KR20220070085A - Preventing or treating of ulcerative colitis by using hot water extract of sprout of Coix lacryma-jobi var. ma-yuen Stapf - Google Patents

Abstract

In animal models of colitis induced by dextran sulfate sodium (DSS), pharmacological effects of a Coix lacryma-jobi sprout hot water extract are clearly suggested. The present invention provides a pharmaceutical composition for prevention or treatment of ulcerative colitis and a food composition for prevention or alleviation of ulcerative colitis, containing the Coix lacryma-jobi sprout hot water extract as an active component.

Description

　Preventing or treating of ulcerative colitis by using hot water extract of sprout of Coix lacryma-jobi var. ma-yuen Stepf}

The present invention relates to a pharmaceutical composition and a food composition for the prevention or treatment of ulcerative colitis, and more particularly, to a pharmaceutical composition and a food composition for the prevention or treatment of ulcerative colitis, comprising an extract of ginseng radish.

Inflammatory bowel disease (IBD) refers to a disease in which an abnormal inflammatory response occurs repeatedly in the small or large intestine (see Non-Patent Document 1). It is known that such IBD can cause severe chronic diarrhea, bloody stool, and abdominal pain (see Non-Patent Document 2). Although the exact cause of IBD is not known, it is known that enteric bacteria, etc. are related to environmental factors, heredity, immunity, and inflammatory response (see Non-Patent Document 3). Representative examples include Crohn's disease (CD) and ulcerative colitis (UC) (see Non-Patent Document 4). Among them, UC is a disease in which symptoms appear in the rectum and colon, and normal colon tissue is divided into mucosa, submucosal tissue, muscle layer, and serous membrane. 5). It mainly occurs in countries that like to eat meat, such as the United States or Europe, and recently, the prevalence is increasing in Korea as well (see Non-Patent Document 6).

In oriental medicine, it belongs to the categories of dysentery (痢疾), diarrhea (泄瀉), bloody stool (便血), and intestinal barrier (腸癖). In the case of diarrhea, it can be said that it is close to fever, and among bloody stools, it can be said that it is closely related to intestinal wind. See Patent Document 3). Causes of the disease include indigestion, non-renal hemorrhage, large intestine damp gas, damage to the spleen and liver, and a range of reversal犯胃) and the like (see Non-Patent Document 7).

On the other hand, Coix lacryma-jobi var. ma-yeun Stapf) is a herbaceous plant belonging to the Grapeaceae, and its origin is Southeast Asia such as China and Japan, and is currently widely cultivated in other countries (see Non-Patent Document 8). In Korea, the seeds have been used for pain relief, diuresis, inflammation, etc., and have an effect of suppressing appetite, and are now widely used as a treatment for obesity (see Non-Patent Document 9).

In oriental medicine, the seeds of yulmu are called uiiin (薏苡仁), and their temperament is yang and smoky, making them dry and draining the air, draining the heat from the air, and swallowing. It is known to treat ailments such as beriberi, non-heuristic diarrhea, edema, and intestinal ailments (see Non-Patent Document 10) because it has the same effect as diarrhoea (除止瀉). .

Currently, many studies have been conducted on Ui-in (薏苡仁), but studies on Sprout of Coix lacryma - jobi var.

[Non-patent literature]

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Therefore, in the present invention, it is intended to clearly suggest that there is a pharmacological effect of the hot water extract of edible radish sprouts in an animal model of colitis induced by dextran sulfate sodium (DSS).

As an aspect for solving the above problems, the present invention provides a pharmaceutical composition for the prevention or treatment of ulcerative colitis, which includes an extract of radish sprouts as an active ingredient.

In addition, the extract provides a pharmaceutical composition for the prevention or treatment of ulcerative colitis, characterized in that the hot water extract.

As another aspect for solving the above problems, the present invention provides a food composition for the prevention or improvement of ulcerative colitis comprising an extract of sprout radish as an active ingredient.

In addition, the extract provides a food composition for the prevention or improvement of ulcerative colitis, characterized in that the hot water extract.

According to the present invention, significant results were confirmed as a result of studying the pharmacological effects of hot water extract of sprouted radish radish in an animal model of dextran sulfate sodium (DSS) induced ulcerative colitis. It can be effectively used as a pharmaceutical composition for prevention or treatment, or a food composition for prevention or improvement.

1 is a photograph and graph showing the result of measuring the length of the large intestine extracted after autopsy in an animal experiment according to an experimental example of the present invention;
2 is a photograph and graph showing the results of measurement of the expression levels of Nrf2, HO-1, SOD, Catalase and GPx-1/2, which are antioxidant-related factors in the colon tissue in the experimental example of the present invention;
3 is a photograph and graph showing the results of measuring the expression levels of NF-κBp65 and p-IκBα, which are inflammatory mediators in an experimental example of the present invention;
4 is a photograph and graph showing the results of measuring the expression levels of COX-2, iNOS, TNF-α and IL-1β, which are inflammation-related factors in an experimental example of the present invention;
5 is a photograph and graph showing the results of measuring the expression levels of IL-4 and IL-10, which are anti-inflammatory factors in the colon tissue in the experimental example of the present invention;
6 is a photograph and graph showing the results of measurement of the expression levels of Bcl2, Bax and Caspase-3, which are factors related to apoptosis in the experimental example of the present invention.

