KR102624035B1 - Anticancer compositions comprising ferments of beet and cabbage extracts - Google Patents

Abstract

The present invention relates to an anti-cancer composition comprising fermented beet and cabbage extract, and specifically to the anti-cancer use of the fermented beet and cabbage extract obtained by fermenting the beet and cabbage extract using a strain.

Description

Anticancer compositions comprising ferments of beet and cabbage extracts

The present invention relates to an anti-cancer composition comprising fermented beet and cabbage extract, and specifically to the anti-cancer use of the fermented beet and cabbage extract obtained by fermenting the beet and cabbage extract using a strain.

Chemical compounds that inhibit the progression to malignant cancer by synthesizing and developing new derivatives with significantly reduced side effects while maintaining the efficacy of existing anticancer drugs through various synthetic methods, or by suppressing the occurrence of cancer itself or delaying or reversing the progression of cancer. It is aimed at preventing cancer.

Recently, developed countries are paying a lot of attention to developing non-toxic and highly effective chemical cancer preventive agents from natural resources such as vegetables, fruits, and medicinal plants. Based on this, research on the development of anticancer materials using various natural products is actively underway.

Cabbage ( Brassica oleracea var. capitata ) is an annual or biennial plant of the mustelid family of the dicotyledonous plant Poppy order. It is native to the Mediterranean coast and Asia Minor and is currently cultivated all over the world.

Beet ( Beta Vulgaris ) is an annual or biennial herb of the dicotyledonous Amaranth family and belongs to a group of representative Western vegetables along with broccoli. It is native to northern Africa and Europe, has an optimal growth temperature of 13~18℃, and contains many useful ingredients such as betacyanin, betaine, and potassium.

Although strong anticancer activity has been confirmed in some natural materials, research on improving the functionality of original natural products through bioconversion of natural products has only recently been conducted.

In particular, there are few studies showing improved anticancer effects through bioconversion of natural products.

Under this technical background, the inventors of the present application confirmed that extracellular polysaccharides were produced when applying Leuconostoc mesenteroides CJNU 0147 strain to beet and cabbage extract and verified the anticancer efficacy of fermented beet and cabbage extract. By doing so, the present invention was completed.

In order to solve the above problems, the purpose of the present invention is to provide an anticancer composition.

Another object of the present invention is to provide a pharmaceutical comprising the composition.

Another object of the present invention is to provide a health functional food containing the above composition.

In order to achieve the above object, the present invention provides an anticancer composition comprising fermented beet and cabbage extracts or fractions thereof.

The present invention also provides a pharmaceutical product comprising the composition.

The present invention further provides a health functional food containing the composition.

Through the composition according to the present invention, it was confirmed that an anti-cancer material can be developed using the biotransformation product produced by applying the extracellular polysaccharide-producing Leuconostoc mesenteroides CJNU 0147 strain to beet and cabbage extract.

Figure 1. Scanning electron microscopy (SEM) image of Leuconostoc mesenteroides CJNU 0147 strain producing extracellular polysaccharide: A, in tryptic soy broth without sucrose, there is no production of extracellular polysaccharide, so the surface is rough; B, When sucrose is added, extracellular polysaccharides are produced in tryptic soy broth, resulting in a smooth surface.
Figure 2. Comparative results of extracellular polysaccharide production of Leuconostoc mesenteroides CJNU 0147 strain with or without addition of 2% sucrose and 3% yeast extract in beet and cabbage extract basic medium.
Figure 3. Results of anticancer effects of biotransformed beet and cabbage extract applied to Leuconostoc mesenteroides CJNU 0147 strain on lung cancer cell line A549: 2-9, other strains producing extracellular polysaccharides.
Figure 4. Results of anticancer effects of biotransformed beet and cabbage extract applied to Leuconostoc mesenteroides CJNU 0147 strain on colon cancer cell line HCT116. TSB (tryptic soy broth).
Figure 5. Results of anticancer effects on lung cancer cell lines A549 and H1299 according to fraction and concentration of biotransformed beet and cabbage extract applied to Leuconostoc mesenteroides CJNU 0147 strain.
Figure 6. Results of anticancer effects on colon cancer cell lines HCT116 and HT29 according to fraction and concentration of biotransformed beet and cabbage extract applied to Leuconostoc mesenteroides CJNU 0147 strain.
Figure 7. Shows the results of identifying the anticancer mechanism of the colon cancer cell line HCT116 according to the biotransformed beet extract fraction applied to the Leuconostoc mesenteroides CJNU 0147 strain.

Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by a person skilled in the art to which the present invention pertains. In general, the nomenclature used herein is well known and commonly used in the art.

In one aspect, the present invention relates to an anticancer composition comprising a fermented beet and cabbage extract or a fraction thereof.

The beet and cabbage extract may be a mixture of extracts of beet or cabbage alone, or may be an extract of a beet and cabbage mixture.

The extraction can be performed using a common extraction solvent, and can be performed by appropriately selecting the solvent without limitation. For example, the extraction may be performed using beets or cabbage alone in water, an alcohol with 1 to 6 carbon atoms, or a mixed solvent of water and an alcohol with 1 to 6 carbon atoms. For example, the alcohol having 1 to 6 carbon atoms may be ethanol. The extracts of the beet or cabbage alone can be mixed at a weight ratio of 1:1.

The extraction may be performed, for example, by using a beet and cabbage mixture with water, an alcohol with 1 to 6 carbon atoms, or a mixed solvent of water and an alcohol with 1 to 6 carbon atoms. For example, the alcohol having 1 to 6 carbon atoms may be ethanol. The beets or cabbage can be mixed at a weight ratio of 1:1.

The fermentation product refers to denaturing or transforming raw materials by physical or chemical methods using strains, and can refer to a metabolic process that can obtain desired results in a short time, which in nature would take a long time to change.

Specifically, the fermented product is a fermented product produced by inoculating and fermenting a strain using the beet and cabbage extract itself as a medium source. For example, the beet and cabbage extract is mixed with a medium component such as yeast extract and It may be fermented by inoculating the strain in the presence of sucrose.

The fermented product not only refers to a product fermented by a strain, but may also include a fermented filtrate filtered after fermentation or a fermented extract obtained by extracting the fermented product with a solvent.

The strain may be Leuconostoc mesenteroides ( Leu. mesenteroides ) CJNU 0147 strain with accession number KCTC 11475BP. The Leuconostoc mesenteroides CJNU 0147 strain with the accession number KCTC 11475BP was used to prepare fermented beet and cabbage extract.

It was confirmed that extracellular polysaccharides were produced by the Leuconostoc mesenteroides CJNU 0147 strain in the presence of the beet and cabbage extracts.

The fermented product of the beet and cabbage extract is a biotransformation product, and the biotransformation product itself can be used in an anticancer composition. In some cases, a composition containing dead cells obtained by centrifuging a biotransformation, a composition containing a supernatant of a biotransformation obtained by centrifuging the biotransformation and extracting only extracellular polysaccharides from the supernatant (extracellular polysaccharides removed), or cells. It may be a composition containing polysaccharides.

The anticancer agent may exhibit anticancer activity through apoptosis of cancer cells. Apoptosis is an important mechanism by which most anticancer drugs exert an inhibitory effect on the proliferation of cancer cells. It refers to active cell death controlled by genes, and plays an important role in maintaining the balance between cell proliferation and cell death in vivo. do.

The composition may exhibit anticancer activity against lung cancer or colon cancer. After fractionating the bioconverted beet and cabbage extracts (dead cells, supernatant, and extracellular polysaccharide), the anticancer activity was measured for lung cancer cell lines (A549, H1299) and colon cancer cell lines (HCT116, HT29) at different concentrations, and the results showed that all three fractions had High activity was confirmed at 500 ppm, and some were confirmed to be active even at 250 ppm. In particular, the activity was relatively high in dead cells and extracellular polysaccharides in lung cancer cell lines, and in the supernatant in colon cancer cell lines, predicting anticancer activity through a different mechanism.

