KR102149007B1 - Method for kimchi using amtak baechu cabbage and uses thereof - Google Patents

Abstract

It relates to a method for preparing kimchi having excellent anticancer effect, and an anticancer composition comprising a kimchi extract prepared according to the above method. Kimchi prepared according to the method according to an aspect is excellent in preventing or treating cancer, and thus can be usefully used in a pharmaceutical composition or food composition for preventing, improving or treating cancer.

Description

Method for preparing kimchi using amtak cabbage and uses thereof {Method for kimchi using amtak baechu cabbage and uses thereof}

It relates to a method for preparing kimchi having excellent anticancer effect, and an anticancer composition comprising a kimchi extract prepared according to the above method.

Unlike other fermented foods, kimchi is a complex fermented food with a lot of seasoning, and foreigners' interest in fresh taste is increasing.Therefore, there is an urgent need to develop a method of industrialization and storage of kimchi for expanding domestic consumption and export. Research has been continued to develop processed foods that can utilize the characteristics of seasoning.

Kimchi is a traditional food rich in nutritionally important vitamins, minerals, fiber, lactic acid bacteria and various organic products, but it is an obstacle to the expansion of consumption due to strong fermentation odor when over-ripe and texture reduction due to softness phenomenon, and various processed products using kimchi are developed. The demand is increasing.

Research on kimchi known to date has mainly focused on preventing rancidity of kimchi, long-term preservation methods, and research on processed kimchi foods using new ingredients.

Also, kimchi is a fermented food, and the fermentation technology of kimchi has traditionally been handed down by housewives, but in recent years, industrialization has been promoted and the use of factory kimchi is increasing. Along with this industrialization, the diversification and functionality of kimchi is being emphasized. However, until now, kimchi with anticancer properties has been mainly studied to obtain the effect by simply adding ingredients with anticancer effect to kimchi. No research has been published on the technology of manufacturing kimchi with anticancer function by controlling the type and content of the ingredients.

In order to solve the above problems, various types of cabbage kimchi were prepared according to the type of ingredients, the mixing ratio and fermentation conditions, and then the anticancer function of these cabbage kimchi was investigated to determine the mixing ratio and fermentation conditions of the cabbage kimchi with the best anticancer effect. By investigation, the present invention was completed.

An aspect is to provide a method for preparing kimchi using Amtak cabbage and an anticancer effect of kimchi prepared according to the method.

One aspect is the step of marinating the black cabbage at 20 to 25°C for 14 to 16 hours by immersing the black cabbage in salt water in which 5 to 15% by weight of salt is dissolved based on the total weight of the black cabbage; And mixing the seasoning with a salinity of 1.5 to 1.8% in an amount of 25 to 35% by weight based on the total weight of the pickled cabbage. And it provides a method for producing kimchi comprising the step of fermenting for 18 to 25 days at 4 to 6 ℃.

In the present specification, the term "amtak cabbage" refers to a cabbage manufactured through intermediate crossing of turnip and cabbage, and may mean that it is supplied from Jeil Seed Bio.

The dark cabbage is known to have a high content of beta-carotene and gluconasturtiin. In the method according to one aspect, it was first confirmed that the anticancer effect was maintained even when kimchi was prepared using the black cabbage, and the manufacturing method showing the best anticancer effect was optimized.

The manufacturing method will be described in detail according to steps.

The manufacturing method includes the step of immersing the black cabbage in brine in which 5 to 15% by weight of salt is dissolved with respect to the total weight of the black cabbage and pickling for 14 to 16 hours at 20 to 25°C.

The salt may be a natural sea salt or a refined salt, and preferably, a natural sea salt. The salt in about 5 to 15% by weight, about 6 to 14% by weight, about 7 to 13% by weight, about 8 to 12% by weight, about 9 to 11% by weight, or about 10% by weight based on the total weight of the black cabbage You can pickle the black cabbage by dipping it in the dissolved brine.

Here, the step of marinating the black cabbage by immersing the black cabbage in salt water dissolved in salt may be referred to as a so-called brine method. In the case of pickling the Amtak cabbage according to the brine method, the anticancer effect is superior to the so-called tendonitis method, which is a method of pickling by directly applying salt to the cabbage. Therefore, in the manufacturing method according to an aspect, the black cabbage may be preferably pickled by the brine method.

The dark table cabbage may be pickled at about 20 to 25°C.

The black cabbage may be pickled for about 14 to 16 hours.

The anticancer effect of the Amtak cabbage may be excellent by the step of pickling the Amtak cabbage in the above range.

In addition, the manufacturing method includes mixing a seasoning with a salinity of 1.5 to 2.0% in an amount of 25 to 35% by weight based on the total weight of the pickled cabbage.

The seasoning is about 6 to 10% by weight, about 7 to 9% by weight, or about 8% by weight of radish, about 1 to 3% by weight, or about 2% by weight of chives, and about 3 to about 2% by weight of kelp based on the total weight of the pickled cabbage. 5 wt%, or about 4 wt%, glutinous rice paste 3 to 5 wt%, or about 4 wt%, red pepper powder 3 to 5 wt%, or about 4 wt%, minced garlic 1 to 3 wt%, or about 2 wt% , Minced ginger 0.1 to 0.5% by weight, or about 0.4% by weight, anchovy powder 0.1 to 0.5% by weight, or about 0.4% by weight, fish sauce 3 to 4% by weight, or about 3.4% by weight, and salted shrimp 0.5 to 1% by weight, Or it may include about 0.8% by weight.

The seasoning may have a salinity of about 1.5 to 2.0%, about 1.7 to 1.9%, or about 1.8%.

The salinity and the content of the constituents of the seasoning are not limited to the above range, but if it is included within the above range, the anticancer effect of kimchi prepared using black cabbage may be maximized.

In addition, the manufacturing method may include fermenting at 4 to 6° C. for 18 to 25 days.

After the fermenting step is completed, the kimchi may have a pH of 4.3±0.2.

The temperature and duration of the fermentation are not limited to the above range, but when fermented within the above range, the anticancer effect of kimchi prepared using black cabbage can be maximized.

Another aspect provides kimchi manufactured according to the kimchi manufacturing method.

Kimchi prepared according to the above manufacturing method may be kimchi having excellent cancer prevention or treatment effect.

Another aspect provides a pharmaceutical composition for preventing or treating cancer comprising kimchi prepared according to the method for producing kimchi or an extract of kimchi prepared according to the method for producing kimchi.

Another aspect provides a food composition for preventing or improving cancer comprising kimchi prepared according to the method for producing kimchi or an extract of kimchi prepared according to the method for producing kimchi.

In the present specification, the extract of kimchi prepared according to the method for producing kimchi is lyophilized and then crushed to prepare kimchi powder; And it may be prepared according to the step of obtaining a kimchi extract by adding and stirring methanol to the crushed kimchi powder.