Hereinafter, the present invention will be described in detail through preferred embodiments. Prior to this, the terms or words used in the present specification and claims should not be construed as being limited to conventional or dictionary meanings, and the inventor should properly understand the concept of the term in order to best describe his invention. Based on the principle that can be defined, it should be interpreted as meaning and concept consistent with the technical idea of the present invention. Therefore, since the configuration of the embodiments described in the present specification is only the most preferred embodiment of the present invention and does not represent all the technical spirit of the present invention, various equivalents and modifications that can be substituted for them at the time of the present application are provided. It should be understood that there may be Also, throughout the specification, when a part "includes" a certain component, it means that other components may be further included, rather than excluding other components, unless otherwise stated.

In the present invention, as a result of studying the pharmacological effects of the hot water extract of edible radish radish in an animal model of ulcerative colitis induced by DSS (dextran sulfate sodium), significant results were confirmed.

Accordingly, the present invention discloses a pharmaceutical composition for the prevention or treatment of ulcerative colitis, comprising an extract of the radish sprouts as an active ingredient, and a food composition for the prevention or improvement of ulcerative colitis, comprising the extract of the radish sprouts as an active ingredient.

There is no limitation on the form or properties of the extract, and it may be a solution or a concentrate, and may be a solid or powder obtained by removing the solvent used to prepare the extract.

The radish sprout extract may be prepared using a conventional extraction method known in the art, for example, a solvent extraction method. The extraction solvent used in preparing the extract using the solvent extraction method is water, a lower alcohol having 1 to 4 carbon atoms (eg, methanol, ethanol, propanol and butanol) or a mixture thereof, which is a hydrous lower alcohol, propylene glycol, 1,3 -Butylene glycol, glycerin, acetone, diethyl ether, ethyl acetate, butyl acetate, dichloromethane, chloroform, hexane and mixtures thereof may be selected from the group consisting of, and the production yield with the prevention or improvement of the therapeutic effect of ulcerative colitis In view of the above, it may be preferably used as a hot water extract.

According to an embodiment of the present invention, as a method for preparing a hot water extract of sprouted radish, 5 to 15 times (v/w) of water is added to dried sprouted radish, boiled at 100° C. for 1 to 5 hours, and the extract is extracted, and the extract is filtered. And it can be concentrated to prepare a powder form.

That is, in the present invention, in order to obtain a meaningful result, the analysis results regarding the amount of body weight change in the ulcerative colitis-induced animal model and the protein expression level of antioxidant-related factors, inflammation-related factors, anti-inflammatory factors, and apoptosis-related factors It was confirmed that it can be implemented through the method.

The composition according to the present invention may be provided in the form of a pharmaceutical composition or a food composition.

When provided in the form of the pharmaceutical composition, it may further contain one or more pharmaceutically acceptable carriers, excipients or diluents in addition to the extract of the present invention. As used herein, "pharmaceutically acceptable" means a non-toxic composition that is physiologically acceptable and does not inhibit the action of the active ingredient when administered to humans and does not normally cause allergic reactions such as gastrointestinal disorders, dizziness, or similar reactions. say

The pharmaceutically acceptable carrier may further include, for example, a carrier for oral administration or a carrier for parenteral administration. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. In addition, various drug delivery materials used for oral administration of the peptide formulation may be included. In addition, the carrier for parenteral administration may include water, a suitable oil, saline, aqueous glucose and glycol, and the like, and may further include a stabilizer and a preservative. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives are benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, etc. in addition to the above components. For other pharmaceutically acceptable carriers and agents, reference may be made to those described in the following literature (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).

The composition according to the present invention may be administered to mammals including humans by any method. For example, it may be administered orally or parenterally. Parenteral administration methods include, but are not limited to, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual or rectal administration. can be

The pharmaceutical composition according to the present invention may be formulated as a formulation for oral administration or parenteral administration according to the administration route as described above.

In the case of a formulation for oral administration, the composition of the present invention may be formulated as a powder, granule, tablet, pill, dragee, capsule, liquid, gel, syrup, slurry, suspension, etc. using methods known in the art. can For example, oral preparations can be obtained by mixing the active ingredient with a solid excipient, grinding it, adding suitable adjuvants, and processing it into a granule mixture to obtain tablets or dragees. Examples of suitable excipients include sugars, including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol and maltitol, and starches, including corn starch, wheat starch, rice starch and potato starch, cellulose, Cellulose, including methyl cellulose, sodium carboxymethylcellulose and hydroxypropylmethyl-cellulose, and the like, fillers such as gelatin, polyvinylpyrrolidone, and the like may be included. In addition, if necessary, cross-linked polyvinylpyrrolidone, agar, alginic acid or sodium alginate may be added as a disintegrant. Furthermore, the pharmaceutical composition may further include an anti-aggregating agent, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent, and a preservative.

