KR101897896B1 - Composition comprising Coumestrol for improving pregnancy - Google Patents

Abstract

The present invention relates to a composition containing quimestrol as an active ingredient, and more particularly to a pharmaceutical composition for promoting pregnancy comprising quimestrol as an active ingredient, a composition for promoting pregnancy containing quimestrol as an active ingredient A composition, and a pharmaceutical composition and food composition for preventing or treating infertility comprising as an active ingredient quimestrol.
The composition comprising quimestrol as an active ingredient according to the present invention can minimize toxicity or side effects in the body compared with chemical compounds by using natural materials as raw materials and can inhibit the expression of proteins in PI3K / AKT and ERK1 / 2 MAPK signaling pathways Can enhance the migration and proliferation ability of the malignant ectodermal cells through activation, and thus can be usefully used in fields related to pregnancy promoters, medicines and functional foods that can prevent and treat female infertility.

Description

[0002] Composition comprising Coumestrol for improving pregnancy,

The present invention relates to a composition containing quimestrol as an active ingredient, and more particularly to a pharmaceutical composition for promoting pregnancy comprising quimestrol as an active ingredient, a composition for promoting pregnancy containing quimestrol as an active ingredient A composition, and a pharmaceutical composition and food composition for preventing or treating infertility comprising as an active ingredient quimestrol.

With the recent aging society, infertility is increasing as maternal age increases. In addition, due to environmental pollution caused by industrialization and the advancement of women into society, the stress of women is increasing, and the pregnancy rate is significantly lowered. Common causes of female infertility include ovulation disorders, transfer of fertilized eggs, and implantation disorders. Infertility due to obstructive ovarian failure and transfer of embryos is largely solved through in vitro fertilization (IVF), but sterilization due to implantation disorder has yet to be clarified.

In order to maintain a successful pregnancy, it is necessary to develop the embryo, implantation, placenta formation, hormonal regulation, and exchange of maternal and fetal nutrients and gases (Spencer TE et al ., Reproduction, 128 (6): 657-668, 2004 ).

Porcine trophectoderm cells (pTr cells) are an in vitro model constructed to understand embryonic development and differentiation, implantation process, and placenta formation process according to various tissue nutrients at the early pregnancy. (Jaeger LA et al ., Endocrinology, 146 (9): 3933-3942, 2005).

The proliferation and migration ability of porcine anterior ectoderm cells from 10 to 12 days after fertilization is an essential process to achieve epithelial chorion placental development through noninvasive implantation into uterine luminal epithelial cells (Geisert RD et al ., Adv. Embryol Cell Biol , ≪ / RTI > 216: 137-163, 2015).

During the peri-implantation period, estrogen secreted from the aquatic product blocks the secretion of prostaglandin F 2α, thereby blocking luteolysis and remodeling the endometrial tissue to form the uterine environment for implantation (Bazer FW and Johnson GA, Differentiation, 87 (1-2): 52-65, 2014).

On the other hand, phytoestrogens or phytoestrogens are classified as isoflavones, lignans, coumestans, stilbenes and affect pregnancy and birth through estrogen-like structure and biological activity (Patisaul HB and Jefferson W, Front Neuroendocrinol , 31 (4): 400-419, 2010).

Coumestrol, a derivative of Coomestan, belongs to the phytoestrogenic estrogen and exhibits various physiological effects in vitro and in vivo through estrogen receptors (Markaverich BM et al ., Environ Health, 103 (6): 574-581 , 1995).

Previous studies have shown that the increased weight of the uterus by cus- sterol in ovariectomized rats suggests that cus- sterol is an estrogen-dependent estrogen receptor (Markaverich BM et al ., Environ Health, 103 (6) 574-581, 1995). It has been known that administration of a chome sterol to mice during pregnancy and lactation increases immunoglobulin A, which improves fetal immune system (Wang M et al ., Anim Sci J , 84 (4): 322-327, 2013), so far there has been no report on the mechanism of promoting implantation using cumestrol.

In addition, studies on compositions for fertilization or fertility-promoting compositions of animals have been reported to induce an increase in pregnancy rate and an increase in implantation rate by about 25% using MMP-9 (metalloproteinase-9) (Patent Document 1) There are no researches related to the related substances derived from natural plant compounds.

Accordingly, the present inventors have made intensive efforts to develop a substance capable of promoting the implantation efficiency in a new pregnancy using a material derived from a natural substance, and as a result, have found that the effect of the cumestrol- And confirming that cumestrol activates PI3K / AKT and ERK1 / 2 MAPK signaling pathways in malignant ectodermal cells, thereby completing the present invention.

