WO2017074159A2 - Coumestrol-containing germinated fermented soybean extract having hepatocyte protective effect and bone density increase effect and composition containing same - Google Patents

Abstract

The present invention relates to a germinated fermented soybean extract containing coumestrol, a preparation method therefor, and a composition containing the germinated fermented soybean extract. It can be seen that the germinated fermented soybean extract of the present invention is effective in the hepatocyte protection against hepatotoxicity through in vitro antioxidative activity and cell viability, and hematological tests and MDA, GST, GSH, SOD, and CAT measurements in animal models. In addition, the germinated fermented soybean extract of the present invention increases the mineral content in bones, thereby significantly increasing the bone density, and has an effect of increasing the thickness and number of spongy bones. Furthermore, the germinated fermented soybean extract of the present invention has an effect of relieving symptoms of menopause.

Description

Cumesterol-containing germinated fermented soybean extract having hepatocellular protective effect and bone mineral density increasing effect and composition comprising same

The present invention relates to cousterol-containing germinated fermented soybean extract having a hepatocellular protective effect and an effect of increasing bone density and a composition comprising the same. More specifically, the in vitro antioxidant activity, cell viability, hematological test of animal models, MDA (malone) for the hepatotoxicity of menopausal disorders using germinated fermented soybean extract as a component that improves lipids and menopausal disorders in natural foods Dialdehyde), GST (glutathione S-transferase), GSH (reduced glutathione), SOD (Superoxide dismutase), and catalase were measured to verify hepatocellular protective effects. In the present invention, the germinated fermented soybean extract as a sample in vivo In order to examine the effect of improving bone loss in menopausal disorders, we examined the concentrations of calcium and phosphorus in blood, ALP activity, bone mineral content, and bone mineral content. It measured using.

Fermented germinated soybean extract of the present invention significantly increases blood estrogen and testosterone concentrations, thereby relieving menopausal symptoms, and increasing bone mineral protection and increasing bone mineral content in vitro and in vivo. Increase, and the thickness and number of spongy bones increase

Didzein in legumes isoflavones contain cumesterol, which regulates female hormone balance and content to prevent breast and uterine cancer, slowing menopause and alleviating many of the symptoms associated with it.

The aging of the population is progressing very rapidly all over the world, and especially in Korea, the progress of aging is very fast compared to other developed countries. As women's average life expectancy is increased and the intellectual level is improved as a result of the health promotion resulting from economic growth, interest in women's menopause and postmenopausal quality of life and health improvement is also increasing. The main symptoms of menopause include hot flashes, anxiety, depression and nervousness. Symptoms such as cardiovascular disease, osteoporosis and memory loss are known. The cause of these symptoms is known to be caused by interrupted production and secretion of estrogen. As a preventive measure, hormone replacement therapy has been proposed, but there are no results on usage, dose, and safety.

Phytoestrogen, a natural food product, is a reproductive hormone-like substance that is beginning to attract attention for the prevention of menopausal symptoms and osteoporosis, and has been studied and reported to be able to substitute estrogen in postmenopausal women. As synthetic estrogen replacement therapy is currently used to treat menopausal symptoms, most women experience side effects such as remenstruation, breast softening and weight gain, and food intake with plant-based estrogen-like substances in natural foods. Relieving menopausal symptoms will help to alleviate the side effects.

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

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

Republic of Korea Patent Publication No. 10-2014-0053471 (composition for the prevention and treatment of menopausal symptoms comprising a soybean extract containing cumestrol as an active ingredient) a menopause containing a soybean extract containing cumestrol as an active ingredient It is the experimental result of the cell experiment (in vitro) on the composition for the prevention of diseases, and to improve the menopausal symptoms improvement and liver cell protection effect through the animal experiment (in-vivo) to confirm the cell experiment in the present invention. And improved germinated fermented soybean extract to achieve advanced methods and results.

{Prior art technical literature}

(Patent Document 1) Korean Patent Publication No. 10-2014-0053471

(Non-Patent Document 1) 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.

(Non-Patent Document 2) 2. Ettinger, B., Overview of estrogen replacement therapy a historical perspective. Proc Soc Exp Biol Med. 217 (1): p. 2-5.

(Non-Patent Document 3) 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.

(Non-Patent Document 4) 4. Boker, L.K., et al., Intake of Dietary Phytoestrogens by Dutch women. J Nutr. 2002. 132 (6): p. 1319-28.

(Non-Patent Document 5) 5. Morito, K., et al., Interaction of Phytoestrogens with estrogen receptors alpha and beta, Biol Pharm Bull. 2001. 24 (4); p.6-11.

