KR20140102867A - A composition for inhibiting cancer metastasis comprising Hwangryunhaedoktang or its product fermented by lactic acid bacteria - Google Patents

Abstract

The present invention provides a pharmaceutical composition for inhibiting cancer metastasis and a functional health food for preventing or alleviating cancer metastasis, comprising hwangryunhaedoktang or a lactic acid bacteria fermentation product thereof as an active ingredient. The hwangryunhaedoktaing or the lactic acid bacteria fermentation product thereof of the present invention effectively inhibits the movement and invasion of cancer cells, and can be used as a composition for preventing or treating cancer metastasis since low cytotoxicity on the normal cells is checked. In addition, since the hwangryunhaedoktang is already confirmed to be harmless to a human body, thereby being used as a functional health food and being immediately applied to a human body.

Description

TECHNICAL FIELD The present invention relates to a composition for inhibiting cancer metastasis, which comprises a fermented product of fermented milk,

The present invention provides a pharmaceutical composition for inhibiting cancer metastasis comprising a fermented product of Huanghin Haitang or its lactic acid bacteria as an active ingredient, and a health functional food for preventing or ameliorating cancer metastasis.

As modernization accelerates, the eating habits of our country are becoming more abundant, the eating patterns are changing to the west, and the lifestyle is also changing a lot. As the level of consciousness increases, expectations of life increase, and to lead a better life, modern people are doing more work than before and are being stressed in various ways. Prior to industrialization, I lived close to nature, but now I can not artificially create a living environment, so I can not get close to nature, and the surrounding environment is also polluted and living in a totally different way from the past. There are many side effects such as the pattern of disease occurrence and the cause of death. According to a statistical survey released by the National Statistical Office (NSO), the death rate per 100,000 Korean population in 2010 was 144.4, the number one cause of death. By type, lung cancer was the most common, followed by gastric cancer, liver cancer, colon cancer, and pancreatic cancer.

Cancer is a group of abnormal cells that are caused by continuous division and proliferation by destroying the balance between cell division and death by various causes, and are also called tumors or neoplasms. It usually develops in more than 100 different parts of the body, including organs, leukocytes, bones, and lymph nodes. It develops into a serious symptom through invasion into surrounding tissues and metastasis to other organs (WHO, 2006). The causes of cancer are environmental or external factors such as chemicals, viruses, bacteria, and ionizing radiation, and internal factors such as congenital gene mutations (Klaunig & Kamendulis, Annu. Rev. Pharmacol. : 239-267).

To overcome these cancers, the incidence of cancer has increased over the past four decades, despite the remarkable development in the search for new targets, including regulation of cell lineage or apoptosis, and carcinogenic genes or cancer-suppressing genes It is increasing as civilization develops. Currently, the treatment of cancer patients depends on surgical treatment, radiation therapy, chemotherapy by administration of 40 kinds of strong cytotoxic anticancer drugs. Most of these treatments are limited to early cancer patients or specific cancer, Of deaths are continuing to increase. On the other hand, the most common cause of life-threatening cancer is cancer metastasis, and most of cancer deaths are explained by cancer metastasis. Although currently the most clinically proven method of treating cancer is surgery, even if the original cancer is removed, it becomes difficult to treat the patient by cancer cells that have metastasized to other tissues. Therefore, cancer conquest is actually a fight against cancer metastasis.

In cancer metastasis, cell migration is involved in several stages: when cancer cells migrate from the original primary carcinoma location through the extracellular matrix (ECM) to the blood vessels, from the second metastatic tissue outside the blood vessels, And the vascular endothelial cell (vascular endothelial cell) is involved in the movement of the process is very complex and has a variety of functions. Therefore, considering that a general anticancer drug has an effect of simply killing cancer cells or inhibiting the proliferation of cancer cells, the preparation for suppressing metastasis should be separately developed.

Thus, anticancer drugs that target cancer cell migration are aimed at inhibiting cancer metastasis. Cell migration inhibitors are not the best target, but they are a realistic approach to prolong the life of cancer patients. In fact, cancer can be removed simply by surgical operation if it finds the site of occurrence, regardless of the site where it occurs. The limitation of such surgical operation is that the cancer cells spread to other parts of the original cancer site, Can be expected to cure through. However, if the cancer is not diagnosed at an early stage, if the cancer cell already spreads through the blood vessel and forms a very small colony at the second or third position, I can not expect it. In many cases, in many cases, the cancer cells are transferred to give up surgical treatment. In such a case, the cancer cells are killed by inhibiting the cancer cell migration so as to prevent further spread of the metastasis. You can help. Therefore, development of cancer cell migration inhibitor can be developed as a new concept of new drug chemotherapeutic agent, and effective inhibitor can be an important data for academic study on cell migration.

