KR20040060323A - Composition for preventing or treating a disease related to the inhibition of gap junctional intercellular communication and imbalance of homeostasis, comprising an extract of the root bark of ulmus davidiana var - Google Patents

Abstract

PURPOSE: Provided is a composition for preventing and treating disease associated with inhibition of gap junctional internal cellular communication and unbalance of Homeostasis, which composition contains Ulmus macrocarpa HANCE extract and effectively recovers the inhibition of gap junctional internal cellular communication and the unbalance of Homeostasis. It is therefore, used for manufacture of medicaments, processed foods, functional foods, beverages and drinks. CONSTITUTION: A composition for preventing and treating disease associated with inhibition of gap junctional internal cellular communication and unbalance of Homeostasis is characterized by containing, as an active ingredient, an effective amount of Ulmus macrocarpa HANCE extract or its active fraction, and pharmaceutically acceptable carriers. The extract is obtained by extracting Ulmus macrocarpa HANCE with 60% ethanol solution or hot water.

Description

FIELD OF PREVENTING OR TREATING A DISEASE RELATED TO THE INHIBITION OF GAP JUNCTIONAL INTERCELLULAR COMMUNICATION AND IMBALANCE OF HOMEOSTASIS , COMPRISING AN EXTRACT OF THE ROOT BARK OF ULMUS DAVIDIANA VAR}

The present invention relates to a composition for preventing or treating diseases related to inhibition of intercellular communication and gap homeostasis imbalance through gap binding, which includes extracts of root bark, ie, Ulmus davidiana var .

Gap junctions are a form of complex that connects adjacent cells and directly connect to the interior of neighboring cells. In mammals, they are distributed in all cells except skeletal muscle fibers, some nerves and circulating blood cells. (Bennett et al., Neuron, 6: 305-320, 1991).

The gap binding pathway consists of two hemichannels called connexons, which consist of a collection of six small proteins called connexins. The diameter of this passage is about 1.2-2 nm, and its size depends on the type of protein that forms the gap bond, which allows small molecules (<2000 Da) or ions (e.g., various ions, water, sugars). , Nucleotides, amino acids, fatty acids, small peptides, drugs and carcinogens) can spread (Loewenstein, Physiol Rev, 61: 829-913, 1981). The intercellular mass flow through these gap binding pathways is called Gap Juntional Intercellular Communication (GJIC), which is achieved by passive diffusion that does not require ATP.

To sum up the function of gap binding, first, it is the most basic and important function to achieve a quick equilibrium of nutrients, ions and fluids between cells (Loewenstein, Physiol Rev, 61: 829-913, 1981); Secondly, it acts as an electrical synapse in electrically excited cells such as cardiomyocytes, smooth muscle cells, and neurons (Bennett et al., Neuron, 6: 305-320, 1991); Thirdly, the passage of secondary transporters such as cyclic nucleotides, calcium, and inositol phosphate enhances the tissue's response to external stimuli, which is related to the tissue's response to hormones (Loewenstein, Physiol Rev, 61: 829-913, 1981); Fourth, in embryonic stage, embryonic development is controlled by chemical and electrical signals (Kalimi et al., J Cell Biol, 107: 241-225, 1988).

As such, gap binding promotes cellular response regulation by homeostasis in tissues and the rapid transport of secondary transporters to adjacent cells. Numerous diseases including cancer, including diabetes, cerebral ischemia, neurological diseases including Chacot-Marie-Tooth disease type 1 (CMT1), spermatogenic disorders, cardiovascular hypertension It has already been reported in various literature that vascular disease, kidney disease, epilepsy and myopathy are caused by homeostatic imbalance resulting from abnormal intercellular communication through gap binding (Nalin et al., Cell, 84: 381-388, 1996; Blanc et al., J. Neurochem., 70: 958-870, 1998; Trosko et al., Toxicol. Lett., 102: 71-78, 1998; Upham et al., Carcinogenesis, 18 : 37-42, 1997).

