KR102223635B1 - Composition for improvementing, preventing or treating intestinal inflammation or leaky gut syndrome comprising fractions or extract of radish leave - Google Patents

Abstract

The present invention relates to a composition for improving, preventing or treating inflammatory bowel disease or leaky intestinal syndrome, and a composition for improving, preventing or treating inflammatory bowel disease or leaky intestinal syndrome containing radish extract as an active ingredient, and a composition for improving intestinal immunity About.

Description

Composition for improvementing, preventing or treating intestinal inflammation or leaky gut syndrome comprising fractions or extract of radish leave}

The present invention relates to a composition for improving, preventing or treating inflammatory bowel disease or leaky intestinal syndrome, and functional food for improving intestinal immunity as well as preventing, improving or treating inflammatory bowel disease or leaky intestinal syndrome by containing radish extract as an active ingredient And it can be usefully used in medicines and the like.

In addition to the basic functions of digestion, absorption, and excretion of food, the intestinal tract of the human body performs an immunological function as a barrier to the intestinal mucosa blocking the inflow of microorganisms, their by-products, antigens, and toxins into the bloodstream. That is, the merit film is a barrier that blocks foreign substances and has a dual function of passing and absorbing them.

Intestinal health is very important because if there is a problem with these intestinal functions, not only does it cause problems in the supply of nutrients to the human body by itself, and symptoms such as repetitive constipation, diarrhea, and abdominal pain greatly interfere with daily life.

Inflammatory bowel disease is a disease in which the intestine is chronically inflamed, and the cause of the disease and the cure method are not yet known. In general, it refers to Crohn's disease and ulcerative colitis. Crohn's disease and ulcerative colitis may show symptoms such as diarrhea, abdominal pain, nausea, fever, loss of appetite, weight loss, and fatigue. Crohn's disease occurs mainly in the small and large intestine, but ulcerative colitis is invented only in the large intestine. In addition to this, long-sized Behcet's disease, which is relatively common in Korea, belongs to this.

Drugs used to treat such inflammatory bowel disease include steroidal immunosuppressants, 5-aminosalicylic acid (5-ASA) drugs that block the production of prostaglandins (e.g., sulfasalazine), Mesalazine and the like are being used, and they cause serious side effects such as fullness, headache, rash, liver disease, leukopenia, agranulocytosis and male infertility, as well as having insignificant therapeutic effect on inflammatory bowel disease. Its use is restricted.

Therefore, if it is possible to develop a therapeutic agent for inflammatory bowel disease that does not cause side effects, it is expected that it will be possible to treat patients with inflammatory bowel disease more safely and effectively, but a therapeutic agent without side effects has not been developed yet.

Under this background, the present inventors have made diligent efforts to develop a therapeutic agent for inflammatory bowel disease derived from a natural product that does not cause side effects, and as a result, a composition comprising radish extract is effective in the treatment of not only inflammatory bowel disease but also intestinal leak syndrome, The present invention was completed by confirming that it enhances the intestinal immune function.

Korean Registered Patent Publication No. 10-1916603

An object of the present invention is to provide a food composition for improving or preventing inflammatory bowel disease or leaky intestinal syndrome using radish extract as an active ingredient.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating inflammatory bowel disease or leaky intestinal syndrome using radish extract as an active ingredient.

Another object of the present invention is to provide a composition for enhancing intestinal immunity using radish extract as an active ingredient.

In order to achieve the above object, the present invention provides a food composition for improving or preventing inflammatory bowel disease or leaky intestinal syndrome using radish extract as an active ingredient.

The radish extract may be extracted with water, an alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof.

The composition may increase immune activity, suppress an inflammatory response, and reduce activation of inflammatory cells.

The composition may be one that enhances IgA expression and inhibits or reduces the expression _{of IL-6 and LTB 4.}

The inflammatory bowel disease may be a disease selected from the group consisting of Crohn's disease, ulcerative colitis, enteric Behcet's disease, infectious enteritis, ischemic bowel disease, and radiation enteritis.

The leaky gut syndrome may be induced by a high fat diet.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating inflammatory bowel disease or leaky intestinal syndrome using radish extract as an active ingredient.

The radish extract may be extracted with water, an alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof.

The composition may increase immune activity, suppress an inflammatory response, and reduce activation of inflammatory cells.

The composition may be one that enhances IgA expression and inhibits or reduces the expression _{of IL-6 and LTB 4.}

The inflammatory bowel disease may be a disease selected from the group consisting of Crohn's disease, ulcerative colitis, enteric Behcet's disease, infectious enteritis, ischemic bowel disease, and radiation enteritis.

The leaky gut syndrome may be induced by a high fat diet.

In order to achieve the above another object, the present invention provides a composition for enhancing intestinal immunity using radish extract as an active ingredient.

The composition of the present invention contains radish extract, thereby suppressing inflammation and activating immunity to improve, prevent or treat inflammatory bowel disease. In addition, the composition of the present invention has the effect of improving, preventing or treating intestinal leak syndrome by enhancing intestinal permeability. In addition, the composition of the present invention has the effect of increasing the immune response to the antigen.

1 is a graph showing the measurement of the concentration of IgA in serum for the normal feed group (NC), the positive control group (GC), the first experimental group (WERL50), and the second experimental group (WERL100).
Figure 2 is a measurement of the expression level of cytokine IL-6 in serum for the normal feed group (NC), high fat diet group (HF), positive control group (GC), the first experimental group (WERL50), and the second experimental group (WERL100). This is the graph shown.
Figure 3 is a normal feed group (NC), high fat diet group (HF), positive control group (GC), the first experimental group (WERL50) and the second experimental group (WERL100) in serum cytokine leukotriene B4 (LTB ₄ ) expression amount It is a graph showing by measuring.
Figure 4 shows the detection concentration of FITC-dextran in the normal feed group (NC), the high fat diet group (HFD), the positive control group (GC), the first experimental group (WERL50) and the second experimental group (WERL100) over time. It is a graph. 5 is a graph showing the area under the curve of the result measured from FIG. 4.
FIG. 6 shows when the radish polymer fraction prepared in Example 2 was treated with 20 µl at concentrations of 100 and 1000 µg/ml, respectively, to Peyer's patch cells isolated from the mouse intestine, IgA and cytokines (IL-6, IL-10 , TGF-β1, GM-CSF) and bone marrow cell proliferation ability are measured and shown as a graph.

