KR101797813B1 - Compositions for preventing or treating bladder cancer comprising citrus fermentd broth with Kombucha as an active ingredient - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for prevention or treatment of bladder cancer comprising a citrus sub-body fermentation broth as an active ingredient, and a food composition for preventing or improving bladder cancer.
The composition comprising the citrus comb secondary fermentation broth of the present invention as an active ingredient induces apoptosis of bladder cancer cells, particularly non-invasive (superficial) bladder cancer cells, without showing toxicity to normal cells, It exhibits excellent killing effect. Therefore, the citrus comb secondary fermentation broth of the present invention cultured in the presence of citrus juice may be useful as a pharmaceutical composition or a food composition for the prevention or treatment of early bladder cancer.

Description

TECHNICAL FIELD [0001] The present invention relates to a composition for preventing or treating bladder cancer comprising an active ingredient of a citrus comb supplementary fermentation broth,

The present invention relates to a pharmaceutical composition for prevention or treatment of bladder cancer comprising a citrus sub-body fermentation broth as an active ingredient, and a food composition for preventing or improving bladder cancer.

The incidence of cancer is steadily increasing globally, and according to a report by the National Statistical Office (NSO) of major deaths in Korea, it is reported that the cause of death in Korea is cancer, cerebrovascular disease, and heart disease. According to a report from the International Cancer Institute under the World Health Organization, 30% of cancer deaths are due to smoking, 30% are due to dietary factors, 18% are due to chronic infections, and other occupational, genetic, drinking, Hormone, radiation, environmental pollution, etc. are also contributing 1 ~ 5% each.

Among them, bladder cancer was ranked as the 9th highest cancer among male teenage cancer patients in Korea in the year of 2012. As a side effect of surgery for bladder cancer treatment, drug therapy and chemotherapy, digestive system, urinary tract infection, blood system, Bone marrow suppression, leukopenia, and systemic side effects have been reported.

Although the exact cause of bladder cancer is not known, age, smoking, exposure to chemical agents, and bladder stones are known to be risk factors for bladder cancer and are highly likely to recur after surgery (Vinod H. Nargund et al. al ., 2012, Seminars in oncology , 39 (5): 559-572).

The progression stage of bladder cancer is divided into non-invasive (superficial) bladder cancer, superficial invasive bladder cancer and metastatic bladder cancer depending on the extent of bladder muscle invasion, and the shape of non-invasive bladder cancer is the most diagnosed form of bladder cancer . In addition, it has been reported that early diagnosis and appropriate follow-up and treatment of bladder cancer are important for non-invasive (superficial) bladder cancer because of the difficulty of metastasis and recurrence rate of 60 ~ 80% , Yonsei University Graduate School Master's Thesis ).

Cancer cells that invade muscular layer of the bladder are invasive bladder cancer, which is known as cancer that penetrates the muscle layer and invades into the surrounding tissues and is metastasized. The cancer of the last stage, metastatic cancer, Bladder cancer is divided into three stages.

Recently, studies on natural products having physiological activities such as anticancer and antioxidation have been conducted, and research results have been reported that compounds found in natural products induce apoptosis of cancer cells and exhibit anticancer effects (OS Frankfurt et al ., 2003, Anti - cancer Drugs , 14 (7): 555-561).

The process of cell death is largely divided into two pathways: necrosis and apoptosis. The cell death is accompanied by an inflammation reaction when the stimulus is given to the cell by inflow of moisture outside the cell, and the cell is self-destructed by an internal factor or external stimulus, so that the nucleus is fragmented and the cell is fragmented or phagocytes By swallowing the fragments, cell death proceeds without inflammation (H Steller, 1995, Science , 267 (5203): 1445-1449).

Apoptosis is known to be an active death caused by regulation of several genes and expression proteins. It plays an important role in maintaining the homeostasis of cells by removing the cells with risk factors from normal cells (WS Simonet et al., 1997, Cell , 89 (2): 309-319).

During the course of apoptosis, the caspase (cysteine aspartate-specific protease) affects many biochemical and structural changes in cells dying from apoptosis and acts as an important regulator of apoptosis. The classes that act as initiator caspases include caspase-2, caspase-8, caspase-9, caspase-10, And caspase-3, caspase-6, and caspase-7, which act as effector caspases. In addition, PARP (poly-ADP ribose polymerase), which is a representative caspase-3 substrate protein, plays an important role in maintaining DNA repair or gene stability of normal cells. In apoptosis induction, caspase- This decomposition is lost as the degradation takes place (V Schreiber et al ., 2006, Nature Reviews Molecular Cell Biology 7: 517-528).

The mechanism of apoptosis is divided into the extrinsic pathway associated with the receptor of the cell and the intrinsic pathway associated with mitochondria and includes the death receptors, the Bcl-2 family, Apoptosis is regulated by interactions such as caspase (Z Jin et al ., 2005, Cancer biology & therapy , 4 (2): 147-171). In addition, in apoptosis mechanism, anti-apoptotic factor that suppresses cell death and apoptotic factor by the action of pro-apoptotic factor that induces cell death apoptotic cell death.

B-cell lymphoma-2, an anti-apoptotic protein, binds to Bax (Bcl-2-associated X protein) to form a heterodimer to inhibit the activity of Bax Have been reported to interfere with apoptosis or affect caspases.

Bax, a pro-apoptotic protein, increases apoptotic protease activating factor 1 (Apopt-1) by increasing the mitochondrial membrane permeabilization (MOMP) and increasing the release of cytochrome c, (SN Farrow et al ., 1996, Curr Opin Genet Dev , 6 (1): 1): < RTI ID = 45-49)

In addition, phytochemicals such as flavonoids and polyphenols, which are compounds found in natural products, have been studied to inhibit cancer cell proliferation and inhibit cancer cell metastasis. In addition, it is required to research and develop new uses and value-added by using natural materials that can be commonly found in the surrounding area as a raw material (OS Frankfurt et al ., 2010, Febs Journal , 277 (16): 3437-3448, et al ., 2003, Korean Journal of Food Science and Nutrition 32 (2): 217-222).

It is known that citrus fruits, which are used as raw materials for oriental medicine and herbal medicine, contain various components such as flavonoids, carotenoids and coumarins. The major citric-derived flavonoid compounds are naringin and hesperidin and their aglycone forms, naringenin and hesperetin, as well as rutin, nobiletin, ), And tangeretin, and it is also effective in improving the vascular disease by blood cholesterol and neutral lipid inhibition effect, improving liver disease including liver disease, anti-cancer effect by inhibiting cancer cell proliferation, antioxidants to inhibit lipid peroxidation, antibacterial action, anti-inflammatory and antiallergic, immune-enhancing activity there are a variety of physiological functions, reported such activity (chajaeyoung et al, 2001, J. Korean Soc . Agric. Chem. Biotchnol., 44 (2): 122-128).

(CW Hesseltine, 1983, Nutr Rev , 41 (10)) was one of the drinks known as traditional fermented beverages in Russia, : 293-301). In the United States, it is known that there is a refreshing taste, detoxification effect, chronic fatigue, constipation, and health improvement and medical treatment effect of the cow bug in the United States. In Korea, fermented beverage known as black tea mushroom is known to have detoxification activity, antimicrobial activity and antioxidant activity have.

