WO2017086572A1 - Kimchi exhibiting preventive and therapeutic activity against helicobacter pylori caused disease - Google Patents

Abstract

The present invention provides kimchi exhibiting a preventive and therapeutic activity against Helicobacter pylori caused disease, comprising a Chinese pepper, a pear, a sea tangle or sea tangle extract, a mushroom or mushroom extract, and any one selected from the group consisting of leaf mustard leaves, mustard leaves and a mixture thereof. It has been shown that when kimchi according to the present invention is consumed, the mutational tendency of Helicobacter decreases, and in the case of prolonged use, the progression of atrophic gastritis caused by the Helicobacter can be improved and thus Helicobacter-related gastric cancer can be prevented and treated.

Description

Kimchi exhibits preventive and therapeutic activity against Helicobacter pylori causative diseases

The present invention relates to kimchi which is a fermented food exhibiting prophylactic and therapeutic activity against Helicobacter pylori causative disease.

In 1983, Australian microbiologists Marshallall and Warren were the first to discover flexible bacteria in the gastric mucosal biopsy tissue of patients with type B gastritis, and then cultivated the bacteria to be a new species associated with the campylobacter genus. Since its discovery, active research has been conducted on the link between Helicobacter pylori and upper gastrointestinal diseases such as gastritis and peptic ulcer. Helicobacter pylori is a Gram-negative bacillus with a thin membrane-enclosed flagella, which is known to release large amounts of urease and propagate through the stool-oral route.In 1994, the World Health Organization (WHO) was a definite carcinogen. It is a pathogen that turns out to be a factor and attracts worldwide attention.

Currently, the pathogenesis of the disease caused by Helicobacter pylori is a large amount of urease, as bacteria entering the gastric lumen along with food penetrate the gastric mucus layer using strong motility flagella and inhabit the upper epithelial cell layer and the junction between cells in the gastric mucus layer. It has been found that the mucous layer is made alkaline by inducing the gastric acid secretion promotion by gastrin abnormally, resulting in inflammation and ulceration.

In addition, according to a recent study that found that Helicobacter pylori is an important risk factor for gastric adenocarcinoma, the International Health Organization designated Helicobacter Pylori as the first group carcinogen.

Infection with Helicobacter pylori has been reported to occur particularly frequently in developing countries, including Korea, and according to Baek et al., It is estimated that more than 80% of normal Koreans are infected with Helicobacter pylori (Baek Seung-cheol et al. Journal, 25: 45-52, 1990). Therefore, the development of treatment and prevention methods for Helicobacter pylori infection is very urgent.

Currently, chemotherapy using antibiotics is mainly used to treat diseases caused by microbial infections. The widespread use of this method may be effective in the short term, but the emergence of antibiotic resistant strains due to the continuous use of antibiotics. There is a problem that there is a risk.

As an antimicrobial therapy for Helicobacter pylori, Rauws et al. Reported the therapeutic results by administering three antimicrobial agents, bismuth, amoxicillin and metronidazole (Rauws EAJ, Langenberg W, Houthoff JH, Zanen HC and Tytgat GHJ, Gastroenterol, 94: 33 ~ 40, 1988), and Borody et al. Reported bi-muth preparations, tetracycline and metronidazole in patients with peptic ulcers and reported about 80% of their results (Borody T, Lynne). j anew Moore-Jones D, Gastroenterol, 98: A24, 1990), from which the combination of these three antimicrobial agents is most commonly used for the treatment of Helicobacter pylori infection.

However, various side effects such as diarrhea, reflux esophagitis, suppressing normal flora in the intestine, and emergence of resistant strains have been reported due to continuous use of antibacterial therapy.

Recently, there has been a growing interest in the function of lactic acid bacteria fermentation materials in the prevention and treatment of gastrointestinal diseases, and in particular, many studies on probiotic Lactobacillus that can inhibit the growth of human pathogens have. These lactic acid bacteria produce lactic acid, acetic acid, and hydrogen peroxide as fermentation products, as well as antibacterial substances associated with bacteriocins.

Marie-Helen Coconier et al. Cultivated Lactobacillus acidophilus strain LB ( LB ) in humans that produce antimicrobial agents against Helicobacter pylori in vitro, and the supernatant of this culture was applied to human intestinal cells in vitro. In addition to inhibiting adhesion and inhibiting the activity of urease, H. felis was confirmed to inhibit the infection of mice (Marie-Helen Coconier et al., Environ.Microbiol, Nov. .4573-4580, 1998).