Korean Patent Publication No. 10-2010-0049153

It is an object of the present invention to provide a pharmaceutical composition for promoting pregnancy which comprises cumestrol as an active ingredient.

Another object of the present invention is to provide a food composition for promoting pregnancy comprising quimestrol as an active ingredient.

The present invention also aims to provide a pharmaceutical composition for preventing or treating infertility comprising as an active ingredient cumestrol.

Another object of the present invention is to provide a food composition for preventing or ameliorating infertility comprising quesmestrol as an active ingredient.

The present invention also aims to provide a method for enhancing the migration and proliferation ability of malignant ectodermal cells using the above composition.

The present invention provides a pharmaceutical composition for promoting pregnancy, which comprises cumestrol as an active ingredient.

The present invention also provides a food composition for promoting pregnancy comprising quimestrol as an active ingredient.

The present invention also provides a pharmaceutical composition for the prophylaxis or treatment of infertility comprising as an active ingredient cumestrol.

The present invention also provides a food composition for the prevention or amelioration of infertility, comprising as an active ingredient, quemestrol.

The present invention also provides a method for enhancing the migration and proliferation ability of malignant ectodermal cells using the composition.

The composition comprising quimestrol as an active ingredient according to the present invention can minimize toxicity or side effects in the body compared with chemical compounds by using natural materials as raw materials and can inhibit the expression of proteins in PI3K / AKT and ERK1 / 2 MAPK signaling pathways Can enhance the migration and proliferation ability of the malignant ectodermal cells through activation, and thus can be usefully used in fields related to pregnancy promoters, medicines and functional foods that can prevent and treat female infertility.

FIG. 1 shows the mechanism of cell proliferation by cumestrol, which is related to the effect of enhancing the migration of porcine malignant ectodermal cells.
FIG. 2 shows the results of analysis of the effect of cumestrol on the cell migration ability of porcine malignant ectodermal cells, wherein (A) is a result of analyzing cell mobility using a Transwell membrane, and (B) The number of malignant ectodermal cells was measured.
FIG. 3 shows the effect of cumestrol on the proliferation signal transduction regulatory mechanism of porcine malignant ectodermal cells, wherein (A) to (E) show the phosphorylation pattern of signaling molecules according to dose-dependent, time-dependent administration of cumestrol The results of the analysis are shown.
FIG. 4 shows the results of measurement of the phosphorylation pattern of AKT and ERK1 / 2 protein by the immunostrophin technique by the quercetrol in the porcine malignant ectodermal cells.
FIG. 5 shows the results of an electrophoretic analysis of porcine malignant ectodermal cells through a mixture of cumestrol, PI3K / AKT, and ERK1 / 2 signal transduction inhibitor.
FIG. 6 shows the migration ability analysis of pig malnutrition exocrine cells through a mixture of cumestrol, PI3K / AKT, and ERK1 / 2 signal transduction inhibitor.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

In the present invention, the mechanism of cell proliferation by cumestrol, which is related to the effect of enhancing the migration of porcine malignant ectodermal cells, is shown (Fig. 1).

The present invention provides a composition for promoting pregnancy comprising quimestrol as an active ingredient.

The composition comprises a pharmaceutical composition food composition.

The term "composition" as used herein is intended to encompass the products comprising the specified ingredients, as well as any products made directly or indirectly by the combination of the specified ingredients.

In the present invention, "pregnancy" is a process in which the fertilized egg is implanted in the inner wall of the uterus, is supplied with nutrition from the mother, and develops into the fetus. The normal pregnancy process proceeds in the order of fertilization, implantation and fetal development.

The term "fertilization" means that an ovum ovulated in a female ovary meets with spermatozoa at the upper part of the oviduct to form an embryo. The ovum has the ability to stay alive for 1-2 days after ovulation and the sperm to remain alive for 2-3 days in the uterus, and the sperm survive for one week after being ejaculated. Sperm are spermatozoa that reach the ovary close to the ovary 2 ~ 3 hours after the ejaculation. In general, the spermatozoon first reaches the upper part of the oviduct, and when ovum is ovulated from the ovary, only one of the spermatozoa .

The "implantation" refers to a phenomenon in which an embryo in which a sperm and an egg are brought together at the upper part of the oviduct is moved to the uterus repeatedly and buried in the uterine wall in a bladder state. After the embryo is conceived, it is called pregnancy. Embryos implanted into the uterus are born after about nine months of age, while being fed from the uterine lining.

