a culture thereof as an active ingredient. A feed composition for preventing or improving breast cancer, liver cancer, lung cancer, or blood cancer in livestock containing Leuconostoc mesenteroides LB-LM1 (Accession No. KCCM 12701P) strain or a culture thereof as an active ingredient. A feed additive composition for preventing or improving breast cancer, liver cancer, lung cancer, or blood cancer in livestock, comprising Leuconostoc mesenteroides LB-LM1 (Accession No. KCCM 12701P) or a culture thereof as an active ingredient.