On the other hand, Oriental medicine has been used for many years to prevent and treat various diseases. Herbal preparations have relatively few side effects in the human body and have various advantages due to the complex interactions of constituent herbal medicines. Thus, interest and demand for herbal preparations have been greatly increased. However, studies on herbal preparations which are still safe and effective for inhibiting cancer treatment or cancer metastasis are in short supply.

In addition, herbal medicines or health foods are orally administered to the intestinal flora until they are absorbed into the body, and many compounds contained therein are produced by assimilation and catabolism of intestinal flora, resulting in various bioconverters (metabolites). Making such diverse bioconverters means that they are catalyzing various reactions. Many of these bioconverters increase or decrease toxicity compared to the original compound. Of these, a compound that has no effect is metabolized by intestinal bacteria, and thus, there are many cases where the drug efficacy is expressed. Thus, it is estimated that the metabolic pathway in which intestinal flora can metabolize herbal medicine including natural products is higher than the metabolic enzyme group present in the liver. When people take herbal medicine, the ingredients in herbal medicine need to be transfused into the blood to manifest a medicinal effect. Most of herbal medicine ingredients are present in the form of glycosides. Therefore, the form of aglycone . However, all of them had different intestinal bacterial guns, so they had the disadvantage that the efficacy of herbal medicine was not uniform due to the different efficiency of converting herbal medicine ingredients into aglycone form.

Accordingly, the present inventors have made intensive researches to find a substance capable of inhibiting cancer metastasis by inhibiting the migration and invasion of cancer cells while having low toxicity to normal cells. As a result, It is possible to increase the secretion of anti-cancer-related cytokines while suppressing the migration and infiltration of cancer cells and inhibiting the lung metastasis of cancer cells in vivo. Thus, the composition is useful as a composition for preventing or treating cancer metastasis And the present invention has been completed.

It is an object of the present invention to provide a pharmaceutical composition for inhibiting cancer metastasis, which comprises a phytophthora or a lactobacillus fermented product thereof as an active ingredient.

Another object of the present invention is to provide a health functional food for preventing or ameliorating cancer metastasis, which comprises Huanghin Hao Yangtang or its fermented product of lactic acid bacteria as an active ingredient.

In order to accomplish the above object, the present invention provides a pharmaceutical composition for inhibiting cancer metastasis, which comprises a phytophthora or a lactobacillus fermented product thereof as an active ingredient.

The term " Huangryun Haidoksoo "of the present invention is made up of Huang Riyuan, Huang Bai, Huang Bai, and Zijia, which are basically used medicines in Chinese traditional medicine, Refers to a herbal medicine derived from a herbal medicine which is efficacious when there is a fever of the body (meaning the body except for the upper body, the middle body, and the lower body). It is prescribed when there is inflammation with fever disease (fever disease), mental anxiety caused by fever and congestion, bleeding in urine, redness in the lower part of the heart (lower part of the heart) and resistance, and also prescribed in hemorrhage, hemoptysis, . Recent studies have reported antioxidative, anti-ulcer, diuretic, hypotensive and antimicrobial effects, and have been reported to be effective in atopic, dermatitis and seborrheic dermatitis associated with the skin .

It is preferable that the above-mentioned Huangliao gangsangtang is hydrothermal extract of Huangliao, golden, yellowish white, and gardenia, as can be deduced from the name.

The above-mentioned "Rhodiola" is an evergreen perennial herbaceous herbaceous perennial herbaceous plant of the dicotyledonous plant, but is widely grown in Korea, do. It is said to be dried in the sun, picking roots of 5 to 6 years old harvested from November to November, and it is said to be dried in the sun. It has the effects of dryness, soothing, anti-inflammation, antibacterial, , Heart palms, burns, mental anxiety, sore throat, blood clots, nosebleeds, hemorrhages and burns. Other names are Wiseng, Gujil, Triangope Huanglia, Ami Yaniyun, Yunnan Huanglia, Huangpeng, Chun Huanglian, Huanghui, Sun Huanglian, Mao Huangli, Chosun Huangyan, and Sanjiao. Actually, many kinds of plants have been used as a generic name for Rhizome, and therefore Plagiorhegma , including species of Coptis , dubium Maxim. And Jeffersonia dubia Bent. Et < RTI ID = 0.0 > Hook. ≪ / RTI >

The "golden ( Scutellaria Baicalensis "is a perennial herb that belongs to the family Lamiaceae and grows in the country of origin or at the foot of mountains around the country and is cultivated as a herb. The bitterness enters the heart and lungs, and the coldness enters the liver and the lungs, eliminating the heat of the humid heat. It is also known to be effective for insomnia, gastroenteritis, large / small bowel, hepatitis, cystitis, urethritis, and asthma. Fragrance aquatic plants, gold, gray gold, golden gold, gold, spruce grass, gold candle, etc.