It is also known that glucuronic acid, vitamins B1, B2, and B2 are effective for pain relief of arthritis, blood pressure reduction, increase of T cells, constipation relief, digestive or metabolic diseases, B6 and antibiotic-active usnic acid, and antioxidant activity (KH Steinkraus et al ., 1996, Acta Biotechnol. , 16 (2-3): 199-205; R Srinivasan et al ., 1997, Journal of general internal medicine , 12 (10): 643-645).

Examples of the Acetobacter sp. Are Acetobacter Xylinum, Saccharomyces sp., Zygosaccharomyces sp., And the like. Examples of the Acetobacter sp. , Yeasts such as Torulopsis sp., Pichia sp., And Brettanomyces sp. Are present.

Acetobacter sp. And yeast are in a symbiotic relationship with each other. These strains interact simultaneously and fermentation proceeds to produce acetic acid, glucuronic acid, Amino acids and cellulose pellicle. Recent researches on comb-bacterium have shown that the effect of down-regulation of prostate cancer neovascularization and the prevention of toxic substances in hepatocytes (SC Chu. Et al., 2006, Food Chem , 98 (3): 502-507; T Srihari et al ., 2013, Biomedicine & Preventive Nutrition , 3 (1): 53-58; S Bhattacharya et al ., 2011, Indian Journal of Experimental Biology , 49: 511-524).

Although tea is mostly used when cultivating kombu tea, green tea has a higher content of tea catechin than fermented tea such as black tea or oolong tea. Tea catechin is used as an antioxidant to prevent food from deteriorating (MWL Koo et al ., 2004, Eur J Pharmacol , 500 (1-3): 177). However, it has been reported that serum lipid components such as body weight, waist circumference, -185; Korean Patent No. 10-0482308).

Under these circumstances, the inventors of the present invention have made efforts to find a natural product having an anticancer effect against bladder cancer and a low side effect, and as a result, it has been found that a citrus fruit supplement solution containing green tea extract and citrus juice is effective in killing bladder cancer cells And the present invention has been completed.

Korean Patent No. 10-0482308

Vinod H. Nargund et al., 2012, Seminars in oncology, 39 (5): 559-572 2006, Yonsei University Graduate School Master's Thesis OS Frankfurt et al., 2003, Anti-cancer Drugs, 14 (7): 555-561 H Steller, 1995, Science, 267 (5203): 1445-1449 WS Simonet et al., 1997, Cell, 89 (2): 309-319 V Schreiber et al., 2006, Nature Reviews Molecular Cell Biology 7: 517-528 Z Jin et al., 2005, Cancer biology & therapy, 4 (2): 147-171 SN Farrow et al., 1996, Curr Opin Genet Dev, 6 (1): 45-49 OS Frankfurt et al., 2010, Febs Journal, 277 (16): 3437-3448 Hwang, Yong-Joo et al., 2003, The Korean Journal of Food Science and Nutrition 32 (2): 217-222) Cha Jae-young et al., 2001, J. Korean Soc. Agric. Chem. Biotchnol., 44 (2): 122-128 CW Hesseltine, 1983, Nutr Rev, 41 (10): 293-301 K.H. Steinkraus et al., 1996, Acta Biotechnol, 16 (2-3): 199-205 R Srinivasan et al., 1997, Journal of general internal medicine, 12 (10): 643-645 SC Chu. et al., 2006, Food Chem, 98 (3): 502-507 T Srihari et al., 2013, Biomedicine & Preventive Nutrition, 3 (1): 53-58 S Bhattacharya et al., 2011, Indian Journal of Experimental Biology, 49: 511-524 MWL Koo et al., 2004, Eur J Pharmacol, 500 (1-3): 177-185

It is an object of the present invention to provide a pharmaceutical composition for preventing or treating bladder cancer, which comprises a citrus comb secondary fermentation broth as an active ingredient.

It is another object of the present invention to provide a food composition for prevention or improvement of bladder cancer, which comprises a citrus comb secondary fermentation broth as an active ingredient.

In one aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating bladder cancer, which comprises a citrus comb secondary fermentation broth as an active ingredient.

The term "Kombucha (K)" in the present invention is a fermented black tea drink, which is one of the traditional fermented beverages in Russia which is fermented by putting a fungus called tea fungus into a black tea extract. / ≪ / RTI > and adding the sugar to the green tea and fermenting it.

In one embodiment of the present invention, the Comvita is prepared by adding a commercial comb supplement and a hot water-extracted green tea extract containing a pellicle, and 5 to 15% (w / v) (V / v) of the total composition, and the pellicle has a volume of 6.25 to 50% (v / v), which is 1/2 to 1/16 of the volume. w / v) at a temperature of 28 to 35 DEG C for 5 to 10 days (Example 1-1, Fig. 1 and Fig. 3).

In the present invention, the term "Citrus Kombucha (CK) fermentation broth" is a fermentation broth fermented by adding citrus juice to the comb culture.

The citrus comb secondary fermentation broth may be prepared by: 1) adding a green tea extract and a 5 to 15% (w / v) sugar of the whole composition to a pre-culture by stirring the mixture; 2) cooling extracting the pre-cultured comb cells of step 1) at 5 to 15 캜; And 3) 5 to 20% (v / v), 0.01 to 0.5% (v / v) acetate, 6.25 to 50% (w / v) pellicle of the comb secondary culture obtained through the pre- (Citric acid solution) and 5 to 50% (v / v) citrus solution for 5 to 10 days (Examples 1-1, 2 and 3).

The term "bladder cancer" in the present invention refers to a malignant tumor that occurs in the bladder. The bladder cancer is largely localized only in the bladder mucosa or the submucosal layer. Therefore, ) Bladder cancer, and bladder cancer is a muscle-invasive bladder cancer that requires bladder removal to completely remove the tumor by invading the muscle layer, and metastatic bladder cancer.

The term "prevention" in the present invention means all actions that inhibit or delay the generation, spread and recurrence of bladder cancer by the administration of the pharmaceutical composition according to the present invention, and "treatment" And all actions that alleviate or ameliorate the symptoms of the affected individuals.

Cancers which can be prevented or treated by the composition of the present invention include, but are not limited to, bladder cancer, preferably non-invasive (superficial) bladder cancer.

Preferably, the prevention or treatment of cancer can be accomplished by the apoptosis of cancer cells induced by the citrus comb secondary fermentation broth according to the present invention. The above-mentioned "apoptosis" is a kind of programmed cell death (PCD), in which abnormal cells, damaged cells and aged cells are killed and killed by the biological program inputted into our body, . Such apoptosis is distinguished from cell death caused by stimulation such as burns, scabs, and poisons, such as necrosis, which is the death or accidental death of a cell. That is, apoptosis is an active death of a cell that is regulated by a protein expressed from a specific gene. While apoptosis occurs chaotically, apoptosis occurs in an orderly fashion in a short time. Specifically, apoptosis begins with the contraction of the cell, creating a gap between the adjacent cells, and DNA, which is a genetic material in the nucleus of the cell in the process of dying, is regularly cleaved into fragments. Ultimately, the entire cell called the apoptotic body is also broken up, and it is killed by other cells around it. It is involved in the formation of the human body during the early development of the human body. In adults, it is responsible for eliminating normal cells when they become senescent, or removing cells with abnormalities. The apoptosis is distinguished from PCD in that it also occurs in cases other than PCD, and is related to cell death due to cancer cell death, viral infection, drug, and radiation.