Formulations for parenteral administration may be formulated in the form of injections, creams, lotions, external ointments, oils, moisturizers, gels, aerosols and nasal inhalants by methods known in the art. These formulations are described in Remington's Pharmaceutical Science, 19th ed., Mack Publishing Company, Easton, PA, 1995, which is a commonly known recipe for all pharmaceutical chemistry.

The total effective amount of the composition of the present invention may be administered to a patient as a single dose, or may be administered by a fractionated treatment protocol in which multiple doses are administered for a long period of time. The pharmaceutical composition of the present invention may vary the content of the active ingredient depending on the severity of the disease. Preferably, the preferred total dose of the pharmaceutical composition of the present invention may be about 0.01 μg to 10,000 mg, more preferably 0.1 μg to 1,000 mg per kg of the patient's body weight per day. However, the dosage of the pharmaceutical composition is determined by considering various factors such as the formulation method, administration route and number of treatments, as well as the patient's age, weight, health status, sex, severity of disease, diet and excretion rate, etc., the effective dosage for the patient is determined. Therefore, in consideration of this point, those of ordinary skill in the art will be able to determine an appropriate effective dosage of the composition of the present invention. The pharmaceutical composition according to the present invention is not particularly limited in its formulation, administration route and administration method as long as the effect of the present invention is exhibited.

When provided in the form of the food composition, "food composition" includes all forms of functional food, nutritional supplement, health food, and food additives. Food compositions of this type can be prepared in various forms according to conventional methods known in the art. For example, as a health food, the composition itself may be prepared in the form of tea, juice, and drink to drink, or may be ingested by granulation, encapsulation, and powder. In addition, functional foods include beverages (including alcoholic beverages), fruits and their processed foods (eg canned fruit, canned fruit, jam, marmalade, etc.), fish, meat, and their processed foods (eg ham, sausage corn beef, etc.); Breads and noodles (eg udon noodles, soba noodles, ramen, spaghetti, macaroni, etc.), fruit juice, various drinks, cookies, syrup, dairy products (eg butter, cheese, etc.), edible vegetable oils and fats, margarine, vegetable protein, retort foods, It can be prepared by adding the extract to frozen food and various seasonings (eg, soybean paste, soy sauce, sauce, etc.).

In addition, in order to use the composition of the present invention in the form of a food additive, it may be prepared and used in the form of a powder or a concentrate. A preferred content of the extract of the sprout radish extract in the food composition may be in the range of 0.001 to 50%, preferably in the range of 0.1 to 50%, based on the total weight of the food composition.

Hereinafter, the present invention will be described in more detail with reference to specific experimental examples.

Manufacture of radish sprout extract

1 L of distilled water was added to 100 g of dried Sprout of Coix lacryma-jobi var. ma-yuen Stapf (supplied from Rural Development Administration, RDA), followed by extraction by boiling at 100° C. for 2 hours. The extract was filtered and concentrated to form a powder, which was named SC (yield 20%, water content 4.615%). SCs were stored at -80°C until just before use in experiments.

Materials and Methods

(1) reagent

Folin-Ciocalteu's phenol reagent, gallic acid, sodium carbonate, diethylene glycol, sodium hydroxide, potassium phosphate monobasic, potassium phosphate dibasic, naringin, 2-Diphenyl-1-picrylhydrazyl (DPPH) and 7 mM 2,2 used in this example '-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) was purchased from Sigma aldrich (St. Louis, MO, USA), and Dextran sodium sulfate (DSS) was obtained from MP Biologicals (Santa Ana, CA, USA). ), primary antibodies phosphorylation of nuclear factor-kappa B p65 (NF-ĸBp65), inhibitor of nuclear factor kappa B alpha (IĸBα), phosphorylation inhibitor of nuclear factor kappa B alpha (p-IĸBα), cyclooxygenase- 2 (COX-2), tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), interleukin-4 (IL-4), interleukin-10 (IL-10), nuclear factor erythroid 2 -related factor 2 (Nrf2), heme oxygenase-1 (HO-1), super oxide dismutase (SOD), Catalase, B-cell lymphoma 2 (Bcl2), Bcl-2-associated X (Bax), cysteine aspartyl-specific proteases-3 (caspase-3), histone, β-actin and secondary antibodies are produced by Santa Cruz Biotechnology (Santa Cruz, CA, USA), Protease inhibitor mixture, DMSO, and ethylenediaminetetraacetic acid (EDTA) were obtained from Wako Pure Chemical Industries, Ltd. (Osaka. Japan), Nitrocellulose membranes were purchased from Amersham GE Healthcare (Little. Chalfont, UK), and 2', 7' Dichlorofluorescein diacetate (DCFH-DA) and Dihydrorhodamine 123 were obtained from Molecular Probes (Eugene, OR, U.S.A. ), ECL Western Blotting Detection Reagents were purchased from GE Healthcare, and BCA protein assay kit for protein quantification was purchased from Thermo Scientific (Rockford, IL, USA).

