KR101706817B1 - Composition comprising radish leave extract for anti-inflammatory and anti-oxidant - Google Patents

Abstract

The present invention relates to a dietary composition for diets containing an extract powder obtained by pulverizing a crude extract having the antioxidative and antiinflammatory activity and the ulcerative colitis-preventing effect of the extract of Aspergillus oryzae, A feed composition for anti-inflammation and antioxidation.

Description

[0001] The present invention relates to an anti-inflammatory and anti-oxidant composition for anti-inflammation and antioxidation,

The present invention relates to a feed composition for anti-inflammation and antioxidation comprising an extract of Wujing, and more particularly to a composition for preventing or treating antioxidative and anti-inflammatory activity and ulcerative colitis of the Wujing extract or an extract powder obtained by pulverizing the Wujing extract To a dietary composition for feed use.

Inflammatory bowel disease (IBD) is a common high-risk disease of colon cancer (see Terzic J, Grivennikov S, Karin E, Karin M. 2010. Inflammation and colon cancer. Gastroenterology 138: 2101-14e5) The ulcerative colitis (UC) and Crohn's disease (CD) are divided according to the lesion and inflammatory symptoms (Fiocchi C. 1998. Inflammatory bowel disease, etiology and pathogenesis. Gastroenterology. 115: 182-205 Reference). Both are difficult to cure and repeat improvement and deterioration, and have similar pathological signs such as weight loss, diarrhea with blood, fever, intestinal motility disorder, and shortening of the colon (Hendrickson BA, Gokhale R, Cho JH 2000. Clinical aspects and pathophysiology of inflammatory bowel diesease. Clin Microbiol Rev. 15 (1): 79-94).

In particular, UC has not been subjected to a large-scale epidemiological survey, but the incidence of UC was 0.2 per 100,000 population in 1986-1988 (Chang DK, Kim YH, Byeon JS, The present state of ulcerative colitis-associated colorectal cancer in Korea: a KASID (Korean Society for Thoracic and Cardiovascular Surgery), Seoul, Korea. study, Korean J Gastroenterol. 46 (4): 276-282), increased to 1.23 persons between 1995 and 1997, and increased to 3.08 persons per 100,000 population between 2001 and 2005 (Yang SK, Hong WS , Min YI, 2000. Incidence and prevalence of uncer- tative colitis in the Songpa-Kangdong District, Seoul, Korea, 1986-1997 J Gastroenterol Hepatol 15: 1037-1042). The prevalence also increased steeply from 7.57 per 100,000 population in 1997 to 30.87 in 2005 (Yang SK, Yun S, Kim JH, Park JY, Kim YH, Chang DK, Kim JS, Song PS, Park JB, Park (KASID Study, Inflammatory Bowel Diseases 14 (4): 542-549). In this study, we compared the incidence of infarcted bowel disease with the incidence of infectious diseases in Korea. In North America, the annual incidence was 6.0-15.6 per 100,000 population and 1.5-20.3 in Europe, and the prevalence was reported to be 21.4-246 according to the region (Loftus E. 2004. Clinical epidemiology of inflammatory Gastroenterology 126 (6): 1504-1517), and the prevalence of UC in Korea is an uncommon disease compared to the incidence in North America and Europe, but the trend is steadily increasing (Yang SK, 2002. Current status and clinical characteristics of inflammatory bowel disease in Korea. Korean J Gastroenterol 40 (1): 1-4).

Many therapeutic agents have been used for this purpose, and glucocorticoids (Boumpas DT, Chrousos GP, Wilder RL, 1993. Glucocorticoid therapy for immune-mediated diseases: basic and therapeutic agents for ulcerative colitis (UC) Clinical correlates. Ann Intern Med 119: 1198-1208) and sulfasalazine (Msiewicz JJ, Lennard-hones JE, Connell AM, 1965. Controlled trial of sulfasalazine in maintenance therapy for ulcerative colitis. Lancet: 185-188) But it also causes side effects such as nausea, vomiting, dyspepsia, anorexia and urticaria, fever, hepatitis, hemolytic anemia, bone marrow suppression, and the like, by killing useful bacterial guns in the intestine (Das KM, Eastwood MA, McManus JP, Sircus W. 1973. Adverse reactions during salicylazosulphapyridine therapy and the relationship with drug metabolism and acetylator phenotype M Engl J Med 289: 491-495; Dignassa, Layer P. 1995. Inflammatory bo ulcerative colitis occurs in 18% of patients within 30 years of the disease (Canavan C, Abrams KR, Mayberry J < RTI ID = 0.0 > 2006. Meta-analysis: colorectal and small bowel cancer risk in patients with Crohn's disease. Aliment Pharmacol Ther 23: 1097-104) have been reported (Eaden JA, Abrams KR, Mayberry JF. The risk of colorectal cancer in ulcerative colitis: a meta-analysis. Gut 2001; 48: 526-35 (Ullman T, Croog V, Harpaz N, Sachar D, Itzkowitz S. 2003. Progression of flat low-grade dysplasia to advanced neoplasia in patients with ulcerative colitis. Gastroenterology 125: 1311 -1319).

