KR101730949B1 - Compositions for preventing or treating infertility comprising an extract of Perilla frutescens - Google Patents

Abstract

The present invention relates to a composition for preventing or treating infertility, a composition for promoting pregnancy or a composition for improving fertility, and more particularly to a composition for preventing or treating infertility comprising Perilla frutescens extract as an active ingredient, A composition for promoting ovarian cancer, or a composition for enhancing the intrauterine implantability of oocytes. According to the present invention, the self-lobular extract according to the present invention increases the expression of leukocyte inhibitory factor (LIF) and integrin [alpha] v [beta] 3 and alpha v [beta] 5 and promotes the binding of maternal endometrial cells to fetal- , It can be usefully used for the prevention or treatment of infertility.

Description

TECHNICAL FIELD The present invention relates to a composition for preventing or treating infertility,

The present invention relates to a composition for preventing or treating infertility, a composition for promoting pregnancy or a composition for improving fertility, and more particularly, to a composition for preventing or treating infertility comprising Perilla frutescens extract as an active ingredient, A composition for promoting pregnancy, or a composition for enhancing the intrauterine implantability of an oocyte.

In general, about 85% of married couples who have a marital relationship after marriage become pregnant within a year, whereas infertility refers to a condition that does not become pregnant after one year. The cause of male infertility is genetic defects, environmental defects, abnormalities of endocrine system, and immunological causes. Male infertility is caused by sperm and semen defects which account for about 30% Respectively.

In women, the cause of infertility is much more varied and complicated than in men. Causes of female infertility include ovulation disorders, abnormalities of the uterine tube, obstruction of transfer of embryos due to obstruction, obstruction or stenosis, abnormal uterine or cervical incision. Female infertility originating in the uterus is caused by congenital abnormalities of the uterus and invasion of the oocyte.

Currently, infertility treatment methods include intrauterine insemination (IU), in vitro fertilization-embryo transfer (IVF-ET), and in vitro fertilization There is a way. Despite much progress, the success rate of this assisted reproductive technology is low to 10 ~ 30%, and there is a disadvantage that the mental and financial troubles of the family are caused by the failure of the procedure. Furthermore, even if fertilization has been done in artificial fertilization and in vitro fertilization, fertilization is not successful if the fertilized embryo fails to conceive in the endometrium of the mother. Currently, development and treatment of implantation disorder treatment are not enough as medical technology, and development of infertility treatment that can increase implantation efficiency is required.

Implantation refers to a state in which a fertilized embryo is attached to the lining of the uterus to allow the fetus to receive oxygen and nutrients from the mother. The fertilization process of the embryo is performed by hatching, which removes the zona pellucida, apposition, which is a loose interaction between the embryo and the endometrium, adhesion which is a strong bond between the embryo and the endometrium, And invasion through the process. This implantation process is regulated by the involvement of various cytokines and growth factors. In particular, leukemia inhibitory factor (LIF), an interleukin-6 (IL-6) 2 (insulin-like growth factor-2) expression is important (Hum Rep Update, 12 (6): 731-746).

In mice lacking the LIF gene, normal progression to apposition leads to defects in the adhesion process (Nature 359 (6390): 76-9; Endocrinology 150 (6): 2915-2923), inhibition of LIF function using an antagonist to the receptor of LIF (LIF-R) does not result in implantation (PNAS 104 (49): 19357-19362). In addition, the expression of integrins αvβ3 and αvβ5, which directly regulate the endometrium's water receptivity, is very important in the implantation of embryos (Mol Hum Reprod. 2 (7): 527-534). Lymphocyte- (BJOG, 109: 610-617), which increase the expression of various adhesion factors such as heparin and CD44, are the most important factors in the implantation process.

Perilla frutescens (L.), on the other hand, is a leaf and end of charcoal or wrinkled lobules belonging to the family Lamiaceae (Labiatae), which is dried and used for medicinal purposes. It has been traditionally used for the treatment of food poisoning caused by colds, fever, cough, asthma, chest, and fish and crab. It has been used for pharmacological effects such as antipyretic action, (Chinese Medicines Dictionary, 3574-4576).

In addition, it has been known that japanese lobules are effective for the treatment of abortion and premature birth, and for the treatment of cold during pregnancy (Donguibogam).