The present invention relates to a composition for improving, preventing or treating inflammatory bowel disease or leaky gut syndrome, and a composition for enhancing intestinal immunity, using radish extract as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present invention relates to a composition for improving, preventing or treating inflammatory bowel disease or leaky intestinal syndrome, using radish extract as an active ingredient. In addition, the present invention relates to a composition for enhancing intestinal immunity using radish extract as an active ingredient.

Radish (Raphanus sativus Leaves) is a single-year-old or monthly-born herb belonging to the mustard family, and has been introduced from China since the Three Kingdoms period, and there are many indigenous species indigenous to each region. Radish contains 93% moisture, 1% crude protein, 3% sugar mainly as glucose, and 19-39 mg% vitamin C. Radish tissue contains fibrin, pectin, various minerals and digestive enzymes, amylase, and has unique aromatic components such as methyl mercaptane and mustard oil.

Radish is the upper part of the radish root, that is, the stem and leaves, and the dried siraegi (Siraegi and/or heaviness; hereinafter, it is unified as radish) is a favorite food that most Koreans like. The harvest period is limited and it is not commercialized for common sense throughout the year, so it is not fulfilling its role as a product like other traditional foods.

In the present invention,'leaky gut syndrome' or'leaky gut syndrome'; LGS' refers to a phenomenon in which intestinal membrane cells maintain a certain gap between cells, and during the process of digestion and absorption, any stimulation or damage is applied, thereby increasing the permeability of the intestinal membrane in which a polymer material can reciprocate through the gap between cells. It collectively refers to symptoms caused by a phenomenon in which a polymer material in the blood leaks into the intestinal lumen due to a failure to function properly in the intestine or a phenomenon in which a polymer material in the lumen directly enters the blood (leaky gut). These symptoms appear in various clinical conditions such as aging, allergies, multiple trauma, rheumatoid arthritis, inflammatory bowel disease, chronic fatigue syndrome, and irritable syndrome. In addition, pathogens, antigens, and decay substances are introduced into the intestinal tract due to increased permeability of the intestinal tract or damage to the intestinal tract, leading to inflammatory reactions, and endotoxins flowing into the bloodstream, resulting in bacterial translocation and intestinal endotoxemia. It causes various inflammatory reactions and immune reactions.

Such leaky gut syndrome complains of various, extensive, and ambiguous symptoms without specific symptoms. Specifically, it has a peculiarity in that it has symptoms that complain in common in various diseases.

The exact cause of the leaky gut syndrome has not been identified, but the use of non-steroidal antiin ammatory drugs (NSAIDs) used as pain relievers for a long time, antibiotics and steroids, anticancer chemistry as an anticancer therapy In the case of receiving therapy or radiation therapy, when a change in the composition of the normal intestinal flora occurs, the reproduction of fungi in the intestine, ingestion of excessive food, ingestion of spoiled food or ingestion of heavy metals or toxic substances, excessively irritating food Ingestion of or hypersensitivity to certain foods, excessive use of alcohol, multiple trauma, exposure to acute or chronic mental stress, or chronic intestinal bacteria, parasites, and yeast infection.

Specifically, the composition for improving, preventing, or treating leaky gut syndrome may be referred to as a composition having an effect of improving, preventing, alleviating, inhibiting and treating the increase in intestinal permeability due to the various causes. This has the effect of improving, preventing, and treating intestinal leak syndrome, which causes various diseases, as high intestinal permeability increases, so that polymeric substances in the blood leak into the intestinal lumen, or polymeric substances in the lumen enter the branched blood. The improvement, prevention, or treatment of leaky gut syndrome may mean suppressing a phenomenon in which the intestinal membrane cell gap is loosened and thus the permeability of the intestinal membrane through which a polymer material can reciprocate is increased.

The inflammatory bowel disease may be a disease selected from the group consisting of Crohn's disease, ulcerative colitis, enteric Behcet's disease, infectious enteritis, ischemic bowel disease, and radiation enteritis.

According to the experimental results of the present invention, it can be seen that when a high-fat diet is administered for a long time, inflammatory factors such as cytokine IL-6 are increased, and the activation of inflammatory cells is increased. This induces inflammation in the intestine over time, and soon enteritis is induced.

At this time, it can be seen that when the radish extract is co-administered, the immune activity is increased, the inflammatory response is suppressed, and the activation of the inflammatory cells is reduced. Therefore, it can be seen that the composition comprising the radish extract according to the present invention as an active ingredient has an effect of preventing or treating inflammatory bowel disease.

In the present specification, "enhancing intestinal immunity" refers to a function of non-specifically promoting an immune response against an antigen during the initial activation of immune cells present in the intestine or a function of enhancing the intestinal immunity by increasing the activity of cells in the immune system. .

The disease due to intestinal immune function decline may be any one or more selected from the group consisting of infectious diseases and cancer caused by pathogenic microorganisms. The composition for enhancing intestinal immunity has an applied effect of improving or preventing human flu, improving or preventing bacterial diarrhea, improving or preventing bacterial infection, and enhancing treatment effect after wounding.

The radish extract is more specifically characterized in that it enhances IgA expression and inhibits or reduces the expression _{of IL-6 and LTB 4.}

In addition, the radish extract is an immune response such as IgA, cytokines (IL-6, IL-10, TGF-β1, GM-CSF), and myeloid cell proliferation ability, which are known as the most important indicators for intestinal mucosal immune responses when exposed to antigens. It was confirmed that it has an effect of promoting.

