Composition for prevention, amelioration or treatment of menopausal syndrome, bone diseases, and / or dyslipidemia in women comprising fermented pueraria lobata root extract and fermented glycine max (l.) merrill extract

Abstract

An aspect relates to a composition for the prevention, amelioration or treatment of menopausal syndrome, bone diseases, and / or dyslipidemia in women, the composition comprising a fermented Pueraria lobata root extract and a fermented Glycine max (L.) Merrill extract. The composition according to an aspect comprises: a Pueraria lobata root extract or a fermented product thereof; and a Glycine max (L.) Merrill extract or a fermented product thereof, which are familiar natural substances, thereby including both glycosides and aglycones of various phytoestrogens. Therefore, the composition has an excellent in vivo absorption rate and excellent efficacy, and thus has an excellent effect of preventing, ameliorating or treating menopausal syndrome, bone diseases, and / or dyslipidemia in women. The composition can be used as a highly palatable, low-dose, high-efficiency product as compared with existing products that have been proven to help menopausal women's health, and can secure excellent competitiveness in the domestic and international markets for women's health functional foods.

Description

Composition for preventing, improving or treating menopausal syndrome, bone disease and / or dyslipidemia in women, comprising fermented galgeun extract and fermented seoritae extract

[0001] The present invention relates to a composition for preventing, improving or treating menopausal syndrome, bone disease and / or dyslipidemia in women, comprising a fermented extract of galgeun and a fermented extract of seoritae.

[0002]

[0003] Conventional techniques have confirmed that phytoestrogens contained in natural extracts can help support the health of menopausal women. Therefore, many cases of incorporating phytoestrogens or natural extracts containing them into health functional foods have emerged. However, most single natural extracts or natural complex extracts are often extracts of unfamiliar herbal medicines. Furthermore, most are primarily composed of glycoside compounds containing sugars, resulting in low bioabsorption rates. Consequently, these methods can lead to excessive intake of health functional foods, sometimes leading to undesirable side effects.

[0004] Therefore, in the present invention, we attempted to increase palatability by using familiar natural products such as galgeun and seoritae. In addition, we attempted to increase the absorption rate in the body by bioconverting the phytoestrogen glycoside form present in natural products into the aglycone form with the sugar removed by utilizing Lactiplantibacillus plantarum, a safe lactic acid bacterium without toxic genes or antibiotic resistance genes. Furthermore, the present invention confirmed that the efficacy for menopausal syndrome, bone disease, and dyslipidemia in menopausal women is enhanced through a combination of various phytoestrogens, and thus it was confirmed that the composition of the present invention can be utilized as a pharmaceutical, health functional food, or food.

[0005]

[0006] One aspect provides a pharmaceutical composition for preventing or treating menopausal syndrome, bone disease or dyslipidemia in women, comprising an extract of Pueraria lobataroot or a fermented product thereof; and an extract of Glycine max(L.) Merrill or a fermented product thereof.

[0007] Another aspect is to provide a health functional food for preventing or improving menopausal syndrome, bone disease or dyslipidemia in women, comprising an extract of Pueraria lobataroot or a fermented product thereof and an extract of Glycine max(L.) Merrill or a fermented product thereof.

[0008] Another aspect provides a method for preventing or treating menopausal syndrome, bone disease or dyslipidemia in women, comprising administering to a subject in need thereof an extract of Pueraria lobataroot or a fermented product thereof and an extract of Glycine max(L.) Merrill or a fermented product thereof.

[0009] Another aspect provides the use of an extract of Pueraria lobataroot or a fermented product thereof; and an extract of Glycine max(L.) Merrill or a fermented product thereof for the manufacture of a medicament for preventing or treating menopausal syndrome, bone disease, or dyslipidemia in women.

[0010]

[0011] One aspect provides a pharmaceutical composition for preventing or treating menopausal syndrome, bone disease or dyslipidemia in women, comprising an extract of Pueraria lobataroot or a fermented product thereof; and an extract of Glycine max(L.) Merrill or a fermented product thereof.

[0012] The term "Pueraria lobataroot" refers to the root of the kudzu plant, a member of the legume family and the genus Kudzu, primarily used as a medicine in Oriental medicine. It is known to relieve fever, diarrhea, and vomiting.

[0013] The term "Glycine max(L.) Merrill" refers to a type of black bean, with a black skin and a bluish interior, hence the name "sokcheong". It is known to have a high sugar content and high anthocyanin pigmentation, which are effective in preventing aging.

[0014] The term "extract" refers to the result obtained by immersing the target substance in various solvents and then extracting it for a certain period of time at room temperature or at a temperature of 100°C, such as a liquid component, or a solid component obtained by removing the solvent from the liquid component. In addition, it can be comprehensively interpreted to include, in addition to the result, a dilution of the result, a concentrate thereof, a controlled substance, a purified substance thereof, etc. Accordingly, an extract according to one aspect can be interpreted to include an extract obtained by extraction treatment, a dilution or concentrate of the extract, a dried product or powder obtained by drying the extract, a controlled substance or purified substance of the extract, or a mixture thereof, such as the extract itself and all formulations that can be formed using the extract.

[0015] The above-mentioned galgeun or seoritae may be the whole, a part thereof, or a material derived therefrom. The galgeun or seoritae used for extraction may be the whole, a part thereof, or a material derived therefrom, ground or chopped, or suitably dried. The galgeun or seoritae may be used in a dried form with a moisture content of less than 20%, less than 15%, or, for example, less than 12%.

[0016] In one specific example, the galgeun or seoritae may be used after undergoing a purification process. The purification process refers to removing foreign substances and particles other than the raw material, and may include, but is not limited to, a washing process and a process of removing rotten or spoiled materials.

[0017] In one specific example, the galgeun or seoritae may undergo a pretreatment process prior to extraction, and the method is not limited to a method commonly used in the art. According to one specific example, the pretreatment may be soaking, grinding, pulverization, or steaming.

[0018] The extract may be extracted with at least one selected from the group consisting of water, alcohols having 1 to 10 carbon atoms, hexane, chloroform, and butylene glycol. In addition, the extract may be extracted with a hydrophilic solvent, for example, alcohol, water, or a combination thereof. The alcohol may be a compound having at least one -OH group having 1 to 10 carbon atoms. The alcohol may be an alcohol having 1 to 6 carbon atoms, a polyhydric alcohol having 3 to 6 carbon atoms. The alcohol may be methanol, ethanol, n-propanol, isopropanol, n-butanol, sec-butanol, isobutanol, tert-butanol, n-pentanol, n-hexanol, or a mixture thereof. The solvent may be, for example, a mixture of water and alcohol, i.e., an aqueous alcohol solution. The alcohol concentration of the alcohol aqueous solution may be 1 to 100% (w / w), for example, 1 to 99.5% (w / w), 10 to 100% (w / w), 20 to 100% (w / w), 30 to 100% (w / w), 40 to 100% (w / w), 50 to 100% (w / w), 60 to 100% (w / w), 70 to 100% (w / w), 75 to 100% (w / w), 60 to 90% (w / w), 60 to 80% (w / w), 65 to 75% (w / w) or 70% (w / w). The alcohol aqueous solution may be a methanol, ethanol or butanol aqueous solution.

[0019] The above extract may be extracted with ethanol. Specifically, the above extract may be extracted with 60% ethanol.

[0020] The above extract may be extracted by a method conventional in the art, such as static extraction, heating extraction, pressure extraction, ultrasonic extraction, hot water extraction, reflux cooling extraction, subcritical extraction, or supercritical extraction. In the present invention, the extraction method of the extract is not particularly limited. It may be performed alone or in combination of two or more methods according to a method conventionally used in the art. Specifically, the extract may be statically extracted.

[0021] The above extraction may include adding the extraction solvent to the galgeun in an amount of 1 to 50 (w / w) times, for example, 1 to 40 (w / w) times, 1 to 30 (w / w) times, 2 to 50 (w / w) times, 2 to 40 (w / w) times, 2 to 30 (w / w) times, 3 to 50 (w / w) times, 3 to 40 (w / w) times or 3 to 30 (w / w) times, and specifically, the extraction may be performed by adding 3 to 5 times.

[0022] In addition, the extraction may include adding the extraction solvent to the frost-like substance in an amount of 1 to 50 (w / w), for example, 1 to 40 (w / w), 1 to 30 (w / w), 2 to 50 (w / w), 2 to 40 (w / w), 2 to 30 (w / w), 3 to 50 (w / w), 3 to 40 (w / w), or 3 to 30 (w / w), and specifically, the extraction may be performed by adding 8 to 12 times.

[0023] The above extraction may be performed at 4°C to 70°C, for example, 4°C to 50°C, 4°C to 40°C, 4°C to 30°C, 10°C to 70°C, 15°C to 70°C, 20°C to 70°C, 4°C to 50°C, 10°C to 50°C, 4°C to 40°C, 4°C to 30°C, 10°C to 40°C, 10°C to 35°C, 10°C to 30°C, or room temperature. Specifically, the above extraction may be performed at room temperature.

[0024] The extraction time may vary depending on the selected temperature and may be from 1 hour to 2 months, for example, from 1 hour to 1 month, from 1 hour to 10 days, from 1 hour to 5 days, from 1 hour to 3 days, from 1 hour to 2 days, from 1 hour to 1 day, from 1 hour to 10 hours, from 2 hours to 1 month, from 2 hours to 15 days, from 2 hours to 10 days, from 2 hours to 5 days, from 2 hours to 3 days, from 2 hours to 2 days, from 2 hours to 1 day or from 2 hours to 10 hours. The extraction may include mixing the whole galgeun or seoritae, a part thereof or a material derived therefrom in the solvent and leaving it for a certain period of time. The leaving it may include appropriate stirring. The extraction may be repeated one or more times, for example, from 1 to 5 times. Specifically, the above extraction may be performed for 72 hours in the case of galgeun and for 24 hours in the case of seoritae.

[0025] The above extraction can be performed by separating the residue and the extract by a known method such as filtration. In addition, the extraction can include removing the solvent from the obtained extract by a known method such as vacuum concentration, rotary evaporation, etc. For example, the galgeun extract can be concentrated to 30 to 40 Brix%, and the seoritae extract can be concentrated to 10 to 20 Brix%. In addition, the extraction can include preparing a dry extract by drying the obtained extract by a method such as freeze-drying and spray-drying.

[0026] The term "fermentation" can mean the process by which microorganisms such as bacteria break down organic compounds to produce useful organic substances, such as alcohols, organic acids, and carbon dioxide, while "fermented product" can mean the process of physically or chemically denaturing or transforming raw materials using lactic acid bacteria.

[0027] According to one specific example, the fermented product may be obtained by fermenting an extract dilution obtained by diluting the extract in water, for example, sterilized distilled water.

[0028] Specifically, the dilution may include adding sterile distilled water to the galangal extract by 1 to 50 (w / w) times, for example, 1 to 40 (w / w) times, 1 to 30 (w / w) times, 2 to 50 (w / w) times, 2 to 40 (w / w) times, 2 to 30 (w / w) times, 3 to 50 (w / w) times, 3 to 40 (w / w) times or 3 to 30 (w / w) times, and specifically, adding 4 to 8 times.

