KR20160108690A - Composition containing benzoic acid for improving pregnancy - Google Patents

Abstract

Provided in the present invention is a composition for promoting pregnancy, comprising benzoic acid as an active ingredient, or a composition for preventing or treating female infertility. The benzoic acid of the present invention increases the expression of LIF related to the implantation in endometrial cells and increase the expression of integrin V3 and V5 as an adhesive molecule related to water solubility of the endometrium, thereby increasing binding force of the endometrial cells with embryo-derived trophoblast cells and having excellent effects in introducing the implantation of a fertilized egg in an effective manner. The composition can be efficiently used in a field related to a functional food and drugs, which can prevent and treat the female infertility including infertility caused by non-implantation of the fertilized egg.

Description

[0001] The present invention relates to a composition for promoting pregnancy comprising benzoic acid as an active ingredient,

The present invention provides a composition for promoting pregnancy or a composition for preventing or treating infertility, which comprises benzoic acid as an active ingredient.

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. Normal pregnancy progresses in the order of fertilization, implantation and fetal development. Quality of fertilized eggs and receptivity of endometrium are very important factors in embryo implantation. The major factors controlling the endometrium's water solubility include growth factors such as cytokine such as leukemia inhibitory factor (LIF), insulin-like growth factor-2 and interleukin-11 (IL-11) is important (Hum Rep Update, 12 (6): 731-746). Among them, the expression of LIF, an interleukin-6 (IL-6) family cytokine, is known to be a key factor controlling the implantation process. In particular, in mice lacking LIF, there are reports of defects in the adhesion process between the embryo and the endometrium, which do not result in implantation and pregnancy (Nature 359 (6390): 76-79; Endocrinology 150 6): 2915-2923). In addition, it has been reported that inhibition of LIF function by using an antagonist for LIF does not result in implantation (PNAS 104 (49): 19357-19362). Such LIF-like cytokines are known to increase the expression of various adhesion factors such as mucin, integrin, carderine and CD44 in the endometrium, and the expression of these adhesion factors directly regulates the water receptivity of the endometrium (BJOG, 109: 610-617). Especially, expression of integrin [alpha] v [beta] 3 and [alpha] v [beta] 5 has been reported to be very important for implantation of embryos (Mol Hum Reprod. 2 (7): 527-534).

Benzoic acid is a simple aromatic carboxylic acid. It is a colorless crystalline compound. It is naturally contained in many kinds of plants and is used as a raw material for biosynthesis of various secondary metabolites in plants Biochem J. 48 (4): 422-425; Mol Plant. Pii: ssu126). The benzoic acid has an antifungal effect and an antioxidative effect and is used as a food preservative and skin fungus treatment agent and is also used as a material of various cosmetics such as a bath agent, a cleaner and a suntan product (Int J Toxicol. 20 (S3): 23-50 ). However, there has been a concern about the toxicity of benzoic acid, but recent studies have shown that unlike other derivatives of benzoic acid, benzoic acid itself has no adverse effects such as oral toxicity, reproductive toxicity, and carcinogenicity Int J Toxicol 20 (S3): 23-50).

Therefore, the inventors of the present invention have found that when benzoin acid induces fertilization of embryos and is effective in promoting pregnancy, it increases the expression of LIF, a key cytokine related to the implantation in endometrial cells, The present invention has been accomplished by confirming that the present invention is effective in promoting pregnancy and infertility caused by non-implantation of oocytes by increasing binding between endometrial cells and embryo-derived trophoblast cells.

It is an object of the present invention to provide a composition for promoting pregnancy comprising benzoic acid as an active ingredient.

It is also an object of the present invention to provide a composition for preventing or treating infertility comprising benzoic acid as an active ingredient.

In order to solve the above-mentioned problems, the present invention provides a pharmaceutical composition for promoting pregnancy comprising benzoic acid as an active ingredient.

The present invention also provides a food composition for promoting pregnancy comprising benzoic acid as an effective ingredient.

The present invention also provides a pharmaceutical composition for preventing or treating infertility comprising benzoic acid as an active ingredient.

The present invention also provides a food composition for preventing or ameliorating infertility comprising benzoic acid as an active ingredient.