According to a specific embodiment of the present invention, it was confirmed that the composition of the present invention, for example, citrus squamous carcass did not show cytotoxicity against normal cells (RAW 264.7) but showed killing effect on bladder cancer cell lines (T24 and 5637) , Especially T24 cells, a non-invasive (superficial) bladder cancer cell line (FIGS. 4 and 5). In addition, it was confirmed that the treatment of the composition induces apoptosis and induces apoptosis. In particular, in the case of non-invasive (superficial) bladder cancer cells (T24), apoptosis is effectively induced in the treatment of citrus subspecies, Activity was further confirmed (FIG. 11). Further, by confirming the expression of proteins involved in the apoptosis of cancer cells, it can be confirmed once again that the composition of the present invention induces apoptosis of cancer cells through the external pathway through the death receptor and the internal pathway in the mitochondria (Fig. 13).

The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier. 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. Such carriers may be used without limitation as long as they are known in the art such as buffers, preservatives, wetting agents, solubilizers, isotonic agents, stabilizers, bases, excipients and lubricants.

In addition, 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 have. Furthermore, it can be used in the form of an external preparation for skin in the form of ointments, lotions, spray agents, patches, creams, powders, suspensions, gels or gels. Examples of carriers, excipients and diluents that can be included in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used.

Solid formulations 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 are also used. Liquid preparations for oral use may include various excipients such as wetting agents, sweetening agents, fragrances, preservatives, etc. in addition to water and liquid paraffin, which are simple diluents commonly used in suspension, liquid solutions, emulsions and syrups have. 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.

Meanwhile, the pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount " of the present invention means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment and not causing side effects, The type of cancer, the severity, the activity of the drug, the sensitivity to the drug, the method of administration, the time of administration, the route of administration and the rate of release, the duration of the treatment, factors including the drugs used concurrently or concurrently and other factors well known in the medical arts . 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, and may 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 side effects, which can be easily determined by those skilled in the art.

In particular, the effective amount of the citrus sub-body fermentation broth contained in the composition of the present invention may vary depending on the age, sex, and body weight of the patient. Generally, the citrus comb sub-composition of the present invention is 0.001 To 100 mg, preferably 1 to 10 mg, more preferably 1 to 5 mg per kg of body weight per day or every other day, or one to three divided doses per day. However, the dosage may not be limited in any way because it may be increased or decreased depending on route of administration, severity of disease, sex, weight, age, and the like.

The composition of the present invention may be administered to mammals such as rats, mice, livestock, humans, and the like in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

In another aspect, the present invention provides a method for preventing or treating bladder cancer, comprising administering a citrus sub-fermentation broth to a subject in need thereof.

The term "individual" of the present invention means a mammal such as a monkey, a cow, a horse, a sheep, a pig, a chicken, a turkey, a quail, a cat, a dog, a mouse, a rat, a rabbit or a guinea pig , And the cancer can be effectively prevented or treated by administering the pharmaceutical composition of the present invention to a subject. The pharmaceutical composition of the present invention can be administered in parallel with existing therapeutic agents.

The term "administering" of the present invention means providing the patient with the desired substance in any suitable manner, and the administration route of the composition of the present invention may be administered through any conventional route so long as it can reach the target tissue have. But are not limited to, intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, intrathecal, rectal. In addition, the pharmaceutical composition of the present invention may be administered by any device capable of moving the active substance to the target cell. The preferred modes of administration and formulations are intravenous, subcutaneous, intradermal, intramuscular, and drip injections. The injectable solution may be a non-aqueous solvent such as an aqueous solvent such as a physiological saline solution or a ring gel solution, a vegetable oil, a higher fatty acid ester (e.g., oleic acid), an alcohol (e.g., ethanol, benzyl alcohol, propylene glycol, glycerin, etc.) (For example, ascorbic acid, sodium hydrogen sulfite, sodium pyrophosphate, BHA, tocopherol, EDTA and the like), an emulsifier, a buffer for pH control, a microbial growth inhibitor And a pharmaceutical carrier such as a preservative (e.g., mercury nitrate, thimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol, etc.).

The pharmaceutical composition of the present invention may further include a known anticancer agent in addition to the citrus comb secondary fermentation liquid as an active ingredient and may be used in combination with other known treatments for the treatment of these diseases. Other treatments include, but are not limited to, chemotherapy, radiation therapy, hormone therapy, bone marrow transplantation, stem cell replacement therapy, other biological therapies, immunotherapy, and the like.

Examples of anticancer agents that can be included in the pharmaceutical composition of the present invention include DNA alkylating agents such as mechloethamine, chlorambucil, phenylalanine, mustard, cyclophosphate, Cyclophosphamide, ifosfamide, carmustine (BCNU), lomustine (CCNU), streptozotocin, busulfan, thiotepa, cisplatin cisplatin and carboplatin; The anticancer antibiotics include dactinomycin (actinomycin D), doxorubicin (adriamycin), daunorubicin, idarubicin, mitoxantrone, Plicamycin, mitomycin C, and bleomycin; And plant alkaloids such as vincristine, vinblastine, paclitaxel, docetaxel, etoposide, teniposide, topotecan, And iridotecan, but are not limited thereto.

In another aspect, the present invention provides a food composition for prevention or amelioration of bladder cancer, which comprises a citrus comb secondary fermentation broth as an active ingredient. When the composition of the present invention is used as a food composition, the citrus subterranean fermentation broth can be used as it is, or can be used in combination with other food or food ingredients, and can be suitably used according to ordinary methods. The composition may contain a food-acceptable food-aid additive in addition to the active ingredient, and the amount of the active ingredient to be mixed may be suitably determined according to the purpose of use (prevention, health or therapeutic treatment).

As used herein, the term "food-aid additive " refers to a component that can be added to foods in a supplementary manner, and is appropriately selected and used by those skilled in the art as added to produce health functional foods of each formulation. Examples of food-aid additives include flavors such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, colorants and fillers, pectic acid and its salts, alginic acid and its salts, organic acids, , a pH adjusting agent, a stabilizer, a preservative, a glycerin, an alcohol, and a carbonating agent used in a carbonated drink. However, the types of the food auxiliary additives of the present invention are not limited by these examples.

A health functional food may be included in the food composition of the present invention. The term "health functional food " as used in the present invention refers to a food prepared and processed in the form of tablets, capsules, powders, granules, liquids and rings using raw materials and components having useful functions in the human body. Here, 'functional' refers to the structure and function of the human body to obtain nutritional effects and obtain useful effects for health use such as physiological action. The health functional food of the present invention can be prepared by a method commonly used in the art and can be prepared by adding raw materials and ingredients that are conventionally added in the art. In addition, the formulations of the above health functional foods may also be manufactured without limitations as long as they are acceptable as health functional foods. The composition for food of the present invention can be manufactured in various forms, and unlike general pharmaceuticals, it has the advantage that there is no side effect that may occur when a drug is used for a long period of time, and is excellent in portability, Can be ingested as an adjuvant to enhance the effectiveness of anticancer drugs.