The term "yellow white" refers to a deciduous tree belonging to the over-acid family, or a medicinal product manufactured by drying the skin thereof, and is also called other names such as yellow blooms, wall walls, The stem skin is light gray or cork layer developed and deeply cracked, and the endothelium with the cork layer removed is yellow. The part mainly used as a medicinal material is generally a bark having a thickness of about 3 mm and a length of about 30 cm. It is also known that it is effective for the treatment of diseases caused by mung bean. It has a peculiar odor, is mucous and stains the needle in yellow. The pharmacological effect is similar to the above-mentioned gold or phthalocyanine, but the site to which the pharmacological effect is applied is different. Known pharmacological actions include antimicrobial action, convergence action, subcutaneous haemorrhagic absorption promotion action, antiinflammatory action, cardiovascular action, blood pressure lowering action, muscle contraction enhancement action, Diarrhea, diarrhea, jaundice, obesity, arthralgia, muscle pain and so on.

The term "gardenia" refers to the fruit of the gardenia belonging to the madder family, which is an evergreen shrub, or the dried fruit. The gardenia grows in the deep mountains of the hot regions such as the southern part of Korea, China, Taiwan, and Japan. The fruit is about 3.5 cm long and ripens in yellowish red in September, and is used not only as medicinal but also as a dye. It is known to have pharmacological effects such as diabetes, hypertension, insomnia, jaundice, urinary disorders, eye irritation, conjunctivitis, hematemesis, hemorrhage, hematuria, bruise. It also heals joint bruises and has a detoxifying effect. It can be used alone or in combination with other medicinal materials for a short time in water, or it can be powdered and put on the affected part by splitting it into water.

The term "lactobacillus fermented product" of the present invention means a cultured product obtained by inoculating lactic acid bacterium into a culture medium on which lactic acid bacteria can grow, or a culture result obtained therefrom. The term "fermentation" of the present invention means, in a narrow sense, a process of obtaining a substance useful for human beings by using microorganisms or fungi in a sugar decomposition process for obtaining energy without using oxygen in a narrow sense. Accordingly, the fermented product of lactic acid bacteria of the present invention may include a substance advantageous to human beings produced through various enzymatic actions by culturing the lactic acid bacteria in a medium containing a substance to be fermented. Preferably, the lactic acid bacterium fermented product is obtained by inoculating lactic acid bacteria into a phthalocyanine solution and fermenting the mixture at a temperature of 35 ° C to 45 ° C for 1 to 3 days under an aerobic atmosphere. In a specific example of the present invention, the culture supernatant obtained by inoculating lactic acid bacteria into Huangryinghaotoktang and culturing was dried to prepare powder.

On the other hand, as the lactic acid bacteria, isolated strains or various commercially available lactic acid bacteria can be used without limitation. Is not particularly limited to, Lactobacillus (Lactobacillus), A Streptococcus (Streptococcus) genus Bifidobacterium (Bifidobacterium) genus Lactococcus (Lactococcus), A Phedi O Rhodococcus (Pediococcus) in or flow Pocono stock (Leuconostoc) Microorganisms can be used.

More preferably, the strain is selected from the group consisting of Lactobacillus casei), Lactobacillus even know Phil Ruth (Lactobacillus acidophilus , Lactobacillus < RTI ID = 0.0 > plantarum , Lactobacillus < RTI ID = 0.0 > fermentum), Lactobacillus keoba tooth (Lactobacillus curvatus), Lactobacillus konpu Seuss (Lactobacillus confusus), Lactobacillus joined Lee (Lactobacillus gasseri), Bifidobacterium breather bracket (Bifidobacterium breve) or Bifidobacterium spent a brush Rum (Bifidobacterium thermophilum ).

Most preferably lactobacillus fermentum.

The term "cancer" of the present invention refers to a condition in which abnormally excessive proliferation occurs due to a problem in regulating functions of normal division, differentiation and death of cells to form lumps in surrounding tissues and organs and destruction or transformation of existing structures it means. The cancer is divided into a primary cancer that is present at the site of development and a metastatic cancer that spreads to other parts of the body from the site of development. The metastatic cancer is formed by spreading cancer cells through blood circulation or lymphatic circulation. In general, it is transferred to other organs after blood circulation and then developed into a new tumor. In contrast, cancer cells are formed by direct migration to neighboring tissues. In the present invention, cancer metastasis is caused by the spread of cancer cells by invasion of cancer cells that directly migrate into and penetrate into neighboring tissues, and the transfer of cancer cells through blood flow to physically form new tumors in the organs not adjacent to primary cancer (metastasis). On the other hand, in the above-mentioned metastasis, cell migration is essential. Therefore, it is clear that inhibiting the movement of cancer cells is a primary method for preventing cancer metastasis.

