KR100523463B1 - Composition comprising the extract of Astilbe rubra or Astilbic acid and Peltoboykinolic acid derivatives having anti-inflammatory or anti-allergic activity - Google Patents

Abstract

The present invention relates to a composition containing an extract of Astilbe rubra having anti-inflammatory and anti-allergic activity. By containing derivatives of lysoPAF-acetyltransferase, an enzyme that synthesizes PAF from lysoPAF, a precursor of platelet activating factor (PAF), which causes various inflammatory diseases It inhibits the production of PAF by inhibiting the activity, while inhibiting the activity of lipoxygenase (5-LO, 5-lipoxygenase) to prevent the production of inflammatory eicosanoids (inflammatory and allergic diseases) It relates to a pharmaceutical composition for the prevention and treatment of.

Description

Composition comprising the extract of Astilbe rubra or Astilbic acid and Peltoboykinolic acid derivatives having anti-inflammatory or anti-allergic activity }

The present invention relates to a composition for preventing and treating inflammatory diseases or allergic diseases containing herbal extracts and compounds isolated therefrom. Specifically, the present invention relates to platelet activating factors and leukotriene C ₄ (related to inflammatory and allergic diseases). The present invention relates to a pharmaceutical composition for treating inflammatory and allergic diseases containing Astilbe rubra HooK f. Et Thomas extract having inhibitory activity of LTC ₄ ) and purified astilbic acid and peltibokinic acid isolated therefrom. .

Inflammation is the defense of biological tissue against local injuries. In other words, in response to various harmful stressors, the biodefense reaction to remove the injury caused by the stimulus and restore the original state is an inflammatory response. Inflammatory stimuli include infection or chemical or physical stimuli. Bioconstituents involved in the inflammatory response include low-molecular or high-molecular chemicals such as free radicals, proteins, sugars, and lipids, and plasma, blood cells, blood vessels, and connective tissue. The process of inflammation is usually divided into two, and can be divided into acute and chronic inflammation. Acute inflammation is a short-term reaction within a few days, and plasma components or blood cells are associated with the removal of foreign bodies by posting a microcirculatory system. Chronic inflammation has a long duration, and tissue growth is seen.

Allergic diseases are most often caused by mast cells in tissues activated by antigen-antibody reactions and by basophil cells in blood and eosinophils (primarily histamine, leukotrienes, TNF-α, cytokines, etc.). Since the use of allergy therapeutic drugs currently used remains symptomatic, there is an urgent need for the development of more fundamental therapeutic drugs.

Key mediators that induce inflammation and allergic diseases include prostaglandindes, leukotriens, and platelet activators (PAFs). Phospholipase A2 and cycloox It is produced from the precursor arachidonic acid by cyclooxygenase and lipoxygenase.

In order to treat the above inflammatory diseases, various types of inflammatory disease treatments have been developed. The inflammatory disease therapeutic substances reported to date are induced by damaged tissue cells, cells involved in inflammation, or chemotactic factors in acute inflammation. It is a drug that inhibits the production of eicosanoids from the cell membrane of the white blood cells. Allergy agents are also drugs that inhibit histamine free inhibition, histamine receptor blockade, leukotriene production inhibition and leukotriene receptor blockade, which are released during antigen-antibody reactions.

On the other hand, drugs recently attracting attention as an allergic agent for treating asthma are drugs having both histamine free inhibition, leukotriene C ₄ production inhibition, and platelet activator inhibitory activity at the same time.

