KR101460417B1 - A new compound and Composition comprising compounds isolated from the fruit extract of Morus alba L for immuno-stimulating activity - Google Patents

Abstract

More particularly, the present invention relates to a composition for enhancing NO production in macrophage Raw 264.7 cells, TNF-a and IL < RTI ID = 0.0 > -12, and exhibits an effect of enhancing the activity in immune cells. Thus, it is possible to provide a food, drug, and feed additive having immunity enhancing effect excellent in prevention, inhibition and treatment of immunosuppression due to excellent immunity enhancement effect .

Description

[0001] The present invention relates to an immunoconjugate composition comprising a novel compound and a separate compound isolated from a heart extract as an active ingredient,

The present invention relates to a composition for immunomodulation comprising a novel compound and a separated compound isolated from a fresh heart extract as an active ingredient.

[Patent Document 1] Richard A. Goldsby, et al., KUBY Immunology , 2000

[2] Ragupathi, G., Yeung, KS, Leung, PC, Lee, M., Lau, CBS, Vickers, A., Hood, C., Deng, G., Cheung, NK, Cassileth, B., Livingston, P., 2008. Evaluation of widely consumed botanicals as immunological adjuvants. Vaccine 26, 4860-4865.

[3] Won, D.P., Lee, J.S., Kwon, D. S., Lee, K.E., Shin, W.C., Hong, E.K., 2011. Immunostimulating activity by polysaccharides isolated from fruiting body of Inonotus obliquus. Molelular Cells 31, 165-173.

[4] Sun, HX, Qin, F., Ye, YP, 2005. Relationship between haemolytic and adjuvant activity and structure of protopanaxadiol-type saponins from the roots of Panax notoginseng . Vaccine 23, 5533-42.

[Literature 5] Wolf, S. F., Sieburth. D., Sypek, J. 1994. Interleukin 12: a key modulator of immune function. Stem Cells 12,154-168.

[Literature 6] Lee, JS, Hong, EK, 2011. Immunostimulating activity of the polysaccharides isolated from Cordyceps militaris . International Immunopharmacology 11, 1226-1233.

[Literature 7] Murray, P. J., Wynn, T. A., 2011. Protective and pathogenic functions of macrophage subsets. Nature Reviews Immunology 11, 723-737.

[Literature 8] Information Sourcing, Illustrated Dictionary of Contemplation, Yeonglim Publishing House, p545-548, 1998)

[9] Hiermann, A., Kedwani, S., Schramm, HW, Seger, C., 2002. A new pyrrole alkaloids from seeds of Castanea sativa Phytotherapy 73, 22-27.

[10] Chin, Y. W., Lim, S. W., Kim, S. H., Shin, D. Y., Suh, Y. G., Kim, Y.B., Kim, Y.C., Kim, J., 2003. Hepatoprotective pyrrole derivatives of Lycium chinensis fruits. Bioorganic & Medicinal Chemistry Letters 13, 79-81.

J., DeGraff, W. G., Gazdar, A. F., Minna, J. D., Mitchell, J. B., Jobgen, W. S., 1987. Evaluation of a tetrazolium-based seminomatous colorimetric assay: assessment of chemosensitivity testing. Cancer Research 47, 936-942.

[Literature 12] Jobgen, W. S., Jobgen, S. C., Li, H., Meininger, C. J., Wu, G., 2007. Analysis of nitrite and nitrate in biological samples using high-performance liquid chromatography. Journal of Chromatography B Analytical Technologies in Biomedical and Life Sciences. 851, 71-82.

[Literature 13] Ledur, A., Fitting, C., David, B., Hamberger, C., Cavaillon, J.M., 1995. Variable estimates of cytokine levels produced by commercial ELISA kits: results using international cytokine standards. Journal of Immunological Methods 186, 171-179.

(IL-10) mediated suppression of IL-12 production in RAW 264.7 cells also involved c-myc, rel transcription factor. Immunology 114, 313-321.

[Literature 15] Cheng, AW, Wan, FC, Wang, JQ, Jin, ZY, Xu, XM, 2008. Macrophage immunomodulatory activity of polysaccharides isolated from Glycyrrhiza uralensis Fish. International Immunopharmacology 8, 43-50.

[16] Chen W., Zhang W., Shen W., Wang K., 2010. Effects of the acid polysaccharide fraction isolated from a cultivated Cordyceps sinensis on macrophages in vitro. Cell Immunol. 262 (1), 69-74.

[Literature 17] Korea Food and Drug Administration, Toxicological Testing Standards for Drugs, etc., Korea Food & Drug Administration Notice 2005-60, 2005

The immune system can be divided into natural resistance, nonspecific immune system and specific immune system. Natural resistance (first line of defense) is an anatomical physiological element that blocks all invaders, including microorganisms, regardless of their type. Nonspecific immunity (second line of defense) is a phagocyte that breaks through natural resistance and removes intruders. A specific immune system is a highly developed immune system with the ability to distinguish between memory and non-immune systems (Richard A. < RTI ID = 0.0 > A. < / RTI > Goldsby, et al., KUBY Immunology , 2000).

Macrophages contain many lysosomes, which contain acid hydrolytic enzymes and peroxidase. It is also strongly attached to the glass surface and plastic surface and actively digests microorganisms and tumor cells. The cell has a cytokine receptor such as IFN-y. They produce cytokines such as complement components, interferons, interleukin-1 and tumor necrosis factors, and can be enhanced by a variety of cytokines produced from T-cells. T-cells, a type of leukocytes, account for about 70% of the bovine lymphocytes in the blood. T-cells differentiate in the thymus and have T-cell antigen receptors (TCRs). Peripheral blood T- cells were divided into CD4-positive T _H cells (helper T-cell) and CD8-positive T _C cells (cytotoxic T-cell), CD4 + T _H cells recognize the antigen binds to MHC II class (class) molecule And helps to stimulate B cells to enable the production of antibodies or help other T cells function. CD4 + T _H cells can be further classified as T _H 1 and T _H 2 based on the cytokines they produce. T _H 1 cells in mice (mouse) is Interleukin -2 (IL-2), interferon - T _H 2 cells, while the secretion of gamma (IFN-γ), etc., IL-4, IL-5, IL-6 , IL-9, IL-10, IL-13 and the like. However, the production of IL-2, IL-6, IL-10 and IL-13 in humans is not strictly classified. T _H 1 cells are involved in cellular immune responses and activate cytotoxic and inflammatory responses. T _H 2 cytokines produced from cells in promoting antibody formation, especially helping to produce IgE because of enhancing the proliferation and function of eosinophils, T _H 2 cytokines is commonly found in antibody production and an allergic reaction. It has been shown that T _H 1 and T _H 2 cytokines can inhibit hepatotoxicity and can alter the progression of the disease with anti-IL-4 and anti-IFN-γ antibodies in animal infectious diseases. In patients with rheumatoid arthritis In addition, IFN-γ has been shown to improve symptoms (Richard A. Goldsby, et al., KUBY Immunology . 2000).