In the present invention, the'extract' includes not only the crude extract obtained by treating the extraction raw material with the extraction solvent, but also the processed product of the crude extract. For example, radish extract may be prepared in a powder state by additional processes such as distillation under reduced pressure and freeze drying or spray drying.

In addition, the'extract' in the present invention also includes a fraction obtained by further fractionating the crude extract. That is, radish extract includes not only those obtained by using an extraction solvent, but also those obtained by additionally applying a purification process thereto. For example, a polymer fraction obtained by passing through an ultrafiltration membrane having a certain molecular weight cut-off value, separation by various chromatography (made for separation according to size, charge, hydrophobicity or affinity), etc. It can be purified through a method.

The polymer fraction may preferably be a fraction obtained by separating only a polymer material having a molecular weight of 10 kDa or more after obtaining the radish extract or the radish extract using an extraction solvent.

In addition, in the present invention, the'radish extract' is an extract of the radish leaves using water, alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof, and the extraction method does not need to be particularly limited. Preferably, the radish extract may be a radish hot water extract or a radish ethanol aqueous solution, for example, radish alcohol extract. Most preferably, it may be a radish hot water extract.

In addition, the radish extract may be processed by the radish extract, and then repeatedly extracted one or more times with water, an alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof.

The radish leaves can be cut into a certain size, dried using a shade or a dryer, and then crushed or used as it is. The drying period is not particularly limited as long as the moisture content in the radish is less than 20%.

In the present invention, the term'contained as an active ingredient' or'contained as an active ingredient' includes an amount sufficient to improve, prevent or treat inflammatory bowel disease or leaky intestinal syndrome of the radish extract of the present invention, and to enhance intestinal immune activity. Means that. Since the radish extract is a natural product and does not have side effects on the human body even if it is administered in an excessive amount, the upper and lower limits of the quantity of the radish extract contained in the composition of the present invention can be selected and carried out by a person skilled in the art within an appropriate range.

In the present invention, when the composition containing the radish extract as an active ingredient is treated, the composition enhances IgA expression when administered on a high fat diet for a long time, and inhibits or reduces the expression _{of IL-6 and LTB 4,} It was confirmed that there is an effect of improving, preventing or treating inflammatory bowel disease, and it was confirmed that it has an effect of inhibiting and reducing intestinal permeability.

In the present invention, when the composition containing the radish extract as an active ingredient is treated, IgA and cytokines (IL-6, IL-10, TGF-β1, GM-CSF, which are known as the most important indicators for the intestinal mucosal immune response) ) And bone marrow cell proliferation ability.

The composition of the present invention is a food composition, and can be used in the form of various foods including the radish extract of the present invention as an active ingredient, such as beverages, gums, teas, vitamin complexes, powders, granules, tablets, capsules, and the like.

The food composition according to the present invention may be formulated in the same manner as the pharmaceutical composition described later to be used as a functional food or added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, alcoholic beverages, confectionery, diet bars, dairy products, meat, chocolate, pizza, ramen, other noodles, gums, ice creams, vitamin complexes, health supplements. Etc.

The food composition of the present invention may include not only radish extract as an active ingredient, but also ingredients that are commonly added during food production, and include, for example, proteins, carbohydrates, fats, nutrients, flavoring agents, and flavoring agents. Examples of the aforementioned carbohydrates include monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, oligosaccharides, and the like; And polysaccharides, for example, common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents, natural flavoring agents [taumatin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.]) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used. For example, when the food composition of the present invention is made of drinks and beverages, in addition to the radish extract of the present invention, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, and various plant extracts may be further included.

The present invention provides a health functional food comprising a food composition for improving, suppressing or preventing inflammatory bowel disease or intestinal leak syndrome comprising the radish extract as an active ingredient, and a food composition for enhancing intestinal immunity. Health functional foods are foods prepared by adding radish extract to food ingredients such as beverages, teas, spices, chewing gums, confectionery, etc., or prepared by encapsulation, powdering, or suspension. However, unlike general drugs, it has the advantage of not having side effects that may occur when taking drugs for a long time by using food as a raw material. The health functional food of the present invention obtained in this way is very useful because it can be consumed on a daily basis. The amount of radish extract added in such health functional foods cannot be uniformly regulated depending on the type of health functional food to be targeted, but it can be added within the range that does not damage the original taste of the food, and is usually 0.01 for the target food. To 50% by weight, preferably 0.1 to 20% by weight. In addition, in the case of health functional foods in the form of pills, granules, tablets or capsules, it is usually added in the range of 0.1 to 100% by weight, preferably 0.5 to 80% by weight. In one embodiment, the health functional food of the present invention may be in the form of a pill, tablet, capsule or beverage.

In another example, it may be variously used as a pharmaceutical composition for preventing or treating inflammatory bowel disease or leaky gut syndrome, and a pharmaceutical composition for enhancing intestinal immunity, using the radish extract as described above as an active ingredient. In addition to the radish extract, which is an active ingredient, it can be prepared using a pharmaceutically suitable and physiologically acceptable adjuvant, and the adjuvant includes excipients, disintegrants, sweeteners, binders, coating agents, expanding agents, lubricants, lubricants, or flavoring agents. Etc. can be used.

For administration, the pharmaceutical composition may contain one or more pharmaceutically acceptable carriers in addition to the above-described active ingredients, and may be preferably formulated into a pharmaceutical composition.

The formulation form of the pharmaceutical composition may be granules, powders, tablets, coated tablets, capsules, suppositories, solutions, syrups, juices, suspensions, emulsions, drops, or injectable solutions. For example, for formulation in the form of tablets or capsules, the active ingredient may be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. In addition, if desired or necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included in the mixture. Suitable binders are, but are not limited to, natural sugars such as starch, gelatin, glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, trackcacanth or sodium oleate, sodium stearate, magnesium stearate, sodium Benzoate, sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like.