Ulcerative colitis is caused by a combination of various environmental factors and genetic factors. However, the main cause of ulcerative colitis is still unclear. However, in Japan, the incidence of ulcerative colitis has increased since the 1960s when consumption of animal proteins such as meat and dairy products began to increase (Yosida Y, Murata Y. 1990. Inflammatory bowel disease in Japan: studies of epidemiology and etiopathogenesis. Med Clin North Am 74: 67-90). In Korea, the incidence of ulcerative colitis increased from 1980 to the mid 1990s when the daily intake of animal protein per person increased (Ministry of Health & Welfare. (See also Report on 1998 national health and nutrition survey, pp. 38-140), but it is more prevalent in industrialized countries such as the US and Europe than in underdeveloped countries, Increasingly, dietary habits have been altered by the consumption of more calories than necessary and the consumption of animal foods and the low intake of vegetable foods, including vegetables and fruits I believe the closely related benign colitis (literature Doll R, Peto R. 1981. The causes of cancer Quantitative estimates of avoidable risks of cancer in the Unitede States today J Natl Cancer Inst 66: 1191-1308, see.).

Rief et al. Reported that fat intake was associated with an increased incidence of ulcerative colitis, and that intake of fruits, vegetables and dietary fiber reduced this (Rief S, Klein I, Lubin F, Farbstein M, Hallake , Gilat T., 1997. Gut 40 (6): 754-760). In another study, a comparative study of patient-control groups showed that excessive sugar intake (Sakamoto N, Kono S, Wakai K, Fukuda Y, Satomi M, Shimoyama T, Inada Y, Miyake Y, Sasaki S, Okamoto K, Kobashi G, Washio M, Yokoyama T. Date C, Tanaka H. 2005. Dietary risk factors for inflammatory bowel disease: a multicenter case-control study in Japan. Inflamm Bowel Dis 11 (2): 154-163).

The inflammatory response of ulcerative colitis is promoted by oxidative stress in the body (Choi, Su-Yeon, 2004. In the general diet and high-fat diets, quercetin or -carotene supplementation is associated with chemically induced colon cancer development and colonic mucosal inflammatory response A study on the effects of the research. Oxidative stress produces reactive oxygen species (ROS), which react with macromolecules such as intracellular DNA, proteins, lipids, and carbohydrates to cause DNA segmentation, inactivation of proteins, and peroxidation of unsaturated fatty acids (Volko M, Leibfritz D, Moncol J, Cronin MT, Mazur M, Telser J, 2007. Free radicals and antioxidants in normal physiological func- tions and human disease. Nt J. Biochem Cell Biol 39: 44 -84). An excess accumulation of ROS causes damage to DNA and various molecules in the cell. Therefore, there is an antioxidant system in the body to improve it (Andersen HR, Nielsen JB, Nielsen F, Grandjean P. 1997. Antioxidative enzyme activities in human erythrocytes Clin. Chem. 43: 562-568). The main components of the antioxidant system are functional substances such as vitamins C, E, A and minerals and carotenoids, most of which can be obtained from plant foods.

That is, the intake of vegetable foods has a positive effect on the antioxidant level of the human body (Liu RH, Finley J. Potential cell culture models for antioxidant research, 2005. J Agric Food Chem 53: 4311-4314) The higher the proportion of animal food is, the more unbalanced the antioxidant system. Therefore, it is important to increase the proportion of vegetable foods in the diet for prevention and treatment of ulcerative colitis.

Despite these efforts, there is still a myriad of unknown antioxidants in the natural world. Therefore, the need for research and development of natural antioxidant materials with better antioxidant activity is increasing day by day.

Radish is one of the major vegetables that contain large amounts of nutrients such as vitamins in roots and leaves. In particular, leafless chlorophyll is widely distributed on the leafless leaves and has a low sugar content, while vitamin A, B1, B2, C, niacin, protein, lipid, iron and calcium are uniformly distributed, It is known that it is good for prevention of adult diseases by food. According to recent research, it is useful for the treatment of obesity and constipation, and it is reported to be beneficial for gastrointestinal because it is contained starch hydrolyzate diastase (diastase) It is occupied. Wuchung is the upper part of the radish root, stem and leaf part, and the dried seagrass (seagrass and / or non-seaweed) is the favorite food of most of the Korean people In addition to being limited in harvest time, it has not been commercialized so that it can be commonly consumed throughout the year. Therefore, it does not fulfill its role as a commodity like other traditional foods. As for the process of manufacturing the seagrass, the raw seaweed collected from radishes is knitted with straw, rope, nylon string, hanging in a proper place, naturally dried, and then relying on a pre-modern method There is a lot of nutrition loss, and because it is unsanitary, the value of food is inevitably falling. In consideration of this point, various food manufacturing methods that can utilize Wuqing have been proposed, but the manufacturing cost is expensive and the commerciality is poor, which is far from popularization.

Korean Patent Laid-Open Publication No. 2004-0011578 describes a method of producing food using mushrooms. Here, a method of producing food using mushrooms is described in which products (dry seaweeds and / or wet dairy cows) (Regularized), can be popularized, and packaged together with spices, according to the taste of the consumer, chikenjyeon, or added to the cooked food is described in the description.

It is an object of the present invention to provide a dietary composition containing an aspartic acid extract having an antioxidative and anti-inflammatory activity and an ulcerative colitis-preventing effect, or an extract powder obtained by pulverizing the aspartame extract.

The composition for anti-inflammation and antioxidation according to the present invention is prepared by extracting the extracts of Wuchung, wherein the extraction ratio is 90 to 99% by weight of ethanol with 70 to 90% (v / v) And 1 to 10% by weight of the crude dry powder, suspending the mixture at room temperature for 40 to 54 hours, further immersing the mixture for 20 to 28 hours, filtering the mixture by repeating 1 to 5 times, And the mixture is concentrated at a temperature of 1 to 20 占 폚.