(2) Antioxidant efficacy evaluation

1) Measurement of total polyphenol content

To measure the total polyphenol content of SC, the Folin-Denis method (see Non-Patent Document 11) was used. Add 50 µL of Folin-Ciocalteu's phenol reagent and 10 µL of SC to 790 µL of distilled water, mix well, add 150 µL of sodium carbonate solution (20% (v/v)), and react at room temperature for 2 hours. After the reaction, absorbance was measured at 765 nm. Gallic acid was used as a standard material.

2) Determination of total flavonoid content

To measure the total flavonoid content of SC, the method described in Lister (see Non-Patent Document 12) was used. Add 10 μL of 1 N sodium hydroxide solution and 100 μL of SC to 1 mL of diethylene glycol, mix well, and react at 37° C. for 1 hour to measure absorbance at 420 nm. As a standard material, naringin was used.

(3) Ulcerative colitis induction and efficacy evaluation

1) Ulcerative colitis induction and drug administration

Seven-week-old Balb/c mice (22-25 g) were purchased from Daehan Biolink (Eumseong, Korea), supplied with sufficient water and solid feed, and acclimatized to the laboratory environment for a week before use. The conditions of the animal breeding room were conventional system, and the light-dark cycle was adjusted to a 12-hour cycle, a temperature of 22±2℃, and a humidity of 50±5%. The experiment was conducted with the approval of the Animal Experiment Ethics Committee of Daegu Haany University (DHU2020-042), and the animal management regulations were followed. Experimental groups were the normal group (Normal), control group (Control), group administered orally with 100 mg/kg/day of sprouted radish hot water extract (SCL), and group administered orally with 200 mg/kg/day of sprouted radish hot water extract (SCH) After dividing into 4 groups of 8 animals in each group, they were acclimatized for a week, and then the experiment was conducted. Except for the normal group, 5% (w/v) DSS was supplied with drinking water for one week to induce ulcerative colitis. An autopsy was performed on the 8th day of induction. After fasting for 24 hours the day before autopsy, blood was collected from the heart by inhalation anesthesia using isoflurane, and serum was separated from the collected blood using a centrifuge at 4,000 rpm for 10 minutes. After that, the large intestine was removed and the length was measured, and the serum and large intestine were stored at -80°C.

2) Measurement of oxidative stress biomarkers

Serum was separated from the blood collected from the heart using a centrifuge at 4,000 rpm for 10 minutes. After mixing 25 mM DCFH-DA to measure reactive oxygen species (ROS) in serum, the emission wavelength is 530 nm and the excitation wavelength 485 nm using a fluorescence photometer at intervals of 5 minutes. was calculated through the values measured from 0 minutes to 30 minutes (see Non-Patent Document 13). And, in order to measure ONOO- ^, it was measured by implementing the method of Kooy et al. (refer to Non-Patent Document 14). Each sample was mixed with rhodamine buffer of pH 7.4 and 5 mM DHR123, shaken at 37°C for 5 minutes, and then measured at 5 minute intervals using emission 535 nm and excitation 480 nm for 30 minutes from 0 minutes. .

3) Measurement of liver toxicity indicators AST and ALT

Serum was separated from the blood collected from the heart using a centrifuge at 4,000 rpm for 10 minutes. Hepatotoxicity indices in serum were measured according to the protocol of alanine aminotransferanse (ALT) and aspartate aminotransferase (AST) assay kit (Wako Pure Chemical Industries, Ltd. Osaka, Japan).

4) Western blotting

For Western blotting analysis, 100 mM Tris-HCl (pH 7.4), 1.5 M sucrose, protease inhibitor cocktail, 0.1 M DTT, 2 mM MgCl ₂ , 15 mM CaCl ₂ and 5 mM Tris-HCl (pH 7.5) were added to colon tissue for Western blotting analysis. After the added buffer A was added, it was ground using a tissue grinder (Bio Spec Product, USA). After grinding, 10% NP-40 solution was added and left on ice for 20 minutes, followed by centrifugation at 12,000 rpm for 2 minutes to separate the supernatant including the cytoplasm. After removing the supernatant, rinse twice in buffer A containing 10% NP-40 and 100 µL of buffer C (0.1 mM EDTA, 1 mM DTT, 50 mM KCl, 50 mM HEPES, 0.3 mM NaCl, 0.1 mM PMSF and 10% glycerol) was added and resuspended, followed by vortexing three times at 10-minute intervals. Thereafter, the nuclei were separated by centrifugation at 4° C. at 12,000 rpm for 10 minutes. After separation, the cytoplasm and nucleus were stored frozen at 80°C. After electrophoresis of 8 to 10 μg of protein on 10 to 12% SDS-polyacrylamide gel to measure protein expression in the cytoplasm and nucleus, the acrylamide gel is transferred to a nitrocellulose membrane made it Then, each primary antibody was treated on the membrane, overnight at 4°C, washed with PBS-T, and the secondary antibody used for each treated primary antibody (diluted 1:3,000 with PBS-T) After use) for 2 hours at room temperature, washed with PBS-T, exposed to an enhanced chemiluminescence (ECL) solution, and after checking protein expression with Sensi-Q2000 Chemidoc, ATTO Densitograph Software (ATTO Corporation, Tokyo) , Japan) program was used to quantify the band, and the expression levels were compared between the experimental groups.