The kimchi or kimchi extract can increase the mRNA or protein expression of Bax, Bad, Bim, Caspase-3, and Caspase-9, which are factors related to apoptosis, and reduce the mRNA or protein expression of Bcl-2, Bcl-w. I can.

Apoptosis is a form of a method in which cells die by being controlled by genes, and refers to a phenomenon in which abnormal cells, damaged cells, and aged cells commit suicide by themselves. Cancer cells, unlike normal cells, occur when cell division and growth are abnormally high and apoptosis is not smoothly performed. Therefore, activating apoptosis of cancer cells can be said to be a method of effectively blocking cancer development and proliferation. Representative factors involved in apoptosis include pro-apoptotic Bcl-2 families such as Bax, Bad, and Bim, and pro-survival regulators such as Bcl-2 and Bcl-w. In addition, when DNA is damaged, the p53 protein is activated, activating the pro-apototic Bcl-2 family. The activated pro-apoptotic Bcl-2 family again activates the caspase family, such as caspase-3 and caspase-9 proteins, and thereby induces critical apoptosis. Therefore, it can be said that when the expression level of pro-apoptotic Bcl-2 family and caspase family such as Bax, Bad, and Bim is increased, apoptosis of cancer cells is induced, leading to cancer cell death.

In addition, caspase exists in the form of a precursor, procaspase, and when stimulated by the apoptosis activation pathway, it becomes a cleaved caspase, which induces apoptosis. Therefore, if the amount of cleaved caspase-3 and caspase-9 is analyzed, the level at which apoptosis is activated can be analyzed.

The kimchi or kimchi extract may increase the mRNA or protein expression of p21 and p53, which are cell cycle related factors.

When a cell proliferates, M -> G1 -> S -> G2 repeats, and cell division occurs. When inhibiting the proliferation of cancer, a method of stopping the cell cycle and preventing the infinite proliferation of cancer cells is used. p21 is activated by p53 and plays a role in inhibiting the activity of cyclins/CDKs. Therefore, it inhibits Cyclin D1, a genetic factor related to Cyclins/CDKs, which causes cell cycle disruption. p53 is a gene involved in cell cycle disruption and apoptosis. When the cell membrane is stressed, it activates cell cycle disruption in relation to p21, activates Bax, a apoptosis-related gene, and inhibits Bcl-2, thereby participating in cell apoptosis. Is done. The p21 and p53 genes are involved in cell division throughout the G1, S, and G2/M phases, and by measuring the amount of these genes, it is possible to determine how much a substance such as an anticancer agent prevents the division of cancer cells.

The kimchi or kimchi extract may alleviate a decrease in the length of colon tissue due to the occurrence of colon cancer.

When colon cancer occurs, the mucous membrane becomes thinner and atrophy as the epithelial cells are damaged by inflammation, and thus the length of the colon is shortened. Therefore, it can be said that measuring the length of the colon is very important in confirming the progression of colon cancer and the anticancer effect of colon cancer.

The kimchi or kimchi extract has an effect of reducing an increase in the weight/length ratio of the large intestine according to the occurrence of colon cancer.

When colon cancer progresses, inflammatory polyps of the colon epithelium are formed, and accordingly, the weight of the colon is increased. Therefore, it is possible to compare the progression of colon cancer by measuring the ratio of the length and weight of the colon.

The kimchi or kimchi extract has an effect of reducing the number of tumors produced by induction of colon cancer.

The kimchi or kimchi extract has an effect of reducing the expression of mRNA or protein of proinflammatory cytokines in colon tissue.

나 TNF-α와 같은 물질들은 전염증성 사이토카인 (pro-inflammatory cytokines)으로써, 다량으로 분비될 경우 prostaglandins, leukotriens, NO와 같은 염증 매개물질들의 발현이 촉진된다. 이로 인하여 조직의 손상을 유도하고 염증을 유발하며, 지속적으로 발생될 경우 암으로 진행된다. 따라서, 전염증성 사이토카인의 발현이나 분비를 감소시키는 경우 항암 효과를 나타낼 수 있다.IL-6, IL-1

Substances such as TNF-α are pro-inflammatory cytokines, and when secreted in large amounts, the expression of inflammatory mediators such as prostaglandins, leukotriens, and NO is promoted. This induces tissue damage and causes inflammation, and if it occurs continuously, it progresses to cancer. Therefore, when the expression or secretion of pro-inflammatory cytokines is reduced, it can exhibit anticancer effects.

The kimchi or kimchi extract may contain a higher level of pyropheophorbide a compared to kimchi or kimchi extract prepared from ordinary cabbage.

The cancer may be colon cancer, but is not limited thereto.

As used herein, the term "treatment" refers to any action in which symptoms of cancer are improved or beneficially changed by administration of the composition.

In the present specification, the term "prevention" refers to any action that suppresses cancer or delays the onset of cancer by administration of the composition.

The pharmaceutical composition may additionally include a pharmaceutically acceptable diluent or carrier. The diluent may be lactose, corn starch, soybean oil, microcrystalline cellulose, or mannitol, and as a lubricant, magnesium stearate, talc, or a combination thereof. The carrier may be an excipient, a disintegrant, a binder, a lubricant, or a combination thereof. The excipient may be microcrystalline cellulose, lactose, low-substituted hydroxycellulose, or a combination thereof. The disintegrant may be calcium carboxymethylcellulose, sodium starch glycolate, anhydrous calcium monohydrogen phosphate, or a combination thereof. The binder may be polyvinylpyrrolidone, low-substituted hydroxypropylcellulose, hydroxypropylcellulose, or a combination thereof. The lubricant may be magnesium stearate, silicon dioxide, talc, or a combination thereof.

The pharmaceutical composition may be formulated as an oral or parenteral dosage form. The parenteral dosage form may be an injection or an external preparation for skin. Solid preparations for oral administration may be prepared by mixing tablets, pills, powders, granules and capsules. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, liquid solutions, emulsions and syrups, and various excipients, such as humectants, sweeteners, fragrances and preservatives, in addition to water and liquid paraffin, which are commonly used simple diluents. I can. Formulations for oral administration may contain an active ingredient of about 0.1 to 1000 mg/kg, 0.1 to 800 mg/kg, 1 to 500 mg/kg or 100 to 300 mg/kg body weight per dosage unit. It can be administered 1 to 6 times a day, 2 to 5 times, 3 to 4 times a day.

The preferred dosage of the pharmaceutical composition depends on the condition and weight of the patient, the degree of the disease, the form of the drug, the route and duration of administration, and may be appropriately selected by those skilled in the art. The pharmaceutical composition may be administered in an amount of 0.0001 mg/kg to 10000 mg/kg per day. Administration of the composition may be administered once a day, or may be divided several times.

