KR20240027226A - Composition for preventing premature birth by simultaneously regulating estrogen receptor and progesterone receptor expression - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating premature birth containing daidzein. In particular, daidzein derived from black soybeans has higher levels of ER (estrogen receptor) and PR (progesterone receptor) than other isoflavones. ) By reducing the rate of expression, premature birth can be prevented and treated more effectively.

Description

Composition for preventing premature birth by simultaneously regulating estrogen receptor and progesterone receptor expression}

The present invention provides a composition for preventing premature birth through simultaneous regulation of estrogen receptor (ER) and progesterone receptor (PR) expression.

Among the complex pathophysiology of preterm birth, cervical softening is essential and sex hormones play an important role in labor. As childbirth approaches, the mother's progesterone function ceases and estrogen increases, so the balance of the estrogen receptor and progesterone receptor is important for maintaining a successful pregnancy, with a decrease in ER levels and an increase in PR levels in the cervix. A subsequent decrease in ER/PR ratio is associated with preterm birth.

Isoflavone is a polyphenol compound that has both estrogen agonist and antagonist properties, and isoflavone from black bean extract is a well-known diphenol-based compound that has a structure similar to estrogen and acts as an estrogen receptor. binds to ERα and ERβ. Soy isoflavones suppress serum estrogen and progesterone levels by reducing luteinizing hormone and follicle-stimulating hormone levels, and the main components of black soy isoflavones, such as genistein and daidzein, act on estrogen receptors. ) and activity on the progesterone receptor. The effects of isoflavones on a variety of reproductive health conditions, including menopausal symptoms, infertility, abnormal uterine bleeding, dysmenorrhea, abortion, and lactation have been reported, but their effect on preventing preterm birth has not been studied. There is also controversy regarding its positive effects on pregnancy and infertility.

Therefore, research on preventing premature birth or infertility by reducing the expression ratio between estrogen receptor and progesterone receptor in human cervical cells is continuously needed.

1. Republic of Korea Patent No. 10-1600751.

The purpose of the present invention is to provide a pharmaceutical composition for preventing or treating premature birth containing daidzein.

Another object of the present invention is to provide a health functional food composition for preventing or improving premature birth containing daidzein.

Another object of the present invention is to provide a reagent composition containing daidzein for inhibiting estrogen receptor (ER) expression and stimulating progesterone receptor (PR).

Another object of the present invention is a) adding powdered black beans to an organic solvent and stirring them; b) removing the organic solvent from step a) by filtering under reduced pressure, then adding the organic solvent again and stirring; c) washing and drying the stirred mixture of step b); d) mixing the dried solid of step c) with an organic solvent and heating it at 50 to 70° C. for 1 to 3 hours; and e) adding an acidic solution to the heated mixture of step d) and heating at 70 to 90° C. for 12 to 20 hours. To provide a method for extracting a black bean extract containing daidzein. It is in

Another object of the present invention is to provide a health functional food composition for preventing or improving premature birth, which is obtained by the above extraction method and contains a black bean extract containing daidzein.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating premature birth containing daidzein.

In addition, the present invention provides a health functional food composition for preventing or improving premature birth containing daidzein.

Additionally, the present invention provides a reagent composition for inhibiting estrogen receptor (ER) expression and stimulating progesterone receptor (PR), containing daidzein.

In addition, the present invention includes the steps of a) adding black beans in powder form to an organic solvent and stirring them; b) removing the organic solvent from step a) by filtering under reduced pressure, then adding the organic solvent again and stirring; c) washing and drying the stirred mixture of step b); d) mixing the dried solid of step c) with an organic solvent and heating it at 50 to 70° C. for 1 to 3 hours; and e) adding an acidic solution to the heated mixture of step d) and heating the mixture at 70 to 90° C. for 12 to 20 hours.

In addition, the present invention provides a health functional food composition for preventing or improving premature birth, which is obtained by the above extraction method and contains a black bean extract containing daidzein.