In addition, Yuji Aida MT et al confirmed that the infection of Helicobacter pylori to sterile murine is inhibited by oral administration of L. salivarius , US Patent No. 5,578,392 (Lactobacillus Johnson Ni ( L. johnsonii ) describes a function that inhibits the adhesion of Helicobacter pylori to intestinal cells.

In addition, Canducci et al. Prescribe the inactivation of human Lactobacillus ecidophilus culture in combination with antimicrobial therapy using labeprazole, clarithromycin, amoxycillin, and the likelihood of eradication of Helicobacter pylori. Increasing results were obtained (Canducci F. et al., Aliment Pharmacol. Ther, 14: 1625-1629, 2000).

In addition, Korean Patent Application No. 10-1999-0040387 discloses Lactococcus sp., A lactic acid bacterium that exhibits inhibitory activity against Helicobacter pylori with an antibody against Helicobacter pylori. Food products containing HY 49, Lactobacillus casei HY 2782, Bifidobacterial long gum HY 8001 and the like are described.

However, the research on how to prevent or treat diseases caused by Helicobacter pylori infection through frequently consumed foods is not complete and is still ongoing.

The problem to be solved by the present invention is to secure the safety as a traditional Korean fermented food that has undergone lactic acid fermentation process using salt, spices and other condiments, and improve the composition of Kimchi, which can be defined as a Korean probiotic food, Helicobacter pylori It is intended to exhibit prophylactic and therapeutic activity against causative diseases.

In order to achieve the above technical problem, the present invention

Providing kimchi exhibiting prophylactic and therapeutic activity against Helicobacter pylori causative diseases, including any one selected from the group consisting of mustard leaf, mustard leaf, and mixtures thereof, acid, pear, kelp or kelp extract, and mushroom or mushroom extract do.

Kimchi showing prophylactic and therapeutic activity against Helicobacter pylori causative diseases according to the present invention is 1.0 to 10.0% by weight, any one selected from the group consisting of the fresh leaf, mustard leaf and mixtures thereof with respect to the total weight of kimchi To 0.05 to 0.5% by weight, 1.0 to 5.0% by weight of the pear, 1.0 to 5.0% by weight of the kelp or kelp extract, may contain 1.0 to 5.0% by weight of the mushroom or mushroom extract.

 In kimchi exhibiting prophylactic and therapeutic activity against Helicobacter pylori causative disease according to the present invention, it is preferable that the mushroom is an extract solvent of shiitake mushroom, kelp extract and mushroom extract.

In addition, Lactobacillus plantarum ( Lactobacillus plantarum ), Leuconostoc mesenteroides ( Luconostoc mesenteroides ) as a starter (starter) in order to manufacture kimchi showing the preventive and therapeutic activity against the Helicobacter pylori causative diseases according to the present invention and these Any one selected from the group consisting of a mixture of may be used.

In addition, the kimchi according to the present invention exhibits a particularly prophylactic and therapeutic activity is gastritis, gastric ulcer, gastric cancer among Helicobacter pylori causative diseases.

The anticancer kimchi according to the present invention is any one selected from the group consisting of mustard leaf, mustard leaf, and mixtures thereof, and the herbaceous, pear, kelp or kelp extract, and mushroom or mushroom extract are added to the original kimchi recipe to reduce the mutation tendency of Helicobacter For a long time, it could improve the progression of atrophic gastritis caused by Helicobacter and prevent and treat Helicobacter related gastric cancer.

Figure 1 shows the processing of kimchi for use in in vitro cells and in vivo animal model.

Figure 2 shows the biological response in the in vitro Helicobacter infected cell model of normal kimchi and anti-cancer kimchi.

Figure 3 shows the improvement effect (24 weeks after Helicobacter infection) in chronic atrophic gastritis due to Helicobacter infection.

Figure 4 illustrates the molecular biological mechanisms for anti-inflammatory action and regeneration of anti-cancer kimchi.

Figure 5 illustrates the prevention of Helicobacter induced gastric tumor formation with long-term intake of anti-cancer kimchi.

Figure 6 illustrates the molecular biological mechanisms that explain the cancer prevention effect of anti-cancer kimchi.

Hereinafter, the present invention will be described in detail.

As a dietary adjustment for Helicobacter pylori infection, we newly studied the recipe of anti-cancer kimchi (cpKimchi), and using a rat model, dietary adjustment of anti-cancer kimchi is typically used to prevent and treat Helicobacter pylori-causing diseases such as gastritis, gastric ulcer and gastric cancer. Hypothesis can be made, and anticancer kimchi was prepared by adding the following five kinds of generalized kimchi.

