KR101093009B1 - Composition for preventing and relieving of menopausal symptoms comprising glyceollin - Google Patents

Abstract

The present invention relates to a composition for preventing and improving menopausal symptoms containing glycerol as an active ingredient. More specifically, the present invention relates to a composition for the prevention and improvement of menopausal symptoms containing glycerol extracted from edible fungal inoculated beans as an active ingredient. Glycerin compound according to the present invention is excellent in antioxidant activity and has an estrogen-like activity to prevent and improve menopausal symptoms caused by aging and estrogen deficiency, and because there are no side effects, hormone replacement therapy for the treatment of menopausal symptoms and It can be used as a material for health functional food.

Glycerin, Menopause, Edible Fungus, Antioxidant, Estrogen

Description

Composition for preventing and improving menopausal symptoms comprising glycerin as an active ingredient

The present invention relates to a composition for preventing and improving menopausal symptoms containing glycerol as an active ingredient. More specifically, the present invention relates to a composition for the prevention and improvement of menopausal symptoms containing glycerol extracted from edible fungal inoculated beans as an active ingredient.

In the modernized industrialized life through the development of science and civilization, human beings are gradually prolonged in life, and at the same time, immunity is lowered and aging progresses due to various stresses, pollution and naturally occurring aging phenomena. In particular, women suffer from physical and mental difficulties due to the menopausal symptoms that begin slowly after their 40s.

On the other hand, menopausal is a type of endocrine syndrome refers to a transition period in which the physiological function and sexual function is reduced or lost due to a decrease in female hormone, ie, estrogen, due to the gradual aging of ovarian function. Symptoms of menopausal symptoms include vascular changes such as hot flashes, tachycardia, sweating or headaches, symptoms of musculoskeletal changes such as myalgia, arthralgia and back pain, and symptoms of urogenital changes such as urinary or urinary incontinence. In addition, there are symptoms caused by changes in the brain's nervous system such as memory loss, depression, concentration decline, and dizziness. In addition, symptoms such as decreased vision and changes in skin and hair occur, and women such as osteoporosis or cardiovascular diseases caused by hormonal changes. It can also cause fatal diseases.

Therefore, in order to improve the physical and mental health and quality of life of middle-aged women, development of treatments that can improve menopausal symptoms was required, and drugs such as hormone replacement therapy and nonsteroidal preparations were developed to improve these menopausal symptoms. It has been. However, most of these drugs are known to have side effects such as headache and weight gain. In particular, estrogen replacement therapy artificially administers hormones to the body, and therefore, rejection of uterine bleeding, stroke, and heart. It is known that the risk of developing seizures, breast and uterine cancers may be increased (Swaran L., et. Al., Obstetrics & Gynecology , 91, 678-684, 1998).

Therefore, due to these problems, there is a growing interest in replacing estrogen therapy with a natural method of ingesting it in the form of foods or additives, and it is required to develop a new menopausal treatment agent that is excellent in alleviating the symptoms of menopausal without side effects. There is a situation.

On the other hand, phytoestrogens collectively refer to various kinds of compounds existing in plants having estrogen activity, and have two phenol rings as common skeletons of chemical structures. Phytoestrogens can be broadly divided into three types: isoflavones, coumestans, and lignans. Among them, isoflavones are biochanin A and formononetin in plants. It exists as an inactive glycoside of human body, and when absorbed into human body, it is converted into genistein and daidzein, which have a structure similar to estrogen by glucosidase, respectively, to show estrogen-like activity. do. Plants containing phytoestrogens include soybean processed foods including soybeans (soy milk, tofu, miso, etc.), various cereals, fruits, and clover, and isoflavones are mainly used in legumes and lignans. It is known that is mainly contained in cereals.

Glycerollins are one of the phytoalexins, such as isoflavones, which are low-molecular-weight substances produced when plants are stressed by substances such as microbial infections or inorganic salts. It has antibacterial action but is known to be harmless to human body (Boue et. Al., J Agric Food Chem. Jun., 48, 6, 2167-72, 2000), and recently, the anti-cancer and anti-inflammatory effects have been known, attracting attention as a new functional substance, for example, resveratrol of grapes, as the most representative phytoalexin In addition to antioxidant and anti-cancer effects, it is known to activate the pathway of sirtuin, a protein deacetylase that is made in some tissues of the body such as the brain, liver and kidneys of the human body, thereby lowering calorie intake and extending lifespan. There is a bar. However, there is little research on other functions of phytoalexin.