(Non-Patent Document 6) 6. Albertazzi, P., et al., The effect of dietary soy supplementation on hot flushes, Obstet Gynecol. 1988. 91 (3): p. 389-94.

(Non-Patent Document 7) 7. Han, K, K., et al., Benefits of soy isoflavone therapeutic regimen on menopausal symptoms, Obstet Gynecol. 2002. 99 (3): p. 389-94.

(Non-Patent Document 8) 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.

(Non-Patent Document 9) 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.

(Non-Patent Document 10) Cheng, G., et al., Isoflavone treatment for acute menopausal symptoms, Menopause. 2007.14 (3Pt1): p.468-73.

(Non-Patent Document 11) 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.

(Nonpatent Document 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.

(Non-Patent Document 13) 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.

(Non-Patent Document 14) 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.

(Non-Patent Document 15) 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 uses fermented germinated soybean extract to relieve menopausal symptoms, to protect the liver cells, and to increase the mineral content of bone.

As women's average life expectancy is increased and the intellectual level is improved as a result of the health promotion resulting from economic growth, interest in women's menopause and postmenopausal quality of life and health improvement is also increasing. The main symptoms of menopause include hot flashes, anxiety, depression and nervousness. Symptoms such as cardiovascular disease, osteoporosis and memory loss are known. The cause of these symptoms is known to be caused by interrupted production and secretion of estrogen. As a preventive measure, hormone replacement therapy has been proposed, but there are no results on usage, dose, and safety. In addition, there is a lack of appropriate drugs or health foods that have hepatoprotective effect and bone mineral density increase effect in postmenopausal women.

The present invention relates to a cousterol-containing germinated fermented soybean extract having an effect of protecting liver cells and increasing bone density in postmenopausal women and a composition comprising the same. The germinated fermented soybean extract containing cumesterol to verify the effect of hepatocellular protection and bone density increase in postmenopausal women. In the present invention, the germinated fermented soybean extract as a sample in vivo In order to examine the effect of improving hepatocellular protection and bone loss in postmenopausal women during menopausal disorders, we examined blood calcium and phosphorus concentrations, ALP activity, bone mineral content, and bone mineral content. Various indicators were measured using Micro-CT.

Significant increases in blood estrogen levels can be expected to replace the role of estrogens in germinated fermented soybeans, thereby alleviating menopausal symptoms. Testosterone concentration was found to be significantly increased (p <0.05) compared to the ovarian control group.

The present invention was found to be effective for the protection of hepatocytes by measuring in vitro antioxidant activity, cell viability and hematological tests, MDA, GST, GSH, SOD, CAT, etc. for hepatotoxicity in menopausal disorders. It was also confirmed that the administration of germinated fermented soybeans increased the mineral content of bone. Bone density was significantly increased in the rats treated with germinated fermented soybean, and the thickness and number of spongy bones were increased. Significant increases in blood estrogen levels can be expected to replace the role of estrogens in germinated fermented soybeans, thereby alleviating menopausal symptoms. In particular, since the concentration of triglycerides and glucose in blood tend to decrease, the administration of germinated fermented soybean is expected to help improve menopausal obesity, and the high concentration of germinated fermented soybean is similar to that of estrogen. It was confirmed that there is a preventive effect of osteoporosis. The oxidative damage of the body to oxidative stress, which is the cause of various diseases in humans, requires antioxidant enzymes such as catalase, SOD, GSH, and GST. As a result of measuring liver enzyme antioxidant activity using an animal model with artificial liver damage using germinated fermented soybean, it was significantly reduced and increased compared to liver damage model administered only t- BHP such as SOD, GSH, GST, and MDA. Indicated. The germinated fermented soybean was found to have a liver protective effect against oxidative stress.

Figure 1 shows the weight gain of the group receiving the estrogen.

Figure 2 shows serum lipids, cholesterol and glucose concentrations

3 shows the change in hormone.

Figure 4 shows the bone metabolic index measurement results.

Figure 5 shows the results of bone salt measurement.

6 shows bone microstructural indicators.

Figure 7 shows a 2D image taken by using the Micro-CT.

Figure 8 shows the separation process using sep-pak of 70% ethanol germinated fermented soybeans.

Figure 9 shows the cell viability of germinated fermented soybean and 70% ethanol extract germinated fermented soybean.

Figure 10 shows the growth rate of t- BHP cells of germinated fermented soybean and 70% ethanol extract germinated fermented soybean.

Figure 11 shows the results of ROS measurement of germinated fermented soybean and 70% ethanol extract germinated fermented soybean.

Figure 12 shows the results of animal model hematology test AST (GOT), ALT (GPT).