The kimchi or kimchi extract may be administered by a method known in the art. Administration is, for example, intraperitoneal, intravenous, intramuscular, transdermal, mucosal, intranasal, intratracheal or subcutaneous administration, such as by any means, directly to the subject. Can be. The administration can be administered systemically or locally.

The food composition may be used with other foods or food ingredients in addition to kimchi or kimchi extract, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined according to the purpose of use (prevention, health or therapeutic treatment). In general, when preparing food or beverage, the health functional food of the present specification may be added in an amount of 15 parts by weight or less based on the raw material. There is no particular limitation on the kind of the health functional food. Among the types of health functional foods, the beverage composition may contain various flavoring agents or natural carbohydrates as an additional component, like a conventional beverage. The natural carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As the sweetener, natural sweeteners such as taumatin and stevia extract, and synthetic sweeteners such as saccharin and aspartame can be used. The food composition is also used in nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonated beverages Carbonation agent, or a combination thereof. The health functional food may also contain natural fruit juice, fruit juice beverage, flesh for the manufacture of vegetable beverages, or a combination thereof.

Another aspect provides a method of preventing or treating cancer in an individual comprising administering an effective amount of the kimchi or kimchi extract to the individual.

Kimchi prepared according to the method according to an aspect is excellent in preventing or treating cancer, and thus can be usefully used in a pharmaceutical composition or food composition for preventing, improving or treating cancer.

1 is a result showing the results of analyzing the components of the kimchi extract prepared from ordinary cabbage and dark cabbage.
2 is a graph measuring the relative frequency of fatigue peoh forbidden a among the components of the kimchi extract prepared from ordinary cabbage and dark cabbage cabbage.
Figure 3 is a graph confirming the colorectal cancer cell growth inhibition efficiency of kimchi extract by MTT assay.
4 is a graph of Bax mRNA expression measured by RT-PCR in colon cancer cells during kimchi extract treatment.
5 is a graph showing the expression of Bad mRNA in colon cancer cells when kimchi extract was treated by RT-PCR.
6 is a graph of Bim mRNA expression measured by RT-PCR in colon cancer cells during kimchi extract treatment.
7 is a graph of the expression of Caspase-3 mRNA in colon cancer cells when kimchi extract was treated by RT-PCR.
8 is a graph showing the expression of Caspase-9 mRNA in colon cancer cells when kimchi extract was treated by RT-PCR.
9 is a graph showing the expression of p21 mRNA in colon cancer cells when kimchi extract was treated by RT-PCR.
FIG. 10 is a graph showing the expression of p53 mRNA in colon cancer cells during kimchi extract treatment by RT-PCR.
11 is a gel photograph confirming the expression of Bax, cleaved caspase-3, cleaved caspase-9, p21, and actin proteins in colon cancer cells during kimchi extract treatment.
12 is a graph measuring the expression of Bax protein in colon cancer cells by Western blotting during kimchi extract treatment.
13 is a graph showing the expression of cleaved caspase-3 protein in colon cancer cells during kimchi extract treatment by Western blotting.
14 is a graph measuring the expression of cleaved caspase-9 protein in colon cancer cells during kimchi extract treatment by Western blotting.
15 is a graph showing the expression of p21 protein in colon cancer cells during kimchi extract treatment by Western blotting.
16 is a protocol for testing the cancer inhibitory effect of kimchi in cancer-causing mice.
17 is a graph showing the weight measurement results of mice in cancer-induced mice.
18 is a photograph and a graph measuring the length of the colon in cancer-causing mice.
19 is a graph showing the results of measuring the weight of the kidney, testis, liver, and spleen of cancer-causing mice, and dividing the weight by the weight.
20 is a graph showing the result of measuring the ratio of the weight of the large intestine/length of the large intestine of a cancer-induced mouse.
21 is a graph measuring the number of tumors in cancer-causing mice.
22 is a photograph showing the results of pathological observation of colon tissue in cancer-induced mice.
23 is a graph of the Bcl-2 gene mRNA measured by RT-PCR in colon tissues of cancer-causing mice.
FIG. 24 is a graph of a Bcl-w gene mRNA measured by RT-PCR in colon tissues of cancer-causing mice.
FIG. 25 is a graph of Bax gene mRNA measured by RT-PCR in colon tissues of cancer-causing mice.
26 is a graph of the mRNA of the p21 gene measured by RT-PCR in colon tissues of cancer-causing mice.
Fig. 27 is a graph of the cyclin D1 gene mRNA measured by RT-PCR in colon tissues of cancer-causing mice.
Fig. 28 is a graph of IL-1β gene mRNA measured by RT-PCR in colon tissues of cancer-causing mice.
29 is a graph of the mRNA of the IL-6 gene measured by RT-PCR in colon tissues of cancer-causing mice.
FIG. 30 is a graph of the TNF-α gene mRNA measured by RT-PCR in colon tissues of cancer-causing mice.
31 is a result of analyzing the expression levels of Bax, cleavedcaspase-3, p21, and actin proteins in colon tissues of cancer-causing mice by Western blotting.
32 is a graph showing the results of measuring the content of proinflammatory cytokines in the serum of cancer-causing mice by ELISA.

It will be described in more detail through the following examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example 1. Kimchi manufacturing

Kimchi was prepared using the tendon salt method and the brine method, respectively. Specifically, general Chinese cabbage and Amtak cabbage were provided by Jeil Seedbio (Jeungpyeong-gun, Korea), and radish, green onion, kelp water, glutinous rice paste, red pepper powder, minced garlic, minced ginger, anchovy used in kimchi production. Powder, salted anchovies, salted shrimp, etc. were provided and used from Bonwoori Chan Kimchi (Namyang-Ju, Korea). Each of the ordinary cabbage and the black cabbage was cut in half, washed three times in fresh water, and then the cabbage was pickled using sea salt. As the pickling method, the tendon salt method and the brine method were used. By the tendon salt method, 4% of the total weight of the cabbage was sprinkled on the roots and stems of the cabbage, and then pickled at 20 to 25℃ for 14 to 16 hours. It was immersed in salt water in which sea salt was dissolved and pickled at the same temperature and time conditions as in the vat method. Pickled cabbage was washed 3 times to remove remaining salt, and then placed on a tray for 3 hours to remove moisture.