In addition, glyceryl De (glyceollins) is common bean is not present, and secure the soybean edible Aspergillus scavenger (Asp. Sogae), Aspergillus duck claim (Asp. Oryzae) as a material derived from soybeans by fungal infection , Aspergillus play bus (Asp. flavus), or separation tank crispus oligonucleotide sports Russ when treated with (R. oligosporus), glyceryl raised and oxo octadecyl Kano acid (oxooctadecadienoic acids) such as Pitot Alec capable of inducing production of the gods Known as (Boue et al., J Agric Food Chem . Jun . 48 (6), 2167-72, 2000, Feng et. al., J Agric Food Chem . Oct., 17, 55 (21), 8589-95, 2007). Recently, it has been reported that such glycerol has the effect of preventing the progression and inhibiting the progression of cancers such as breast cancer and ovarian cancer. However, nothing else has been revealed about the other functions of glycerol in addition to the above functions.

Therefore, the present inventors are developing a new menopausal symptom improving composition which is excellent in improving the symptoms of menopausal symptoms without any side effects, and the glycerol compound isolated from the inoculated soybean has excellent antioxidant activity and estrogen-like activity. The present invention was completed by confirming that there is an effect that can improve symptoms of menopause.

Accordingly, an object of the present invention is to provide a composition for the prevention and improvement of menopausal symptoms containing a glycerin compound or a pharmaceutically acceptable salt thereof as an active ingredient.

Another object of the present invention to provide a composition for the prevention and improvement of menopausal symptoms containing an extract extracted with water or an organic solvent of soybean inoculated with fungi as an active ingredient.

Another object of the present invention to provide a food for preventing and improving menopausal symptoms comprising the composition.

Furthermore, another object of the present invention is to provide a medicament for the prevention and treatment of menopausal symptoms comprising the composition.

In order to achieve the object of the present invention as described above, the present invention provides a composition for the prevention and improvement of menopausal symptoms containing a glycerin compound or a pharmaceutically acceptable salt thereof as an active ingredient.

The present invention also provides a composition for the prevention and improvement of menopausal symptoms containing the extract extracted with soybean fungus inoculated with fungi as an active ingredient.

The present invention also provides a food for the prevention and improvement of menopausal symptoms comprising the composition.

Furthermore, the present invention provides a medicament for the prevention and treatment of menopausal symptoms comprising the composition.

Since the glycerin compound according to the present invention has excellent antioxidant activity and has estrogen-like activity, it has an effect of preventing and improving menopausal symptoms caused by aging and estrogen deficiency, and since there are no side effects, hormone replacement therapy for treating menopausal symptoms and It can be used as a material for health functional food.

The present invention is characterized by providing a composition for the prevention and improvement of menopausal symptoms containing a glycerin compound as an active ingredient.

The glycerol compound according to the present invention may be a glycerol compound I, II or III represented by the following formula (I) to (III).

<Formula I>

<Formula II>

<Formula III>

In addition, the glycerol compound according to the present invention may be used in the form of a salt, preferably a pharmaceutically acceptable salt. The salt is preferably an acid addition salt formed by a pharmaceutically acceptable free acid, and an organic acid and an inorganic acid may be used as the free acid. The organic acid is not limited thereto, citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, fumaric acid, formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, metasulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, Glutamic acid and aspartic acid. In addition, the inorganic acid includes, but is not limited to, hydrochloric acid, bromic acid, sulfuric acid and phosphoric acid.

The glycerol compounds according to the invention can be isolated from nature or prepared by chemical synthesis methods known in the art.

Preferably, the glycerol compounds of the present invention can be obtained from fungal inoculated soybeans using methods of extracting and separating conventional materials. At this time, the soybean can be used to dehydrate properly, macerated or only dehydrated to obtain the desired extract, preferably dehydrated and then pulverized and then ground. In addition, the mold as is available the edible fungus, as a kind of such fungi include, but are not limited to, Aspergillus scavenger (Asp. Sogae), Aspergillus duck claim (Asp. Oryzae), Aspergillus Asp. Flavus or R. oligosporus can be used, and preferably Aspergillus sojae can be used. As described above, the soybean seed inoculated with the fungus is fermented for a predetermined time and then extracted with a suitable solvent, and the desired extract can be purified using purification methods known to those skilled in the art.