The present invention relates to a pharmaceutical composition for preventing and treating preterm birth containing daidzein or a black bean extract containing daidzein, which stimulates estrogen receptor (ER) and progesterone receptor (PR) By reducing the rate of expression, it can be used as a medicine or treatment method to prevent premature birth.

Figure 1 shows chromatograms of genistein, genistin, and daidzein from black bean ( Glycine max (L.) Merrill) extract.
Figure 2 shows data confirming the effect of genistein, genistin, daidzein, and black bean extract on the expression of ecdysis-triggering hormone (ETH).
Figure 3 shows data confirming whether estrogen receptor (hereinafter referred to as ER) is reduced when treated with genistein, genistin, daidzein, and black bean extract at each concentration.
Figure 4 shows data confirming whether the progesterone receptor (hereinafter referred to as PR) increases when treated with genistein, genistin, daidzein, and black bean extract at each concentration.
Figure 5 shows data confirming whether the expression ratio between ER and PR (hereinafter referred to as ER/PR) decreases when treated with genistein, genistin, daidzein, and black bean extract at each concentration. represents.
Figure 6 shows data confirming CCK when ME-180 cells were treated with genistein, genistin, daidzein, and black bean extract at each concentration.
Figure 7 shows data confirming CCK when primary cervix stromal cells were treated with genistein, genistin, daidzein, and black bean extract at each concentration.
Figure 8 shows data comparing ER expression by concentration of genistein, genistin, and daidzein.

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

Progesterone and estrogen play an important role during childbirth, and it is known that premature birth can be prevented by controlling their expression levels. Therefore, the present inventors were able to control the estrogen receptor (ER) and progesterone receptor (PR) simultaneously. This invention has been completed.

The present invention provides a pharmaceutical composition for preventing or treating premature birth containing daidzein.

The daidzein may be derived from black bean extract, and the black bean extract may be hot water extracted.

The daidzein can simultaneously regulate the expression levels of estrogen receptor (ER) and progesterone receptor (PR).

The daidzein may have an expression level ratio (ER/PR) between estrogen receptor (ER) and progesterone receptor (PR) of 0.00001 to 0.05, preferably 0.00003 to 0.01. .

In another embodiment of the present invention, the pharmaceutical composition may contain suitable carriers, excipients, disintegrants, sweeteners, coating agents, bulking agents, lubricants, lubricants, flavoring agents, antioxidants, buffers, bacteriostatic agents, etc. commonly used in the preparation of pharmaceutical compositions. It may further include one or more additives selected from the group consisting of diluents, dispersants, surfactants, binders, and lubricants.

Specifically, carriers, excipients, and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, and microcrystalline. Cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil can be used. Solid preparations for oral administration include tablets, pills, powders, granules, and capsules. agents, etc., and such solid preparations can be prepared by mixing the composition with at least one or more excipients, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium styrate and talc can also be used. Liquid preparations for oral use include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients may be included, such as wetting agents, sweeteners, fragrances, and preservatives. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, etc. Non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao, laurin, glycerogeratin, etc. can be used.

According to one embodiment of the present invention, the pharmaceutical composition is intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, intrasternal, transdermal, intranasal, inhalational, topical, rectal, oral, intraocular or It can be administered to a subject in a conventional manner via the intradermal route.

The dosage of the active ingredient according to the present invention may vary depending on the subject's condition and weight, type and degree of disease, drug form, administration route and period, and may be appropriately selected by a person skilled in the art, and the daily dosage is 0.01 mg. /kg to 200 mg/kg, preferably 0.1 mg/kg to 200 mg/kg, more preferably 0.1 mg/kg to 100 mg/kg. Administration may be administered once a day or divided into several administrations, and the scope of the present invention is not limited thereby.

In addition, the present invention provides a health functional food composition for preventing or improving premature birth containing daidzein.

The health functional food includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavors, colorants and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, It may contain organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc.