[0029] In addition, the dilution may include adding sterilized distilled water to the frost extract by 1 to 50 (w / w) times, for example, 1 to 40 (w / w) times, 1 to 30 (w / w) times, 2 to 50 (w / w) times, 2 to 40 (w / w) times, 2 to 30 (w / w) times, 3 to 50 (w / w) times, 3 to 40 (w / w) times or 3 to 30 (w / w) times, and specifically, 9 to 13 times.

[0030] In one specific example, the fermented product may be fermented with a strain of the genus Lactiplantibacillus.

[0031] The above Lactiplantibacillus strains are Lactiplantibacillus argentoratensis, Lactiplantibacillus daoliensis, Lactiplantibacillus daowaiensis, Lactiplantibacillus dongliensis, Lactiplantibacillus fabifermentans, Lactiplantibacillus garii, Lactiplantibacillus herbarum, Lactiplantibacillus modestisalitolerans, Lactiplantibacillus Lactiplantibacillus mudanjiangensis, Lactiplantibacillus nangangensis, Lactiplantibacillus paraplantarum, Lactiplantibacillus pentosus, Lactiplantibacillus pingfangensis, Lactiplantibacillus plajomi, Lactiplantibacillus plantarum, Lactiplantibacillus songbeiensis, Lactiplantibacillus xiangfangensis, and specifically, The strain of the genus Lactiplantibacillus may be Lactiplantibacillus plantarum.

[0032] In one specific example, the Lactobacillus genus strain may be Lactobacillus plantarum CCHR1 deposited under accession number KCCM13337P or Lactobacillus plantarum CCHR2 deposited under accession number KCCM13338P.

[0033] In one specific example, the Lactobacillus plantarum CCHR1 deposited under the accession number KCCM13337P may include a 16s rRNA gene consisting of a base sequence of SEQ ID NO: 1. In addition, the Lactobacillus plantarum CCHR2 deposited under the accession number KCCM13338P may include a 16s rRNA gene consisting of a base sequence of SEQ ID NO: 2.

[0034] More specifically, the fermented galgeun may be manufactured by fermenting the galgeun extract with the Lactobacillus plantarum CCHR1, and the fermented seoritae may be manufactured by fermenting the seoritae extract with the Lactobacillus plantarum CCHR2.

[0035] For the above fermentation, any one selected from the group consisting of a Lactiplantibacillus plantarum strain, a culture medium of the strain, a fraction of the strain, and a mixture thereof may be inoculated into the galgeun or seoritae extract. Specifically, the fermentation may be performed by inoculating a culture medium of the strain.

[0036] In one specific example, the inoculation concentration (%) of the strain culture may be 0.01 to 10%, 0.02 to 9%, 0.02 to 8%, 0.05 to 7%, 0.1 to 6%, 0.1 to 5%, 0.5 to 4%, 0.5 to 3%, 0.9 to 3%, or 0.9 to 2%. Specifically, the inoculation concentration of the strain may be 1%.

[0037] In one specific example, the fermentation temperature may be 20 to 40°C, for example, 30 to 40°C or 35 to 40°C. Specifically, the fermentation temperature may be 37°C.

[0038] In one specific example, the fermentation pH may be 5 to 9, for example, 5.5 to 9, 5.5 to 8.5, 6 to 8.5, 6 to 8, 6.5 to 8, 6.5 to 7.5. Specifically, the fermentation pH may be 7.

[0039] In one specific example, the fermentation time may be 10 to 100 hours, for example, 10 to 90 hours, 20 to 80 hours, 20 to 70 hours, 30 to 60 hours, 40 to 50 hours, or 45 to 50 hours. Specifically, the fermentation time may be 48 hours.

[0040] The above-mentioned culture temperature, pH, and time can be selected to be sufficient for fermentation to occur, and these can be appropriately selected by those skilled in the art by considering the amount of fermentation raw material, type of strain, etc.

[0041] In addition, the fermented product according to the aspect may be interpreted to include the fermented product itself and all forms of fermented product that can be formed using the fermented product, such as a fermented liquid obtained by fermenting an extract, a diluted or concentrated liquid of the fermented liquid, a dried product or powder obtained by drying the fermented liquid, a controlled or purified product of the fermented liquid, or a mixture thereof.

[0042] Additionally, in one specific example, the composition may include a glycoside and an aglycone of phytoestrogen.

[0043] In one specific example, the fermented product may have an increased content of non-glycoside isoflavones compared to the extract before fermentation due to bioconversion caused by fermentation.

[0044] As used herein, the term "bioconversion" refers to the process of converting an added substance into a chemically modified form by utilizing the physiological functions of an organism. Specifically, the bioconversion may refer to the conversion of glycoside isoflavones in extracts of galgeun or seoritae into aglycone isoflavones.

[0045] The term "isoflavone" refers to a subgroup of flavonoids, plant secondary metabolites found primarily in the legume family (Fabaceae / Leguminosae), which includes soybeans. They are also called "phytoestrogens" because they possess functions similar to the female hormone estrogen.

[0046] The term "glycoside" refers to a general term for a substance widely distributed in the plant kingdom that can be broken down into a sugar portion and a non-sugar portion called aglycone or genin by the action of acid, alkali, or appropriate hydrolytic enzymes.

[0047] The above glycoside isoflavone may be at least one selected from the group consisting of baicalin, wogonoside, daidzin, genistin, glycitin, saponin, procyanidin, naringenin, rutinose, hesperidin, mogroside V, amygdalin, and 3-phenyl coumarin, and specifically, may be at least one selected from the group consisting of puerarin, daidzin, and genistin.

[0048] The term "aglycone" refers to a substance from which the sugar portion has been removed from a glycoside, which can be obtained by hydrolysis of the glycoside.

[0049] The above non-glycosidic isoflavone may be at least one selected from the group consisting of baicalein, wogonin, genistein, daidzein, glycitein, sapogenin, 3,4-hydroxyphenylacetic acid, 4-HPA, m-coumaric acid, p-coumaric acid, O-beta-D-glucuronides, stilbenoids, rutin, hesparetin, mogroside IIIE, mendelonitrile and benzaldehyde, and specifically, one selected from the group consisting of daidzein and genistein. It could be strange.

[0050] In one specific example, the extract of galangal or a fermented product thereof in the composition; And the mixing ratio of the above-mentioned frost extract or its fermented product may be 1:20 to 20:1, for example, 1:20 to 20:1, 1:20 to 15:1, 1:20 to 10:1, 1:20 to 9:1, 1:20 to 8:1, 1:20 to 7:1, 1:20 to 6:1, 1:20 to 5:1, 1:20 to 4:1, 1:20 to 3:1, 1:20 to 2:1, 1:20 to 1:1, 1:15 to 20:1, 1:15 to 15:1, 1:15 to 10:1, 1:15 to 9:1, 1:15 to 8:1, 1:15 to 7:1, 1:15 to 6:1, 1:15 to 5:1, 1:15 to 4:1, 1:15 to 3:1, 1:15 to 2:1, 1:15 to 1:1, 1:10 to 20:1, 1:10 to 15:1, 1:10 to 10:1, 1:10 to 9:1, 1:10 to 8:1, 1:10 to 7:1, 1:10 to 6:1, 1:10 to 5:1, 1:10 to 4:1, 1:10 to 3:1, 1:10 to 2:1, 1:10 to 1:1, 1:9 to 20:1, 1:9 to 15:1, 1:9 to 10:1, 1:9 to 9:1, 1:9 to 8:1, 1:9 to 7:1, 1:9 to 6:1, 1:9 to 5:1, 1:9 to 4:1, 1:9 to 3:1, 1:9 to 2:1, 1:9 to 1:1, 1:8 to 20:1, 1:8 to 15:1, 1:8 to 10:1, 1:8 to 9:1, 1:8 to 8:1, 1:8 to 7:1, 1:8 to 6:1, 1:8 to 5:1, 1:8 to 4:1, 1:8 to 3:1, 1:8 to 2:1, 1:8 to 1:1, 1:7 to 20:1, 1:7 to 15:1, 1:7 to 10:1, 1:7 to 9:1, 1:7 to 8:1, 1:7 to 7:1, 1:7 to 6:1, 1:7 to 5:1, 1:7 to 4:1, 1:7 to 3:1,1:7 to 2:1, 1:7 to 1:1, 1:6 to 20:1, 1:6 to 15:1, 1:6 to 10:1, 1:6 to 9:1, 1:6 to 8:1, 1:6 to 7:1, 1:6 to 6:1, 1:6 to 5:1, 1:6 to 4:1, 1:6 to 3:1, 1:6 to 2:1, 1:6 to 1:1, 1:5 to 20:1, 1:5 to 15:1, 1:5 to 10:1, 1:5 to 9:1, 1:5 to 8:1, 1:5 to 7:1, 1:5 to 6:1, 1:5 to 5:1, 1:5 to 4:1, 1:5 to 3:1, 1:5 to 2:1, 1:5 to 1:1, 1:4 to 20:1, 1:4 to 15:1, 1:4 to 10:1, 1:4 to 9:1, 1:4 to 8:1, 1:4 to 7:1, 1:4 to 6:1, 1:4 to 5:1, 1:4 to 4:1, 1:4 to 3:1, 1:4 to 2:1, 1:4 to 1:1, 1:3 to 20:1, 1:3 to 15:1, 1:3 to 10:1, 1:3 to 9:1, 1:3 to 8:1, 1:3 to 7:1, 1:3 to 6:1, 1:3 to 5:1, 1:3 to 4:1, 1:3 to 3:1, 1:3 to 2:1, 1:3 to 1:1, 1:2 to 20:1, 1:2 to 15:1, 1:2 to 10:1, 1:2 to 9:1, 1:2 to 8:1, 1:2 to 7:1, 1:2 to 6:1, 1:2 to 5:1, 1:2 to 4:1, 1:2 to 3:1, 1:2 to 2:1, 1:2 to 1:1, 1:1 to 20:1, 1:1 to 15:1, 1:1 to 10:1, 1:1 to 9:1, 1:1 to 8:1, 1:1 to 7:1, 1:1 to 6:1, 1:1 to 5:1, 1:1 to 4:1, 1:1 to It can be 3:1 or 1:1 to 2:1.

[0051] In addition, in one specific example, the mixing ratio of the galgeun extract or a fermented product thereof and the seoritae extract or a fermented product thereof in the composition may be a weight ratio or a volume ratio, and specifically, may be a weight ratio.

[0052] The composition comprises both the extract of galgeun or a fermented product thereof and the extract of seoritae or a fermented product thereof, thereby including both glycosides and aglycones of various phytoestrogens. Accordingly, the composition has excellent in vivo absorption rate and efficacy, and thus has excellent efficacy in preventing, improving, or treating menopausal syndrome, bone disease, and / or dyslipidemia in women.

[0053] In addition, in one specific example, the composition may be obtained by mixing galgeun and seoritae and then extracting or fermenting them. In addition, according to one specific example, the composition may be obtained by mixing extracts or fermented products of galgeun or seoritae, respectively. When the composition is prepared by extracting each raw material and then mixing the extracted extracts, not only can the efficacy of the raw materials be maintained, but the superior efficacy can be increased compared to preparing the extract by mixing the raw materials and then extracting them.