There is no limitation on the kind of health food to which the composition of the present invention can be used. In addition, the composition comprising the citrus comb secondary fermentation broth of the present invention as an active ingredient may be prepared by mixing other suitable auxiliary ingredients and known additives, which may be contained in health functional foods, according to the selection of a person skilled in the art. Examples of foods that can be added include dairy products, such as meat, sausage, bread, chocolates, candies, snacks, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, Vitamin complex, and the like, and they may be added to juice, tea, jelly, juice and the like prepared from the citrus comb supplementary fermentation broth according to the present invention as a main ingredient.

The citrus comb secondary fermentation broth of the present invention can be used as a pharmaceutical composition or a food composition because it can be less harmful than general synthetic compounds and thus can be safely used in pharmaceutical compositions and health functional foods have.

The composition comprising the citrus comb secondary fermentation broth of the present invention as an active ingredient induces apoptosis of bladder cancer cells, particularly non-invasive (superficial) bladder cancer cells, without showing toxicity to normal cells, It exhibits excellent killing effect. Therefore, the citrus comb secondary fermentation broth of the present invention cultured in the presence of citrus juice may be useful as a pharmaceutical composition or a food composition for the prevention or treatment of early bladder cancer.

Fig. 1 is a flow diagram schematically showing a step of producing a Kombucha fermentation broth of the present invention.
FIG. 2 is a flow diagram schematically showing a step of producing a citrus Kombucha fermentation broth of the present invention.
FIG. 3 is a photograph showing a sample of a fermented beverage of the present invention.
FIG. 4 is a graph showing the results of cytotoxicity test according to the concentration of the comb and carnitine secondary to the present invention (RAW 264.7 cell). FIG.
FIG. 5 is a graph showing the results of inhibition of cell proliferation according to the concentration of comb and carnitine secondary to the non-invasive (superficial) bladder cancer cell (T24 cell) of the present invention.
FIG. 6 is a graph showing the results of inhibiting cell proliferation according to the concentration of comb and carnitine secondary to the present invention on high-risk superficial bladder cancer cells (5637 cells).
FIG. 7 is a photograph showing a morphological change of a T24 cell of a non-invasive (superficial) bladder cancer cell line according to the concentration of the Comvatus (A) and Citrus subunit (B) of the present invention under a microscope.
FIG. 8 is a photograph showing a morphological change of 5637 cells of a high-risk superficial bladder cancer cell line according to the concentration of the Comvatus (A) and the Citrus subunit (B) of the present invention under a microscope.
FIG. 9 is a graph showing the degree of migration of non-invasive (superficial) bladder cancer cell line T24 cells according to the concentration of the combus subtype (A) and the citrus combus subtype (B) of the present invention through a wound healing assay It's a picture.
FIG. 10 is a microscopic photograph showing the degree of migration of high-risk superficial bladder cancer cell line 5637 cells according to the concentration of the combus subtype (A) and the citrus combus subtype (B) of the present invention through a wound healing assay to be.
FIG. 11 is a graph showing the degree of apoptosis by quantifying the degree of apoptosis of non-invasive (superficial) bladder cancer cell line T24 cells according to the concentration of a comb and a citrus sub-anchor of the present invention.
FIG. 12 is a graph showing quantitation of apoptosis-induced apoptosis of high-risk superficial bladder cancer cell line 5637 cells according to the concentration of comb and carnitine secondary soma of the present invention.
FIG. 13 is a graph showing the ability of the non-invasive (superficial) bladder cancer cell line T24 cells according to the present invention to control the expression of apoptosis-related proteins according to the concentrations of the comb and carnitine secondary soma.
FIG. 14 is a graph showing the ability of the present invention to control the expression of apoptosis-related protein on the high-risk superficial bladder cancer cell line 5637 cells according to the concentrations of comb and carnitine secondary.

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

Example  One: Kom Bucha  Culture and sample preparation

Example  1-1: Kom Bucha  culture

The Kombucha used in the present invention is a domestic domestic Kombucha, which is purchased and sold in the private sector through the Internet (www.auction.co.kr).

In the case of Comvuka, 900 ml of green tea extract and 90 g of sugar were added and stirred, and then pre-cultured, 100 ml of the origin kombucha culture obtained after the pre-culture and 18 g of the pellicle produced by the pre-culture fermentation were added at a volume of 1/4 of the volume and cultured at 30 ° C for 10 days. In addition, as shown in FIG. 2, the Citrus comb subspecies were prepared by pre-incubation after mixing 700 ml of green comb extract and 90 g of sugar, which contains a pellicle for commercial use, and incubated at the origin 100 μl of pellicle, 200 ml of citrus juice and 500 μl of acetate were added and cultured under the same conditions as those of Komatsu.

The citrus juice used in the present invention was obtained by using the one produced in Ilhwa, Korea.

Example  1-2: Sample Preparation

Kombicha and Citrus Kombucha samples were prepared for antioxidant and total polyphenol contents measurement and for cell experiments.

Specifically, for the antioxidation and total polyphenol contents measurement, supernatants obtained after centrifugation of comb and carnitine at 20,000 rpm for 12 minutes were prepared. In the cell experiments, Each supernatant obtained after the minute centrifugation was filtered using a syringe filter unit (pore size 45 μm) and diluted in medium (RPMI-1640).

Example  2: Culture of cell line

T24 cells, which are non-invasive (superficial) bladder cancer cell lines used in the present invention, and 5637 cells that are high-risk superficial bladder cancer cells were purchased from Korean Cell Line Bank. The T24 cells and 5637 cells were cultured at 37 ° C in 5% (Sigma-Aldrich) supplemented with fetal bovine serum (FBS) and 1% penicillin (GIBCO Inc.).

RAW 264.7 cells used in the present invention were purchased from ATCC (American Type Culture Collection, Manassas, Va., USA) and cultured in RPMI 1640 supplemented with 10% fetal bovine serum (Hyclone Laboratories, Logan, UT, USA) glutamine, and 100 mg / L penicillin-streptomycin (Hyclone Laboratories).

Example  3: Kombucha  Citrus Kombucha  Antioxidant activity measurement

DPPH radical scavenging activity and ABTS radical cation scavenging activity were measured to determine the antioxidant activity of Kombucha K and Citrus Kombucha CK. In addition, the antioxidant ability of the sample is not directly measured, but since the antioxidant ability contained in the sample can be predicted, the total polyphenol content was measured as one of widely used methods for antioxidant research.

Example  3-1: DPPH Radical Scatters  Measure

DPPH (1,1-diphenyl-2-picryl hydrazyl) is a dark purple colored ganic nitrogen free radical. It shows absorbance in the ethanol solution state. When it comes into contact with a substance having antioxidative activity, It is characterized by the irreversible conversion of a stable compound that is reduced while accepting electrons of hydrogen radicals, and the deep violet is thinned.

