KR100516192B1 - Health improving food containing hirsutenone - Google Patents

Abstract

The present invention relates to a health-promoting food comprising hirsutenone, and the health-promoting food according to the present invention effectively suppresses intercellular information transmission and homeostasis imbalance through gap coupling and thus prevents diseases related thereto. It can be useful to

Description

Health-Promoting Foods Containing Immune Tenone {HEALTH IMPROVING FOOD CONTAINING HIRSUTENONE}

The present invention relates to foods for health promotion for maintaining intercellular communication and homeostasis through gap binding, including hirsutenone.

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 dysfunctions, 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).

Immunophenone represented by the following formula (1) has not been reported as yet for pharmacological action.

The inventors of the present invention, while searching for a composition for preventing or treating diseases related to an imbalance between homeostasis and inhibition of intercellular communication through gap binding derived from natural foods or plants that ensure human safety, The present invention has been completed by discovering that imaginary hexatenone obtained by acid hydrolysis of oregonin) can effectively restore intracellular information transmission and gaps in homeostasis through gap binding.

Accordingly, it is an object of the present invention to provide a composition for preventing or treating diseases related to the suppression of intercellular information transmission through gap binding and an imbalance of homeostasis, and processed foods containing the same, as an active ingredient of imaginary tenon. .

In accordance with the above object, in the present invention, an effective amount of isohydrogentenone as an active ingredient together with a pharmaceutically acceptable carrier, the prevention of diseases associated with the inhibition of intercellular communication and gaps of homeostasis through gap junctions Or a therapeutic pharmaceutical composition, and a processed food added thereto.

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

Isotenone used in the present invention can be produced by separating a teal or its dried product from an extract obtained by solvent extraction or by chemically synthesizing.

Specifically, oregonine is obtained from a teal tree according to the process as shown in FIG. In detail, the fresh material of the teal bark was extracted and filtered three times at room temperature with an acetone aqueous solution, for example, an 80% acetone aqueous solution. The extract was concentrated under reduced pressure, suspended in water, filtered, and then separated into four fractions (Fr) according to the concentration of methanol in the eluate using Sephadex LH-20 (H ₂ O → MeOH gradient system) column chromatography. .1, Fr.2, Fr.3 and Fr.4). MCI-gel CHP 20P column chromatography (H ₂ O → MeOH, gradient system) and low pressure liquid column chromatography (column: YMC ODC-gel, 10%) for Fr. ₂ obtained in the methanol section at a concentration of 20% to 40%. MeOH-> 50% MeOH, gradient system) is repeated, and oregonin (1.2g) is obtained as compound 3.

To remove the sugar from the oregonin obtained as described above to obtain the isotenone, oregonin was acid-hydrolyzed by heating at 80 to 90 ℃ for 1 hour in H ₂ SO ₄ or H ₂ SO ₄ + dioxane solution , Extracted with ethyl acetate to obtain hexatenonone. It can be confirmed by thin layer chromatography and mass spectrometry that the obtained material is imaginary tenon.

Ischentenone of the present invention effectively suppresses intracellular information transmission through gap binding and restores an imbalance of homeostasis, thereby preventing cancer, diabetes, cerebral ischemia, neurological diseases including Chaco-Marie-Tud type 1, spermatogenic disorders, and cardiovascular system. Relational hypertension can be used to prevent and treat diseases associated with imbalances of homeostasis and suppression of intercellular communication through gap binding such as vascular disease, kidney disease, epilepsy and muscle disease. In particular, the isotenenone of the present invention is a method of intracellular signal transduction through gap binding generated by H ₂ O ₂ irrespective of the direct antioxidant activity against hydrogen peroxide (H ₂ O ₂ ), a major disease causing agent present in vivo. By specifically restoring only inhibition, the prophylactic and therapeutic effects of the related disease are exhibited.

The imaginary isontenone 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. 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 imaginary isontenone of the present invention range from 0.1 to 100 mg / kg body weight, preferably 1 to 10 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 intercellular information transmission and homeostasis through gap binding, comprising an effective amount of imaginary tenone. 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.

In the preparation of the food, the isotenone can be added to the raw material during the manufacture of the food or mixed appropriately in the cooked food to prepare the above-mentioned health-promoting food or drink, in which case the content of 0.01 to 50% by weight.

Hereinafter, the effect of preventing or treating diseases related to the inhibition of intercellular communication and the disproportionation of homeostasis through gap binding of imaginary tenon will be 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 Immuno-Tenon with Efficacy and Prevention of Diseases Associated with Inhibition of Information Transmission and Disproportionation of Homeostasis through the Gap Binding from Tealwood

In this experiment, the entire process of obtaining oregonin from a teal, which has been wildly collected in Cheonggyesan and undergoing a botanical assay, is shown in FIG. 1. In detail, 2.5 kg of the fresh material of the teal bark was cut and then extracted three times with an aqueous 80% acetone solution and filtered. The extract was concentrated under reduced pressure, suspended in water, filtered, and then separated into four fractions using Sephadex LH-20 (H ₂ O → MeOH gradient system) column chromatography (Fr. 1, Fr. 2, Fr). .3 and Fr.4), Fr.1 is 100% water-20% MeOH section, Fr.2 is 20% MeOH-40% MeOH section, Fr.3 is 40% MeOH-70% MeOH section, Fr. 4 eluted in 70% MeOH-100% MeOH sections, respectively. Repeat MCI-gel CHP 20P column chromatography (H ₂ O → MeOH, gradient system) and low pressure liquid column chromatography (column: YMC ODC-gel, 10% MeOH → 50% MeOH, gradient system) for Fr.2. The oregonin (1.2 g) of the following formula (2) was obtained.