Five things are added

First, any one selected from the group consisting of mustard leaf, mustard leaf, and mixtures thereof,

Second, Sancho,

Third,

Fourth, kelp or kelp extract,

Fifth, mushroom or mushroom extract

to be.

As described above, the content of the five additional ingredients is not particularly limited, but the pharmacological effect of kimchi should be considered as well as the taste as food. Any one selected from the group consisting of leaves and mixtures thereof 1.0 to 10.0% by weight, 0.05 to 0.5% by weight of the herbaceous acid, 1.0 to 5.0% by weight of the pear, 1.0 to 5.0% by weight of the kelp or kelp extract, the mushroom or mushroom Kimchi is prepared so that the extract contains 1.0 to 5.0% by weight.

In addition, the mushroom can be used without any particular limitation as long as the mushroom can be used for edible production of kimchi using shiitake mushrooms as a result of various tests of the present inventors is effective in preventing and treating the taste of kimchi and Helicobacter pylori causative disease.

And the mushrooms and kelp can be used as such, by extracting the active ingredient with water may be used an extraction solution in which the active ingredient is dissolved.

When preparing kimchi according to the present invention is fully effective in the production by natural fermentation without the inoculation of the separate kimchi lactic acid bacteria, but contains a lot of kimchi lactic acid bacteria having resistance to gastrointestinal digestive enzymes and gastric acid and bile acid, among various kimchi lactic acid bacteria The effect can be doubled when selected from the group consisting of Lactobacillus plantarum , Leuconostoc mesenteroides and mixtures thereof, which have been found to have excellent resistance from various tests. Kimchi may be prepared by further including any one as a starter.

Kimchi according to the present invention shows an excellent preventive and therapeutic effect against Helicobacter pylori causative disease, but in the specific gastritis, gastric ulcer, gastric cancer.

Hereinafter, specific details will be described in order to practice the present invention. First, the present invention will be described with reference to the manufacturing process, test method, statistics, and the like as the introduction of the description.

<Kimchi Preparation: Anti-cancer Kimchi (CPKimchi) Production>

Kimchi's preparation was based on the generalized kimchi recipe (sKimchi) of Pusan National University Kimchi Research Institute. Ingredients and contents of the general kimchi (sKimchi) and the anticancer kimchi (cpKimchi) according to the present invention is a control as shown in Table 1 below.

As shown in Table 1 below, general kimchi is made of salted cabbage, red pepper powder, garlic, ginger, anchovy sake, radish, green onion, and some sugar, and it is prepared by fermenting for a while to produce kimchi lactic acid bacteria such as L. plantarum. It was.

In addition, in addition to these essential components for the production of general kimchi (sKimchi) was added to the fresh leaf, pepper, pear, shiitake mushroom extract, kelp extract was prepared in anti-cancer kimchi (cpKimchi) according to the present invention. Taste and appearance could not be distinguished between regular kimchi and anticancer kimchi.

The shiitake mushroom extract was prepared by placing 5 pieces of shiitake mushrooms in 1 l of water, and then left overnight, followed by heating and filtering for about 5 seconds to remove the shiitake mushrooms, and using the filtered extract, and the kelp extract was 2 × 3 cm ^2. Five pieces of kelp of size were placed in 1 L of water and left overnight, followed by heating for about 5 seconds and then filtered to remove the kelp.

ingredient	General kimchi	Anticancer kimchi
cabbage	100.0	100.0
Chili Powder	3.5	2.5
garlic	1.4	2.8
ginger	0.6	0.6
Anchovy fish sauce	2.2	-
radish	13.0	11.0
wave	2.0	2.0
Sugar	1.0	1.0
Leaf	-	7.5
Sancho	-	0.1
stomach	-	2.8
Shiitake and Kelp Extract	-	5.0
Final salinity (%)	2.5	2.2

In Table 1, the content of each component is a relative weight ratio when the weight of the Chinese cabbage is 100, shiitake mushroom and kelp are put together in water and used to extract the weight ratio of anti-cancer kimchi according to the invention 5: 5 It can be seen that it contains.

In addition, in Table 1, without using mustard leaves, only fresh leaves were used.Gad is a perennial herb of the dicotyledonous poppy family Mustardaceae, and it is a variant of mustard. Practically, the mustard and mustard are freezing, but they have the same effect.