In Korea, roots of Yu-geun have been traditionally used as medicine and variously used in the form of health food. It is known that the root skin contains flavonoids, saponins, mucus, β-Sitosterol, Phytosterol, Sigmasterol, Resin, Fattyoil, etc. (Herbal component: Ilwol Seokwi), medicinal effect and application include sediment (淋 濁), species, single improvement (Chinese medicine standard: Ministry of Health and Welfare), cough, astringent, anti-inflammatory, insect repellent, antibacterial (herbal ingredient: wolwolseo), diuresis, small species It is listed as a medicine such as poison, cure, embroidery supplement (herbology: changchangsu), and the oral remedy has been known to cure boils and swelling. It is known that it is effective in various ulcers such as gastric ulcer and duodenal ulcer.

The inventors of the present invention, while searching for a composition for preventing or treating a disease related to an intracellular information transfer inhibition and homeostatic imbalance through gap binding derived from a natural food or a plant, which has secured human safety, extracts from the roots of the roots of the cells through gap binding The present invention has been completed by discovering that inhibition of liver communication and homeostasis imbalance can be effectively recovered.

Accordingly, the first object of the present invention is to provide a composition for preventing or treating diseases related to the imbalance of inhibition of homeostasis and homeostasis through gap binding, which contains the extract of the myrtle root.

It is a second object of the present invention to provide a food for maintaining intercellular information transmission and homeostasis through gap binding, which has the extract of E. coli as an active ingredient.

Figure 1 shows the results of measuring the efficacy to restore the inhibition of intercellular information transmission through gap binding generated by the extract of hydrogen root peroxide (H ₂ O ₂ ).

Figure 2 shows the results of measuring the change in the amount of H ₂ O ₂ after reacting the extracts and fractions with H ₂ O ₂ according to the FOX2 measurement method.

In accordance with the above object, the present invention comprises an effective amount of the extract of the myrtle root extract or fraction with a pharmaceutically acceptable carrier, the inhibition of intercellular communication and the homeostatic imbalance through GapJunction Prophylactic or therapeutic pharmaceutical compositions, and foods for maintaining intercellular communication and homeostasis.

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

The root extract of the roots used in the present invention can be produced by ethanol extraction or high pressure hot water extraction of root roots or dried products thereof.

Specifically, the root skin or its dried product is extracted with an aqueous ethanol solution, preferably 50% ethanol solution for 5 hours at 80-90 ° C. (“ethanol extraction”), or hot water extraction at a temperature of 100-110 ° C. for 5 hours. ("High-Pressure Hot Water Extraction") After repeating the extraction process two to three times, the obtained extract can be filtered, concentrated under reduced pressure, and dried to obtain the root extract.

In addition, adsorption column chromatography can be used from these extracts to obtain fractions with further enhanced efficacy in preventing or treating diseases associated with the inhibition of intercellular communication through gap binding and the imbalance of homeostasis. The adsorption column chromatography was carried out by adsorbing the extract of the roots of the roots on a column filled with a hydrophobic filler, for example, a styrene-based porous resin such as XAD-4 (Sigma, USA) or HP-20 (Mitsbish, Japan). It can be obtained by fractionation using an aqueous solution of ethanol with a stepwise increase in concentration as an eluent. Preferably, an ethanol aqueous solution in which the concentration of ethanol is increased in steps of 20, 40, 60, 80 and 100% may be used, and in particular, fractions having enhanced activity may be obtained when using 60% and 80% ethanol aqueous solutions.

The root extract and fraction of the present invention effectively suppresses intracellular information transmission through gap binding and restores imbalance of homeostasis, thereby preventing cancer, diabetes, cerebral ischemia, neurological diseases including Chaco-Marie-Tud type 1, and spermatogenic disorders. It can be used to prevent and treat diseases related to the imbalance of inhibition of intracellular communication and homeostasis through gap binding such as vascular disease, kidney disease, epilepsy and muscle disease, including cardiovascular hypertension. In particular, the extracts and fractions of the myofifolia extract of the present invention are intercellular signals through gap binding generated by H ₂ O ₂ regardless of the direct antioxidant activity against hydrogen peroxide (H ₂ O ₂ ), a major disease-causing substance present in vivo. By specifically restoring the inhibition of delivery, the prophylactic and therapeutic effects of the relevant diseases are exhibited.