As acceptable pharmaceutical carriers in the composition formulated as a liquid solution, as sterilization and biocompatible, saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added as needed. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to prepare injection formulations such as aqueous solutions, suspensions, emulsions, etc., pills, capsules, granules, or tablets.

Further, it can be preferably formulated according to each disease or ingredient using a method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA as an appropriate method in the field.

The pharmaceutical composition of the present invention may be administered orally or parenterally, and in the case of parenteral administration, it may be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, etc., preferably oral administration.

A suitable dosage of the pharmaceutical composition of the present invention varies depending on factors such as formulation method, mode of administration, age, weight, sex, pathological condition, food, administration time, route of administration, excretion rate and response sensitivity of the patient, and is usually As such, a skilled physician can easily determine and prescribe an effective dosage for the desired treatment or prophylaxis. According to a preferred embodiment of the present invention, the daily dosage of the pharmaceutical composition of the present invention is 0.001-10 g/kg.

The pharmaceutical composition of the present invention may be prepared in unit dosage form by formulation using a pharmaceutically acceptable carrier and/or excipient, or may be prepared by incorporating it into a multi-dose container. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, granule, tablet or capsule, and may additionally include a dispersant or a stabilizer.

In addition, the present invention provides a method for improving, preventing or treating inflammatory bowel disease or leaky intestinal syndrome, comprising administering an effective amount of radish extract to a mammal, and a method for enhancing intestinal immunity.

The term "mammal" as used herein refers to a mammal that is an object of treatment, observation or experiment, and preferably refers to a human.

The term "effective amount" as used herein refers to the amount of an active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal or human, which is considered by a researcher, veterinarian, doctor or other clinician, which Contains an amount that induces relief of symptoms. The effective amount and the number of administrations for the active ingredient of the present invention may be changed according to the desired effect. Therefore, the optimal dosage to be administered can be easily determined by those skilled in the art, and the degree of symptoms of inflammatory bowel disease, degree of intestinal permeability to intestinal leak syndrome, degree of activation of immune cells, degree of immune response to antigens, and effectiveness contained in the composition. Depending on various factors, including the content of ingredients and other ingredients, the type of formulation, and the patient's age, middle, general health condition, sex and diet, administration time, route of administration and secretion rate of the composition, treatment period, and drugs used concurrently. Can be adjusted. In the prevention, treatment, or improvement method of the present invention, in the case of adults, when the radish extract is administered once to several times a day, it is preferable to administer the radish extract at a dose of 0.001 g/kg to 10 g/kg.

In the treatment method of the present invention, the composition comprising radish extract as an active ingredient is in a conventional manner through oral, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, topical, intraocular or intradermal routes. It can be administered, and can be preferably administered orally or intraperitoneally.

Hereinafter, a preferred embodiment is presented to aid in the understanding of the present invention, but it is obvious to those skilled in the art that various changes and modifications are possible within the scope of the present invention and the scope of the technical idea, but the following examples are only illustrative of the present invention, It is natural that such modifications and modifications fall within the appended claims.

Example 1. Radish hot water extract

Korean radish leaves (daikon leaves) produced by Wanju Nonghyup in Jeonbuk were purchased and used. The radish was dried in a drying oven at 50° C. for 24 hours and then pulverized to a size of 1.0 mm or less with a grinder to prepare radish leaf powder.

Distilled water equivalent to 20 times the volume was added to 5 g of radish leaf powder, followed by reflux extraction at 80° C. for 3 hours to obtain a hot water extract. After filtering the hot water extract, it was concentrated under reduced pressure using a rotary vacuum evaporator, and lyophilized for 72 hours. The radish hot water extract obtained by the above method was described as WERL (Water-soluble extract from radish leaves).

Example 2. Radish polymer fraction

Korean radish leaves (daikon leaves) produced by Wanju Nonghyup in Jeonbuk were purchased and used. Radish (radish leaves) was dried in a drying oven at 50° C. for 24 hours, and then pulverized to a size of 1.0 mm or less with a grinder to prepare radish powder.

Distilled water corresponding to 20 times the volume was added to 100 g of radish powder, followed by reflux extraction at 80° C. for 3 hours to obtain a hot water extract. After filtering the hot water extract, it is concentrated under reduced pressure to be 1/10 of the extract using a rotary vacuum evaporator, and 80% alcohol corresponding to 4 times the volume (v/v) of the hot water extract concentrated under reduced pressure Was added to the hot water extract and precipitated for 24 hours. This was centrifuged (6000 rpm, 635 g, 30 minutes) to obtain a precipitate. A small amount of distilled water was added thereto to redissolve the precipitate and a dialysis membrane (MW cut off 12,000, Sigma-Aldrich Co., St. Louis, MO, USA) was performed for 2 to 3 days to remove the low molecular weight material, and lyophilized for 72 hours to obtain a radish polymer fraction. The radish polymer fraction obtained by the above method was named HFPL (High-molecular fraction from radish leaves).

Comparative Example 1. Garcinia cambogia extract

Garcinia cambogia extract was purchased and used from Unicon Natural Products PVT, India. At this time, the garcinia cambogia extract was used that contains 600 mg/g or more of HCA.