According to one embodiment of the present invention, the above-mentioned cumestrol is characterized by promoting the implantation of malnutrition ectodermal cells.

Herein, the malnutrition-derived ectodermal cells may be cells derived from a mammal, and the mammal may be selected from the group consisting of a set (for example, a mouse, a mouse, a hamster, a gerbil and a guinea pig) (For example, dogs, cats, tigers, wolves, foxes, lions, cheetahs, leopards, horses, Rabbits and crying rabbits), insects (eg hedgehog, mole and selenodon) and primates (eg, chimpanzees, orangutans, gorillas, bonobos, Japanese monkey, Rhesus monkey).

According to one embodiment of the present invention, the above-mentioned cumestrol has an effect of activating the PI3K / AKT and ERK1 / 2 MAPK signal transduction mechanism and thus enhances the implantation efficiency through the enhancement of migration and proliferation ability of malignant ectodermal cells .

The composition may further comprise, as an active ingredient, a substance which is effective for promoting pregnancy, or may contain, in addition to the above-mentioned effective ingredient, an additional ingredient, Acceptable or nutritionally acceptable carrier, excipient, diluent or subcomponent.

More specifically, the pharmaceutical composition for promoting pregnancy comprising the above-mentioned cumestrol may further contain, in addition to the above-mentioned active ingredients, a nutrient, a vitamin, an electrolyte, a flavoring agent, a colorant, a thickening agent, a pectic acid and its salt, , Protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like.

Examples of the carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, Calcium stearate, mineral oil, calcium carbonate, dextrin, propyleneglycol, liquid paraffin, sodium carboxymethylcellulose, sodium carboxymethylcellulose, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, And physiological saline, but is not limited thereto. The components can be added to the active ingredient, i.e., cumestrol, either independently or in combination.

The composition according to the present invention can be used singly for promoting pregnancy or in combination with surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers.

The composition according to the present invention may be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically) according to a desired method, and the dosage may be appropriately selected according to the weight, age, sex, The range varies depending on the condition, diet, time of administration, method of administration, excretion rate and severity of the disease. The daily dose of cumestrol of the present invention is 0.01 to 10,000 mg / kg, preferably 1 to 20 mg / kg, and is preferably administered once to three times a day.

In addition, the present invention provides a food composition for promoting pregnancy comprising quimestrol as an active ingredient.

The composition according to the present invention may be included in a health functional food for the purpose of promoting pregnancy. When the active ingredient of the present invention is used as a food additive, And can be suitably used according to a conventional method. The amount of the active ingredient to be mixed may be appropriately adjusted depending on the intended use (prevention, health or therapeutic treatment).

There is no particular limitation on the kind of the food. Examples of the food to which the above substance can be added include dairy products including meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, ice cream, soups, drinks, tea, , An alcoholic beverage and a vitamin complex, and includes all the healthy foods or health functional foods in a conventional sense.

Various additives such as flavors or natural carbohydrates can be used as the food-aid additive of the present invention. The above-mentioned natural carbohydrates are sugar saccharides such as monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like.

In addition to the above, the composition according to the present invention may further contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid concentrating agents, pH adjusting agents, stabilizers, preservatives, , A carbonating agent used in carbonated drinks, and the like. In addition, the composition according to the present invention may contain flesh for the production of natural fruit juice, fruit juice beverage and vegetable beverage. These components may be used independently or in combination.

In addition, the present invention provides a composition for preventing or treating infertility comprising as an active ingredient cumestrol.

The composition comprises a pharmaceutical composition or a food composition.

The infertility may be female infertility.

The female infertility is associated with nonimplantation of ovum, female infertility associated with anovulation associated with anovulation, female infertility of tubal origin originating from the uterine tube, Tubal occlusion, Tubal stenosis, Female infertility of the uterine origin, Infertility of the uterus, Infertility of the uterus, Infertility associated with male factors, female infertility associated with cervical origins, female infertility associated with male factors, and other factors related to congenital anomaly of uterus. Female infertility of other origin and unspecified female infertility (female infertility, unspecified). But is not limited thereto.

In the present invention, the above-mentioned "Nonimplantation of ovum" is a representative cause of female infertility. During fertilization, the embryo moves while dividing cells into the uterus, and when it reaches the blastocyst stage, it is buried in the endometrium and seated. However, when embryos are not thick enough to be implanted or stiff due to damage, it is difficult to obtain implants, and abortion is often caused by lack of space due to adhesion of the uterine wall. Endometrial hyperplasia due to intrauterine inflammation caused by spontaneous abortion, mastectomy, abortion, endometritis, pelvic tuberculosis and intrauterine device, or uterine myopathy, Or if the uterine endometrium is incomplete due to congenital abnormal uterine hormone secretion. The oocyte implantation has been identified as the major cause of artificial insemination and failure of in vitro procedures.