On the other hand, Astilbe rubra HooK f. Et Thomas is commonly found in valleys throughout the country, and is a perennial herb distributed in Japan, Manchuria, China, Amur and Ussuri, 30-70cm tall, with long brown hairs and roots. The stem is thick and thinly cut sideways. The leaves are divided 2-3 times in 3, small leaves are oval, 3-8cm long, 2-4cm long, thin like paper, and have claws or symmetrical serrates on the edges, and petioles are long. Flowers bloom in July-August, many flowers grow in red inflorescences, cones at the end of stems, brown hairs in flower shafts, calyxes are divided into 5, cracks are egg-shaped, 5 petals are linear, and stamens are 10 Dogs with 2 styles and 3-4 mm long. It is also called horse riding or red riding horse. It is effective in improving blood circulation, releasing blood, releasing heat, releasing poison, relieving cramps, and relieving pain, resulting in overworked illnesses, sore muscles and bones, soreness and bruises. , Arthralgia, pain after surgery, snake bite poison (Korean medicinal plant, Kyohaksa, 2000, p204, ventilated bed)

Korean Patent Registration No. 1996-14989 discloses a 3-beta-hydroxyolean-12ene-27 pentaphosphate compound having analgesic effect isolated from the rooting of roe deer urine. However, there is no teaching or disclosure in the literature that extracts of roe deer urine have anti-inflammatory or anti-allergic activity.

Therefore, the inventors of the present invention, lysoPAF-acetyltransferase, which is a synthase of platelet activator in RBL 2H3 cells of rat mast mast cell line, which is purified by hexane soluble extract and hexane-soluble extract from them. The present invention was completed by finding out that it has potent inhibition of activating activity and at the same time strongly inhibiting leukotriene C ₄ (LTC ₄ ) production.

It is an object of the present invention to provide a roe deer urinary tract extract and astilbic acid and feltokininolic acid purified therefrom that have an anti-inflammatory effect. It is also to provide an anti-inflammatory, anti-allergic pharmaceutical composition containing the compound as an active ingredient.

In order to achieve the above object, the present invention is a pharmaceutical composition for the prevention and treatment of inflammatory diseases or allergic diseases, containing a crude extract of Astilbe rubra showing the inhibition of PAF production and 5-lipoxygenase inhibitory activity as an active ingredient. To provide.

The crude extract refers to an extract soluble in lower alcohols such as water, methanol, ethanol, or a mixed solvent thereof.

In another aspect, the present invention provides a pharmaceutical composition for the prevention and treatment of inflammatory diseases or allergic diseases containing a roe deer urinary nonpolar solvent soluble extract showing inhibitory activity of PAF production and 5-lipoxygenase inhibitory activity.

The non-polar solvent soluble extract means a non-polar solvent such as hexane, ethyl acetate, chloroform, preferably an extract soluble in hexane.

The roe deer urine includes roe deer urine (全 草), roots, leaves or stems.

In addition, the present invention is directed to the treatment of inflammatory diseases or allergic diseases containing astilbic acid or feltokininolic acid derivative compounds exhibiting PAF production inhibitory activity and 5-lipoxygenase inhibitory activity, and pharmaceutically acceptable salts thereof as an active ingredient. It provides a pharmaceutical composition for the prevention and treatment.

The inflammatory disease or allergic disease includes general inflammatory disease, bronchial asthma, allergic rhinitis, allergic asthma, allergic dermatitis and the like.

It is also to provide an anti-inflammatory or anti-allergic agent containing the compound as an active ingredient.

Crude extracts, non-polar solvent soluble extracts and astilbic acid or feltokininolic acid derivative compounds from the roe deer urine of the present invention can be prepared as follows.

Hereinafter, the present invention will be described in more detail.

The roe deer urine crude extract of the present invention is cut into the root or ground portion (leaf, stem) of dried roe urine, about 3 to 20 times the weight (kg), preferably about 5 to 10 times the water, C Lower alcohol of ₁ to C ₄ or a mixed solvent thereof, preferably methanol, at an extraction temperature of 20 to 50 ° C., preferably at 20 to 30 ° C. for about 1 to 10 days, preferably about 3 to 8 days Extraction obtained by cold needle, hot water extraction, ultrasonic extraction, reflux cooling extraction, preferably cold needle extraction method can be obtained by filtration, concentration under reduced pressure or vacuum rotary concentration to obtain crude extract as a polar solvent soluble extract.

The present invention provides a pharmaceutical composition for treating inflammatory diseases or allergic diseases, including crude extracts obtained in the extraction process.