Natural products have been used in the treatment of various diseases in Asian countries. Plants that have been widely used have been used as tonic for immunity enhancement, health maintenance and long life, and have been used in a variety of compositions, especially polysaccharides,? -Glucans, saponins, Curcuminoids have been proposed as a broad-spectrum immunomodulatory agent in clinical and drug applications (Ragupathi et al., 2008; Won et al., 2011; Sun et al., 2005).

Immunostimulation is one of the important therapeutic strategies to reinforce the body defense mechanisms for various diseases such as cancer and inflammatory diseases. Macrophages play a major role in the immune response. Phagocytosis, a major role in macrophages, has been shown to absorb microbes and other exothermic particles and also to inhibit the production of many cytokines, such as tumor necrosis factor (TNF) -α, interleukin (IL) -1β, 1994, Lee and Hong (2002), stimulate the immune response by secreting cytokines and cytotoxic and inflammatory substances such as nitric oxide (NO) and reactive oxygen species (ROS) , 2011; Murray and Wynn, 2011).

Saddened chairs (桑子) is Mulberry (Morus belonging to Moraceae (Moraceae) alba ) , which contains sugars, tannic acid, malic acid, vitamins B1, B2, C, carotene, linolic acid, stearic acid and oleic acid. , And has been used for the treatment of constipation, such as dyspareunia (Information,

However, none of the above references discloses that compounds isolated from heartburn extracts are effective in enhancing the immune system.

Therefore, we aimed to clarify the immune enhancement effect and the mechanism of action of natural products used as medicinal herbs by using modern objective immune activity indicators.

Therefore, the present inventors have found that the compounds isolated from the sickle cell extract have an effect of increasing NO production in macrophage Raw 264.7 cells, and enhancing secretion ability of immunity enhancing factors such as TNF-α and IL-12, And excellent immunosuppressive effect, and it is confirmed that it is excellent in prevention, suppression and treatment of hypoimmunities, and thus it can be effectively used as food, medicine and feed additive having immunity enhancing effect.

In order to achieve the above object,

The present invention relates to a novel alkaloid compound (4- (2-formyl) -5 - (((3-hydroxybutane- 2-yl) oxy) methyl) -1H-pyrrole-1-yl) butanoic acid (= morole A :, Morrole A), an isomer thereof or a pharmacologically Acceptable salts are provided:

Morrolee

The present invention also provides a pharmaceutical composition for enhancing immunity, which comprises a compound isolated from a topical extract as an active ingredient.

The present invention provides a health functional food for immunity enhancement containing as an active ingredient a compound isolated from a topical extract.

In addition, the present invention provides an animal feed additive for immunity enhancement, which comprises a compound isolated from a root extract as an active ingredient.

The extracts as defined herein include water, lower alcohols having 1 to 4 carbon atoms, or a mixed solvent thereof, preferably water-soluble extracts.

The compounds isolated from the above extracts as defined herein may be selected from the group consisting of 5- (hydroxymethyl) -1 H -pyrrole-2-carboxaldehyde (Compounds 1), 2-formyl ) -1 H - pyrrole (pyrrole) -1- butanoate acid (butanoic acid) (Compound 2) , 2- formyl (formyl) -5- (hydroxymethyl; hydroxymethyl) -1 H - pyrrole (pyrrole) - Butanoic acid (Compound 3), 2-formyl-5- (methoxymethyl) -1 H -pyrrole-1-butanoic acid ( Compound 4) and a novel alkaloid compound (4- (2-formyl) -5 - ((hydroxybutan-2-yl) oxy) methyl) Pyrrol-1-yl) butanoic acid. ≪ / RTI >

The compounds of the present invention may be prepared by pharmaceutically acceptable salts and solvates according to methods conventional in the art.

Pharmaceutically acceptable salts include acid addition salts formed by free acids. The acid addition salt is prepared by a conventional method, for example, by dissolving the compound in an excess amount of an acid aqueous solution, and precipitating the salt using a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. The same molar amount of the compound and the acid or alcohol (e.g., glycol monomethyl ether) in water may be heated and then the mixture may be evaporated to dryness, or the precipitated salt may be filtered by suction.

As the free acid, organic acids and inorganic acids can be used. As the inorganic acids, hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, tartaric acid and the like can be used. Examples of the organic acids include methanesulfonic acid, p -toluenesulfonic acid, acetic acid, trifluoroacetic acid Citric acid, lactic acid, glycollic acid, gluconic acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, manderic acid, propionic acid, citric acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanillic acid and hydroiodic acid can be used.

In addition, bases can be used to make pharmaceutically acceptable metal salts. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving a compound in an excess amount of an alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the non-soluble compound salt, and evaporating and drying the filtrate. At this time, it is preferable for the metal salt to produce sodium, potassium or calcium salt in particular, and the corresponding silver salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (for example, silver nitrate).