The benzoic acid of the present invention increases the expression of LIF associated with the implantation in endometrial cells and increases the expression of integrin alpha v beta 3 and alpha v beta 5, which are adhesion molecules involved in endometrial water receptivity, so that the binding strength between endometrial cells and fetal- And induce the implantation of fertilized eggs effectively. Therefore, the benzoic acid according to the present invention can be usefully used in fields related to medicines for the prevention and treatment of female infertility including ovarian non-implantation infertility, and functional foods.

FIG. 1 is a graph showing the results of confirming cytotoxicity according to the treatment concentration of benzoic acid. FIG.
FIG. 2 is a graph showing the results of RT-PCR for the increase in the expression of LIF mRNA according to the concentration of benzoic acid treatment.
FIG. 3 is a graph showing the results of western blot analysis for the increase in the expression of LIF protein according to the treatment concentration of benzoic acid.
Fig. 4 is a graph showing the results of RT-PCR for the expression of integrin [alpha] v [beta] 3 and [alpha] v [beta] 5 upon treatment with benzoic acid.
Fig. 5 is a graph showing the results of fluorescence microscopy for adhesion of sperm cells in Ishikawa cells treated with benzoic acid and in the untreated control group. Fig.
Fig. 6 is a graph showing the results of counting the number of adhesion of sperm cells in Ishikawa cells treated with benzoic acid and in the control group without treatment. Fig.
FIG. 7 is a photograph showing the results of hysterectomy to confirm the pregnancy rate of the control group (A), the contraceptive (RU486) (B) treated group, the control group treated with the contraceptive pill and the group administered with orally administered benzoic acid Fig.
FIG. 8 is a chart showing the results of counting the number of mouse embryos in the positive control group, the negative control group treated with the contraceptive pill, the contraceptive treatment group and the group administered orally with benzoic acid for a certain period.

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

The composition comprises a pharmaceutical composition or a food composition.

In the present invention, the above-mentioned "Benzoic acid" is a substance having the formula C ₆ H ₅ COOH and having a molecular weight of 122.13, represented by the following formula (1).

[Chemical Formula 1]

The benzoic acid is a colorless crystal, which is difficult to melt in cold water but melts well in hot water. It dissolves well in organic solvents such as acetone, ethanol and ether, and has a characteristic of being insoluble in petroleum ether. Benzoic acid produces various metals and salts, making alcohols and esters.

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 benzoic acid has an activity of increasing the expression of leukemia inhibitory factor (LIF) and increasing the expression of integrin? V? 3 and? V? 5, It has an excellent effect of increasing the binding force of trophoblast and effectively inducing fertilization of embryos.

The composition may contain a benzoic acid alone or a substance effective for promoting pregnancy as an effective ingredient. In addition to the above-mentioned effective ingredients, the composition may contain additional ingredients, that is, pharmaceutically acceptable Or a nutritional acceptable carrier, excipient, diluent or subcomponent.

More specifically, the pharmaceutical composition for promoting pregnancy comprising the above-mentioned benzoic acid may further contain, in addition to the above-mentioned active ingredients, a nutritional supplement, a vitamin, an electrolyte, a flavoring agent, a colorant, a thickening agent, a pectic acid and a salt thereof, Preservatives, colloidal thickeners, pH adjusting agents, stabilizers, antiseptics, 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., benzoic acid, either independently or in combination.

The composition according to the present invention can be used alone 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 the benzoic acid of the present invention is 0.01 to 10000 mg / kg, preferably 1 to 20 mg / kg, and is preferably administered once to three times a day.

The present invention also provides a food composition for promoting pregnancy comprising benzoic acid as an effective ingredient.

The composition according to the present invention may be included in a health food for the purpose of promoting pregnancy. When the active ingredient of the present invention is used as a food additive, the composition isolated from the synthesis or the extract may be added as it is, 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, , Alcoholic beverages and vitamin complexes, and includes all health 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.

The present invention also provides a composition for preventing or treating infertility comprising benzoic acid as an active ingredient.

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 a pharmaceutical preparation or a food preparation containing the above-mentioned benzoic acid as an active ingredient, the contents overlapping with the composition of the present invention described above can be applied to the composition of the present invention Its description is omitted in order to avoid excessive complexity.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention as defined by the appended claims. It will be obvious to you.

Example  One. Benzoic  Cytotoxicity Assessment

To confirm the toxicity of benzoic acid to cells by MTT (3- (4,5-dimethylthiazole-2-yl) -2,5-diphenyltetrazoli-umbromide) assay, the following experiment was conducted.