The composition according to the invention can be used as a pharmaceutical composition. Based on this, the present invention relates to a pharmaceutical comprising the composition.

The composition may include a ‘pharmaceutically acceptable carrier’. Each can be formulated in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterile injection solutions according to conventional methods.

The pharmaceutically acceptable carriers include those commonly used in the art, such as lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, Including, but not limited to, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition, the pharmaceutical composition of the present invention includes diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants, and other pharmaceutically acceptable additives.

When the composition is formulated as a solid preparation for oral use, it includes tablets, pills, powders, granules, capsules, etc., and such solid preparations contain at least one excipient such as starch, calcium carbonate, sucrose ( sucrose) or lactose, gelatin, etc., and includes, but is not limited to, lubricants such as magnesium stearate and talc.

When the composition is formulated as a liquid formulation for oral use, it includes suspensions, oral solutions, emulsions, syrups, etc., and includes, but is not limited to, diluents such as water and liquid paraffin, humectants, sweeteners, fragrances, and preservatives.

When the composition is formulated for parenteral use, it includes sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, and olive oil. Including, but not limited to, vegetable oil, injectable esters such as ethyl oleate, etc. As a base for suppositories, witepsol, macrogol, tween 61, cacao, laurel, glycerogelatin, etc. can be used, but are not limited to these.

The composition can be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) depending on the desired method, and the dosage depends on the patient's condition and weight, degree of disease, and drug form. , It varies depending on the route and time of administration, but can be appropriately selected by a person skilled in the art.

The composition is administered in a pharmaceutically effective amount. “Pharmaceutically effective amount” means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type and severity of the subject, age, gender, activity of the drug, and It can be determined based on factors including sensitivity, time of administration, route of administration and excretion rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. It may be administered singly or in multiple doses. Considering all of the above factors, it is important to administer an amount that can achieve maximum effect with the minimum amount without side effects, and can be easily determined by a person skilled in the art.

The composition can be used as a food composition. Based on this, the present invention relates to a health functional food containing the above composition.

“Health functional food” refers to food manufactured and processed using raw materials or ingredients with functionality useful to the human body in accordance with Act No. 6727 on Health Functional Food. “Functional” refers to food that is related to the structure and function of the human body. It means ingestion for the purpose of controlling nutrients or obtaining useful health effects such as physiological effects.

The composition may contain common food additives, and its suitability as a “food additive” is determined in accordance with the general provisions of the Food Additive Code and General Test Methods approved by the Ministry of Food and Drug Safety, unless otherwise specified. Judgment is made according to specifications and standards.

Items listed in the "Food Additives 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, high-liquid pigment, and guar gum; Examples include mixed preparations such as sodium L-glutamate preparations, noodle additive alkaline preparations, preservative preparations, and tar coloring preparations.

The food composition can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc.

The above-mentioned health functional food in the form of tablets is made by granulating a mixture of excipients, binders, disintegrants, and other additives in addition to the active ingredients in a conventional manner, then adding a lubricant, etc., and compression molding, or directly compression molding the mixture. You can. In addition, the health functional food in the form of tablets may contain a flavoring agent, etc., if necessary, and may be peeled with a suitable peeling agent, if necessary.

Among the capsule-type health functional foods, hard capsules can be manufactured by filling a regular hard capsule with a mixture of additives such as active ingredients and excipients, or their granules or peeled granules, and soft capsules contain active ingredients and excipients. It can be manufactured by filling a mixture with additives such as gelatin into a capsule base. The soft capsule may contain plasticizers such as glycerin or sorbitol, colorants, preservatives, etc., if necessary.

Health functional foods in the form of pills can be prepared by molding a mixture of active ingredients, excipients, binders, disintegrants, etc. in an appropriate manner, and if necessary, can be coated with white sugar or other suitable coating agents, or with starch, talc, or other suitable substances. You can also wear a fancy dress.