Thus, a DPPH assay (DPPH assay) was conducted to measure the rapid and simple free radical scavenging ability as a widely used method for measuring antioxidant ability. Vitamin C (ascorbic acid) was used as a control group.

More specifically, 4 ml of a 0.2 mM DPPH solution dissolved in ethanol was added to 1 ml of each sample of the comb-tertiary and citrus subcombinations, followed by reaction at room temperature for 20 minutes. The absorbance values at 517 nm wavelength were measured using an ELISA microplate reader (Versa Max, USA) and the results were shown by the following equation.

(DPPH radical scavenging activity, (%)) = [1- (absorbance of control group without addition of combs or citrus sub-samples)] × 100

As a result, the DPPH radical scavenging activity of citrus squam carcinoma supplemented with the Comvatus culture medium and the citrus juice supplement was 1 mg / ml of squid carcass and citrus squam carbeta as shown in Table 1 below, and the DPPH radical scavenging activity was 87.9 % And 91.2% respectively.

As a result, the antioxidant activity of both the comb and the citrus combs was superior to that of the control group, vitamin C. In particular, the free radical scavenging activity of the citrus combs supplemented with citrus fluids versus comb , The antioxidant activity of citrus squam carcinoma was found to be superior to that of comb carcinoma.

Example  3-2: ABTS Radical  Cation Scatters  Measure

ABTS (3-ethylbenzothiazoline-6-sulphonic acid) assay is a method to measure the abilities of antioxidants to cations (ABTS · +). Potassium persulfate persulfate to form a green ABTS radical, and the resulting ABTS radical is reduced to a colorless substance by receiving electrons from the antioxidant substance.

Thus, the ABTS assay capable of measuring the antioxidant activity was performed on the basis of the above principle, and the ABTS radical cation scavenging activity of the present invention was measured. Vitamin C (ascorbic acid) was used as a control group.

Specifically, ABTS radical cationic scavenging activity was measured by incubating 7.4 mM ABTS and 2.6 mM potassium persulfate in a dark place overnight to form ABTS cations. The solution was adjusted to have an absorbance value of 1.4 to 1.5 at a wavelength of 735 nm Diluted with distilled water and used.

100 μl of each sample of Kombucha and Citruscomb and 900 μl of ABTS solution were added and left for 30 minutes, and then measured at 735 nm wavelength using an ELISA reader. The results are shown in the following formula.

Absorbance of ABTS radical cation scavenging activity (%) = [1- (absorbance of control group without addition of combs or citrus sub-sample)] / 100

As a result, the ABTS radical cation-scavenging activity of citrus squam carcinoma supplemented with the comb-secondary culture and the citrus juice was found to be 1 mg / ml of the comb and carnitine secondary metabolites as shown in Table 2 below, and the ABTS radical cation- 78.78% and 94.59%, respectively.

The antioxidative activity of citrus squam carcinoma was found to be superior to that of the carcinoma squam carcinoma by confirming that the free radical scavenging activity of citrus squam carcinoma supplemented with citrus juice was higher than that of combus carota. But also the antioxidant activity of vitamin C, which is similar to the control vitamin C.

Example  3-3: Total polyphenol content (total polyphenol  content measurement

Since several hydroxyl groups substituted on the benzene ring of the polyphenol compound participate in the reduction reaction with free radicals, the antioxidative activity is exhibited. Therefore, as the total polyphenol content in the sample increases, the antioxidant activity (Y Park et al ., 2008, Food Science and Biotechnology 17 (2): 251-256).

Therefore, the antioxidant ability of the sample is not directly measured. However, since the antioxidant ability contained in the sample can be predicted, the total polyphenol content was measured as a widely used method for antioxidant research.

Specifically, 1 ml of a sample of each of Combacca and Citruscomb subunit was mixed with 1 ml of a 10% folin-ciocalteu's phenol regent and 1 ml of a 2% sodium carbonate solution, Absorbance was measured at a wavelength of 750 nm using an ELISA reader.

The total phenolic content was calculated using a standard curve prepared using tannic acid, and the total polyphenol content was expressed as 쨉 g / ml per sample weight.

As a result, as shown in Table 3 below, the total polyphenol contents of Comvatus and Citrus subunit were 17.82 μg / ml and 28.62 μg / ml, respectively, when expressed as equivalents of tannic acid per μg.

The total polyphenol content of Citrus buccal supplemented with citrus juice was higher than that of.

The results of the antioxidant activity measurement showed that the total polyphenol content of citrus squam carcinoma supplemented with citrus juice was higher than that of Combauchi, and the antioxidant activity was excellent. Thus, cell membrane destruction, enzyme inactivation, lipid oxidation, DNA denaturation, And reactive oxygen species (ROS), which cause a serious pathological disorder such as atherosclerosis, autoimmune disease, including cancer, by causing potential cell damage such as cell senescence, It can be efficiently removed.

Example  4: Kombucha  Citrus Kombucha  Cytotoxicity measurement

(MTT assay, 3- (4,5-dimethylthiazol-2-yl) -2,5-dimethyl-thiazol-2-yl) -acetic acid was used to measure the inhibition of cell proliferation by treatment with Kombucha, K and Citrus Kombucha diphenyltetrazolium bromide assay).

Example  4-1: For normal cells (RAW 264.7) Kombucha  Citrus Kombucha  Cytotoxicity measurement

First, MTT assay was performed to determine the effect of Kombucha and Citrus Kombucha on the cell viability of normal cell RAW 264.7 cells.

Specifically, RAW 264.7 cells were seeded in a 96-well plate at a density of 1 × 10 ⁴ cells / well, and the somatic and somatic sub-samples were divided into 0, 1, 2, 4, 6, 8 mg / ml, and cultured for 24 hours. After culturing, 10 쨉 l of 5 mg / ml MTT reagent (Thiazolyl Blue Tetrazolium Bromide) was added to each well and cultured for 1 hour, and the medium was removed. 100 쨉 l of DMSO (dimethyl sulfoxide) was dispensed into each well from which the medium had been removed. All of the formazan produced by the MTT reagent was dissolved in each well, and the absorbance at a wavelength of 560 nm was measured with an ELISA reader Were measured. At this time, the cell viability was expressed as a percentage of the absorbance of the control group (untreated group) in the respective samples of the comb and carcass sub-samples.

As shown in FIG. 4, when the toxin of the comb and the citrus comb subspecies were treated at the concentrations of 0, 1, 2, 4 and 6 mg / ml, respectively, the survival rate of 100 %, Respectively. In the case of treating each of the comb and carcinoma subculture samples at a concentration of 8 mg / ml, the survival rate was 80% or more and the toxicity was about 20% or less, which greatly influenced the cell survival rate of normal cells RAW 264.7 cells Respectively.

Example  4-2: Nasal invasion (Superficial) for bladder cancer cells (T24 cell) Kombucha  Citrus Kombucha  Cytotoxicity measurement

Of non-invasive (superficial) bladder cancer cells (T24 cells) following treatment with Kombucha and Citrus Kombucha MTT assay was performed to measure cell proliferation inhibition. At this time, the treatment concentrations of the comb and carnitine subunits were 0, 1, 2, 4, 6 and 8 mg / ml, which do not significantly affect the cell viability of the normal cell RAW 264.7 cells in Example 4-1 Respectively.