Pharmaceutically acceptable salts of the compounds of the present invention include, unless otherwise indicated, salts of acidic or basic groups that may be present in the compounds of the present invention. For example, pharmaceutically acceptable salts include sodium, calcium and potassium salts of hydroxy groups, and other pharmaceutically acceptable salts of amino groups include hydrobromide, sulfate, hydrogen sulfate, phosphate, hydrogen phosphate, dihydrogen phosphate phosphate, acetate, succinate, citrate, tartrate, lactate, mandelate rate, methane sulfonate (mesylate) and p - toluene sulfonate (tosylate) and a salt, the salt manufacturing method or manufacturing process known in the art ≪ / RTI >

Further, the novel compounds having the structure of the above structural formula (5) can exist in different enantiomeric forms because they have an asymmetric center, and all optical isomers and R or S type stereoisomers and mixtures thereof are also included within the scope of the present invention . The present invention includes the use of racemates, one or more enantiomeric forms, one or more diastereoisomeric forms, or mixtures thereof, and includes methods and processes for the isolation and isomerization of isomers known in the art.

Compounds isolated from the extract of the present invention can be prepared by the following production process or commercially available.

Dried bulkheads may be prepared according to conventional extraction methods and may be prepared from C ₁ to C ₄ lower alcohols, such as water, methanol, etc., in a volume from 1 to 20 times, preferably from 5 to 15 times, And the mixture is stirred for 0.5 to 10 hours, preferably 2 to 5 hours, at 70 to 100 ° C for 1 to 10 times, preferably 2 to 5 times, in a mixed solvent of these, preferably water and methanol, The extract is preferably subjected to extraction by hot water extraction such as extraction, hot water extraction, ultrasonic extraction and reflux cooling extraction. The extract is filtered under reduced pressure through a filter paper, and the filtrate is concentrated and lyophilized to obtain a fresh extract of the present invention after that, the suspended water was added to the resulting methanol extract, take advantage of this, successively performed, the fraction with CHCl _3, EtOAc and n-BuOH, and targeting the EtOAc fractions, CH ₂ Cl ₂ -MeOH eluant ? Silica gel performing a column chromatography of 6 fractions (MFE1-MFE6) obtained and filling the Sephadex (Sephadex) LH-20 and the resulting MFE2 fraction and a developing solvent _{(MeOH-H 2 O; 1} : 1) for using Column chromatography was performed to obtain six fractions (MFE21-MFE26), and semipreparative HPLC was performed on the MFE23 fraction of the fractions to obtain the compounds of the present invention, 5- (hydroxymethyl) -1 H -pyrrole-2-carboxaldehyde (Compounds 1), 2-formyl-1 H -pyrrole-1-butanoic acid (Compound 2 ), 2-formyl (formyl) -5- (hydroxymethyl; hydroxymethyl) -1 H - pyrrole (pyrrole) -1- butanoate acid (butanoic acid) (Compound 3), 2-formyl (formyl) - 5-methoxymethyl- 1H -pyrrole-1-butanoic acid (Compound 4) and the novel alkaloid compound Morro A = (4- (2- (formyl) -5 - (((3- Hydroxybutan-2-yl) oxy) methyl) -1H-pyrrole-1-yl) butanoic acid A (Compound 5) Respectively.

The present invention provides a pharmaceutical composition for enhancing immunity, which comprises, as an active ingredient, a compound isolated from the extract of the upper and lower heights obtained by the above production method and the above-mentioned production method.

The pharmaceutical composition for immunostaining according to the present invention contains the above extract in an amount of 0.1 to 50% by weight based on the total weight of the composition.

The pharmaceutical composition of the present invention can be used for the treatment of diseases caused by immunotherapy such as chemotherapy and radiotherapy such as reduction of immune function or bone marrow transplantation, immunosuppression due to immune system damage, ≪ / RTI >

The pharmaceutical compositions containing the compounds isolated from the extract of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the preparation of pharmaceutical compositions.

The pharmaceutical composition containing the compound isolated from the extract according to the present invention may be administered orally or parenterally in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups and aerosols, external preparations, suppositories, Examples of the carrier, excipient and diluent which can be contained in the composition including the compound isolated from the extract include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol , Starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate And mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid form preparations for oral administration include tablets, pills, powders, granules, capsules and the like. These solid preparations may contain at least one excipient such as starch, calcium carbonate, Sucrose, lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The preferred dosage of the compound isolated from the extract of the present invention varies depending on the condition and body weight of the patient, the degree of disease, the drug form, the administration route and the period, but can be appropriately selected by those skilled in the art. However, for the desired effect, the compound isolated from the extract of the present invention is preferably administered at 0.0001 to 100 mg / kg per day, preferably 0.001 to 10 mg / kg per day. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.

The compound isolated from the extract of the present invention can be administered to mammals such as rats, mice, livestock, humans, and the like in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

The present invention provides a health functional food for immunity enhancement containing a compound isolated from the above-mentioned core extract.

Examples of foods to which the compound isolated from the extract of the present invention can be added include various foods, beverages, gums, tea, vitamin complexes, and health functional foods.

It can also be added to foods or beverages for the purpose of enhancing the immunity. In this case, the amount of the compound isolated from the extract in the food or beverage may generally be 0.01 to 50% by weight, preferably 0.01 to 15% by weight, of the total food weight of the health functional food composition of the present invention, The composition may be added at a rate of 0.02 to 5 g, preferably 0.3 to 1 g, based on 100 ml.

The health functional food of the present invention includes forms such as tablets, capsules, pills, and liquids.

The health beverage composition of the present invention has no particular limitations on other ingredients other than those containing the compound isolated from the extract as an essential ingredient in the indicated ratio and contains various flavors or natural carbohydrates as an additional ingredient . Examples of the above-mentioned natural carbohydrates include monosaccharides such as disaccharides such as glucose and fructose, polysaccharides such as maltose, sucrose and the like, such as dextrin, cyclodextrin and the like And sugar alcohols such as xylitol, sorbitol and erythritol. Natural flavors (tau martin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

In addition to the above, the extract of the present invention can also be used as a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and aging agents (cheese, chocolate, etc.), pectic acid and its salts, Salts, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages and the like. In addition, the compounds isolated from the extract of the present invention may contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The proportion of such additives is not so critical, but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

In addition, the present invention provides an animal feed additive for immunity-enhancing animal containing a compound isolated from the above-mentioned bulkhead extract and a feed comprising the animal feed additive.

The composition for animal feed additive may be 20 to 90% high concentrate or may be in the form of powder or granules.