Then, for 5000 g of pickled cabbage, 400 g (8%) radish, 100 g (2%) chives, 200 mL (4%) kelp water, 200 g (4%) glutinous rice paste, 200 g (4%) red pepper powder, 100 g (2%) chopped garlic ), minced ginger 20g (0.4%), anchovy powder 20g (0.4%), anchovy sauce 170mL (3.4%), and salted shrimp 40g (0.8%) to make seasoning. The salinity of the seasoning was 1.8%, and the specific weight was shown in Table 1. After pickling, dried cabbage was cut into 5~8 cm size, and the prepared seasoning was mixed to make kimchi, and the prepared kimchi was fermented at 4~6℃ for 3 weeks. After the fermentation was completed, as a result of measuring the pH, it was confirmed that the fermented kimchi was pH 4.3±0.2.

ingredient content cabbage 5000 g Sea salt 500 g radish 400 g Chives 100 g Kelp water 200 mL Glutinous rice paste 200 g chili powder 200 g chopped garlic 100 g Minced ginger 20 g Anchovy powder 20 g Salted anchovy 170 mL Salted shrimp 40 g

Example 2. Preparation of Kimchi Extract

After fermenting the kimchi prepared in Example 1 for 3 weeks, it was completely frozen at -20°C, and then completely dried using a freeze dryer (FD5512, Ilshin BioBase CO., Korea). The dried kimchi was ground using a blender. Methanol in an amount equivalent to 20 times that of kimchi powder was added and stirred with a stirrer for 48 hours to obtain a kimchi methanol extract. This extract was completely concentrated using a vacuum concentrator (EYELA, Tokyo Rikakikai Co., Tokyo, Japan), and dissolved in 4 times the amount of the concentrate with dimethyl sulfoxide (DMSO) to prepare a kimchi extract.

Kimchi extract made from ordinary Chinese cabbage and pickled by the tendon salt method is prepared with SK-D, Kimchi extract made by tendon salt method is made with AK-D, and Kimchi extract made by pickling by the salt water method is made with SK-B. , A kimchi extract prepared by marinating and pickled by a salt water method was named AK-B.

Experimental example 1. Analysis of ingredients of kimchi extract

The components of the kimchi extract prepared as in Example 2 were analyzed as follows. In order to extract metabolites from kimchi, the freeze-dried sample powder was mixed with 80% methanol containing terfenadine, a standard material, and extracted with a bullet blender (Next Advance, NY, USA). After centrifugation, the supernatant was analyzed by UPLC-Q-TOF MS (Waters, USA).

The kimchi metabolite extract was analyzed using Waters Acquity UPLC-Q-TOF. The sample extract was injected into an Acquity UPLC BEH C18 column (2.1 mm Х 100 mm, 1.7 μm; Waters), and the mobile phase was flow rate with water (A) containing 0.1% formic acid and ACN (B) containing 0.1% formic acid. The (flow rate) was 0.35 mL/min, the analysis time was 9 minutes, and the column temperature was 40°C. The eluent passed through the column was analyzed by Q-TOF MS with positive electrospray ionization (ESI) mode. The scan range of TOF MS data is 100-1500 m/z, scan time is 0.2 s, capillary and sampling cone voltages are 3 kV and 40 V, desolvation flow rate is 800 L/h, desolvation temperature is 400 ℃, source temperature is 100°C. Leucine-enkephalin ([M+H] = 556.2771) was used as a standard compound for lack mass and was analyzed per 10 s. Quality control (QC) samples made by mixing all samples were analyzed after every 10 analysis. MS/MS spectra were obtained under the conditions of collision energy ramp (10 to 30 eV) and m/z 50-1500. Mass spectrophotometer data processing including m/z, retention time, and ion intensity was performed using MakerLynx software (Waters). The analysis results are shown in FIG. 1. SK refers to the kimchi extract prepared by the brine method of general Chinese cabbage, and AK refers to the kimchi extract prepared by the salt water method of Amtak cabbage.

As shown in Fig. 1, as a result of the analysis of metabolites, pyrophophorbide a, which is a chloroplast decomposition product, and erucic acid, which is a fatty acid, were analyzed as major substances, and lysophosphatidylcholine: LPC) family was identified. Among the identified substances, it was confirmed that the change in fatigue peofobide a was the largest, and the results of comparing the relative frequencies of the identified substances are shown in FIG. 2.

As shown in Figure 2, it was confirmed that the fatigue peofobide a was significantly higher in the kimchi extract prepared from the black cabbage compared to the kimchi extract prepared from the ordinary cabbage.

Therefore, it can be predicted that the fatigue peophobide a contained in a large amount of Amtak cabbage is the cause of the excellent anticancer effect of the kimchi extract prepared from Amtak cabbage.

Experimental example 2. Evaluation of anticancer effect of kimchi extract

In order to evaluate whether the four kinds of kimchi extracts prepared as in Example 2 have an effect of inhibiting the growth of cancer cells, an MTT assay was performed. Specifically, HT-29 (human colorectal cancer cells) was sold and used by the Korean Cell Line Bank (KCLB, Seoul, Korea). It was cultured in RPMI 1640 medium, and each medium was used by mixing 10% Fetal Bovine Serum (FBS) and 1% penicillin-streptomycin. The medium was changed and cultured 2-3 times per week, followed by periodic subculture. The cultured cells were passaged 3-7 times, and then the number of cells was counted using a cell counter (Luna automated cell counter; Logos Biosystems, Gyunggi, Korea), and 1.0 × 10 ⁵ cells per well in a 96-well plate/ The cells were aliquoted into mL and cultured for 24 hours. Thereafter, the cells were treated with kimchi extract at concentrations of about 1, 1.5, 2, and 2.5 mg/mL for 48 hours, respectively. Then, 100 μL of a medium mixed with MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) at a concentration of 5 mg/mL was added per well and reacted for 4 hours. After the reaction, the formed formazan was dissolved using DMSO and reacted for 30 minutes in the dark. Afterwards, the absorbance was measured at 540 nm using a Wallac Victor3 1420 Multilabel Counter (Perkin-Elmer, Wellesley, MA, USA). The measurement results are shown in FIG. 3.

As shown in FIG. 3, at all concentrations, the kimchi group prepared through the saline method showed a significantly higher cancer inhibition rate by about 10% than the kimchi group prepared through the tendonitis method (p<0.05). In addition, the pattern of kimchi groups made with this brine method was about 20% higher in kimchi groups made with black cabbage than those made with ordinary cabbage (p<0.05).

Therefore, it was confirmed that the anticancer effect against colorectal cancer was remarkably high when kimchi was prepared from pickled black cabbage using the brine method.

Experimental example 3. Analysis of expression of colon cancer cells by kimchi extract treatment

In order to check the mechanism by which the anticancer effect of the kimchi extract as confirmed in Experimental Example 2 appears, the expression of colon cancer cells according to the kimchi extract treatment was analyzed.