The inventors of the present invention have been studying effective substances for the promotion of pregnancy and infertility, and have found that the extract of L. japonica promotes the expression of leukocyte inhibitory factor (LIF) and integrin αvβ3 and αvβ and promotes the binding of maternal endometrial cells to fetal- The present inventors completed the present invention by confirming that there is an effect of preventing or treating female infertility by increasing fertilization efficiency of embryo.

It is an object of the present invention to provide a composition for the prevention or treatment of infertility comprising an inner lobule extract as an active ingredient.

It is also an object of the present invention to provide a composition for promoting pregnancy which comprises a self-lyophilized extract as an active ingredient.

It is also an object of the present invention to provide a composition for enhancing the intrauterine implantability of an oocyte comprising an inner lobule extract as an active ingredient.

In order to accomplish the above object, the present invention provides a pharmaceutical composition for preventing or treating infertility comprising an extract of a natural lobule as an active ingredient.

In addition, the present invention provides a food composition for preventing or ameliorating infertility, which comprises a self-leaf extract as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for promoting pregnancy, comprising a self-leaf extract as an active ingredient.

In addition, the present invention provides a food composition for promoting pregnancy, comprising a self-leaf extract as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for enhancing the intrauterine implantability of an oocyte comprising an extract of a natural lobule as an active ingredient.

In addition, the present invention provides a food composition for enhancing the intrauterine implantability of an oocyte comprising a self-lobular extract as an active ingredient.

According to the present invention, the self-lobular extract according to the present invention increases the expression of leukocyte inhibitory factor (LIF) and integrin [alpha] v [beta] 3 and alpha v [beta] 5 and promotes the binding of maternal endometrial cells to fetal- May be useful for the prevention or treatment of infertility, especially female infertility due to implantation disorders.

Fig. 1 shows the cytotoxicity of Ishikawa cells, a human endometrium-derived cell of the endoplasmic reticulum extract, by MTT assay (Pf in Fig. ).
FIG. 2 is a graph showing the increase in expression of LIF mRNA according to the concentration of the self-lyophilized extract by RT-PCR.
FIG. 3 is a graph showing the results of western blot analysis for the increase in LIF protein expression according to the concentration of the self-leaf extract.
FIG. 4 is a graph showing the results of RT-PCR analysis of the increase in integrin αvβ3 and αvβ5 mRNA expression by the treatment of a child's leaf extract (hITGAV in FIG. 4 represents integrin αv, hITGB3 represents integrin β3, and hITGAB5 represents integrin β5 ).
Fig. 5 is a graph showing the results of confirming the increase in the adhesion of Ishikawa cells, which are endometrial cells derived from endothelial cells, and JAR cells, which are fetal cells derived from endothelial cells.
Fig. 6 is a graph showing the results of evaluating the degree of increase of adherence between Ishikawa cells, endometrial-derived cells, and JAR cells, fetal-derived trophoblast cells.

The present invention provides a composition for preventing or treating infertility comprising an extract of a natural lobule as an active ingredient.

In addition, the present invention provides a composition for promoting pregnancy comprising a self-leaf extract as an active ingredient.

In addition, the present invention provides a composition for enhancing the intrauterine implantability of an oocyte comprising a self-lobular extract as an active ingredient.

The composition comprises a pharmaceutical composition or a food composition.

Hereinafter, the present invention will be described in detail.

The term " extract " in the present invention means a preparation obtained by squeezing a herbal medicine with an appropriate leaching solution and concentrating by evaporating the leaching solution. The extract is obtained by extracting the diluted solution or concentrate of the extract, It may be a product or a purified product.

The self-lobular extract of the present invention includes not only an extract obtained by adding an extraction solvent to the true lobes but also a fraction obtained by adding a fraction solvent to a crude extract prepared by extracting with an extraction solvent.

For example, the active ingredient Perilla frutescens extract of the present invention is prepared by a separation and purification method comprising the following steps:

1) extracting the child lobules with an extraction solvent;

2) filtering the self-leaf extract of step 1);

3) Concentrating the filtered extract of step 2) under reduced pressure and drying.