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The concentrate can be subsequently lyophilized to produce an extract powder, and the lyophilized extract powder can be used by dissolving in ethanol of 70 to 90% (v / v) again.

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According to the present invention, functional ingredients having antioxidative activity of Wujing extract reduce the oxidative stress caused by inflammation, so that the extract of Wuchuan, which can reduce the blood, weight loss and dehydration accompanying the ulcerative colitis inflammation, And a composition for anti-inflammation and antioxidation.

FIG. 1 is an electrophoresis image showing various intestinal flora obtained by electrophoresis on denatured washing gels in normal group, control group, Wuqing class group, and Pseudomonas group.
2 is a photograph showing the appearance of inflammatory cells in the colon.

Hereinafter, specific embodiments of the present invention will be described in detail with reference to the accompanying drawings.

In particular, the extract of Wuchuan is selected from the group consisting of alcohols selected from the group consisting of lower alcohols having 1 to 4 carbon atoms, preferably ethanol, more preferably 50 - , And 90% (v / v) ethanol, most preferably 70 to 90% (v / v) ethanol as an extraction solvent. Particularly, the mulberry extract used in the production of the extract of Wuchuan is a part of the upper part of radish root, that is, stem and leaf part, except for the radish root which is mainly used for cooking, and the chlorophyll is widely distributed, While it is low in vitamin A, B1, B2, C, niacin, protein, lipid, iron and calcium, and is rich in fiber foods. The Wuchung extract may be obtained by extracting the dried powder obtained by washing with water, drying, preferably lyophilization, and pulverization, with an extraction solvent, preferably ethanol. The freeze-drying and pulverization may be performed by a person skilled in the art The present invention is not limited to the method of lyophilization and pulverization or the processing conditions.

In order to prevent this problem, the conventional method of making a mushroom is to dry the mushroom through freeze - drying, which is a drying method, because the amount of minerals and nutrients contained in the mushrooms is small and the contamination degree of the sample is large. The temperature is the most important factor in the extraction and concentration of dried Sagae through lyophilization. When heat is applied during the extraction and concentration process, the usable component of the wheat germ is destroyed, and the amount of the remaining useful ingredient is also reduced. Therefore, in this experiment, the concentration never exceeded 20 ° C and the possibility of the change of the extracted useful ingredient was reduced.

In the present invention, the term " silken " means that leaves and roots other than rootless roots are included in both biological state and dry state, and it should be understood that the term " roots "

In addition to the anticancer components such as glyconasturtin, glycosinolate, methyl mercaptane and mustard oil, as well as anti-cancer components such as harmful active oxygen scavenging Beta-carotene, anthocyanin (Malisorn C, Suntornsuk W. 2008. Improved-carotene production of Rhodotorula glutinis in fermented radish brine by continuous cultivation, Biochem Engineer J doi: 10.1016 / j.bej.2008.08. 005), chlorophyll (see Yu BP, 1996. Aging and antioxidative stress: Modulation by dietary restriction, Free Rad Biol Med 21: 651-668), vitamin C (literature Anatol K, Ulrike M, Sonke A, In the present study, we found that more than 35% of the dry matter was found in the dietary intake of Alzheimer's disease (Alzheimer's disease, Free Rad Biol Med 31: 345-354) Fiber, and 2 It is a good quality food containing 0% protein, iron, calcium, etc. (Ku KH, Lee KA, Kim YL, Lee YW 2006. Quality characteristics of hot-air dried radish (Raphanus sativus L.) leaves. Korean Soc Food Sci Nutr 34: 780-550).

The results of this study are as follows: 1) Changes in mineral content of commercial vegetables according to cooking methods (Han, JS, Kim, MS, Choi, YH 1999. Changes in minerals contents of vegetable by various cooking methods. Korean J Soc Food Sci 15: 382- 387), protein extraction from aquaculture for use as feed (Yasui T. 1996. Effect of the pretreatment with dilute hydrochloric acid solutions on the extractability of grass proteins. Nippon Eiyo Shokuryo Gakkaishi 49: 29-37; Yasui T. et al. (See, for example, Yim HB, Lee GS, Chae et al., 1995), and the effect of ethanol extract of Seagrass on lung cancer HJ. 2004. Cytotoxicity of ethanol extracts of Raphanus sativus on a human lung cancer cell line, J Korean Soc Food Sci Nutr 33: 287-290), the gastrointestinal stimulation of uterus extract and the uterine contraction activity (Ghayur MN, Gilani AH 2005. Ga (Gilani AH, Ghayur MN, 2004. Pharmacological basis for the gut stimulatory activity of Raphanus sativus leaves. . J Ethnopharmacol 95: 169-172) and physiological activity (Ku KH, Lee KA, Kim YE. 2008. Physiological Activity of Extracts from Radish (Raphanus sativus L.) Leaves. J Korean Soc Food Sci Nutr 37 390-395) have been reported. In addition, antihypertensive effects of Sira powder (Kim BR, Park JH, Kim SH, Cho KJ, Chang MJ. 2010. Antihypertensive Properties of Dried Radish Leaves Powder in Spontaneously Hypertensive Rats. J Nutr 43 (6): 561 ~ 569 ) And cholesterol diet-fed rats (Rhee SJ, Ahn JM, Ku KH, Choi JH, 2005. Effects of Radish Leaves Powder on Hepatic Antioxidative System in Rats Fed High Cholesterol Diet. J Korean Soc Food Sci Nutr 34 8): 1157-1163) and improvement of bowel function and lipid metabolism (Jang HS, Ahn JM, Ku KH, Rhee SJ, Kang SK, Choi JH 2008. Effect of Radish Leaves Powder on Gastrointestinal Function and Fecal Triglyceride Sterol Excretion in Rats Fed a Hypercholesterolemic Diet. J Korean Soc Food Sci Nutr 37 (10): 1258-1263). However, there have been few studies on direct colonic inflammation.

