KR102100295B1 - Germinated Fermented Soybean Extract Having Effect of Protecting Liver Cells and Increasing Bone Density Containing Coumesterol, And Composition Containing The Extract - Google Patents

Abstract

The present invention relates to a germinated fermented soybean extract containing coumesterol, a preparation method thereof, and a composition comprising the germinated fermented soybean extract. It can be seen that the germinated fermented soybean extract of the present invention is effective for hepatocyte protection by measuring in vitro antioxidant activity and cell viability against hepatotoxicity and hematological examination of animal models, MDA, GST, GSH, SOD, CAT, and the like. In addition, by increasing the mineral content of bone, the density of bone is significantly increased, and it has the effect of increasing the thickness and number of spongy bone. It also has the effect of relieving menopausal symptoms.

Description

Germinated Fermented Soybean Extract Having Effect of Protecting Liver Cells and Increasing Bone Density Containing Coumesterol, And Composition Containing The Extract}

The present invention relates to a germinated fermented soybean extract containing cousterol having a hepatocyte protection effect and an increase in bone density and a composition comprising the same. In more detail, in the natural foods, in vitro antioxidant activity, cell viability and hematological examination of animal models for hepatotoxicity occurring during menopausal disorder using the germinated fermented soybean extract according to the present invention as a component having the effect of improving the lipid and climacteric disorder in the body , MDA (malone dialdehyde), GST (glutathione S-transferase), GSH (reduced glutathione), SOD (Superoxide dismutase), and to measure the protective effect of hepatocytes. In addition, in the present invention, in order to examine the improvement effect of bone loss that may occur during menopausal disorders through in vivo experiments using the germinated fermented soybean extract according to the present invention as a sample, the concentration of calcium and phosphorus in the blood, ALP activity as a bone formation index, Bone mineral content was checked, and various indicators of spongy bone were measured using Micro-CT.

Fermented germinated soybean extract of the present invention significantly increases the concentration of estrogen and testosterone in the blood, thereby alleviating menopausal symptoms, increasing the hepatocellular protective effect in in vitro and in vivo experiments, and increasing the mineral content of bone, thereby increasing bone density. Enemies, and the thickness and number of spongy bones increases.

Didzein in the isoflavones of legumes contains cousterol, and regulates female hormone balance and content to prevent breast cancer and uterine cancer, slowing menopause and alleviating a number of symptoms associated with it.

The aging of the population is progressing very rapidly all over the world, especially in Korea, the aging progress rate is very fast compared to other developed countries, which is a big problem. As a result of the increase in health due to economic growth, the interest in improving the quality of life and health of women after menopause and postmenopausal women is also increasing. The main symptoms of menopause are hot flashes, anxiety, depression, and nervousness. Symptoms such as cardiovascular disease, osteoporosis, and memory loss are typical, and it is known that the cause of these symptoms is that estrogen production and secretion are stopped. As a preventative measure, hormone replacement therapy has been suggested, but there are no results on usage, dose, and safety.

Phytoestrogen in natural foods is a reproductive hormone-like substance that has been studied and reported as an alternative to estrogen in postmenopausal women. As synthetic estrogen replacement therapy is currently used to treat menopausal symptoms, most women are experiencing side effects such as remenstrual symptoms, breast softening and weight gain, and through food intake with vegetable estrogen analogs in natural foods. I think the relief of menopausal symptoms will help a lot of side effects.

Phytoestrogens include isoflavones and lignans, and are compounds similar to estrogens naturally occurring in plants such as beans, fruits, vegetables, peanuts, and grains. Isoflavones are attracting attention as a kind of phytoestrogens that have various physiological effects by weakly binding to estrogen receptors in the body. Isoflavones extracted from soybean are mainly present as glycosides in plants, and when absorbed into the human body, they are converted into aglycone forms of genistein and daidzein by glycosidase to have estrogenic properties.

In relation to women's health, intake of isoflavones has been reported to alleviate menopausal symptoms without side effects and reduce cardiovascular risk and blood lipids and oxides in women with serum lipid levels and hypercholesterolemia, unlike conventional estrogen treatments. There is a bar.

Republic of Korea Patent Publication No. 10-2014-0053471 (composition for the prevention and treatment of menopausal symptoms including soybean extract containing coumestrol as an active ingredient) climacteric containing soybean extract containing coumestrol as an active ingredient Results of cell experiments ( in vitro ) on the composition for the prevention of diseases, etc., and improved to check the improvement of menopausal symptoms and the effect of hepatocyte protection through animal experiments ( in-vivo ) to confirm the cell experiments in the present invention And the purpose of obtaining advanced methods and results with improved germinated fermented soybean extract.