Specifically, T24 cells were seeded in a 96-well plate at a density of 1 × 10 ⁴ cells / well, and the cells were inoculated with 0, 1, 2, 4, 6 , 8 mg / ml, and cultured for 24 hours. After culturing, 10 쨉 l of 5 mg / ml MTT reagent was added to each well, followed by incubation for 1 hour, and the medium was removed. 100 쨉 l of DMSO (dimethyl sulfoxide) was dispensed into each of the wells from which the medium had been removed. All of the formazan produced in each well was dissolved and the absorbance at 560 nm was measured with an ELISA reader. At this time, the cell proliferation rate was compared with the cell proliferation according to the concentration of the test group treated with each of the comb and carcass sub-samples, based on 100% of the cell viability of the control group (untreated group).

As shown in FIG. 5, the T24 cells of bladder invasion (superficial) bladder cancer cells were treated with the comb cells and the citrus comb cells at the concentrations of 1, 2, 4, 6 and 8 mg / Survival rates were 82.57%, 69.01%, 50.12%, 33.47% and 26.09%, respectively, and the survival rate was 81.96%, 72.77%, 50.36%, 19.49% and 13.44% respectively when treated with Citrus squamous carcass p < 0.05).

These results suggest that both the comb and the citrus sub - cultures inhibit the proliferation of non - invasive (superficial) bladder cancer cells as the treatment concentration increases. Especially, when the citrus comb sub - (Superficial) bladder cancer cell proliferation compared to the control group.

Example  4-3: High-risk superficial bladder cancer cells (5637 cells) Kombucha  Cytotoxicity measurement of citrus squam carcinoma

High-risk superficial bladder cancer cells (5637 cells) following treatment with Kombucha and Citrus Kombucha MTT assay was performed to measure cell proliferation inhibition. At this time, the treatment concentrations of the comb and carnitine subunits were 0, 1, 2, 4, 6 and 8 mg / ml, which do not significantly affect the cell viability of the normal cell RAW 264.7 cells in Example 4-1 Respectively.

Specifically, 5637 cells were seeded in a 96-well plate at a density of 1 × 10 ⁴ cells / well, and the cells were inoculated with 0, 1, 2, 4, 6 , 8 mg / ml, and cultured for 24 hours. After culturing, 10 쨉 l of 5 mg / ml MTT reagent was added to each well, followed by incubation for 1 hour, and the medium was removed. 100 쨉 l of DMSO (dimethyl sulfoxide) was dispensed into each of the wells from which the medium had been removed. All of the formazan produced in each well was dissolved and the absorbance at 560 nm was measured with an ELISA reader. At this time, the cell proliferation rate was compared with the cell proliferation according to the concentration of the test group treated with each of the comb and carcass sub-samples, based on 100% of the cell viability of the control group (untreated group).

As shown in FIG. 6, when the comb secondary cells were treated with high-risk superficial bladder cancer cells 5637 cells at a concentration of 1, 2, 4, 6 and 8 mg / ml, Survival rates were 84.22%, 84.71%, 85.63%, 80.63% and 68.22%, respectively, and 82.5%, 74.24%, 76.03%, 65.71% and 35.87% survival rates of citrus subspecific treatment were respectively ( p < 0.05).

As a result, it was found that high-risk superficial bladder cancer cells (TGF-β1) and high-dose superficial bladder cancer cells (TGF-β) 5637 cells also inhibited cell proliferation. In addition, it was found that the treatment of citrus squam carcinoma significantly inhibited the proliferation of superficial superficial bladder cancer cells compared to the case of treatment of squamous cell carcinoma.

The results of cytotoxicity measurements of the comb and carnitine mutants show that both comb and carnitine are not toxic to normal cells, and that T24 cells, which are non-invasive (superficial) bladder cancer cells, and high-risk superficial bladder cancer cells 5637 cells were inhibited. In addition, the bladder cancer cells were found to have a higher cell proliferation inhibition rate than the humps.

In addition, the inhibition rate of cell proliferation was higher for the T24 cells of the non-invasive (superficial) bladder cancer cells than the 5637 cells of the high-risk superficial bladder cancer cells, and the inhibition rate of the cell proliferation of the citrus sub- . Thus, it can be seen that the citrus comb secondary of the present invention can be advantageously used for the prevention or treatment of bladder cancer which is an early bladder cancer.

Example  5: Kombucha, K and  Citrus To Citrus Kombucha (CK)  Observation of cell morphology of bladder cancer

Example  5-1: Kombucha  Citrus Kom Bucha  Following Nasal invasion (Superficiality) Observation of cell morphology of bladder cancer cells (T24 cell)

In order to confirm the cell morphology of non-invasive (superficial) bladder cancer cells (T24 cells) according to the concentration of the comb and carnitine secondary metabolites of the present invention, 6 × 10 ⁵ / well lt; RTI ID = 0.0 &gt; T24 cells were subcultured, and each of Combacca and Citrus subspecies samples were treated at 0, 1, 2, 4, 6, 8 mg / ml and cultured for 24 hours. After culturing for 24 hours, the cells were observed with a 100 × (0.25) PhP magnification using an inverted microscope (CK × 40-CPG30, Olympus, Japan).

As a result, as shown in Fig. 7A, when T4 cells as non-invasive (superficial) bladder cancer cells were treated with Comvatus at a concentration of 8 mg / ml, the number of cancer cells was decreased, but morphological changes were not observed. On the other hand, as shown in FIG. 7B, it was observed that when the citrus subsp. Subspecies were treated at a concentration of 6 and 8 mg / ml, the shape including the decrease of the number of cancer cells was changed.

Example  5-2: Kombucha  Citrus Kom Bucha  Of high-risk superficial bladder cancer cells (5637 cells)

To examine the cell morphology of the high-risk superficial bladder cancer cells (5637 cells) according to the concentration of the comvatus and citrus sub-aberration of the present invention, 6 × 10 ⁵ cells / well in a 6-well plate ) As the number of cells 5637 cells were subcultured, and the cells were subjected to the treatment with a solution of 0, 1, 2, 4, 6, and 8 mg / ml of each of Combacca and Citrus subspecies for 24 hours. After culturing for 24 hours, the cells were observed with a 100 × (0.25) PhP magnification using an inverted microscope (CK × 40-CPG30, Olympus, Japan).

As a result, as shown in FIG. 8A, when cancer cells were treated with high-risk superficial bladder cancer cells, 5637 cells, the number of cancer cells decreased in a concentration-dependent manner, but no significant morphological changes were observed. On the other hand, as shown in FIG. 8B, it was observed that when the citrus squam carcass was treated at a concentration of 8 mg / ml, the morphology was slightly changed, including the decrease in the number of cancer cells.

As a result of observing the cell morphology of bladder cancer according to the present invention, the concentration and adherence of cancer cells are lost by the treatment of high concentration (6 and 8 ㎎ / ㎖) of citrus fruits, And the morphological changes of cancer cells were observed.