Since the pharmaceutical composition or the food composition includes the pharmaceutical preparation or the food preparation containing the above-mentioned quimestrol as an active ingredient, the contents overlapping with the composition of the present invention described above can be used in the present specification The description thereof is omitted in order to avoid excessive complexity.

The present invention also provides a method for enhancing the migration and proliferation ability of malnutrition ectodermal cells using the composition comprising the above-mentioned cumestrol.

[Example]

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for illustrating the present invention and that the scope of the present invention is not construed as being limited by these embodiments. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Example

<Experimental Method>

1. Experimental animal and cell culture

The pregnant sows were separated from the pregnant sows at 12 days after fertilization, and the nutritional ectoderm cell lines were identified and primary cultures were performed. Fetal bovine serum (FBS) and ZellShield, an antibiotic, were mixed together in DME / F12 1: 1 medium for monolayer cultures of porcine malignant ectodermal cells.

2. Experimental material

In order to confirm the mechanism of signal transduction by cucumbersterol, the candidate compound Cummistrol was purchased from Sigma and phospho-AKT, ERK1 / 2, P70S6K, P90RSK, S6 protein and total AKT, ERK1 / 2, P70S6K , P90RSK, and S6 proteins were purchased from Cell Signaling Techonology. In addition, PI3K inhibitor LY294002 was purchased from Cell Signaling Technology, and ERK1 / 2 inhibitor U0126 was purchased from Enzo Life Science in order to investigate the effect of suppressing the target signal transduction process.

3. Transwell  Measurement of cell migration ability

To examine the effect of cumestrol on the cell migration ability of porcine malignant ectodermal cells, 1 × 10 ⁵ pig malignant ectodermal cells cultured in FBS starvation conditions and 100 μl of DME / F12 1: 1 medium were inoculated into 8-mm Transwell inserts (Corning, Inc., Corning, NY, USA) and cultured for 12 hours at a concentration of 0 μM, 5 μM, 10 μM, 20 μM, 50 μM and 100 μM, Cells trans-membrane transferred for 10 min are fixed, dried and stained with hematoxylin (catalog number: HHS32, Sigma Aldrich, Inc.) for 30 min and washed. The membrane was immersed in a glass slide and immersed in a Permount solution, and the cell migration ability was observed, photographed, and calculated using an optical microscope DM3000 (Leica).

4. Immunofluorescence

3 × 10 ⁴ malignant ectodermal cells were cultured in a confocal dish (catalog number: 100350, SPL Life Science, Republic of Korea) with 300 μl of DME / F12 1: 1 medium containing 10% Time FBS starvation and treated with 20 μM of cumestrol for 15 minutes, then fixed with methanol for 10 minutes, treated with 1: 200 diluted p-AKT, p-ERK1 / 2 antibody, Was treated with rabbit IgG and incubated at 4 ° C for 16 hours. After 2 washes with PBS containing 0.1% BSA (bovine serum albumin), goat anti-rabbit IgG Alexa 488 (catalog number: A-11008, Invitrogen, Carlsbad, CA, USA) diluted 1: 200 in antibody dilution buffer, and incubated at room temperature for 1 hour. The malignant ectodermal cells were washed with 0.1% BSA-PBS and then DAPI staining was performed to observe the nuclei as well as the target proteins in the malignant ectodermal cells. After completion of the experiment, cells were observed and photographed using a confocal microscope of LSM710 (Carl Zeiss, Thornwood, NY, USA).

5. Protein expression analysis ( Western blot )

Protein was quantified by Bradford protein assay (Bio-Rad, Hercules, Calif., USA) after the total protein was extracted from the malignant ectodermal cells after treating the pig nourishment ectodermal cells with a mixture of cumestrol or a cell signaling inhibitor. Then, the extracted proteins were denatured at 95 ° C for 5 minutes, electrophoresed using a 10% SDS / PAGE gel, transferred to nitrocellulose membrane, incubated with primary antibody and secondary antibody, and then subjected to chemiluminescence detection (SuperSignal West Pico, Pierce, Rockford, Ill., USA) reagents were used to analyze the expression of target proteins using the ChemiDoc EQ system and Quantity One software (Bio-Rad)

6. Statistical Analysis

The mean and standard error were calculated using a statistical analysis system (SAS) statistical program and one-way ANOVA was performed. Significance test was performed at P <0.05 level.