Korean Patent Publication No. 10-2014-0053471

1.Starzec, J, J, and D.F. Berger, Effects of stress and ovariectomy on the plasma cholesterol, serum triglyceride, and aortic cholesterol levels of female rats, Physiol Behav. 1986.37 (1): p.99-104. 2. Ettinger, B., Overview of estrogen replacement therapy a historical perspective. Proc Soc Exp Biol Med. 217 (1): p.2-5. 3. Baker, M.E., K.L. Medlock, and D.M. Sheehan, Flavonoids inhibit estrogen binding to rat alpha-fetoprotein. Proc Soc Exp Biol Med. 1998. 217 (3): p.317-21. 4. Boker, L.K., et al., Intake of Dietary Phytoestrogens by Dutch women. J Nutr. 2002. 132 (6): p.1319-28. 5. Morito, K., et al., Interaction of Phytoestrogens with estrogen receptors alpha and beta, Biol Pharm Bull. 2001. 24 (4); p.6-11. 6. Albertazzi, P., et al., The effect of dietary soy supplementation on hot flushes, Obstet Gynecol. 1988. 91 (3): p. 389-94. 7.Han, K, K., et al., Benefits of soy isoflavone therapeutic regimen on menopausal symptoms, Obstet Gynecol. 2002. 99 (3): p. 389-94. 8. Petri Nahas, E., et al., Benefits of soy germ isoflavones in postmenopausal women with contraindication for conventional hormone replacement therapy, Maturitas. 2004. 48 (4): p.372-80. 9. Kaari, C., ey al., Randomized clinical trial comparing conjugated equine estrogens and isoflavones in postmenopausal women: a pilot study, Maturitus. 2006. 53 (1): p.49-58. 10. Cheng, G., et al., Isoflavone treatment for acute menopausal symptoms, Menopause. 2007.14 (3Pt1): p.468-73. DJ., Et al., Effect of soy protein foods on low-density lipoprotein oxidation and ex vivo sex hormone receptor activity--a controlled crossover trial. Metabolism. 2000.49 (4): p.537-43. 12. Kotsopoulos, D., et al., The effects of soy protein containing phytoesterolemic and mildly hypercholesterolemic postmenopausal women. Am J Clin Nutr. 2001.55 (21): p.8589-95. 13. Wangen, K.E., et al., Soy Isoflavones improve plasma lipids in normocholesterolemic and mildly hypercholesterolemic postmenopausal women. Am J Clin Nutr. 2001.73 (2): p.225-31. 14.Feng, S., et al., Fungal-stressed germination of black soybeans leads to generation of oxooctadecadienoic acids in addition to glyceollins, J Agric Food Chem. 2007.55 (21): p.8589-95. 15. Rosalki, S.B. and A.Y.Foo. Two new methods for seperating and quantifying bone and liver alkaline phosphatase isoenzymes in plasma, Cli Chem. 1984. 30 (7): p. 1182-6.

The present invention is to alleviate menopausal symptoms by using fermented germinated soybean extract, protect hepatocytes, and increase the mineral content of bone.

As a result of the increase in health due to economic growth, the interest in improving the quality of life and health of women after menopause and postmenopausal women is also increasing. The main symptoms of menopause are hot flashes, anxiety, depression, and nervousness. Symptoms such as cardiovascular disease, osteoporosis, and memory loss are typical, and it is known that the cause of these symptoms is that estrogen production and secretion are stopped. As a preventative measure, hormone replacement therapy has been suggested, but there are no results on usage, dose, and safety. Moreover, there is a lack of decent drugs or health foods for menopausal women with hepatocyte protection and bone density increase.

The present invention relates to a germ-fermented bean sprout containing cousterol according to the present invention having a hepatocyte protection and an increase in bone density in postmenopausal women and a composition comprising the same. Using the germinated fermented soybean extract containing cousterol above, it is intended to verify the effect of hepatocyte protection and bone density increase in postmenopausal women. In addition, in the present invention, using the germinated fermented soybean extract according to the present invention as a sample, in vivo experiments to improve bone loss that may occur during menopausal disorders and to examine hepatocellular protective effects in postmenopausal women, the concentration of calcium and phosphorus in the blood, bone The formation index ALP activity, bone mineral content was checked, and various indicators of spongy bone were measured using Micro-CT.

Through a significant increase in blood estrogen concentration, isoflavone of the germinated fermented soybean according to the present invention can replace the role of estrogen, and it can be expected that menopausal symptoms are alleviated. It was confirmed that the testosterone concentration resulted in a significant increase (p <0.05) compared to the ovarian ablation control group.

The present invention was found to be effective in protecting hepatocytes through measurement of in vitro antioxidant activity, cell viability, and hematologic examination of animal models, MDA, GST, GSH, SOD, CAT, etc., against hepatotoxicity in menopausal disorders. In addition, it was confirmed that the administration of germinated fermented soybean extract according to the present invention increases the mineral content of bone. In the rats to which the germinated fermented soybean extract according to the present invention was administered, the bone density was significantly increased, and the thickness and number of spongy bones were increased. Through a significant increase in the concentration of estrogen in the blood, the isoflavone of the germinated fermented soybean extract according to the present invention can replace the role of estrogen, and it can be expected that menopausal symptoms are alleviated. In particular, it is expected that the concentration of triglyceride and glucose in the blood tends to decrease, so that administration of germinated fermented soybean extract according to the present invention is expected to help improve menopausal obesity, and germination according to the present invention at a high concentration. It was confirmed that administration of fermented soybean extract had a prophylactic effect on osteoporosis at a level similar to that of estrogen administration. The oxidative damage of the body against oxidative stress, which is the cause of various diseases in humans, requires antioxidant enzymes such as catalase, SOD, GSH, and GST. Therefore, as a result of measuring the liver enzyme antioxidant activity with an animal model having artificial liver damage using the germinated fermented soybean extract according to the present invention, it is significant compared to the liver damage model in which only t- BHP such as SOD, GSH, GST, MDA was administered. It showed decrease and increase. Accordingly, it was found that the germinated fermented soybean extract according to the present invention has a liver protective effect against oxidative stress.

Figure 1 shows the weight gain of the group administered estrogen.
Figure 2 shows the serum lipid, cholesterol and glucose concentration
3 shows a change in hormones.
Figure 4 shows the results of the bone metabolism indicator measurement.
Figure 5 shows the results of the measurement of bone salt.
6 shows bone microstructure indicators.
7 shows a 2D image taken using Micro-CT.
Figure 8 shows the separation process using sep-pak of germinated fermented soybean extract according to the present invention.
Figure 9 shows the cell viability of germinated fermented soybean extract (control sample) and germinated fermented soybean extract (test sample) according to the present invention.
Figure 10 shows the t- BHP cell viability of germinated fermented soybean extract (control sample) and germinated fermented soybean extract (test sample) according to the present invention.
Figure 11 shows the results of the ROS measurement of germinated fermented soybean extract (control sample) and germinated fermented soybean extract (test sample) according to the present invention.
FIG. 12 shows the results of AST (GOT) and ALT (GPT) measurements of animal model hematology.