In addition, the non-polar solvent soluble extract of the present invention is suspended by distilled water, the suspension is added to a non-polar solvent such as hexane, ethyl acetate, chloroform of about 1 to 100 times, preferably about 1 to 5 times the volume of 1 It can be obtained by extracting and separating the nonpolar solvent soluble layer from times to 10 times, preferably from 2 times to 5 times. It is also possible to further carry out conventional fractionation processes (Harborne JB Phytochemical methods: A guide to modern techniques of plant analysis. 3rd Ed. Pp 6-7, 1998).

In a preferred embodiment of the present invention, the crude methanol extract of roe deer urine is suspended in distilled water, and fractionated in the order of n-hexane, ethyl acetate, n-butanol, and water to prepare each solvent fraction. The hexane layer can be recrystallized to prepare astilbic acid and feltokininolic acid.

The compound of the present invention is subjected to adsorption chromatography, such as silica gel column chromatography with the non-polar solvent soluble extract, preferably a hexane / ethyl acetate mixed solvent or a chloroform: methanol mixture, preferably 1: 0 in a silica gel column. To a developing solvent having a ratio of 10: 1 (w / w) to a column, and then fractionated, and then reverse-phase chromatography or recrystallization, such as silica gel column chromatography or Rp-18 column chromatography, was repeated to give the astil of Formula 1 below. The big acid and the feltokininolic acid of formula (2) can be separated.

Dichloromethane, chloroform, hexane, ethyl acetate, methanol, distilled water or a mixed solvent thereof may be used instead of the developing solvent.

The present invention provides a crude extract and a non-polar solvent soluble extract of roe deer urine having PAF production inhibitory activity or 5-lipoxygenase inhibitory activity obtained by the above-mentioned method from roe deer urine.

The present invention is an inflammatory disease or allergy containing a crude extract or a non-polar solvent soluble extract of the roe deer urine having a PAF production inhibitory or 5-lipoxygenase inhibitory activity obtained by the above method as an active ingredient and a pharmaceutically acceptable carrier. It provides a pharmaceutical composition for the prevention and treatment of sexual diseases.

The present invention is a 5-lipoxygenase (5-LO, 5-lipoxygense) containing the compound represented by the formula (1) or (2) and their pharmaceutically acceptable salts as an active ingredient obtained from the roe deer urine by the above method. ) As a inhibitor.

In addition, the present invention is an inflammatory disease or allergy containing the compound represented by the formula (1) and (2) having the inhibitory activity of PAF production or 5-lipoxygenase obtained by the above production method as an active ingredient and a pharmaceutically acceptable carrier Provided is a pharmaceutical composition for preventing and treating sexual diseases.

Inflammatory diseases or allergic diseases that can be prevented and treated using the pharmaceutical composition of the present invention include atopic dermatitis, contact dermatitis, insect allergy, food allergy, drug allergy, hypersensitivity, urticaria, bronchial asthma, allergic rhinitis, allergy Sexual asthma, allergic dermatitis, and the like.

In order to investigate the efficacy of the astilbic acid of the general formula (1) of the present invention, that is, to determine the inhibitory ability of lysoPAF acetyltransferase related to platelet activator (PAF) production, astilbic acid is a dose-dependent platelet activator Production was inhibited and the concentration (IC ₅₀ ) required for 50% inhibition was 20.5 μg / ml.

In addition, the inhibitory activity of 5-lipoxygenase, which is involved in the synthesis of leukotriene C ₄ (LTC ₄ ), which causes bronchial asthma and allergic reactions, showed that dose-dependently inhibited the production of LTC ₄ by 50%. The required concentration (IC ₅₀ ) was 1 μg / ml.

In addition, the roe deer urine has been used as a herbal medicine for a long time, the compounds of the present invention extracted and separated from them also have no problems such as toxicity and side effects. Therefore, it is possible to prepare a pharmaceutical composition for the prevention and treatment of inflammatory diseases or allergic diseases containing the compounds of the present invention as an active ingredient.