<Test Example>

Test Example 1. Analysis of the yield, sugar and protein content of the radish hot water extract of Example 1 and the radish polymer fraction of Example 2

For the analysis of neutral sugars, the method of Albersheim et al. was partially modified to derivatize each constituent sugar with alditol acetate and aldonolactone after hydrolysis, and GC (Gas Chromatography ACME-6100, Young-Lin Co. Ltd. Anyang, Korea) ) Was used. The sample of Example 1 or 2 was hydrolyzed by reacting for 1.5 hr at 121° C. in 2 M TFA (trifluroacetic acid), and then dissolved in 1 ml of 1 M NH ₄ OH (ammonia solution) and 10 mg of NaBH ₄ It was reduced to 4 hr. An appropriate amount of acetic acid was added _{to remove the remaining NaBH 4,} and then, methanol was added thereto, followed by repeated drying to remove excess acetic acid, which was converted to alditol. 1 ml of acetic anhydride was added to the alditol and reacted at 121° C. for 30 min to convert to alditol acetate, which was _{separated and extracted with a chloroform/H 2} O two-phase solvent system. After drying, the extract was dissolved in a small amount of acetone and used as a sample for GC analysis.

Protein quantification was measured according to the Bradford method, and BSA (Bovine Serum Albumin, sigma aldrich) was used as a standard and diluted at a constant multiple with 1 mg/ml as the highest concentration. After adding 20 µl of each standard and sample to 980 µl of Bradford reagent, the absorbance was measured at 595 nm using a microplate reader. Protein quantification was converted into an amount for BSA.

Total sugar was measured according to the phenol-sulfuric acid method, and after stirring the same amount of 5% phenol solution to 0.5 mL of the sample solution, 2.5 mL of concentrated sulfuric acid (98%, v/v) was added and reacted at room temperature for 20 minutes to make a microplate. Absorbance was measured at 470 nm using a reader. For the determination of total sugar, a calibration curve was prepared using a glucose standard (sigma aldrich) and converted into the amount.

The total polyphenol content was analyzed according to the Folin Denis method using gallic acid (sigma aldrich).

In the case of constituent sugar, the oligosaccharides of the sample were hydrolyzed to monosaccharides, and then HPAEC-PAD (high-performance anion-exchange chromatography-pulsed amperometric detector) was used, and the data were analyzed using Chromelon 6.80 software (Dionex). Monosaccharides (fucose, rhamnose, arabinose, galactose, glucose, xylose) were used as standard solutions.

The physicochemical properties, such as the content of major components of the radish hot water extract prepared from Example 1 and the radish polymer fraction of Example 2, were analyzed, and the results are shown in Table 1 below.

Example 1 (WERL) Example 2 (HFRL) Extraction yield (%) 34.3 7.9 Chemical composition (%) Protein 4.5 ± 0.2 0.7 ± 0.1 Neutral sugar 72.4 ± 1.2 70.8 ± 0.7 Uronic acid 10.9 ± 0.1 22.3 ± 0.4 KDO-liked material - 1.6 ± 0.1 polyphenol 12.2 ± 0.2 4.5 ± 0.3 Component sugar (PMP) (Mole %) Rhamnose 6.6 ± 0.1 5.4 ± 0.3 Fucose 0.6 ± 0.0 5.0 ± 0.7 Arabinose 5.7 ± 0.1 22.9 ± 2.3 Xylose 0.4 ± 0.1 0.4 ± 0.1 Mannose 1.7 ± 0.1 2.0 ± 0.2 Galactose 8.7 ± 0.1 40.7 ± 3.6 Glucose 55.8 ± 0.6 2.1 ± 0.1 Glucuronic acid - 1.9 ± 0.1 Galacturonic acid 3.8 ± 0.3 12.7 ± 1.9

* The content ratio (%) of the chemical component represents the weight% in the dry sample.

As shown in Table 1, the radish hot water extract prepared from Example 1 was found to have a yield of 34.3%, a protein content of 4.5% by weight, and a neutral sugar content of 72.4% by weight. That is, it was analyzed that most of the radish hot water extract of Example 1 was occupied by sugar.

The radish extract prepared from Example 1 contained the most glucose (55.8 Mole%), followed by galactose, rhamnose, and arabinose at 8.7, 6.6, and 5.7 Mole%, respectively, galacturonic acid, mannose , Fucose, xylose (3.8, 1.7, 0.6, 0.4 Mole %, respectively) was confirmed to contain trace amounts.

The extraction yield of the radish polymer fraction prepared in Example 2 was 7.9%, the protein content was 0.7% by weight, and the neutral sugar content was 70.8% by weight, which was confirmed to have a high sugar content. The main constituent sugar was galactose (40.7 Mole%), followed by arabinose and galacturonic acid, which accounted for 22.9 and 12.7 Mole%, respectively, and rhamnose, fucose, glucose, mannose, glucuronic acid, and xylose (each 5.4. , 5.0, 2.1, 2.0, 1.9, 0.4 Mole %) was confirmed to exist in trace amounts.

Test Example 2. Analysis of Factors Related to Inflammation of Radish Hot Water Extract of Example 1

1) Experimental animals

First, 45 6-week-old male C57BL/6J mice were purchased through a central laboratory animal. The experimental animals were divided into five groups as follows. Nine experimental animals were used for each group, and after 8 weeks of diet, the intestinal immune modulating efficacy was evaluated.

-Normal feed group (NC): An experimental group fed with normal feed (AIN-93G) and administered only the vehicle orally

-High fat diet group (HFD): Experimental group that fed high fat (60% kcal fat) feed and administered only vehicle orally

-Positive control group (GC): An experimental group fed with a high-fat feed and orally administered 50 mg/kg of garcinia cambogia extract

-Experimental group 1 (WERL50): an experimental group in which 50 mg/kg of the radish hot water extract prepared in Example 1 was administered orally after feeding a high-fat feed

-2nd experimental group (WERL100): a high-fat feed was fed, and 100 mg/kg of the radish hot water extract prepared in Example 1 was administered orally.

At this time, for the high fat data, a modified AIN 76A purified rodent diet (60 cal% fat) in which Lard was added to increase the caloric value by 60 cal% was used.