Suitable solvents for the extraction may be water or an organic solvent, preferably purified water, methanol (methanol), ethanol (ethanol), propanol (isopropanol), butanol (butanol), acetone (acetone) ), Or a mixture of various solvents such as ether, benzene, chloroform, ethyl acetate, methylene chloride, hexane and cyclohexane Can be used. More preferably ethanol can be used.

In addition, the separation and purification of the glycerol compound of the present invention from the soybean seed inoculated with the fungus is characterized by column chromatography and high-speed liquid chromatography filled with various synthetic resins such as silica gel or activated alumina. Graphy (HPLC) and the like can be used alone or in combination. However, the extraction and separation and purification method of the active ingredient is not necessarily limited to the above method.

The glycerol compound of the present invention may be used in the form of a purely isolated and purified compound, and may also be used in the form of an extract containing the same. The extract can be obtained by extracting the soybean inoculated with the fungus as water or an organic solvent as described above.

On the other hand, glycerol compound is one of the phytoalexin (isoflavone), such as phytoalexin has been found to have anti-cancer activity (Payton Stewart F, et. Al., Mol Carcinog . Mar., 4, 2009, Salvo VA, et. al., Clin Cancer Res . , 12, 7159-64, 2006), has not been reported that the glycerol compound can improve the menopausal symptoms by having antioxidant activity and estrogen-like activity.

On the other hand, the present invention was first identified that the soybean extract containing the glycerol compound and glycerol compound of the present invention has excellent antioxidant activity and estrogen-like activity.

According to one embodiment of the present invention, a soybean extract containing a glycerol compound prepared according to the method of the present invention, that is, a soybean extract inoculated with Aspergillus powder, which is known in the art, the method of ferric reducing ability As a result of measuring the antioxidant activity through the method of plasma, it was confirmed that the antioxidant activity was significantly superior to the soybean extract containing no glycerol compound, that is, the soybean extract not inoculated with Aspergillus powder. Phosphorus phenol content was also higher in soybean extracts containing glycerol compounds (see FIGS. 3A and 3B).

Therefore, through the above results, the present inventors found that the glycerol compound and the soybean extract containing the glycerol compound have excellent antioxidant activity, and when inoculated with soybean with Aspergillus bacteria, the production of glycerol compound is promoted. The antioxidant activity was also increased further by.

On the other hand, active oxygen refers to oxygen that is harmful to the body, oxygen is an indispensable substance for humans, it is absorbed into the body when we breathe to generate energy in the cells. At this time, some of the absorbed oxygen causes chemical reactions in the body to denature to toxic components, which is oxygen that is toxic. The denaturation is caused by freon gas, exhaust gas, food additives, food peroxides, chemicals, ultraviolet rays, electromagnetic waves, stress, and chemicals made of synthetic synthetic chemicals in the air pollutants. It has a strong oxidizing power that makes your body sick and promotes aging.

Therefore, the glycerol compound of the present invention and the soybean extract containing the glycerol compound has an antioxidant activity to remove the active oxygen which is one of the causes to promote the aging of the human body, thus preventing diseases caused by oxidative stress, It can be used to improve and treat.

The present inventors also found that the glycerol compound and glycerol of the present invention in the MCF-7 breast cancer cell line having an estrogen receptor in order to confirm whether the glycerol compound and the glycerol compound containing the glycerol compound of the present invention has estrogen-like activity As a result of treating the soybean extract containing the compound, it was confirmed that the cell proliferation of the MCF-7 cell line was promoted (see Example <3-1>).

In addition, in one embodiment of the present invention, the result of measuring the estrogen activity of the soybean extract containing the glycerol compound and glycerol compound of the present invention in a recombinant yeast strain having a human estrogen receptor In addition, the glycerol compound of the present invention was shown to be superior in estrogen activity than Dyzezein and Genistein, known as estrogen hormones estradiol and estrogen analogs used as a positive control (see Fig. 4), further estrogen-like activity As a result of measuring the expression level of pS2 used as an indicator, it was shown that the soybean extract containing the glycerol compound and glycerol compound of the present invention increased the expression of pS2 more strongly than the estradiol hormone (see FIG. 5).