The pharmaceutical composition for preventing and treating inflammatory diseases or allergic diseases of the present invention comprises 0.01 to 50% by weight of the extract or compound, preferably 0.1 to 10% by weight, based on the total weight of the composition.

Compositions comprising extracts or compounds of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

Pharmaceutical dosage forms of the extracts or compounds of the present invention may be used in the form of their pharmaceutically acceptable salts, and may be used alone or in combination with other pharmaceutically active compounds as well as in a suitable collection.

Compositions comprising extracts according to the invention are formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterile injectable solutions, respectively, according to conventional methods. The extract or compound of the present invention may be formulated into an ointment or cream for topical application. Carriers, excipients and diluents that may be included in the composition comprising the extract include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations may contain at least one excipient such as starch, calcium carbonate, sucrose, or the like in the extract or fraction. (sucrose), lactose (lactose), gelatin, etc. are mixed and prepared. In addition to simple excipients, lubricants such as magnesium styrate and talc are also used. Liquid preparations for oral use may include various excipients, such as wetting agents, sweeteners, fragrances, preservatives, etc., in addition to water and liquid paraffin, which are commonly used to include suspensions, solutions, emulsions, and syrups. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

Preferred dosages of the extracts or compounds of the present invention vary depending on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. However, for the desired effect, the extract or compound of the present invention is preferably administered at 0.0001 to 100 mg / kg, preferably at 0.001 to 100 mg / kg. Administration may be administered once a day or may be divided several times.

The pharmaceutical composition of the present invention can be administered to various mammals such as rats, mice, livestock, humans, and the like. All modes of administration can be expected, for example by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

The present invention provides a health functional food containing an extract of roe deer urine and a food supplement acceptable food supplement additive which have an effect of preventing and improving an inflammatory disease or an allergic disease. Examples of the food to which the roe deer urine extract can be added include various foods, beverages, gums, teas, vitamin complexes, and health functional foods.

It may also be added to foods or beverages for the purpose of preventing and ameliorating inflammatory or allergic diseases. At this time, the amount of the extract in the food or beverage, in general, in the case of the health functional food composition of the present invention can be added to 0.1 to 15% by weight, preferably 1 to 10% by weight of the total food weight, the health beverage composition It can be added at a ratio of 1 to 30 g, preferably 3 to 10 g based on 100 ml.

The health beverage composition of the present invention has no special limitation except for containing the roe deer urinary extract as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates, etc. as additional ingredients, like ordinary drinks. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.), and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of natural carbohydrates is generally about 1-20g, preferably about 5-12g per 100ml of the composition of the present invention.

In addition to the above, the composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, alginic acid and its Salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. The compositions of the present invention may also contain pulp for the production of natural fruit juices and fruit juice beverages and vegetable beverages. These components can be used independently or in combination. The proportion of such additives is not so critical but is generally selected from the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

Hereinafter, the present invention will be described in detail by Examples and Experimental Examples.

However, the following Examples and Experimental Examples are only illustrative of the present invention, and the content of the present invention is not limited to the following Examples and Experimental Examples.

Example 1. Preparation of roe deer urine crude extract

1-1. Crude extract preparation example 1

1.3 kg of dried roe deer urine roots purchased from Gyeongdong market were chopped and immersed in 10 l of methanol, and then extracted at room temperature. Then, 5 l of methanol was added to extract one more time, and each extract was filtered and then filtered. The liquids were mixed. The methanol extract was depressurized, concentrated and dried to give 100 g of the total extract.

1-2. Crude extract preparation example 2

1.0 kg of dried ground roe urine (stem, leaf) purchased from Gyeongdong market was prepared in the same manner as in Example 1-1, to obtain a total extract 78g.

1-3. Crude extract preparation example 3

1.0 kg of dried roe deer urine (stem, leaf) purchased from Gyeongdong market was chopped and immersed in 10 l of 70% methanol, extracted once with reflux cooling at about 60 ° C. for 12 hours, and then with 5 L of 70% methanol. Extracted twice under the same conditions, the total extract was filtered through Whatman filter paper, the filtrate was concentrated under reduced pressure at 65 ℃ to remove methanol, and dried to obtain 110 g of Tobacco crude extract.