In addition, it may contain pulp for the production of natural fruit juice, synthetic fruit juice, and vegetable drinks. These ingredients can be used independently or in combination. In addition, the health functional food composition may be in the form of any one of meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, gum, ice cream, soup, beverages, tea, functional water, drink, alcohol, and vitamin complex. It can be.

In addition, the above-mentioned health functional food may additionally contain food additives, and its suitability as a “food additive” is determined according to the general provisions and general test methods of the Food Additives Code approved by the Food and Drug Administration, unless otherwise specified. Determination is made according to relevant standards and standards.

Items listed in the "Food Additives Code" include, for example, chemical compounds such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamic acid, natural additives such as subchromic pigment, licorice extract, crystalline cellulose, cold pigment, and guar gum, L -Mixed preparations such as sodium glutamate preparations, noodle-added alkaline preparations, preservative preparations, and tar color preparations are included.

At this time, in the process of manufacturing health functional foods, the content of the active ingredients added to the food can be appropriately adjusted as needed, and is preferably added in an amount of 1 to 90 parts by weight per 100 parts by weight of the food. .

Additionally, the present invention provides a reagent composition for inhibiting estrogen receptor (ER) expression and stimulating progesterone receptor (PR), containing daidzein.

In addition, the present invention includes the steps of a) adding black beans in powder form to an organic solvent and stirring them; b) removing the organic solvent from step a) by filtering under reduced pressure, then adding the organic solvent again and stirring; c) washing and drying the stirred mixture of step b); d) mixing the dried solid of step c) with an organic solvent and heating it at 50 to 70° C. for 1 to 3 hours; and e) adding an acidic solution to the heated mixture of step d) and heating the mixture at 70 to 90° C. for 12 to 20 hours.

The organic solvent in steps a) and b) may be n-hexane, and the organic solvent in step d) may be an aqueous ethanol solution.

The acidic solution in step e) may be a weak acid with a pH of 5.5 to 6.5.

The black bean extract may contain 15 to 30 mg/L of daidzein.

In addition, the present invention provides a health functional food composition for preventing or improving premature birth, which is obtained by the above extraction method and contains a black bean extract containing daidzein, preferably where the daidzein is 15 It may contain from 30 mg/L.

Hereinafter, to aid understanding of the present invention, it will be described in detail through examples. However, the following examples only illustrate the content of the present invention, and the scope of the present invention is not limited to the following examples. Examples of the present invention are provided to more completely explain the present invention to those with average knowledge in the art.

[Experimental Example 1] Black bean extract extraction

Black beans ( Glycine max (L.) Merrill) were made into powder using a blender and then sieved through a sieve (150um). 48 g of sieved Seoritae powder was placed in a 2 L glass Erlenmeyer flask, 1.5 L of n-hexane was added, and the mixture was stirred using a stirrer while blocking light. When the color of n-hexane changed to yellow, stirring was stopped, and when the solids settled below the triangular plast, n-hexane was removed using a vacuum filter. After this, n-hexane was refilled and stirred, and the process was repeated until the color of n-hexane became transparent. When n-hexane was transparent rather than yellow, it was washed with 95% ethanol. After washing with ethanol, the solid was dried naturally in a hood. 96% ethanol was added to the dried solid at a ratio of 1:20, and then heated at 60°C for 2 hours. After filtering, 37% hydrochloric acid (1.1 N) was added and heated at 80°C for 16 hours. The final product, black bean extract, was diluted 1:1 with distilled water (DW) and stored at -20°C.

[Experimental Example 2] HPLC (High-performance liquid chromatography)

The black bean extract extracted according to Example 1 was analyzed for genistein, genistin, and daidzein using a Waters HPLC system (Alliance e2695, Waters) at the Korea Basic Research Institute (Seoul, Korea). analyzed. The column used in the experiment was a YMC-Pack ODS-AM-303 column (250 mm × 4.6 mm i.d., S-5 μm, 120 Å, YMC Co., Kyoto, Japan), and the column temperature was 35 °C. 0.1% acetic acid (A) and 0.1% acetic acid/acetonitrile (B) were used as mobile phases. Elution conditions were programmed as follows: 0 min, 87% A, 13% B; 70 minutes, 70% A, 30% B; 75 minutes, 87% A, 13% B; 80 minutes, 87% A and 13% B; and the injection volume was 10 μL. Analysis was performed at a wavelength of 260 nm.

