WO2023229356A2 - Composition for preventing, alleviating or treating menopausal symptoms in women, containing, as active ingredient, mixture of fermented product of soybeans and lettuce - Google Patents

Abstract

The present invention relates to a composition for preventing, alleviating or treating menopausal symptoms in women, containing, as an active ingredient, a mixture of fermented product of soybeans and lettuce. In the fermented product of the present invention, nitric oxide metabolites are fixated and stabilized, and the mixture of the present invention has excellent menopause alleviation effects in women, such as those of osteoblast differentiation induction and bone density formation.

Description

Composition for preventing, improving or treating female menopausal symptoms comprising a mixture of fermented soybeans and lettuce as an active ingredient

The present invention relates to a composition for preventing, improving, or treating female menopausal symptoms, comprising a mixture of fermented soybeans and lettuce as an active ingredient.

Female menopause is called menopause, including the menopause transition, menopause, and post-menopause period. During this period, female hormones change, and in particular, the level of the female hormone, estrogen, decreases. Along with these hormonal changes, you may experience a variety of menopausal symptoms, and the type, severity, and duration of symptoms experienced vary depending on the individual.

During the first five years after menopause, more than 30% of collagen is lost, affecting skin and epithelial cells, and more than one-third of menopausal women suffer from urinary system diseases, and menopausal osteoporosis directly affects women's lives in middle age. It is a threatening disease that can cause serious complications due to decreased bone mass and bone strength due to continued lack of female hormones after menopause. In addition, metabolic changes caused by changes in female hormones during menopause result in a decrease in basal metabolic rate and an increase in body fat, making it highly likely that menopausal obesity will occur. During menopause, basic energy consumption decreases, body fat mass decreases, and after menopause, the resting metabolic rate decreases and physical activity decreases due to the loss of luteal phase energy consumption during menstruation and the decrease in female hormones, resulting in an increase in body fat mass and body fat distribution. changes and develops into obesity, which makes it easier to be exposed to fatal adult diseases such as coronary artery disease, heart disease, stroke, diabetes, and high blood pressure.

To improve these menopausal symptoms, drug treatments such as hormone replacement therapy and non-steroidal agents have been developed, and hormone replacement therapy is currently used as the most effective method. However, long-term administration of hormones is known to cause side effects such as cancer risk, headaches, and weight gain. Therefore, the development of safe and effective natural product-derived menopausal and menopausal symptom improvement agents is being attempted and is still required.

Soybeans are attracting attention as an ingredient in functional foods as research on not only nutritional aspects but also bioactive substances such as saponin, isoflavone, and lecithin has become more active. In particular, isoflavone is a health functional food ingredient notified by the Ministry of Food and Drug Safety and can help with bone health after menopause. The recommended daily intake is set at approximately 24-27 ㎍/g in the form of non-glycoside isoflavone (aglycone). It is done.

Meanwhile, Korean Patent No. 1824147 discloses 'Soybean fermented composition with enhanced physiological activity and health functional food containing the same for improving menopausal or menopausal symptoms', and Korean Patent No. 1917363 discloses 'Soybean sprout embryo extract'. 'Pharmaceutical composition for preventing or treating female menopausal diseases containing' is disclosed, but 'composition for preventing, improving or treating female menopausal symptoms containing a mixture of fermented soybeans and lettuce as an active ingredient' of the present invention is not described. There is no bar.

The present invention was derived from the above-mentioned needs. In order to develop a functional fermented product containing natural nitric oxide metabolites using local resources, the present inventors used fermented soybean products in an ovariectomized animal model to develop a functional fermented product containing natural nitric oxide metabolites using local resources. Based on previous research results that confirmed the effect of improving blood lipids, the effect of improving menopausal symptoms using the mixture of fermented soybeans and lettuce was examined, and the results showed that the mixture of fermented soybeans and lettuce induced differentiation of osteoblasts and The present invention was completed by confirming that it was effective in forming bone density.

In order to solve the above problems, the present invention provides a health functional food composition for preventing or improving female menopausal symptoms containing a mixture of fermented soybean products and fermented lettuce as an active ingredient.