The composition for animal feed additive of the present invention is a composition containing an organic acid such as citric acid, fumaric acid, adipic acid, lactic acid and malic acid, a phosphate such as sodium phosphate, potassium phosphate, acid pyrophosphate, polyphosphate (polymerized phosphate), polyphenol, catechin ), Natural antioxidants such as alpha-tocopherol, rosemary extract, vitamin C, green tea extract, licorice extract, chitosan, tannic acid and phytic acid.

Animal feed additive containing the composition of the present invention and feeds containing the composition of the present invention may be supplemented with various adjuvants such as amino acids, inorganic salts, vitamins, antibiotics, antimicrobials, antioxidants, antifungal enzymes, living microbial agents, For example, crushed or shredded wheat, oats, barley, corn and rice; Vegetable protein feedstuffs, for example, based on rapeseed, soybeans and sunflower; Animal protein feeds such as blood, meat, bone meal and fish meal; It is preferable to mix all of the dry ingredients comprising the sugar and the milk product such as the various powdered milk and the whey powder and the dry additive and then mix the liquid ingredient with the ingredient to be liquid after heating, In addition to the main components such as fat and vegetable fat, can be used together with materials such as nutritional supplements, digestion and absorption enhancers, growth promoters, disease prevention agents and the like.

The composition for the animal feed additive can be administered alone to the animal in combination with other feed additives in the edible carrier.

In addition, the composition for animal feed additives can be easily administered as top dressing or they can be mixed directly with animal feed, or separately from feed, in separate oral formulations, by injection or percutaneously, or in combination with other ingredients. Typically, a single daily dose or a divided daily dose can be used as is well known in the art.

When the composition for animal feed additives is administered separately from an animal feed, the dosage form of the composition, as is well known in the art, may be prepared in an immediate release or sustained release formulation in combination with their non-toxic pharmaceutically acceptable excipient . Such edible carriers may be solid or liquid, for example corn starch, lactose, sucrose, soy flakes, peanut oil, olive oil, sesame oil and propylene glycol. When a solid carrier is used, the dosage form of the compound may be a tablet, capsule, powder, troche or emulsion, or a top-dressing in finely divided form. When a liquid carrier is used, it may be in the form of a soft gelatin capsule, or a syrup or liquid suspension, emulsion or solution. In addition, the dosage form may contain adjuvants such as preservatives, stabilizers, wetting or emulsifying agents, solution promoting agents and the like.

In addition, the animal feed in which the compound of the present invention is included as an animal feed additive can be any protein-containing organic grain fraction commonly used to meet animal dietary needs. These protein-containing flours usually consist mainly of corn, soy flour or corn / soy flour mix.

The animal feed additive may be added to the animal feed by immersion, spraying or mixing.

The present invention is applicable to a number of animal diets including mammals, poultry, and fish. More specifically, the diets include, but are not limited to, commercially important mammals such as pigs, cows, sheep, goats, laboratory animals (rats, mice, hamsters and gerbils), fur- Zoo animals (eg, monkeys and tailless monkeys), as well as livestock (eg, cats and dogs). Common commercially important poultry include chicken, turkey, duck, goose, pheasant and quail. Commercial breeding fish such as trout can also be included.

The animal feed compounding method comprising the composition according to the present invention is such that the composition is incorporated into animal feed in an amount of about 1 g to 100 g per kg of feed on a dry weight basis.

Also, after the feed mixture is thoroughly mixed, lightly viscous granulation or granulation material is obtained depending on the degree of crushing of the components. It is supplied as a mesh or is formed into a desired discrete shape for further processing and packaging. At this time, in order to prevent separation during storage, it is preferable to add water to the animal feed, followed by a conventional pelleting, expansion, or extrusion process. Excess water can be dried off.

Compounds isolated from the extracts of the present invention exhibit an effect of enhancing NO production in macrophage Raw 264.7 cells, and enhancing the secretion ability of immunological enhancers such as TNF-α and IL-12, thereby enhancing activity in immune cells Which is superior in immunity enhancement efficacy and is excellent in prevention, inhibition and treatment of hypoimmune, and thus can be usefully used as a food, medicine and feed additive having immunity enhancing effect.

Figure 1 shows the immunomodulating activity of a sample in Raw 264.7 cells { (A) cell viability test results; (B) NO production, (C) TNF-α, (D) IL-12 secretion, and (E) phagocytic activity in macrophages. Data were mean (mean ± SD) UN) ( ^* p <0.05, ^** p <0.01).

Below, The present invention will be described in detail by the following examples and experimental examples.

However, the following examples and experimental examples are illustrative of the present invention, and the content of the present invention is not limited by the following examples and experimental examples.

Example  One. Heartbreaker  Isolation of Compounds from Extracts

1.1. Methanol extract

1.0 kg of 50% MeOH in 1 L was added to Moru alba (purchased from the University of Korea) and 1 kg of CBNU201009-MF from the Herbal Farm of Chungbuk National University, The mixture was extracted with an extraction method and concentrated under reduced pressure to obtain a methanol extract (131.8 g). The obtained methanol extract was suspended in 200 ml of purified water and fractionated successively with CHCl ₃ , EtOAc and n-BuOH.

The resulting EtOAc fraction (2.7 g) was subjected to silica gel column chromatography using CH ₂ Cl ₂ -MeOH developing solvent to obtain six fractions (MFE 1 -MFE 6), and the resulting MFE 2 fraction was purified by Sephadex LH -20 were charged and column chromatography using a developing solvent (MeOH-H ₂ O; 1: 1) was carried out to obtain six fractions (MFE21-MFE26).

1.2. Separation of material from methanol extract

Semipreparative HPLC using an elution solvent (MeOH-water with 0.1% acetic acid; 43:57, F = 2 min / ml, Rt = 11.4, 14.0 and 15.21 min, respectively) gave the pyrrole alkaloid based compounds which are (pyrrole alkaloids), 2- formyl (formyl) -1 H showing the following physical-pyrrole (pyrrole) -1- butanoate acid (butanoic acid) (compound 2, 4.2mg),

Butanoic acid (Compound 4. 13.9 mg) and a novel alkaloid compound, moles A {(methoxymethyl) -1 H -pyrrole-1-butanoic acid (4- (2-formyl) -5 - ((3-hydroxybutane-oxy) methyl) -1H-pyrrole- ) Butanoic acid, 3.9 mg), respectively.