<Example 1>; Materials and methods

1.1. Experimental material

The germinated fermented soybean used in this experiment was provided by Hubio. Soybeans obtained from Jeju Agricultural Research and Extension Services are obtained by germination according to previous studies and inoculated with Aspergillus oryzae . After an extraction process using water washing, pickling, and ultrasound, drying was used as an experimental sample.

1.2. Preparation of samples

Soybeans were filtered through a fermentation process using water washing, pickling, germination, and Aspergillus oryzae, and then dried at 60 ° C. 80% (v / v) ethanol was added to 10 g of the dried sample at 7 mL / g for decomposition, followed by hot water extraction using ultrasound. Thereafter, a germinated fermented soybean extract was obtained by centrifugation, filtered through a PTFE membrane, and dried to obtain a sample. The above sample was dissolved in tertiary distilled water and used for animal experiments.

1.3. Experimental animals

Experimental animals were purchased from 8-week-old Sprague-Dawley-based female rats from Daehan Biolink (Daejeon) and reared with solid feed (Purina) in this laboratory, and were adjusted to a control diet for 2 weeks before the start of the experiment. According to each group, 6 groups of 7 animals (Negative control, non-ovarian ablation control (Sham), estrogen administration group (OVX-estrogen), germinated fermented soybean extract according to the present invention) so that the average weight of each group is 289.72 ± 0.86 g 0.5 g / kg administered group (OVX-0.5), germinated fermented soybean extract according to the invention 1.0 g / kg administered group (OVX-1.0) and germinated fermented soybean extract according to the invention 2.0 g / kg administered group (OVX-2.0)] Divided and tested for 6 weeks. The laboratory animal uses a breeding cage (20 × 26 × 13 cm) to maintain the laboratory temperature of 22-24 ℃ and a humidity of 60 ± 5%, and considers the day and night cycle (12 hours light / 12 hours dark) to be controlled by an automatic control device. After breeding in the University Animal Room and receiving approval from the Korea University Animal Experiment Ethics Committee, it was conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals (NRC). Water and diet were freely supplied, and the experimental sample was dissolved in tertiary distilled water and administered orally 3 ml daily, and the control (Negative-control, sham) was orally administered with the same amount of tertiary distilled water daily.

1.4. Ovarian resection

The ovarian resection procedure was performed by dividing the group by the randomized complete block design by adapting to the surrounding environment for 2 weeks. After surgery, anesthesia was performed with isoflurane, and when the cardiac anesthesia was reached, the dorsal flank was dissected to remove the ovaries and the incision was closed. In the case of the non-ovarian ablation control group (Sham), the fat next to the ovary was removed in the same way and the incision was closed. Samples were administered orally after 2 weeks of recovery after surgery.

1.5. Serum lipid concentration and enzyme activity analysis

Triglyceride (TG), total cholesterol (TCHO), high-density lipoprotein cholesterol (HDL), low-density lipoprotein cholesterol (LDL) and glucose (GLU) of the separated serum were measured using FUJI DRI-CHEM 3500. Glutamate oxaloacetate transaminase (GOT) and Glutamate pyruvate transmaminase (GPT) activity were also measured in the same manner.

1.6. Hormone measurements

The amount of estradiol and testosterone in the plasma was measured by an ELISA method using rat estradiol Elisa kit (Enzo life science, NY, USA) and rat testosterone Elisa kit (Enzo life science, NY, USA), respectively.

1.7. Bone metabolism indicator measurement

Plasma calcium and phosphorus concentrations and plasma alkaline phosphatase (ALP) concentrations were measured with FUJI DRI-CHEM 3500 and BONE-alkaline phosphatase (BONE-ALP) was measured according to previous studies. The femur was painted through a drying and lean process, after which the amount of osteoarthritis of the femur was measured.

1.8. Bone tissue morphology measurement: micro-CT imaging

The area of 1 mm from the growth plate of the tibia of the sacrificial rat was photographed using a Micro-CT (Skyscan1072, SKYSCAN, Antwerpen, Belgium) at a height of 2 mm. The tube voltage was 50 kVp, the tube current was 500 μA, 0.5 mm aluminum filtration was used, the shooting time was 210 ms, and the pixel size was 18 μm. In order to separate the bone soju and bone marrow from the individual images, the threshold value was fixed at 58 and 3D images were reconstructed. From the images taken with Micro-CT, Skyscan ^TM CT-analyzersoftware was used to analyze bone microstructure indicators. The indicators analyzed were Percent bone volume (BV / TV (%)), trabecular number (Tb.N (mm ^-1 )), trabecular thickness (Tb.Th (μm)), trabecular separation (Tb.Sp (μm)) ), trabecular pattern factor (Tb.pf (mm ^-1 )), and structure model index (SMI).

1.9. Statistics processing

The data obtained as a result of the study were processed using a statistical package for SPSS (statistical package for social science, version 12.0). T-test was conducted on the negative-control group to examine the significant differences between groups. The measured values are expressed as the average standard error, and p <0.05, p <0.01, and p <0.001 are indicated as ^{*, **, and ***} , respectively.