The extract of Wuchuan is prepared by extracting the extracts with a ratio of 90 to 99% by weight of ethanol with 70% to 90% (v / v) of the extract and 1 to 10% , Suspending the mixture at room temperature for 40 to 54 hours, immersing again for 20 to 28 hours, filtering the mixture 1 to 5 times, and concentrating the extracts at a temperature of 1 to 20 ° C.

The concentrate thus obtained may be subsequently freeze-dried to produce an extract powder.

The lyophilized extract powder can be used again by dissolving in 70% to 90% (v / v) ethanol.

The Wuchuan extract may be lyophilized to make a granular dietary powder.

Hereinafter, preferred embodiments and comparative examples of the present invention will be described.

The following examples are intended to illustrate the invention and should not be construed as limiting the scope of the invention.

1. Preparation of extract and dietary powder

1) Preparing wuchu extract

In order to prevent the destruction of pollutants and nutrients, the domestic aquaculture purchased from general marts was thoroughly washed with running water, lyophilized, and then pulverized with a blender to obtain a dry powder in the form of particles. Then, 80% (v / v) (EtOH) as a solvent to a concentration of 5%. At this time, more specifically, 5% by weight of non-woven dry powder was mixed with 95% by weight of 80% (v / v) ethanol, suspended at room temperature for 48 hours, allowed to stand for another 24 hours and then filtered. This process was repeated three times, and the obtained extracts were collected and concentrated under reduced pressure at a temperature of 20 DEG C or less. The obtained concentrate was freeze-dried to obtain an extract powder (solid), which was used in the subsequent experiments.

The lyophilized extract powder of the freeze-dried extract powder was dissolved in ethanol at a ratio of 1: 19 (19 parts by weight of 99.9% (v / v) ethanol as a unit used to dissolve the powder in the liquid) , And used for the antioxidant activity test.

The lyophilized extract powder of the lyophilized extract powder was pulverized and used as a dietary powder in an animal experiment.

2) Experimental animal feed of granular dietary powder containing Wujing extract

Fifty - four week old three - week - old imprinting control regions (ICR) mice were divided into normal, irritated, and inflammatory feed groups. The prescription is summarized in Table 1 below. Experimental animals were maintained under controlled environmental conditions of 50% humidity and 25 ℃.

ingredient N, DN DV Corn starch 38 33 Sucrose 20 20 Casein 20 20 L-cystein 0.3 0.3 Soybean oil 10 10 Cellulose powder 5 5 Vitamin mixture One One Mineral mixture 3.7 3.7 Choline chloride 2 2 Dietary powder - 5 * N: Normal group
* DN: inflammation-inducing group (inflammation induced by AIN-93G feed + DSS)
* DV: Inflammation-induced dietary powder (AIN-93G feed + powdered diet + inflammation induced by DSS)
Unit: wt%

3) Experimental animals and breeding conditions

Seventy-four week old ICR mice were divided into normal group, inflammation induction group and inflammation induction group, and AIN-93G feed was provided. In the inflammatory induction powder group, each of the above dietary powders was mixed at a ratio of 5% To provide feeds. The preparation method is shown in Table 1, and the experimental animals were bred at a controlled humidity of 50% at a temperature of 25 ° C, and the light-dark cycle was adjusted at a cycle of 12 hours.

4) Schedule of experiment and induction of colitis

The duration of the experiment was divided into 5 weeks of total, 4 weeks of non-inflammatory period of colonic inflammation and 5 days of colon inflammation induction period. According to the method of Fukata et al., 3% of Dextran sulfate sodium (molecular weight 36,000 to 50,000 daltons (Da)) to induce colitis.

For 5 days, all groups except the normal group were fed with drinking water containing 3% DSS because of macrophages, and macrophages were uniformly distributed throughout the intrinsic plate and submucosal layer, but inflammation from the 6th day after exposure to DSS The number of macrophages increased in the infiltrated area, so inflammation was induced for 5 days in order to obtain the best accuracy of inflammation - induced colon damage.

5) Measurement of weight gain, dietary and drinking water intake

Weight gain, dietary and drinking water intake were measured once a week before inflammation induction and daily after inflammation. Based on this, the dietary efficiency and DSS intake by population were calculated by the following equation (1). Blood counts and weights were also collected daily for 5 days during which colitis was induced by 3% DSS.

[Equation 1]

Food Efficiency Ratio = Weight gain (g / day) / Dietary intake (g / day)

DSS load = (total negative volume (ml) * DSS concentration (mg / ml)) / initial weight (g)

6) Sacrifice and sample collection

① Measure length of colon

The animals were fasted for 12 hours on the day before sacrifice and then anesthetized with carbon dioxide (CO ₂ ), and then the colon was removed from the cecum to the anus immediately before the anus. The extracted organs were washed with physiological saline to remove blood and fat, and the water was sucked into the absorbent paper and then the length was measured.