The fraction (30.3 g) of the n- BuOH fraction was subjected to XAD-2 column chromatography using a solvent (methanol-water; 0%, 25%, 50%, 75%, 100% MeOH in water) MFB4 fractions were subjected to Sephadex LH-20 column chromatography using a solvent (MeOH-H ₂ O; 1: 1) to obtain three fractions (MFB41-MFB43) MFB43 fractions were subjected to semipreparative HPLC using a solvent (MeOH-water with 0.1% acetic acid; 45:55, F = 2 min / ml, Rt = 4.8 and 6.2 min, respectively) 5- (hydroxymethyl) -1 H -pyrrole-2-carboxaldehyde (Compounds 1, 2.1 mg) and 2-formyl-5- (hydroxymethyl; hydroxymethyl) -1 H -pyrrole-1-butanoic acid (Compound 3, 12.0 mg) were separated and their structures were compared with the physical properties described in the existing literature. (Hiermann et al., 2002; Chin et al., 2003)

5- ( Hydroxymethyl ; hidroxymethyl )-One H - pyrrole ( pyrrole ) -2-carboxaldehyde ( carb oxaldehyde) ( Compounds  One)

^{1 H NMR (400 MHz, CD} 3 OD): δ 4.64 (2H, s, CH 2 -6), δ 6.60 (1H, d, J = 3.5 Hz, CH-5), 7.40 (1H, d, J = 3.5 Hz, CH-4), 9.56 (1H, s, CH-1) ppm;

^{13 C NMR (100 MHz, CD} 3 OD): δ 56.8 (C-6), 108.5 (C-4), 125.7 (C-3), 132.6 (C-2), 142.0 (C-5), 178.1 ( C-1)

ESI-MS (positive mode) m / z : 148.15 [M + Na] &lt; ^{+ &}

2- Formyl ( formyl )-One H - pyrrole ( pyrrole )-One- Butanoic acid ( butanoic acid ) ( Compound  2)

^{1 H NMR (400 MHz, CD} 3 OD): δ 2.04 (2H, p, J = 7.2 Hz, CH 2 -H2'), 2.26 (2H, t, J = 7.2 Hz, CH2-H3'), 4.39 ( 2H, t, J = 7.2 Hz , CH 2 -H1'), 6.27 (1H, dd, J = 2.4, 4.0, CH-H4), 7.05 (1H, dd, J = 1.6, 4.0, CH-H3), 7.18 (1H, s, CH-5), 9.47 (1H, d, J = 0.8, CH-1) ppm;

^{13 C NMR (100 MHz, CD} 3 OD): δ 26.3 (C-2'), 30.1 (C-3'), 45.6 (C-1'), 108.7 (C-4), 125.3 (C-3) , 128.1 (C-5), 132.3 (C-2), 175.1 (COOH), 179.4 (CHO)

ESI-MS (positive mode) m / z : 261.01 [2M-H] ^-

2-formyl-5- (hydroxymethyl) -1 H Pyrrole-1-butanoic acid (Compound 3)

^{1 H NMR (400 MHz, CD} 3 OD): δ 2.04 (2H, m, H-2'), 2.33 (2H, m, H-3'), 4.42 (2H, m, H-1'), 4.66 (2H, s, H-6), 6.28 (1H, d, J = 4.0 Hz, H-4), 7.01 (1H, d, J = 4.0 Hz), 9.44 (1H, s, H-1) ppm;

^{13 C NMR (100 MHz, CD} 3 OD): δ 26.2 (C-2'), 30.4 (C-3'), 44.4 (C-1'), 55.0 (C-5), 110.8 (C-4) , 125.9 (C-3), 132.1 (C-2), 143.2 (C-5), 175.3 (COOH), 179.5

ESI-MS (positive mode) m / z : 234.19 [M + Na] &lt; ^{+ &}

2-formyl-5- (methoxymethyl) -1 H Pyrrole-1-butanoic acid (Compound 4)

^{1 H NMR (400 MHz, CD} 3 OD): δ 2.01 (2H, m, H-2'), δ 2.34 (2H, t, J = 7.6, H-3'), 3.38 (3H, s, H- _{OCH 3), 4.39 (2H,} m, H-1'), 4.51 (2H, s, H-6), 6.30 (1H, d, J = 4.0 Hz, H-4), 7.00 (1H, d, J = 4.0 Hz, H-3), 9.47 (1H, s, H-1) ppm;

^{13 C NMR (100 MHz, CD} 3 OD): δ 26.2 (C-2'), 30.3 (C-3'), 44.4 (C-1'), 56.8 (OCH 3), 64.9 (C-6), (C-2), 139.6 (C-5), 175.1 (COOH), 179.7 (CHO)

ESI-MS (positive mode) m / z : 248.21 [M + Na] &lt; ^{+ &}

4- (2- Formyl ( formyl ) -5 - (((3- Hydroxybutane ; hydroxybutane -2- yl ) Oxy ; oxy ) methyl ; methyl ) -1H-pyrrole ( pyrrol )-One- yl ) Butanowic  mountain( butanoic acid ) ( Compound  5)

pale yellowish gum;

UV (MeOH)? _Max (log?): 293 (3.85); IR _max 3407, 2938, 1703, 1408 cm &lt; ^-1 &gt;

^{1 H NMR (400 MHz, CD} 3 OD): δ 1.15 (3H, d, J = 6.4 Hz, CH 3 -4 "), 1.18 (3H, d, J = 6.4 Hz, CH 3 -3", 2.03 ( M, H-3 '), 2.36 (2H, t, J = 7.2 Hz, H-2'), 3.47 (2H, t, J = 7.6 Hz, H-1 '), 4.62 (1H, d, J = 12.4 Hz, H-6a), 4.67 (1H, d, J = 12.4 Hz, H-6b), 6.31 ( 1H, d, J = 4.0 Hz, H-4), 7.01 (1H, d, J = 4.0 Hz, H-3), 9.46 (1H, s, CHO) ppm;

¹³ C (100 MHz, CD ₃ OD):? 15.0 (C-3 ", 18.6 (C-4", 27.6 C-6), 70.9 (C-2), 80.6 (C-1), 112.6 (C-4), 126.0 ), 181.0 (CHO) ESI- MS (positive mode) m / z: 306 [M + Na] +

HRESIMS (positive mode) m / z :. 306.1312 [M + Na] + (Calc for [C 14 H 21 NO 5 + Na] ⁺ 306.1317).