The root extract of the present invention may be mixed with a suitable pharmaceutically acceptable carrier or excipient or diluted with a diluent according to a conventional method to prepare a pharmaceutical composition having the above function. Examples of suitable carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, ziitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, Microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. The pharmaceutical composition may further include fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives and the like. The pharmaceutical compositions of the invention can be formulated using methods well known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. The formulations may be in the form of tablets, pills, powders, sachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, sterile powders and the like.

The pharmaceutical compositions of the invention can be administered via several routes including oral, transdermal, subcutaneous, intravenous or intramuscular. Typical daily dosages of the myofibril extract and fractions of the present invention range from 1 to 1,000 mg / kg body weight, preferably 10 to 100 mg / kg body weight, and may be administered once or in several doses. However, it is to be understood that the actual dosage of the active ingredient should be determined in light of several relevant factors such as the route of administration, the age, sex and weight of the patient, and the severity of the disease, and therefore the dosage should in any way be regarded as the present invention. It does not limit the scope of.

In another aspect, the present invention provides a food or beverage composition for health promotion for maintaining the intercellular information transmission and homeostasis through gap binding, comprising an effective amount of the extract of the roots. Foods to which the extract of the present invention may be added to exhibit the above effects include, for example, various foods, meats, beverages, chocolates, snacks, confections, pizzas, ramen noodles, other noodles, gums, ice creams, alcoholic beverages, Alcohol, vitamin complexes and other health supplements, but are not limited thereto.

The root extract of the present invention may be added to the raw material during food preparation or properly mixed with the cooked food to prepare the above-mentioned health promoting food or beverage, and in this case, the content of the root root extract in the finally prepared food or beverage is In the range from 0.1 to 80% by weight.

Hereinafter, the effects of preventing or treating diseases related to the inhibition of intercellular communication and the disproportionation of homeostasis through gap binding of the extracts or fractions of the rhizome are described in more detail with reference to Examples. However, these Examples are only for illustrating the present invention, the present invention is not limited to these.

In addition, in the examples below, the following percentages for solids and solid mixtures, liquids and liquids, and liquids and solids are based on weight / weight, volume / volume and weight / volume, respectively, and all reactions are carried out at room temperature unless otherwise noted. It was.

Example 1 Preparation of Extracts and Fractions Having Efficacy in Preventing and Treating Diseases Associated with Inhibition of Information Transmission and Disproportionation of Homeostasis through Gap Binding from Radical Skin

<1-1> Preparation of Radix Cortex Extract by Hot Water Extraction and Solvent Extraction

50 parts of ethanol (Duksan, Korea) or 100-110 ℃ of hot water was added to about 1-10 parts by weight of dried root skin, and the mixture was extracted under reflux for 5 hours using a stirrer at room temperature. The extracts were centrifuged at 8,000 rpm for 30 minutes at 4 ° C. (Vision, Korea) to obtain a supernatant, and the remaining residue was repeatedly extracted 1-2 times in the same manner. The extracts thus obtained were collected, filtered through a filter paper (Whatmann 41), and then freeze-dried to obtain root extracts.

<1-2> Preparation of Extracts from Root-derived Skin by Adsorption Column Chromatography

From the ethanol extract obtained in Example 1-1, fractions were prepared by adsorption column chromatography in order to prepare fractions with enhanced efficacy of preventing and treating diseases related to imbalance between homeostasis and homeostasis through gap binding. In other words, an extract was added to a glass column (150 mm × 6 mm) filled with XAD-4 (Sigma, USA) or HP-20 (Mitsbish, Japan), which is a hydrophobic polyaromatic filler, followed by adsorption with 20% ethanol. A fraction containing the nutritional component ("fraction 1") was obtained. Subsequently, 40, 60, 80 and 100% ethanol was flowed in order to obtain fractions by each eluate. These fractions were named fractions 2, 3, 4 and 5, respectively.