[Experimental Example 3] Drosophila stock and experimental design using it

Wild-type yellow fruit fly ( Drosophila melanogaster ) Canton-S strain was obtained from the Bloomington Drosophila Stock Center, Department of Biology, Indiana University (Indiana, USA). Drosophila stocks were maintained in an incubator (25 °C, 50 to 60% humidity) on standard food. Each fruit fly sample was collected under carbon dioxide anesthesia 2 to 5 days prior to treatment.

Drosophila were divided into four groups: normal parturition group; lead-induced preterm birth group; The normal delivery group was treated with isoflavones or black bean extract; and a lead-induced preterm birth group treated with isoflavones or black bean extract. Drosophila , an invertebrate melanogaster ) was exempted from Korea University IACUC review.

[Experimental Example 4] Preparation of medium for Drosophila model of premature birth

Wild-type yellow fruit fly ( Drosophila melanogaster ) Canton-S strain was used in the experiment and was stored in flasks with commonly used commercial media. The medium contained sucrose, yeast, cornmeal, agar, methylparaben (tegosept), propionic acid, and lead (II) nitrate. . Twenty pairs of male and female flies were transferred to bottles for mating and screened for lead(II) nitrate concentrations ranging from 2 to 10 mg. During breeding, experimental groups were exposed to lead for 3, 6, 9, 12, 15, 18, and 24 hours.

[Experimental Example 5] Fruit fly eggs

The Drosophila model of premature birth according to Experimental Example 4 was derived using flies reared at the lowest effective concentration of lead nitrate (4 mg). Flies were exposed to lead nitrate for 24 hours and the number of eggs was counted. To determine the effect of isoflavones or extracts on preterm birth in fruit flies, the number of eggs was examined, and the preterm birth rate over time was analyzed based on the number of eggs.

[Experimental Example 6] Expression of shedding-promoting hormone

Expression of ecdysis-triggering hormone (ETH) was analyzed by quantitative PCR in each group of Drosophila. Total RNA was isolated using TRIzol® reagent (Invitrogen, CA, USA). RNA was used to generate cDNA using oligo(dT) primers (Superscript® III Reverse Transcriptase Kit; Invitrogen). Cycling conditions for real-time PCR were 50°C for 2 minutes, 95°C for 10 minutes, 95°C for 15 seconds, and 60°C for 1 minute, repeated 40 times. Quantitative analysis was performed using StepOne plus Software V. 2.0 (Applied Biosystems). The following target genes were used for qRT-PCR: ribosomal protein L32 (RPL 32, NM_00144655.3), glutathione S-transferase D1 (gstD1, NM_079602.5), and ecdysis-promoting hormone (ETH).

[Experimental Example 7] Collection of human cervix

Under Korea University Anam Hospital Institutional Review Board approval (No.2020AN0474, approved on October 26, 2020), informed consent was obtained from women who underwent hysterectomy for benign uterine body pathology without cervical malformation. Cervical stroma was collected aseptically in 1X calcium- and magnesium-free Hank's balanced salt solution (Gibco Laboratories, Grand Island, NY, USA) immediately after hysterectomy. All procedures were performed under sterile conditions.

[Experimental Example 8] Cytotoxicity test

The cell line was cultured in a 96-well plate at a cell number of 5x10 ³ per well for 24 hours. After culturing in a 37°C CO ₂ incubator for 24 hours, ME-180 cells and primary cervix stromal cells were treated with genistein, genistin, and daidzein (Sigma-Aldrich, St. Louis, MO, USA). Then, the black bean extract was cultured in α-MEM or RPMI-1640 medium containing each concentration for 24 hours, and then each well was treated with 10 μl of CCK-8 solution. And after culturing in a 37°C CO ₂ incubator for 4 hours, the absorbance was measured at 450 mm using a microplate reader.