2) analysis

After the 8-week diet period was over, the experimental animals of each group were fasted from 6 hours before analysis. Next, the experimental animals were anesthetized and the abdomen was opened. After collecting blood from the inferior vena cava using a sterile syringe, it was stored in a sterile tube treated with heparin. The obtained blood was centrifuged (12,000 × g, 10 minutes, 4° C.), and the supernatant was taken and used for analysis. The concentrations of immunoglobulin A, IL-6, and leukotriene B4 in blood were determined using Mouse IgA ELISA kit (Abcam, ab157717), Mouse IL-6 Quantikine ELISA kit (R&D systems, M6000B), LTB4 Parameter Assay Kit (R&D systems, KGE006B), respectively. Quantified.

3) Analysis result

Intestinal inflammation-related indicators for the radish hot water extract prepared from Example 1 were confirmed, and the results are shown in FIGS. 1 to 3.

1 is a graph showing the measurement of the concentration of IgA in serum for the normal feed group (NC), the positive control group (GC), the first experimental group (WERL50), and the second experimental group (WERL100). As shown in FIG. 1, in the normal feed group (NC), the blood IgA concentration was 16.25 µg/ml, but in the positive control group (GC), it was confirmed that the intestinal immunity activity was increased to 26.17 µg/ml. In the first experimental group in which the radish hot water extract prepared in Example 1 was orally administered at a concentration of 50 mg/kg, 28.63 µg/ml of IgA was confirmed. In addition, 32.32 µg/ml IgA was detected in the second experimental group in which the radish hot water extract prepared in Example 1 was orally administered at a concentration of 100 mg/kg.

Taken together, it can be seen that the radish hot water extract of Example 1 also enhances intestinal immune activity like the positive control (GC). In particular, it was confirmed that the intestinal immune activity was also increased as the dose concentration of the radish hot water extract (Example 1) increased, and it was found that the radish hot water extract according to the present invention has the effect of reducing inflammation by enhancing the intestinal immune activity. I can.

Figure 2 is a measurement of the expression level of cytokine IL-6 in serum for the normal feed group (NC), high fat diet group (HF), positive control group (GC), the first experimental group (WERL50), and the second experimental group (WERL100). This is the graph shown.

IL-6 is a representative cytokine known to be increased in inflammatory bowel disease. In the present invention, while inducing inflammatory bowel disease through a long-term high-fat diet for 8 weeks, it was intended to investigate the improvement, prevention, or treatment effect of inflammatory bowel disease by mixing and administering the radish hot water extract of Example 1. As shown in Figure 2, it can be seen that the concentration of IL-6 was significantly increased to 12.37 pg/ml in the high fat diet group (HF) in which inflammatory bowel disease was induced through a high fat diet for 8 weeks. In the case of the first and second experimental groups in which the high-fat diet and the radish hot water extract of Example 1 were co-administered at various concentrations, 5.32 pg/ml and 5.35 pg/ml of IL-6 were detected, respectively. This is lower than the concentration of IL-6 (2.89 pg/ml) in the positive control group (GC), which was co-administered with Garcinia cambogia extract, which is known to have a therapeutic effect on inflammatory bowel disease, and lower than that of the normal feed group (NC) (2.78 pg/ml). It's a shame. That is, it can be seen that the radish hot water extract prepared from Example 1 significantly inhibits IL-6 production, like the normal feed group or garcinia cambogia extract. Through this, it can be seen that the radish hot water extract according to the present invention has an improvement, prevention or treatment effect on inflammatory bowel disease by suppressing the inflammatory reaction in the intestine. Garcinia cambogia is known to accompany various side effects such as dry mouth, dizziness, headache, or diarrhea when ingested.However, the radish hot water extract according to the present invention has no known side effects as a food that can be consumed on a daily basis, as well as in cytotoxicity experiments at all. It has a great advantage in that it is a stable substance with no toxicity detected.

Figure 3 is a normal feed group (NC), high fat diet group (HF), positive control group (GC), the first experimental group (WERL50) and the second experimental group (WERL100) in serum cytokine leukotriene B4 (LTB ₄ ) expression amount It is a graph showing by measuring.

leukotriene B4 (LTB ₄ ) is known to be involved in the activity of inflammatory cells by inducing an inflammatory response in immune cells. Therefore, the level of inflammation was analyzed by measuring the _{LTB 4} concentration from experimental animals in which inflammatory bowel disease was induced by a long-term high fat diet. As shown in FIG. 3, when inflammatory bowel disease was induced through a high-fat diet, it was _{found that the concentration of LTB 4} was significantly increased to 1357 pg/ml compared to the normal feed group (NC). _{On the other hand, it was confirmed that the concentration of LTB 4} decreased to 976 pg/ml in the positive control group (GC) in which garcinia cambogia extract was administered concurrently with a high fat diet.

_{It was also confirmed that the concentration of LTB 4} decreased to 1162 pg/ml and 996 pg/ml, respectively, in experimental groups 1 and 2 in which the radish hot water extract prepared from Example 1 was administered in parallel with a high fat diet.

In addition, the radish hot water extract according to the present invention _{exhibited a reduction in the concentration of LTB 4} similar to that of the positive control group and the normal feed group, through which it can be seen that it has an effect of inhibiting _{LTB 4} production equivalent to that of the garcinia cambogia extract.

Although the 1st and 2nd experimental groups to which radish hot water extract was administered had less _{decrease in LTB 4} concentration than the positive control, garcinia cambogia extract had a problem that various side effects could occur, whereas the radish hot water extract of the present invention was very As a stable substance, it can be seen that it has a remarkably excellent effect in that it exhibits a remarkable anti-inflammatory effect without showing any cytotoxicity.

It was confirmed that the radish hot water extract according to the present invention can exhibit an effect in improving, preventing or treating inflammatory bowel disease by inhibiting the activity of inflammatory cells. Further, for LTB ₄ production, it is preferable that the radish hot water extract is administered at a concentration of 100 mg/kg in order to obtain a remarkable activity of inhibiting it to the level of a normal feed group.