Additionally, the present invention provides a pharmaceutical composition for preventing or treating female menopausal diseases containing a mixture of fermented soybean and fermented lettuce as an active ingredient.

The mixture of fermented soybean and fermented lettuce according to the present invention is excellent in inducing differentiation of osteoblasts and forming bone density, and is also effective in improving facial flushing and alleviating symptoms such as insomnia, nervousness, and depression, and is derived from natural products. Because it is safe, it can be widely used for the prevention, improvement or treatment of female menopausal symptoms caused by female hormone deficiency, or for the development of food and medicine.

Figure 1 shows compounds identified in soybean fermentation products.

Figure 2 shows the effect of fermented soybean products on improving blood lipids using an ovariectomized animal model. SHAM: sham-operated group, OVC-C: Ovariectomized + D.W., OVX-MP: Ovariectomized + Whey-fermented extract, OVX-SB: Ovariectomized + Soybean-fermented extract.

Figure 3 shows the results of analyzing cell viability (A) and cytotoxicity (B) after treating MG-63 osteoblasts with fSLE to analyze the osteoblast toxicity of the fermented soybean-lettuce mixture (fSLE).

Figure 4 shows the results of analyzing alkaline phosphatase (ALP) activity to investigate the effect of fermented soybean-lettuce mixture (fSLE) on bone growth and osteocyte differentiation activity of osteoblasts.

Figure 5 shows the results of examining the effect of phosphorylation of ERK and p38 MAPKs on promoting osteoblast differentiation by fermented soybean-lettuce mixture (fSLE).

Figure 6 confirms the effect of fermented soybean-lettuce mixture (fSLE) on menopausal syndrome in ovariectomized rats. A is the result of analyzing body weight change, and B is the bone volume of trabecular bone (BA). /TA), and C is the result of examining the bone density of the tibia using μ-CT.

In order to achieve the object of the present invention, the present invention provides a health functional food composition for preventing or improving female menopausal symptoms containing a mixture of fermented soybean and fermented lettuce as an active ingredient.

In the health functional food composition according to the present invention, the soybean fermentation product or lettuce fermentation product is a fermentation product in which the nitric oxide metabolite is fixed and stabilized, and the fermentation strain is added to the soybean or lettuce at a temperature of 18 to 35 ° C. and 0.03 to 0.03 ° C. It may be manufactured by a manufacturing method including fermentation for 2 to 45 days at a dissolved oxygen concentration of 0.1 mg/L, but is not limited thereto, and detailed manufacturing methods can be referred to Korean Patent No. 2129038.

The fermentation strain used to produce the fermented soybean product or the fermented lettuce product according to the present invention is preferably Bacillus subtilis - Chun Hyun Su, whose accession number is KCTC12501BP, but is not limited thereto.

In an example according to one embodiment of the present invention, the mixture of fermented soybean and fermented lettuce may preferably be a mixture of fermented soybean and fermented lettuce at a weight ratio of 6 to 9: 4 to 1, More preferably, the fermented soybean product and the fermented lettuce product may be mixed at a weight ratio of 8:2, but the mixture is not limited thereto.

The health functional food composition according to the present invention may further include glutathione, and the glutathione may be pure glutathione or an extract containing glutathione, preferably a yeast extract containing glutathione, and more preferably may be a glutathione-containing yeast extract with a glutathione content of 7 to 50% by weight based on the total weight of the extract, but is not limited thereto.

In addition, the health functional food composition of the present invention includes, in addition to the mixture of the fermented soybean product and the fermented lettuce product, thistle, flower oil, buckwheat, Cheongung, cow's knee, mulberry, bee flower, Schisandra chinensis, Osuyu, Jeju bamboo, Cheonmundong, Baeksuo, pomegranate, It may additionally include one or more extracts selected from the group consisting of red ginseng and locust fruit, but is not limited thereto.

In addition, the health functional food composition for preventing or improving female menopausal symptoms containing a mixture of fermented soybean and fermented lettuce as an active ingredient is available in pills, tablets, capsules, powders, powders, granules, candies, It can be manufactured with any one selected from syrup and beverage, or can be manufactured by adding it as an ingredient in food, and can be manufactured appropriately according to conventional methods.