Wherein R is xylose.

Acid hydrolysis was carried out as follows to remove saccharides from oregonin obtained as above to obtain hexatenenone. First, oregonine was heated for 1 hour at 90 ° C. in 2 NH ₂ SO ₄ or 2 NH ₂ SO ₄ + 60% dioxane, followed by acid hydrolysis, followed by extraction with ethyl acetate to give hydrated tenone. It was confirmed by thin layer chromatography and mass spectrometry that the obtained material was imaginary tenon.

In the following examples to determine the efficacy of inhibiting the intercellular communication and the prevention of related diseases of the imaginary hexatenone and fractions obtained as described above, and to show that it is involved in intercellular signal transmission irrelevant to the antioxidant activity The amount of change in H ₂ O ₂ by the treatment was measured.

Example 2 Efficacy Determination of Restoring Imbalance of Information Transmission Through Gap Binding of Immunotenone

In order to confirm the effect of restoring the inhibition of gap junctional intercellular communication through gap binding of the imaginary hexatenone obtained in Example 1, the SL / DT assay (Scrape Loding / Dye Transfer assay: Upham, 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 Incubated in a 5% CO ₂ , 37 ° C. incubator (Forma Scientific Co., Marjetta, OH, USA) using no D-medium. 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, when the cells were filled in a dish about 90%, the cells cultured in each dish were treated with a culture solution containing 1-20 μg / ml of hexatenone 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 using lucifer yellow stain was measured by confocal microscopy (BioRad, Hercules, CA, USA).

The results are shown in Table 1 and FIG. 2, in FIG. 2, FIG. 2A is an untreated control group, FIG. 2B is a 500 μM H ₂ O ₂ treated group, and FIG. 2C is 5 μg / ml isotenone + 500 μM H ₂ O. ₂ treatment group, FIG. 2D is a 10 μg / ml isotenone + 500 μM H ₂ O ₂ treatment group.

In Table 1, the percent increase in intercellular communication through gap binding of imaginary tenone is {(stained cell number of sample treated group-number of stained cell of H ₂ O ₂ treated group) / (control group stained cell number). -The number of stained cells in the H ₂ O ₂ treated group)} × 100, each is the average value of the values obtained through three repeated experiments.

As can be seen in Table 1 and FIG. 2, isotenone exhibited the effect of completely restoring intercellular communication through gap binding inhibited by H ₂ O ₂ at a concentration of 5 μg / ml or more. Therefore, it can be seen that the imaginary tenone has the effect of preventing and treating diseases related to the imbalance between intracellular information transmission and homeostasis through gap binding.

Example 3: H after imaginary tenon treatment ₂ O ₂  Change measurement

To show that the main cause of the imaginary Tenon recover the inhibition of the transfer cells between the information through the gap coupling caused by the H ₂ O ₂ by the intracellular signal transduction than the antioxidant activity, the amount of H ₂ O ₂ as follows: The FOX2 assay (Nourooz-Zadeh, J., Tajaddini-Sarmadi, J., & Wolff, SP Analytical Biochemistry 220: 403-409, 1994), which is a well-known method for measuring the amount of HCl, was performed.

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. Mix the sample containing 500 uM of H ₂ O ₂ and the working reagent in a 1: 9 volume ratio in a 96-well microplate under the same conditions as in Example 2, and 37 ° C. incubator (Forma Scientific Co., Marjetta, OH, USA) After reacting for 1 hour at, absorbance was measured at 560 nm. Here, in the imaginary group and catalase group, with the concentration of 0 to 20 μg / ml, the sample was used with 500 uM H ₂ O ₂ as a sample, and the control group was 500 uM H _2. Only cultures containing 0 ₂ were used in place of the samples.

As a result, as shown in Figure 3, 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 H ₂ O ₂ is added. In addition, it was confirmed that to reduce the H ₂ O of 500 uM for Catalyst raised well known to ₂ remove H ₂ O ₂ in a concentration-dependent manner. However, in the case of imaginary tenons, the amount of H ₂ O ₂ was rather increased. These results can be confirmed from this that the imaginary Tenon to revive the GJIC inhibition by H ₂ O ₂ relating to the signaling between cells is not due to the antioxidant effect of clearing the H ₂ O _2.

In the above results, it has been confirmed that the imaginary ison has a prophylactic and inhibitory effect of a disease associated with the inhibition of intercellular communication and gaps in homeostasis through gap binding. In addition, by measuring the amount of change in H ₂ O ₂ after treatment with isotenone, it was found that the recovery of intercellular information transmission through gap binding by the sample is not related to the antioxidant activity of isotenonone and is involved in intercellular signal transmission. Confirmed.

The health promoting food containing the imaginary tenone of the present invention can restore the imbalance of intercellular information transmission through gap binding.

1 is a schematic diagram showing a process for separating oregonin from a teal.

Figure 2 shows the results of measuring the efficacy of the recovery of the suppression of intercellular communication through gap binding generated by hydrogen peroxide (H ₂ O ₂ ).

Figure 3 shows the result of measuring the change in the amount of H ₂ O ₂ after reacting the imaginary hexatenone with H ₂ O ₂ according to the FOX2 measurement method.

Claims (1)

Foods for promoting health to maintain intercellular information transmission and homeostasis through gap binding, containing the hexatenone of formula (I): Formula 1