<Example 1> Experimental results

2.1. Weight change and dietary intake

During the 6-week experiment, compared to the non-ovaryectomy control group, it was confirmed that the tendency to increase body weight in all groups induced menopause by ovarian resection (FIG. 1). The weight gain of the non-ovarian ablation control group was 20.4 ± 3.3 g, compared with this, the weight gain of the group administered with the ovarian ablation control group, germinated fermented soybean extract according to the present invention at 0.5 g / kg, 1.0 g / kg, 2.0 g / kg It was confirmed that 39.8 ± 11.1 g, 42.2 ± 4.5 g, 34.0 ± 5.6 g, and 36.8 ± 4.3 g, respectively. The weight gain of the estrogen-administered group was 12.1 ± 8.7 g, which tended to decrease relatively, but there was no significant difference in the germinated fermented soybean extract-administered group according to the present invention. There was no significant difference in dietary intake. The ovarian ablation control group, the non-ovarian ablation control group, and the estrogen-administered group were denoted as Neg.C, Sham, and Estradiol, and the germinated fermented soybean extract 0.5g / kg, 1.0g / kg, and 2.0g / kg administered group according to the present invention were 0.5 respectively. , 1.0, 2.0. Values are expressed as mean ± standard error.

2.2. Serum lipid concentration and enzyme activity analysis

Estrogen secretion decreases sharply in ovarian resection groups, as in female menopause. At this time, the body accumulates a lot of fat cells capable of producing estrogen in order to reinforce the reduced estrogen secretion, thereby causing menopausal obesity. Lipid, cholesterol, and glucose concentrations in blood were identified as indicators of menopausal obesity, and the results are shown in FIG. 2.

The triglyceride levels of the group administered with the germinated fermented soybean extract according to the present invention at 0.5 g / kg, 1.0 g / kg, and 2.0 g / kg were 62.5 ± 21.3 mg / dl, 59.0 ± 8.5 mg / dl, 55.8 ± 8.8, respectively. In mg / dl, it was confirmed that it decreased to a level similar to that of the non-ovarian ablation control group 66.8 ± 12.6 mg / dl.

In the case of total cholesterol, when the germinated fermented soybean extract according to the present invention was administered at 1.0 g / kg and 2.0 g / kg, it was confirmed that the values increased to 139.5 ± 9.6 mg / dl and 136.7 ± 6.5 mg / dl, respectively. As a result, the cholesterol pool increased, and it seems that similar effects were observed in high-density lipoprotein cholesterol and low-density lipoprotein cholesterol.

Looking at Figure 2, the concentration of glucose in the blood was also 98.3 ± 16.1 mg / dl, 123.3 ± 14.4 mg / dl, 112.8 ± 6.6 mg / dl in all groups to which the germinated fermented soybean extract according to the present invention was administered, in the blood of the ovarian ablation control group It was confirmed that a tendency to decrease was observed compared to the glucose concentration of 162.7 ± 18.6 mg / dl.

Since the concentration of triglyceride and glucose in the blood tends to decrease, administration of germinated fermented soybeans is expected to help improve menopausal obesity.

GOT and GPT are normal enzymes produced by the liver. As liver cells break when the liver is damaged, these enzymes are released into the blood and increase more than normal, so they are used as a representative indicator of liver toxicity. Looking at Table 1 below, serum GOT and GPT activity analysis do not have a significant difference for each group. Therefore, it can be seen that the treatment of the sample does not show liver toxicity.

Toxicity Sham Neg.C Estradiol 0.5 1.0 2.0 GOT (U / L) 92.00 ± 10.42 76.57 ± 7.28 63.71 ± 5.89 62.71 ± 6.35 74.00 ± 3.91 69.14 ± 5.11 GPT (U / L) 28.43 ± 3.90 23.86 ± 1.58 31.00 ± 3.72 22.00 ± 6.25 23.86 ± 0.59 26.43 ± 5.42

2.3. Hormone measurements The cause of various menopausal symptoms is reduced estrogen production and secretion due to aging of the ovaries. Therefore, it is important to check the increase in the concentration of estrogen in the blood in order to see the effect of alleviating climacteric symptoms of the administration of the germinated fermented soybean extract according to the present invention.

2.3.1. Estrogen

The estrogen concentration of the ovarian ablation group was 500.3 ± 27.8 pg / ml compared to the estrogen concentration of the non-ovarian ablation control group 1020.0 ± 63.6 pg / ml. Can be confirmed. In the case of the estrogen-administered group, it was confirmed that the concentration of estrogen in the blood increased to 1134.1 ± 205.8 pg / ml, which is a similar value to the non-ovarian ablation control group (FIG. 3). In the three groups to which the germinated fermented soybean extract of the present invention was administered, the concentration of estrogen in the blood was significantly increased (1577.1 ± 341.6 pg / ml, 2358.6 ± 191.8 pg / ml, 938.5 ± 51.2 pg / ml, respectively) compared to the ovarian ablation control group ( Fig. 3). Through a significant increase in the concentration of estrogen in the blood, the germinated fermented soybean extract according to the present invention can replace the role of estrogen, which can be expected to relieve menopausal symptoms.

2.3.2. Testosterone

Testosterone is a representative anabolic hormone and is involved in muscle metabolism and fat metabolism. When the level of this hormone decreases, the amount of muscle in the body decreases, leading to a decrease in basal metabolism and easily causing obesity. It also induces keratinization of the skin, one of the typical symptoms of menopause. Referring to FIG. 3, the concentration of testosterone in the blood was reduced in all groups subjected to ovarian ablation compared to the non-ovarian ablation control group. Among these, the estrogen-administered group and the group in which the germinated fermented soybean extract according to the present invention were administered at 1.0 g / kg were able to confirm testosterone concentrations of 2797.8 ± 517.3 pg / ml and 1969.8 ± 164.2 pg / ml, respectively. It was confirmed that the testosterone concentration was significantly (p <0.05) increased compared to the ovarian ablation group.