Figure 13 shows the results of MDA measurement in Liver of the group administered t- BHP after the administration of germinated fermented soybean.

Figure 14 shows the results of GST measurement in Liver of the group administered t- BHP after administration of germinated fermented soybeans.

Figure 15 shows the results of GSH measurement in Liver of the group administered t- BHP after administration of germinated fermented soybean.

Figure 16 shows the results of SOD measurement in Liver of the group administered t- BHP after the administration of germinated fermented soybean.

Figure 17 shows the results of CAT measurements in Liver of the group administered t- BHP after the administration of germinated fermented soybeans.

<Example 1>; Materials and methods

1.1. Experimental material

Germinated fermented soybean used in this experiment was provided by Hubio. Soybeans obtained from Jeju Agricultural Research and Extension Services are germinated and fermented by inoculation with Aspergillus oryzae according to previous studies. After the extraction process using water washing, choice and ultrasonic, it was dried and used as a test sample.

1.2. Preparation of Sample

Soybeans wash, select, germinate and Aspergillus After the fermentation process using oryzae filtered and dried at 60˚C. 80% ethanol was added to 7 mL / g of 10 g of the dry sample, and the hydrothermal extract was extracted using ultrasonic waves. Thereafter, germinated fermented soybean extract was obtained by centrifugation, which was filtered through a PTFE membrane and dried to obtain a sample. The sample was dissolved in tertiary distilled water and used for animal experiments.

1.3. Laboratory animals

The experimental animals were purchased from Korea Biolink (Daejeon), 8-week-old Sprague-Dawley female rats, and were bred as solid feed (Purina) in the laboratory. Therefore, the average body weight of each group was 289.72 ± 0.86 g, and 7 animals of 6 groups (Negative control, Non-ovarian control group (sham), Estrogen group (OVX-estrogen), Germinated fermented soybean extract 0.5 g / kg Administered group (OVX-0.5), germinated fermented soybean extract 1.0 g / kg administered group (OVX-1.0) and germinated fermented soybean extract 2.0 g / kg administered group (OVX-2.0)]. The experimental animals were kept in a laboratory temperature of 22-24 ° C, humidity 60 ± 5% using a breeding cage (20 × 26 × 13 cm), and the day and night cycle (12 hours light / 12 hours dark) was controlled by an automatic control device. It was raised in the university animal room and approved by Korea University's Animal Experiment Ethics Committee, and managed according to the 'Guide for the care and use of Laboratory Animals' (NRC). Water and diet were freely supplied, and the experimental samples were dissolved in tertiary distilled water and orally administered 3 ml daily, and the control group (Negative-control, sham) was orally administered daily with the same dose of tertiary distilled water.

1.4. Ovarian excision procedure

For 2 weeks, ovarian resection was performed by dividing the group by randomized complete block design according to body weight. Surgery was performed with isoflurane and after anesthesia, the dorsal flank was incised to remove the ovary and the incision was closed. In the case of non-ovarian resection control (Sham), fat was removed from the ovary in the same way and the incision was closed. Samples were administered orally after having a recovery period of 2 weeks after surgery.

1.5. Serum Lipid Concentration and Enzyme Activity Analysis

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

1.6. Hormone measurement

Plasma levels of estradiol and testosterone were measured by the rat estradiol Elisa kit (Enzo life science, NY, USA) and rat testosterone Elisa kit (Enzo life science, NY, USA) by ELISA.

1.7. Bone metabolism indicator measurement

Plasma calcium and phosphorus concentrations and alkaline alkaline phosphatase (ALP) concentrations were measured by FUJI DRI-CHEM 3500 and BONE-alkaline phosphatase (BONE-ALP) as bone markers. The femur was dried and crushed through the fat removal process, and then the bone salt in the femur was measured.

1.8. Bone Morphology Measurement: Micro-CT

A 2 mm high part of the tibia of the sacrificial rats was photographed using Micro-CT (Skyscan1072, SKYSCAN, Antwerpen, Belgium). The imaging tube voltage was 50 kVp, the tube current was 500 μA, 0.5 mm aluminum filtration, the imaging time was 210 ms, and the pixel size was 18 μm. The threshold value was fixed at 58 to separate the bone column and bone marrow from the individual images, and the 3D image was reconstructed. Bone microstructure indicators were analyzed using Skyscan ^™ CT-analyzersoftware from images taken with Micro-CT. The indicators analyzed were Percent bone volume (BV / TV (%)), trabecular number (Tb.N ()), trabecular thickness (Tb.Th (μm)), trabecular separation (Tb.Sp (μm)), trabecular pattern factor (Tb.pf ()), and structure model index (SMI).