<Experimental Results>

One. Cumestrol  Pig Nutritional ectoderm  Effect of cell migration on cell migration

In order to confirm the migration ability of porcine malignant ectodermal cells, transwell membrane was added to growth medium supplemented with dose-dependent cumestrol, and cultured for 12 hours. The number of malignant ectodermal cells migrated through the transwell membrane was examined. (Fig. 2). As shown in Fig.

2. Pig Nutritional ectodermal cells  of mine To cumestrol  Regulatory signal transduction regulator

Pancreatic malignant ectodermal cells were treated with 0 μM, 5 μM, 10 μM and 20 μM of cumestrol, and then total protein was extracted from malignant ectodermal cells. Western blot analysis revealed that AKT, ERK1 / 2, P70S6K, P90RSK Indicating that the phosphorylation of the protein increases in a dose-dependent manner (Fig. 3A). In addition, AKT, P70S6K, and S6, which are down-signaling molecules of PI3K / AKT, were phosphorylated in a short time and incubated with ERK1 / 2 MAPK signal transduction in time-dependent incubation of porcine malignant ectodermal cells in growth medium containing 20 μM of cumestrol ERK1 / 2, and P90RSK, the molecules, showed a relatively long duration of phosphorylation. Thus, it was possible to deduce that the migration of porcine natriuretic cells enhanced by quimestrol is regulated by the PI3K / AKT, ERK1 / 2 MAPK signaling pathway (FIG. 3B-3F).

Afterwards, AKT and ERK1 / 2 protein phosphorylation, which is the main target signaling molecule of cumestrol, was stained with immunofluorescence technique in order to identify the expression position in porcine malignant ectodermal cells. As a result, it was confirmed that phosphorylation of AKT was induced in the nucleus of the malignant ectodermal cells and phosphorylation of the ERK1 / 2 was induced in the nucleus and cytoplasm of the malignant ectodermal cells when quesamestrol was treated in comparison with the malignant ectodermal cells of the control group ).

3. With cumestrol PI3K / AKT , ERK1 / 2 signaling pathway inhibitor mixture Pigs through Nutritional ectodermal cells  My Mechanism Analysis

The PI3K pathway inhibitor, LY294002 (20 μM), and the ERK1 / 2 pathway inhibitor U0126 (20 μM) were injected in order to confirm the regulatory mechanism of PI3K / AKT and ERK1 / 2 MAPK signal transduction mechanism by cumestrol in porcine malignant ectodermal cells. Mixed with mestrol, incubated in porcine malignant ectodermal cells, and then Western blot confirmed the phosphorylation pattern of signaling molecules. As a result, it was confirmed that phosphorylation of AKT, P70S6K, S6 protein activated by cumestrol was inactivated by LY294002 and U0126, and that ERK1 / 2 and P90RSK were inactivated only by U0126. These results suggest that the mechanism by cumestrol in pig eutrophic cells is regulated by PI3K / AKT and ERK1 / 2 MAPK, and that ERK1 / 2 protein acts as an upper signaling molecule of AKT and phosphorylates AKT, P70S6K, S6 (Fig. 5).

4. With cumestrol PI3K / AKT , ERK1 / 2 signaling pathway inhibitor mixture Pigs through Of malignant ectodermal cells  Migration ability analysis

In order to confirm whether the migration ability of porcine malignant ectodermal cells, which were increased by cumestrol, was regulated by PI3K / AKT and ERK1 / 2 MAPK signaling regulation, cumestrol and LY294002 and U0126 were mixed, And the results were confirmed using a transwell membrane. It was confirmed that the migration of the malignant ectodermal cells, which was increased by about 170% by cumestrol, was reduced again by treating with LY294002 and U0126, and showed migration ability similar to that of the control group (FIG. 6). This demonstrates that cumestrol plays a role in activating the PI3K / AKT and ERK1 / 2 MAPK pathways and thus greatly enhances the migration of pig trophoblast cells.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will readily appreciate that many modifications are possible in the exemplary embodiments without materially departing from the novel teachings and advantages of this invention. something to do. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (9)

delete delete delete delete delete delete delete delete A method for enhancing migration and proliferation ability of a mammal-derived nutritional ectodermal cell other than a human using a composition for promoting pregnancy comprising cumestrol as an active ingredient.

Images