Health functional foods in the form of granules can be manufactured into granules from a mixture of active ingredients, excipients, binders, disintegrants, etc. by an appropriate method, and may contain flavoring agents, flavoring agents, etc. as needed. For health functional foods in granular form, use No. 12 (1680 μm), No. 14 (1410 μm), and No. 45 (350 μm) sieves. When the next particle size test is performed, the entire amount passes through the No. 12 sieve and what remains on the No. 14 sieve is the total amount. Less than 50%, and what passes through No. 45 sieve may be less than 150% of the total amount.

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

Manufacturing example.

One) Leuconostoc mesenteroides Confirmation of extracellular polysaccharide production by strain CJNU 0147

Scanning electron microscopy (SEM) photographs were taken to confirm the production of extracellular polysaccharides by the Leuconostoc mesenteroides CJNU 0147 strain. Leuconostoc mesenteroides CJNU 0147 strain was inoculated into tryptic soy broth (TSB) and TSB containing 2% sucrose, respectively, and cultured at 37°C for 24 hours. Afterwards, 20㎕ of each culture was taken, centrifuged, and the supernatant was removed. 50%, 60%, 70%, 90%, 95%, and 100% EtOH were added and removed in that order and finally suspended in 20㎕ of 100% EtOH, 2㎕ of which was taken and filtered through a membrane filter (0.22ㅅm). Instill in Scanning electron microscope (JSM-6700F, JEOL, Japan) observation was performed according to the manufacturer's instructions.

2) Leuconostoc mesenteroides Anticancer effect of biotransformed beet and cabbage extract applied with CJNU 0147 strain

To produce bioconverted beet and cabbage extract, beet and cabbage powder were mixed with primary distilled water to a final concentration of 2%, followed by hot water extraction at 100°C for 1 hour, followed by centrifugation (4,000 rpm, 4°C, 10 minutes) and filtration ( 0.45ㅅm membrane filter). Sucrose (2%) and yeast extract (3%) were added to the filtrate, sterilized (100°C, 30 minutes), and then Leuconostoc mesenteroides CJNU 0147 strain was inoculated. This was cultured at 25°C for 24 hours, then sterilized (100°C, 30 minutes), freeze-dried, and used as biotransformation material (including dead cells). The biotransformate was centrifuged to remove dead cells, and the fraction was lyophilized and used as the supernatant of the biotransformate, and used together with the biotransformate for initial anticancer activity screening for lung cancer cell line (A549). Afterwards, for additional fractionation, dead cells (obtained by centrifugation) and biotransformate supernatant were fractionated into biotransformate supernatant (extracellular polysaccharide removed) and extracellular polysaccharide from which only extracellular polysaccharides were extracted. This was used in anticancer tests on lung cancer and colon cancer cell lines according to fraction and concentration. To extract extracellular polysaccharides, isopropanol was added in twice the volume of the supernatant and left at room temperature for 24 hours. This was centrifuged (5,000 rpm, 4°C, 15 minutes) to remove the supernatant, dried overnight in a 50°C dry oven, then dissolved in water and finally freeze-dried to obtain extracellular polysaccharide. The supernatant containing isopropanol was used as a supernatant fraction through rotary concentration and freeze-drying.

For anti-cancer tests, lung cancer cell lines A549 and H1299 cells were incubated in a CO ₂ incubator (36.5 m) using DMEM medium (Welgene, Daegu, South Korea) containing 10% FBS and 1% antibiotic-antimycotic agent (Corning Inc., USA). ℃), and colon cancer cell lines HCT116 and HT29 cells were cultured in a CO ₂ incubator (36.5℃) using RPMI medium (Corning Inc.) containing 10% FBS and 1% antibiotic-antimycotic agent. After dispensing ^1x104 cells/100ul per well into a 96-well microplate, samples such as bioconverted beets and cabbage extract were added and further cultured. After adding 10ul of Ez-cytox solution (DoGen, Seoul, Korea) per well, reacting in an incubator for 1 hour, absorbance was measured at 450nm.