On the other hand, estrogen is a type of endocrine hormone that is synthesized from cholesterol in the body, the secretory sources are mainly follicles and corpus luteum of the ovary, and during pregnancy are also secreted in the fetal placenta, adrenal glands and testes. Estrogen secretion in the ovary secretes follicle stimulating hormones and luteinizing hormones, which are hormones that regulate menstrual cycle and enable pregnancy, when gonadotropins secreted from the anterior pituitary gland transmit signals to the reproductive organs, which in turn act on the ovaries Releases estrogen and progesterone Estrogen stimulates the endometrium in the first half of the menstrual cycle to thicken the endometrium and to increase blood supply so that the fertilized egg can be implanted. In other words, estrogen is a representative female hormone involved in endometrial proliferation, uterine muscle development, secondary sexual manifestations, mediation of menstrual cycle, maternal change during pregnancy, and stimulation of proliferation of the mammary gland. It functions as described above via. However, in postmenopausal women, estrogen secretion is reduced, causing a variety of symptoms.

Therefore, substances with improved estrogen deficiency, promoted estrogen secretion, or similar activity of estrogen can be used to prevent or treat menopausal symptoms. Therefore, the soybean extract containing the glycerol compound and the glycerol compound according to the present invention is very excellent in estrogen-like activity can be used to prevent, improve and treat the symptoms of menopausal symptoms caused by estrogen deficiency.

Therefore, the present invention provides a composition for the prevention and improvement of menopausal symptoms containing a glycerol (Clyceollin) compound or a pharmaceutically acceptable salt thereof as an active ingredient.

In addition, the composition of the present invention can be used as a pharmaceutical composition for the prevention and treatment of menopausal symptoms.

The "menopausal symptoms" refers to the symptoms appearing in women before and after menopause as the secretion of estrogen decreases due to aging of the ovary, such as fatigue, excitement, insomnia, decreased concentration, depression, memory loss, headache, anxiety , Cardiovascular problems such as nervousness, timidity, hot flashes, dizziness, paresthesia, palpitations, tachycardia, nausea, constipation, diarrhea, joint pain, myalgia, cold hands and feet, weight gain, osteoporosis and heart disease, hypertension, stroke, etc. (Kalin MF & Zumoff B., Steroids , 55, 330-352, 1990).

The composition for preventing and improving menopausal symptoms according to the present invention may include a pharmaceutically effective amount of a glycerol compound alone or may include one or more pharmaceutically acceptable carriers, excipients or diluents. The pharmaceutically effective amount herein refers to an amount sufficient to prevent, ameliorate and treat menopausal symptoms.

The pharmaceutically effective amount of glycerol according to the present invention is 0.5 to 100 mg / day / kg body weight, preferably 0.5 to 5 mg / day / kg body weight. However, the pharmaceutically effective amount may be appropriately changed according to the degree of menopausal symptoms, the age, weight, health condition, sex, route of administration and duration of treatment of the patient.

In addition, the pharmaceutically acceptable refers to a composition that is physiologically acceptable and does not cause an allergic reaction such as gastrointestinal disorders, dizziness or the like when administered to humans. Examples of such carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, Polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In addition, fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers and preservatives may be further included.

In addition, the compositions of the present invention may be formulated using methods known in the art so as to provide rapid, sustained or delayed release of the active ingredient after administration to the mammal. The formulations may be in the form of powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatine capsules, sterile injectable solutions, sterile powders.

The composition for preventing and improving menopausal symptoms according to the present invention may be administered through various routes including oral, transdermal, subcutaneous, intravenous or muscle, and the dosage of the active ingredient is determined by the route of administration, the age, sex, weight and It may be appropriately selected depending on various factors such as the severity of the patient. In addition, the composition for preventing and improving menopausal symptoms of the present invention can be administered in parallel with a known compound having the effect of preventing, improving or treating menopausal symptoms.

Therefore, the present invention can provide a medicament for preventing and treating menopausal symptoms, including a composition containing a glycerin compound or a pharmaceutically acceptable salt thereof as an active ingredient.

Furthermore, the composition for preventing and improving menopausal symptoms according to the present invention not only provides an effect of relieving menopausal symptoms through excellent antioxidant effect and estrogen-like effect, but also has no toxicity and side effects for drugs, so it can be used with confidence even for long-term use. In addition, the fungus used to obtain the soybean extract containing glycerol in the present invention, that is, the Aspergillus dust can also be used as a food grade, so the composition of the present invention has a very stable characteristic to the body.

Therefore, the composition for prevention and improvement of menopausal symptoms of the present invention can be added to food for the purpose of preventing and improving menopausal symptoms, the composition of the present invention can be used as a composition for food for the prevention and improvement of menopausal symptoms Can be.