1.9. Statistical processing

The data obtained were processed using the statistical package for social science (version 12.0) statistical program. T-test was performed on the negative-control group to identify the significant differences among the groups. The measured values are expressed as average standard error, and p <0.05, p <0.01, and p <0.001 are indicated by ^{*, **, and ***} , respectively.

Example 1 Experimental Results

2.1. Weight change and dietary intake

During the six-week experiment, body weight gain was observed in all groups induced by menopause by ovarian resection compared to the non-ovarian resection control (FIG. 1). The weight gain of the non-ovarian control group was 20.4 ± 3.3 g, whereas the weight gain of the ovarian control group and the germinated fermented soybean at 0.5 g / kg, 1.0 g / kg and 2.0 g / kg were 39.8 ± 11.1, respectively. g, 42.2 ± 4.5 g, 34.0 ± 5.6 g, 36.8 ± 4.3 g was confirmed. The weight gain of the estrogen-treated group was 12.1 ± 8.7g, which tended to decrease relatively, but there was no significant difference in the germinated fermented soybean group. There was no significant difference in dietary intake changes. The ovarian resection control group, non-ovarian resection control group, and the estrogen-administered group were labeled as Neg.C, Sham, and Estradiol, and the germinated fermented soybean 0.5g / kg, 1.0g / kg, and 2.0g / kg groups were 0.5, 1.0, and 2.0, respectively. Notation. The figures are expressed as mean ± standard error.

2.2. Serum Lipid Concentration and Enzyme Activity Analysis

As in female menopause, estrogen secretion decreases rapidly in the ovarian resection group. At this time, the body accumulates a lot of fat cells capable of producing estrogen in order to enhance the reduced estrogen secretion, which leads to menopausal obesity. Lipid, cholesterol, and glucose concentrations in blood were identified as indicators of menopausal obesity, and the results are shown in FIG. 2.

Neutral fat levels of the groups administered with 0.5 g / kg, 1.0 g / kg and 2.0 g / kg of germinated fermented soybean were 62.5 ± 21.3 mg / dl, 59.0 ± 8.5 mg / dl and 55.8 ± 8.8 mg / dl, respectively. The decrease was similar to that of the non-ovarian ablation control 66.8 ± 12.6 mg / dl.

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

Referring to Figure 2, the blood glucose concentration was also 98.3 ± 16.1 mg / dl, 123.3 ± 14.4 mg / dl, 112.8 ± 6.6 mg / dl in all groups administered with germinated fermented soybeans, the blood glucose concentration of 162.7 ± It was confirmed that the tendency to decrease compared to 18.6 mg / dl.

Given that the concentration of triglycerides and glucose in the blood tend to decrease, the administration of germinated fermented soybean is expected to help improve menopausal obesity.

GOT and GPT are normal enzymes made in the liver. When the liver is damaged, these cells are broken down and the enzymes are released into the blood, increasing more than normal, so they are used as representative indicators of liver toxicity. Looking at Table 1 below, serum GOT, GPT activity analysis does not have a significant difference between groups. 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 measurement

The cause of various menopausal symptoms is a decrease in estrogen production and secretion due to aging of the ovary. Therefore, it is important to confirm the increase of blood estrogen concentration in order to alleviate the menopausal symptoms of the administration of germinated fermented soybeans.

2.3.1. Estrogen

The estrogen concentration of the ovarian resection control group was 500.3 ± 27.8 pg / ml compared to the estrogen concentration of 1020. ± 63.6 pg / ml of the non-ovarian resection group, resulting in a significant (p <0.001) decrease in estrogen concentration. You can see that. In the estrogen-administered group, it was confirmed that blood estrogen concentration was increased to 1134.1 ± 205.8 pg / ml, which is similar to that of the non-ovarian ablation control group (FIG. 3). All three groups that received germinated fermented soybeans significantly increased blood estrogen levels in 1577.1 ± 341.6 pg / ml, 2358.6 ± 191.8 pg / ml, and 938.5 ± 51.2 pg / ml, respectively, as compared to the ovarian ablation control (FIG. 3). Significantly increased blood estrogen levels can be expected to reduce the role of estrogens by extracts containing cousterol from germinated fermented soybeans, thereby alleviating menopausal symptoms.