Test Example 3. Effect of radish hot water extract on improving, preventing or treating leaky gut syndrome

1) Experimental animals

First, 45 6-week-old male C57BL/6J mice were purchased through a central laboratory animal. The experimental animals were divided into five groups as follows. Nine experimental animals were used for each group, and after 8 weeks of diet, the efficacy of improvement, prevention and treatment for intestinal leak syndrome were evaluated.

-Normal feed group (NC): An experimental group fed with normal feed (AIN-93G) and administered only the vehicle orally

-High fat diet group (HFD): Experimental group that fed high fat (60% kcal fat) feed and administered only vehicle orally

-Positive control group (GC): An experimental group fed with a high-fat feed and orally administered 50 mg/kg of garcinia cambogia extract

-Experimental group 1 (WERL50): an experimental group in which 50 mg/kg of the radish hot water extract prepared in Example 1 was administered orally after feeding a high-fat feed

-2nd experimental group (WERL100): a high-fat feed was fed, and 100 mg/kg of the radish hot water extract prepared in Example 1 was administered orally.

At this time, for the high fat data, a modified AIN 76A purified rodent diet (60 cal% fat) in which Lard was added to increase the caloric value by 60 cal% was used.

2) analysis

After the 8-week diet period was over, the experimental animals of each group were fasted from 6 hours before analysis. In order to confirm the intestinal permeability from each experimental animal, FITC-dextran 400 mg/kg body weight dissolved in sterile saline was administered orally, followed by microvenous blood collection 0, 2, and 4 hours later. The obtained 100 μL of blood was centrifuged (12,000 × g, 10 min, 4 ℃) to take the supernatant, and the excitation wavelength was 485 nm and the emission wavelength was 535 nm using a microplate reader. It was set and the absorbance was measured. The concentration was calculated using the standard curve of FITC-dextran, and the concentration of FITC-dextran by time was expressed as an area under the curve (AUC).

3) Analysis result

Figure 4 shows the detection concentration of FITC-dextran in the normal feed group (NC), the high fat diet group (HFD), the positive control group (GC), the first experimental group (WERL50) and the second experimental group (WERL100) over time. It is a graph. 5 is a graph showing the area under the curve of the result measured from FIG. 4.

As shown in FIGS. 4 and 5, when the normal feed group (NC) (normal control) was set to 100%, the high fat diet group (HFD) caused by leaky gut syndrome through a high fat diet was 399.61%, and the positive control group (normal control) was 399.61%. GC) was detected as 271.71%. That is, it can be seen that even if a high-fat diet was provided, intestinal permeability was suppressed when garcinia cambogia extract was administered in combination.

However, in the 1st and 2nd experimental groups to which the radish hot water extract prepared from Example 1 was co-administered, it was measured as 80.16% and 242.79%, respectively, and it can be seen that the intestinal permeability was suppressed and decreased to a value substantially similar to that of the normal feed group. have. In particular, it was confirmed that the most effective radish hot water extract prepared from Example 1 was administered less than 100 mg/kg and 50 mg/kg or more.

Intestinal permeability decreased by 5 and 1.6 times, respectively, in the first and second experimental groups compared to the high fat diet group (HFD). Therefore, it can be seen that the radish hot water extract according to the present invention has a remarkably excellent improvement, prevention or treatment effect for leaky gut syndrome.

Test Example 3. Intestinal immunity modulating effect of the radish polymer fraction of Example 2

1) Experimental animals

In this experiment, cells recovered from Peyer's patch existing on the outer wall of the small intestine of C3H/He mice (6 weeks old, female) ^{were adjusted to 2×10 6} cells/ml and plated 180 µl on a 96-well plate. To this, 20 µl of the radish polymer fraction (100 µg/ml or 1000 µg/ml concentration) of Example 2 was added and incubated for 5 days in _{a 37°C, 5% CO 2 incubator.} Bone marrow cells were recovered from the femoral bone of the same species ^{and adjusted to a concentration of 2.5×10 5} cells/ml, and then 100 μl was dispensed into a 96-well plate, and Peyer's patch cells mentioned above and the radish polymer fraction of Example 2 50 µl each of the supernatant (conditioned medium) and 10% FBS/RPMI-1640 medium recovered from the reaction of were added at 37° C. and 5% CO ₂ incubator for 6 days.

The negative control (NC) was not treated with anything, and the positive control (PC) was treated with 20 µl of LPS having a concentration of 10 µg/ml instead of the radish polymer fraction of Example 2.

2) analysis

Bone marrow cells cultured for 6 days were added with 20 µl of CCK-8 kit solution, and after 4 hours, absorbance was measured at 450 nm, and this was calculated as relative activity (%) to the absorbance value of the negative control group (NC) to proliferate bone marrow cells. (Bone marrow proliferation. %) was measured. On the other hand, immunoglobulin A (IgA) (Bethyl laboratories, Montgomery, Texas, USA), which is a systemic immune activation cytokine derived from Peyer's patch cells for 5 days, transforming growth factor (TGF)-β1 (eBioscience, San Diego, CA), interleukin (IL)-6, IL-10, granulocyte macrophage-colony stimulating factor (GM-CSF) (BD bioscience, San Diego, CA) content was measured according to the manufacturer's instructions using a sandwich ELISA kit. I did.

3) Analysis result

The results of the intestinal immunity regulating efficacy of the radish polymer fraction of Example 2 are shown in FIG. FIG. 6 shows IgA and cytokines (IL-6, IL-10, when 20 μl of each of Peyer's patch cells isolated from the mouse intestine was treated with the radish polymer fraction prepared in Example 2 at concentrations of 100 and 1000 μg/ml. TGF-β1, GM-CSF) and bone marrow cell proliferation ability were measured and shown as a graph.