1-4. Crude extract preparation example 4

1.0 kg of dried roe urine ground parts (stem, leaf) purchased from Gyeongdong Market was added to 10 liters of distilled water and stirred well, and then the mixture was extracted under reflux for 3 hours at an extraction temperature of 90 to 95 ° C. The filtrate was collected three times by the method, and the crude drug extract was concentrated under reduced pressure at 55-65 ° C., and then lyophilized to obtain a crude drug composition powder of 115 g.

Example 2. Preparation of Fractions of Roe Deer Urinary Extract

2-1. Preparation of roe deer urine hexane fraction

60 g of the roe deer urine root crude extract of Example 1-1 or the roe deer urine ground crude extract of Example 1-2 were suspended in 1 L of distilled water, respectively, to obtain a hexane soluble fraction and a water soluble fraction, Concentration and drying gave 32 g of hexane soluble fraction and 28 g of water soluble fraction.

2-2. Preparation of roe deer urine ethyl acetate fraction

Suspension in the roe deer urine water-soluble fraction of Example 2-1 was suspended in 1 L of distilled water, and then extracted three times by adding 10 L of ethyl acetate. The ethyl acetate soluble fraction and the water-soluble fraction were each obtained under reduced pressure and concentration. And dried to obtain 12.5 g of ethyl acetate soluble fraction and 15.5 g of water soluble fraction.

2-3. Preparation of roe deer urethane butanol fraction

Each of the roe deer urine water-soluble fractions of Example 2-2 was suspended in 1 L of distilled water, and then extracted three times by adding 10 L of butanol. Each of the butanol-soluble fractions and the water-soluble fractions was obtained. 9.0 g of soluble fraction and 6.5 g of water soluble fraction were obtained.

Example 3 Isolation of Astilic Acid and Feltoboikinic Acid Derivatives

3-1. Fraction of roe deer hexane soluble extract

30 g of the soluble urethane hexane soluble part of Example 2-1 was adsorbed onto the same amount of silica gel (Merck), followed by silica gel column chromatography (10 x 90 cm) at 1000 ml per hour. At this time, the eluent was eluted with n-hexane: ethyl acetate mixed solvent as an initial solvent, gradually increasing polarity (n-hexane: ethyl acetate (9: 1) → chloroform: methanol (7: 3)). The polar fractions were washed with methanol to elute the PAF production and the lipoxygenase inhibitors.

Silica gel chromatography, Rp-18 (ODS) column chromatography, or recrystallization was repeatedly performed for each fraction to obtain the following two compounds.

Figure 1 shows a simple process for separating astilbic acid and feltokininic acid from roe deer urine.

(1) Compound 1: Astilic acid (ACH53c)

Compound 1 astilbic acid was subjected to silica gel column chromatography (10 x 100 cm, developing solvent; n-hexane: ethyl acetate (9: 1)-> chloroform: methanol (7: 3)) in the hexane fraction of Example 2-1. 8 fractions were obtained at 1000 ml / hr. The fifth fraction (1.1 g) was again subjected to silica gel column chromatography at 300 mL / hour under the above conditions to obtain four fractions. The third fraction of this was recrystallized to obtain 0.2 g of astilbic acid, and the compound was analyzed by NMR analyzer as follows.

¹³ C-NMR (75MHz, MeOH- d ₄ )

δ: 17.9 (C-25), 18.1 (C-24), 20.3 (C-26), 23.1 (C-7), 23.6 (C-11), 23.8 (C-30), 28.5 (C-16) , 28.5 (C-23), 28.5 (C-2), 28.6 (C-28), 31.2 (C-20), 33.4 (C-29), 33.4 (C-17), 34.7 (C-21), 37.1 (C-22), 37.3 (C-10), 39.6 (C-8), 40.4 (C-4), 41.5 (C-1), 44.5 (C-19), 45.0 (C-15), 48.1 (C-9), 50.0 (C-18), 56.1 (C-5), 56.9 (C-14), 67.4 (C-6), 78.5 (C-3), 125.9 (C-12), 138.1 ( C-13), 178.7 (C-27)