2.4. Bone metabolism indicator measurement

Referring to Figure 4, the calcium concentration in the blood did not show a significant difference between the groups, in the case of phosphorus, the germinated fermented soybean extract according to the present invention showed a significant difference in the group administered 1.0 g / kg (p <0.05)).

Alkaline phosphatase (ALP) is a biomarker of osteoblasts involved in bone metabolism. When there is a disease in which bone metabolism increases rapidly, such as osteoporosis, the level of ALP also increases. The ovarian ablation group's blood ALP level was 454.0 ± 69.9 U / L, which was higher than the non-ovarian ablation group's blood ALP level of 340.2 ± 33.31 U / L. On the other hand, in the group to which the germinated fermented soybean extract 0.5 g / kg according to the present invention was administered was 260.0 ± 646.9 U / L, which showed significantly lower ALP activity than the control group (p <0.05). In addition, compared to the ovarian ablation control group, the level of Bone-ALP was 140.8 ± 58.5 U / L in the group administered with 0.5 g / kg of germinated fermented soybean extract according to the present invention, which significantly (p <0.05) lowered. I could confirm that.

Referring to FIG. 5, after 6 weeks of oral administration, it was confirmed that the amount of osteoarthritis was significantly decreased (p <0.05) compared to the non-ovarian ablation control group in the ovarian ablation group. In the group administered with the germinated fermented soybean extract according to the present invention at 1.0 g / kg and 2.0 g / kg, the respective bone salts were found to be 61.3 ± 0.36% and 60.8 ± 0.31%. This is a significant increase (p <0.05) of osteoarthritis compared to the ovarian ablation control group. Through this, it was confirmed that administration of the germinated fermented soybean extract according to the present invention increases the mineral content of bone.

2.5. Bone tissue morphology measurement: micro-CT imaging

The values of percent bone volume (BV / TV (%)), trabecular number (Tb.N (mm ^-1 )), trabecular thickness (Tb.Th (μm)) in all ovarian ablation groups compared to the non-ovarian ablation group It can be seen that is decreased. In the estrogen-administered group and the group administered with 2.0 g / kg of germinated fermented soybean extract according to the present invention, the values were significantly increased compared to the ovarian ablation group. In the case of BV / TV, it decreased to 13.7 ± 1.3% in the ovarian ablation control group, and the BV / TV of the group administered 2.0 g / kg of germinated fermented soybean extract according to the present invention was 23.3 ± 1.3%, showing a significant difference. It was seen (p <0.01) (Figure 6). In the case of Tb.N, the ovarian ablation control group showed a low value of 1.17 ± 0.09 mm ^-1 , but the group administered with 2.0 g / kg of germinated fermented soybean extract according to the present invention was 1.69 ± 0.15 mm ^-1 with the estrogen administration group. It was confirmed that the increase was similar (p <0.05). In the case of Tb.Th, the ovarian ablation control group showed a low value of 0.12 ± 0.00 mm ^-1 , but Tb.Th was significantly increased at all concentrations administered with estrogen and germinated fermented soybean extract according to the present invention (Fig. 6). This means that in the rats to which the germinated fermented soybean extract according to the present invention was administered, the bone density increased significantly, and the thickness and number of cancellous bone increased.

6, trabecular separation (Tb .Sp (μm)), trabecular pattern factor (Tb.pf (㎜ ^-1 )) and structure model index (SMI) in the non-ovarian ablation control group in all groups undergoing ovarian ablation. Compared, the numbers increased. In the group administered with the germinated fermented soybean extract 2.0 g / kg according to the present invention, the Tb.Sp value was 0.70 ± 0.10, which was significantly decreased (p <0.05). Through this result, it can be seen that the dispersion degree of spongy bone, which is closely related to osteoporosis, was decreased in the group administered with the germinated fermented soybean extract according to the present invention compared to the ovarian ablation control group. In the case of Tb.pf, the estrogen-administered group and the 2.0 g / kg germinated fermented soybean extract group according to the present invention significantly decreased (p <0.01, p <0.05) compared to the ovarian control group. The values were 0.76 ± 0.48 mm ^-1 and 0.63 ± 1.25 mm ^-1 respectively. Finally, in the case of the structure model index (SMI), the estrogen-administered group and the 2.0 g / kg germinated fermented soybean extract group according to the present invention were 1.54 ± 0.07, 1.53 ± 0.10, respectively, compared to the ovarian ablation control group significantly ( p <0.01). Therefore, it is expected that the germination fermented soybean extract 2.0 g / kg administration according to the present invention has a prophylactic effect on osteoporosis at a level similar to that of estrogen administration.

3. Antioxidative effect of liver toxicity model

3.1. In vitro antioxidant activity (DPPH, ABTS, Polyphenol) measurement results

Looking at the following Table 2, as a result of measuring the antioxidant activity of each sample, the homeostasis activity is the antioxidant activity and the polyphenol content of the germinated fermented soybean extract (test sample) according to the present invention than the germinated fermented soybean extract (control sample) provided by Hubio. This was measured higher.

In vitro antioxidant activity (DPPH, ABTS, Polyphenol) measurement results IC ₅₀ Valued ABTS (mg / ml) DPPH (mg / ml) Polyphenol (μg / ml) GFS extract (control sample) 4.2936 ± 0.03 2.4272 ± 0.04 324.09 ± 9.43 Ethanol-decomposed GFS ultrasound hydrothermal extract (test sample) 2.522 ± 0.01 0.7366 ± 0.19 684.61 ± 8.94 Ascorbic acid (IC50, μg / ml) 68.2545 ± 0.95 30.6857 ± 1.87 -

3.2 Separation and Purification of Experimental Materials (Germinated Fermented and Soybeans: GFS) Looking at FIG. 8, the antioxidant activity after separating the substance using Sep-pak using hydrothermal extract using ethanol decomposition GFS ultrasonic wave with high antioxidant activity was measured. The results showed high polyphenol content in Metanol 30% and 70%. In addition, looking at Table 3, ABTS, a method for measuring antioxidant activity, showed the highest antioxidant activity in 70% of methanol. Table 3 below is a table showing the results of in vitro antioxidant activity (ABTS IC50 value, Polyphenol) measurement.