According to this, as a result of measuring the production amount of IgA, which is known as the most important indicator for the intestinal mucosal immune response, when 20 µl of the radish polymer fraction of Example 2 was used at a concentration of 1,000 µg/ml, it was used as a positive control (PC). Jun-ha showed excellent production ability, and it could be seen that an immune response similar to that of the positive control was induced in the results of IL-6 and GM-CSF.

In addition, when the radish polymer fraction of Example 2 was used, it was confirmed that the production ability of IL-10 also reached half of that of the positive control group, and the bone marrow cell proliferation ability was also achieving a relatively high level. Therefore, it was found that the radish polymer fraction of Example 2 exhibited excellent intestinal immune activity via Peyer's patch cells. It can be seen that the radish polymer fraction of Example 2 is effective in improving, preventing or treating inflammatory bowel disease and leaky intestinal syndrome.

Hereinafter, examples of the formulation of the composition containing the powder of the present invention will be described, but the present invention is not intended to limit it, but is intended to be described in detail.

Formulation Example 1. Preparation of powder

Radish hot water extract obtained in Example 1 or 500 mg of radish polymer fraction obtained in Example 2

100 mg lactose

Talc 10 mg

The above ingredients are mixed and filled in an airtight cloth to prepare a powder.

Formulation Example 2. Preparation of tablets

Radish hot water extract obtained in Example 1 or 300 mg of radish polymer fraction obtained in Example 2

Corn starch 100 mg

100 mg lactose

2 mg of magnesium stearate

After mixing the above ingredients, tablets are prepared by tableting according to a conventional tablet manufacturing method.

Formulation Example 3. Preparation of Capsule

Radish hot water extract obtained in Example 1 or 200 mg of radish polymer fraction obtained in Example 2

Crystalline cellulose 3 mg

Lactose 14.8 mg

Magnesium stearate 0.2 mg

According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare a capsule.

Formulation Example 4. Preparation of injection

Radish hot water extract obtained in Example 1 or 600 mg of radish polymer fraction obtained in Example 2

Mannitol 180 mg

2974 mg of sterile distilled water for injection

Na ₂ HPO _4, 12H ₂ O 26 mg

It is prepared in the amount of the above ingredients per ampoule according to a conventional injection preparation method.

Formulation Example 5. Preparation of liquid formulation

Radish hot water extract obtained in Example 1 or 4 g of radish polymer fraction obtained in Example 2

10 g of isomerized sugar

5 g of mannitol

Purified water appropriate amount

According to the usual preparation method of liquid formulations, add and dissolve each component in purified water, add an appropriate amount of lemon flavor, mix the above ingredients, add purified water and add purified water to adjust the total to 100 g, then fill in a brown bottle for sterilization. To prepare a liquid formulation.

Formulation Example 6. Preparation of granules

Radish hot water extract obtained in Example 1 or 1,000 mg of radish polymer fraction obtained in Example 2

The right amount of vitamin mixture

Vitamin A acetate 70 ㎍

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

Vitamin B2 0.15 mg

Vitamin B6 0.5 mg

Vitamin B12 0.2 ㎍

Vitamin C 10 mg

Biotin 10 ㎍

Nicotinic acid amide 1.7 mg

Folic acid 50 ㎍

0.5 mg of calcium pantothenate

Suitable amount of inorganic mixture

Ferrous sulfate 1.75 mg

Zinc oxide 0.82 mg

Magnesium carbonate 25.3 mg

Potassium monophosphate 15 mg

Dicalcium phosphate 55 mg

Potassium citrate 90 mg

100 mg of calcium carbonate

Magnesium chloride 24.8 mg

The composition ratio of the vitamin and mineral mixture is relatively suitable for granules, but it is possible to arbitrarily modify the mixing ratio. After mixing the above ingredients according to a conventional granule preparation method, the granules And can be used to prepare a health functional food composition according to a conventional method.

Formulation Example 7. Preparation of functional beverage

Radish hot water extract obtained in Example 1 or 1,000 mg of radish polymer fraction obtained in Example 2

Citric acid 1,000 mg

100 g oligosaccharides

2 g of plum concentrate

1 g taurine

Total 900 ml by adding purified water

After mixing the above ingredients according to the normal health drink manufacturing method, stirring and heating the mixture at 85°C for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed and sterilized, and then stored in a refrigerator. It is used to prepare the functional beverage composition of the present invention.

The composition ratio is a mixture of ingredients suitable for a relatively preferred beverage in a preferred embodiment, but the mixing ratio may be arbitrarily modified according to regional and ethnic preferences such as the demand class, the country of demand, and the purpose of use.

Claims (13)

A food composition for improving or preventing leaky gut syndrome using radish extract as an active ingredient. The method of claim 1,
The radish extract is a food composition for improving or preventing leaky gut syndrome, characterized in that it is extracted with water, alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof. delete The method of claim 1,
The composition is a food composition for improving or preventing leaky gut syndrome, characterized in that it enhances IgA expression in a high-fat diet _{and inhibits or reduces the expression of IL-6 and LTB 4.} delete The method of claim 1,
The leaky gut syndrome is a food composition for improving or preventing leaky gut syndrome, characterized in that induced by a high fat diet. A pharmaceutical composition for preventing or treating leaky intestinal syndrome using radish extract as an active ingredient. The method of claim 7,
The radish extract is a pharmaceutical composition for preventing or treating intestinal leak syndrome, characterized in that it is extracted with water, alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof. delete The method of claim 7,
The composition is a pharmaceutical composition for preventing or treating leaky gut syndrome, characterized in that it enhances IgA expression in a high fat diet _{and inhibits or reduces the expression of IL-6 and LTB 4.} delete The method of claim 7,
The leaky gut syndrome is a pharmaceutical composition for preventing or treating leaky gut syndrome, characterized in that induced by a high fat diet. delete

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