[0054] The term "menopause" refers to the period of physical and psychological changes associated with menopause, which occurs as ovarian function declines due to disease or aging. It encompasses both the premenopausal and postmenopausal stages, and decline in ovarian function and the decrease in the female hormone estrogen are known to be the primary contributing factors.

[0055] In one specific example, the composition may increase the concentration of estradiol in the blood.

[0056] Additionally, in one specific example, the composition may increase the expression amount of ERβ (Estrogen Receptor β), and specifically, may increase the expression amount of ERβ (Estrogen Receptor β) without changing the expression amount of ERα (Estrogen Receptor α).

[0057] In one specific example, the menopausal syndrome may be at least one selected from the group consisting of facial flushing, vasomotor disorders, sweating, depression, nervousness, dizziness, fatigue, sleep disorders, anxiety, concentration disorders, memory loss, nervousness, cardiovascular disease, bone disease, obesity, muscle pain, headache, palpitations, feeling of being uneasy, dry skin, cystitis, dysuria, vaginitis, and vaginal dryness.

[0058] Additionally, in one specific example, the bone disease or dyslipidemia may be due to menopausal syndrome in women.

[0059] In one specific example, the composition may promote differentiation, activation, and calcification of osteoblasts. Specifically, the composition may increase ALP (Alkaline phosphatase) activity and increase the expression levels of ALP, Runx2 (Runt-related transcription factor 2), or OCN (Osteocalcin), which are factors related to osteoblast formation and calcification. The term "expression level" includes not only protein expression level but also mRNA expression level.

[0060] In addition, the composition may inhibit the activity of osteoclasts. Specifically, the composition may regulate the RANKL / OPG signaling pathway by reducing the concentration of RANKL (Receptor activator of nuclear factor kappa-B ligand) in the blood, thereby reducing the RANKL / OPG ratio. In addition, the composition may reduce the concentrations of CTx (C-telopeptide of collagen type 1) and NTx (N-telopeptide of collagen type 1) in the blood. In addition, in one specific example, the composition may increase bone density.

[0061] In one specific example, the bone disease may be at least one selected from the group consisting of osteoporosis, osteomalacia, osteopenia, bone defect, bone atrophy, osteonecrosis, fibrous dysplasia, osteitis fibrosa cystica, Paget's disease, fracture, osteolysis, osteoarthritis, osteogenesis imperfecta, rickets, rheumatoid arthritis, hip dysplasia, and osteodystrophy.

[0062] In one specific example, the composition may reduce at least one selected from the group consisting of total cholesterol, low-density lipoprotein cholesterol (LDL-cholesterol), and triglycerides in the blood, and increase high-density lipoprotein cholesterol (HDL-cholesterol) in the blood. Specifically, the composition may reduce at least one selected from the group consisting of total cholesterol and low-density lipoprotein cholesterol (LDL-cholesterol).

[0063] Additionally, in one specific embodiment, the composition may increase relaxation of blood vessels. Specifically, the composition may increase at least one selected from the group consisting of blood nitric oxide concentration, blood endothelial nitric oxide synthase (eNOS) concentration, blood cyclic guanosine monophosphate (cGMP) concentration, and blood cyclic GMP-dependent protein kinase (cGMP-dependent protein kinase, protein kinase G, PKG) concentration, and more specifically, may increase all of the blood nitric oxide concentration, blood endothelial nitric oxide synthase (eNOS) concentration, blood cyclic guanosine monophosphate (cGMP) concentration, and blood cyclic GMP-dependent protein kinase (cGMP-dependent protein kinase, protein kinase G, PKG) concentration.

[0064] Additionally, in one specific example, the dyslipidemia may be at least one selected from the group consisting of hyperlipidemia, hyper-LDL cholesterolemia, hypertriglyceridemia, and hypo-HDL cholesterolemia.

[0065] The term "dyslipidemia" refers to a vascular disease caused by an increase in total cholesterol, low-density lipoprotein cholesterol (LDL-cholesterol), and triglycerides in the plasma, or a decrease in high-density lipoprotein cholesterol (HDL-cholesterol). It also refers to symptoms such as blood circulation disorders in which blood supply to various organs and extremities is not smooth due to the deposition of cholesterol, etc. on the inner walls of the arteries, narrowing the inner surface of the arteries.

[0066] The above term “prevention” may mean any action of suppressing or delaying menopausal syndrome, bone disease and / or dyslipidemia in a subject woman by administering a pharmaceutical composition according to one aspect.

[0067] The above term “treatment” may mean any action that improves or beneficially changes the symptoms of menopausal syndrome, bone disease and / or dyslipidemia in a subject woman by administering a pharmaceutical composition according to one aspect.

[0068] The term "administration" means introducing a given substance into a subject in an appropriate manner, and "subject" means any living organism, including humans, rats, mice, cows, horses, pigs, dogs, sheep, goats, cats, or any livestock that may have menopausal syndrome, bone disease, and / or dyslipidemia in women. A specific example may be a mammal, including humans.

[0069] The term “pharmaceutical composition” may refer to a molecule, compound, protein, extract, fermentation, mixture, or the like that, when administered to a subject, imparts several beneficial effects. Beneficial effects may include enabling diagnostic determination; improving a disease, symptom, disorder, or condition; reducing or preventing the onset of a disease, symptom, disorder, or condition; and generally counteracting a disease, symptom, disorder, or condition.

[0070] In addition, the pharmaceutical composition may be provided as a pharmaceutical composition comprising, as active ingredients, the extract of Pueraria lobataroot or a fermented product thereof; and the extract of Glycine max (L.) Merrill or a fermented product thereof, alone or together with one or more pharmaceutically acceptable carriers, excipients, or diluents.

[0071] The above term "including as an active ingredient" may mean that the fermented product is added to an extent that it can exhibit the above-mentioned effect, and may include formulation in various forms by adding various components as auxiliary components for drug delivery and stabilization, etc.

[0072] Specifically, the carrier may be, for example, a colloidal suspension, powder, saline, lipid, liposome, microspheres, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, or nano-spheres. These may form a complex with or be associated with a carrier vehicle, and may be delivered in vivo using a carrier system known in the art, such as lipids, liposomes, microparticles, gold, nanoparticles, polymers, condensation agents, polysaccharides, polyamino acids, dendrimers, saponins, adsorption enhancing substances, or fatty acids. In addition, other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added as needed. Furthermore, the carrier may be formulated according to each disease or component using an appropriate method in the art.

[0073] When the above pharmaceutical composition is formulated, it can be formulated using diluents or excipients such as commonly used lubricants, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, fillers, bulking agents, binders, wetting agents, disintegrating agents, surfactants, anticoagulants, pH regulators, nutrients, vitamins, electrolytes, alginic acid and its salts, pectic acid and its salts, protective colloids, glycerin, or preservatives. Solid preparations for oral administration may include tablets, pills, powders, granules, soft or hard capsules, and such solid preparations can be formulated by mixing at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc., with the composition. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups, etc. In addition to commonly used simple diluents such as water and liquid paraffin, they may contain various excipients such as wetting agents, sweeteners, fragrances, and preservatives. Preparations for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories, creams, lotions, ointments, pastes, aerosols, fluid extracts, elixirs, instillations, sachets, patches, or injections. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As a suppository base, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin, etc. can be used, and when manufacturing in the form of eye drops, known diluents or excipients can be used.

[0074] In one aspect, the pharmaceutical composition may further include a pharmaceutical composition for preventing or treating menopausal syndrome, bone disease and / or dyslipidemia in women other than the extract of Pueraria lobataroot or a fermented product thereof and the extract of Glycine max (L.) Merrill or a fermented product thereof.

[0075] Additionally, the pharmaceutical composition may be provided mixed with another pharmaceutical composition for preventing or treating menopausal syndrome, bone disease and / or dyslipidemia in women.

[0076] The pharmaceutical composition for preventing or treating menopausal syndrome, bone disease and / or dyslipidemia in other women includes a pharmaceutical composition for preventing or treating menopausal syndrome, bone disease and / or dyslipidemia in women known in the art or a newly developed pharmaceutical composition for preventing or treating menopausal syndrome, bone disease and / or dyslipidemia in women.

[0077] When the above pharmaceutical composition further comprises a pharmaceutical composition for preventing or treating menopausal syndrome, bone disease and / or dyslipidemia in other women or is provided mixed with a pharmaceutical composition for preventing or treating menopausal syndrome, bone disease and / or dyslipidemia in other women, it is important to mix an amount that can obtain the maximum effect with the minimum amount without causing side effects, which can be easily determined by a person skilled in the art.

[0078] The pharmaceutical composition may be administered alone or in combination with another pharmaceutical composition for the prevention or treatment of menopausal syndrome, bone disease, and / or dyslipidemia in women. That is, the composition may be administered in parallel without being mixed with other pharmaceutical compositions for the prevention or treatment of menopausal syndrome, bone disease, and / or dyslipidemia in women other than the Pueraria lobataroot extract or its fermented product; and the Glycine max (L.) Merrill extract or its fermented product, and may be administered simultaneously, separately, or sequentially, and may be administered singly or in multiple doses. It is important to administer an amount that can achieve the maximum effect with the minimum amount without side effects by taking all of the above factors into consideration, and this can be easily determined by those skilled in the art.

[0079] The pharmaceutical composition may be administered orally or parenterally, and when administered parenterally, it may be administered by external application to the skin or by intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, intramuscular injection, intraarterial injection, intramedullary injection, intracardiac injection, intrathecal injection, percutaneous injection, intranasal injection, intraenteric injection, local injection, sublingual injection, or intrathoracic injection.

[0080] The pharmaceutical composition is administered in a pharmaceutically effective amount. The term “pharmaceutically effective amount” means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment. The effective dosage level may be determined based on the type and severity of the patient’s disease, the activity and sensitivity of the drug, the time of administration, the route of administration and excretion rate, the duration of treatment, concomitant medications, and other factors well known in the medical field. For example, the pharmaceutical composition may be administered at a dose of 0.001 to 10,000 mg / kg / day. The administration may be administered once a day or in several divided doses. For example, it may be administered every other day or once a week.

[0081]

[0082] Another aspect provides a health functional food for preventing or improving menopausal syndrome, bone disease or dyslipidemia in women, comprising an extract of Pueraria lobataroot or a fermented product thereof; and an extract of Glycine max(L.) Merrill or a fermented product thereof.

[0083] The above “galgeun”, “seoritae”, “extract”, “fermented product”, “menopause”, “menopausal syndrome”, “bone disease”, “dyslipidemia”, “prevention”, etc. may be within the aforementioned scope.

[0084] The term "improvement" may refer to any action that at least reduces the severity of a parameter associated with the condition being treated, such as symptoms. In this case, the health functional food may be used prior to or after the onset of the disease, concurrently with or separately from a therapeutic agent, to prevent or improve menopausal syndrome, bone disease, and / or dyslipidemia in women.

[0085] The above term "health functional food" refers to a food manufactured or processed using a specific ingredient as a raw material or a specific ingredient contained in a food raw material through extraction, concentration, purification, mixing, etc. for the purpose of health supplementation, and refers to a food designed and processed so that the above ingredient can sufficiently exert bioregulatory functions such as biodefense, regulation of biological rhythm, prevention and recovery of disease, etc. on the body. The above health functional food composition can perform functions related to the prevention and improvement of menopausal syndrome, bone disease, or dyslipidemia in women. The above health functional food includes all health foods in the conventional sense.