Experimental Example  1. Cells On survival  Effect on

In order to confirm the effect of the compounds of the above examples on the viability of the cells, MTT assays were performed as follows (Carmichael et al., 1987) using the method described in the literature.

RAW 264.7 cells (ATCC, USA) were cultured in DMEM medium (Gibco, USA) supplemented with 10% heat-inactivated fetal bovine serum and penicillin-streptomycin at 37 ° C in 5% CO2 and 95% % Air in the humidified condition. RAW 264.7 cells were treated with 3, 10, 30 and 100 mM concentrations for 24 hours and cultured.

As a result of the experiment, it was confirmed that the compounds of the present invention did not show significant cytotoxicity as compared with the negative control group (the group not treated with the compound) as shown in Fig. 1-A.

Experimental Example  2. Experiment of immune enhancement.

2.1. NO , TNF -α and IL -12 Increased effect of secretion

In order to confirm the increasing effect of the compounds of the above examples on the secretion of NO, TNF-α and IL-12, the method described in the literature was applied using the method described in the literature (Jobgen, WS, Jobgen, SC, Li, H., Meininger, CJ, Wu, G., 2007. Analysis of nitrite and nitrate in biological samples using high-performance liquid chromatography. Journal of Chromatography B Analytical Technologies in Biomedical and Life Sciences 851, 71-82 ).

RAW 264.7 cells (ATCC, USA) and then by 1 × 10 per well in a 96-well microplate ⁵ frequency divider, and the process of the invention compounds with a 3, 10, 30 and a concentration of 100 mM, 37 ℃, 5% CO 2 (Griess reagent, 0.1% w / v N- (1-naphthyl) ethylenediamine dihydrochloride + 1% (w / v)) was added to 50 μl of the cell culture supernatant. / v) sulfanilamide in 5% (v / v) phosphoric acid) was added and mixed at room temperature for 10 minutes. Absorbance was then measured at 540 nm using an Infinite 200 spectrophotometer (Tecan, Switzerland) . Lipopolysaccharide (LPS, 100 ng / ml) was used as a positive control.

In addition, 50 [mu] l of the cell culture supernatant was subjected to ELISA test for inflammatory mediators TNF-alpha (tumor necrosis factor-alpha) and IL-12 (Interlukin 12) under the same conditions (R & D Systems Quantikine Mouse TNF-α / IL-12 kit; Ledur et al., 1995; Rahim et al., 2005).

As a result, as shown in FIG. 1B, the concentration of nitrite ions (NO ₂ ^- ) in Compounds 3 and 4 increased 1.8 and 1.5-fold, respectively, compared to the control group in RAW 264.7 cells. . In the RAW 264.7 cells, TNF-α was 2.37 and 2.31-fold higher than that of the control group, and the increase in NO secretion was similar to that of the LPS alone treatment group, which was used as a positive control. As IL-12 secretion also showed a significant increase effect compared with the control group, it was confirmed that the compounds 3 and 4 increase the measured TNF-a, IL-12 and NO secretion (see FIGS. 1C and 1D).

2.2. RAW  In 264.7 On phagocytosis  Active effect on

Analysis was carried out using the Neutral Red Uptake method described in the literature (Cheng, WX et al., Cellular Immunology, 262, pp69-74 2010.).

RAW 264.7 cells (ATCC, USA) were dispensed into 96-well microplates at a rate of 1 × 10 ⁵ cells per well, and the compounds of the present invention were added at 37 ° C., 5% CO ₂ , And 100 μl of 0.075% Neutral Red was added. After culturing in an incubator at 37 ° C for 1 hour, 200 μl of a solution containing 0.01% acetic acid and 100% ethanol in an equal volume was treated at room temperature for 2 hours, Absorbance was measured at 570 nm using a 200 spectrophotometer (Tecan, Switzerland). Lipopolysaccharide (LPS, 100 ng / ml) was used as a positive control.

As a result, as shown in FIG. 1E, the sowing ability of RAW 264.7 cells according to the treatment with Compound 3 increased 2.29-fold in the 30-mM treatment group compared to the control group, and the compound 4 also significantly increased the spermatogenesis in all concentrations. Thus, it was confirmed that the compounds 3 and 4 improve the phagocytic ability of macrophages (see FIG. 1B).

2.3. Experiment result

As a result, compounds 3 and 4 increased NO production without significant cytotoxicity up to 100 mM (Tables 1 and 2, FIGS. 1A and B). The effects on TNF-a and IL-12 production were similar (Tables 3 and 4, Figs. 1C and D).

In addition, phagocyte activity in RAW 264.7 cells treated with 100 mM Compound 3 was about 1.93 times higher than the non-treated group (Table 4, Fig. 1E). Compound 4 also showed activity to enhance the phagocytosis significantly.