Example  6: Kombucha, K and  Citrus Citrus Kombucha, CK  Evaluation of effect on bladder cancer cell mobility

Example  6-1: Kombucha  Citrus Kombucha Nasal invasion (Superficiality) Evaluation of effect on bladder cancer cell (T24 cell) mobility

In order to confirm the mobility of non-invasive (superficial) bladder cancer cells (T24 cells) according to the concentrations of the comb and carnitine secondary cells of the present invention, a scratch was performed to examine the wound healing phenomenon healing assay. At this time, the time point at which the cells were removed was 0 hours.

Specifically, in a 6-well plate, cells were seeded at 2 × 10 ⁵ cells / well T24 cells were dispensed and cultured until the cells reached a subconfluence state. When the cells reached the subconfluence state, the cells were scratched by scraping to the end of a 200 l pipet tip to remove the cells. Subsequently, the upper layer medium was removed from wells with scratched T24 cells. The supernatant of the supernatant was removed from the supernatant and the supernatant was added to the supernatant. The supernatant of the supernatant and the supernatant of the supernatant were added to the supernatant, and the supernatant was diluted to a concentration of 0, 1, 2, 4, 6 and 8 mg / ml. After 24 hours, changes in cell mobility were observed at a magnification of 40 × (0.13) PhP using an inverted microscope (CK × 40-CPG30, Olympus, Japan).

As a result, as shown in Fig. 9, when the T24 cells as non-invasive (superficial) bladder cancer cells were treated as a control group (no sample added, untreated sample treated, 0 mg / ml) However, it was confirmed that migration of cells was suppressed with increasing concentration of comb and carnitine. In particular, it was confirmed that the cell mobility was more remarkably suppressed when the citrus subtilis was treated at the concentrations of 4, 6 and 8 mg / ml compared to the comb (supernatant) (Fig. 9B).

Example  6-2: Kombucha  Citrus Kombucha  Evaluation of effects on high-risk superficial bladder cancer cell (5637 cell) mobility

In order to confirm the mobility of the high-risk superficial bladder cancer cells (5637 cells) according to the concentration of the comb bacterium and the citrus sub-bacterium of the present invention, a wound healing assay ) Were performed. At this time, the time point at which the cells were removed was 0 hours.

Specifically, in a 6-well plate, cells were seeded at 2 × 10 ⁵ cells / well 5637 cells were seeded and cultured until the cells reached a subconfluence state. When the cells reached the subconfluence state, the cells were scratched by scraping to the end of a 200 l pipet tip to remove the cells. Subsequently, the upper layer of the medium was removed from the wells containing the scratched 5637 cells. The supernatant of the supernatant was removed from the supernatant and the supernatant was added to the supernatant. The supernatant of the supernatant and the supernatant of the supernatant were added to the supernatant, and the supernatant was diluted to a concentration of 0, 1, 2, 4, 6 and 8 mg / ml. After 24 hours, changes in cell mobility were observed at a magnification of 40 × (0.13) PhP using an inverted microscope (CK × 40-CPG30, Olympus, Japan).

As a result, as shown in FIG. 10, when 5637 cells of high-risk superficial bladder cancer cells were treated as control (no sample added, untreated sample treated 0 mg / ml), migration of cells after 24 hours was significantly increased , But it was confirmed that migration of the cells was inhibited when the comb-carcass was treated at a concentration of 8 mg / ml. In particular, it was confirmed that the cell mobility was more remarkably suppressed in a concentration-dependent manner when Citrus quadratus was treated as compared to Comb (Fig. 10B).

As a result of the evaluation of the effect on the bladder cancer cell mobility of the above-mentioned Comvatus and Citrus subsp. Carcass, it was confirmed that both of the somatic invasive (superficial) bladder cancer cell T24 cells and the high-risk superficial bladder cancer cell 5637 cell Mobility was suppressed. In addition, it was found that the citrus comb subunit compared to comb secondary to bladder cancer cells significantly inhibited cell migration.

In addition, it was confirmed that T24 cells, which are non-invasive (superficial) bladder cancer cells, have better inhibitory effect on cell mobility than that of 5637 cells, which are high-risk superficial bladder cancer cells. Thus, it can be seen that the citrus comb secondary of the present invention can be advantageously used for the prevention or treatment of bladder cancer which is an early bladder cancer.

Example  7: Kombucha  Citrus Kom Bucha  Treatment of bladder cancer cells Apoptosis (apoptosis) effect analysis

To determine whether the inhibition of growth and cell morphology of bladder cancer cells by treatment with Kombucha (K) and Citrus Kombucha (CK) was due to apoptosis, the cells were treated with non-invasive (superficial) bladder cancer cells T24 cells and high-risk superficial bladder cancer cells, 5637 cells, were treated for 24 hours with 0, 4, 6, 8 mg / PE-Annexin V Kit was used to quantitatively determine the induction of apoptosis-induced apoptosis.

When Annexin V is apoptotic, the membrane structure is destroyed at an early stage and phospholipids such as phosphatidylserine (PS) exclusively inside the cell are exposed to the outside of the cell. Annexin V binds to them Moxi flow cytometry was used to confirm that the cells are in the early stage of apoptosis.

Example  7-1: Kombucha  Citrus Kom Bucha  By treatment Nasal invasion (Superficial) on bladder cancer cells (T24 cell) Apoptosis (apoptosis) effect analysis

In 6 well plates (6-well plate) to 2 × 10 ⁵ cells of the number / well (well) T24 cells were subcultured, and each of the combus and citrus subspecies samples were treated at 0, 4, 6 and 8 mg / ml and cultured for 24 hours. After culturing for 24 hours, the medium and cells were transferred to a tube and centrifuged at 1000 rpm for 2 minutes. Separated cells were washed with cold PBS (phosphate buffer saline), centrifuged again, and supernatant was removed. After removal of the supernatant, 1 × cold binding buffer was added to dilute the cells to 1 × 10 ⁵ cells. 5 μl of PE-Annexin V was added thereto and vortexed vigorously. After incubation at 25 ° C for 15 minutes, 400 μl of 1 × binding buffer was added and the degree of induction of apoptosis was measured by FACS (Moxi flow cytometry, ORFLO, Cambridge, USA).

As shown in FIG. 11, the T24 cells as non-invasive (superficial) bladder cancer cells were treated with the comb cells and the citrus comb cells at the concentrations of 0, 4, 6 and 8 mg / ml, respectively. As a result, Apoptosis was induced in the order of 4.47%, 4.07%, 6.4%, and 11.1%, respectively. In the case of treatment with Citrus subtilis, apoptosis was observed in 3.5%, 6.87%, 30.97% and 97.4% I could. Especially, when treated with Citrus squamous carcass at a concentration of 8 mg / ml, the apoptotic cell ratio was 97.4%, and apoptosis-induced apoptosis was significantly increased ( p <0.05).