EtOH decomposition GFS ultrasonic hydrothermal extract Fraction 1 Fraction 2 Fraction 3 Fraction 4 Fraction 5 Fraction 6 ABTS (mg / ml) 2.96 ± 0.07 1.16 ± 0.01 1.09 ± 0.03 0.68 ± 0.01 5.62 ± 0.07 1.09 ± 0.09 Polyphenol (µg / ml) 383.1396 ± 7.61 256.7509 ± 1.63 298.9451 ± 2.65 261.6921 ± 1.73 92.8187 ± 1.55 988.1484 ± 9.67

(Ascorbic acid (IC ₅₀ ug / ml): 2.13 ± 0.06) 3.3. Measurement of the protective activity of samples from damage caused by t-BHP using HepG2 cells 3.3.1. Cell viability

The MTT assay is an experimental method using the principle that the yellow soluble tetraz olium salt is reduced to the blue insoluble formazan product by the mitochondrial succina dehydrogenase of living cells. The absorbance of formazan is maximized at a wavelength of 540 nm.

Absorbance measured at this wavelength reflects the concentration of live and metabolically active cells, and is used as an indicator of mitochondrial activity. MTT is an experiment that measures the activity of mitochondria to show how toxic the test substance is by examining the cytotoxicity. When the test substance is toxic and the mitochondria is destroyed, the absorbance value is low.

 Referring to FIG. 9A, when only the germinated fermented soybean extract (control sample) was treated in HepG2 cells, 0.0312 (98.52 ± 3.64) in the group treated with the germinated fermented soybean extract when compared to the cells not treated with the sample (100%). , 0.0625 (97.08 ± 2.93), 0.125 (94.44 ± 1.72), 0.25 (96.46 ± 2.86), 0.5 (96.19 ± 3.03) mg / ml concentrations showed significant toxicity.

In addition, looking at Figure 9B, the cell viability when treated with cells using a hot water extract (test sample) using EtOH-decomposed germinated fermented soybean of the present invention was not different from the group not treated with the sample.

In addition, as shown in A of FIG. 10, when the germinated fermented soybean extract (control sample) and t-BHP were treated using HepG2 cells, compared to cells that did not process anything, in the case of cells treated only with t-BHP It was found to be about 60% lower, and when treated with germinated fermented soybean samples, there was no significant difference compared to cells treated with t-BHP only when compared to cells treated with t-BHP only. However, looking at Figure 10B, when compared to the EtOH-decomposed germinated fermented soybean hot water extract (test sample) using t-BHP only cells (64.67 ± 0.7 8%), the concentration of the cells is not significant but the survival rate of the cells You can see that this increases. 0.0625 (80.11 ± 0.90%), 0.125 (82.67 ± 0.84%), 0.25 (83.52 ± 1.98%), 0.5 (82.54 ± 2.58%) mg / ml.

3.4. Measurement of reactive oxygen species (ROS) using HepG2 cells

DCFH-DA (dichlorofluorescin diacetate) is converted to DCF (dichlorofluorescein), a strong fluorescent substance by reacting with free radicals (ROS) in the cell. Therefore, the relative amount of ROS can be estimated by measuring the fluorescence by DCF.

To confirm the ROS effect on the hot water extract (test sample) using EtOH-decomposed germinated fermented soybean of the present invention, the hot water extract using EtOH-decomposed germinated fermented soybean of the present invention was incubated for 12 hours at a concentration without cytotoxicity and t- BHP 1mM was treated for 1 hour to cause oxidative stress. After adding 25 μM of DCFH-DA for 60 minutes, the fluorescence of DCF was measured.

The results are shown in FIG. 11. Looking at Figure 11, compared to the normal (100 ± 1.00%) compared to the t-BHP treatment (267.62 ± 2.68%) increased fluorescence, EtOH decomposition germination fermented soybean according to the present invention 0.5mg by pretreatment with hot water extract / ML (195.24 ± 1.95%), 0.25mg / mL (195.24 ± 1.95%), 0.125mg / mL (240 ± 2.41%), 0.0625mg / mL (252.80 ± 2.53%) As a result, it was confirmed that the effect of reducing free radicals by hot water extract using EtOH-decomposed germinated fermented soybean of the present invention.

3.5. Hematological examination using animal models

The blood biochemical test results of the group administered with the hot water extract using EtOH-decomposed germinated fermented soybean of the present invention and administered t-BHP are shown in Table 4 below. As a result of the measurement of Total Cholestrol, LDH, Total Bilirubin, ALP, and ALT, there was a significant difference (Table 4) when compared with the control group. LDH is a glycolytic enzyme and is widely distributed in each tissue in the body. In particular, LDH in serum present in the heart, liver, kidneys and muscles increases in heart disease, liver disease malignancies and leukemia, but has low specificity. In acute hepatitis, as in AST and ALT, it escapes from hepatocytes and increases in early acute hepatitis. In chronic hepatitis and cirrhosis, it increases in normal or mild. Significant differences were found in AST and ALT, one of the indicators of liver failure.