(2) Compound 2: Felttokininolic acid (10-hydroxy-2,2,4a, 6b, 9,9,12a-heptamethyl-2,3,4,4a, 5,6,6b, 7,8 , 8a, 9,10,11,12,12a, 12b, 13,14b-octadecahydro-1H-picene-6a-carboxylic acid methyl ester)

Compound 2 was prepared by using the hexane fraction of Example 2-1 in silica gel column chromatography (10 x 100 cm, developing solvent; n-hexane: ethyl acetate (9: 1)-> chloroform: methanol (7: 3)). Was carried out at 1000 mL per hour to obtain 8 fractions. The fifth fraction (1.1 g) was again subjected to silica gel column chromatography at 300 mL / hour under the above conditions to obtain four fractions. The first fraction of this was recrystallized to obtain 0.1 g of peltoboykinolic acid, and the result of analyzing the compound by an NMR analyzer is as follows.

¹³ C-NMR (75MHz, MeOH- d ₄ )

δ: 15.8 (C-25), 16.5 (C-24), 18.1 (C-26), 18.3 (C-6), 22.3 (C-15), 22.9 (C-11), 23.8 (C-30) , 27.1 (C-2), 27.6 (C-16), 28.2 (C-28), 28.3 (C-23), 31.2 (C-20), 33.0 (C-16), 33.0 (C-17), 33.4 (C-29), 34.4 (C-21), 36.4 (C-22), 36.7 (C-8), 37.1 (C-10), 38.8 (C-1), 39.9 (C-4), 44.0 (C-19), 47.3 (C-18), 49.2 (C-9), 50.8 (-OCH ₃ ), 55.4 (C-5), 56.0 (C-14), 79.3 (C-3), 125.9 ( C-12), 137.9 (C-13), 179.5 (C-27)

Experimental Example 1. Investigation of the influence on platelet activator biosynthesis

The following experiment was carried out to examine the effects on the PAF biosynthesis of the roe deer urinary extract and the purified astilbic acid and feltokininolic acid in the above Example.

The RBL cell extract was pulverized and homogenized by culturing RBL2H3 cells (provided by Professor Kudo Ichiro, Pharmacy, Showa University, Japan), and pellets obtained by centrifugation at 100,000 × g were used as enzyme sources.

Specific experimental method was added to the RBL cell lysate, [ ³ H] acetyl CoA (Sigma), lysoPAF (Sigma) and 100 mM Tris-HCl (pH 6.9) (Sigma), at 37 ℃ [ ³ H] acetyl PAF produced by the reaction for 10 minutes was extracted with an organic solvent method, and then measured by a liquid scintillation counter (Beckman). At this time, astilbic acid was pretreated for 10 minutes to measure the amount of [ ³ H] PAF production.

As a result, Table 1 shows the inhibition rate of each PAF production of the extracts and fractions, the astilbic acid dose-dependently inhibited the biosynthesis of PAF, the concentration required to inhibit 50% was 20.5 ㎍ / ㎖ (see Figure 2) ).

Experimental Example 2. Analysis of the effect on the production of leukotriene

The following experiment was carried out to examine the effects of leukotriene C ₄ (LTC ₄ ) production of the roe deer urine extract prepared in the above example and the purified astilbic acid and feltokininolic acid.

Mouse bone marrow-derived mast cells (BMMC) were derived from BALB / C mice by Murakami et al. [Murakami et al .; J. Biol. Chem. 269 , pp22269-22275, (1994)], medium under 50% WEHI-3, containing culture supernatant of WEHI-3 cells (provided by Professor Kudo Ichiro, Pharmacy, Showa University, Japan), which are IL-3 producing cells. (conditioned medium, containing 10% FCS).

After 3 weeks of culture, mast cells were treated with SCF / LPS mixed stimulant (Sigma) for 30 minutes. LTC ₄ Determination of the supernatant after cell stimulation using the LTC ₄ assay kit (Cayman Inc.) were measured by EIA (Enzyme linked immuno assay). Asylbic acid was pretreated for 15 minutes in advance, and the amount of LTC ₄ produced by adding a stimulant was measured.