In the following Examples to measure the efficacy of inhibiting the intercellular communication of the extracts and fractions obtained as described above and to prevent the treatment associated with the disease, and to show that it is involved in intracellular signal transmission irrelevant to the antioxidant activity The amount of change in H ₂ O ₂ by the treatment was measured.

Example 2: Efficacy measurement of restoring imbalance of information transmission through gap binding of extracts

In order to confirm the efficacy of restoring the inhibition of gap junctional intercellular communication through gap binding of the extracts and fractions obtained from Example 1, the SL / DT assay (Scrape Loding / Dye Transfer assay: Upham, BL et al., Carcinogenesis 18: 37-42, 1997).

WB-F344 cells (University of Michigan, USA) contain 10% fetal bovine serum (FBS), penicillin 7.5 mg / L, streptomycin 7.5 mg / L, neomycin 15 mg / L and phenol red Cells were cultured in a 5% CO ₂ , 37 ° C. incubator (Forma Scientific Co., Marjetta, OH, USA). Media compositions were all used by GIBCO BRL (Grand Island, NY, USA). The cultured cells were dispensed at 1 × 10 ⁵ per ml in a 2 ml plastic dish and incubated for about 48 hours. After culturing, the cells were filled in about 90% of the dish, and the cells cultured in each dish were treated with a culture solution containing 1-20 μg / ml of the root extract and fractions prepared in the above example, and 500 μM of H ₂ O ₂ . . The control group was divided into two groups, the group treated with the culture medium only and the group treated with the culture medium and 500 μM H ₂ O ₂ . After 1 hour, the degree of intercellular communication through gap binding was measured using lucifer yellow staining solution by confocal microscopy (BioRad, Hercules, CA, USA).

The results are shown in Table 1 and FIG. 1, in FIG. 1, FIG. 1A is an untreated control group, FIG. 1B is a 500 μM H ₂ O ₂ treated group, and FIG. 1C is a 20 μg / ml rooted skin extract + 500 μM H ₂ O ₂ treatment group, FIG. 1D is a 5 μg / ml root skin fraction 3 (60% ethanol fraction) +500 μM H ₂ O ₂ treatment group.

In Table 1, the percentage increase in intercellular communication through gap binding of the myofibril extract and fractions was shown as {(stained cell number of sample treated group-stained cell number of H ₂ O ₂ treated group) / (control stained group). Cell number-the number of stained cells in the H ₂ O ₂ treated group)} × 100, each of which is the average of the values obtained from three replicates.

Increasing rate of intercellular information transfer through gap binding of extracts and fractions Concentration of extract (1 μg / ml) (5 μg / ml) (10 μg / ml) (20 μg / ml) extract Boiling water extract <20 <20 <0 60 50% Ethanol Extract <20 <20 60 80 Fraction Fraction 1 <20 <20 <20 <20 Fraction 2 <20 <20 <20 60 Fraction 3 60 100 100 100 Fraction 4 40 100 100 100 Fraction 5 <20 <20 <20 50

As can be seen in Table 1 and Figure 1, the 50% ethanol extract of the root bark showed a significant effect of restoring intercellular communication through gap binding inhibited by H ₂ O ₂ at a concentration of 10 ㎍ / ㎖ or more . Therefore, it can be seen that the extract of E. coli has the effect of preventing and treating diseases related to imbalance between homeostasis and homeostasis through gap binding. In addition, it can be seen that the fractions by column chromatography in Table 1 and FIG. 1, in particular, fractions 3 and 4, have further enhanced efficacy in restoring intercellular communication inhibition through gap binding.