Processing of Kimchi for Use in In Vitro Cell and In Vivo Animal Models

The two kimchi prepared above was freeze-dried and made into fine kimchi powder, then stirred overnight for 20 times with methanol (methanol) to prepare a kimchi methanol extract, these were concentrated by evaporation (BRE 111 rotavapor, Switzerland) 4 Store at ° C and use for testing.

In general, the amount of kimchi is about 30g-100g / day according to individual taste, and about 90% of general kimchi is composed of water, so in this test, the volume of kimchi was reduced by freeze drying and evaporation. Kimchi and Anticancer Kimchi was mixed daily with two bottles of drinking water, 1.7g / kg / day and 5.0g / kg / day, equivalent to the usual Kimchi intake for animals. In addition, anti-cancer kimchi and general kimchi extract was dissolved in 2.5mg / ml, 5mg / ml, 7.5mg / ml in order to perform in vitro experiments.

The machining process as described above is shown in FIG. 1.

<Bacteria culture>

The Helicobacter pylori strain ATCC 43504 (American Type Culture Collection, strains cag A + and vac A s1-m1) was used in in vitro models, and the Sydney strain (suitable for infection with SS1, cag A + and vac As2-m2 mice) was used in vitro. Used for model.

Helicobacter pylori 3 at 37 ° C. under aerobic conditions (BD GasPaK EZ Gas Generating Systems, BD Biosciences) in BBL's Trypticase soy (TS) agar medium (TSAII; BD Biosciences, Franklin Lakes, NJ) with 5% sheep blood Incubated for days. Bacteria were harvested from clean TS medium and centrifuged at 3000 X g for 5 minutes and resuspended in medium to give a final concentration of 10 ⁹ colony-forming units (CFUs) / ml. All experiments used cultures grown for 72 hours in TS agar medium.

Mice infected with Helicobacter pylori

Animals: Five-week-old female C57BL / 6 rats (Charles River Japan) fed commercially available sterile granulated feed (Biogenomics, South Korea), sterile water was allowed to drink freely, and the air conditioner was maintained at a temperature of 24 ° C. Live in a biohazardous space equipped. One week after adaptation, 20 mg / kg pantoprazole was injected three times to lower gastric acid to form a Helicobacter pylori colony. Each rat was then inoculated into the stomach using a gastric intubation needle in an equal volume (0.1 ml) of Helicobacter pylori suspension containing 10 ⁹ CFUs / ml.

All groups received Helicobacter pylori four times a week. Ten rats were injected with clean TS medium to remove uninfected groups. To collect worse data, rats were fed a special grain diet based on AIN76 containing 7.5% NaCl for 4 weeks. Thereafter, mice that positively responded to Helicobacter pylori were randomly divided into four groups (n = 20). In all infected rats, grain feed AIN76 containing 7.5% NaCl was given for 24 and 36 weeks to promote Helicobacter pylori-induced carcinogenesis. All animal studies were conducted according to a plan approved by the Institutional Animal Care and Use Committee (IACUC) at the Cha Cancer Institute of the Secondary School after IRB approval. The stomach was removed and further biopsies, ELISA, Western blotting, RT-PCR were performed.

Total figures: After killing the rats, the detached stomach was incised along a large curvature and washed with saline cold ice. The mucosal layer of the stomach was photographed with a digital camera to investigate the extent of total mucosal damage, and a portion of the mucosa was immediately fixed in 10% formalin solution. The total damage to the gastric mucosa was assessed by three gastroenterologists who did not know what treatment was done using the gross ulcer index.

Their lesion types were defined as follows;

Type I, edema, hyperemia, or the presence of a single pointed bleeding under the mucosa;

Type II, the presence of hemorrhagic lesions under the mucosa with mild gastritis;

Type III, presence of deep ulcers and invasive lesions with gastritis.

Histopathology: The stomach was fixed in 10% neutralized formalin for pathological analysis, processed using standard methods and placed in paraffin. The slices were then stained with hematoxylin and eosin (H & E). Histologic examination of the gland mucosa and gastric cavity of the stomach was performed. Pathological changes in Helicobacter pylori infections, such as inflammatory cell invasion, sore lesions, ulcers, dysplasia, and adenoma formation (pre-cancerous lesions), do not know what treatment was done using the index of histologic injury. Classified by three gastroenterologists.

In this study, inflammation was defined as the score of invasion of inflammatory cells.

0: nothing,

1: below the mucosa-specific layer,

2: half of the mucosa,

3: up to epithelial gland layer (all mucous membranes). act

Atmosphere was defined as the percentage of sores lesions.