In the above-mentioned preparation method, it may be used as it is, or may be dried and used as it is in step 1). As the drying method, it is possible to use any of the methods of biped, shade, hot air drying and natural drying. In order to increase the extraction efficiency, it may be pulverized by a self-lobing mill. The self-lyophilized leaf can be used without limitation such as cultivated or commercially available.

In the above production method, in step 1), an appropriate amount of extraction solvent is added to the true lobes so as to be completely immersed. As the extraction solvent, inorganic or organic solvents generally used in the art may be used without limitation, and for example, solvents selected from water, alcohols having 1 to 4 carbon atoms, or mixed solvents thereof are preferable, It is not. The amount of the extraction solvent may be 2 to 20 times, preferably 3 to 5 times, in order to increase the extraction efficiency. The extraction method may be performed at room temperature or warmed, preferably at 20 to 70 ° C for 1 to 10 days, by ultrasonic extraction or reflux-cooling extraction, but is not limited thereto. The number of extraction times is not limited to 1 to 5 times, preferably 1 to 3 times.

In the above production method, the filtration in step 2) is preferably performed using a paper filter or a gauze, but is not limited thereto.

In the above production method, it is preferable to use a vacuum decompression concentrator or a vacuum rotary evaporator for the decompression concentration in step 3), but the present invention is not limited thereto. The drying is preferably performed under reduced pressure, vacuum drying, boiling, spray drying or freeze drying, but is not limited thereto.

In addition, the production method may further include a step 4) further fractionating the concentrate of step 3) with an organic solvent.

In the above production method, the organic solvent in step 4) may be n-hexane, methylene chloride, chloroform, ethyl acetate, dichloromethane, butanol or a mixture thereof, but is not limited thereto.

In one embodiment of the present invention, a self-leaf extract is obtained through the following procedure. First, lobules are washed with water to remove foreign matters, and then dried and crushed in the shade. 100 g of the crushed white lobate powder was added to 1 L of distilled water and heated for 2 hours at 100 ° C by reflux cooling. The solution was filtered through a paper filter, and ethanol was added thereto at 70% by volume. It precipitates polysaccharides. After removing the polysaccharide by centrifugation, the supernatant is filtered, and the filtrate is concentrated under reduced pressure and dried to obtain an endogenous leaf extract.

The term " infertile " in the present invention means a state in which a couple who does not have contraception fails to reach the pregnancy within one year despite the normal marital relationship.

In the present invention, the term " pregnancy " refers to a process in which an embryo created by combining an oocyte and a sperm is implanted into the uterus and develops into a fetus. The normal pregnancy process proceeds in the order of fertilization, implantation and fetal development.

In the present invention, the term " implantation " refers to a method in which the embryo and sperm meet at the upper part of the oviduct and the fertilized egg is formed, and then the embryo is transferred to the uterus, State. At this time, blastocysts and endometrial epithelial cells are attached and adhered through various receptors on the cell surface, and then the nutrient penetrates into the endometrium and is implanted.

The term " infertility " of the present invention is preferably female infertility.

The female infertility can be classified into two categories: nonimplantation of ovum, female infertility 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. But are not limited to, female infertility of other origin and unspecified female infertility (unspecified) Preferably from an infertility due to failure of the egg implantation.

The above-mentioned "Nonimplantation of ovum" is a representative cause of female infertility. When fertilized, the embryo moves while dividing cells into the uterus, and when it reaches the stage of blastocyst, it is buried in the endometrium and seated. If the thickness of the endometrium is not enough, or if it is stiff due to injury, it is difficult to be implanted, and if the space is short due to adhesion of the uterine wall, it is often aborted. 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.

According to one embodiment of the present invention, the self-lyophilized extract has an activity of increasing the expression of leukemia inhibitory factor (LIF) and integrins alpha v beta 3 and alpha v beta 5, It has an excellent effect of promoting the binding and inducing the fertilization of the embryo effectively. Therefore, the baby leaf extract may be useful as a medicine and a health functional food for prevention or treatment of infertility, promotion of pregnancy and improvement of fertilization ability of oocyte in uterus.

Accordingly, the present invention provides a pharmaceutical composition for the prevention or treatment of infertility comprising an extract of a natural lobule as an active ingredient.