3.1 mRNA Expression analysis

The cultured colon cancer cells were counted using a cell counter (Luna automated cell counter; Logos Biosystems, Gyunggi, Korea), and cells of 1.0 × 10 ⁶ cells/mL per well were dispensed into a 6-well plate and 24 Incubated for hours. A medium containing kimchi extract at a concentration of about 2.0 mg/mL was added to the cultured cells and reacted for 48 hours to be used in the experiment. RNA was isolated from the cells using Trizol (Invitrogen, Carlsbad, CA, USA), and dissolved in 0.1% DEPC (diethyl-pyrocarbonate). The dissolved total RNA was quantified using NanoDrop ND-1000 (NanoDrop Technologies Inc., Wilmington, DE, USA), and cDNA was synthesized using Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA, USA). The synthesized cDNA was amplified with the thermal cycler BioRad CFX-96 real time system (BioRad, USA), and fluorescence detected using FIREPol PCR Mix (Solis BioDyne, Tartu, Estonia) to identify the gene expressed from the amplified cDNA Was measured. The mRNA expression of Bax, Bad, Bim, Caspase-3, Caspase-9, which are genes related to apoptosis, and P21 and P53, which are related to cell cycle, were analyzed, and the primer sequences used are shown in Table 2.

gene order 18S rRNA Forward direction 5’-CAGCCACCCGAGATTGAGCA-3’ Reverse 5’-TAGTAGCGACGGGCGGTGTG-3’ Bax Forward direction 5'-TGCTTCAGGGTTTCATCCAG-3' Reverse 5’-GGCGGCAATCATCCTCTG-3’ Bad Forward direction 5’- CGGAGGATGAGTGACGAGTT-3’ Reverse 5’- GATGTGGAGCGAAGGTCACT-3’ Bim Forward direction 5’- AGATCCCCGCTTTTCATCTT-3’ Reverse 5’- TCTTGGGCGATCCATATCTC-3’ P21 Forward direction 5’-ATGTCAGAACCGGCTGGGG-3’ Reverse 5’-GCCGGGGCCCCGTGGGA -3’ P53 Forward direction 5’-ATGGAGGAGCCGCAGTCAGA-3’ Reverse 5’-TGCAGGGGCCGCCGGTGTAG -3’ Caspase-3 Forward direction 5’-TTTTTCAGAGGGGATCGTTG-3’ Reverse 5’-CGGCCTCCACTGGTATTTTA-3’, Caspase-9 Forward direction 5’-CTAGTTTGCCCACACCCAGT-3’ Reverse 5’-CTGCTCAAAGATGTCGTCCA-3’

Figure 4 is a graph measured by RT-PCR of mRNA expression of Bax in colorectal cancer cells during kimchi extract treatment. FIG. 5 is a graph showing the expression of Bad mRNA in colorectal cancer cells during kimchi extract treatment by RT-PCR.

6 is a graph of Bim mRNA expression measured by RT-PCR in colon cancer cells during kimchi extract treatment.

7 is a graph of the expression of Caspase-3 mRNA in colon cancer cells when kimchi extract was treated by RT-PCR.

8 is a graph showing the expression of Caspase-9 mRNA in colon cancer cells when kimchi extract was treated by RT-PCR.

9 is a graph showing the expression of p21 mRNA in colon cancer cells when kimchi extract was treated by RT-PCR.

FIG. 10 is a graph showing the expression of p53 mRNA in colon cancer cells during kimchi extract treatment by RT-PCR.

As shown in Figures 4 to 8, for all genes of the Bcl-2 family that induce apoptosis of HT-29, significantly higher levels in the kimchi group prepared through the saline method than in the kimchi group prepared through the tendonitis method. The mRNA gene expression rate of was shown, and this pattern was higher in Kimchi group of Amtak cabbage than that of Kimchi group of general cabbage. In addition, as a result of analyzing the mRNA expression levels of caspase-3 and caspase-9, the subfactors that are affected by the apoptosis inducing factor, the mRNA was significantly higher in kimchi group prepared by saline method than in kimchi group prepared by tendonitis method. Gene expression rate was shown, and this pattern was higher in the Kimchi group of Amtak cabbage than the Kimchi group of ordinary cabbage.

Therefore, it can be said that kimchi made with Amtak Chinese cabbage induces apoptosis of cancer cells better than kimchi made with general Chinese cabbage, and cabbage kimchi pickled with saline method induces apoptosis better than kimchi pickled with tendon salt method. , It can be seen that the anticancer effect is excellent.

In addition, as shown in Figures 9 to 10, for all genes that induce the inhibition of cell cycle progression of HT-29, significantly higher levels of kimchi group prepared through the saline method than in kimchi group prepared through the tendonitis method. The mRNA gene expression rate was shown, and this pattern was higher in Kimchi group of Amtak cabbage than that of Kimchi group of general cabbage.

Therefore, it can be seen that kimchi made with Amtak cabbage has a higher ability to stop the cell cycle than kimchi made with ordinary cabbage, and thus has a better anticancer effect.

3.2 Protein expression analysis

The cultured colon cancer cells were counted using a cell counter (Luna automated cell counter; Logos Biosystems, Gyunggi, Korea), and cells of 1.0 × 10 ⁶ cells/mL per well were dispensed into a 6-well plate and 24 Incubated for hours. A medium containing kimchi extract at a concentration of about 2.0 mg/mL was added to the cultured cells and reacted for 48 hours to be used in the experiment. To measure protein expression from cells, SDS-polyacrylamide gel electrophoresis and Western blotting analysis were performed. Cells were selected for each group and lysed using a radio-immunoprecipitation assay (Invitrogen, Carlsbad, CA, USA) buffer, and the total protein in the supernatant was separated by centrifugation for 15 minutes at 12000 rpm. Protein concentration in the supernatant was quantified by Bio-Rad protein assay die reagent concentration (Bio-Rad, USA), mixed with the same amount of Laemmli sample buffer (Bio-Rad, USA), and reacted at 94°C for 10 minutes to form a protein Released. The same amount of sample was separated by SDS-polyacrylamide gel electrophoresis, and then transferred to a PVDF (polyvinylidene fluoride) (BioRad, USA) membrane. 10% skim milk was reacted to the protein-transferred membrane at room temperature for 1 hour to block non-specific proteins and washed with PBS-T (3 times for 5 minutes). Then, the primary antibody was treated and reacted overnight at 4°C, washed with PBS-T (3 times for 5 minutes), and reacted for 2 hours at room temperature using a secondary antibody suitable for the treated primary antibody, and then PBS-T Washed with (3 times for 5 minutes). After washing, photosensitization with a LAS-4000 luminescent image analyzer (Fujifilm Life Science, Tokyo, Japan) was performed to analyze the expression levels of Bax, cleaved caspase-3, cleaved caspase-9, p21, and actin proteins.

11 is a gel photograph confirming the expression of Bax, cleaved caspase-3, cleaved caspase-9, p21, and actin proteins in colon cancer cells during kimchi extract treatment.

12 is a graph measuring the expression of Bax protein in colon cancer cells by Western blotting during kimchi extract treatment.