More specifically, the endometrial cell line, Ishikawa cell, was treated with benzoic acid at 0, 1, 50, 100 and 500 μM concentration. The MTT solution (2 mg / ml) was reacted at 37 ° C for 4 hours in a CO ₂ incubator. After removing the supernatant, the viable cells were reduced with intracellular redox enzyme and the violet dye (Formazan) was dissolved in DMSO. And measured with a microplate reader at a wavelength of 540 nm. Live cells were expressed as percentage compared to the control group. The results are shown in Fig.

As shown in FIG. 1, it was confirmed that no significant cytotoxicity was confirmed even when benzoic acid was treated to a concentration of 500 μM. Therefore, the concentrations below 50 μM without toxicity were used in the experiments below.

Example  2. Benzoic  Leukemia inhibitory factor (leukemia inhibitory factor; LIF ) mRNA Confirmation of expression of

In order to confirm the expression level of LIF mRNA according to the treatment concentration of benzoic acid by RT-PCR, the following experiment was conducted.

More specifically, Ishikawa cells were treated with benzoic acid at concentrations of 0, 10, 30 and 50 μM and cultured for 24 hours. Then, the RNAs of the cells treated at the respective concentrations were extracted and primers specific for LIF were used. The sequence of the primer was 5'-ACGCCACCTGTGCCATACGC-3 '(SEQ ID NO: 1) in the forward direction and 5'-GATGTCGGCGGTTGGCGTTGA-3' (SEQ ID NO: 2) in the reverse direction and the primer sequence used for the amplification of β- 3 '(SEQ ID NO: 3) and 5'-TCCTTCTGCATCCTGTCGGCA-3' (SEQ ID NO: 4) in the reverse direction. CDNA was synthesized by reacting with 1 μg total RNA, oligo-dT primer and M-MLV reverse transcriptase, which were the same amount of each sample, at 42 ° C for 1 hour. The cDNA was amplified by PCR using 30 cycles of denaturation at 95 ° C for 30 sec, annealing at 60 ° C for 30 sec and extension at 72 ° C for 30 sec. And the amount of mRNA expression was confirmed by polymerase chain reation. The amplified DNA was electrophoresed and photographed under ultraviolet light (UV), and then the intensity of the band was measured using densitometry. In addition, the expression level of LIF mRNA and the ratio of β-actin mRNA expression level as a control standard were confirmed.

As shown in FIG. 2, when the endometrial cells were treated with benzoic acid at a concentration of 50 μM, the expression of LIF mRNA was 2.40 higher than that of the control.

Therefore, it was confirmed that the expression of LIF mRNA was increased depending on the concentration of benzoic acid .

Example  3. Benzoic  Depending on treatment concentration LIF  Identification of Protein Expression

In order to confirm the expression of LIF protein according to the treatment concentration of benzoic acid by Western blotting, the following experiment was conducted.

More specifically, Ishikawa cells were treated with benzoic acid at a concentration of 0, 10, 30 and 50 μM and cultured for 24 hours. Proteins were extracted from the cells and electrophoresed by SDS-PAGE. Subsequently, the separated proteins were transferred to a nitrocellulose membrane by electrophoresis and then sequentially bound to LIF-specific antibodies (SantaCruz) and secondary antibodies to which HRP (Horseradish peroxidase) had been added ECL (enhanced chemiluminescence) solution. The intensity of the band was measured using Densitomycetes and the ratio of the expression level of LIF protein and the expression level of GAPDH protein as a control standard was confirmed. The results are shown in Fig.

As shown in FIG. 3, when the endometrial cells were treated with benzoic acid at 10, 30 and 50 μM, the amount of LIF protein was increased to about 1.20, 2.18 and 3.78, respectively, as compared with the control.

Therefore, it was confirmed that the expression of LIF protein was increased depending on the concentration of benzoic acid.

Example  4. Benzoic  Depending on the treatment Integrin  Increased expression of? V? 3 and? V? 5

The following experiment was carried out to confirm whether the expression of integrin [alpha] v [beta] 3 and [alpha] v [beta] 5, which are adhesion factors that regulate the water receptivity of endometrium in Ishikawa cells according to treatment with benzoic acid, is increased by RT-PCR.