Example  7-2: Kombucha  Citrus Kom Bucha  The high risk of treatment was also found in superficial bladder cancer cells (5637 cells) Apoptosis ( apoptosis ) Effect analysis

In 6 well plates (6-well plate) to 2 × 10 ⁵ cells of the number / well (well) 5637 cells were subcultured, and the combus carotid and citrus subspecies samples were treated at 0, 4, 6, and 8 mg / ml, respectively, for 24 hours. After culturing for 24 hours, the medium and cells were transferred to a tube and centrifuged at 1000 rpm for 2 minutes. Separated cells were washed with cold PBS (phosphate buffer saline), centrifuged again, and supernatant was removed. After removal of the supernatant, 1 × cold binding buffer was added to dilute the cells to 1 × 10 ⁵ cells. 5 μl of PE-Annexin V was added thereto and vortexed vigorously. After incubation at 25 ° C for 15 minutes, 400 μl of 1 × binding buffer was added and the degree of induction of apoptosis was measured by FACS (Moxi flow cytometry, ORFLO, Cambridge, USA).

As shown in FIG. 12, when the cells were treated with high-risk superficial bladder cancer cells, 5637 cells, at a concentration of 0, 4, 6 and 8 mg / ml, ) Were 3.87%, 5.57%, 6.73% and 7.83%, respectively, and 3.87%, 6.27%, 7.3% and 15.73% respectively when treated with Citrus squamous cell carcinoma ( p < 0.05).

Through the analysis of apoptosis effect on the bladder cancer cells by the treatment of the comb and carcinoma cells, the cell death rate of the comb cells in the non-invasive (superficial) bladder cancer cell T24 cells was 11 %, Respectively. On the other hand, in the case of treatment with Citrus squamous carcass at the concentrations of 6 and 8 mg / ml, apoptosis induced apoptosis induction was 38% and 97%, respectively. On the other hand, in both high-risk superficial bladder cancer cells 5637 cells, both the comb and the citrus comb subunits were found to be less than 11% in apoptosis-induced apoptosis regardless of treatment concentration. Thus, it can be seen that the citrus comb subcomponent of the present invention can be usefully used for the prevention or treatment of bladder cancer of nasally invasive (superficial) bladder cancer, which is an early bladder cancer.

Example  8: Kombucha  Citrus Kom Bucha  Treatment of bladder cancer cells Apoptosis (apoptosis) related protein

In order to examine the apoptosis-related proteins and genes of bladder cancer cells by Kombucha (K) and Citrus Kombucha (CK), proteins were separated by size and then subjected to antigen-antibody reaction Western blot analysis was performed on the principle of detecting only specific proteins in whole proteins. The types of antibodies used in the Western blot analysis are shown in Table 4, and β-actin is a mixture of T24 cells of bladder cancer cells treated with Combusa and citrus subsp. And used as a control (internal control).

Specifically, T24 cells as non-invasive (superficial) bladder cancer cells and 5637 cells as high-risk superficial bladder cancer cells were divided into Petri dishes at a cell number of 1 × 10 ⁶ cells / ml, Sub-samples were treated at 0, 4, 6, and 8 mg / ml, respectively, and cultured for 24 hours. After culturing for 24 hours, the cells treated with the comb and the citrus sub-samples were collected using a scraper. The recovered cells were washed with phosphate buffered saline (PBS). After washing, the cell lysate was added with protein lysis buffer to each cell sample, and the cells were disrupted and centrifuged at 1000 rpm for 2 minutes. The protein of each supernatant obtained after centrifugation was quantitated using BCA (bicinchoninic acid) and used as a sample.

The proteins in each of the supernatants obtained above were loaded equally into 12% SDS-PAGE (12% gradient sodium dodecyl sulfate polyacrylamide gel electrophoresis) gel and proteins were separated for 3 hours. The separated proteins were transferred to a polyvinylidene fluoride (PVDF) membrane of 0.45 μm size for 2 hours and then the membrane was blocked with 5% skim milk blocking. After completion of the blocking, the membrane was washed with TBS-T (Tris Buffered Saline-Tween 20), and the primary antibody was reacted overnight at 4 ° C. After the overnight reaction, the membrane was washed with TBS-T 3 times for 10 minutes and the secondary antibody reacted for 1 hour. After secondary antibody reaction, the cells were reacted with ECL solution (ECL (enhanced chemiluminescence) -solution) and analyzed using a protein ECL image analysis system (Fusion-FX7 Advance, VILBER, Europe) About The degree of apoptosis related protein expression was measured.

As a result, as compared with the case of treatment with comb adjunct as shown in FIG. 13, when T24 cells, which are non-invasive (superficial) bladder cancer cells, were treated with citrus combs at a concentration of 6 and 8 mg / The expression of Bcl-2, an anti-apoptotic protein, was decreased and the expression of Bax, a pro-apoptotic protein, was not significantly different. In addition, the expression of the active cleaved caspase-3, cleved caspase-8 and cleved caspase-9 was increased in a concentration-dependent manner, Expression of the inactive forms of Pro caspase-3, Cleved caspase-8 and Cleved caspase-9 was decreased in a concentration-dependent manner, and caspase-3 it was confirmed that the cleaved PARP (cleaved PARP) expression was increased by cleavage of the tumor-promoting complex protein (PARP, poly-ADP ribose polymerase) by caspase-3. On the other hand, the apoptosis-related protein did not show any difference in expression when treated with combat.

As shown in FIG. 14, the expression levels of the apoptosis-related proteins in the high-risk superficial bladder cancer cells, 5637 cells, were compared with those of the control group. There was no difference in expression between the two groups.

In this study, we investigated the effect of citrus juice supplemented with citrus juice on the apoptosis of caspase-mediated T24 cells, a non-invasive (superficial) bladder cancer cell with low malignancy, compared with 5637 cells with high-risk superficial bladder cancer cells apoptosis), it can be seen that the citrus comb secondary of the present invention can be usefully used for the prevention or treatment of bladder cancer, which is an early bladder cancer.

In summary, through the above results, it was possible to confirm the antioxidant ability and the total polyphenol content of citrus squam carcinoma cultured with the addition of citrus juice, compared with that of the common squid carcinoma, and the growth of T24 cells as a non-invasive (superficial) bladder cancer Inhibition and regulation of apoptosis - inducing protein expression, it was found that the citrus subspecies can inhibit the progression of early bladder cancer and is effective for prevention and treatment.

Example  9: Statistical processing

The experiments performed in the present invention were repeated 3 times or more independently. The results of the experiments were analyzed using t -test analysis and ANOVA analysis using the SPSS Version 18.0 package program for statistical analysis The significance of the treatment group was verified using Dancan's multiple range test at P = 0.05 level.

Claims (5)

The broth containing the pellicle and the green tea extract and the sugar were pre-cultured and then the culture broth of the origin kombucha and the pellicle, citrus juice and acetate (superficial) bladder cancer comprising the citrus comb secondary fermentation broth as an active ingredient, which is cultivated by adding acetic acid (acetate).
delete The pharmaceutical composition according to claim 1, wherein the prophylactic or therapeutic treatment of non-invasive (superficial) bladder cancer is achieved by inducing apoptosis of cancer cells.
delete The broth containing the pellicle and the green tea extract and the sugar were pre-cultured and then the culture broth of the origin kombucha and the pellicle, citrus juice and acetate (superficial) bladder cancer, comprising the citrus comb secondary fermentation broth as an active ingredient.

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