13 is a graph showing the expression of cleaved caspase-3 protein in colon cancer cells during kimchi extract treatment by Western blotting.

14 is a graph measuring the expression of cleaved caspase-9 protein in colon cancer cells during kimchi extract treatment by Western blotting.

15 is a graph showing the expression of p21 protein in colon cancer cells during kimchi extract treatment by Western blotting.

11 to 15, the expression level of Bax, a factor that induces apoptosis of cancer cells, was higher in the protein level of the kimchi extract of Amtak cabbage than that of the kimchi extract of general cabbage. The case was higher than that of kimchi extract pickled by tendonitis method. In addition, as a result of analyzing the expression of the cleaved form of caspase-3 and caspase-9 proteins that appear when stimulated by the apoptosis activation pathway, the expression level of the proteins in Amtak cabbage kimchi extract rather than general cabbage kimchi extract The results were significantly higher, and this pattern was significantly higher when pickled by the brine method than when pickled by the tendonitis method (p<0.05). As for the levels of p53 and p21, which are proteins related to the cell cycle, black cabbage was significantly higher than that of normal cabbage, and when pickled by saline method was significantly higher than when pickled by tendonitis method (p<0.05).

Therefore, it was confirmed that kimchi was prepared with Amtak cabbage and the cabbage was pickled by the saline method, and the anticancer effect against colon cancer cells was excellent, which induces the death of cancer cells by promoting apoptosis against colon cancer cells. It is confirmed that the proliferation is suppressed by stopping the cycle.

Experimental example 4. Confirmation of anticancer effect of kimchi extract in colon cancer mice

4.1 Feed preparation

According to the results of the above experimental examples, kimchi extract prepared by the brine method and aged for 3 weeks was prepared by adding 10% weight of feed. Diets of the normal group and the control group were prepared according to the AIN-93G diet preparation method, and the prepared diet was made into a solid, dried, and then supplied while refrigerated at 4°C. Both general feed and feed containing kimchi extract samples were provided and used by Doo-Yeol Biotech, Seocho-gu, Seoul, Korea.

4.2 Preparation of experimental animals and Experimental group Configuration

The mouse used in the animal experiment was a 6-week-old C57BL/6 with a body weight of 20±2g, and was sold and tested by Orient Bio (Seong-nam, Gyeonggi, Korea), and the temperature was 23±2℃ and the relative humidity was 55±5. %, it was reared under the condition that the light-dark cycle was maintained at a cycle of 12 hours. After distribution, an adaptation period was given for 1 week, and each group was given an adaptation period for one week, and each group was given a normal diet, AOM/DSS to induce colorectal cancer and AIN-93G normal diet, AOM/ The experimental group (SK) inducing colon cancer with DSS and ingesting a feed containing 10% of ordinary cabbage kimchi extract, and the experimental group (SK) inducing colorectal cancer with AOM/DSS and ingesting a feed containing 10% of black cabbage kimchi extract ( AK). Prior to conducting this animal experiment, it was approved by the Animal Experimental Ethics Committee (approval number: IACUC170089) of Cha Medical University.

4.3 Sample administration and colon cancer induction

To induce colorectal cancer in C57BL/6 mice, azoxymethane (AOM, Sigma, St Louis, MO, USA) was mixed with water and administered intraperitoneally at 10 mg/kg, and 3 weeks after AOM administration. From this point, 2% DSS (dextran sulfate sodium, MPBio, Santa Ana, CA, USA) was administered in negative water for 1 week, and then a rest period was carried out. Test substances were supplied in diet from 1 week after AOM induction, and autopsies were performed after a total of 8 weeks. The experimental protocol is shown in Figure 16.

4.4 weight measurement

After obtaining all mice, the body weight of each individual was measured once a week during the 8 weeks of acclimatization period, once a week during group separation and during the test period. In particular, weight measurement during the test period was measured using the same scale on the same day of the week (Wednesday) and the same time period (around 2 pm) to minimize errors during each measurement. The weight measurement results are shown in FIG. 17.

As shown in FIG. 17, the weight of all experimental animals immediately before the experiment was 20 ± 2 g, and there was no significant difference between all groups. In the case of the normal group that did not induce colorectal cancer, the body weight of the experimental animals gradually increased as the test period elapsed. Showed. In the control group and the kimchi sample-administered group, weight gain was shown before DSS supply, but the body weight was suppressed or the rate of increase was decreased during the recovery period of 1 week after DSS supply. It is considered that colon inflammation and colorectal cancer are caused by DSS treatment, and the increase in body weight decreases by showing symptoms of bloody stool and diarrhea. This pattern was noticeable from the 3rd week of the experiment, and until the 8th week, the end of the experiment, both the control group and the kimchi group showed significantly lower body weight than the normal group (p<0.05).

4.5 Colon Length Measurement

When colon cancer occurs, the mucous membrane becomes thinner and atrophy as epithelial cells are damaged by inflammation, and thus the length of the colon is shortened. Therefore, measuring the length of the colon is very important in confirming the progression of colon cancer or the anticancer effect of colon cancer.

After the end of the test period (8 weeks), the large intestine was removed, and the length of the large intestine was measured with a ruler. The shorter the length of the large intestine, the more colon cancer was induced. Fig. 18 shows the results of comparing the length of the large intestine.

As shown in Figure 18, the control group (7.00 ± 0.34 cm) compared to the normal group (8.39 ± 0.48 cm), the length of the colon was significantly decreased, it was found that colon cancer was significantly advanced, SK group (7.12 ± 0.38 cm) was not significantly different from the control group (7.73 ± 0.45 cm). On the other hand, the colon length of the AK group that ingested the Kimchi extract from Amtak cabbage was longer than that of the normal group.

4.6 organ weight measurement

After the end of the experiment period, the experimental animals were sacrificed, and the kidneys, testis, liver and spleen were collected and weights were compared, and each result was divided by body weight and shown in FIG. 19 as a ratio of organ weight/body weight.

As shown in FIG. 19, as a result of measuring the weight of the kidney and testis, there was no significant difference between the groups for these two organs. The two organs were not affected in this experiment, and were used as a control, and there was no difference between the two, so it was confirmed that there was no major problem with this colon cancer model experiment. On the other hand, when toxicity appears in the liver and spleen, inflammation progresses and swelling occurs, and accordingly, the liver becomes somewhat larger. The control group showed significantly higher liver weight compared to the normal group, and the kimchi diet group had lower liver and spleen weights than the normal group. Therefore, it is thought that the levels of toxicity and inflammation were alleviated in the two groups that were fed kimchi compared to the control group.

In addition, when colon cancer progresses, inflammatory polyps of the colon epithelium are formed, and accordingly, the weight of the colon is increased. Therefore, it is possible to compare the progression of colon cancer by measuring the ratio of the length and weight of the colon.