Cell viability (%) conc. (mM) 3 10 30 100 UN 100.0 ± 1.0 LPS 98.0 ± 1.0 Compound 1 98.3 ± 1.0 96.4 ± 1.0 96.0 ± 1.0 97.1 ± 1.0 Compound 2 96.4 ± 1.0 96.9 ± 1.0 97.2 ± 1.0 95.7 ± 1.0 Compound 3 96.0 ± 1.0 97.3 ± 1.0 96.9 ± 1.0 98.3 ± 1.0 Compound 4 97.1 ± 1.0 96.3 ± 1.0 96.3 ± 1.0 95.9 ± 0.9 Compound 5 98.0 ± 1.0 98.0 ± 0.9 97.0 ± 0.8 98.0 ± 0.9

NO (mM) production conc. (mM) 3 10 30 100 UN 10.7 ± 0.75 LPS 21.8 ± 1.00 Compound 1 11.6 ± 0.78 12.9 ± 0.43 12.4 ± 1.30 13.9 ± 1.40 Compound 2 12.6 ± 0.78 12.9 ± 0.43 12.4 ± 1.30 13.9 ± 1.40 Compound 3 15.3 ± 0.19 18.4 ± 1.06 * 18.3 ± 0.80 * 19.3 ± 0.78 * Compound 4 11.2 ± 0.52 15.5 ± 0.81 * 16.3 ± 0.35 * 14.2 ± 0.27 * Compound 5 10.5 ± 0.43 10.6 ± 0.77 10.0 + 1.43 10.0 + - 0.71

TNF-a (ng / ml) secretory ability conc. (mM) 3 10 30 100 UN 0.86 + 0.13 LPS 2.80 ± 0.13 Compound 1 0.89 ± 0.08 0.79 + - 0.01 1.16 ± 0.02 1.24 ± 0.05 Compound 2 0.75 + 0.03 0.99 + 0.04 0.69 + 0.07 0.89 + 0.03 Compound 3 1.06 ± 0.16 1.97 + 0.02 * 2.03 0.04 * 1.80 + - 0.05 * Compound 4 1.28 + 0.04 1.90 + - 0.20 * 1.98 ± 0.06 * 1.99 + 0.04 * Compound 5 0.86 ± 0.24 0.90 + - 0.43 0.87 ± 0.39 0.98 + 0.40

IL-12 (pg / ml) secretory ability conc. (mM) 3 10 30 100 UN 70.0 + - 23.0 LPS 593.0 + - 67.0 Compound 1 95.0 + - 19.0 103.0 + - 21.0 98.0 ± 30.0 95.0 + - 21.0 Compound 2 89.0 + - 13.0 105.0 ± 33.0 90.0 ± 32.0 101.0 ± 33.0 Compound 3 88.0 + - 40.0 133.0 + - 12.0 * 160.0 + - 40.0 * 155.0 + - 62.0 Compound 4 93.0 ± 35.0 103.0 + - 40.0 133.0 + - 20.0 * 124.0 + - 30.0 Compound 5 98.0 ± 40.0 108.0 ± 20.0 93.0 + - 40.0 112.0 + - 30.0

Phytogenic activity (%) conc. (mM) 3 10 30 100 UN 100.0 + - 0.67 LPS 284.1 ± 1.90 Compound 1 109.6 ± 0.73 90.9 ± 0.61 90.1 ± 0.60 90.9 ± 0.61 Compound 2 99.1 + - 0.66 141.1 ± 0.94 * 123.4 ± 0.83 111.2 ± 0.74 Compound 3 90.7 ± 0.61 162.7 ± 1.09 * 228.9 ± 1.53 ** 192.6 ± 1.29 * Compound 4 140.8 ± 0.94 * 152.2 ± 1.02 * 145.0 ± 0.97 * 175.8 ± 1.18 * Compound 5 78.6 ± 0.69 87.3 ± 0.33 85.9 ± 1.03 81.0 ± 0.98

Statistical test

Statistical significance was considered to be statistically significant at p <0.05 on the Student's t- test.

Experimental Example  3. Oral dose toxicity test

In this experiment, the mice were fasted for 4 hours before the administration of the compounds of the present invention using ICR mice (Orient Bio, Japan) at 7 weeks of age, and their weights were measured. The test results were evaluated by the Food and Drug Administration Kg, 50 mg / kg and 500 mg / kg, respectively) were diluted with purified distilled water to a maximum dose of 2,000 mg / kg according to the Korea Food and Drug Administration Notice 2005-60 A test substance was prepared. The administration was made by oral administration, which is the main route of application in the clinic. The method of administration was as follows: The animal was fixed by the adriamycin fixation method and then injected directly into the stomach using a metal oral administration unit. 10 ml per kg of body weight was administered once a day on the basis of body weight on the day of feeding. Feed was restricted for 2 hours after administration of the test substance to the test animal, and then feed and water were continuously supplied during the test period.

In accordance with the Toxicological Testing Standards of the Korea Food and Drug Administration and the guidelines of the International Organization for Economic Cooperation and Development (OECD) Guidelines for the Testing Chemicals revised draft guideline 420 (OECD, 2001) During the first 6 hours, with special attention, the test animal's skin, hair, eyes, mucous membrane, respiratory system, circulatory system, autonomic nervous system, central nervous system, physical activity, behavioral form and tremors of test animals, convulsions, We observed the presence of salivation, diarrhea, lethargy, sleep, and coma. The general clinical symptoms were observed once a day for 14 days after the administration and the individual body weight of the test animals was measured once a day before and after administration of the test substance and during the test period.

ICR mice were anesthetized with ethyl ether on the 14th day after the injection, and all the organs were removed by bled lethal injection to observe gross lesions of individual test animals and weigh the organs.

As a result of the acute toxicity test by single oral administration of the compounds of the present invention showing the effect on the immune enhancement, no mortality was observed at the highest dose of 2,000 mg / kg administered in the test animals and conditions used in the present study, No toxicity by administration of the compounds of the present invention was observed in any of the indicators observed according to the Food and Drug Administration's toxicity test standards at any dose used. In conclusion, the compounds of the present invention were found to be safe without toxicity at the maximum recommended dose of 2,000 mg / kg of acute toxicity test.

Formulation examples of the pharmaceutical compositions containing the compounds of the present invention are illustrated, but the present invention is not intended to be limited thereto but is specifically described.

Formulation example  One. Sanje  Produce

Compound 1 ---------------------------------------- 20 mg

Lactose ---------------------------------------------- 100 mg

Talc ---------------------------------------------- 10 mg

The above components are mixed and filled in airtight bags to prepare powders.

Formulation Example 2. Preparation of tablets

Compound 2 ---------------------------------------- 10 mg

Corn starch ---------------------------------------- 100 mg

Lactose ---------------------------------------------- 100 mg

Magnesium stearate ------------------------------- 2 mg

After mixing the above components, tablets are prepared by tableting according to the usual preparation method of tablets.