More specifically, the same method as in Example 3 was used. In order to confirm the expression of integrin alpha V, 5'-ATGCTCCATGTAGATCACAAGAT-3 '(SEQ ID NO: 5) was used as the primer used for the RT- -TTCCCAAAGTCCTTGCTGCT-3 '(SEQ ID NO: 6). The primer sequence used for confirming the expression of integrin? 3 was 5'-CTGCCGTGACGAGATTGAGT-3 '(SEQ ID NO: 7) and the reverse direction was 5'-TGCCCCGGTACGTGATATTG-3' (SEQ ID NO: 8) (SEQ ID NO: 9), 5'-AAAAGATGCCGTGTCCCCAA-3 '(SEQ ID NO: 10) in the reverse direction and 5'-CAAGAGATGGCCACGGCTGCT-3' No. 3) and 5'-TCCTTCTGCATCCTGTCGGCA-3 '(SEQ ID NO: 4) in the reverse direction. The results are shown in Fig.

As shown in Fig. 4, it was confirmed that mRNA of integrin [alpha] v [beta] 3 and integrin [alpha] v [beta] 5 was expressed in Ishikawa cells treated with benzoic acid, and it was confirmed that treatment with benzoic acid can regulate the water solubility of endometrium.

Example  5. Benzoic acid  Of the JAR cells to the treated cells Attachment  Confirm

The following experiment was carried out in order to confirm the adherence ability of the fetal kidney cell line to the endocardial Ishikawa cell line treated with benzoic acid.

More specifically, Ishikawa cells, a human endometrial cell line derived from human endometrium, were cultured in a single layer so as to have an 80% confluence, followed by treatment with benzoic acid to a concentration of 50 μM, followed by culturing for 72 hours to obtain 100% Respectively. Then, the pre-cultured fetal-derived cell line was detached from the culture dish, and then suspended well to obtain a single cell. Using 4 ', 6-diamidino-2-phenylindole (DAPI), which is a fluorescent substance for dyeing DNA, The nuclei were stained. The cells were then sprinkled on a single layer of Ishikawa cells and cultured in an incubator at 37 ° C for 30 minutes with stirring at a speed of about 60 rpm. The cultured cells were washed twice with phosphate buffered saline (PBS) to remove unattached cells, and the adhered cells were fixed with 3.7% formalin solution. When confirmed by fluorescence microscopy, the number of cells that fluoresce by DAPI is the number of spermatocytes attached to the Ishikawa cells at the bottom. Thereafter, the total number of cells counted in photographs of five or more different regions was compared with a control group not treated with benzoic acid, and then the mean and standard deviation of the values repeated three times were determined (FIG. 6). The results are shown in Fig. 5 and Fig.

As shown in Fig. 5 and Fig. 6, it was confirmed that the adhesion capacity with sperm cells was about three times higher than that of the control group not treated with Ishikawa cells treated with benzoic acid.

Example  6. Benzoic acid  Identification of increased implantation sites from orally administered rats

In order to confirm whether or not the intrauterine implantation site increased and the number of fetuses in the rats increased, the following experiment was conducted.

More specifically, 7-8 week old mice of C57BL / 6 were purchased and fed with general diet and water for 12 hours in a light and dark cycle, and were kept for a week. Twenty-four female rats were randomly divided into three groups (positive control, negative control with contraceptives, contraceptive treatment and benzoic acid). In the group administered with benzoic acid, 100 μl of 50 μg of benzoic acid per mouse (20 g) was orally administered, and the same amount of PBS was orally administered to the positive control and the negative control. After 7 days, the male and female pairs were mated for a week at a ratio of (female: male = 2: 1). After 4 days of mating, 0.08mg / 0.1ml of the contraceptive was injected into the abdominal cavity of each group in the negative control and the benzoin phosphate group, and the positive control group injected the same amount of cone oil as the contraceptive drug into the abdominal cavity. Seven days after the contraceptive treatment, both uterine corners were cut out and counted (Fig. 7) in order to determine the number of implantation sites in the rats by the number of fetal rats. Then, the mean and standard deviation of the number of fetuses counted in 8 rats of each group were determined (Fig. 8). The results are shown in Fig. 7 and Fig.

As shown in FIGS. 7 and 8, the oral administration group of benzoic acid showed that the number of injected mouse embryos was lower than that of the positive control group but higher than that of the control group fed with the birth control pills. This confirms that benzoic acid induces implantation of embryos in rats even after treatment with birth control pills.