② Histological evaluation of colitis

The colon tissue fixed to the neutral formalin was taken into the paraffin through a general tissue treatment process after taking one piece of the proximal, middle, and distal portions within 48 hours, and the section was cut to a thickness of 5 to 6. Hematoxylin and eosin Hematoxylin & Eosin) solution, and the degree of ulceration and inflammation were calculated as the sum of the scores.

③ Reading standard

The findings of DSS-induced colonic inflammation were read and scored according to Yuan's criteria (Table 2). Scores were expressed as the mean and standard deviation for each group, and the degree of inflammation was divided into 6 groups.

Rating Criteria for score of mucosal injury Degree of rating 0 Intact crypts (intact crypts) 0% One Cystic dilatation 1 to 20% 2 Basal 1/3 loss of celiac gland 20 to 40% 3 Basal 2/3 loss of celiac gland 40 to 60% 4 Loss of whole cecal vessels with epithelial surface remaining intact 60 to 80% 5 Loss of whole cortex and epithelial surface 80 to 100%

7) Comparison of intestinal microbial changes in colitis induced animals

The control group and the dietary powder-fed group were separated and subjected to animal experiments. The colon tissues were extracted and sectioned into a rice grain size of about 3 to 5 쨉 m. Then, a DNA miniprep kit for tissue (manufactured by Kia Motors Corporation DNA was isolated using Qi cube (Qiagen Co.) and QIAcube (Kia Kogen Co., USA), amplified only 16S rDNA gene of bacterial genomic DNA with 338f-GC and 518r primer pair, (denaturing detergent gel) to analyze intestinal microbial patterns.

2. Antioxidant component

1) Total polyphenol content

2 μl of 2% Na ₂ CO ₃ was added to 200 μl of the Wuchung extract and mixed with 1M FC reagent (50% Folin-ciocalteu's phenol regent, 2N 100 mg / ml in primary distilled water (Distilled water)) (200 mu l of Sigma Co., USA) was added and reacted at room temperature for 30 minutes, and absorbance was measured at 750 nm. Tannin acid (Sigma Co., USA) was used as a control group And the calibration curve was calculated by the following equation (2).

&Quot; (2) "

Calibration curve formula: Y = aX + b

Where a is the slope and b is the intercept. These a and b are the concentration of the sample stock solution (X ) From the equation created for the inhibition concentration (Y). The total polyphenol content in the samples was calculated by the following equation (3).

&Quot; (3) "

Total polyphenol content in the sample = Y * V / S

Y is the Y value obtained by substituting the absorbance value of the sample into the calibration curve, V is the volume of the extraction solvent used in the preparation of the sample extract, and S is the weight of the extracted sample.

2) Total flavonoid content

10 ml of diethylenglycol was added to 1 ml of the extract and 1 ml of 1 N sodium hydroxide (NaOH) was added. After reacting at room temperature for 1 hour, the absorbance was measured with a spectrophotometer at 420 nm. Gallic acid (Sigma, USA) was used as a control. The calibration curve formula is shown in Equation 4 below.

&Quot; (4) "

Calibration curve formula: Y = aX + b

Where a is the slope and b is the intercept, these a and b are the inhibition concentrations for the stepwise concentration (X) of the sample stock solution, and X is the absorbance value for each concentration of the sample liquid, Y is the concentration of the sample liquid, (Y). ≪ / RTI >

The total flavonoid content in the sample was calculated by the following equation (5).

&Quot; (5) "

Total flavonoid content in the sample = Y * V / S

Where Y is the Y value obtained by substituting the absorbance value of the sample into the calibration curve, V is the volume of the extraction solvent used in the step of preparing the Wuchuan extract, and S is the weight of the extracted sample.

2) Antioxidant activity

(1) Electronic Difficulty

Electron donating ability (EDA) is measured by the electron donating effect of DPPH (1,1-diphenyl-2-picrydrazyl) on the sample. (Chu SM, 2007. Antioxidative and antimicrobial activities of medicinal plants collected from Nepal, Dankook University, Master's thesis). 100 μl of each of the wells of the 96-well plate diluted with the concentration was diluted, and 150 μl of a solution of 150 μM DPPH (59.145 mg / ml in methanol (MeOH): Sigma Co., USA) And the mixture was reacted at room temperature for 30 minutes, and absorbance was measured at 518 nm. Ascorbic acid (Sigma, USA) was used as a control. The inhibitory concentration was calculated by the following equation (6).

&Quot; (6) "

Inhibitory concentration (%) = (1-D / C) * 100

Y = aX + b

IC ₅₀ = (0.5-b) / a

Where a is the slope and b is the intercept, these a and b are the inhibitory concentrations (X) for the stepwise concentration (X) of the sample stock solution, D is the absorbance of the Wuchsia extract solution, C is the blank absorbance, Y). ≪ / RTI > IC ₅₀ is the concentration required to inhibit the activity of free radicals by 50%.

(2) Superoxide dismutase (SOD) -like activity

SOD-like activity was measured by modification of the method of Marklund et al. (Marklund S and Marklund G. 1974. Involvement of superoxide anion radicals in pyrogallol and a convenient assay for superoxide dismutase. Eur J Biochem 47: 468). Dilution buffer (Dilution Buffer) and Enzyme Working Solution (20 μl) were added to 20 μl of the Wuchung Extract, and reacted at room temperature for 20 minutes. Absorbance was measured at 450 nm. Blank was added to the third distilled water instead of the sample, and a calculation formula for calculating the SOD activity inhibition rate was obtained by the following equation (7).