Figure 20 shows the results of measuring the weight/length ratio of the large intestine.

As shown in FIG. 20, in the case of the control group, the ratio was significantly higher than that of the normal group (p<0.05). It was found that the SK group had a significantly lower weight/length ratio than the control group, and the AK group was lower than that of the SK group (p<0.05).

Therefore, it seems that the condition of the large intestine due to ulcerative colorectal cancer induced by AOM/DSS was improved by the kimchi sample. In particular, the effect was higher in the AK group than in the SK group, and it seems that the inhibitory effect of kimchi on colorectal cancer is increased due to the dark-tack cabbage compared to the general cabbage.

4.7 Tumor Count Measurement

After 8 weeks of the experiment, the large intestine was removed to measure the number of tumors in the large intestine, and the results are shown in FIG. 21.

As shown in FIG. 21, since the normal group did not induce colorectal cancer, it was found that there was no tumor, and the control group was 6.3 ± 1.9, indicating the largest number of tumors compared to all groups. On the other hand, the SK group was confirmed to be 4.8 ± 1.3, and the AK group was 3.5 ± 0.9, indicating that the two experimental groups that consumed the kimchi sample significantly reduced the incidence of tumors compared to the control group that did not consume the kimchi sample (p <0.05). In particular, it was found that the number of tumors in the AK group using dark black cabbage was significantly smaller than that of the SK group using normal cabbage.

Therefore, it seems that the antitumor effect of kimchi is increased due to the dark turquoise cabbage rather than the general cabbage.

4.8 Pathological observation of colon tissue

After the end of the experiment period, the colon was excised, washed with PBS, and fixed in a neutral 10% formalin solution. Subsequently, paraffin tissue samples were prepared for pathological evaluation, and each tissue sample was sectioned to a thickness of 4 μm. The sectioned tissue was stained with hematoxylin & eosin (H&E) dye for pathological examination and observed at x100 magnification using an optical microscope. The observation results are shown in FIG. 22.

As shown in FIG. 22, in the case of general colon tissue, inflammation does not occur, and normal villus (villus, hairy hair) can be confirmed, but when inflammation and tumor in the large intestine occurs, it is stained blue when tissue staining is observed. It can be seen that it collapsed differently academically. As a result of tissue staining observation, no inflammation or tumor was observed in the normal group. However, colon invasion and tumor were observed in the control group. In the SK group, it was confirmed that inflammation and tumor were significantly relieved than the control group. The AK group showed a tissue morphology comparable to that of the normal group, and it was confirmed that inflammation and tumors were significantly reduced.

4.9 RT- qPCR Used colon tissue mRNA Expression measurement

The large intestine tissue extracted from animal experiments was cut and collected by about 5 mm under frozen conditions, RNA was isolated using Trizol, and then dissolved in 0.1% DEPC. The dissolved total RNA was quantified using NanoDrop ND-1000 (NanoDrop Technologies Inc., Wilmington, DE, USA), and cDNA was synthesized using Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA, USA). The synthesized cDNA was amplified with the thermal cycler BioRad CFX-96 real time system (BioRad, Hercules, CA, USA), and FIREPol PCR Mix (Solis BioDyne, Tartu, Estonia) was used to confirm the gene expressed from the amplified cDNA. The detected fluorescence was measured. Table 3 shows the primers used to measure the mRNA expression of colon tissue.

gene order 18S rRNA Forward direction 5'-TCGAGGCCCTGTAATTGGAA-3' Reverse 5'-CCCTCCAATGGATCCTCGTT-3' Bcl-2 Forward direction 5'-GATGCTGGAGATGCGGA-3' Reverse 5'AGACGTCCTGGCAGCCA-3' Bcl-w Forward direction 5'-AACAATAGCGAATGCCCTTG-3' Reverse 5'-TCTTGGCCTTGAAACATTCC-3' Bax Forward direction 5'-GGGCCTTTTTGCTACAG-3' Reverse 5'-GACACTCGCTCAGCTTC-3' p21 Forward direction 5'-GTGAGCAGTTGCGCCGT-3' Reverse 5'-GCTCCCAGACGAAGTTG-3' Cyclin D1 Forward direction 5'-CAACGACCGGGTGCTGC-3' Reverse 5'-CAGGTGGCCACGATTTT-3' IL-1β Forward direction 5'-AAGGGCTGCTTCCAAAC-3' Reverse 5'-CTCCACAGCCACAATGA-3' IL-6 Forward direction 5'-ATGAAGTTCCTCTCTGCAA-3' Reverse 5'-AGTGGTATCCTCTGTGAAG-3' TNF-α Forward direction 5'-ATGGGGGGCTTCCAGAA-3' Reverse 5'-CCTTTGGGGACCGATCA-3'

4.9.1 Cell Self-destruction Related gene expression measurement

The results of measuring the expression of the apoptosis-related genes Bcl-2, Bcl-w, and Bax genes are shown in FIGS. 23, 24, and 25, respectively.

As shown in Figs. 23 to 25, in the case of the pro-survival regulators Bcl-2 and Bcl-w, the SK group and the AK group were significantly lower than those of the control group (p<0.05). On the other hand, the expression level of Bax, which induces apoptosis, was significantly higher in the SK group than in the control group, and the AK group was found to have the highest expression level (p<0.05).

4.9.2 Cell cycle related gene expression measurement

The results of measuring the expression of the cell cycle-related genes p21 and cyclin D1 genes are shown in FIGS. 26 and 27, respectively.

As shown in FIGS. 26 to 27, in the case of the mRNA expression level of p21, a protein important for cell cycle interruption, it can be seen that the expression level of p21 mRNA in the control group was significantly lowered compared to the normal group (p<0.05). On the other hand, the two groups treated with kimchi samples were significantly higher than those of the control group, and in particular, the AK group showed higher expression levels than the SK group (p<0.05). And, as a result of analyzing the mRNA expression level of Cyclin D1 regulated by p21, it was found that the mRNA expression level of the control group was significantly increased compared to the normal group, indicating that the cell cycle was continuously occurring. On the other hand, the two groups (SK, AK) treated with kimchi samples showed significantly lower expression than the control group, and in particular, the AK group showed a lower expression level than the SK group (p<0.05).

Therefore, it was confirmed that kimchi made from dark tactile cabbage at the gene level in the colon tissue regulates the growth of cancer cells.

4.9.3 Proinflammatory Measurement of cytokine related gene expression

The results of measuring the expressions of the proinflammatory cytokine-related genes IL-1β, IL-6, and TNF-α are shown in Figs. 28, 29, and 30, respectively.

As shown in Figs. 28 to 30, it was found that the mRNA expression levels of all proinflammatory cytokines of the control group were significantly increased compared to the normal (p<0.05). On the other hand, in the two groups treated with kimchi extract, the expression was significantly lower than that of the control group, and in particular, the AK group showed a lower level of expression than that of the SK group (p<0.05).