Formulation Example 3. Preparation of capsules

Compound 3 ---------------------------------------- 10 mg

Crystalline cellulose - 3 mg

Lactose ------------------------------------------ 14.8 mg

Magnesium stearate 0.2 mg &lt; RTI ID = 0.0 &gt;

The above components are mixed according to a conventional capsule preparation method and filled in gelatin capsules to prepare capsules.

Formulation Example 4. Preparation of injection

Compound 4 ---------------------------------------- 10 mg

Mannitol -------------------------------------------- 180 mg

Sterile sterilized distilled water for injection - 297 mg

Na ₂ HPO ₄ .12H ₂ O ------------------------------------- 26 mg

(2 ml) per ampoule in accordance with the usual injection method.

Formulation Example 5. Preparation of a liquid preparation

Compound 5 ---------------------------------------- 20 mg

Isomerization Party ------------------------------------------ 10 g

Mannitol -------------------------------------------- 5 g

Purified water --------------------------------------------

Each component was added to purified water in accordance with the usual liquid preparation method and dissolved, and the lemon flavor was added in an appropriate amount. Then, the above components were mixed, and purified water was added thereto. The whole was adjusted to 100 ml with purified water, And sterilized to prepare a liquid preparation.

Formulation Example 6. Preparation of Healthy Foods

Compound 4 ---------------------------------------- 1000 mg

Vitamin mixture -------------------------------------

Vitamin A Acetate ------------------------------- 70 μg

Vitamin E ------------------------------------------ 1.0 mg

Vitamin B ₁ ------------------------------------------ 0.13 mg

Vitamin B ₂ ------------------------------------------ 0.15 mg

Vitamin B ₆ ------------------------------------------ 0.5 mg

Vitamin B ₁₂ ----------------------------------------- 0.2 g

Vitamin C ------------------------------------------ 10 mg

Biotin -------------------------------------------- 10 μg

Nicotinic acid amide 1.7 mg

Folic acid ---------------------------------------------- 50 g

Calcium pantothenate ------------------------------------- 0.5 mg

Inorganic mixture -------------------------------------

Ferrous sulfate - 1.75 mg &lt; RTI ID = 0.0 &gt;

Zinc oxide ------------------------------------------ 0.82 mg

Magnesium carbonate -------------------------------------- 25.3 mg

Potassium phosphate monohydrate 15 mg

Secondary calcium phosphate --------------------------------------- 55 mg

Potassium citrate ---------------------------------------- 90 mg

Calcium carbonate ------------------------------------------ 100 mg

Magnesium chloride -------------------------------------- 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with a composition suitable for health food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional method for producing healthy foods , Granules can be prepared and used in the manufacture of health food compositions according to conventional methods.

Formulation example  7. Manufacture of health drinks

Compound 1 ---------------------------------------- 100 mg

Vitamin C ------------------------------------------ 15 g

Vitamin E (powder) ------------------------------------ 100 g

Lactic Acid Iron -------------------------------------------- 19.75 g

Zinc oxide ------------------------------------------ 3.5 g

Nicotinic acid amide 3.5 g

Vitamin A ------------------------------------------ 0.2 g

Vitamin B ₁ ------------------------------------------ 0.25 g

Vitamin B ₂ ------------------------------------------ 0.3g

Water ------------------------------------------------ Quantitative

The above components were mixed according to a conventional health drink manufacturing method, and the mixture was stirred and heated at 85 ° C for about 1 hour. The resulting solution was filtered to obtain a sterilized 2-liter container, which was sealed and sterilized, It is used in the production of the health beverage composition of the invention.

Although the compositional ratio is relatively mixed with a component suitable for a favorite drink, it is also possible to arbitrarily modify the compounding ratio according to the regional or national preference such as the demand class, the demanding country, and the use purpose.

Claims (7)

(4- (2-formyl) -5 - (((3-hydroxybutane; hydroxybutane), which is a novel alkaloid compound having a structure of the following formula (5) -2-yl) oxy) methyl) -1H-pyrrole-1-yl) butanoic acid or a pharmacologically acceptable salt thereof:

Morrolee ( 5) : Formyl-1 H -pyrrole-1-butanoic acid (Compound 2), 2-formyl-5- (hydroxymethyl) ; hydroxymethyl) -1 H - pyrrole (pyrrole) -1- butanoate acid (butanoic acid) (Compound 3) , 2- formyl (formyl) -5- (methoxymethyl; methoxymethyl) -1 H - pyrrole (pyrrole Butanoic acid (Compound 4) and (4- (2-formyl) -5 - (((3-hydroxybutane; hydroxybutan-2-yl) oxy) Methyl-1H-pyrrol-1-yl) butanoic acid as an active ingredient. The pharmaceutical composition according to claim 2, wherein the extract is an extract soluble in a solvent selected from water, lower alcohols having 1 to 4 carbon atoms, or a mixed solvent thereof. Formyl-1 H -pyrrole-1-butanoic acid (Compound 2), 2-formyl-5- (hydroxymethyl) ; hydroxymethyl) -1 H - pyrrole (pyrrole) -1- butanoate acid (butanoic acid) (Compound 3) , 2- formyl (formyl) -5- (methoxymethyl; methoxymethyl) -1 H - pyrrole (pyrrole Butanoic acid (Compound 4) and (4- (2-formyl) -5 - (((3-hydroxybutane; hydroxybutan-2-yl) oxy) Methyl-1H-pyrrole-1-yl) butanoic acid as an active ingredient. The health functional food according to claim 4, which is a powder, a granule, a tablet, a capsule or a beverage. Formyl-1 H -pyrrole-1-butanoic acid (Compound 2), 2-formyl-5- (hydroxymethyl) ; hydroxymethyl) -1 H - pyrrole (pyrrole) -1- butanoate acid (butanoic acid) (Compound 3) , 2- formyl (formyl) -5- (methoxymethyl; methoxymethyl) -1 H - pyrrole (pyrrole Butanoic acid (Compound 4) and (4- (2-formyl) -5 - (((3-hydroxybutane; hydroxybutan-2-yl) oxy) Methyl-1H-pyrrole-1-yl) butanoic acid as an active ingredient. A feed comprising the animal feed additive of claim 6.

Images