As described above, metastasis excluding invasion causes cancer cells to move in blood flow. Therefore, the pattern of the blood flow acts as an important factor for determining the site of metastasis. On the other hand, the most frequently occurring tissue, regardless of the form of primary cancer, is the lung. This is related to the fact that blood that has passed through most tissues returns to the heart through the lungs.

The composition of the present invention is characterized by inhibiting the cancer metastasis by inhibiting the movement of cancer cells.

Non-limiting examples of the cancer, that is, primary cancer, include colon cancer, breast cancer, cervical cancer, cervical cancer, ovarian cancer, prostate cancer, brain tumor, head and neck carcinoma, melanoma, myeloma, leukemia, lymphoma, Pancreatic cancer, kidney cancer, renal cancer, renal cell carcinoma, gastric cancer, lung cancer, pancreatic cancer, non-small cell lung cancer, liver cancer, esophageal cancer, small intestine cancer, anorectal cancer, fallopian tube carcinoma, endometrial carcinoma, vaginal carcinoma, vulvar carcinoma, , Pituitary adenocarcinoma, soft tissue sarcoma, urethral cancer, penile cancer, central nervous system (CNS), pancreatic cancer, pancreatic cancer, pancreatic cancer, kidney pelvic carcinoma, bone cancer, skin cancer, head cancer, CNS) tumors, primary CNS lymphoma, spinal cord tumor, brainstem glioma, or pituitary adenoma.

In particular, the suppression of metastasis can be achieved by inhibiting the metastasis of cancer to the lungs. As described above, the lungs in the blood flow pattern are tissues in which cancer metastasis frequently occurs regardless of the type of primary cancer.

According to a specific embodiment of the present invention, it has been confirmed that the migration and infiltration of high-dose heterologous cancer cells can be suppressed in the test tube by treating the fermented product of Huangryinghae or its lactic acid bacteria (Fig. 3) (Fig. 4). The results are shown in Fig. The fermented product according to the present invention and fermented lactic acid bacteria thereof also have the ability to inhibit and kill cancer cells (FIG. 1). In other words, it has the ability to kill cancer cells and inhibits the migration and infiltration of cancer cells. Therefore, it can be used in combination with the treatment for primary cancer, and thus it can exert an excellent effect of treating primary cancer while suppressing cancer metastasis do.

Treatment of the primary cancer can be performed by any known method without limitation. For example, surgical surgery, chemotherapy, biological therapy, radiation therapy and the like may be used.

The composition for suppressing cancer metastasis of the present invention may further comprise a pharmaceutically acceptable carrier, excipient or diluent.

The term "pharmaceutically acceptable" of the present invention means that it exhibits properties that are not toxic to the cells or humans exposed to the composition. The carrier may be any of those known in the art such as a buffer, a preservative, an anhydrous agent, a solubilizer, an isotonic agent, a stabilizer, a base, an excipient, and a lubricant. Examples of pharmaceutically acceptable carrier excipient diluents that can be used in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin , Calcium phosphate, calcium silicate, calcium carbonate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidine, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, have.

The pharmaceutical composition of the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterilized injection solutions according to a conventional method . In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants and the like which are generally used. Typical usable surfactants that facilitate membrane-mediated migration include those derived from steroids or those derived from N- [1- (2,3-dioloyl) propyl-N, N, N-trimethylammonium chloride] ), Or cholesterol hemi-succinate and the like. Solid form preparations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient, such as starch, calcium carbonate, sucrose, Lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. Various excipients such as wetting agents, sweeteners, fragrances, preservatives, etc. in addition to water and liquid paraffin, . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The composition of the present invention may further comprise an anticancer agent.

Non-limiting examples of the anticancer agent may be DNA alkylating agents, anti-cancer antibiotics or plant alkaloids. Specifically, the anticancer agent may be selected from the group consisting of mechloethamine, but are not limited to, chlorambucil, phenylalanine, mustard, cyclophosphamide, ifosfamide, carmustine (BCNU), lomustine (CCNU), streptozotocin streptozotocin, busulfan, thiotepa, cisplatin, carboplatin, dactinomycin (actinomycin D), doxorubicin (adriamycin), daunorubicin Idarubicin, mitoxantrone, plicamycin, mitomycin C, bleomycin, and the like; Such as, for example, vincristine, vinblastine, paclitaxel, docetaxel, etoposide, teniposide, topotecan or iridotecan. have.