Example 3: Determination of H ₂ O ₂ change after treatment with extracts and fractions

In order to confirm that a major cause of the yugeunpi extract or a fraction recovering the inhibition of the transfer cells between the information through the gap coupling caused by the H ₂ O ₂ by intracellular signaling, not antioxidative effects, H ₂ O, as follows: ₂ change the known method of measurement for measuring FOX2: was performed (Nourooz-Zadeh, J., Tajaddini -Sarmadi, J., & Wolff, SPAnalytical Biochemistry 220 403-409, 1994). Xylenol orange and ammonium ferrous sulfate were added to 250 mM H ₂ SO ₄ at 1 mM and 2.5 mM, respectively. The reagent thus prepared was mixed with HPLC-grade methanol containing 4.4 mM butylated hydroxyltoluene (BHT) in a volume ratio of 1: 9 to prepare a working reagent. Under the same conditions as in Example 2, a sample containing 500 uM of H ₂ O ₂ and a working reagent were mixed at a volume ratio of 1: 9 in a 96-well microplate at 37 ° C. (Forma Scientific Co., Marjetta, OH, USA) After reacting for 1 hour at, absorbance was measured at 560 nm. Herein, 50% ethanol extract and fraction 3 group was used as a sample with 500 uM H ₂ O _{2 at} a concentration of 0 to 200 ㎍ / ml, respectively, and the control group was 500 uM. Only the culture solution containing H ₂ O ₂ was used in place of the sample.

As a result, it can be seen that the amount of H ₂ O ₂ is not changed in the case of the control group to which only 500 uM of H ₂ O ₂ is added as shown in FIG. 2. In addition, it was confirmed that to reduce the H ₂ O _{2 2} of 500 uM when the well-known that catalase to remove H ₂ O in a concentration dependent manner. However, the amount of H ₂ O ₂ slightly increased for each 50% ethanol extract and fraction 3. From these results, it can be seen that the recovery of GJIC inhibited by H ₂ O ₂ by 50% ethanol extract and fraction 3 is not due to the antioxidant effect of removing H ₂ O ₂ but by intercellular signaling.

In the above results, it was confirmed that the extracts and fractions of the root bark have a prophylactic and inhibitory effect on diseases related to the imbalance of inhibition of intercellular information transmission and homeostasis through gap binding. In addition, by measuring the amount of change of H ₂ O ₂ after treating the extracts and fractions of the roots, recovery of intercellular information through gap binding by the sample is independent of the antioxidant activity of the sample and is involved in intercellular signal transduction. It can be confirmed.

The extracts and fractions of the present invention can be used as a prophylactic and therapeutic agent for diseases caused by the imbalance of intercellular communication through gap binding, and also by the imbalance of intercellular communication through gap binding with simple separation process. It is very economical as it can produce effective prophylactic and therapeutic agents for the diseases that occur.

Claims (5)

Prevention of diseases related to the inhibition of intercellular communication and gaps in homeostasis through gap binding, comprising, as an active ingredient, an effective amount of extract of dermis roots or an active fraction obtained by adsorption column chromatography therefrom, together with a pharmaceutically acceptable carrier. Or therapeutic pharmaceutical compositions. The method of claim 1, Pharmaceutical composition, characterized in that the extract of the roots obtained by ethanol extraction or boiling water extraction of the roots or dried products thereof. The method of claim 1, A pharmaceutical composition, characterized in that the active fraction is obtained by adsorption column chromatography using a hydrophobic filler and 20-100% aqueous ethanol solution from the root extract. The method of claim 1, The disease is selected from the group consisting of cancer, diabetes, cerebral ischemia, neurological diseases including Chaco-Marie-Tud type 1, spermatogenic disorders, vascular diseases including cardiovascular hypertension, kidney disease, epilepsy and muscle disease Pharmaceutical composition characterized by. A health-promoting food for maintaining intercellular information transmission and homeostasis through gap binding, which contains an extract of Yugeun-pi or an active fraction obtained by adsorption column chromatography therefrom.