0: nothing,

1: loss of epithelial gland (1/3 part),

2: 2/3 part (2/3 part) of mucous membrane,

3: all mucous membranes (3/3 part)

Weston Blot (Western blot) analysis: The extracted stomach tissue with PBS, washed twice, and then, 1mM of phenylmethylsulfonyl fluoride cold cell lysis solution containing (PMSF, Sigma Aldrich, St Louis , MO) (Cell Signaling Technology, Denver, MA )). After reacting for 20 minutes, the samples were centrifuged at 10,000 X g for 10 minutes.

The supernatants were then collected, and the proteins in the lysate were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and polyvinylidene fluoride (polyvinylidene). fluoride) (PVDF) membrane, which was reacted with the primary antibody, washed with water, reacted with a peroxidase-conjugated secondary antibody, and washed again. This was then visually confirmed using an enhanced chemiluminescence (ECL) system (GE Healthcare, Buckinghamshire, UK).

Immunohistochemistry (Immunohistochemical staining): removing the paraffin block, and then rehydrated with alcohol (alcohol), the organization side, the heat for 10 minutes in the microwave in a pressure bottle filled with citric acid (citrate) solution of 10mM / L It was. The slides were cooled in water for 15 minutes and washed with PBS. The slides were reacted with the primary antibody overnight. After the reaction, the subsequent reaction was used VECTOR kit (Vector Laboratories, Inc, Burlingame, CA). Finally, the slides were reacted with 3,3′-diaminobenzidine (Invitrogen Life Technologies) and counter-stained with hematoxylin (Sigma-Aldrich). The number of cells that responded positively to the antibody was determined at five sites of randomly selected mesothelial cells in each rat, and observed at a magnification of 100 times. Values are given as mean-to-average error.

PAS staining: Histochemical staining of glycoconjugate for staining Periodic acid and Schiff was performed in the dark for 20 minutes using 2% Periodic acid and Schiff (PAS) reagent. It was performed by the method of Pandurangan.

This was concluded with a PAS staining score between 10 (good protection) and 0 (low protection).

TUNEL Analysis: For detection of apoptosis, the stomach tissues were stained by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) method using DeadEnd ™ Fluorometric TUNEL system (G3250 #, Promega, USA).

In vitro cell model infected with Helicobacter pylori

Cell culture and cell toxicity (cytotoxicity) Analysis: gastric epithelial cell line (RGM-1) in rats is H. Matsui (. Univ Tsukuba, Japan) received from Professor, AGS cells (DNA that can be proven to be appropriate management capabilities and rarity Purchase from ATCC (Manassas, VA). After resuscitation in the laboratory, all cells were not used for more than six months. AGS cells were cultured in RPMI-1640 medium (Gibco BRL, Gaithersburg, MD) and RGM-1 cells were cultured in DMEM medium.

All media were added with 10% fetal bovine serum (Gibco BRL) at 37 ° C., 5% CO ₂ . Cell viability was evaluated using the MTT colorimetric assay.

MTT [3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazoliumbromide] was purchased from Sigma Chemical Company (St. Louis, MO). The filtrate (50g) was mixed with 1L of water and lyophilized. Cells were placed in 96-well plates at a concentration of 10 ⁴ cells / ml and allowed to attach for 24 hours. Kimchi extract was used in experimental wells at various concentrations for 24 hours.

Observation of cell migration in the image of living cells: AGS cells treated according to the concentrations of normal kimchi and anti-cancer kimchi extract were scraped with a pipette tip and 18 hours of cell migration under ScopeTek MDC200 (CHA University, Seoul, Korea) Observed until. Observations were made in three groups: no treatment, normal kimchi concentration, and anticancer kimchi concentration. The average rate of cell migration was calculated according to the group with the picture after 18 hours, showing the average value of cell migration.

ELISA analysis: The cells were harvested and homogenized in 10 mM sodium phosphate solution at pH 7.4 (1 ml). After centrifugation at 9000 × g, the PGE ₂ levels of the supernatants were measured by ELISA, and the concentrations were expressed in pg / mg protein. The treatment was performed according to the original Prostaglandin E ₂ express EIA kit (Cayman, Ann Arbor, MI).

HO-1, Bax , PARP , Western for cleaved capspase - 3 Blot (Western blot): After washing twice the extracted cells with PBS, dissolved in cold cell lysis solution containing phenylmethylsulfonyl fluoride (PMSF, Sigma Aldrich, St Louis, MO) in 1mM (Cell Signaling Technology, Denver, MA) It was. After reacting for 20 minutes, the samples were centrifuged at 10,000 X g for 10 minutes before collecting the supernatant.