Therefore, the composition for food for the prevention and improvement of menopausal symptoms of the present invention can be easily utilized as foods, such as the main ingredients, secondary ingredients, food additives, functional foods or beverages that are effective in preventing and improving menopausal symptoms.

As used herein, the term “food” refers to a natural product or processed product containing one or more nutrients, and preferably means a state in which it can be directly eaten through a certain processing step, It includes all foods, food additives, functional foods and drinks.

Foods to which the composition for preventing and improving menopausal symptoms according to the present invention can be added include, for example, various foods, beverages, gums, teas, vitamin complexes, functional foods, and the like. In addition, in the present invention, food includes special nutritional products (e.g., formulated milk, young, infant food, etc.), processed meat products, fish products, tofu, jelly, noodles (e.g. ramen, noodles, etc.), bread, health supplements, seasonings. Foods (e.g. soy sauce, miso, red pepper paste, mixed soy sauce), sauces, confectionery (e.g. snacks), candy, chocolates, gums, ice creams, dairy products (e.g. fermented milk, cheese, etc.), other processed foods, kimchi, Pickled foods (various kimchi, pickles, etc.), beverages (e.g., fruit drinks, vegetable drinks, soy milk, fermented beverages, etc.), natural seasonings (e.g. ramen soup, etc.) are not limited thereto. The food, beverage or food additive may be prepared by a conventional production method.

In addition, the term "functional food" refers to the control of biological defense rhythms and disease prevention of food groups or food compositions that have added value to the food by using physical, biochemical, or biotechnological techniques to act and express the function of the food for a specific purpose. It means a food that is designed and processed to fully express the body's regulatory function regarding recovery and the like, and specifically, it may be a health functional food. The functional food may include food acceptable food additives, and may further include appropriate carriers, excipients and diluents commonly used in the manufacture of functional foods.

In addition, in the present invention, the "beverage" refers to a generic term for drinking to quench thirst or to enjoy a taste and includes a functional drink. The beverage contains, as essential ingredients, a composition for the prevention and amelioration of the menopausal symptoms as an essential ingredient, and there are no special limitations on the other ingredients, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. Can be.

Furthermore, in addition to the above, foods containing compositions for preventing and improving menopausal symptoms of the present invention include various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavoring agents such as flavoring agents, colorants and fillers (cheese). , Chocolate and the like), pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. The components can be used independently or in combination.

In a food containing a composition for preventing and improving menopausal symptoms of the present invention, the amount of the composition according to the present invention may include 0.001% to 90% by weight of the total food weight, preferably 0.1% by weight to 40% by weight, and in the case of a beverage, may be included in a ratio of 0.001g to 2g, preferably 0.01g to 0.1g based on 100ml, but for health and hygiene purposes or for health control purposes. In the case of long-term ingestion may be below the above range, the active ingredient is not limited to the above range because it can be used in an amount above the above range because there is no problem in terms of safety.

Hereinafter, the present invention will be described in detail by way of examples. However, these examples are intended to illustrate the present invention in more detail, and the scope of the present invention is not limited to these examples.

< Example  1>

Glycerin  Separation and Purification

The present inventors conducted the following experiment to separate glycerol, a type of phytoalexin. That is, after sterilizing the surface of the beans for 3 minutes with 70% ethanol, washed twice with deionized water for 2 minutes and immersed in water for 4 hours. The sterilized gauze moistened with water was then placed in a culture vessel, soybean divided into 1 / 2-1 / 8, and 10 μl of Aspergillus spore suspension of fungal fungi were inoculated on one side of the divided beans. At this time, the control group was used that was not inoculated with the fungus divided. After the fungus was inoculated into the beans by the above method, the culture vessel was packaged with black vinyl and left in a dark chamber at 25 for 3 days. Since beans 2 inoculated with the fungus was refluxed with 8L of 80% ethanol for 3 hours from 78. The reflux extract was concentrated under reduced pressure, and then the concentrated extract was dispersed with water, followed by partition extraction with normal-hexane to remove fatty acids. The aqueous layer was partitioned and extracted under ethyl acetate, and then concentrated under reduced pressure to obtain about 25 g of ethyl acetate fraction extract. Got it. The extract was then purified using MPLC chromatography. The filler was then eluted with silica gel 9385 (30x300 mm, Merck), changing the hexanes: (ethylacetate / methanol (1: 1)) from 50: 1 to 5: 1 at 254 nm at a flow rate of 4 mL / min. The collected fractions were collected, and after confirming the presence of glycerol in the second fraction (Fr. 2) of the five fractions by TLC and HPLC, the second fraction was concentrated under reduced pressure. 340 mg of fraction 2 was obtained, This was confirmed to be a glycerol compound through analysis using a mass spectrometer (see FIG. 2), and the glycerol compounds separated by the method of the present invention were glycerol I, II, and III having the structures of Formulas I to III. In addition, the separation process of separating the glycerol compound from the soybean seed inoculated according to the method of the present invention is shown in FIG.