In another aspect, the present invention provides a health functional food for preventing or ameliorating cancer metastasis, which comprises as an active ingredient, a fermented product of phytophthora infestans or its lactic acid bacteria.

Specifically, the fermented lactic acid bacteria according to the present invention may be added to foods or beverages for the purpose of suppressing cancer metastasis. The type of food is not particularly limited and includes, for example, various foods such as confectionery, bakery products, , Drinks such as soft drinks and fruit drinks, gum, tea, vitamin complex, seasoning, and health functional foods. At this time, the amount of the fermented lactic acid bacteria in the food or beverage may generally be 0.01 to 15% by weight, preferably 0.1 to 5% by weight, of the total food weight in the case of the health functional food composition of the present invention, May be added in a proportion of 0.01 to 5.0 g, preferably 0.01 to 1.0 g based on 100. [ The health functional food of the present invention thus obtained contains the fermented product of the lactic acid bacterium of the present invention and is thus a food which can sufficiently utilize the effect of inhibiting the cancerous fungus of the fermented product of lactic acid bacteria.

The health functional food of the present invention can be easily produced by adding the lactic acid bacteria fermented product of the present invention described above to the base material or by adding the lactic acid bacteria fermented product of the present invention described above after the production of the food . At this time, a taste and odor corrector may be added as needed.

In addition, the health functional food may contain flavoring agents such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, colorants and heavies (cheese, chocolate, etc.), pectic acid and its salts, Salts, organic acids, protective colloid concentrating agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages and the like. In addition, the health functional food of the present invention may contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks. Such components may be used independently or in combination. The proportion of such additives is generally selected within a range of about 20 parts by weight or less per 100 parts by weight of the health functional food of the present invention.

In another aspect, the present invention provides a method for preventing or treating cancer metastasis in an animal other than human and human. The method comprises the step of administering to a subject in need of treatment a pharmacologically effective amount of a phytophthora or a lactobacillus fermented product thereof.

The term "individual" in the present invention means all animals, including humans, from which metastasis has occurred or may occur, and the cancer metastasis can be effectively inhibited by administering the pharmaceutical composition of the present invention to a subject. The pharmaceutical composition of the present invention can be administered in combination with existing anticancer drugs.

The term "administering" in the present invention means introducing a predetermined substance into an individual by any appropriate method, and the administration route of the pharmaceutical composition according to the present invention may be administered orally, May be administered parenterally. In addition, the phytophthora infestans of the present invention or lactobacillus fermented products thereof can be administered by any device capable of moving the active ingredient to the target cells.

The term "pharmaceutically effective amount " as used herein means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level will vary depending on the sex, age, The activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of the treatment, factors including co-administered drugs, and other factors well known in the medical arts. The fermented lactic acid bacteria of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents. In addition, the fermented product of the present invention and its fermented lactic acid bacteria can be administered singly or multiply. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without adverse effect, and can be easily determined by those skilled in the art. Specifically, the pharmaceutical composition of the present invention is preferably administered orally or intravenously.

The preferable dosage of the fermented soybean flour according to the present invention and its lactic acid fermented product depend on the condition and body weight of the patient, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art. However, for the desired effect, the fermented lactic acid bacteria of the present invention can be administered at 50 to 1000 mg / kg per day, preferably 100 to 500 mg / kg per day. The administration can be done once a day, or divided into several times.

The phthalocyanine fermentation product of the present invention and lactic acid fermented product thereof effectively inhibited the migration and infiltration of cancer cells, while confirming the low cytotoxicity against normal cells, and thus can be usefully used as a composition for preventing or treating cancer metastasis. In addition, the above-mentioned Hwangryeonhaeokgang can be used as a health functional food because it is a herbal medicine material which has already proved to be harmless to human body, and it can be applied to human body immediately.

Brief Description of the Drawings Fig. 1 is a graph showing the inhibitory effect of the fermented soybean flour according to the present invention on the survival of cancer cells in fermented lactic acid bacteria.
FIG. 2 is a graph showing changes in the amount of anticancer-associated cytokine expressed by the phytophthora capsules according to the present invention and its lactic acid fermentation treatment and their cytotoxicity.
FIG. 3 is a graph showing effects of inhibiting the migration and infiltration of cancer cells by the fermented soybean flour according to the present invention and its fermented lactic acid bacteria.
FIG. 4 is a graph showing the effect of inhibiting the lung metastasis of cancer cells in an animal model experiment with a fermented sea tangle according to the present invention and fermented lactic acid bacteria thereof.