&Quot; (7) "

SOD activity inhibition rate = [(Blank 1 - Blank 3) - (Sample - Blank 2)] / (Blank 1 - Blank 3) * 100

In the above equation, Blank 1 is a mixture of tertiary distilled water + 2- (4-iodophenyl) -3- (4-nitrophenyl) -5- (2,4- disulfo-phenyl) -2H- tetrazolium monosodium salt + Enzyme working solution and Blank 2 is a sample solution (a substance to be used as a control) + 2- (4-iodophenyl) -3- (4-nitrophenyl) -5- ) -2H-tetrazolium monosodium salt + diluting buffer, and Blank 3 is a solution of 2- (4-iodophenyl) -3- (4-nitrophenyl) -5- ) -2H-tetrazolium monosodium salt) + diluent buffer.

(3) ABTS + radical scavenging activity

The ABTS (2,2'-azinobis- (3-ethylbenzthiazoline-6-sulphonic acid)) radical scavenging activity of the dietary powder (2,2'-azinobis- (Van den Berg, Haenen GR, Van den Berg H. Bast A. 1999. Applicability of an improved trolox equivalent antioxidant capacity (TEAC) assay for evaluation 7.4 mM ABTS reagent and 2.6 mM potassium persulfate were dissolved in tertiary distilled water and reacted and stabilized in the dark for 14 hours. , And the absorbance was measured at 735 nm at a dilution of 50 × to obtain an absorbance value of between 1.4 and 1.5. After mixing 10 μl of the sample and 200 μl of the ABTS reagent, the mixture was allowed to react at room temperature for 30 minutes, The ABTS radical scavenging activity was measured by the following equation It is calculated through Equation 8.

&Quot; (8) "

ABTS radical scavenging ability (%) = ((blank optical density-sample optical density) / blank optical density) * 100

In this equation, the blank optical density (blank OD) is the absorbance value of the ascorbic acid + ABTS reagent.

3. Statistical Analysis

The results were statistically analyzed using SAS (Satisical Analysis System) and the mean standard deviation was obtained. One-way analysis of variance was performed to test the difference between the mean values among the experimental groups, and the difference between the variables was verified by Duncanmultiple range test. All statistical significance was performed at a level of less than 0.05. The relationship between total polyphenol, total flavonoid content and antioxidant activity was examined by linear regression analysis.

4. Results

1) Antioxidant activity test of Wuchung extract

(1) Electronic Difficulty

The IC ₅₀ value of the extract of Wuchuan according to the present invention was found to be higher than that of vitamin C (ascorbic acid) used as a control (see Table 3). In particular, considering that the heat of the similar system is 5608.1 μg / ml, it is quite high as a general vegetable (see Lim HW, Yoon JH, Kim YS, Lee MW, Park SY, Choi HK 2007. Free radical scavenging and inhibition of nitric oxide production by four grades of pine mushroom (Tricholoma matsutake Sing.), Food Chem 103: 1337-1342). This result is due to the fact that the total polyphenol content in the wheat germ is among the vegetables used for food.

It is well known that DPPH radical scavenging ability is changed in proportion to the total polyphenol content through previous studies (Kim OK, Lee MW, Park WB, Ham SS, Kung SS 1992. The Nutritional Components of Aerial Whole Plant and Juice of Therefore, the high activity of DPPH radical scavenging ability of Wakun extract is related to polyphenol content.

plant Antioxidant activity (IC _50,占 퐂 / ml) Wuchuan extract 183.37 + - 48.17 Ascorbic acid (vitamin C) 221.87 ± 34.77 1) Each value is expressed as the mean standard deviation of triplicate experiments.
2) Significant difference between groups by Duncan multiple test (p <0.05).
3) IC: The inhibitory activity is expressed as the mean of the 50% inhibitory concentration of triplicates obtained by extrapolation of the concentration-inhibition curve.

(2) Total polyphenol content

The total polyphenol content of the Wako extract according to the present invention was found to be 2.84 g / 100 g (see Table 4). In a previous study (see Jin SY, 2011. Study on Antioxidant Activities of Extracts from Different Parts of Korean and Iranian Pomegranates, J Korean Soc Food Sci Nutr 40 (8): 1063-1072), commercial vegetables such as broccoli, spinach, (80.8 1.2, 79.6 8.4, 68.9 1.8, 23.3 5.8, 14.4 1.5 mg / 100g) of potato and cucumber were found to contain a large amount of polyphenols.

plant Total polyphenol content (g / 100 g, dry weight) Wuchuan extract 2.84 0.11 Tannic acid
(Tannin acid) 0.02 ± 0.00 1) Each value is expressed as the mean ± standard deviation of triplicate experiments.
2) Significant difference between groups by Duncan multiple test (p <0.05).

(3) Measurement of total flavonoid content

Flavonoids have been shown to have potential as anti-inflammatory agents in many inflammatory studies, particularly in the LPS-stimulated rat fibroblasts, and the activity of NF-kB and the intestinal epithelial cells IEC18 and myeloid dendritic cells Inhibited the IkB kinase activity and TNF-secretion and also suppressed inflammation in the Crohn's disease model (Kim JS, Jobin C. 2005. The flavonoid luteolin inhibits lipopolysaccharide-induced NF-kappaB cignalling and gene expression by blocking IkappaB kinase Kang SS, Flavonoids: Potential anti-inflammatory agents. Natural Products Sci 58 (9): 371-377. Arch Pharm Res 16 (1): 25-35, 1992. The anti-inflammatory activity of naturally occurring flavonol and flavonol glycosides is shown in Table 1. [ 28). Therefore, the total flavonoid content of Wuchung extract is 0.19g per 100g, which is higher than that of Galic acid used as a control, and these components are useful for relieving symptoms of ulcerative colitis And the other.

plant Total flavonoid content (g / 100 g, dry matter) Wuchuan extract 0.19 ± 0.00 Gallan 0.00 ± 0.00 1) Each value is expressed as the mean standard deviation of triplicate experiments.
2) Significant difference between groups by Duncan multiple test (p <0.05).