[0086] In the above health functional food, the active ingredient can be added directly to the food or used in combination with other foods or food ingredients, and can be used appropriately according to conventional methods. The amount of the active ingredient mixed can be appropriately determined depending on the intended use (prevention or improvement). Generally, when manufacturing a food or beverage, the health functional food can be added in an amount of about 15% by weight or less, more specifically about 10% by weight or less, based on the raw material. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount may be below the above range.

[0087] Specifically, the extract of Pueraria lobataroot or a fermented product thereof in the health functional food; And the content of the extract of Glycine max (L.) Merrill or its fermented product is 0.00001 wt% to 80 wt%, for example, 0.00001 wt% to 60 wt%, 0.00001 wt% to 40 wt%, 0.00001 wt% to 30 wt%, 0.00001 wt% to 20 wt%, 0.00001 wt% to 10 wt%, 0.00001 wt% to 5 wt%, 0.05 wt% to 60 wt%, 0.05 wt% to 40 wt%, 0.05 wt% to 30 wt%, 0.05 wt% to 20 wt%, 0.05 wt% to 10 wt%, 0.05 wt% to 5 wt%, 0.1 wt% to 60 It can be 0.1 wt% to 40 wt%, 0.1 wt% to 30 wt%, 0.1 wt% to 20 wt%, 0.1 wt% to 10 wt%, or 0.1 wt% to 5 wt%. It can be suitably determined depending on the intended use.

[0088] The above health functional food may be manufactured in the form of tablets, pills, powders, granules, powders, capsules, liquids, gels, jellies, suspensions, emulsions, syrups, tea bags, infused teas, or health drinks, by further including one or more of carriers, diluents, excipients, and additives. Foods to which extracts, fermented products, or mixtures thereof may be added according to one aspect include various foods, powders, granules, tablets, capsules, syrups, beverages, gums, teas, vitamin complexes, and health functional foods.

[0089] Specific examples of the carrier, excipient, diluent and additive may include at least one selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, erythritol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium phosphate, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, polyvinylpyrrolidone, methylcellulose, water, sugar syrup, methylcellulose, methyl hydroxy benzoate, propyl hydroxy benzoate, talc, magnesium stearate and mineral oil.

[0090] In addition to containing the above-mentioned effective ingredient, the above-mentioned health functional food may contain other ingredients as essential ingredients without special restrictions. For example, it may contain various flavoring agents or natural carbohydrates as additional ingredients, as in conventional beverages. Examples of the above-mentioned natural carbohydrates may include conventional sugars such as monosaccharides, such as glucose, fructose, etc.; disaccharides, such as maltose, sucrose, etc.; and polysaccharides, such as dextrin, cyclodextrin, etc.; and sugar alcohols, such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (thaumatin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) may be advantageously used. The proportion of the above-mentioned natural carbohydrates may be appropriately determined by a person skilled in the art.

[0091] In addition to the above, health functional foods according to one aspect may contain various nutrients, vitamins, minerals (electrolytes), flavorings such as synthetic flavorings and natural flavorings, coloring agents and thickeners (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, they may contain fruit pulp for the production of natural fruit juices and vegetable beverages. These ingredients may be used independently or in combination, and the ratio of these additives may also be appropriately selected by those skilled in the art. In addition, the health functional foods may be in the form of any one of meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, gum, ice cream, soup, beverage, tea, functional water, drink, alcohol, and vitamin complex.

[0092] In addition, the above health functional food may additionally contain food additives, and its suitability as a “food additive” may be determined by the specifications and standards for the relevant item in accordance with the general provisions and general test methods of the Food Additives Codex approved by the Ministry of Food and Drug Safety, unless otherwise provided.

[0093] Items listed in the above "Food Additives Codex" include, for example, chemically synthesized products such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamic acid; natural additives such as persimmon pigment, licorice extract, crystalline cellulose, kohlrabi pigment, and guar gum; and mixed preparations such as sodium L-glutamate preparations, alkaline agents for noodles, preservative preparations, and tar color preparations.

[0094] In one aspect, the health functional food may further include a health functional food for preventing or improving menopausal syndrome, bone disease and / or dyslipidemia in women, in addition to a Pueraria lobataroot extract or a fermented product thereof and a Glycine max (L.) Merrill extract or a fermented product thereof.

[0095] In addition, the above health functional food may be provided in combination with other health functional foods for preventing or improving menopausal syndrome, bone disease, and / or dyslipidemia in women.

[0096] The health functional food for preventing or improving menopausal syndrome, bone disease and / or dyslipidemia in other women includes a health functional food for preventing or improving menopausal syndrome, bone disease and / or dyslipidemia in women that is known in the past or a health functional food for preventing or improving menopausal syndrome, bone disease and / or dyslipidemia in women that is newly developed.

[0097] When the above health functional food further includes a health functional food for preventing or improving menopausal syndrome, bone disease and / or dyslipidemia in women, or is provided mixed with another health functional food for preventing or improving menopausal syndrome, bone disease and / or dyslipidemia in women, it is important to mix in an amount that can achieve the maximum effect with the minimum amount without causing side effects, and this can be easily determined by a person skilled in the art.

[0098] In addition, in one aspect, the health functional food may be consumed alone or in combination with other health functional foods for preventing or improving menopausal syndrome, bone disease, and / or dyslipidemia in women.

[0099] The above health functional food can be taken in conjunction with a known composition or a newly developed health functional food for the prevention or improvement of menopausal syndrome, bone disease, and / or dyslipidemia in women, and can be taken simultaneously, separately, or sequentially, and can be taken singly or in multiple doses. It is important to take all of the above factors into consideration and take the amount that achieves the maximum effect with the minimum amount without causing side effects, and this can be easily determined by those skilled in the art.

[0100]

[0101] Another aspect provides a method for preventing or treating menopausal syndrome, bone disease or dyslipidemia in women, comprising administering to a subject in need thereof an extract of Pueraria lobataroot or a fermented product thereof and an extract of Glycine max(L.) Merrill or a fermented product thereof.

[0102] The above “galgeun”, “seoritae”, “extract”, “fermented product”, “entity”, “administration”, “menopause”, “menopausal syndrome”, “bone disease”, “dyslipidemia”, “prevention”, “treatment”, etc. may be within the scope described above.

[0103]

[0104] Another aspect provides the use of an extract of Pueraria lobataroot or a fermented product thereof; and an extract of Glycine max(L.) Merrill or a fermented product thereof for the manufacture of a medicament for the prevention or treatment of menopausal syndrome, bone disease, or dyslipidemia in women.

[0105] The above “galgeun”, “seoritae”, “extract”, “fermented product”, “menopause”, “menopausal syndrome”, “bone disease”, “dyslipidemia”, “prevention”, “treatment”, etc. may be within the aforementioned scope.

[0106]

[0107] Another aspect provides a method for preparing a pharmaceutical composition for preventing or treating menopausal syndrome, bone disease or dyslipidemia in women, comprising the steps of extracting Pueraria lobataroot or Glycine max(L.) Merrill to obtain a Pueraria lobata extract or a Pueraria lobata extract; fermenting the Pueraria lobata extract or the Pueraria lobata extract to obtain a fermented Pueraria lobata product or a fermented Pueraria lobata product; and mixing the Pueraria lobata extract or the fermented product thereof and the Pueraria lobata extract or the fermented product thereof.

[0108] The above “galgeun”, “seoritae”, “extract”, “fermented product”, “menopause”, “menopausal syndrome”, “bone disease”, “dyslipidemia”, “prevention”, “treatment”, etc. may be within the aforementioned scope.

[0109]

[0110] A composition according to one aspect comprises a familiar natural product, a galgeun extract or a fermented product thereof; and a seoritae extract or a fermented product thereof, thereby including both glycosides and aglycones of various phytoestrogens. Accordingly, the composition has excellent in vivo absorption rate and efficacy, and thus has excellent efficacy in preventing, improving, or treating menopausal syndrome, bone disease, and / or dyslipidemia in women. Compared to existing products proven to be helpful for the health of menopausal women, the composition can be utilized as a low-dose, high-efficiency product with high palatability, and can secure excellent competitiveness in the domestic and international markets for women's health functional foods.

[0111]

[0112] Figure 1 is a diagram showing the results of confirming the effect of the mixtures of Examples 1 to 3 on ALP activation.

[0113] Figures 2 and 3 are diagrams showing the results of confirming the effects of the mixtures of Examples 1 to 3 on the mRNA expression levels of factors related to osteoblast formation and calcification.

[0114] Figure 3 is a diagram showing the results of confirming the effect of the mixtures of Examples 1 to 3 on the mineralization of osteoblasts.

[0115] Figure 4 is a diagram showing the results of confirming the effects of the mixtures of Examples 1 to 3 on the blood estradiol concentration.

[0116] Figures 5 and 6 are diagrams showing the results of confirming the effects of the mixtures of Examples 1 to 3 on the expression levels of ERα and ERβ in the uterus.

[0117] Figure 7 is a diagram showing the results of confirming the effects of the mixtures of Examples 1 to 3 on femoral bone density.

[0118] Figure 8 is a diagram showing the results of confirming the effects of the mixtures of Examples 1 to 3 on the concentrations of RANKL and OPG in blood.

[0119] Figure 9 is a diagram showing the results of confirming the effects of the mixtures of Examples 1 to 3 on the concentrations of CTx and NTx in blood.

[0120] Figure 10 is a diagram showing the results of confirming the effects of the mixtures of Examples 1 to 3 on blood lipid levels.

[0121] Figure 11 is a diagram showing the results of confirming the effects of the mixtures of Examples 1 to 3 on the concentration of nitric oxide in the blood.

[0122] Figure 12 is a diagram showing the results of confirming the effects of the mixtures of Examples 1 to 3 on the concentrations of aortic vascular endothelial cell-type NOS, cyclic guanosine monophosphate, and cyclic GMP-dependent protein kinase.

[0123]

[0124] The present invention will be described in more detail below through examples. However, these examples are intended to exemplify the present invention and the scope of the present invention is not limited to these examples.

[0125]

[0126] Manufacturing example - Manufacturing of extract, fermented product and fermented product powder of galgeun or seoritae

[0127] Manufacturing Example 1. Manufacturing of galgeun and seoritae extracts

[0128] (1) Preparation of galgeun extract

[0129] Domestically produced dried galangal was purchased from Gwangmyeongdang Pharmaceutical Co., Ltd. (Ulsan, Korea) and an extract was prepared. 120 g of the selected galangal was placed in a non-woven mesh strainer and 4 times the volume of the original weight of 60% ethanol (480 mL) was added. The ethanol extract of galangal was obtained by stationary extraction at room temperature for 72 hours. For fermentation, the ethanol extract of the galangal was concentrated to 36.6 Brix% using a rotary evaporator (N-1300V-W, EYELA, Japan) to obtain a fermentable galangal extract.