Hereinafter, the constitution and effects of the present invention will be described in more detail through examples. These embodiments are only for illustrating the present invention, and the scope of the present invention is not limited by these embodiments.

Example  1: Culture of fermenting bacteria

A total of 45 Lactobacillus spp., 2 Bifidobacterium spp. And 1 Lactobacillus sp. Were purchased from Korea Food Research Institute Food Microbiology Gene Bank. The cultured strain was subcultured in a standard medium (aerobic bacteria: MRS medium, anaerobic bacteria: Reinforced clostridial medium), and the initial number of bacteria was adjusted to 10 ⁵ to 10 ⁶ .

MRS broth (Difco ^™ Lactobacilli MRS Broth, Becton Dickinson, Franklin Lakes, NJ, USA) was used as the liquid medium for the growth of Lactobacillus strains. MRS broth medium (Bacto ^™ Lactobacilli MRS AGAR, Becton Dickinson). The composition of the MRS medium for the growth of Lactobacillus strains is as follows: 10 g / L peptone, 10 g / L juice, 5 g / L yeast extract, 20 g / L glucose, 1 ml / L Tween 80, 2 g / LK ₂ HPO ₄ , 5 g / L sodium acetate, 2 g / L triammonium citrate, 0.2 g / L MgSO ₄ .7H ₂ O, 0.2 g / L MnSO ₄ .4H ₂ O, pH 6.2-6.6.

Reinforced Clostridial medium (Becton Dickinson) was used as a liquid medium for the growth of Bifidobacterium strains. The composition of the Reinforced Clostridial medium for Bifidobacterium strain propagation is as follows: 10 g / L bacto-tryptose, 10 g / L bacterium-juice, 3 g / L bacto-yeast extract, 5 g / 5 g / L sodium chloride, 1 g / L water-soluble starch, 0.5 g / L L-cystine hydrochloric acid, 3 g / L sodium acetate, 0.5 g / L bacto-agar medium, pH 7.6.

Example  2: Hwang Ryun Ha  And its lactic acid bacteria Fermented  Produce

1 L of water was added to the medicines prepared by mixing 5 g of Rhizome, 5 g of gold, 5 g of Porphyra and 5 g of Gardenia as a base, and the mixture was extracted with hot water for 3 hours. ; OR HRT). 1 M NaOH was added to the prepared spinach to adjust the pH to 8.0. The mixture was sterilized at 121 ° C for 15 minutes under high pressure and then cooled to room temperature to prepare a medium for fermentation.

The fermentation strains cultivated and cultivated in Example 1 were inoculated at a concentration of 1% (v / v) in a sterilized herbal preparation after the pH was adjusted, and fermented at 37 ° C for 12 hours, 24 hours and 48 hours, Respectively. Based on the results, 10 strains with high pH - lowering ability were selected and used for fermentation. 128 and 217 are lactobacillus acidophilus, 144 and 348 are lactobacillus plantarum, 164 is lactobacillus fermentum, 166 < RTI ID = 0.0 > Lactobacillus cabbatus, Lactobacillus communis 227, and Bifidobacterium breve strain 744.

In order to prepare the fermented phthalocyanine solution, 700 ml of the fermentation broth was inoculated with 1% (v / v) of the above selected strains, and cultured at 37 ° C for 48 hours. Then, the culture supernatant was centrifuged, , And the obtained culture supernatant was lyophilized to prepare a fermented hwanryunhaedoktang (FHRT) of the present invention.

Example  3: Effect on cancer cell survival rate

(OR HRT) and fermentation broth (FHRT) 164 and 227 prepared in Example 2 were added to HCT-116 cell line, human colon cancer cells, at concentrations of 9, 18, 37, 75, 150, 300 [mu] g / ml) for 48 hours and MTT assay was performed to measure the survival rate of the cancer cells.

As a result, as shown in Fig. 1, it was confirmed that the growth and inhibition of survival of cancer cells in a dose-dependent manner were inhibited by the fermentation of fermentation broth and fermentation. Also, it was confirmed that the fermented hot water had a higher effect than the raw hot water.

Example  4: Evaluation of cytotoxicity and cytotoxicity of anti-cancer-related cytokine expression

In order to confirm the effect of the phytophthora according to the present invention on the expression of anticancer-related cytokines in the fermented product of lactic acid bacteria, macrophages were isolated from BALB / c mice, Respectively. In addition, the viability of these cells was measured, and the cytotoxicity of the above fermented soybean flour and fermented lactic acid bacteria to normal cells was evaluated.