(4) Statistical analysis

In vitro and in vivo values were expressed as mean and standard deviation. After using a one-way analysis of variance (ANOVA) test using SPSS (Version 25.0, IBM, Armonk, NY, USA), a post hoc test was performed with the least-significant differences (LSD) test to determine the significance level as a p-value It was verified at <0.05.

Experiment result

(1) Determination of total polyphenol and total flavonoid content

To measure the antioxidant capacity of SC, total polyphenol and total flavonoid contents were measured, and the results are shown in Table 1 below.

Sample Total polyphenol (mg/g) Total flavonoids (mg/g) SC 74.35±0.18 17.31±0.04

Referring to Table 1, the total polyphenol content of SC was measured to be 74.35±0.18 mg/g, and the total flavonoid content was measured to be 17.31±0.04 mg/g.

(2) body weight and drinking water

Table 2 below shows the results of measuring the amount of change in body weight during the experiment.

Group Initial body weight (g) Final body weight (g) Body weight gain (g) 5% DSS intake (mL/day) Normal 23.18 ± 1.35 23.61 ± 1.48 0.63 ± 0.22 3.25 ± 0.26 Control 23.09 ± 1.03 19.64 ± 1.12 ^### -3.29 ± 0.13 ^### 3.28 ± 0.16 SCL 23.07 ± 1.28 20.39 ± 0.77 ^* -2.65 ± 0.25 ^* 3.21 ± 0.11 SCH 23.16 ± 1.04 21.25 ± 1.66 ^** -1.98 ± 0.27 ^*** 3.31 ± 0.19 [significance level] ^### p<0.001 compared vs. Normal group, ^* p<0.05, ^** p<0.01 and ^*** p<0.001 vs. control group

Referring to Table 2, the Control group significantly decreased to -3.29±0.13 g (p<0.001) compared to 0.63±0.22 g in the Normal group, and -2.65±0.25 g (p<0.05) in the SCL group compared to the Control group. , SCH group significantly increased to -1.98±0.27 g (p<0.001). During the experimental period, the daily average drinking water amount was 3.25±0.26 mL/day in the Normal group, 3.28±0.16 mL/day in the Control group, 3.21±0.11 mL/day in the SCL group, and 3.31±0.19 mL/day in the SCH group, indicating no significance between groups.

(3) effect of SC on visible damage

The results of measuring the length of the large intestine removed after the autopsy are shown in FIG. 1 .

Referring to FIG. 1 , compared to the Normal group, the colon length decreased by 32.90% in the Control group, and the SCL group and the SCH group increased by 6.69% and 13.33%, respectively, compared to the Control group.

(4) oxidative stress biomarker measurement results

Table 3 below shows the results of measuring ROS and ONOO- ^, which are biomarkers of oxidative stress in serum.

Group ROS (Fluoresence/min/mL) ONOO ^- (Fluoresence/mL) AST (IU/L) ALT (IU/L) Normal 581 ± 98 1515 ± 140 33.99 ± 1.91 3.83 ± 0.29 Control 1299 ± 240 ^## 1892 ± 430 ^## 41.04 ± 2.63 4.38 ± 0.68 SCL 743 ± 88 ^* 1591 ± 139 ^** 36.31 ± 3.96 4.07 ± 0.26 SCH 650 ± 113 ^** 1508 ± 105 ^** 37.01 ± 3.08 3.97 ± 0.46 [significance level] ^## p<0.01 compared vs. Normal group, ^* p<0.05 and ^** p<0.01 vs. control group

Referring to Table 3, ROS increased by 123.50% in the Control group compared to the Normal group (p<0.01), and compared to the Control group, the SCL group and the SCH group were significantly 42.79% (p<0.05) and 49.92% (p<0.05) and 49.92% ( p<0.01) decreased, and ONOO ^- was significantly increased in the Control group compared to the Normal group (p<0.01), and the reduction rate was significantly decreased to 15.89% and 20.30% in the SCL group and the SCH group compared to the Control group, respectively. .

(5) liver toxicity indicators AST and ALT measurement results

Table 3 shows the results of measuring AST and ALT, which are indicators of liver toxicity in serum.

Referring to Table 3, the AST increased by 20.74% in the Control group compared to the Normal group, and the SCL group decreased by 11.51% and the SCH group compared to the Control group by 9.81%, and the ALT increased by 14.43% in the Control group compared to the Normal group. Compared with the Control group, the SCL group decreased by 7.16% and the SCH group decreased by 9.32%.

(6) Analysis of expression level of antioxidant-related factors in colon tissue

The expression levels of Nrf2, HO-1, SOD, Catalase and GPx-1/2, which are antioxidant-related factors in colon tissue, were measured, and the results are shown in FIG. 2 .