The pharmaceutical composition according to the present invention may further contain, in addition to the above-mentioned active ingredients, additional ingredients, that is, pharmaceutically acceptable or nutritionally acceptable carriers, excipients, diluents or subcomponents depending on the formulation, method of use and purpose of use .

More specifically, the composition may further contain, in addition to the active ingredient, a nutrient, a vitamin, an electrolyte, a flavoring agent, a coloring agent, a filler, a pectic acid and a salt thereof, an alginic acid and a salt thereof, an organic acid, a protective colloid thickening agent, Preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages, 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 silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, calcium carbonate, dextrin, propylene glycol, Paraffin, and physiological saline, but is not limited thereto. The components may be added to the active ingredient, i.e., a self-leaf extract, either independently or in combination.

The pharmaceutical composition of the present invention may be selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze- Or < / RTI >

The pharmaceutical composition of the present invention may be administered to the individual by various routes. For example, the compositions of the present invention may be administered by intravenous, intraperitoneal, intramuscular, intraarterial, oral, intracardiac, intramedullary, intradermal, transdermal, intestinal, subcutaneous, sublingual or topical administration.

The therapeutically effective dose of the pharmaceutical composition of the present invention will vary depending upon the species, body weight, age and individual condition of the subject, the disorder or disease being treated, or their severity. The daily dose of the composition of the present invention is 0.0001 to 1000 mg / kg, preferably 0.001 to 100 mg / kg, based on the amount of the self-leaf extract, and can be administered 1 to 6 times a day, And are not intended to limit the scope of the invention in any way.

In addition, the present invention provides a food composition for preventing or ameliorating infertility, which comprises a self-leaf extract as an active ingredient.

The food composition of the present invention can be provided as a food composition by mixing with a pharmaceutically acceptable carrier. The self-leaf extract is preferably contained in a weight ratio of 0.01 to 99.99% with respect to the total food composition, but is not limited thereto.

When the active ingredient of the present invention is used as a food or beverage additive, the self-leaf extract can be directly added or used together with other food or food ingredients, and can be suitably used according to a conventional method. The amount of the active ingredient to be mixed may be determined by suitably adjusting the amount of the active ingredient according to its intended use (prevention, health or therapeutic treatment).

For long-term consumption intended for health or hygiene purposes or for health control purposes, the food composition of the present invention has no problem in terms of safety and can be taken for a long period of time.

There is no particular limitation on the kind of the food. Examples of the food to which the above substances can be added include meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, drinks, tea, , Alcoholic beverages and vitamin complexes. When formulated into beverage, the liquid ingredient to be added in addition to the above-mentioned food composition is not limited, but may include various flavors or natural carbohydrates such as ordinary beverages as an additional ingredient. Examples of the above-mentioned natural carbohydrates include, but are not limited to, monosaccharides (e.g., glucose and fructose), disaccharides (e.g., maltose, sucrose, etc.), and polysaccharides (e.g., dextrin, cyclodextrin, Phosphorus), and sugar alcohols such as xylitol, sorbitol and erythritol. The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention. Natural flavors (taumarin, stevia extract) and synthetic flavorings (e.g., saccharin, aspartame, etc.) other than those described above can be used.

The food composition of the present invention can be used as a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, colorants and enhancers (cheese, chocolate etc.), pectic acid and salts thereof, PH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like. The food composition of the present invention may also contain pulp for the production of fruit and vegetable drinks. These components may be used singly or in combination, and the proportion of such additives is generally selected in the range of 0.001 to 50 parts by weight based on the total weight of the composition.

In addition, the present invention provides a pharmaceutical composition for promoting pregnancy, comprising a self-leaf extract as an active ingredient.

In addition, the present invention provides a food composition for promoting pregnancy, comprising a self-leaf extract as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for enhancing the intrauterine implantability of an oocyte comprising an extract of a natural lobule as an active ingredient.

In addition, the present invention provides a food composition for enhancing the intrauterine implantability of an oocyte comprising a self-lobular extract as an active ingredient.

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

Hereinafter, the present invention will be described in detail with reference to Examples, Experimental Examples and Preparation Examples.

However, the following Examples, Experimental Examples and Preparation Examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following Examples, Experimental Examples and Preparation Examples.