Examples of foods to which the mixture of fermented soybean and lettuce of the present invention can be added include dairy products including meat, sausages, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, and ice cream. , it may be in any form selected from various soups, beverages, teas, drinks, alcoholic beverages, and vitamin complexes, and includes all health functional foods in the conventional sense.

The health functional foods include various nutrients, vitamins, minerals (electrolytes), synthetic and natural flavors, colorants and enhancers (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, It may contain pH adjusters, stabilizers, preservatives, glycerin, alcohol, and carbonating agents used in carbonated beverages. Additionally, it may contain pulp for the production of natural fruit juice and vegetable drinks. These ingredients can be used independently or in combination.

The health functional food composition of the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients. The natural carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As a sweetener, natural sweeteners such as thaumatin and stevia extract or synthetic sweeteners such as saccharin and aspartame can be used.

The present invention also provides a pharmaceutical composition for preventing or treating female menopausal diseases containing a mixture of fermented soybean products and fermented lettuce as an active ingredient.

In the pharmaceutical composition according to the present invention, the mixture of fermented soybean product and fermented lettuce product is the same as described above.

In the pharmaceutical composition of the present invention, the female menopausal diseases include facial flushing, sweating, dry skin, vaginal dryness, vaginal atrophy, lower urethral atrophy, vaginitis, cystitis, urinary pain, urgent urination, concentration disorder, short-term memory disorder, anxiety, and nervousness. , memory loss, muscle pain, joint pain, and osteoporosis, but is not limited thereto.

The pharmaceutical composition of the present invention may be in various oral or parenteral dosage forms. When formulated, in addition to the above active ingredients, pharmaceutically acceptable carriers, excipients, or diluents may be further included. Commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, or surfactants may be used. It can be prepared. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations contain one or more compounds and at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. It is prepared by mixing. Additionally, in addition to simple excipients, lubricants such as magnesium stearate, talc, etc. are also used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. there is. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspension solvents include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, wethepsol, macrogol, Tween 61, cacao, laurin, glycero gelatin, etc. can be used.

The pharmaceutical composition of the present invention can be administered orally or parenterally, and when administered parenterally, external application to the skin or intraperitoneal, rectal, intravenous, muscle, subcutaneous, intrauterine dura, or intracerebrovascular injection can be selected. The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, “pharmaceutically effective amount” means an amount sufficient to treat the disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type, severity, and activity of the patient's disease. , can be determined based on factors including sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, drugs used simultaneously, and other factors well known in the field of medicine. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art.

The dosage of the composition of the present invention varies depending on the patient's weight, age, gender, health condition, diet, administration time, administration method, excretion rate, and severity of the disease. The daily dosage is comprised of fermented soybean products and fermented lettuce. Based on the amount of water mixture, it is 0.01 to 3,000 mg/kg, preferably 1,000 to 2,000 mg/kg, and can be administered 1 to 6 times a day. The pharmaceutical composition of the present invention can be used alone or in combination with surgery, radiation therapy, hormone therapy, chemotherapy, and methods using biological response regulators.

Hereinafter, the present invention will be described in detail by examples. However, the following examples only illustrate the present invention, and the content of the present invention is not limited to the following examples.

1. Soybean fermentation and composition analysis

For soybean fermentation, Human Enos Co., Ltd. (formerly HTHO Life Co., Ltd.) produced fermented soybeans using soybeans ranging from 370 kg in 2017 to 490 kg in 2018, 1,780 kg in 2019, and 4,100 kg in 2020. Component analysis of soybean fermentation was performed through LC/MS and ¹ H - NMR.

As a result of analyzing the fermented soybean product, it was confirmed that isoflavones daidzin, glycitein, and malonylgenistin were detected (Figure 1).