 <Formula I> <Formula II> <Formula III>

< Example  2>

Determination of Antioxidant Activity of Glycerin Compounds According to the Present Invention

In Example 1 to examine whether the fraction of the glycerol compound, that is, the fraction of the soybean extract containing all of the glycerin I, II and III has an antioxidant activity inoculated with the Aspergillus fungus in Example 1 Folin-Ciocalteu assay was performed on the fractions extracted from soybean (containing glycerol). At this time, a fraction of the soybean extract that was not inoculated with Aspergillus granules was used as a control. The Folin-Ciocalteu Assay method is a method for measuring the amount of phenolic compounds having antioxidant activity well known in the art, using a mixture of tungstate and molybdate in an alkaline solution (5-10% water soluble sodium carbonate) If the phenolic compound to be oxidized, O ₂ ^- to form a molybdate and reaction are oxidized molybdenum (MoO ₄ ^-) there is the formation of so measuring the formed material at 750nm and it gallic acid dangryangga (gallic acid equivalent: GAE ).

In addition, the antioxidant activity of glycerol according to the present invention was measured using a method of ferric reducing ability of plasma (FRAP) (Parker, L. 1999. Method in Enzymology , 293. Oxidants and antioxidants part A. pp. 15- 27) The FRAP method is divalent by the oxidation of ferric 2,4,6-trippyridyl-s-triazine (TPTZ) reagent. The iron was converted into a trichromate product by oxidizing to trivalent. A 1 mM solution (0.278 g FeSO ₄ 7H ₂ O / L) was prepared using ferrous sulfate, and this was used as a standard solution. The FRAP solution was prepared using 300 mM acetate buffer ( pH 3.6) 200 mL, 20 mL of 10 mM TPTZ solution, 20 mM FeCl ₃ 20mL and 24mL of the solution was prepared by mixing sterile water and then maintained at 37 ° C. After the assay solution was prepared as described above, the mixture was mixed with 170 μL of sterile water per well, 7 μL of the fraction of the standard solution and the soybean extract, and 30 μL of the FRAP solution in a 96-well plate, and then measured for absorbance at 593 nm after 4 minutes. The degree of FRAP was calculated by linear regression analysis of the standard solution of absorbance values.

As a result, as shown in Fig. 3, the fraction of soybean inoculated with Aspergillus fungus, that is, the fraction containing glycerol, was found to have more phenolic content than the fraction of soybean without inoculation. As a result of measuring the antioxidant activity through analysis, it was confirmed that the fraction of soybean containing glycerol by fungal inoculation was superior to the fraction of soybean not inoculated with mold.

Therefore, the results indicate that the glycerol compound of the present invention has excellent antioxidant activity.

<Example 3>

Determination of Estrogen-like Activity of Glycerin Compounds According to the Present Invention

In order to confirm whether the glycerol compound of the present invention isolated in Example 1 has estrogen-like activity, the following experiment was performed.

<3-1> Proliferation Measurement of MCF-7 Cells

 In order to measure the estrogen-like activity of the glycerin compound according to the present invention, the MCF-7 breast cancer cell line, which is an estrogen-like substance assay cell line, was used. In other words,

MCF-7 cells with estrogen receptors were cultured at 37 ° C. with EMEM medium containing 10% charcoal script-FBS at 2 × 10 ⁴ cell counts / well in 24-well plates. After 24 hours, the medium was removed and replaced with a medium containing a glycerol compound of the present invention in the concentration range of 0.01-500 μg / mL. Then, after the culture period of 2 days, the degree of cell proliferation was measured by MTT assay method. At this time, the control cell line was an MDA-MB-231 cell line without estrogen receptor.

As a result, the MCF-7 breast cancer cell line containing the estrogen receptor treated with the glycerol compound according to the present invention promoted cell proliferation compared with the glycerol compound treated with the MDA-MB-231 cell line used as a control group. (Not shown).