(4) peroxide-eliminating enzyme (SOD) -like activity

SOD-like activities of the extracts were 63% and 36%, respectively.

(5) ABTS + radical scavenging activity

As a result of measuring ABTS cation radical scavenging ability of Wuchung extract and Chinese persimmon extract at 100 μg / ㎖ concentration, ascorbic acid used as a control group had the highest activity of 17%, and 13% of ascorbic acid used as a control group.

(6) Correlation between antioxidant activity and antioxidant activity

There was a significant correlation between flavonoid components and experimental results such as DPPH and ABTS. In a previous study, it was reported that flavonoid compounds showed 3 to 5 times more antioxidative activity than vitamin E. (Kim DO, Lee KW, Lee HJ, Lee CY 2002. Vitamin C equivalent antioxidant capacity (VCEAC) of phenolic phytochemicals. J Agric Food Chem 50: 3713-3717). The conclusion was that the amount of flavonoid compounds was greater in this experiment than in other polyphenols, and that they had a significant effect on ulcerative colitis inhibitory activity I can get off.

Total polyphenol content Total flavonoid content DPPH radical scavenging ability SOD-like activity ABTS radical scavenging ability Total polyphenol content One Total flavonoid content 0.0796 One DPPH radical scavenging ability 0.0578 0.9611 One SOD-like activity 0.0101 0.1813 0.2367 One ABTS radical scavenging ability 0.1438 0.9727 0.9233 0.0921 One 1) Significance at p <0.05 between groups by linear regression analysis and correlation was between -1 and 1.

2) Identification of the anti-ulcerative colitis inhibitory effect

(1) Weight gain, dietary intake, dietary efficiency, drinking water and DSS intake before and after induction of colonic inflammation (see Tables 7 to 9)

Dietary intake and body weight gain before and after induction of inflammation in rats fed with Wuchung extract were higher than those of the control group.

After induction of colonic inflammation with DSS, body weight, dietary intake, and dietary efficiency were decreased in the control group except the control group.

Particularly noteworthy was the decrease in both the dietary intake and the body weight gain in the control group, while the dietary intake in the dietary supplement group with wuchu extract decreased to a value close to that before induction of colitis, and the weight increase was also similar to that before induction of colitis. This is closely related to the antioxidant activity of Wuqing. In the previous study, weight, organ and dietary intake of AFB1 and antioxidant vitamins were compared. In the AFB1 alone group, body weight was decreased, but in the mixed group of antioxidant vitamins C and E, The intake was also significantly decreased in AFB1, but in the antioxidant vitamin-treated group, the dietary intake was also not decreased (Park SJ, Kim HK, Chung DH 2002. The effect of antioxidant vitamins on residual serum Alfatoxin B1 in mice. Assoc 28 (1) 81-88).

Therefore, it was hypothesized that the functional ingredients with anti-oxidant activity of wheat germs relieve weight loss and dehydration by inflammation.

Table 7 below shows the weight gain, food intake and food efficiency of mice during the period of colitis infestation.

group Weight gain
(g / day) Food intake
(g / day) Food Efficiency (FER) Normal group 0.29 0.26 4.06 ± 0.33 0.059 + 0.046 Control group 0.32 ± 0.26 4.08 ± 0.40 0.075 ± 0.057 Wuchuan extract. 0.26 ± 0.93 4.72 + 0.76 0.033 0.157 1) Average standard deviation
2) Significant difference between groups by Duncan multiple test (p <0.05).
3) Food efficiency = weight gain (g) / food intake (g)
6) The experimental group (n = 52) was as follows:
N (13): The control diet is rapid.
C (13): 3% DSS + 5% control diet
Wuqing (13): 3% DSS + 5% Wuchuan extract powder

Table 8 below shows the weight gain, food intake and food efficiency of mice during the induction period of colitis.

group Weight gain
(g / day) Food intake
(g / day) Food Efficiency (FER) Normal group 0.15 0.21 3.66 ± 0.71 0.07 ± 0.08 Control group 0.10 0.27 3.44 ± 0.59 0.01 ± 0.11 Wuchuan extract. 0.65 ± 0.68 4.50 + - 0.87 0.09 0.23 1) Average standard deviation
2) Significant difference between groups by Duncan multiple test (p <0.05).
3) Food efficiency = weight gain (g) / food intake (g)
4) The experimental group (n = 52) was as follows:
N (13): The control diet is rapid.
C (13): 3% DSS + 5% control diet
Wuqing (13): 3% DSS + 5% Wuchuan extract powder

Table 9 below shows the DSS / water uptake during the 5 day colitis induction period.

group DSS / water intake
(Ml / 5 days) DSS / water intake
(Mg / g) Control group 11.99 ± 0.16 9.23 + - 0.11 Wuchuan extract. 10.67 ± 0.07 9.28 ± 0.11 1) Average standard deviation
2) DSS load = total beverage intake (ml) DSS concentration (mg / ml) / initial weight (g)
3) Significant difference between groups by Duncan multiple test (p <0.05).