2. Prior research on fermented soybean products

The effect of fermented natural products on improving blood lipids was studied in an ovariectomized animal model. Fermented soybean product (SB) was administered to an ovariectomized animal model, and total cholesterol (TC) and triglyceride (TG), indicators of blood lipid components, were measured using a dedicated kit (Roche, Germany) using Modular analytics (PE, Roche, Germany) was measured using the Enzymatic Colorimetri Assay method.

As a result of the measurement, the neutral fat and total cholesterol levels of the group administered soybean fermentation (OVX-SB) were approximately 30 mg/dL lower than the control group (OVX-C), and the difference compared to the sham surgery group (SHAM) It was measured to be 7 mg/dL higher in neutral fat and about 33 mg/dL higher in total cholesterol, showing that administration of fermented soybeans was effective in improving blood lipids (Figure 2).

3. Preparation of soybean-lettuce fermentation mixture

Beans and lettuce were mixed with distilled water in a 1:1 ratio and then ground. Afterwards, about 1% ⁽ 1.0 The temperature of the fermentation process was set at 30°C, and fermentation was performed for 15 to 20 days for lettuce and 35 to 45 days for soybeans. All factors that can affect the fermentation process, such as ventilation, temperature, and pH, were managed throughout the process to maintain optimal fermentation conditions. After fermentation, the solids and fermentation broth are separated using a centrifuge (1.5 t/Hr, disk separator, Alfatechkorea Corp, Korea), and the fermentation broth is concentrated with a concentrator (1.5 t, Vacuum Evaporator, BDMPLANT, Korea) to 3~4 Brix. did.

For preclinical tests and human application experiments, each concentrated fermented product was frozen at -40°C for 24 hours, then vacuum-dried using a freeze dryer (1.5 t, Vacuum Freeze Drier, Ajin E.S.R Co. Ltd., Korea) to form powder. After preparing, fermented soybean powder and fermented lettuce powder were mixed and used in a ratio of 8:2.

4. Verification of the efficacy of natural fermentation using cells in alleviating menopausal symptoms

MG-63 cells were treated with fermented soybean-lettuce mixtures at different concentrations, and the medium was replaced every two days to induce osteoblast differentiation for 5 days. After 5 days, the differentiated cells are washed with PBS (pH 7.4). For ALP staining, cells were fixed using 10% formalin, BCIP/NBT substrate (Sigma-Aldrich Chemical, St, Louis, MO, USA) solution was added, and reaction was performed for 1-2 hours in the absence of light. Osteoblast differentiation was confirmed through the degree to which cells were stained blue. ALP enzyme activity was measured using the Fluorometric Alkaline Phosphatase Assay Kit (Abcam, Cambridge, MA, USA).

5. Verification of the efficacy of natural fermented products in relieving menopausal symptoms using animal models

The experimental animals used in this experiment were female wistar rats purchased from Samtaco (Korea) and used after an acclimatization period of one week. The average weight of rats was 200-300 g, and they were fasted for about 24 hours from 9 a.m. the day before the experiment to 9 a.m. the day of the experiment. In the experimental animal breeding room, light and dark were adjusted at 12-hour intervals, and the temperature was maintained at 23±2°C and humidity at 50-60%.

(1) OVX rat model production

Before surgery, the experimental animals were anesthetized using isoflurane inhalation anesthesia. The surgical area of the experimental animals was disinfected with 10% Povidone-iodine (PVP-I) solution, and then the skin of about 15 cm from the lower abdomen on one side was removed. The peritoneum and abdominal muscles were incised, the ovaries were exposed, the fallopian tubes were ligated with silk thread, and the ovaries were excised on both sides. After this, the abdominal muscles, peritoneum, and skin were sutured with silk thread and nylon thread. After surgery, the wound area was disinfected once daily for 1 week.

(2) Separation of experimental group and sample administration

For biological efficacy evaluation (wistar rats, n=5) in the menopausal induction model (OVX rat), group separation was performed as follows: normal group that did not induce menopause (CON), pseudo-surgery group that did not induce menopause. (SHAM), a control group in which menopause was induced by removing the ovaries (OVX-C), a soybean fermentation group (OVX-FB), and a sodium nitrite group (OVX-NO2) were administered orally once daily.