Example 1: Perilla frutescens  (L.)) extract

The baby leaflets are washed with water to remove foreign matter, then dried and shredded in the shade. 100 g of the crushed white lobate powder was added to 1 L of distilled water and heated for 2 hours at 100 ° C by reflux cooling. The solution was filtered through a paper filter, and ethanol was added at 70% To precipitate the polysaccharide. After removing the polysaccharide by centrifugation, the supernatant is filtered, and the filtrate is concentrated under reduced pressure and dried to obtain an endogenous leaf extract.

EXPERIMENTAL EXAMPLE 1: Toxicity test of the self-leaf extract

In order to confirm the toxicity of the endothelial cell extract obtained in Example 1, the endothelial cell extract was applied to Ishikawa cells, which are the endometrial cells derived from human endometrium, at 0, 10, 30, 50, 100, 500 mu] m and the viability of Ishikawa cells was measured by a conventionally known MTT assay method. The viable cells were expressed as percentage compared to the control group, and the results are shown in Fig.

As shown in Fig. 1, 90% to 100% of the Ishikawa cells survived even when the extract was treated to a concentration of 50 [mu] M, confirming that the self-leaf extract had no significant cytotoxicity. In the following experiments, concentrations of 50 μM or less without toxicity were used.

Experimental Example 2: Expression of leukemia inhibitory factor (LIF) mRNA according to the concentration of self-leaf extract

In order to confirm the expression level of LIF mRNA according to the treatment concentration of the self-leaf extract obtained in Example 1, the self-leaf extract was treated with Ishikawa cells at the concentrations of 0, 10, 30 and 50 μM, respectively Cells were cultured for 24 hours. RNA was extracted from the cultured cells and subjected to denaturation at 95 ° C for 30 seconds using an LIF specific primer, annealing at 60 ° C for 30 seconds, extension at 72 ° C . The amount of LIF mRNA expression was confirmed by RT-PCR, which was repeated 35 times for 30 seconds. The sequence of primers used for PCR amplification for confirmation of LIF expression is 5'-ACGCCACCTGTGCCATACGC-3 'in the forward direction and 5'-GATGTCGGCGGTGGCGTTGA-3' in the reverse direction. The DNA amplified by the RT-PCR was electrophoresed and photographed under ultraviolet light (UV), and then the intensity of the band was measured using densitometry. The expression level of LIF mRNA and the ratio of β-actin mRNA expression level as a control standard were confirmed, and the results are shown in FIG. The primers used for the PCR amplification were 5'-CAAGAGATGGCCACGGCTGCT-3 'in the forward direction and 5'-TCCTTCTGCATCCTGTCGGCA-3' in the reverse direction for confirmation of expression of the β-actin mRNA used as the control standard.

As shown in Fig. 2, the expression of LIF mRNA in Ishikawa cells was found to be increased depending on the concentration of the self-leaf extract.

Experimental Example 3: Comparison of LIF protein expression level according to the concentration of the self-leaf extract

Western blotting was performed according to a conventionally known method in order to confirm the expression of LIF protein according to the treatment concentration of the self-leaf extract obtained in Example 1 above. First, the extracts of Liliaceae were treated with Ishikawa cells at concentrations of 0, 10, 30, and 50 μM, respectively, and cultured for 24 hours. Proteins were then extracted from the cells. The extracted proteins were separated by size by SDS-PAGE. In order to analyze the amount of LIF protein expression, the separated protein was electrophoretically transferred to a nitrocellulose membrane. Then, the separated protein was first bound to an antibody specific for LIF (Santa Cruz), and the second , Followed by secondary immunization with HRP, followed by sensitization with ECL solution to analyze the amount of LIF protein expressed. The intensity of the band was measured using Densitomycetes, and the expression level of the LIF protein and the ratio of the expression level of the GAPDH protein as a control standard were confirmed, and the results are shown in FIG.

As shown in Fig. 3, it was confirmed that the expression of LIF protein in Ishikawa cells was increased in a concentration dependent manner of the self-leaf extract.