Referring to FIG. 2 , compared to the Normal group, the control group significantly decreased (p<0.001), and the expression level was increased in the SC administration group compared to the Control group. In particular, Nrf2, Catalase, and GPx-1/2 increased significantly (p<0.01) in the SCH group compared to the Control group by 27.19%, 25.70%, and 44.52% (Fig. 2).

(7) Analysis of expression level of inflammation-related factors in colon tissue

1) NF-κBp65 and p-IκBα expression level analysis

The expression levels of NF-κBp65 and p-IκBα, which are inflammatory mediators, were measured, and the results are shown in FIG. 3 .

Referring to FIG. 3 , as a result of measuring the expression level of NF-κBp65, there was a significant (p<0.001) increase by 30.11% in the Control group compared to the Normal group, and 13.44% in the SCL group and the SCH group compared to the Control group, respectively. and 21.60% significantly (p<0.01 and p<0.001) decrease. As a result of measuring the expression level of p-IκBα, the control group increased significantly (p<0.001) by 31.87% compared to the normal group, and the SCL group and the SCH group were significantly increased by 14.85% and 18.83% compared to the Control group, respectively (p<0.001). 0.05, p<0.01) decreased.

2) COX-2, iNOS, TNF-α and IL-1β expression level analysis

The expression levels of COX-2, iNOS, TNF-α and IL-1β, which are inflammation-related factors, were measured, and the results are shown in FIG. 4 .

Referring to FIG. 4 , it could be confirmed that the expression level was significantly increased in the Control group compared to the Normal group, and all significantly decreased in the SC administration group compared to the Control group. Among them, COX-2 and iNOS decreased to 31.15% and 68.43% in the SCH group compared to the Control group, respectively, to the level of the Normal group.

(8) Measurement of expression level of anti-inflammatory factors in colon tissue

The expression levels of IL-4 and IL-10, which are anti-inflammatory factors in colon tissue, were measured, and the results are shown in FIG. 5 .

Referring to FIG. 5 , compared to the Normal group, the control group significantly (p<0.001) decreased, and compared to the Control group, the SC administration group significantly increased. Among them, it was confirmed that the SCH group increased IL-4 and IL-10 to 80.85% and 80.17%, respectively, compared to the Control group.

(9) Measurement of expression level of apoptosis-related factors in colon tissue

Expression levels of Bcl2, Bax and Caspase-3, which are factors related to apoptosis, were measured, and the results are shown in FIG. 6 .

Referring to FIG. 6 , as a result of measuring the expression level of Bcl2, there was a significant (p<0.001) decrease of 37.32% in the Control group compared to the Normal group, and 22.88% and 48.13% in the SCL group and the SCH group compared to the Control group, respectively. Significantly (p<0.05, p<0.001) increased. As a result of measuring the expression levels of Bax and Caspase-3, it was confirmed that the reduction rates of Bax and Caspase-3 in the SCH group were significantly reduced to 32.51% and 23.88%, respectively, compared to the Control group.

Coix lacryma-jobi var. ma-yeun Stapf) is a plant belonging to the Grapeaceae and is known to be native to Southeast Asia (see Non-Patent Document 8). Adulmu contains ingredients such as phenolic acids, lignan, benzoxazinold, and phenolic amides, and these ingredients are known to have anti-cancer, anti-inflammatory, anti-allergic, and whitening effects (see Non-Patent Document 15). These yulmu are used in many places for each part. The seeds are used as herbal medicines or food. Eui-in (薏苡仁) with the seed coat removed from the seeds is used for diseases such as cancer and arthritis, the roots are used for jaundice and neuralgia, and the stems and leaves are widely used as feed for livestock. known (refer to Non-Patent Documents 16 and 17).

Colitis is an inflammation of the mucous membrane or submucosal layer that invades from the rectum to the proximal part of the large intestine and causes symptoms such as abdominal pain and bloody diarrhea. It is not supported (refer to Non-Patent Documents 18 and 19). When looking at colitis from the point of view of oriental medicine, hwaseol (fire 泄), bloody stool (便血), intestinal wall (腸), intestinal wind (腸酸), diarrhea (泄瀉), hyeolri (膿血痢), redness (赤痢) and dysentery (痢疾) (see Non-Patent Document 20), and clinical studies include Seokhongjeongamibang (惜紅煎加味方), Guanggeonbitangagambang (補腸健脾湯加減方), and Pyeongwijiyutangami Bang(平胃地楡湯加味方), etc. Experimental studies include thief jiyutang (導赤地楡湯), jeoksodudangguisan (赤小豆當歸散), sodamjeongjangtang (消炎整腸湯),槐花散) and the like (see Non-Patent Document 3).

Sprout of Coix lacryma-jobi var. ma-yuen Stapf is a dried leaf part of Uiyin (薏苡仁). Currently, there are many studies on Uiyin (薏苡仁), but research on Sprout Yulmu is incomplete. Therefore, in the present invention, ulcerative colitis was analyzed by analyzing the expression of ROS, ONOO ^- , AST, ALT and antioxidative, inflammation, anti-inflammatory, and apoptosis-related proteins in colonic tissues in an animal model in which Ulcerative Colitis was induced by DSS in the present invention. It was investigated whether there was an improvement effect.