(2) Weight change before and after colitis induction

group Normal group Control group Wuchuan extract. Before colitis induction 2.00 ± 0.06 2.37 ± 0.09 1.51 + - 0.25 After induction of colitis 1.77 + - 0.45 0.42 ± 0.63 3.44 ± 1.48 1) Average standard deviation
2) Significant difference between groups by Duncan multiple test (p <0.05).

In the case of intestinal extract, the weight gain was smaller than that of the normal group and the control group. However, after the onset of colon inflammation, the weight gain and the dietary intake were observed to be influential on the average weight. This is because the antioxidant component of WuQi extract inhibits inflammation and decomposes dietary fiber monosaccharide of WuQi extract, which is used as a fuel for intestinal cells, thereby relieving the burden of bowel, thus positively affecting the improvement of intestinal function of experimental animals 2008. Effect of Radish Leaves Powder on Gastrointestinal Function and Fecal Triglyceride and Sterol Excretion in Rats Fed A Hypercholesterolemic Diet J Korean Soc Food Sci See Caroline Byrd-Bredbenner, Gail Moe, Donna Beshgetoor, and Jacqueline Berning. Nutr 37 (10): 1258-1263.

3) Change in length of colon

In contrast, the DSS-treated group showed a longer colon length than the normal group, and the DSS-treated control group showed a reduced size of the colon compared to the normal group, indicating that the DSS was directly toxic to the mucosal epithelium, (Jung HC 2001. Animal models for inflammatory bowel disease. Korean J Gastroenterol 37 (2): 69-75; Wirtz S, Neurath MF 2007. Mouse models of inflammatory bowel disease. Adv Drug Deliv Rev 59 (11): 1073-1083).

Antioxidant components of blue extract inhibited inflammation and improved intestinal function. Table 11 below shows changes in the colon.

group Length of colon (cm) Normal group 7.7 ± 0.5 Control group 7.2 ± 1.6 Wuqing 8.7 ± 1.1 1) Average standard deviation
2) Significant difference between groups by Duncan multiple test (p <0.05).

(4) Comparison of intestinal microbial changes

A fragment of approximately 180 bp was obtained by polymerase chain reaction (PCR) of bacterial genomic DNA with 338 f-GC and 518 r primer pair targeting 16S rDNA V3, A variety of intestinal flora was identified by electrophoresis on the washing gel. In the photograph (FIG. 1), normal group, control group, and wuchu extract-fed group from the left.

The first band is 80% of the uncultured bacterium, the second band is 97% of the H. canadensis , the third is 80% of the non-cultured bacteria, the 4th is the uncultured bacterium, Firmicutes bacterium ) 93%, and band 5 was found to be 92% identical to Helicobacter pylori ( H. pylori ). The second and fifth bands were found to be Helicobacter strains, which are harmful bacteria, and showed only 5 times in Wuqing, and decreased the expression of Helicobacter strains in Wuqing extract.

(5) Histological Findings of Intestinal Inflammatory Cells

As shown in Fig. 2 (appearance of inflammatory cells in the colon), A was a normal group and the inflammation stage was grade 2 or clean, while the colon tissue was randomly selected and histologically examined. , And the control group was grade 4, and many infiltrating inflammatory cells were observed in the submucosal layer and mucosal epithelial proliferation was clearly observed. There was a small number of inflammatory cell infiltration in the mucosal layer of the mucosa, and C mucosal inflammation was observed in the mucosal epithelium with weak hyperplasia.

In FIG. 2, A is the control group, B is the 3% DSS + control group, and C is the 3% DSS + 5%

(6) Measurement of the number of inflammatory cells in the colon

As shown in Table 12, the appearance of inflammatory cells was clearly recognized in most of the tissue sections of the colon of the control group, and the inflammatory cells were decreased in the extracts of Wuchung, compared to the control group. Therefore, we could confirm the protective effect of Wuchung extract on intestinal mucosa.

group Colitis cell number Normal group 5.00 ± 2.31 Control group 10.75 ± 0.00 Wuchuan extract. 9.25 ± 0.00 1) Average standard deviation
2) Significant difference between groups by Duncan multiple test (p <0.05).

5. Conclusion

The functional components with antioxidant activity of the extracts of Wuchung can reduce the oxidative stress caused by the inflammation, and thus it is possible to alleviate the accompanying blood loss, weight loss and dehydration in the case of ulcerative colitis.

The present invention can be used in an animal husbandry industry in which diets and the like can be expected to have antioxidative effects and the like and are distributed.

While the invention has been shown and described with reference to certain exemplary embodiments thereof, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention as defined by the appended claims.

(Without reference numerals)

Claims (9)

delete delete delete delete A feed composition for anti-inflammation and antioxidation comprising an extract of Wuchuan as an active ingredient,
The extract of Wuchuan is prepared by extracting the extracts, and the extraction ratio is 90 to 99% by weight of ethanol of 70% to 90% (v / v) and 1 to 10% At a temperature of 1 to 20 DEG C, and the extracts obtained by repeating the step of filtration for 1 to 5 times are mixed and concentrated at a temperature of 1 to 20 DEG C. And a feed composition for antioxidation. delete The method of claim 5,
Wherein the concentrate is lyophilized to prepare an extract powder, and the lyophilized extract powder is dissolved again in 70% to 90% (v / v) ethanol to be used.
delete delete

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