(3) Measurement of osteoporosis indicators through micro CT imaging

At 1, 6, and 12 weeks after the start of the experiment, Micro-CT scans were taken of the leg bones to observe osteoporosis-related indicators, and anesthesia was maintained by supplying 4% isoflurane and oxygen in a rat chamber. The captured images were calculated using an image analyzer and the amount of change was compared and observed.

(4) Blood collection and blood lipid measurement

The rats used in the experiment were fasted for 12 hours, anesthetized with isoflurane, and then blood was collected from the abdominal vena cava. Of the collected blood, 0.5 ml of anticoagulant (3.2% sodium citrate) was placed in a vacuum collection tube (BD Vacutainer ^® Blood Collection Tube). , BD Franklin Lakes NJ, USA) and the remaining 0.5 ml of blood was left at room temperature for 30 minutes and then centrifuged at 3,000 rpm for 15 minutes to obtain serum.

Metabolites that are major indicators of blood lipid components include total cholesterol (TC), triglyceride (TG), high density lipoprotein cholesterol (HDL), and low density lipoprotein cholesterol (LDL). ) was measured by the Enzymatic Colorimetric Assay method using a dedicated kit (Roche, Germany) using a Modular analytics (PE, Roche, Germany) device.

In addition, estradiol in the separated blood was analyzed using the ECLIA (Electrochemiluminescence Immunoassay) method (Modular Analytics E170, Germany).

(5) Uterine weight measurement

The weight of the experimental animals was measured, blood was collected, an autopsy was performed, the uterus was removed, and the weight was measured. Uterine Index was calculated as follows.

Uterine Idex = (uterine weight/body weight) × 100

6. Verification of efficacy in alleviating menopausal symptoms through human application testing of natural fermented products

(1) Kupperman index (KI)

It is a survey tool that scores representative menopausal symptoms to identify the degree and characteristics of menopausal disorders, including facial flushing, paresthesia (numbness in hands and feet), insomnia, nervousness, depression, dizziness, fatigue, joint or muscle pain, headache, heart palpitations, and clothing. Evaluate 11 menopausal symptoms of persimmon (feeling of ants crawling on the skin, itching). Each symptom is scored on a 4-point scale from 0 (no symptoms) to 3 (extremely distressing), with facial flushing having a weighting of 4 points, paresthesia (numbing hands and feet), insomnia, and nervousness having a weighting of 2 points. The rest are evaluated with a weight of 1 point, for a total of 51 points.

(2) Human application experimental design

This study was conducted as a 4-week randomized, double-blind clinical trial (registration number: KCT0007316), and eligible patients with a Kuperman index of 15 or higher received a test drug containing a mixture of soybean-lettuce fermentation or a placebo. , the control group received a placebo under the same conditions as the experimental group, that is, the Kuperman index was 15 or higher, and the condition was the same as the experimental group. After 4 weeks of pill supplementation, the Kupferman index index was evaluated on the first and last days. Of the 40 participants enrolled, 39 participants completed the study without major violations, with one discontinuing. Participants were instructed to lead a normal life and not to consume any other nutritional or health supplements, and to avoid foods containing soy such as tofu and cheonggukjang, nutritional supplements containing soy, lettuce in salads, and supplements containing lettuce extract. .

The composition of the test drug and placebo used in the human experiment are as follows.

Composition of test drug and placebo

Ingredient	Content (mg)
	Experiment Pill	Placebo Pill
Fermented Soybean/Lettuce Powder	270	0
crystalline cellulose	80	350
corn starch	110	110
Bamboo Sap Extract Powder	10	10
Cottonseed Oil Powder	10	10
dry yeast	20	20
Total	500	500

(3) Participants

The clinical trial included adult women aged 45 to 70 years who had reached menopause, were diagnosed with menopausal syndrome, and had a Kupperman index of 15 or higher. Forty volunteers were recruited and randomly assigned to two groups. Patients who have taken hormone replacement therapy (HRT) or undergone surgical menopause within 6 weeks, patients contraindicated in hormonal treatment, acute and severe cardiovascular disease (heart failure, myocardial infarction, stroke), people with serious liver function abnormalities, drugs and Those with a history of clinically significant hypersensitivity to functional foods, those with a history of psychiatric treatment within 2 months prior to screening, and those with a history of drug or alcohol abuse were excluded. Forty applicants gave informed consent before the test.