As described above, oxidative stress biomarkers ROS and ONOO ⁻ and liver toxicity indicators AST and ALT were measured through the serum obtained after the end of the experiment. If ROS is not normally eliminated and accumulates in the body, it puts cells in a state of oxidative stress, and ONOO ^- causes various chronic diseases such as inflammation in this state of oxidative stress (see Non-Patent Document 21) . As a result of measuring these ROS and ONOO ⁻ , it was confirmed that they were significantly decreased in the group administered with the extract of Sprout radish hot water compared to the Control group. As a result of measuring AST and ALT, which are indicators of hepatotoxicity, compared to the control group, it was confirmed that there was a decrease, although not significant, in the group administered with the hot water extract.

The expression levels of antioxidant, inflammation, anti-inflammatory and apoptosis-related proteins in colonic tissues obtained after the end of the experiment were measured using western blot. Nrf2, known as a transcription factor regulating oxidative stress, binds to the antioxidant response element (ARE) and activates important antioxidant enzymes. These activated antioxidant enzymes suppress the production of free radicals (see Non-Patent Document 22). As a result of measuring the protein expression levels of Nrf2 and antioxidant enzymes HO-1, SOD, Catalase and GPx-1/2, it was confirmed that there was a significant increase in the group administered with the hot water extract of sprout radish compared to the control group.

Transcriptional regulators NF-κBp65 and p-IκBα activated by various inflammatory cytokines, inflammatory cytokines and mediators COX-2, iNOS, TNF-α and IL-1β, and anti-inflammatory proteins IL-4 and IL The expression level of -10 was analyzed. Inactive NF-κB is present in the cytoplasm and inhibitor IκB. Such NF-κB moves to the nucleus when inhibitor IκB is stimulated and activated by various cytokines and is degraded through phosphorylation, thereby causing transcription of target genes (see Non-Patent Documents 23 to 26). As a result of measuring the expression levels of these inflammation-related proteins, it was confirmed that the expression levels of the hot water extract-administered group of sprouted radish radish were decreased compared to the control group, and in particular, COX-2 and iNOS decreased to the level of the Normal group. As a result of measuring the expression levels of IL-4 and IL-10, which are anti-inflammatory-related proteins, it was confirmed that the expression levels were increased to the level of the Normal group in the group administered with the hot water extract of sprouted radish compared to the Control group, which is an animal model in which colitis was induced.

Ulcerative colitis is a representative disease causing cancer due to inflammation, and it is known that the incidence of colorectal cancer is 5.7 times higher than that of a group without colitis symptoms (see Non-Patent Documents 27 and 28). Therefore, as a result of measuring the expression levels of Bcl2, Bax, and Caspase-3, which are factors related to apoptosis in the colon tissue, the expression level of Bcl2 increased in the group administered with the hot water extract of Sprout radish compared to the control group, and the expression levels of Bax and Caspase-3 decrease could be observed.

Through these results, it could be confirmed that the hot water extract of sprouted radish radish had an improvement effect in the DSS-induced ulcerative colitis animal model.

From the above, the present invention confirmed the improvement effect of ulcerative colitis by orally administering the DSS ulcerative colitis-induced animal sprout radish hot water extract, and the following results were obtained.

As a result of measuring the amount of change in body weight during the experiment, it was confirmed that the hot water extract of sprout radish significantly increased the body weight compared to the control group.

As a result of measuring ROS and ONOO-, which are oxidative stress biomarkers in the serum of an animal model inducing colitis, the reduction rate of ROS 49.92% and ONOO- 20.30% in the group administered with high concentration of the hot water extract of sprout radish was 49.92% and ONOO ^- 20.30%, the level of the Normal group ^. It can be seen that the decrease to

As a result of measuring AST and ALT, which are indicators of liver toxicity in serum, it was confirmed that the group administered with sprouted radish hot water extract decreased compared to the control group.

As a result of measuring the protein expression levels of Nrf2 and the antioxidant enzymes HO-1, SOD, Catalase and GPx-1/2 in the colon tissue, the high concentration of the hot water extract of sprout sprouts was significantly increased compared to the control group.

As a result of measuring the expression level of inflammation-related factors in the colon tissue, it was confirmed that the phosphorylation of IκBα was decreased in the group administered with the extract of sprouted radish radish compared to the control group, thereby significantly reducing the expression of NF-κBp65. In addition, it was confirmed that the expression levels of COX-2, iNOS, TNF-α and IL-1β were significantly decreased in the group administered with the hot water extract of Sprout radish.

As a result of measuring the expression of anti-inflammatory factors in the colon tissue, the protein expression levels of IL-4 and IL-10 were significantly increased in the colitis-induced animal model due to the administration of the hot water extract of sprouted radish.