Proteins in the lysates were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. It was reacted with the primary antibody, washed with water, reacted with a secondary antibody bound to peroxidase, and washed again. It was then visually observed using an enhanced chemiluminescence (ECL) system (GE Healthcare, Buckinghamshire, UK).

Statistical analysis

The results are expressed as mean standard deviation (SD). Statistical analysis was performed using GraphPad Prism (GraphPad Software, La Jolla, CA, USA) and SPSS software (version 12.0; SPSS Inc., Chicago, IL, USA). Statistical significance between groups was determined by the Mann- Whitney U test, and statistical significance was recognized at p <0.05 .

Next, the preventive and therapeutic effects of the anticancer kimchi according to the present invention will be described through the test results.

<Result>

< Differences in Biological Functions between Normal Kimchi and Anticancer Kimchi in Helicobacter-Infected Cell Models>

Figure 2 illustrates the biological response in the in vitro Helicobacter infected cell model of normal kimchi and anti-cancer kimchi will be described with reference to this.

Two types of gastric cancer cells, AGS cells (left of FIG. 2A) and unmodified gastric epithelial cells, RGM-1 cells (right of FIG. 2A), were extracted with 1, 2.5, 5, and 7.5 mg of normal kimchi and anticancer kimchi, respectively. Four concentrations of / ml were administered, and the cell viability was confirmed according to the concentration.

Kimchi extract concentrations of 5 mg / ml or more showed significant apoptosis in gastric cancer cells ( p <0.05 ), but no apoptosis in all normal gastric cells. Anti-cancer kimchi extract in gastric cancer cells showed a superior effect compared to the general kimchi extract ( p <0.01 ). Similar results were obtained with other gastric cancer cell lines, MKN28 and SNU-719.

The question arises whether the anti-cancer kimchi extract selectively causes apoptosis only in gastric cancer cells and the apoptosis does not occur in gastric normal cells, suggesting that the anti-cancer kimchi extract has superior and selective cell death effects only in cancer cells. To demonstrate the expression of HO-1 showing a cell protective effect after 6 hours and 18 hours, respectively (Fig. 2B).

The expression of HO-1 (heme oxygenase-1) was significantly increased in gastric normal cells and gastric cancer cells treated with anticancer kimchi extract, and HO-1 expression was more prominent in normal gastric cells. The expression of HO-1 also appeared after the general kimchi extract treatment, but was less than the anticancer kimchi extract. Apoptosis of anti-cancer kimchi extract was assumed to be attenuated by vigorous induction of cytoprotective genes in cells without cancer. It was hypothesized that the increased apoptosis only in cancer cells after the anticancer kimchi extract treatment was related to the selective apoptosis of the anticancer kimchi. For this, the expression of Bax and cleaved capspase-3 was confirmed (FIG. 2C).

As shown in FIG. 2C, the anticancer kimchi extract showed increased expression of Bax and cleaved capspase-3 in gastric cancer cells compared to the general kimchi extract. As expected, anti-cancer kimchi extracts showed no increase in Bax expression in gastric normal cells.

In addition to the above experimental results, the anti-cancer kimchi extract selectively induces apoptosis only in cancer cells, it was demonstrated by a cell healing assay to determine the cell migration restriction associated with anti-tumor development, an ability imposed on the anti-cancer kimchi extract. (FIG. 2D).

As shown in Fig. 2D, after 18 hours, the wound disappeared due to active cell migration in the control gastric cancer cells, but 50% at the concentration of 5mg / ml of general Kimchi extract and 35% at the concentration of 7.5mg / ml Only cells migrated ( p <0.005 ), which showed superior cell migration inhibition compared to the control ( p <0.01 ).

However, at the concentration of 5mg / ml anticancer kimchi extract, only 30% of the cells migrated at a concentration of 30% and 7.5mg / ml ( p <0.005 ), and the anticancer kimchi extract treatment showed more delayed cell migration. It showed better cell migration inhibition. Interestingly, the anti-cancer kimchi showed no delayed cell migration in the normal cells of the stomach, as described above.

It was found that anti-cancer kimchi reduced the expression of inflammation-related factors such as Helicobacter-induced COX2 and TNF-α (FIG. 2E). Long-term administration of anti-cancer kimchi may be a significant rescue measure for Helicobacter-induced gastric injury, including tumor development.