Therefore, when benzoic acid is used, the expression of LIF, a key cytokine related to the implantation, is increased in endometrial cells, and the expression of integrin αVβ3 and αVβ5, which are adhesion molecules involved in endometrial water receptivity, is increased. The results showed that the binding of endometrial cells to embryo-derived trophoblast cells was increased, and that the implantation of the embryos in rats was effectively induced.

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 pharmaceutical compositions

1-1. Sanje  Produce

Benzoic acid 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

Benzoic acid 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

Benzoic acid 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

Benzoic acid 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. Liquid  Produce

Benzoic acid 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. Health food manufacturing

100 mg of a vitamin mixture, 70 g of vitamin A acetate, 1.0 mg of vitamin E, 0.13 mg of vitamin B1, 0.15 mg of vitamin B2, 0.5 mg of vitamin B6, 0.2 g of vitamin B12, 10 mg of vitamin C, 10 g of biotin, 50 mg of folic acid, 0.5 mg of calcium pantothenate, a suitable amount of inorganic mixture, 1.75 mg of ferrous sulfate, 0.82 mg of zinc oxide, 25.3 mg of magnesium carbonate, 15 mg of potassium phosphate monobasic, 55 mg of calcium phosphate dibasic, 100 mg of calcium carbonate and 24.8 mg of magnesium chloride were mixed and granules were prepared and a health food was prepared according to a conventional method. At this time, although the composition ratio of the vitamin and mineral mixture is relatively mixed with the ingredient suitable for health food, it may be arbitrarily modified.

2-2. Health drink manufacturing

According to a conventional method for producing healthy beverages, 100 mg of benzoic acid, 15 g of vitamin C, 100 g of vitamin E (powder), 19.75 g of iron lactate, 3.5 g of zinc oxide, 3.5 g of nicotinic acid amide, 0.2 g of vitamin A, B2 was mixed with a predetermined amount of water, and the mixture was heated at 85 ° C for about 1 hour. The resulting solution was filtered to obtain a sterilized 2L container, sealed sterilized, and stored in a refrigerator to prepare a health drink. At this time, although the composition ratio of the ingredients suitable for the beverage is comparatively mixed, the mixture ratio may be arbitrarily varied according to the demand, the demanded country, the intended use, and the regional or national preference.

<110> Pusan National University Industry-University Cooperation Foundation <120> Composition containing benzoic acid for improving pregnancy <130> 1-214 <160> 10 <170> Kopatentin 2.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LIF forward primer <400> 1 acgccacctg tgccatacgc 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LIF reverse primer <400> 2 gatgtcggcg gtggcgttga 20 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> beta-actin forward primer <400> 3 caagagatgg ccacggctgc t 21 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> beta-actin reverse primer <400> 4 tccttctgca tcctgtcggc a 21 <210> 5 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> integrin-alpha-V forward primer <400> 5 atgctccatg tagatcacaa gat 23 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> integrin-alpha-V reverse primer <400> 6 ttcccaaagt ccttgctgct 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> beta 3 forward primer <400> 7 ctgccgtgac gagattgagt 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> beta 3 reverse primer <400> 8 tgccccggta cgtgatattg 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> beta 5 forward primer <400> 9 acctggaaca acggtggaga 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> beta 5 reverse primer <400> 10 aaaagatgcc gtgtccccaa 20

Claims (8)

A pharmaceutical composition for promoting pregnancy comprising benzoic acid as an active ingredient. The method according to claim 1,
Wherein the benzoic acid enhances the expression of a leukemia inhibitory factor (LIF). The method according to claim 1,
Wherein said benzoic acid increases expression of intracellular integrin [alpha] V [beta] 3 and [alpha] V [beta] 5. Benzoic acid as an active ingredient. A pharmaceutical composition for preventing or treating infertility comprising benzoic acid as an active ingredient. 6. The method of claim 5,
Wherein said infertility is female infertility. &Lt; Desc / Clms Page number 24 &gt; The method according to claim 6,
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 unspecified, unspecified female infertility, and unspecified female infertility. A pharmaceutical composition for preventing or treating infertility, characterized in that the infertility. A food composition for preventing or ameliorating infertility comprising benzoic acid as an active ingredient.

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