[0130]

[0131] (2) Preparation of frost extract

[0132] Domestically produced dried seoritae was purchased from Sangju Ijang Farm (Sangju, Republic of Korea) and an extract was prepared. 120 g of the selected seoritae was added to sterilized distilled water (240 mL) twice the amount of the original amount. The seoritae was then soaked for more than 14 hours at room temperature, steamed at 121°C for 40 minutes, and crushed. 1,200 mL of 60% ethanol (10 times the amount of the original amount) was added to the crushed seoritae, and the mixture was allowed to stand at room temperature for 24 hours to obtain a seoritae ethanol extract. For fermentation, the seoritae ethanol extract was concentrated to 13.3 Brix% using a rotary evaporator to obtain a seoritae extract for fermentation.

[0133]

[0134] Manufacturing Example 2. Cultivation of fermenting microorganisms

[0135] Lactiplantibacillus plantarum CCHR1 (KCCM13337P) or Lactiplantibacillus plantarum CCHR2 (KCCM13338P), strains deposited at the Korea Center for Microorganisms (KCCM), were cultured in MRS medium for 24 hours. Afterwards, the culture broth from which the strains grew was inoculated at a concentration of 1% and subcultured for 4 hours, and the OD 600Subcultures with values ​​ranging from 0.5 to 1.0 were used to produce fermented products.

[0136]

[0137] Manufacturing Example 3. Manufacturing of fermented galgeun and seoritae

[0138]

[0139] (1) Production of fermented galgeun product

[0140] The kudzu root extract prepared in the above Manufacturing Example 1-(1) was diluted with sterilized distilled water, and the subculture of Lactiplantibacillus plantarum CCHR1 cultured by the method of Manufacturing Example 2 was inoculated at a concentration of 1%. The kudzu root extract inoculated with the strain was fermented for 48 hours at 37°C, pH 6.5, and 100 rpm using a shaking incubator (KSI-300, KBT Co., Ltd., Republic of Korea), and then the kudzu root fermentation product was obtained.

[0141]

[0142] (2) Production of fermented frostfish

[0143] The frost extract prepared in the above Manufacturing Example 1-(2) was diluted with sterilized distilled water and inoculated with a subculture of Lactiplantibacillus plantarum CCHR2 cultured by the method of Example 2.3 at a concentration of 1%. The frost extract inoculated with the above strain was fermented using a shaking incubator at 37°C for 48 hours, pH 6.5, and 100 rpm, and then a frost fermented product was obtained.

[0144]

[0145] Manufacturing Example 4. Establishment of Optimal Fermentation Conditions for Fermented Galangal Root

[0146] (1) Selection of the optimal dilution concentration of galangal extract

[0147] In the method for producing a fermented galgeun product using the above Manufacturing Example 3-(1), the content of non-glycosides (daidzein, genistein) according to the dilution ratio with sterilized distilled water was analyzed. Specifically, the analysis of daidzein and genistein, which have phytoestrogen effects, was performed using high pressure liquid chromatography (HPLC, high press liquid chromatograph, Agilent 1200 / MS, Agilent Technologies, USA). The analytical column used was Phenomonex Kinetex C18, and the analysis was performed under the gradient conditions shown in Table 1 below. The results are shown in Table 2 below.

[0148]

[0149] Column: Phenomonex Kinetex C18 (4.6 x 250 mm) Mobile phase: A: Water B: Acetonitrile + 0.1% acetic acid Flow rate: 1.0 mL / min Injection volume: 50 μL Gradient elution time (min) Solvent (% B) 0-10 10-10 10-60 10-25 60-70 25-10 Wavelength: 254 nm

[0150]

[0151] 6-fold dilution of aglycone extract in fermented galgeun extract 4-fold dilution of extract 3-fold dilution of daidzein (mg / mL) 1.5 1.0 0.8

[0152]

[0153] The extract of the root was diluted 3-, 4-, and 6-fold with sterilized distilled water and cultured. The content of daidzein, an aglycone, was confirmed to be the highest in the 6-fold diluted fermented root. Therefore, the extract of the root was diluted 6-fold and fermented for subsequent fermentation.

[0154]

[0155] (2) Selection of optimal fermentation conditions for fermented galgeun

[0156] During the fermentation of the extract of galgeun, the optimal fermentation conditions were selected by changing the physical composition, such as pH adjustment and oxygen demand, and changing the chemical composition, such as adding a carbon source or nitrogen source. As shown in Tables 3 to 5 below, the fermented galgeun was produced by the method of Manufacturing Example 3-(1) by changing the physical and chemical composition, and the content of non-glycosides was analyzed in the same manner as in Manufacturing Example 4-(1).

[0157] The fermented Puerariae radix produced by the method of Manufacturing Example 3-(1), which was used as a control, contained nothing other than the Pueraria radix extract and CCHR1 strain. 1 mol / L-Sodium hydroxide (NaOH) solution or 1 mol / L-Hydrochloric acid (HCl) solution was used to adjust the pH, and Gas-pak culture was performed to create an anaerobic condition. In addition, 0.1% (w / v) L-cysteine ​​and / or 0.1% (w / v) L-arginine were used after filtration sterilization as a nitrogen source, and 1% (w / v) D-(+)-Glucose and / or 1% (w / v) D-(+)-Lactose were used after filtration sterilization as a carbon source.

[0158]

[0159] Experimental group Daidzein (mg / mL) Manufacturing example 3-(1) (Control group) 1.546 pH 7.0 control 1.569 pH 7.5 control 1.513 pH 8.0 control 1.468 Anaerobic culture 1.495 pH 7.0 control + anaerobic culture 1.557 pH 7.5 control + anaerobic culture 1.566 pH 8.0 control + anaerobic culture -

[0160]

[0161] Experimental group Daidzein (mg / mL) Manufacturing example 3-(1) (Control group) 1.534 Glucose 1.480 Lactose 1.467 Arginine 1.566 Cysteine ​​1.474 Glucose + Lactose 1.470 Glucose + Arginine 1.490 Glucose + Cysteine ​​- Lactose + Arginine 1.522 Lactose + Cysteine ​​1.468 Arginine + Cysteine ​​1.518 Glucose + Lactose + Arginine 1.483 Glucose + Lactose + Cysteine ​​- Glucose + Arginine + Cysteine ​​1.510 Lactose + Arginine + Cysteine ​​1.500 Glucose + Lactose + Arginine + Cysteine ​​1.485

[0162]

[0163] Experimental group Daidzein (mg / mL) Manufacturing example 3-(1) (Control group) 1.520 pH 7.0 adjustment 1.536 Arginine 1.518 Arginine + pH 7.0 adjustment 1.519

[0164]

[0165] As shown in Tables 3 to 5 above, the content of daidzein was confirmed to be high at pH 7, and through this, the optimal fermentation conditions for the fermented galgeun were established, and thereafter, the fermentation was performed at pH 7 when producing the fermented galgeun.

[0166]

[0167] Manufacturing Example 5. Establishment of Optimal Fermentation Conditions for Fermented Seoritae

[0168] (1) Selection of the optimal dilution concentration of the frost extract

[0169] In the method for producing fermented seoritae using the above Manufacturing Example 3-(2), the content of non-glycosides according to the dilution ratio of sterilized distilled water was analyzed using the same method as the above Manufacturing Example 4-(1). The results are shown in Table 6 below.

[0170]

[0171] 11-fold dilution of aglycone extract in fermented frost radish kimchi 6-fold dilution of extract 4-fold dilution of daidzein + genistein (㎍ / mL) 102.865.763.9

[0172]

[0173] The extract of Seoritae was diluted 4-fold, 6-fold, and 11-fold with sterilized distilled water and cultured. As a result, the content of non-glycosides daidzein and genistein was confirmed to be the highest in the 11-fold diluted Seoritae fermented product. Therefore, the Seoritae extract was diluted 11-fold and fermented for subsequent fermentation.

[0174] (2) Selection of optimal fermentation conditions for fermented frostfish

[0175] The optimal fermentation conditions were selected by changing the physical composition, such as pH adjustment and oxygen demand change, and chemical composition, such as adding carbon or nitrogen sources, during the fermentation of the Seoritae extract. As shown in Tables 7 and 8 below, the physical and chemical composition changes were applied to produce a Seoritae fermented product using the method of Manufacturing Example 3-(2), and the content of non-glycosides was analyzed using the same method as Manufacturing Example 4-(1).

[0176] The fermented product of Seoritae manufactured by the method of Manufacturing Example 3-(2), which was used as a control, contained nothing other than Seoritae extract and CCHR 2 strain. 1 mol / L-Sodium hydroxide (NaOH) solution or 1 mol / L-Hydrochloric acid (HCl) solution was used to adjust the pH, and Gas-pak culture was performed to create an anaerobic condition. In addition, 0.1% (w / v) L-cysteine ​​and / or 0.1% (w / v) L-arginine were used after filtration sterilization as a nitrogen source, and 1% (w / v) D-(+)-Glucose and / or 1% (w / v) D-(+)-Lactose were used after filtration sterilization as a carbon source.

[0177]

[0178] Experimental group Daidzein + Genistein (㎍ / mL) Manufacturing example 3-(2) (Control group) 143.3 pH 7.0 Control 118.7 pH 7.5 Control 107.6 pH 8.0 Control 115.8 Anaerobic culture 117.5 pH 7.0 Control + Anaerobic culture 110.8 pH 7.5 Control + Anaerobic culture 118.7 pH 8.0 Control + Anaerobic culture 107.2

[0179]

[0180] Experimental group Daidzein + Genistein (㎍ / mL) Manufacturing example 3-(2) (Control group) 143.3 Glucose 87.7 Lactose 115.6 Arginine 110.2 Cysteine ​​96.7 Glucose + Lactose 89.0 Glucose + Arginine 87.8 Glucose + Cysteine ​​87.4 Lactose + Arginine 107.3 Lactose + Cysteine ​​127.6 Arginine + Cysteine ​​112.5 Glucose + Lactose + Arginine 91.5 Glucose + Lactose + Cysteine ​​86.6 Glucose + Arginine + Cysteine ​​87.6 Lactose + Arginine + Cysteine ​​100.7 Glucose + Lactose + Arginine + Cysteine ​​28.3

[0181]

[0182] As shown in Tables 7 and 8 above, in the case of frost, the content of daidzein and genistein was confirmed to be the highest under the conditions of manufacturing example 3-(2) in which no treatment was performed, and through this, the optimal fermentation conditions for the frost fermented product were established, and thereafter, no treatment was performed in the manufacturing process as in 3-(2) during the frost fermentation.

[0183]

[0184] Manufacturing Example 6. Manufacturing of fermented powder

[0185] (1) Preparation of fermented galgeun powder

[0186] The fermented galangal product according to the above Manufacturing Example 4 was pulverized using a freeze dryer (FDS-7012, Operon, Korea) to obtain a fermented galangal powder. Hereinafter, the fermented galangal powder is also referred to as “FPE.”

[0187]

[0188] (2) Preparation of fermented frostfish powder

[0189] The fermented seoritae according to the above manufacturing example 5 was pulverized using a freeze dryer (FDS-7012, Operon, Korea) to obtain a fermented seoritae powder. Hereinafter, the fermented seoritae powder is also referred to as “FGE.”

[0190]

[0191] Comparative Example - Production of fermented galgeun or seoritae

[0192] Comparative Examples 1 to 6. Preparation of fermented galgeun under different fermentation conditions

[0193] A fermented galangal product was manufactured under the same optimal fermentation conditions established in Manufacturing Example 4, except that the fermented galangal product was manufactured under the conditions shown in Table 9 below.