As a result, Table 1 shows the inhibitory rate of LTC ₄ production of the extracts and fractions, Astilbic acid inhibited the production of LTC ₄ dose-dependently, the concentration required to inhibit 50% was 1 ㎍ / ㎖ (Fig. 3).

Inhibition of Platelet Activation Factor (PAF) and Leukotriene C ₄ (LTC ₄ ) Formation of Root Extracts, Astilbic Acid and Feltoboykinic Acid Derivatives sample Fraction Platelet activator inhibition Leukotriene production inhibition Final concentration  50 µg / ml 25 µg / ml Inhibition (%) Root Hexane fraction 26.5 98.4 Ethyl acetate fraction 69.7 0 Butanol fraction 81.7 0 Water fraction 20.6 0 Methanol fraction 71.1 0 Ground Hexane fraction 35.6 99.6 Ethyl acetate fraction 34.4 97.5 Butanol fraction 29.6 0 Water fraction 0.0 0 Methanol fraction 16.9 0 Astilic acid 73.4 98.3 Feltoboikinic acid derivative 50.7 99.5

Experimental Example 3. Anti-allergic inhibitory effect in animal model

The experiment was conducted to know the anti-allergic ability of the in vivo (as vivo) to the above-mentioned astilic acid. Passive cutaenous anaphylaxis (Katayama S, Shionoya H and Ohtake) for rats, a typical type I allergy model, using male Sprague-Dawley rats (Hyochang Science) S, Microbiol Immunol 22, pp89-101, (1978).

Monoclonal anti DNP mouse IgE (Sigma, Inc., 100 μl, 1000-fold diluted solution) was administered to Sprague-Dawley rats 48 hours prior, followed by antigen (DNP, Sigma). Thirty minutes before, 50 mg / kg / 100 μl of astilbic acid (ACH53C, dissolved in CMC) was orally administered. At this time, 50 mg / kg / 100 μl of prednisolone (Sigma) was orally administered as a positive control group. Then 1 mg DNP-BSA (antigen solution) / 1% Evans blue solution was injected into the tail vein. After 30 minutes, the pigment spilled on the skin was measured, and the result is shown in FIG. 4.

As can be seen in Figure 4, the substance of the present invention showed a significant anti-allergic action in the allergy model in vivo.

Experimental Example 4. Acute Toxicity

1. Oral administration

       ICR-based mice and Sprague-Dawley rats were divided into four groups of 10 rats each, and the oral urinary tract extracts of the present invention were orally administered at doses of 500, 725, 1000 and 5000 mg / kg, respectively, and observed for 2 weeks. In all cases, no deaths occurred in all four groups, and apparently no symptoms were found.

2. Intraperitoneal administration

       ICR mice (weight 25 ± 5 g) and Sprague dooli rats were divided into four groups of 10 rats each, followed by intraperitoneal administration of the roe deer urine extract of the present invention at doses of 25, 250, 500 and 725 mg / kg, respectively. During the time period, no toxicity was observed in all four groups, and no symptoms were apparent from the control group.

       As a result, it was confirmed that the roe deer urinary extract of the present invention has little acute toxicity.

The roe deer urinary extract or astilbic acid or feltokininolic acid of the present invention may be administered in the following formulations, and the following formulation examples are merely illustrative of the present invention, thereby not limiting the contents of the present invention. .

Formulation Example 1 Preparation of Injection

Astilic acid ......................................... 100 mg

Sodium metabisulfite .................. 3.0mg

Methylparaben .................. 0.8mg

Propylparaben ...... 0.1mg

Sterile distilled water for injection ..............

The above ingredients are mixed and made into 2 ml by a conventional method, and then filled into 2 ml ampoules and sterilized to prepare an injection.