Example 1. Effect of soybean-lettuce fermentation mixture on MG-63 osteoblast differentiation

1) Evaluation of osteoblast cytotoxicity

MG-63 cells, a human osteosarcoma cell line, express various osteogenesis-related factors and are widely used as a replacement for primary osteoblast cells in osteogenesis research. To confirm the effect of the soybean-lettuce fermentation mixture (hereinafter referred to as fSLE) on the proliferation and toxicity of MG-63 osteoblasts, cell viability and cytotoxicity were measured after treatment with fSLE for 24 and 48 hours.

As a result, cell viability and cytotoxicity decreased and increased, respectively, in a concentration-dependent manner of fSLE (Figure 3). At concentrations of 1 to 20 mg/L NO ₂ ^- in fSLE, the decrease in cell viability was less than 20% and cytotoxicity was less than 15%. Therefore, to investigate the effect of fSLE on MG-63 osteoblast differentiation, experiments were conducted at a concentration of 1 to 20 mg/L NO ₂ ^- , which shows low cytotoxicity.

2) ALP activity evaluation

Alkaline phosphatase (ALP) is distributed in almost all tissues, and in particular in bone tissue, ALP activity is known to increase when bone growth occurs. Therefore, ALP activity was measured as a marker of osteoblast activity to investigate the effect of fSLE on osteoblast bone growth and osteocyte differentiation activity.

When fSLE was treated for 6 days at a concentration of 1 to 20 NO ₂ ^- mg/L, ALP enzyme activity increased in a concentration-dependent manner (FIG. 4). ALP staining to confirm ALP enzyme activity showed that ALP enzyme activity was high in fSLE at concentrations of 10 and 20 NO ₂ ^- mg/L, 171.8 and 182.6%, respectively, compared to the negative control group (non-fSLE-treated group). ALP enzyme activity increased. Therefore, fSLE increases the ALP activity of MG-63 osteoblasts, thereby promoting differentiation of osteoblasts, and its potential use as a functional material that can help promote bone growth was confirmed.

3) Effect on phosphorylation of ERK and p38 MAPKs

ERK and p38 MAPKs are cell signaling pathways important for osteoblast differentiation. Therefore, we investigated the effect of phosphorylation of ERK and p38 MAPKs on promoting osteoblast differentiation by fSLE.

As a result of the analysis, fSLE increased the phosphorylation of ERK and p38 MAPKs in a concentration-dependent manner, and compared to the negative control group, the p-ERK/ERK ratio in fSLE increased 2.62-fold at the maximum concentration of fSLE, 20 NO ₂ ^- mg/L. , the p-p38/p38 ratio increased 1.69-fold (Figure 5A). When the phosphorylation of ERK and p38 MAPKs caused by fSLE was inhibited using respective activity inhibitors, ALP enzyme activity was decreased by the ERK and p38 activity inhibitors, and a significant decrease in ALP enzyme activity was observed with the p38 activity inhibitor (Figure 5B, C). Therefore, fSLE appears to promote MG-63 osteoblast differentiation through activation of the ERK and p38 MAPKs signaling pathways.

Example 2. Effect of fermented soybean and fermented lettuce mixture on menopausal syndrome in ovariectomized rats.

1) Weight change

Weight gain is one of the symptoms of menopause and is a phenomenon caused by hormonal imbalance, and is different from general metabolic diseases caused by excessive energy intake or low consumption. After inducing menopause by performing oophorectomy and following a normal diet for 6 weeks, typical menopausal symptoms of weight gain were observed, and the fSLE treatment group (OVX+fSLE) showed no difference in body weight compared to the positive control group (OVX) for up to 2 weeks. However, after 3 weeks, the difference in weight began to appear. After 3 weeks, the body weight of the fSLE administration group decreased compared to the positive control group, and at 6 weeks after the end of the experiment, the body weight of the fSLE administration group was 432.3 ± 22.2 g, a decrease of about 21.7 g compared to the positive control group (454.0 ± 17.3 g) (Figure 6A ).