Therefore, it can be seen that the treatment of kimchi extract reduces the expression of pro-inflammatory cytokines, thereby suppressing the induction of inflammation and cancer.

Therefore, compared to general Chinese cabbage, the expression of apoptosis-related factors was high in the colon tissue of mice fed with kimchi extract made from black cabbage cabbage, and the mRNA expression of cell cycle-related factors was low, so the anticancer effect was excellent in the large intestine. Was confirmed.

4.10 Western Blotting Measurement of protein expression in colon tissue using

The tissues extracted from animal experiments were cut and collected by about 5 mm under frozen conditions. This was dissolved using a radio-immunoprecipitation assay (Invitrogen, Carlsbad, CA, USA) buffer and centrifuged at 12000 rpm for 15 minutes to separate the total protein in the supernatant. Protein concentration in the supernatant was quantified by Bio-Rad protein assay die reagent concentration (Bio-Rad, USA), mixed with the same amount of Laemmli sample buffer (Bio-Rad, USA), and reacted at 95°C for 10 minutes to obtain protein structure. Released. The same amount of sample was separated by SDS-polyacrylamide gel electrophoresis, and then transferred to a PVDF (BioRad, Hercules, CA, USA) membrane. 10% skim milk was reacted to the protein-transferred membrane at room temperature for 1 hour to block non-specific proteins and washed with PBS-T (3 times for 5 minutes). Then, the primary antibody was treated and reacted overnight at 4°C, washed with PBS-T (3 times for 5 minutes), and reacted for 2 hours at room temperature using a secondary antibody suitable for the treated primary antibody, and then PBS-T Washed with (3 times for 5 minutes). After washing, photosensitization with LAS-4000 luminescent image analyzer (Fujifilm Life Science, Tokyo, Japan) was performed to analyze the expression levels of Bax, p21, cleaved caspase-3, and actin proteins. The analysis results are shown in FIG. 31.

As shown in FIG. 31, as in the mRNA expression level analysis, the expression levels of Bax and cleaved caspase-3, which are factors that induce apoptosis of cancer cells, were prepared by the group treated with kimchi extract prepared from black cabbage. The protein level was higher than that of the kimchi extract-treated group.

Likewise, the expression level of p21, a protein that stops the cell cycle, was significantly higher in the AK group than in the control or SK group. Therefore, it was confirmed that the mice ingesting kimchi made from Amtak cabbage were excellent in inhibiting colon cancer by inducing apoptosis or cell cycle arrest of colon cancer.

4.11 mouse serum My pro-inflammatory Measurement of cytokine content

After sacrifice of the experimental animal, blood was collected from the abdominal vein, and this was centrifuged at 3000 rpm for 15 minutes. The centrifuged blood supernatant was separated to obtain serum, which was used in the experiment. To confirm the content of IL-1β, IL-6, and TNF-α in the collected serum, an ELISA kit (BioLegend, San Diego, CA, USA) was used. It was carried out according to the experimental method provided by the manufacturer. The target antibody against mouse cytokines was diluted in a coating buffer in advance in a 96 well plate, and 100 μL was dispensed, and then left at 4° C. overnight. The next day, after washing the plate 4 times with the washing buffer, 200 μL of the assay diluent was dispensed and left at room temperature for 1 hour. After washing 4 times with the washing buffer, 100 μL of the detection antibody was dispensed, and then left at room temperature for 1 hour. After washing 4 times with a washing buffer, 100 μL of avidin-horseradish peroxidase was inoculated, left for 30 minutes at room temperature, and washed 5 times with a washing buffer. 100 μL of the substrate solution containing the TMB substrate solution was inoculated, allowed to stand at room temperature for 15 minutes, and then treated with 100 μL of the stop solution to terminate the reaction. The absorbance (Optical density) was measured at 450 nm with a Wallac Victor3 1420 Multilabel Counter (Perkin-Elmer, Wellesley, MA, USA). The measurement results are shown in FIG. 32.

As shown in FIG. 32, it was found that the concentration levels of proinflammatory cytokines in all plasma of the control group were significantly increased compared to the normal group (p<0.05). On the other hand, the two groups treated with kimchi samples showed significantly lower expression than the control group. In particular, the AK group showed a lower level than that of the normal group compared to the SK group (p<0.05).

Therefore, it was confirmed that the mice ingesting kimchi made from Amtak cabbage inhibit colon cancer by inhibiting the production of pro-inflammatory cytokines that cause colon cancer.

Statistical processing

The experimental results for RT-qPCR used the standard error, and all experimental results except for this were expressed using the standard deviation. To confirm the significant differences between groups, Duncun's multiple range test and One-way analysis of variance (ANOVA) were used. Students' t-test method was used to confirm the significant difference between the two groups, and IBM SPSS ver.23 (SPSS Inc.) statistical software was used for all statistical analysis. For the experimental results, it was judged that there was statistical significance when p<0.05 level.

Claims (9)

Marinating the black cabbage for 14 to 16 hours at 20 to 25°C using the brine method of immersing the black cabbage in brine obtained by dissolving 6 to 14% by weight of salt based on the total weight of the black cabbage;
As a step of mixing the seasonings having a salinity of 1.5 to 2.0% in an amount of 25 to 35% by weight based on the total weight of the black cabbage pickled by the brine method,
The seasoning is 6 to 10% by weight of radish, 1 to 3% by weight of chives, 3 to 5% by weight of kelp, 3 to 5% by weight of glutinous rice paste, 3 to 5% by weight of red pepper powder, minced garlic 1 To 3% by weight, 0.1 to 0.5% by weight of chopped ginger, 0.1 to 0.5% by weight of anchovy powder, 3 to 4% by weight of fish sauce, and 0.5 to 1% by weight of salted shrimp; And
Fermenting the mixture at 4 to 6° C. for 18 to 25 days,
At least one mRNA or protein selected from the group consisting of Bax, Bad, Bim, Caspase-3, and Caspase-9 with an increased content of pyrophorbide a compared to kimchi not prepared by the above method The method for producing kimchi having anticancer function for colon cancer by increasing the expression and reducing the expression of the mRNA or protein of Bcl-2 or Bcl-w. delete Kimchi prepared according to the method of claim 1. A pharmaceutical composition for preventing or treating colon cancer, comprising kimchi prepared according to the method of claim 1 or an extract of kimchi prepared according to the method of claim 1. delete The pharmaceutical composition for preventing or treating colon cancer according to claim 4, wherein the extract is a methanol extract. A food composition for preventing or improving colon cancer, comprising the kimchi prepared according to claim 1 or an extract of kimchi prepared according to claim 1. delete The food composition according to claim 7, wherein the extract is a methanol extract.

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