3) Leuconostoc mesenteroides Identification of anticancer mechanism for colon cancer cell line HCT116 according to bioconversion beet extract fractionation applied to CJNU 0147 strain

After treating 1x10 ⁶ cells with each substance for 24 hours, the apoptosis efficacy was evaluated using Annexin V-FITC apoptosis staining/detection kit (Abcam, Cambridge, UK). Additionally, to confirm apoptosis, western blot was performed using antibodies against cleaved-PARP and cleaved Caspase-3 (Cell Signaling, Danvers, MA, USA) after treating the cells with each substance for 24 hours.

Example 1. Leuconostoc mesenteroides Confirmation of extracellular polysaccharide production by strain CJNU 0147

As a result of scanning electron microscopy, it was confirmed that the surface of Leuconostoc mesenteroides CJNU 0147 strain cultured in TSB was rough, whereas the surface of Leuconostoc mesenteroides CJNU 0147 strain cultured in TSB containing sucrose was smooth, indicating the production of extracellular polysaccharides. It was confirmed that this was happening (Figure 1).

In addition, it was confirmed that mucous was formed around colonies in a medium containing 2% sucrose and 3% yeast extract in beet and cabbage extract, indicating that extracellular polysaccharides are normally produced in the extract medium source. Confirmed (Figure 2).

Example 2. Leuconostoc mesenteroides Anticancer effect of biotransformed beet and cabbage extract applied with CJNU 0147 strain

As a result of analyzing the anticancer activity of the biotransformed beet and cabbage extract applied to the Leuconostoc mesenteroides CJNU 0147 strain against the lung cancer cell line A549, the anticancer activity was excellent enough to correspond to doxorubicin (dox) used as a positive control (Figure 3).

On the other hand, other strains producing extracellular polysaccharides (Figure 3, #2-9) and the control group (beet and cabbage extract) did not have confirmed anticancer activity. In addition, anticancer activity corresponding to dox was confirmed in the anticancer activity against colon cancer cell line HCT116 using biotransformed beet and cabbage extract containing dead cells (Figure 4).

After fractionating the bioconverted beet and cabbage extracts (dead cells, supernatant, and extracellular polysaccharide), the anticancer activity was measured for lung cancer cell lines (A549, H1299) and colon cancer cell lines (HCT116, HT29) at different concentrations, and the results showed that all three fractions had High activity was confirmed at 500 ppm, and some were confirmed to be active even at 250 ppm (Figures 5 and 6). In particular, the activity was relatively high in dead cells and extracellular polysaccharides in lung cancer cell lines, and in the supernatant in colon cancer cell lines, predicting anticancer activity through a different mechanism.

Example 3. Leuconostoc mesenteroides Identification of anticancer mechanism for colon cancer cell line HCT116 according to bioconversion beet extract fractionation applied to CJNU 0147 strain

Supernatant, dead cells, and extracellular polysaccharides were fractionated from the biotransformed beet extract and treated with 500 ppm for 24 hours. Cell suicide was confirmed through flow cytometry. As a result, all treatment groups caused apoptosis. In particular, dead cells are found to cause the most apoptosis of the colon cancer cell line HCT116 (top of Figure 7).

To verify apoptosis, cleavage of PARP and Caspase-3, which can confirm the apoptosis pathway, was confirmed by western blot. As a result, cleavage of PARP and Caspase-3, which indicates apoptosis, was confirmed in all treatment groups, unlike the control group (Figure 7 under).

As above, specific parts of the content of the present invention have been described in detail, and for those skilled in the art, it is clear that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. It will be obvious. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Korea Research Institute of Bioscience and Biotechnology Biological Resources Center (KCTC) KCTC11475BP 20090313

Claims (8)

Anticancer treatment for lung cancer or colon cancer containing fermented product or fractions thereof obtained by inoculating beet and cabbage extract with Leuconostoc mesenteroides ( Leu. mesenteroides ) CJNU 0147 strain with accession number KCTC 11475BP, which produces extracellular polysaccharides. Composition.
delete delete delete The composition according to claim 1, which exhibits anti-cancer activity through apoptosis of the lung cancer or colon cancer cells.
delete A pharmaceutical product containing the composition according to paragraph 1 or 5.
A health functional food containing the composition according to paragraph 1 or 5.

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