[Experimental Example 9] Statistical analysis

The results of the CCK test were analyzed by one-way analysis of variance and Duncan's multi-range test using the Statistical Package for Social Sciences version 12.0 software (SPSS, Chicago, IL). It has been done. Statistical analysis of real-time PCR results was performed using the Excel statistical data analysis program t-test: heteroscedasticity assumption two-group test. Statistical significance was set at P < 0.05.

[Example 1] Isoflavone HPLC analysis of black bean extract

The black bean extract of Experimental Example 1 was analyzed using an HPLC system. Both aglycone and non-aglycone in the extract were analyzed using genistein, genistin, and daidzein standards. Isoflavone samples were injected at a concentration of 50 mg/mL, and black bean extract was injected at a concentration of 20 mg/mL.

As a result, according to Figure 1, genistein, genistin, and daidzein had retention times (RT) of 20.015, 43.378, and 62.467 minutes and concentrations of 7.67, 26.56, and 15.91 mg/mL, respectively. was detected.

[Example 2] Effect on the expression of Drosophila molting-promoting hormone

The effects of isoflavones and black bean extract on the expression of ecdysis-promoting hormone (ETH) were confirmed in the normal delivery group or the lead-induced premature birth group. Regardless of the type of isoflavone, two concentrations that showed positive effects were selected. In the case of black bean extract, it was concentrated to the extent possible and analyzed because it is a mixture of isoflavones rather than a single substance.

As a result, according to Figure 2, in general, the level of ecdysis-promoting hormone (ETH) expression increased after isoflavone or extract treatment in both the normal delivery group and the lead-induced premature delivery group. In the isoflavone-treated normal delivery group, the genistin (GI; 0.6 mg) and daidzein (DA; 1 mg) treatment groups showed the highest expression, and the level of expression increased in the black bean extract (20 mg) treatment group. Even in the lead-induced premature birth group, the expression level was high in all groups treated with isoflavones and black bean extract. In particular, the daidzein (1 mg) treatment group showed the highest expression, and when black bean extract was administered, the expression level significantly increased in both the 10 mg and 20 mg treatment groups.

[Example 3] Effect on expression of fruit fly egg production

The effect of isoflavone or black bean extract treatment on the number of eggs in the normal delivery group or lead-induced preterm birth group was tested.

As a result, according to Table 1, in the case of the normal group, the number of eggs in the isoflavone-treated group increased significantly over time after 6 hours compared to the eggs in the control group, but after 18 hours, the number of eggs in the control group increased significantly. The number of eggs decreased compared to the number of eggs, and unlike other isoflavone samples, the number of eggs in the black bean extract increased after 15 hours compared to the initial time. Meanwhile, in the lead-induced preterm birth group, the group containing genistin was the largest from the beginning to 15 hours, the group treated with daidzein was the most after 15 hours, and in the case of using black bean extract, the group was the largest group containing genistin from the beginning to 15 hours. Regardless of time, the number of participants learned more than the control group.

[ Example 4] Comparison of ER and PR gene expression in primary cervical stromal cells and ME-180 cells

Primary cervix stromal cells were treated with genistein, genistin, and daidzein. CCK to determine the cytotoxicity of genistein, genistin, daidzein, and black bean extract in ME-180 cells, a cervical epithelial cell line, and primary cervical stromal cells, before confirming the expression of ER. Cell proliferation was confirmed by analysis. After confirming the concentration of isoflavone at which ER expression was reduced according to the CCK results according to Figures 6 and 7, the decrease in ER and increase in PR were confirmed for each cell.