[0194]

[0195] Experimental group Fermentation conditions Temperature pH Time Manufacturing example 4 Fermented galangal product 37℃ 748 Comparative example 115℃ 748 Comparative example 245℃ 748 Comparative example 337℃ 5.548 Comparative example 437℃ 8.548 Comparative example 537℃ 720 Comparative example 637℃ 760

[0196]

[0197] Comparative Examples 7 to 12. Preparation of fermented seoritae with different fermentation conditions Except that the fermented seoritae was prepared under the conditions according to Table 10, the fermented seoritae was prepared under the same optimal fermentation conditions established in Manufacturing Example 5.

[0198]

[0199] Experimental group Fermentation conditions Temperature pH Time Manufacturing example 5 Frost fermented product 37℃ 6.548 Comparative example 7 15℃ 6.548 Comparative example 8 45℃ 6.548 Comparative example 9 37℃ 5.548 Comparative example 10 37℃ 8.548 Comparative example 11 37℃ 6.520 Comparative example 12 37℃ 6.560

[0200]

[0201] Experimental Example 1 - Comparison of the aglycone content of fermented galgeun or seoritae according to fermentation conditions 1. Comparison of the aglycone content of fermented galgeun according to fermentation conditions

[0202] In order to compare the aglycone content of fermented galangal products manufactured under different fermentation conditions, the aglycone content of fermented galangal products of Manufacturing Example 4 and Comparative Examples 1 to 6 was analyzed using the same method as Manufacturing Example 4-(1). The results are shown in Table 11 below.

[0203]

[0204] Experimental group Daidzein (mg / mL) Fermented galangal of manufacturing example 4 1.532 Comparative example 10.854 Comparative example 2 - Comparative example 30.728 Comparative example 40.891 Comparative example 51.075 Comparative example 61.541

[0205]

[0206] As shown in Table 11 above, it was confirmed that galgeun fermentation did not proceed well when the temperature or pH was outside the appropriate range, and it was confirmed that the daidzein content did not increase significantly even though the fermentation time was extended. Through this, the conditions were established to carry out fermentation for 24 to 52 hours under the temperature conditions of 20 to 40℃ and the pH conditions of 6 to 8, which are the conditions in which the daidzein content is the highest.

[0207]

[0208] 2. Comparison of the non-glycoside content of fermented seoritae according to fermentation conditions

[0209] In order to compare the aglycone content of fermented products of Seoritae manufactured under different fermentation conditions, the aglycone content of fermented products of galgeun of Manufacturing Example 5 and Comparative Examples 7 to 12 was analyzed using the same method as Manufacturing Example 4-(1). The results are shown in Table 12 below.

[0210]

[0211] Experimental group Daidzein + Genistein (㎍ / mL) Fermented seoritae of Manufacturing Example 5 143.8 Comparative Example 778.1 Comparative Example 8 - Comparative Example 984.5 Comparative Example 1086.7 Comparative Example 11102.6 Comparative Example 12149.0

[0212] As shown in Table 12 above, it was confirmed that the fermentation of frostfish did not proceed well when the temperature or pH was outside the appropriate range, and it was confirmed that the contents of daidzein and genistein did not increase significantly even though the fermentation time was extended. Through this, the conditions were established to carry out the fermentation for 24 to 52 hours under the temperature conditions of 20 to 40℃ and the pH conditions of 6 to 8, which are the conditions in which the contents of daidzein and genistein are the best.

[0213]

[0214] Example

[0215] Example 1. Preparation of a mixture of fermented galgeun powder and fermented seoritae powder

[0216] The fermented galgeun powder of Manufacturing Example 6-(1) and the fermented seoritae powder of Manufacturing Example 6-(2) were mixed in a weight ratio of 1:9 to prepare the mixture of Example 1.

[0217]

[0218] Example 2. Preparation of a mixture of fermented galgeun powder and fermented seoritae powder

[0219] The fermented galgeun powder of Manufacturing Example 6-(1) and the fermented seoritae powder of Manufacturing Example 6-(2) were mixed in a weight ratio of 3:7 to prepare a mixture of Example 2.

[0220]

[0221] Example 3. Preparation of a mixture of fermented galgeun powder and fermented seoritae powder

[0222] The fermented galgeun powder of Manufacturing Example 6-(1) and the fermented seoritae powder of Manufacturing Example 6-(2) were mixed in a weight ratio of 9:1 to prepare a mixture of Example 3.

[0223]

[0224] Experimental Example 2 - In vitro efficacy confirmation of the mixtures of Examples 1 to 3

[0225] 1. Confirmation of differentiation and activation efficacy of osteoblasts (MC3T3-E1)

[0226] (1) Check whether ALP is activated

[0227] Since osteoblasts have the characteristic of exhibiting ALP (Alkaline phosphatase) activity, we attempted to measure the differentiation activity of osteoblasts by measuring ALP activity.

[0228] Specifically, for cell culture for ALP activity measurement, a medium containing 1% 100 units / mL penicillin-streptomycin (WELEGENE) and 10% FBS was used in MEMα (Gibco, 2451171) excluding ascorbic acid, and a 5% CO2 incubator was used. After the cells were stabilized, 2 x 10 were seeded in 24 wells. 4 Cells were divided into 10 cells / mL. After culturing for 24 hours, when it was confirmed that more than 70% proliferation had occurred, the cells were treated with a medium containing 10 mM β-glycerophosphate (Sigma, G9422-10G) and 50 μg / mL ascorbic acid (Sigma, 49725-10G) and a mixture of the fermented powder of galgeun (FPE) of Preparation Example 6-(1), the fermented powder of seoritae (FGE) of Preparation Example 6-(2), and the mixture of Example 1 (1 to 9), Example 2 (3 to 7), and Example 3 (9 to 1). The medium was changed every 3 days, and differentiation was performed for 120 hours. E2 (17β-Estradiol) was used as a positive control, and ALP activity was measured using an ALP assay kit (Sigma, MAK447) according to the experimental method provided by the manufacturer.

[0229] As a result, the degree of ALP activity was as shown in Fig. 1. Specifically, a mixture of galgeun and seoritae fermented powder (1 to 9, 3 to 7, 9 to 1; Examples 1 to 3) showed higher ALP activity than a single powder of galgeun or seoritae fermented powder (Preparation Example 6-(1) and (2)), and the significance was highest in the 1 to 9 and 3 to 7 mixtures (Examples 1 and 2).

[0230]

[0231] (2) Confirmation of expression level of differentiation factors related to osteoblast formation

[0232] Additionally, the mRNA expression levels of ALP, Runx2, and OCN, which are differentiation factors related to osteoblast formation, were also confirmed.

[0233] Specifically, cells were seeded into 6 wells in the same manner as in the second experimental example-1-(1). After culturing for 24 hours, when it was confirmed that the cells had proliferated by more than 70%, they were treated with a medium containing 10 mM β-glycerophosphate (Sigma, G9422-10G) and 50 ㎍ / mL ascorbic acid (Sigma, 49725-10G) and a mixture of the fermented galgeun powder (FPE) of Preparation Example 6-(1), the fermented seoritae powder (FGE) of Preparation Example 6-(2), and the mixtures of Example 1 (1 to 9), Example 2 (3 to 7), and Example 3 (9 to 1). Thereafter, the medium was changed every 3 days, and differentiation was performed for 168 hours. Thereafter, mRNA was extracted using Trizol. 20 ㎕ DEPC water was added to the pellet, and the mRNA concentration was measured using Nanodrop. In addition, cDNA was synthesized according to the ReversTra Ace qPCR RT Master mix with gDNA remover (FSQ-301; TOYOBO, Osaka, Japan) kit protocol, and qRT-PCR was performed using the primers shown in Table 13 below. Meanwhile, E2 (17β-Estradiol) was used as a positive control, and the expression level compared to GAPDH was presented in a graph.

[0234]

[0235] Gene primer sequence (5'→3') Sequence number GAD PHA GGG CAT CTG GGG CTA CACT 3 CACC CTG TTG GCT ALP CAG CAG CAG TTT CTC TGG 5 GGA ATG TTC CCAT GGA GTT G6 Runx 2 CCC TGA ACT CTG CAC CAA GT 7 TGG AGT GGA TGG ATG GGG AT 8 OCN AGC AGC TTG GCC CAG ACC TA 9 TAG CGC CGG AGT CTG TTC ACT AC 10

[0236]

[0237] As a result, the mRNA expression levels of osteoblast formation and calcification-related factors were as shown in Fig. 2. Specifically, it was confirmed that all osteoblast formation and calcification-related factors ALP, Runx2, and OCN significantly increased in the 3:7 mixture (Example 2).

[0238]

[0239] (3) Sintering

[0240] According to the results of the above figures 1 and 2, the mixtures of Example 1 (1 to 9), Example 2 (3 to 7), and Example 3 (9 to 1) had significant effects on osteoblast formation, and in particular, the 3 to 7 mixture (Example 2) had the most outstanding significant effect, so it could be judged that it is a raw material that can help prevent and alleviate osteoporosis or bone disease in women, especially menopausal women.

[0241]

[0242] 2. Confirmation of mineralization of osteoblasts

[0243] Alizarin red S staining was used to confirm osteoblast mineralization of the mixtures of Example 1 (1 to 9), Example 2 (3 to 7), and Example 3 (9 to 1). This is a method used to colorimetrically quantify bone tissue calcification.

[0244] Specifically, cell culture was performed using a medium containing 1% 100 units / mL penicillin-streptomycin (WELEGENE) and 10% FBS in MEMα (Gibco, 2451171) without ascorbic acid, and a 5% CO2 incubator. After the cells were stabilized, 1 x 10 were seeded in 24 wells. 5 Cells were divided into 10 cells / mL. After 24 hours of culture, when it was confirmed that more than 70% proliferation had occurred, the cells were treated with a medium containing 10 mM β-glycerophosphate (Sigma, G9422-10G) and 50 μg / mL ascorbic acid (Sigma, 49725-10G) and a mixture of the fermented galgeun powder (FPE) of Preparation Example 6-(1), the fermented seoritae powder (FGE) of Preparation Example 6-(2), and the mixtures of Example 1 (1 to 9), Example 2 (3 to 7), and Example 3 (9 to 1). Afterwards, the medium was changed every 3 days, and calcification was performed for 28 days. Calcification quantification was performed according to the experimental method provided by the manufacturer using an Alizarin Red S Quantification assay (ScienCell, 8678). Meanwhile, E2 (17β-Estradiol) was used as a positive control, and a medium with all components necessary for differentiation removed was used as a negative control.

[0245] As a result, it was as in Fig. 3, and it was confirmed that calcification occurred significantly, especially in the mixtures of Example 1 (1 to 9), Example 2 (3 to 7), and Example 3 (9 to 1).

[0246] In menopausal women, as the blood estrogen concentration decreases, the speed of bone destruction and bone formation changes, resulting in a decrease in bone density, and osteoporosis occurs in approximately 30% of women after menopause. According to the above results, the powder mixture of fermented galgeun and seoritae (Example 1 (1 to 9), Example 2 (3 to 7), and Example 3 (9 to 1)) promotes the formation of osteoblasts and has the function of calcifying bone cells, so it can be judged to be a raw material that can help prevent and alleviate osteoporosis or bone diseases in women, especially menopausal women.