Regeneration Effect and Mechanism of Anticancer Kimchi in Chronic Atrophic Gastritis Caused by Helicobacter Infection (24 Week Model)

Intake of anti-cancer kimchi for 24 weeks in mild chronic gastritis

Figure 3 shows the improvement effect (24 weeks after Helicobacter infection) in chronic atrophic gastritis due to Helicobacter infection, referring to the high-saline diet to C57BL / 6 mice infected with Helicobacter induced chronic atrophic gastritis (FIG. 3A). This resulted in gastritis lesions, erythema of the gastric mucosa, changes in the nodular mucosa and gastric mucosa in the Helicobacter infected group (Fig. 3B).

Gross lesion score was significantly reduced by administration of anticancer kimchi extract ( p <0.01 , FIG. 3C). As shown in Figures 3B and 3D, chronic atrophic gastritis was prominent in 24 weeks of Helicobacter infection, showing a decrease in gastric mucosa cells and inflammatory cells such as monocytes, lymphocytes, and macrophages replacing gastric mucosal and gastritis mucosal changes. .

However, the above change was decreased in the anti-cancer kimchi extract group treated with 5 mg / kg ( p <0.05 ). The excellent result is based on the regeneration effect of anticancer kimchi.

Anti-inflammatory and Regeneration Effects of Anticancer Kimchi

Figure 4 illustrates the molecular biological mechanisms for the anti-inflammatory action and regeneration of anti-cancer kimchi will be described based on this.

Increased expression of COX-2 after infection with Helicobacter is one of the important inflammatory mechanisms, as shown in FIG. 4A, the expression of COX-2 was significantly increased in the control group ( p <0.01 ). In Helicobacter infection, immunostaining of F4 / 80 antibody increased macrophages and monocytes. However, the expression of COX-2 was decreased in the group treated with anticancer kimchi extract ( p <0.01 ).

After macrophage invasion, NF-kB p65 immunostaining was performed because inflammatory regulators were involved in oxidative-reduction transcriptional activation, whereas the control group found significantly increased NF-kB expression in Helicobacter-infected gastric mucosa. , The expression was significantly decreased in the group treated with anticancer kimchi extract ( p <0.01 ).

In Western blot analysis of gastric mucosal COX-2 expression, COX-2 was significantly increased after Helicobacter infection ( p <0.01 ), but COX-2 expression was significantly decreased in the anti-cancer kimchi extract treated group. ( p <0.05 ).

IL-6 and STAT3 activity was significantly increased in the control group, but IL-6 and STAT3 activity was significantly decreased in the anti-cancer kimchi-treated group ( p <0.05 ).

PGE2, a product of Cox-2, was measured by ELISA, and gastric mucosal PGE2 concentration was significantly reduced in the group treated with anticancer kimchi extract at 5 mg / ml ( p <0.01 ).

The oxidative stress associated with NF-kB activation was determined by the concentration of MDA, a lipid peroxide, which was significantly increased due to Helicobacter infection, but the concentration of MDA could be significantly decreased by anticancer kimchi extract. p <0.05 ).

Expression of HO-1 and HSP70, a representative antioxidant protein, was measured, respectively. As a result, the expression of HO-1 and HSP70 was significantly increased in the anti-cancer kimchi-treated group ( p <0.05 ).

Lastly, gastric neutral mucin was decreased by atrophic gastritis changes or chronic Helicobacter infection, which was significantly decreased after Helicobacter infection by PAS staining. However, mucin levels were not decreased in the anticancer kimchi extract group. ( P <0.01 ).

Helicobacter induced by long-term intake of anticancer kimchi Stomach tumor  Formation Prevention (36 Week Model)>

< Anticancer due to intake of anticancer kimchi for 36 weeks>

FIG. 5 illustrates the prevention of Helicobacter induced gastric tumor formation by prolonged intake of anticancer kimchi, which will be described with reference to this.

A 36-week animal model experiment was conducted to study the effect of anticancer kimchi on tumor formation.

Although gastric pathology associated with Helicobacter significantly reduced the weight of rats after about 7 weeks after Helicobacter infection, statistically significant reductions in weight loss from 2 weeks after and 29 weeks ( p <0.001 ) were found to be 5 mg / kg. Concentrations of anticancer kimchi extract were identified in the group administered ( p <0.05 ).

