KR100517340B1 - Health improving food containing an extract of the leaves of abies nephrolepis - Google Patents

Abstract

The present invention relates to a health promoting food comprising a secretion leaf extract, the health promoting food according to the present invention effectively prevents the disease associated with the inhibition of intercellular information transmission and homeostasis imbalance through gap coupling. It can be usefully used.

Description

HEALTH IMPROVING FOOD CONTAINING AN EXTRACT OF THE LEAVES OF ABIES NEPHROLEPIS}

The present invention relates to foods for promoting health for maintaining intercellular communication and homeostasis through gap binding, including secretion leaf extract.

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 to 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 six small proteins called connexins. The diameter of this passageway is about 1.2 to 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 be diffused (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); Third, 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, regulate embryonic development as chemical and electrical signals in the embryonic stage (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 due to 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).

On the other hand, Abies nephrolepis is an evergreen conifer tree belonging to the pine family, whose leaves are similar to firs, but their leaf ends are divided into two, thinner and thinner than the globular tree, and the leaves of the branches are arranged horizontally like a comb. The flowers bloom in May, the conifers ripen in September, and the alpine species germinate well, making it easy to update the natural species. However, little has been reported about the pharmacological action of secretory leaves.

The inventors of the present invention, while searching for a composition for preventing and treating diseases related to an imbalance between homeostasis and suppression of intercellular communication through gap binding derived from natural foods or plants, which have secured human safety, extracts from the leaves of the secretory tree have gap binding. The present invention has been completed by discovering that inhibition of intercellular communication and effective recovery of acidic imbalance.

It is an object of the present invention to provide a composition for preventing and treating diseases related to the imbalance of suppression of intracellular information and the homeostasis through gap binding, which are used as secretory leaf extracts.

It is also an object of the present invention to provide a food for maintaining intracellular information transmission and homeostasis through gap binding, which is the secretion leaf extract as an active ingredient.

In accordance with the above object, in the present invention, a disease associated with the imbalance of suppression and homeostasis of intercellular communication through gap junction, which comprises an effective amount of secretory leaf extract as an active ingredient together with a pharmaceutically acceptable carrier. A pharmaceutical composition for the prophylaxis and treatment, and a food for intercellular communication and homeostasis.

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

Secretory leaf extract used in the present invention can be produced by solvent extraction of secretory leaves or dried products thereof.

The secretory leaf extract of the present invention is added to the extract solvent 2 to 20 times, preferably 5 to 10 times with respect to the volume of the dried secretion leaves, warmed to 50 to 100 ℃, preferably 80 to 90 ℃ After stirring for 1 to 6 hours, preferably 2 to 3 hours while stirring, cooling and filtration, and concentrated under reduced pressure using a rotary evaporator to obtain a solvent extract from the leaves of the secretion, the filtration process for the remaining residue after filtration It may be repeated one or more times. As the extraction solvent, methanol, aqueous methanol solution, ethanol, aqueous ethanol solution, normal propanol, iso-propanol, normal butanol, or a mixture thereof may be used, and an aqueous 30-80%, preferably 50-60% aqueous ethanol solution is most preferred. Do.

Specifically, in the preferred embodiment of the present invention, after extracting the secretion leaves or dried products thereof with 50% aqueous ethanol solution at room temperature for 2 hours, the resulting extract is filtered, concentrated under reduced pressure and dried to obtain secretion leaf extract.

Secretory leaf extract of the present invention by effectively restoring the imbalance of intracellular information transmission and homeostasis through gap binding, cancer, diabetes, cerebral ischemia, nervous system diseases including Chaco-Marie-Tud type 1, spermatogenic disorders It may be used to prevent and treat homeostasis-related diseases such as vascular diseases, kidney disease, epilepsy and muscle diseases, including cardiovascular hypertension.

In particular, the secretory leaf extract of the present invention is an intercellular signal through gap binding generated by H ₂ O ₂ irrespective of 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 secretory leaf extract of the present invention may be mixed with a suitable pharmaceutically acceptable carrier or excipient according to conventional methods or diluted with a diluent 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 may 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 formulation 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 secretory leaf extract of the invention range from 1 to 1,000 mg / kg body weight, preferably from 10 to 100 mg / kg body weight, and can be administered once or in several divided 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 addition, the present invention provides a functional food or beverage composition for intercellular communication and homeostasis maintenance through gap binding, comprising an effective amount of secretion leaf extract. Foods to which the extract of the present invention may be added to exhibit the above effects include, for example, various foods, meat, beverages, chocolate, snacks, confectionery, pizza, ramen, other noodles, gums, ice creams, alcoholic beverages, Vitamin complexes and other health supplements, but are not limited thereto.

The secretory leaf extract of the present invention can 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, in which case the secretory leaf in the finally prepared food or beverage The content of the extract ranges from 0.1 to 80% by weight.

Hereinafter, the effect of preventing and treating diseases related to the inhibition of intercellular communication and the disproportionation of homeostasis through gap binding of the secretory leaf extract 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 following examples solids and solid mixtures, liquids and liquids, and percentages for liquids and solids are based on weight / weight, volume / volume and weight / volume, respectively, and all reactions were carried out at room temperature unless otherwise noted. .

Example 1 Preparation of Extracts with Efficacy in Preventing and Treating Diseases Associated with Imbalance of Inhibition and Homeostasis through Gap Binding from Secretory Leaves

First, to about 1 part by weight of dried secretion leaves, 50% ethanol (Duksan, Korea) was added at about 5 to 10 times at room temperature and then heated to 80 to 90 ℃ to extract the reflux for 2 hours while stirring. The extract was collected by centrifugation (Vision, Korea) at 8,000 rpm, 4 ° C. for 30 minutes (Vision, Korea), and the remaining residue was extracted two to three times in the same manner. The extract thus obtained was filtered through filter paper (Whatmann 41) and then freeze-dried to obtain secretory leaf extracts.