Therefore, the present inventors have found that the glycerol compound of the present invention induces estrogen activity in response to the estrogen receptor as an estrogen-like compound.

<3-2> Estrogen Activity Measurement Using Yeast

In addition to the above <3-1> method, the present inventors measured the estrogen activity of the glycerol compound using a recombinant yeast strain having a gene of the human estrogen receptor. The measurement was carried out while stirring a recombinant yeast strain ( Saccharomyces cerevisiae ER + LYS 8127) containing a human estrogen receptor at 300 rpm in an incubator maintained at 30 ℃ temperature, wherein the medium was yeast nitrogen base (6.74) g / L), dextrose (20 g / L), L-lysine (36 mg / L) and L-histidine (24 mg / L) were used. After two days of incubation, the yeast strains were allowed to reach _1.0-2.0 at OD _{600 nm} . 50 μM of CuSO ₄ was then added to produce estrogen receptors from the yeast. The added medium was diluted to 0.03 at OD _{600 nm} . Then, in a 50 mL conical tube, 5 mL of the diluted yeast and each sample (ie, the fraction of soybean extract inoculated with mold, the fraction of soybean extract not inoculated with mold, the glycerol compound isolated in the present invention, Dyzedein, Genistein, estradiol as a positive control group and DMSO as a negative control group were added at different concentrations (0.1, 1.0, 10.0 μg / ml), and then incubated with stirring at 300 rpm in an incubator maintained at 30 ° C. . Yeast treated with each material as described above was diluted to a value of 0.25 at OD _{600 nm} and added to the 96-well plate by 100. Then, absorbance was measured at _{590 nm} to measure the color of each medium, and β-Galactosidase activity was 2 mg / ml of O -nitrophenyl-β-D-galactopyranoside (ONPG), 0.1% SDS. (sodium dodecyl sulfate), 50 mM β-mercaptoethanol and 25 U / ml of zymolase added 100 buffer (60 mM Na ₂ HPO ₄ , 40 mM NaH ₂ PO ₄ , 10 mM KCl, 1 mM MgSO ₄ , pH 7.0). Then, 20 minutes incubation at room temperature, was measured at OD _420nm, to calculate the OD _420nm -OD _590nm values were divided for the remaining values as a negative control, and the results are shown in Fig.

As a result, the estradiol hormone, a positive control group, was treated with a fraction of the glycerol-producing soybean extract and the glycerin compound isolated in the present invention by inoculation of the recombinant yeast containing the gene of the human estrogen receptor. Estrogen activity was higher than that of the treatment, and estrogen-like substances such as genistein and diazein also showed estrogen activity, but the activity was lower than that of the glycerol compound of the present invention. appear. On the other hand, the fraction of the soybean extract not inoculated with the fungus was found to have a very low estrogen activity (see FIG. 4).

<3-3> pS2 of mRNA  Determination of estrogen activity according to expression

pS2 protein is the first protein expressed when a human breast cancer cell line is stimulated by estrogen. In the art, the expression level of the protein is used as an index indicating the activity of estrogen. In this regard, the present inventors conducted an experiment to confirm whether the expression of the pS2 protein is promoted in the estrogen activity of the glycerol compound of the present invention. To this end, the cells of the MCF-7 breast cancer cell line used in Example <3-1> were dispensed on a plate of 56 cm ² , and then, as a fungus, a fraction of the soybean extract inoculated with the fungus in the culture medium. Fractions of the inoculum soybean extract, the glycerol compound isolated in the present invention, estradiol and DMSO were treated respectively and incubated for 6 hours. Then, the medium was removed and total RNA was isolated from the MCF-7 cells using RNeasy mini kit (Qiagen), and the pS2 gene was performed by performing reverse transcription polymerase chain reaction (RT-PCR) with the primers described in Table 1 below. The expression level of was measured, wherein beta-tubulin primer was used as a control. The PCR reaction was carried out in a PCR thermocycler (MJ Research), the first strand synthesis conditions of cDNA was subjected to initial denaturation for 2 minutes at 95 ^o C, then 1 minute at 94 ^o C, 60 ^o C for amplification reaction The reaction was repeated 30 times for 1 minute at 72 ^o C at 30 minutes, and the PCR reaction was completed by 10 minutes at 72 ^o C. Then, the PCR reaction product was confirmed by electrophoresis using 1% agarose gel.