As a result of measuring the protein expression levels of Bcl2, Bax, and Caspase-3, which are factors related to apoptosis, it was confirmed that Bcl2 was significantly increased in the hot water extract-treated group, and the expression levels of Bax and Caspase-3 were significantly decreased. could

Therefore, through the results of measuring the change in body weight and the protein expression levels of antioxidant-related factors, inflammation-related factors, anti-inflammatory factors, and apoptosis-related factors in DSS ulcerative colitis-induced animal models, the effect of hot water extract on ulcerative colitis improvement It can be confirmed that there is

Hereinafter, an application preparation example of the pharmaceutical composition comprising the hot water extract of buds chrysanthemum according to the present invention as an active ingredient will be described, but the present invention is not limited thereto.

Preparation Example 1: Preparation of powder

Table 4 below exemplifies the composition of the powder containing the hot water extract of sprouted radish according to the present invention as an active ingredient. The powder may be prepared by mixing the following ingredients and filling the airtight cloth.

ingredient content Sprout radish hot water extract 200mg lactose 100mg talc 10mg

Preparation Example 2: Preparation of tablets

Table 5 below exemplifies the tablet composition containing the hot water extract of sprouted radish according to the present invention as an active ingredient. Tablets can be prepared by mixing the following ingredients and then tableting according to a conventional tablet manufacturing method.

ingredient content Sprout radish hot water extract 200mg corn starch 100mg lactose 100mg magnesium stearate 2mg

Preparation Example 3: Preparation of capsules

Table 6 below exemplifies the composition of capsules containing the above-mentioned hot water extract of sprouted radish according to the present invention as an active ingredient. Capsules can be prepared by mixing the following ingredients according to a conventional capsule preparation method and filling the gelatin capsules.

ingredient content Sprout radish hot water extract 200mg crystalline cellulose 3mg lactose 14.8mg magnesium stearate 0.2mg

Preparation Example 4: Preparation of liquid preparation

Table 7 below exemplifies the composition of the liquid formulation containing the hot water extract of buds saprum radish according to the present invention as an active ingredient. The liquid preparation is prepared by adding and dissolving each of the following ingredients in purified water according to a conventional liquid preparation method, adding an appropriate amount of lemon flavor, mixing the following ingredients, adding purified water to adjust the total to 100 ml, filling and sterilizing a brown bottle can

ingredient content Sprout radish hot water extract 200mg Lee Seonghwadang 10g mannitol 5g Purified water appropriate amount

Preparation Example 5: Manufacturing of health food

Table 8 below shows the composition of a functional health food containing the hot water extract of Sprout radish according to the present invention as an active ingredient. In the following composition, the composition ratio of the vitamin and mineral mixture indicates that a composition suitable for relatively healthy food is mixed as a preferred formulation example, but the mixing ratio may be arbitrarily modified, and each component is mixed according to a conventional health food manufacturing method Then, the granules can be prepared and used in the preparation of a health food composition according to a conventional method.

ingredient content ingredient content Sprout radish hot water extract 1000mg folic acid 50 μg vitamin mixture appropriate amount Calcium Pantothenate 0.5mg vitamin A acetate 70 μg mineral mixture appropriate amount vitamin E 1.0mg ferrous sulfate 1.75mg vitamin B1 0.13mg zinc oxide 0.82mg vitamin B2 0.15mg magnesium carbonate 25.3mg vitamin B6 0.5mg monobasic potassium phosphate 15mg vitamin B12 0.2 μg Dibasic Potassium Phosphate 55mg vitamin C 10mg potassium citrate 90mg biotin 10 μg calcium carbonate 100mg Nicotinamide 1.7mg magnesium chloride 24.8mg

Preparation Example 6: Health Beverage Manufacturing

Table 9 below shows the composition of a functional health drink containing, as an active ingredient, the hot water extract of sprouted radish according to the present invention. For health drink production, for example, the following ingredients are mixed according to a conventional health drink production method, then stirred and heated at 85° C. for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2L container, sealed and sterilized, and then refrigerated. It can be stored and used. The composition ratio below was prepared by mixing ingredients suitable for relatively favorite beverages in a preferred embodiment, but the mixing ratio may be arbitrarily modified according to regional and national preferences such as demand class, demanding country, and use.

ingredient content Sprout radish hot water extract 1000mg citric acid 1000mg oligosaccharide 100g Plum Concentrate 2g Taurine 1 g purified water mix Total 900ml

Preferred embodiments of the present invention described above are disclosed to solve the technical problem, and various modifications, changes, additions, etc. will be possible within the spirit and scope of the present invention by those of ordinary skill in the art to which the present invention pertains. , such modifications and changes should be regarded as belonging to the following claims.

Claims (4)

A pharmaceutical composition for the prevention or treatment of ulcerative colitis, comprising the extract of sprout radish as an active ingredient. The method of claim 1,
The extract is a pharmaceutical composition for the prevention or treatment of ulcerative colitis, characterized in that the hot water extract. A food composition for the prevention or improvement of ulcerative colitis, comprising the extract of sprout radish as an active ingredient. The method of claim 1,
The extract is a food composition for preventing or improving ulcerative colitis, characterized in that it is a hot water extract.

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