As a result, according to Figures 3 and 4, in genistein (20 μM), the ER of primary cervical stromal cells was 0.001 and the PR was 0.02, and the ER of ME-180 cells was 0.02 and the PR was 0.09. . In genistin (20 μM), the ER of primary cervical stromal cells was 0.0002 and the PR was 0.05, and the ER of ME-180 cells was 0.02 and PR was 0.004. In daidzein (10 μM), the ER of primary cervical stromal cells was 0.00009 and the PR was 0.005, and the ER of ME-180 cells was 0.01 and the PR was 0.00004. In black bean extract (3 ng/ml), the ER of primary cervical stromal cells was found to be 0.00009 and the PR was 0.05, and the ER of ME-180 cells was found to be 0.09 and PR was 0.005.

[ Example 5] Comparison of expression ratios between ER and PR in primary cervical stromal cells and ME-180 cells

In the same manner as Example 4, ER and PR gene expression was examined and ER/PR values were examined.

As a result, according to Figure 5, in the case of genistein (20 μM), it is 0.002 in primary cervical stromal cells and 0.08 in ME-180 cells, and in the case of genistin (20 μM), it is 0.08 in primary cervical stromal cells. for daidzein (10 μM), 0.01 in primary cervical stromal cells and 0.00003 in ME-180 cells, and for black bean extract (3 ng/ml). It was found to be 0.0009 in primary cervical stromal cells and 0.04 in ME-180 cells.

[Example 6] Confirmation of decrease in ER at specific isoflavone concentration

In order to suppress preterm birth, a decrease in the ER/PR ratio is essential due to a decrease in ER and an increase in PR at the same time, and the use of genistein, genistin, and daidzein at the same specific concentration (10 μM) is essential. We confirmed whether ER expression was decreased.

As a result, according to Figure 8, in the case of genistein and genistin, a decrease in ER expression could not be confirmed when treated with 10 μM, but in the case of daidzein, the expression of ER was decreased. It was effectively reduced, and it was confirmed that compared to other isoflavones, the concentration that reduces ER/PR values was only half of that.

The above description of the present invention is for illustrative purposes, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential features of the present invention. will be. Therefore, the embodiments described above should be understood in all respects as illustrative and not restrictive.

The scope of the present invention is indicated by the claims described below, and all changes or modified forms derived from the meaning and scope of the claims and their equivalent concepts should be construed as being included in the scope of the present invention.

Claims (13)

A pharmaceutical composition for preventing or treating premature birth containing daidzein. The pharmaceutical composition according to claim 1, wherein the daidzein is derived from black bean extract. The pharmaceutical composition according to claim 2, wherein the black bean extract is hot water extracted. The pharmaceutical composition according to claim 1, wherein daidzein simultaneously regulates the expression levels of estrogen receptor (ER) and progesterone receptor (PR). The pharmaceutical composition according to claim 4, wherein daidzein inhibits estrogen receptor (ER) expression and promotes progesterone receptor (PR) expression. The pharmaceutical composition of claim 4, wherein the daidzein has an expression level ratio (ER/PR) between estrogen receptor (ER) and progesterone receptor (PR) of 0.00001 to 0.05. . A health functional food composition for preventing or improving premature birth containing daidzein. A reagent composition for inhibiting estrogen receptor (ER) expression and promoting progesterone receptor (PR), containing daidzein. a) adding black beans in powder form to an organic solvent and stirring them;
b) removing the organic solvent from step a) by filtering under reduced pressure, then adding the organic solvent again and stirring;
c) washing and drying the stirred mixture of step b);
d) mixing the dried solid of step c) with an organic solvent and heating it at 50 to 70° C. for 1 to 3 hours; and
e) A method of extracting a black bean extract containing daidzein, comprising the step of adding an acidic solution to the heated mixture of step d) and heating it at 70 to 90 ° C. for 12 to 20 hours. The extraction method according to claim 9, wherein the organic solvent in steps a) and b) is n-hexane, and the organic solvent in step d) is an aqueous ethanol solution. The method of claim 9, wherein the acidic solution in step e) is a weak acid with a pH of 5.5 to 6.5. The method of claim 9, wherein the black bean extract contains 15 to 30 mg/L of daidzein. A health functional food composition for preventing or improving premature birth, obtained by the extraction method according to claim 9 and containing black bean extract containing 15 to 30 mg/L of daidzein.