Specifically, 1 ml of 3% thioglycollate was intraperitoneally injected into BALB / c mice and the macrophages collected in the abdominal cavity after 3 days were collected. The macrophages were divided into 6 × 10 ⁵ wells, And 10 kinds of lactic acid bacteria selected from the above Example 2 were treated with 100 μg / ml of fermentation broth (denoted by the respective strain numbers on the figure) for 24 hours, and then the culture broth was recovered. The amount of IL-12 expressed in the recovered culture was measured using a cytokine ELISA kit (Biolegend) according to the protocol provided by the manufacturer, and the cell viability was measured through the MTT assay using the cultured cells, The cytotoxicity of the samples was confirmed. Cells treated with 1 μg / ml of LPS (lipopolysaccharide), which acts as an endotoxin to induce a strong immune response, were used as positive control (LPS) cells and negative control cells (CTL) Untreated cells were used.

The results are shown in Fig. As shown in FIG. 2, the amount of IL-12 expression was significantly increased in the experimental group treated with the fermented phthalocyanine solution, unlike the control. On the other hand, the survival rate of macrophages showed a high cell survival rate of 90% or more in the raw or fermented phthalocyanine solution. Therefore, it was confirmed that the cells were not cytotoxic to normal cells.

Example  5: Cancer cell migration and invasion inhibitory effect

In order to confirm the effects of the phytophthora according to the present invention and the fermented product of lactic acid bacteria in cancer cell migration and infiltration inhibition, cell migration and invasion essays using transwell were performed. As a cell line, a B16F10 cell line which is a highly metastatic mouse melanoma cell line was used. 2 x 10 < ⁵ > B16F10 cells were added to each well and cultured for 24 hours or 48 hours, respectively. For infiltration experiments, not only the incubation time was increased but also the cell infiltration through the matrigel membrane was observed using Matrigel coating. Other experimental conditions were the same as described below.

A trans-well was placed on a 24-well plate and 2x10 ⁵ cells / 100 μl of cells were added. While it wontang goldthread using an open area lying on the trans-well 24-well plates doktang or addition of DMEM medium containing 10% serum in 650 μl containing the fermented product thereof, and 12 to 36 hours at 37 ℃ CO ₂ Lt; / RTI > Α-MSH, a drug inhibiting the metastasis of B16F10 cell line, was treated with 1 μM and used as a positive control. After the culture, 500 μl of crystal violet (2 mg / ml in 20% methanol) was added to each well of the 24-well plate after the transwell was overturned on the hand towel and the transwell was replaced with 30 Minute CO ₂ incubator. The stained trans-wells were washed with PBS and topped up on a hand towel to soak the remaining solution. The membrane was placed on the cover slip with the side to which the cell did not migrate upward, and the unmigrated cells were removed with a cotton swab. A little nail polish was applied to the edge of the cover slip and fixed on the slide glass. The samples thus prepared were photographed with an inverted microscope equipped with a digital camera (CKX41, OLYMPUS, Japan), and the number of transferred cells was counted.

The results are shown in Fig. (HR-OR) or fermented hot water (HR-164) at a concentration of 50 μg / ml compared to the control group (control), the inhibition of migration and invasion of cancer cells was remarkably inhibited. Therefore, it has been confirmed that the phytophthora according to the present invention or the fermented product thereof can be usefully used for inhibiting cancer cell metastasis.

Example  6: Suppression of metastasis in cancer-induced animal models

In order to confirm whether Bongwolryeongtangtang or its fermented product, which has an effective inhibitory effect on the movement and infiltration inhibition of cancer cells, can effectively inhibit cancer metastasis in an animal model through the in vitro experiment of Example 5, . Specifically, a B16F10 cell line (3 x 10 ⁵ cells / ml), a highly metastatic cancer cell, was inoculated into a C57BL / 6 mouse at 6 weeks of age via a tail vein, and fermented for 16 days with phosgene or its fermented lactic acid bacteria The dose of 200 mg / kg was administered orally and the degree of lung metastasis was observed. The dose of the formulation was determined in consideration of the amount of three doses per day of an adult having a body weight of 60 kg and the yield of the herbal preparation, and the negative control group was a physiological saline-administered group.