Experimental Example 4: Expression of Integrin αvβ3 and αvβ5 mRNA Expression According to Concentration of Mulberry Leaf Extract

In order to confirm the expression level of integrin αvβ3 and αvβ5 mRNA according to the treatment concentration of the self-leaf extract obtained in Example 1, the self-leaf extract was added to Ishikawa cells at concentrations of 0, 10, 30 and 50 μM And the cells were cultured for 24 hours. RNA was extracted from the cultured cells and subjected to denaturation at 95 ° C for 30 seconds, annealing at 60 ° C for 30 seconds, and extension using a primer specific for integrin αvβ3 and αvβ5 RT-PCR was repeated 30 times at 72 ° C for 30 seconds to confirm the expression levels of integrin αvβ3 and αvβ5 mRNAs. The sequences of the primers used for the PCR amplification for confirming expression of the integrin alpha v mRNA are 5'-ATGCTCCATGTAGATCACAAGAT-3 'in the forward direction and 5'-TTCCCAAAGTCCTTGCTGCT-3' in the reverse direction. In order to confirm the expression of the integrin beta 3 mRNA, the sequence of primers used for PCR amplification was 5'-CTGCCGTGACGAGATTGAGT-3 'in the forward direction and 5'-TGCCCCGGTACGTGATATTG-3' in the reverse direction. The sequences of the primers used for the PCR amplification for confirming the expression of the integrin [beta] 5 mRNA were 5'-ACCTGGAACAACGGTGGAGA-3 'in the forward direction and 5'-AAAAGATGCCGTGTCCCCAA-3' in the reverse direction. The DNA amplified by the RT-PCR was electrophoresed and photographed under ultraviolet light (UV), and then the intensity of the band was measured using densitometry. The expression levels of integrin [alpha] v [beta] 3 and [alpha] v [beta] 5 mRNA and the ratio of expression amount of [beta] -actin mRNA as a control standard were confirmed and the results are shown in Fig. The primer sequence used for confirmation of β-actin mRNA expression used as the control standard is 5'-CAAGAGATGGCCACGGCTGCT-3 'in the forward direction and 5'-TCCTTCTGCATCCTGTCGGCA-3' in the reverse direction.

As shown in Fig. 4, it was confirmed that integrin αvβ3 mRNA and αvβ5 mRNA expression of Ishikawa cells were increased in a concentration dependent manner of the self-leaf extract.

Experimental Example 5: Confirmation of adherence of JAR cell to Ishikawa cells treated with JAEA extract

In order to confirm the adherence of the embryo-derived trophoblast-derived Ishikawa cells to the endometrial-derived Ishikawa cells obtained in Example 1, 80% confluence of Ishikawa cells, a human endometrium-derived cell, . After culturing the cells in a single layer, the cell lobule extract of 50 μM / ml obtained in Example 1 was treated. Thereafter, the cells were cultured for 72 hours so that Ishikawa cells became 100% clusters. Then, pre-cultured embryo-derived cells were detached from the culture dish and suspended well so that the Ishikawa cells and the sperm cells became single cells. The nuclei were fluorescently stained with DAPI (4 ', 6-diamidino-2-phenylindole), sprayed onto a single layer of Ishikawa cells, and incubated at 37 ° C for 30 minutes in an incubator Lt; / RTI > The unattached cells were then washed twice with phosphate buffered saline (PBS) and the adhered cells were fixed with a 3.7% formalin solution. Fixed cells were photographed by fluorescence microscopy and the results are shown in FIG. The number of sperm cells attached to the Ishikawa cells at the bottom of the fluorescence by DAPI was then measured and the total number of cells counted in photographs of 5 or more different areas was compared with the untreated control group, The mean and standard deviation of the repeated values are shown in FIG.

As shown in Figs. 5 and 6, it was confirmed that the self-cell adhesion to Ishikawa cells was significantly increased about twice as much as that of the control group when treated with the self-lobular extract.

From these results, it can be concluded that the expression of LIF and integrin αvβ3 and αvβ5, which are key cytokines involved in embryo implantation, is promoted by using the self-lyophilized extract and the binding of endometrial cells to fetal-derived nutrient is promoted. It has been confirmed that a lobule extract can be used as a composition for preventing or treating infertility, a composition for promoting pregnancy, and a composition for enhancing the fertilization ability of oocytes in uterus.