[0247]

[0248] Experimental Example 3 - In vivo efficacy confirmation of the mixtures of Examples 1 to 3

[0249] 1. Preparation of menopausal animal model and oral administration of mixtures of Examples 1 to 3

[0250] The menopausal animal model induced by ovariectomy used in the present invention was purchased from Jabio Co., Ltd. (Suwon, Korea) and was used in accordance with experimental methods approved by the Institutional Animal Care and Use Committee (IACUC) of the CHA Bio Complex Laboratory Animal Center (Seongnam, Korea).

[0251] The administration concentration of the mixture of fermented galgeun and seoritae powders (Examples 1 to 3) was established as 5.2 mg / kg by dividing the average daily intake of 24 to 27 mg / day of soybean isoflavones in the standards and specifications for health functional foods of the Ministry of Food and Drug Safety by the conversion factor for a specific animal according to the Food and Drug Administration (FDA) guidelines to convert it into the human equivalent dose (HED). The fermented galgeun powder and the fermented seoritae powder were mixed at the weight ratios of 1:9, 3:7, and 9:1 at the established administration concentrations, and then dissolved in PBS and orally administered for 8 weeks, and the following experiments were conducted. Meanwhile, in the following experiments, instead of the powder mixture of fermented galgeun and seoritae, estradiol (17β-Estradiol, E2) was dissolved in PBS at a concentration of 10 μg / kg and administered intraperitoneally for 8 weeks as a positive control group, and PBS was administered orally for 8 weeks to the normal control group (Sham operation, Sham) and the negative control group (Ovariectomy, OVX).

[0252]

[0253] 2. Measurement of blood estradiol concentration

[0254] After oral administration, the experimental animals were anesthetized by inhalation, blood was collected from the abdominal vena cava, and the animals were sacrificed. After sacrifice, the whole blood was quickly added to a tube treated with heparin anticoagulant and gently shaken to mix the mouse whole blood with the heparin. After centrifugation at 4°C and 13,000 rpm for 20 minutes, plasma was collected and used for the measurement of estradiol (17β-estradiol, E2). Estradiol was measured using an enzyme-linked immunosorbent assay (ELISA) kit (Cusabio, USA) according to the experimental method provided by the manufacturer.

[0255] As a result, it was as in Fig. 4. Specifically, after administering the mixture of fermented galgeun and seoritae powder (Example 1 (1 to 9), Example 2 (3 to 7), and Example 3 (9 to 1)) for 8 weeks, the blood estradiol concentrations of Examples 1 to 3 increased by 46.7%, 40.6%, and 35.0%, respectively, compared to the control group, the OVX group, and increased to a level similar to that of the positive control group, the E2 group.

[0256] Bone diseases including osteoporosis and dyslipidemia that occur in menopausal women are caused by a decrease in blood estradiol concentration due to a decrease in ovarian function. When the powder mixture of fermented galgeun and seoritae (Example 1 (1 to 9), Example 2 (3 to 7), and Example 3 (9 to 1)) was ingested, the blood estradiol concentration was confirmed to increase. Therefore, it was determined that the powder mixture of fermented galgeun and seoritae (Example 1 (1 to 9), Example 2 (3 to 7), and Example 3 (9 to 1)) acts as a phytoestrogen in the body and is a raw material that can help prevent and alleviate various menopausal syndromes.

[0257]

[0258] 3. Measurement of ERα and ERβ expression levels in the uterus

[0259] After sacrificing the experimental animals after oral administration, the uterus and vagina were rapidly removed and separated and stored in a -80℃ deep freezer. The frozen uterine tissue was added to RIPA buffer (Biosesang, Korea) containing a protease inhibitor (Complete Tablet, Roche, Germany) and a phosphatase inhibitor (PhosSTOP EASYpack, Roche, Germany), and chopped using surgical scissors. The tissue was then homogenized using a Handy vortex mixer (Kimble Chase, USA). The homogenized sample was centrifuged at 4℃ and 3,000 rpm for 20 minutes, and the supernatant was used for protein quantitative analysis. In addition, the supernatant was subjected to quantitative analysis using a Bradford protein assay kit (Biosesang, Korea), and then 5X SDS-PAGE loading buffer (Seorin Bioscience, Korea) was added and heat-treated at 100°C for 15 minutes. The heat-treated sample was electrophoresed at 120 V for 1 hour and 30 minutes using a 10% gel (Invitrogen, USA). After electrophoresis, proteins ERα and ERβ separated on the gel were analyzed by Clarity TM After reacting for 1-5 minutes using Western ECL Substrate (Bio-Rad, USA), the expression levels of uterine ERα, ERβ, and the expression levels of the housekeeping gene β-actin were image analyzed using an ImageQuant LAS 500 (Invitrogen, USA). After quantitative analysis using the image analysis program Image J (a Java-based image processing program developed by the Optical and Computational Instrumentation Laboratory of the National Institutes of Health, USA), the expression levels of uterine ERα and ERβ were quantitatively analyzed relative to the expression levels of β-actin.

[0260] The results were as shown in Figures 5 and 6.

[0261] As shown in Fig. 5, after administering a mixture of fermented galgeun and seoritae powder (Example 1 (1 to 9), Example 2 (3 to 7), and Example 3 (9 to 1)) for 8 weeks, there was no significant difference in the uterine ERα expression levels of the three groups, Example 1 (1 to 9), Example 2 (3 to 7), and Example 3 (9 to 1), compared to the control group, the OVX group. On the other hand, when 17-β-estradiol (E2) was administered, the uterine ERα expression level increased by 266.4% compared to the control group, the OVX group.

[0262] In addition, as shown in Fig. 6, after administering the mixture of fermented galgeun and seoritae powder (Example 1 (1 to 9), Example 2 (3 to 7), and Example 3 (9 to 1)) for 8 weeks, the uterine ERβ expression levels of the three groups, Example 1 (1 to 9), Example 2 (3 to 7), and Example 3 (9 to 1), increased by 109.5%, 110.1%, and 115.3%, respectively, compared to the control group, OVX group, which was a similar level to the positive control group, E2 group.

[0263] In conclusion, it was found that the powder mixture of fermented galgeun and seoritae (Example 1 (1 to 9), Example 2 (3 to 7), and Example 3 (9 to 1)) did not affect the expression level of uterine ERα, which increases the risk of uterine myoma and endometrioma, which are side effects of hormone replacement therapy (HRT), a common treatment method for menopausal women, but significantly increased the expression level of uterine ERβ, a potent tumor suppressor that neutralizes the effect of ERα. Therefore, it could be determined that the powder mixture of fermented galgeun and seoritae (Example 1 (1 to 9), Example 2 (3 to 7), and Example 3 (9 to 1)) can be a raw material that can help prevent and alleviate various menopausal syndromes with a low risk of side effects from HRT by inducing an increase in the expression level of ERβ in the body.

[0264]

[0265] 4. Analysis of femoral bone mineral density (BMD) using micro-CT

[0266] After sacrificing the experimental animals that had completed oral administration, the femurs were quickly removed and fixed in a 4% paraformaldehyde solution. Afterwards, to measure each bone mineral density (BMD), the femurs were photographed using a micro-CT scanner (Xradia Versa 620 imaging system Micro-CT, Carl Zeiss, USA) at the Korea Basic Science Institute (Gwangju, Republic of Korea), and the bone density of the trabecular bone and three-dimensional image analysis were performed.

[0267] The results of the analysis are shown in Fig. 7. Specifically, as shown in Fig. 7, after administering the mixture of fermented galgeun and seoritae powder (Example 1 (1 to 9), Example 2 (3 to 7), and Example 3 (9 to 1)) for 8 weeks, the femur bone density of the three groups, Example 1 (1 to 9), Example 2 (3 to 7), and Example 3 (9 to 1), increased by 40.4%, 29.3%, and 25.3%, respectively, compared to the control group, the OVX group, which was a similar level to the positive control group, the E2 group.

[0268] Finally, it was confirmed that the femoral bone density increased when the mixture of fermented galgeun and seoritae powder (Example 1 (1 to 9), Example 2 (3 to 7), and Example 3 (9 to 1)) was consumed, and it could be judged that it is a raw material that can help prevent and alleviate osteoporosis or bone disease caused by a decrease in blood estradiol concentration due to decreased ovarian function in menopausal women.

[0269]

[0270] 5. Measurement of blood RANKL / OPG signaling pathway protein concentration

[0271] After sacrificing the experimental animals that had completed oral administration, the whole blood of the mice was quickly added to a tube treated with heparin anticoagulant and gently shaken to mix with the heparin. After centrifugation at 4°C and 13,000 rpm for 20 minutes, the plasma was collected and used for measuring RANKL and OPG (Osteoprotegerin). RANKL and OPG were measured using an enzyme-linked immunosorbent assay (ELISA) kit (Abcam, UK) according to the experimental method provided by the manufacturer.

[0272] The measurement results are shown in Fig. 8. Specifically, as shown in Fig. 8, after administering the fermented powder mixture of galgeun and seoritae (Example 1 (1 to 9), Example 2 (3 to 7), and Example 3 (9 to 1)) for 8 weeks, the blood RANKL concentrations of the three groups, Example 1 (1 to 9), Example 2 (3 to 7), and Example 3 (9 to 1), decreased by 18.1%, 30.3%, and 30.3%, respectively, compared to the control group, the OVX group, and decreased to a level similar to that of the positive control group, the E2 group.

[0273] On the other hand, in the case of Example 1 (1 vs. 9), Example 2 (3 vs. 7), and Example 3 (9 vs. 1), the blood OPG concentration did not increase significantly, but showed a tendency to increase like the positive control group E2.

[0274] Finally, the RANKL / OPG ratio was reduced by 41.9%, 46.0%, and 39.8% in Example 1 (1 to 9), Example 2 (3 to 7), and Example 3 (9 to 1), respectively, and was reduced to a level similar to that of the positive control group, E2.

[0275] RANKL is expressed on the surface of bone marrow stromal cells of the osteoblast lineage, and is a protein that strongly influences the differentiation, proliferation, and activation of osteoclasts by binding to RANK (Receptor activator of nuclear factor kappa-B) expressed in pre-osteoclasts. In addition, OPG is a decoy receptor of RANKL and is a protein that inhibits the differentiation and proliferation of osteoclasts by interfering with the binding between RANKL and RANK. Therefore, a decrease in the RANKL / OPG ratio means that bone resorption has decreased, and through the above results, by confirming the specific mechanism of action, it was confirmed that the results of Experimental Example 3-4 were supported.

[0276] In conclusion, it was confirmed that the intake of the fermented powder mixture of galgeun and seoritae (Example 1 (1 to 9), Example 2 (3 to 7), and Example 3 (9 to 1)) reduced the blood RANKL / OPG ratio. Therefore, it could be judged that the fermented powder mixture of galgeun and seoritae (Example 1 (1 to 9), Example 2 (3 to 7), and Example 3 (9 to 1)) is a raw material that can help prevent and alleviate osteoporosis or bone disease by regulating the RANKL / OPG signaling pathway.