Primer sequence  Kinds  order Sequence Listing β-tubulin forward 5'-CATACCTTGAGGCGAGCAAA-3 ' One β-tubulin reverse 5'-TGAGAAGCCTGAGGTGATGG-3 ' 2 pS2 forward 5'-TTTGGAGCAGAGAGGAGGCAATGG-3 ' 3 pS2 reverse 5'-TGGTATTAGGATAGAAGCACCAGGG-3 ' 4

As a result, the MCF-7 cell line was treated with the glycerin compound of the present invention, the expression of pS2 was higher than the treatment with estradiol hormone, and the fraction of the soybean seed inoculated with the fungus (glycerol PS2 expression was higher than that treated with estradiol hormone. On the other hand, the fraction of the soybean extract that was not inoculated with the fungus was found to have a lower expression level of pS2 than that of the negative control DMSO (see FIG. 5).

Therefore, from the above results, it was found that the glycerol compound according to the present invention acts to increase the expression of pS2, and the glycerol compound has a very good estrogen-like activity. In addition, the estrogen activity of glycerol was confirmed to be superior to the estrogen hormone estradiol, it was found that the glycerol compound of the present invention can be used for the prevention, improvement and treatment of menopausal symptoms.

So far I looked at the center of the preferred embodiment for the present invention. Those skilled in the art will appreciate that the present invention can be implemented in a modified form without departing from the essential features of the present invention. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the scope will be construed as being included in the present invention.

1 is a schematic view showing a process for separating and purifying a glycerol compound according to the present invention.

Figure 2 shows a graph confirming the glycerol compound separated and purified by the method of the present invention using a mass spectrometer.

Figure 3a is a graph comparing the content of the total phenolic antioxidant component contained in the soybean extract (containing glycerol) and the non-inoculated soybean extract inoculated with Aspergillus dust.

Figure 3b is a graph comparing the antioxidant activity degree using a ferric reducing ability of plasma (FRAP) method for soybean extract (containing glycerol) and inoculated with Aspergillus agent.

4 is a glycerol compound according to the present invention, a soybean extract (containing glycerol) inoculated with a Aspergillus agent, a soybean extract, estradiol, inoculated with a recombinant yeast strain having a gene of a human estrogen receptor. It is a graph comparing estrogen activity after treatment with idzein, genistein and DMSO, respectively.

Figure 5 shows the estrogen-like activity after treating the MCF-7 breast cancer cell line with the glycerol compound, soybean extract (containing glycerol), vaccinated soybean extract, estradiol and DMSO, respectively, inoculated with a glycerol compound according to the present invention. It shows the result of comparing the expression level of pS2, which is an index of through RT-PCR.

<110> Kyungpook national university industry academy cooperation foundation <120> Composition for preventing and relieving of menopausal symptoms          comprising glyceollin <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> beta-tubulin F <400> 1 cataccttga ggcgagcaaa 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> beta tubulin R <400> 2 tgagaagcct gaggtgatgg 20 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> pS2 F <400> 3 tttggagcag agaggaggca atgg 24 <210> 4 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> pS2 R <400> 4 tggtattagg atagaagcac caggg 25  

Claims (8)

Composition for the prevention and improvement of menopausal symptoms containing a glycerin compound or a pharmaceutically acceptable salt thereof as an active ingredient. The composition for preventing and improving menopausal symptoms according to claim 1, wherein the glycerol compound is selected from the group consisting of glycerol I, II and III represented by the following formulas (I) to (III).

Composition for the prevention and improvement of menopausal symptoms containing the extract extracted with soybean fungus inoculated with fungi as an active ingredient. According to claim 3, wherein the fungus is Aspergillus duck ( Asp . Oryzae ), Aspergillus dust ( Aspergillus) sojae ), Aspergillus placeus ( Asp . flavus ) and Rhizopus oligosporus ( Rhizopus oligosporus ) composition for the prevention and improvement of menopausal symptoms, characterized in that selected from the crowd. The menopausal symptoms according to claim 3, wherein the organic solvent is selected from the group consisting of methanol, ethanol, propanol, isopropanol, butanol, acetone, ether, benzene, chloroform, ethyl acetate, methylene chloride, hexane and cyclohexane. Prevention and improvement composition for. According to claim 3, wherein the extract is a composition for preventing and improving menopausal symptoms, characterized in that it comprises a glycerol (glyceollin) compound. Food for the prevention and improvement of menopausal symptoms comprising the composition of any one of claims 1 to 6. A drug for preventing and treating menopausal symptoms comprising the composition of any one of claims 1 to 6.

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