Hereinafter, the pharmaceutical composition of the present invention and the preparation example of the food composition will be described, but the present invention is not intended to be limited but is specifically described.

Formulation Example 1. Preparation of a pharmaceutical composition

1-1. Manufacture of Powder

Lobster extract 20 mg

Lactose 100 mg

Talc 10 mg

The above components are mixed and filled in airtight bags to prepare powders.

1-2. Manufacture of tablets

Lobster extract 10 mg

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

After mixing the above components, tablets are prepared by tableting according to the usual preparation method of tablets.

1-3. Preparation of capsules

Lobster extract 10 mg

Crystalline cellulose 3 mg

Lactose 14.8 mg

Magnesium stearate 0.2 mg

The above components are mixed according to a conventional capsule preparation method and filled in gelatin capsules to prepare capsules.

1-4. Injection preparation

Lobster extract 10 mg

180 mg mannitol

Sterile sterilized water for injection 2974 mg

Na ₂ HPO ₄ 2H ₂ O 26 mg

(2 ml) per 1 ampoule in accordance with the usual injection preparation method.

1-5. Manufacture of liquid agent

Lobster extract 20 mg

10 g per isomer

5 g mannitol

Purified water quantity

Each component was added and dissolved in purified water according to the usual liquid preparation method, and the lemon flavor was added in an appropriate amount. Then, the above components were mixed, and purified water was added thereto. The whole was added with purified water to adjust the total volume to 100 ml, And sterilized to prepare a liquid preparation.

Formulation Example 2. Preparation of Food Composition

2-1. Manufacture of health food

Lobster extract 20 mg

Vitamin mixture quantity

Vitamin A acetate 70 μg

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

0.15 mg of vitamin B2

Vitamin B6 0.5 mg

Vitamin B12 0.2 μg

Vitamin C 10 mg

Biotin 10 μg

Nicotinic acid amide 1.7 mg

Folic acid 50 μg

Calcium pantothenate 0.5 mg

Mineral mixture quantity

1.75 mg of ferrous sulfate

0.82 mg of zinc oxide

Magnesium carbonate 25.3 mg

Potassium monophosphate 15 mg

Secondary calcium phosphate 55 mg

Potassium citrate 90 mg

Calcium carbonate 100 mg

Magnesium chloride 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with a composition suitable for health food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional method for producing healthy foods , Granules can be prepared and used in the manufacture of health food compositions according to conventional methods.

2-2. Manufacture of health drinks

Leaf lily extract 100 mg

Vitamin C 15 g

Vitamin E (powder) 100 g

19.75 g of ferrous lactate

3.5 g of zinc oxide

Nicotinic acid amide 3.5 g

Vitamin A 0.2 g

Vitamin B1 0.25 g

Vitamin B2 0.3 g

Water quantification

The above components were mixed according to a conventional health drink manufacturing method, and the mixture was stirred and heated at 85 DEG C for about 1 hour. The solution thus prepared was filtered and sterilized in a sterilized 2 L container, It is used in the production of the health beverage composition of the invention.

Although the compositional ratio is relatively mixed with a component suitable for a favorite drink, it is also possible to arbitrarily modify the compounding ratio according to the regional or national preference such as the demand class, the demanding country, and the use purpose.

Claims (9)

A pharmaceutical composition for preventing or treating female infertility comprising an extract of Perilla frutescens (L.) as an active ingredient.
delete 2. The pharmaceutical composition according to claim 1, wherein the female infertility is caused by an implantation disorder.
The pharmaceutical composition according to claim 1, wherein the Perilla frutescens (L.) extract is extracted with at least one solvent selected from the group consisting of water, alcohols having 1 to 4 carbon atoms, and mixtures thereof.
(Perilla frutescens (L.)) extract as an active ingredient.
A pharmaceutical composition for promoting pregnancy comprising an extract of Perilla frutescens (L.) as an active ingredient.
A composition for promoting pregnancy comprising an extract of Perilla frutescens (L.) as an active ingredient.
A pharmaceutical composition for enhancing the intrauterine implantability of an oocyte comprising Perilla frutescens (L.) extract as an active ingredient.
The present invention relates to a food composition for promoting in vitro fertilization of an oocyte comprising Perilla frutescens (L.) extract as an active ingredient.

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