[0277]

[0278] 6. Measurement of blood CTx and NTx concentrations

[0279] After sacrificing the experimental animals that had completed oral administration, whole blood was quickly added to a tube treated with heparin anticoagulant and gently shaken to mix. Afterwards, the tube was centrifuged at 4℃ and 13,000 rpm for 20 minutes, and the plasma was collected and used for the measurement of CTx (C-telopeptide of collagen type 1) and NTx (N-telopeptide of collagen type 1). CTx and NTx were measured using an enzyme-linked immunosorbent assay (ELISA) kit (Cusabio, USA) according to the experimental method provided by the manufacturer.

[0280] The measurement results are shown in Fig. 9. Specifically, as shown in Fig. 9, after administering the fermented powder mixture of galgeun and seoritae (Example 1 (1 to 9), Example 2 (3 to 7), and Example 3 (9 to 1)) for 8 weeks, the blood CTx concentrations of the three groups, Example 1 (1 to 9), Example 2 (3 to 7), and Example 3 (9 to 1), decreased by 40.3%, 40.4%, and 25.5%, respectively, compared to the control group, OVX group, which was a similar level to the positive control group, E2 group.

[0281] In addition, for the blood NTx concentration, Example 1 (1 vs. 9), Example 2 (3 vs. 7), and Example 3 (9 vs. 1) showed a decrease of 77.4%, 87.6%, and 80.5%, respectively, which was similar to the Sham group, which was a normal control group.

[0282] CTx and NTx are indicators that are released into the blood and urine when the bone resorption activity of osteoclasts at the terminal ends of the collagen that forms bone increases. Therefore, an increase in blood CTx and NTx indicates an increase in bone resorption. Through the above results, it was confirmed that the results of confirming the mechanism of action in Experimental Example 3-5 were supported by evaluating the degree of osteoclast activity.

[0283] In conclusion, it was confirmed that when a powder mixture of fermented galgeun and seoritae (Example 1 (1 to 9), Example 2 (3 to 7), and Example 3 (9 to 1)) was consumed, NTx and CTx in the blood were reduced. Therefore, it could be judged that a powder mixture of fermented galgeun and seoritae (Example 1 (1 to 9), Example 2 (3 to 7), and Example 3 (9 to 1)) is a raw material that can help prevent and alleviate osteoporosis or bone disease by reducing osteoclast activity.

[0284]

[0285] 7. Blood lipid analysis using an automatic biochemical analyzer

[0286] After sacrificing the experimental animals after oral administration, whole blood from the mice was quickly added to a tube treated with heparin anticoagulant and gently shaken to mix. After centrifugation at 4°C and 13,000 rpm for 20 minutes, plasma was collected and total cholesterol, low-density lipoprotein cholesterol (LDL-cholesterol), high-density lipoprotein cholesterol (HDL-cholesterol), and triglycerides were measured. Blood lipid analysis was performed using an automatic biochemical analyzer (ACCUTE, Toshiba, Japan) at the CHA Bio Complex Experimental Animal Center (Seongnam, Republic of Korea).

[0287] The analysis results are shown in Fig. 10. Specifically, as shown in Fig. 10, after administering the mixture of fermented galgeun and seoritae powder (Example 1 (1 to 9), Example 2 (3 to 7), and Example 3 (9 to 1)) for 8 weeks, only the Example 2 (3 to 7) group showed a significant decrease in blood total cholesterol and low-density lipoprotein cholesterol concentrations by 17.0% and 40.0%, respectively, compared to the control group, the OVX group, which was a similar level to the positive control group, the E2 group.

[0288] On the other hand, high-density lipoprotein cholesterol and triglycerides did not show significant differences among the three groups of Example 1 (1 vs. 9), Example 2 (3 vs. 7), and Example 3 (9 vs. 1).

[0289] Through the above results, it was confirmed that when the powder mixture of fermented galgeun and seoritae (Example 2 (3 to 7)) was consumed, the concentrations of total cholesterol and low-density lipoprotein cholesterol in the blood, which are involved in dyslipidemia, were reduced. Therefore, it could be determined that the powder mixture of fermented galgeun and seoritae is a raw material that can help prevent and alleviate dyslipidemia caused by a decrease in the concentration of estradiol in the blood due to decreased ovarian function in menopausal women.

[0290]

[0291] 8. Measurement of blood and abdominal aorta NO signaling pathway protein concentrations

[0292] After the first experimental animal that had completed oral administration was sacrificed, the whole blood of the mouse was quickly added to a tube treated with heparin anticoagulant and gently shaken to mix. After centrifugation at 4°C and 13,000 rpm for 20 minutes, plasma was collected and nitric oxide (NO) was measured. Nitric oxide was measured using the Nitric Oxide Plus Detection kit (Intron Biotechnology, Republic of Korea) according to the experimental method provided by the manufacturer.

[0293] The measurement results are shown in Fig. 11. Specifically, as shown in Fig. 11, after administering the mixture of fermented galgeun and seoritae powder (Example 1 (1 to 9), Example 2 (3 to 7), and Example 3 (9 to 1)) for 8 weeks, only the Example 2 (3 to 7) group showed a significant increase of 133.9% in blood nitric oxide concentration compared to the control group, the OVX group, which was a similar level to the positive control group, the E2 group.

[0294] Meanwhile, after the second sacrifice of the experimental animals that had completed oral administration, the abdominal aorta was quickly removed and stored in a -80 ℃ deep freezer. Afterwards, endothelial nitric oxide synthase (eNOS) and cyclic guanosine monophosphate (cGMP) were measured using enzyme-linked immunosorbent assay (ELISA) kits (Abcam, UK), and cyclic GMP-dependent protein kinase (protein kinase G, PKG) was measured using enzyme-linked immunosorbent assay (ELISA) kits (Lifespan Biosciences, USA), and pretreatment was performed according to the experimental method provided by the manufacturer.

[0295] The measurement results are shown in Fig. 12. Specifically, as shown in Fig. 12, after administering the mixture of fermented galgeun and seoritae powder (Example 1 (1 to 9), Example 2 (3 to 7), and Example 3 (9 to 1)) for 8 weeks, only the Example 2 (3 to 7) group showed a significant increase in the concentrations of abdominal aortic endothelial cell-type NOS, cyclic guanosine monophosphate, and cyclic GMP-dependent protein kinase by 49.1%, 63.7%, and 37.9%, respectively, compared to the control group, the OVX group, which was a similar level to the positive control group, the E2 group.

[0296] Nitric oxide is produced by endothelium-type NOS in endothelial cells of blood vessels. This nitric oxide induces an increase in the concentration of cyclic guanosine monophosphate in vascular smooth muscle, ultimately causing vasorelaxation through the action of cyclic GMP-dependent protein kinase.

[0297] Meanwhile, dyslipidemia is a condition in which excessive lipids are present in the blood. These lipids accumulate on blood vessel walls, inducing inflammation and hindering the proper production of nitric oxide. Therefore, an increase in the concentration of NO signaling pathway proteins indicates increased vasorelaxation and a decrease in dyslipidemia. Therefore, by confirming the mechanism of action through the above results, the results of Experimental Example 3-7 were confirmed to be supported.

[0298] In conclusion, it was confirmed that when the fermented powder mixture of galgeun and seoritae Example 2 (3 to 7) was consumed, the NO signaling pathway protein increased. Therefore, it could be determined that the fermented powder mixture of galgeun and seoritae Example 2 (3 to 7) is a raw material that can help prevent and alleviate dyslipidemia by regulating the NO signaling pathway.

[0299]

[0300] [Accession number]

[0301] Name of depositor: Korea Center for Microbiological Conservation (KCCM)

[0302] Accession number: KCCM13337P

[0303] Date of acceptance: 20230324

[0304]

[0305]

[0306] Name of depositor: Korea Center for Microbiological Conservation (KCCM)

[0307] Accession number: KCCM13338P

[0308] Date of acceptance: 20230324

[0309]

Claims

1. A pharmaceutical composition for preventing or treating menopausal syndrome, bone disease or dyslipidemia in women, comprising an extract of Pueraria lobataroot or a fermented product thereof and an extract of Glycine max(L.) Merrill or a fermented product thereof.

2. A pharmaceutical composition according to claim 1, wherein the extract is extracted with at least one selected from the group consisting of water, alcohol having 1 to 10 carbon atoms, hexane, chloroform, and butylene glycol.

3. A pharmaceutical composition according to claim 1, wherein the fermented product is fermented with a strain of the genus Lactiplantibacillus.

4. A pharmaceutical composition according to claim 1, wherein the composition comprises a glycoside and an aglycone of phytoestrogen.

5. A pharmaceutical composition according to claim 1, wherein the mixing ratio of the galgeun extract or a fermented product thereof and the seoritae extract or a fermented product thereof in the composition is 1:20 to 20:

1.

6. A pharmaceutical composition according to claim 5, wherein the glycoside is at least one selected from the group consisting of Puerarin, Daidzin, and Genistin.

7. A pharmaceutical composition according to claim 5, wherein the non-glycosylated compound is at least one selected from the group consisting of daidzein and genistein.

8. A pharmaceutical composition according to claim 1, wherein the composition increases the concentration of estradiol in the blood.

9. A pharmaceutical composition according to claim 1, wherein the composition does not change the expression level of ERα (Estrogen Receptor α) but increases the expression level of ERβ (Estrogen Receptor β).

10. A pharmaceutical composition according to claim 1, wherein the menopausal syndrome is at least one selected from the group consisting of facial flushing, vasomotor disorders, sweating, depression, nervousness, dizziness, fatigue, sleep disorders, anxiety, concentration disorders, memory loss, nervousness, cardiovascular disease, bone disease, obesity, muscle pain, headache, palpitations, feeling of restlessness, dry skin, cystitis, dysuria, vaginitis, and vaginal dryness.

11. A pharmaceutical composition according to claim 1, wherein the bone disease or dyslipidemia is caused by menopausal syndrome in women.

12. A pharmaceutical composition according to claim 1, wherein the composition promotes differentiation, activation, and calcification of osteoblasts.

13. A pharmaceutical composition according to claim 1, wherein the composition inhibits the activity of osteoclasts.

14. A pharmaceutical composition according to claim 1, wherein the composition increases bone density.

15. A pharmaceutical composition according to claim 1, wherein the bone disease is at least one selected from the group consisting of osteoporosis, osteomalacia, osteopenia, bone defect, bone atrophy, osteonecrosis, fibrous dysplasia, osteitis fibrosa cystica, Paget's disease, fracture, osteolysis, osteoarthritis, osteogenesis imperfecta, rickets, rheumatoid arthritis, hip dysplasia, and osteodystrophy.

16. A pharmaceutical composition according to claim 1, wherein the composition reduces at least one selected from the group consisting of total cholesterol and low-density lipoprotein cholesterol (LDL-cholesterol) in the blood.

17. A pharmaceutical composition according to claim 1, wherein the composition increases relaxation of blood vessels.

18. A pharmaceutical composition according to claim 1, wherein the dyslipidemia is at least one selected from the group consisting of hyperlipidemia, hyper-LDL cholesterolemia, hypertriglyceridemia, and hypo-HDL cholesterolemia.

19. Health functional food for preventing or improving menopausal syndrome, bone disease or dyslipidemia in women, comprising an extract of Pueraria lobataroot or a fermented product thereof and an extract of Glycine max(L.) Merrill or a fermented product thereof.

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