After 16 days, the mice were sacrificed and the lungs were harvested and fixed with Bouin's solution to confirm the number and pattern of metastatic B16F10 tumor colonies. Photographs of lungs taken from 5 control and experimental groups, respectively, are shown in FIG. Black spots on the drawing represent cancer cell colonies. As shown in Fig. 4, a significantly smaller number of cancer cell colonies were observed in the lungs extracted from the experimental group administered with the Wangtang Huangryinghaotang or the fermented Huanghilhaeyoktang in comparison with the negative control (control) administered with physiological saline. As a result of counting colonies formed in the lungs, about 495 colonies were found in the negative control group, and about 291 colonies and 172 colonies were observed in the control and fermentation broth. From these results, it was shown that the antioxidant inhibition effect was about 40% compared to the negative control, and the fermentation antioxidant test group showed about 65% of the inhibition effect on the transition compared to the negative control. In addition, fewer cancer cell colonies were observed in the lungs of the experimental group treated with fermented phytonyl phthalate than those of the experimental group treated with the phytophagous phytonyl phthalate, which showed an increase of about 40% . Therefore, it was confirmed that the phytophthora according to the present invention and the lactobacillus fermented product thereof were excellent in the suppression of cancer metastasis, and the cancer cell metastasis was strongly inhibited by the fermentation of the fermentation broth. In other words, it was confirmed that the phytophthora according to the present invention has a cancer-suppressing activity and the efficacy is increased by lactic acid fermentation.

Claims (13)

A pharmaceutical composition for inhibiting cancer metastasis comprising a fermented product of Lactic acid bacteria (LAB) as an active ingredient.
The method according to claim 1,
Wherein the lactic acid bacteria fermented product is fermented at a temperature of 35 ° C to 45 ° C and for a time of 1 to 3 days under an aerobic atmosphere by inoculating lactic acid bacteria into a fermented soybean flour.
3. The method according to claim 1 or 2,
The lactic acid bacteria Lactobacillus (Lactobacillus), A Streptococcus (Streptococcus) genus Bifidobacterium (Bifidobacterium) genus Lactococcus (Lactococcus) in, Peddie Oh Caucus (Pediococcus) genus and kind Kono Stock (Leuconostoc) spp ≪ / RTI >
The method of claim 3,
The lactic acid bacterium may be selected from the group consisting of Lactobacillus casei), Lactobacillus even know Phil Ruth (Lactobacillus acidophilus , Lactobacillus plantarum , Lactobacillus < RTI ID = 0.0 > fermentum), Lactobacillus keoba tooth (Lactobacillus curvatus), Lactobacillus konpu Seuss (Lactobacillus confusus), Lactobacillus joined Lee (Lactobacillus gasseri , Bifidobacterium breve and Bifidobacterium thermophilum strains. < RTI ID = 0.0 > 21. < / RTI >
5. The method of claim 4,
Wherein the lactic acid bacterium is lactobacillus fermentum.
The method according to claim 1,
Wherein the composition inhibits the migration of cancer cells.
The method according to claim 1,
The cancer is selected from the group consisting of colon cancer (colon cancer, rectal cancer), breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, brain tumor, head and neck carcinoma, melanoma, myeloma, leukemia, lymphoma, stomach cancer, pancreatic cancer, Renal cell carcinoma, kidney cell carcinoma, renal pelvic carcinoma, bone cancer, skin cancer, head cancer, endometrial carcinoma, endometrial carcinoma, vulvar carcinoma, Hodgkin's disease, bladder cancer, kidney cancer, Cancer of the central nervous system (CNS), primary CNS lymphoma, spinal cord tumor, cervical cancer, endometrial carcinoma, endometrial carcinoma, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, Brain glioma glioma, and pituitary adenoma.
The method according to claim 1,
Wherein said suppression of metastasis is achieved by inhibiting cancer metastasis to the lung.
The method according to claim 1,
Wherein the composition further comprises a pharmaceutically acceptable carrier, excipient or diluent.
The method according to claim 1,
Wherein the composition further comprises an anticancer agent.
11. The method of claim 10,
Wherein the anticancer agent is selected from the group consisting of DNA alkylating agents, anti-cancer antibiotics and plant alkaloids.
12. The method of claim 11,
The anticancer agent may be selected from the group consisting of mechloethamine, chlorambucil, phenylalanine, mustard, cyclophosphamide, ifosfamide, carmustine (BCNU , Streptozotocin, busulfan, thiotepa, cisplatin, carboplatin, dactinomycin (D), actinomycin D, and the like), lomustine (CCNU), streptozotocin, , Doxorubicin (adriamycin), daunorubicin, idarubicin, mitoxantrone, plicamycin, mitomycin C, bleomycin ); Which is composed of vincristine, vinblastine, paclitaxel, docetaxel, etoposide, teniposide, topotecan and iridotecan. ≪ / RTI >
A health functional food for preventing or ameliorating cancer metastasis, which comprises a fermented product of L. acidophilus or its lactic acid bacteria as an active ingredient.

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