 ALT and AST are hepatocellular leak enzymes, which are utilized in liver toxicity studies because the enzymes are released into the blood as necrosis of liver cells and destruction of liver tissue progress. 12 is a result of measuring the AST (GOT) and ALT (GPT) activity. As for AST, the concentration of hydrothermal extract using EtOH-degraded germinated fermented soybean of the present invention was 0.5 g / kg (83.20 ± 2.86 U / L) and 1 g / kg (81.80 ± 6.72 U / L), but there was no significant change in concentration. The treatment group was significantly lower compared to the t-BHP treatment group (94.25 ± 7.37 U / L). The concentration of hot water extract using EtOH-decomposed germinated fermented soybean of the present invention was 0.5 g / kg (33.71 ± 5.59 U / L) and 1 g / kg (38.17 ± 4.49 U / L), but there was no significant change in concentration of ALT. The treatment group significantly decreased compared to the t-BHP treatment group (48.25 ± 16.19 U / L).

Treatment TCHO (mg / dl) LDH (U / Liter) TBIL (mg / dl) Normal 80.00 ± 7.81 ^abc 123.33 ± 21.83 ^b 0.48 ± 0.05 ^ab t-BHP Control 85.25 ± 3.77 ^a 227.0 ± 46.66 ^a 0.60 ± 0.22 ^a Silymarin 80.8 ± 6.87 ^ab 141.33 ± 24.34 ^b 0.54 ± 0.05 ^ab GFS (Test Sample) 0.5g / kg + t-BHP 66.29 ± 9.89 ^bc 127.0 ± 27.38 ^b 0.49 ± 0.07 ^bc GFS (test sample) 1g / kg + t-BHP 64.63 ± 7.89 ^c 102.20 ± 16.83 ^b 0.41 ± 0.04 ^c

3.6. As a result of MDA measurement using an animal model, lipid peroxidation refers to a phenomenon in which polyunsaturated fatty acids in the cell membrane are peroxidized by various free radicals derived from superoxide anion radicals that can occur naturally in living organisms. Unsaturated fatty acids are decomposed through the peroxidation process to produce MDA, so this is used as an index to measure lipid peroxidation. In the case of liver tissue, the group administered with t- BHP after administering the hot water extract using EtOH-decomposed germinated fermented soybean of the present invention is 0.5 g / kg (34.25 ± 5.80), 1 g / kg (32.68 ± 12.57) of sample administration concentration. There was no significant difference in each group, but it decreased as the concentration increased, compared to the t- BHP only group (44.66 ± 1.94).

3.7. GST measurement results using animal models

GST is an enzyme that is present in almost every cell in the body and is involved in many metabolisms as well as detoxification of the liver. In particular, GST serves to catalyze the reaction of binding the electron-affinity substrate to GSH. As a result of GST measurement, after administration of hot water extract using EtOH-decomposed germinated fermented soybean of the present invention, t- BHP was administered 0.5g / kg, 1g / kg, compared with t- BHP alone (25.68 ± 4.28) When the sample concentration was increased at 0.5 g / kg (33.65 ± 12.97) and 1 g / kg (48.43 ± 8.90), the significant difference from the t-BHP-administered group was only at 1 g / kg.

3.8. GSH measurement results using animal models

GSH is a very important detoxifying agent and a powerful antioxidant in the human body, which plays a key role in detoxification, immune function and antioxidant function in the body. As a result of GSH measurement, the group administered t- BHP (0.5 g / kg, 1 g / kg) and the group administered t- BHP only (21.07 ± 8.33) after administration of the hydrothermal extract using EtOH-decomposed germinated fermented soybean of the present invention. ), There was no significant increase in sample concentration at 0.5g / kg (25.12 ± 2.44) and 1g / kg (24.40 ± 2.52). In addition, there was no significant difference between the t- BHP-administered group and the sample-administered group, but a slight increase was observed in the sample-administered group.

3.9. SOD measurement results using animal models

SOD is an enzyme that acts to reduce superoxide anion (O ₂ ) to H ₂ O ₂ and is present in the cytosol and mitochondria. Cytosol only contains Cu / Zn-SOD, but mitochondria has Mn-SOD in the matrix and a small amount of Cu / Zn-SOD between the inner and outer membranes. As a result of SOD measurement, t- BHP was administered (0.5 g / kg, 1 g / kg) after administration of hot water extract using EtOH-decomposed germinated fermented soybean of the present invention and t- BHP alone (23.11 ± 1.71) ), There was no significant increase in sample concentration at 0.5g / kg (27.35 ± 3.32) and 1g / kg (25 .67 ± 0.02). In addition, there is a significant difference between the group administered t -BHP and the group administered sample. However, it did not show a concentration-dependent effect in the group administered with the sample.

3.10. CAT measurement results using animal models

Catalase is the reduction of the H ₂ O ₂ produced by the reduction of the SOD action and O ₂ as a non-toxic, of H ₂ O was present in most of the enzyme as a peroxisome protecting the living body from the venom of a small amount of oxygen is present in the mitochondria. As a result of CAT measurement, after administration of hot water extract using EtOH-decomposed germinated fermented soybean of the present invention, t- BHP was administered 0.5g / kg, 1g / kg, compared with t- BHP alone (182.24 ± 19.29) When the sample concentration was 0.5 g / kg (204.49 ± 41.51) and 1 g / kg (293.19 ± 61.02), the concentration increased as the sample concentration increased. In addition, there was a significant difference between the t- BHP-administered group and the sample-administered group, and the concentration-dependent effect was shown in the sample-administered group.

<Application example>

The present invention is in the group consisting of excipients (disintegrants, binders, lubricants), stabilizers, emulsifiers, additives, etc., which are allowed under the pharmacological, health functional food method, and food hygiene method, in addition to the hot water extract using ethanol decomposition germinated fermented soybean of the present invention It may include one or more selected. In addition, the composition may be used for oral use, such as warning agents, granules, powders, tablets, or capsules. The dosage of the composition should be adjusted according to the criteria (application, purpose of use, patient's condition, age, sex, weight, prescription drugs, disease) prescribed by the relevant law.