Formulation Example 2 Preparation of Tablet

Dry roe deer urine extract of Example 2-1 ....... 200 mg

Lactose ......................................... 100 mg

Starch ......................................... 100 mg

Magnesium stearate proper amount

The above components are mixed and tableted according to a conventional method for producing tablets to produce tablets.

Formulation Example 3 Preparation of Capsule

Astilic acid ............... 100 mg

Lactose ...... 50 mg

Starch ........................ 50 mg

Talc .................................. 2mg

Magnesium stearate .....

Capsules are prepared by mixing the above ingredients and filling into gelatin capsules according to a conventional method for preparing capsules.

Formulation Example 4 Preparation of Liquid

Roe deer urine extract of Example 1-1 ... 1000 mg

Sugar ... 20g

Isomerized sugar ............... 20g

Lemon flavor ......................

Purified water was added to make a total of 1000 ml. According to the conventional method for preparing a liquid, the above components are mixed, and then filled into a brown bottle and sterilized to prepare a liquid.

Although the composition ratio is a composition suitable for a preferred beverage in a preferred embodiment, the composition ratio may be arbitrarily modified according to regional and ethnic preferences such as demand hierarchy, demand country, use purpose.

Formulation Example 5 Preparation of Healthy Food

Dry extract of roe deer urine extract of Example 1-1 ..... 1000 mg

Vitamin Blend ........................

Vitamin A Acetate ............... 70 μg

Vitamin E ........... 1.0 mg

Vitamin B1 ......... 0.13 mg

Vitamin B2 ........... 0.15 mg

Vitamin B6 ............ 0.5 mg

Vitamin B12 ........................ 0.2 μg

Vitamin C ......................................... 10 mg

Biotin ............... 10 μg

Nicotinic Acid Amide ... 1.7 mg

Folic acid ............... 50 ㎍

Calcium Pantothenate ..................... 0.5 mg

Inorganic mixtures ...............

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

Calcium Carbonate ... 100 mg

Magnesium Chloride ............... 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixtures is mixed with a component suitable for a health food in a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional health food manufacturing method. The granules may be prepared and used for preparing a health food composition according to a conventional method.

Formulation Example 6 Preparation of Healthy Drink

Dry extract of roe deer urine extract of Example 1-1 ............. 1000 mg

Citric Acid ........................................ 1000 mg

Oligosaccharide ......................... 100 g

Plum concentrate ........................... 2 g

Taurine ......................................... 1 g

Purified water is added ............................................. 900 ml

After mixing the above components in accordance with a conventional healthy beverage production method, and stirred and heated at 85 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed sterilization and then refrigerated and stored in the present invention For the preparation of healthy beverage compositions.

Although the composition ratio is a composition suitable for a preferred beverage in a preferred embodiment, the composition ratio may be arbitrarily modified according to regional and ethnic preferences such as demand hierarchy, demand country, use purpose.

As described above, the composition containing the roe deer urinary extract of the present invention and the purified astilbic acid and peltibokinic acid from these compounds are effective in preventing the production of platelet activating factors and leukotriene C ₄ related to inflammation, allergic reactions and various diseases. By inhibiting, it shows anti-inflammatory and anti-allergic effects, and also shows significant anti-allergic effects in vivo and can be used significantly in various allergic inflammatory diseases.

1 is a diagram illustrating the whole process of separating astilbic acid and feltokininolic acid derivatives from the roe deer urine extract of the present invention,

Figure 2 is a test result of the inhibitory activity of lyso-PAF acetyltransferase, a biosynthetic enzyme of platelet activator of astilbic acid isolated from the roe deer urine extract of the present invention,

3 is an experimental result of the inhibitory activity of 5-lipoxygenase involved in the synthesis of leukotriene C ₄ causing bronchial asthma and allergic reactions,

FIG. 4 is a result of passive cutaenous anaphylaxis inhibition of rats, a representative type I allergy model, to know the anti-allergic ability of in vivo against astilbic acid.

Claims (7)

delete delete delete delete delete delete A health functional food comprising a roe deer urine extract and a food acceptable food supplement additive for the prevention or improvement of an inflammatory disease or an allergic disease.