A model infected with Helicobacter spp. And a high salinity diet, as shown in Figures 5C and 5D, produced a significant degree of chronic atrophic gastritis as well as significant gastric tumors at 36 weeks, with changes in nodular mucosa, thinned gastric mucosa and adenoma polyps. Tumor lesions were found with the formation of central ulcers. In pathological confirmation, the lesions are those seen in severe chronic atrophic gastritis, gastric ulcer, pericarditis gastritis, adenoma, adenocarcinoma.

The total gross lesion index was significantly increased in the control group, but significantly decreased in the group receiving the anticancer kimchi extract ( p <0.05 ). Similar results were also found in gross appearance from pathological score analysis ( p <0.05 ).

The Helicobacter infection model showed significant tumor formation in the overall identification of the control group, which proved to be adenocarcinoma and gastric adenocarcinoma, but significantly reduced gastric tumor formation in the group receiving the anticancer kimchi extract ( p <0.01 ).

Mechanism of Cancer Prevention Effect of Anticancer Kimchi

6 illustrates a molecular biological mechanism for explaining the cancer prevention effect of anti-cancer kimchi, which will be described with reference to this.

The control group infected with Helicobacter pylori showed a significant increase in the expression of COX-2 and F / 80 after 24 weeks. Focusing on COX-2, COX-2 mRNA expression, as well as COX-2, was significantly increased in Group II infected with Helicobacter pylori, but significantly decreased in Groups III and IV fed anti-cancer kimchi ( p < 0.01 ).

In addition, macrophage invasion was significantly increased in the Helicobacter infection group ( p <0.01 ), but decreased in the group receiving the anti-cancer kimchi ( p <0.05 ).

Western blot and RT-PCR analysis of musocal COX-2 expression also showed a significant decrease in the anti-cancer kimchi-treated group, unlike the Helico-infected group.

As a result, COX-2 increased in the Helicobacter-infected group, but decreased in the group treated with anti-cancer kimchi, and decreased IL-1β, VEGF, and IL-6 in the RNA and protein expression levels. Inflammation and cancer metastasis factors such as MMP-2 were also reduced.

Cox-2 IHC analysis showed the same results, and macrophage invasion was also improved. The expression of inflammatory and transcription factor regulatory proteins, such as STAT3 and IkB-a, also showed the same trend, and apoptosis by Helicobacter was reduced by kimchi. And it was confirmed that the kimchi increased the antioxidant protein HO-1, HSP70, NQO-1.

According to the description as described above, the anti-cancer kimchi according to the present invention is any one selected from the group consisting of mustard leaf, mustard leaf, and mixtures thereof, acid, pear, kelp or kelp extract, and mushroom or mushroom extract is originally made kimchi recipe In addition, it has been shown to reduce the propensity of mutation of Helicobacter and to improve the progression of atrophic gastritis caused by Helicobacter when prolonged anticancer kimchi, and to prevent Helicobacter-related gastric cancer.

Claims (6)

1. Kimchi exhibiting prophylactic and therapeutic activity against Helicobacter pylori causative diseases, including any one selected from the group consisting of mustard leaf, mustard leaf, and mixtures thereof, acid, pear, kelp or kelp extract, and mushroom or mushroom extract.
2. The method of claim 1,

   The total weight of kimchi 1.0 to 10.0% by weight, any one selected from the group consisting of mustard leaves, mustard leaves, and mixtures thereof, 0.05 to 0.5% by weight of the herb, 1.0 to 5.0% by weight pear, the kelp or kelp extract 1.0 To 5.0% by weight, the kimchi showing the preventive and therapeutic activity for Helicobacter pylori causative disease, characterized in that it comprises 1.0 to 5.0% by weight of the mushroom or mushroom extract.
3. [Claim 3] The kimchi exhibiting preventive and therapeutic activity against Helicobacter pylori causative disease, characterized in that the mushroom is shiitake mushroom.
4. [Claim 3] The kimchi exhibiting prophylactic and therapeutic activity against Helicobacter pylori causative disease according to claim 1 or 2, wherein the extraction solvent of the kelp extract and the mushroom extract is water.
5. According to claim 1 or 2, Lactobacillus plantarum ( Lactobacillus plantarum ), Leuconostoc ( Leuconostoc) mesenteroides ) and kimchi showing the prophylactic and therapeutic activity against Helicobacter pylori causative disease, characterized in that it further comprises any one selected from the group consisting of a starter (starter).
6. According to claim 1 or 2, Kimchi showing the preventive and therapeutic activity against the Helicobacter pylori causative disease, characterized in that the disease is gastritis, gastric ulcer, gastric cancer.

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