<Application Example 1>; Formulation

The formulation was prepared by mixing a hot water extract using ethanol-decomposed germinated fermented soybean of the present invention with an excipient or a carrier allowed by the pharmacist method to prepare powders, tablets, capsules, pills, and granules.

<Acid>

2 g of hot water extract using ethanol-decomposed germinated fermented soybean of the present invention and 1 g of lactose were mixed and filled in an airtight fabric to prepare a powder.

<Tablet>

100 mg of hot water extract using ethanol-decomposed germinated fermented soybeans of the present invention, 100 mg of corn starch, 100 mg of lactose and 2 mg of magnesium stearate were mixed, and tablets were prepared.

<Capsule>

It was prepared by mixing 100 mg of hot water extract using ethanol-decomposed germinated fermented soybeans of the present invention, 100 mg of corn starch, 100 mg of lactose, and 2 mg of magnesium stearate, then filling into a gelatin capsule.

<Hwan>

After mixing the ethanol-decomposed germinated fermented soybean of the present invention with 1 g of hot water extract, lactose 1.5 g, glycerin 1 g, and xylitol 0.5 g, a ring was prepared with a size of 4 g.

<Granules>

After premixing 150 mg of hot water extract using ethanol-decomposed germinated fermented soybean of the present invention, 50 mg of soybean extract, 200 mg of glucose, and 600 mg of starch, 100 mg of 30% ethanol solution was added and dried at 60 ° C. to form granules. After filling the fabric.

<Application Example 2>; food

The food was prepared by mixing an ethanol-decomposed germinated fermented soybean hot water extract of the present invention with an excipient or carrier permitted by the Food Sanitation Act or the Health Functional Food Act to prepare a health food and beverage.

<Healthy Food>

1 g of hot water extract using ethanol-decomposed germinated fermented soybean of the present invention, 70 µg of vitamin A, vitamin E 1.0 mg, vitamin B1 0.13 mg, vitamin B2 0.15 mg, vitamin B6 0.5 mg, vitamin B12 0.2 µg, vitamin C 10 mg, 10 μg of biotin, 1.7 mg of nicotinamide, 50 μg of folic acid, 0.5 mg of pantothenate, appropriate amount of mineral mixture, 1.75 mg of ferrous sulfate, 0.82 mg of zinc oxide, 25.3 mg of magnesium carbonate, 15 mg of potassium diphosphate, calcium phosphate dibasic A health food was prepared by mixing 55 mg, potassium citrate 90 mg, calcium carbonate 100 mg, and magnesium chloride 24.8 mg.

In the above application example, the ratio of mixing vitamins and minerals in the hot water extract using ethanol-decomposed germinated fermented soybean of the present invention can be variously modified according to the formulation or consumer layer.

<Drink>

The ethanol-decomposed germinated fermented soybean according to the present invention was mixed with sterilized by mixing sterilized water with 1% of hot water extract, 0.5% of liquid fructose, 2% of oligosaccharides, 2% of sugar, 0.5% of salt, and the rest of the purified water and the packaging material to prepare a beverage.

In the above application example, the ratio of saccharides, dietary fiber, vitamins and minerals to the hot water extract using ethanol-decomposed germinated fermented soybean of the present invention can be variously modified according to the formulation or consumer layer.

The hydrothermal extract using ethanol-decomposed germinated fermented soybean of the present invention of the present invention significantly increases the concentration of estrogen and testosterone in the blood, thereby alleviating menopausal symptoms and increasing the mineral content of the bone to significantly increase bone density. Increases the thickness and number of spongy bones. Therefore, there is industrial availability.

Claims (11)

As a pharmaceutical composition for alleviating and reducing menopausal disorders containing germinated fermented soybean extract containing cousterol as an active ingredient,
The germinated fermented soybean extract containing cousterol was obtained by decomposing the fermented fermented soybean germ using Aspergillus oryzae into an aqueous ethanol solution having a concentration of 80% (v / v) or higher, and then extracting it with hot water using ultrasound. It characterized in that the extract, a pharmaceutical composition for alleviating and alleviating menopausal disorders in women.
The pharmaceutical composition for alleviating and alleviating female menopausal disorder according to claim 1, characterized in that it alleviates and alleviates hepatocyte damage caused by menopausal disorder.
The pharmaceutical composition for alleviating and alleviating female menopausal disorder according to claim 1, which is characterized by alleviating and alleviating a decrease in bone density caused by menopausal disorder.
delete According to claim 1, Characterized in that it further comprises an excipient that is permitted by the pharmacist, pharmaceutical composition for the alleviation and alleviation of menopausal disorders in women.
The method according to any one of claims 1 to 3 and 5, characterized in that it has any one formulation selected from the group consisting of liquids, granules, pills, and capsules, alleviating and alleviating female menopausal disorders Pharmaceutical composition.
As a health functional food composition for alleviating and improving menopausal disorders containing germinated fermented soybean extract containing cousterol as an active ingredient,
The germinated fermented soybean extract containing cousterol is decomposed by fermenting soybean germination using Aspergillus oryzae into an aqueous solution of ethanol having a concentration of 80% (v / v) or higher, and extract obtained by hot water extraction using ultrasound. A health functional food composition for relieving and improving menopausal disorder in women.
The composition of claim 7, wherein alleviating and improving hepatic cell damage, bone mineral density, or both of the liver cell damage and bone mineral density caused by climacteric disorder, is improved.
delete The method of claim 7, further comprising an excipient that is permitted by the Health Functional Food Act, health functional food composition for alleviating and improving menopausal disorders.
The alleviation and improvement of female menopausal disorder according to any one of claims 7, 8, and 10, characterized in that it has any one formulation selected from the group consisting of liquids, granules, pills, and capsules. Health functional food composition.

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