By measuring the efficacy of inhibiting intercellular information transmission and preventing and treating diseases related to the secretion of the secretory leaf extract obtained as described above, and to show that this is involved in intercellular signaling independent of antioxidant activity, ₂ O ₂ change amount was measured.

<Example 2> Efficacy measurement of restoring imbalance of information transmission through gap binding of secretory leaf extract

In order to determine the effect of restoring the inhibition of the gap junctional intercellular communication assay of the secretion leaf extract obtained in 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) contained 10% fetal bovine serum (FBS), penicillin 7.5 mg / l, streptomycin 7.5 mg / l, neomycin 15 mg / l and no phenol red The medium was incubated 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 culture dish, and the culture medium containing the secretion leaf extract prepared in Example 1 of 1 to 40 µg / ml was cultured in each dish and 500 μM of H ₂ O ₂ . Treated. 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 H ₂ O ₂ together. 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. 1, in which A is an untreated control group, B is a 500 μM H ₂ O ₂ treated group, and C is a 20 μg / ml secretory leaf methanol extract + 500 μM H ₂ O ₂ treatment group, D is 40 μg / ml secretion leaf methanol extract + 500 μM H ₂ O ₂ treatment group.

In Table 1, the percentage increase in intercellular communication through gap binding of the secretion leaf extract is {(number of stained cells of sample treated group-number of stained cells of H ₂ O ₂ treated group) / (untreated) Number of stained cells in control group—number of stained cells in H ₂ O ₂ treated group)} × 100.

As can be seen in Table 1 and Figure 1, the 50% ethanol extract of the secretion leaves showed an effect of completely restoring the intercellular information transmission inhibited by H ₂ O ₂ at a concentration of 40 ㎍ / ㎖. Thus, it can be seen that the secretory leaf extract has the effect of preventing and treating diseases related to intracellular communication inhibition and an imbalance of homeostasis.

Example 3 Measurement of H ₂ O ₂ Change after Secretion Leaf Extract Treatment

In order to confirm that the main cause of restoring suppression of intercellular information transmission through gap binding caused by H ₂ O ₂ is that the secretory leaf extract of the present invention is due to intracellular signaling rather than an antioxidant effect, as follows. A FOX2 assay (Nourooz-Zadeh, J., Tajaddini-Sarmadi, J., & Wolff, SP Analytical Biochemistry 220: 403-409, 1994), which is a known method of measuring the amount of H ₂ O ₂ , 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 BHT (butylated hydroxyltoluene) in a volume ratio of 1: 9 to prepare a working reagent.

Samples containing 500 μM of H ₂ O ₂ and a working reagent were mixed in a 1: 9 volume ratio in 96-well microplates under the same conditions as described in Example 2 above. The mixture was reacted in a 37 ° C. incubator (Forma Scientific Co., Marjetta, OH, USA) for 1 hour and then absorbance was measured at 560 nm. Herein, the secretion leaf extract treatment group and the catalase treatment group used 500 μM of H ₂ O ₂ as a sample and the secretion leaf extract and catalase were used at concentrations of 0 to 200 ㎍ / ml, respectively, and the control group was 500 Only cultures containing only μM H ₂ O ₂ were used in place of the samples.

As a result, as shown in Figure 2, in the case of the control group added only 500 μM H ₂ O ₂ It can be seen that the amount of H ₂ O ₂ does not change. In the case of the well-known that catalase to remove H ₂ O ₂ concentration of 500 μM of H ₂ O ₂ was found to reduce the dependent aspects. However, the amount of H ₂ O ₂ was slightly increased as a result of measuring the change in H ₂ O ₂ after treatment with the secretion leaf extract. Therefore, it can be seen that the effect of restoring the inhibition of gap binding intercellular communication by H ₂ O ₂ of secretory leaf extract is independent of antioxidant activity.

As shown in the above results, it was confirmed that the extracts of the leaves of the secretory tree have a prophylactic and inhibitory effect on diseases related to the inhibition of gap binding intercellular communication and the imbalance of homeostasis regardless of their antioxidant activity. In addition, by measuring the amount of change in H ₂ O ₂ after treatment with the secretory leaf extract, it is predicted that the recovery of gap-coupled intercellular communication by the sample is independent of the antioxidant activity of the sample and is involved in intercellular signal transmission. Can be.

As described above, the health-promoting food containing the secretion leaf extract used in the present invention can restore the imbalance of intercellular information transmission through gap binding.

Figure 1 shows the results of measuring the efficacy of the secretion leaf extract to restore the inhibition of intercellular communication through gap binding generated by hydrogen peroxide (H ₂ O ₂ ),

A: untreated control,

B: 500 μM H ₂ O ₂ treatment group,

C: 20 μg / ml secretory leaf methanol extract + 500 μM H ₂ O ₂ treatment group,

D: 40 mg / ml secretory leaf methanol extract + 500 μM H ₂ O ₂ treatment group

Figure 2 shows the results of measuring the change in the amount of H ₂ O ₂ after reacting the secretion leaf extract with H ₂ O ₂ according to the FOX2 measurement method.

Claims (2)

A health promoting food containing secretory leaf extract, for maintaining intercellular communication and homeostasis through gap binding. The method of claim 1, Secretion leaf extract is a health promoting food, characterized in that obtained by extracting the secretion leaf or its dried matter at 50 to 60% aqueous solution of ethanol at 80 to 90 ℃ for 2 to 3 hours.