2) Tibia bone density analysis

A typical symptom that appears in menopausal women is osteoporosis due to abnormal bone metabolism. This is a phenomenon in which bone resorption accelerates after menopause as osteoblast function declines and osteoclast function becomes more active. In particular, it is known that the biggest cause of osteoporosis in women is a decrease in estrogen due to menopause, so bone health, including bone density, is an important factor in menopausal women's health. It is used as a key indicator. In the present invention, bone density of the tibia was investigated using μ-CT, and as a result, it was confirmed that the bone density of the ovariectomized OVX group was lower than that of the sham group through pictures taken through μ-CT, but bone density recovered in the fSLE administration group. showed a tendency to become (Figure 6C). Bone volume (BA/TA) of trabecular bone was reduced to 22.3% in OVX compared to Sham (37.3%), but increased to 27.5% by fSLE administration (Figure 6B). Therefore, it is expected that fermented soybean-lettuce mixture (fSLE) can be used as a health functional food material related to the prevention and treatment of osteoporosis, a bone metabolic disease, in ovariectomized rats.

Example 3. Effect of fermented soybean and fermented lettuce mixture on menopausal syndrome through human experiments

As a result of analyzing the Kupperman index (KI) after four weeks of pill supplementation, there was no difference in Kupperman index between the two groups before taking the pill, but there was a difference between the two groups after taking the pill. KI values were observed to be significantly reduced in the test drug treatment group containing the soybean-lettuce fermentation mixture.

Kuperman menopausal index analysis results before and after clinical trial

	Before			After
	Experiment	Placebo	p	Experiment	Placebo	p
Kupperman index	27.16±6.52	26.45±6.45	0.735	19.68±5.09	25.4±5.63	0.002

The above results showed that the soybean-lettuce fermented product mixture according to the present invention can be used to prevent, improve, or treat female menopausal symptoms or as a material for food and medicine.

Claims (8)

1. A health functional food composition for preventing or improving female menopausal symptoms, containing a mixture of fermented soybean and fermented lettuce as an active ingredient.
2. The method of claim 1, wherein the fermented soybean product or lettuce product is fermented for 2 to 45 days at a temperature of 18 to 35 ° C. and a dissolved oxygen concentration of 0.03 to 0.1 mg / L by adding fermentation bacteria to soybean or lettuce. A health functional food composition, characterized in that it is manufactured by a manufacturing method comprising.
3. The health functional food composition according to claim 1, wherein the fermented soybean product or fermented lettuce product is a fermented product in which nitric oxide metabolites are immobilized and stabilized.
4. The method of claim 1, wherein the composition is one or more selected from the group consisting of thistle, flower balm, buckwheat, Cheongung, soybean knee, mulberry, bee flower, Schisandra chinensis, Osuyu, Jeju seaweed, Cheonmundong, Baeksuo, pomegranate, red ginseng, and locust fruit. A health functional food composition, characterized in that it additionally contains an extract.
5. A pharmaceutical composition for preventing or treating female menopausal diseases, comprising a mixture of fermented soybean and fermented lettuce as an active ingredient.
6. The method of claim 5, wherein the female menopausal diseases include facial flushing, sweating, dry skin, vaginal dryness, vaginal atrophy, lower urethral atrophy, vaginitis, cystitis, urinary pain, urgent urination, concentration disorder, short-term memory disorder, anxiety, nervousness, and memory impairment. A pharmaceutical composition characterized by at least one selected from the group consisting of muscle pain, muscle pain, joint pain, and osteoporosis.
7. The pharmaceutical composition according to claim 5, wherein the composition further comprises a pharmaceutically acceptable carrier, excipient, or diluent.
8. The pharmaceutical composition according to claim 5, wherein the composition is prepared in any one formulation selected from capsules, powders, granules, tablets, suspensions, emulsions, syrups, and aerosols.

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