2.3.2. Testosterone

Testosterone is a representative anabolic hormone that is involved in muscle and fat metabolism. Lower levels of these hormones reduce muscle mass in the body, which leads to lower basal metabolism and easier obesity. It also induces skin keratinization, one of the typical symptoms of menopause. Referring to FIG. 3, the concentration of testosterone in blood was decreased in all the groups that underwent ovarian ablation compared to the non-ovarian ablation control group. Among them, the testosterone concentrations of 2797.8 ± 517.3 pg / ml and 1969.8 ± 164.2 pg / ml were found in the estrogen-administered group and 1.0 g / kg of germinated fermented soybean, respectively. It was confirmed that the results were significantly increased (p <0.05) compared to the control group.

2.4. Bone metabolism indicator measurement

4, there was no significant difference in blood calcium levels among the groups, and in the case of phosphorus, there was a significant difference in the group administered with 1.0 g / kg of germinated fermented soybeans (p <0.05)).

Alkaline phosphatase (ALP) is a biomarker of osteoblasts involved in bone metabolism. In other diseases, such as osteoporosis, in which bone metabolism increases rapidly, the level of ALP also increases. The blood ALP level of the ovarian control group was 454.0 ± 69.9 U / L, which was higher than that of the non-ovarian control group (340.2 ± 33.31 U / L). On the other hand, in the group administered 0.5 g / kg of germinated fermented soybeans, 260.0 ± 646.9 U / L, significantly lower ALP activity than the control group (p <0.05). In addition, the Bone-ALP level was 140.8 ± 58.5 U / L. In the group administered with 0.5 g / kg of germinated fermented soybean compared to the control group of ovarian resection, indicating that it was significantly lower (p <0.05). .

Referring to FIG. 5, after oral administration for 6 weeks, it was confirmed that the bone inflammation was significantly lower (p <0.05) in the ovarian resection group compared with the non-ovarian resection control group. In the groups administered with 1.0 g / kg and 2.0 g / kg of germinated fermented soybeans, the bone salts were 61.3 ± 0.36% and 60.8 ± 0.31%, respectively. This is a significant (p <0.05) increase in bone salt compared to ovarian resection control. This confirmed that the germination of fermented soybeans increased the mineral content of bone.

2.5. Bone Morphology Measurement: Micro-CT

The values of percent bone volume (BV / TV (%)), trabecular number (Tb.N (mm ^-1 )), and trabecular thickness (Tb.Th (μm)) in all ovarian sections compared to non-ovarian sections It can be seen that decreases. The estrogen-treated group and the 2.0 g / kg germinated fermented soybean group increased significantly compared to the ovarian ablation control group. BV / TV was decreased to 13.7 ± 1.3% in the control group of ovarian ablation, and BT / TV of the group treated with 2.0 g / kg of germinated fermented soybean was 23.3 ± 1.3%, showing a significant difference (p <0.01 ) (FIG. 6). In the case of Tb.N, the ovarian control group showed a low value of 1.17 ± 0.09 mm ^-1 , but increased to 1.69 ± 0.15 mm ^-1 similar to that of the estrogen group in the 2.0 g / kg germinated fermented soybean group. It could be confirmed (p <0.05). In the case of Tb.Th, the ovarian control group showed a low value of 0.12 ± 0.00 mm ⁻¹ , but Tb.Th was significantly increased at all concentrations of estrogen and germinated fermented soybeans (FIG. 6). This means that the bone density of the germinated fermented soybeans increased significantly, and the thickness and number of spongy bones increased.

6, in the case of trabecular separation (Tb.Sp (μm)), trabecular pattern factor (Tb.pf (mm ⁻¹ )) and structure model index (SMI), all groups with ovarian resection were compared to the non-ovarian resection control group. The figures increased. In the 2.0 g / kg group of germinated fermented soybean, the Tb.Sp level was 0.70 ± 0.10, which was significantly decreased (p <0.05). These results show that the degree of dispersal of spongy bone, which is closely related to osteoporosis, was decreased in the group to which the germinated fermented soybeans were administered compared to the ovarian control group. In the case of Tb.pf, estrogen-treated and 2.0 g / kg of germinated fermented soybeans were significantly decreased (p <0.01, p <0.05) compared to the ovarian control group. The figures were 0.76 ± 0.48 mm ^-1 and 0.63 ± 1.25 mm ^-1 , respectively. Finally, the structure model inde x (SMI) was 1.54 ± 0.07, 1.53 ± 0.10 in the estrogen-administered group and 2.0 g / kg of germinated fermented soybean, respectively (p <0.01). Decreased. Therefore, the germination fermented soybean 2.0 g / kg administration is expected to be effective in preventing osteoporosis to a level similar to the administration of estrogen.

3. Antioxidant Effect of Liver Toxicity Model

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

Referring to Table 2 below, the antioxidant activity of each sample was measured, and the homeostasis activity of the germinated fermented soybeans extracted with 70% EtOH was higher than the germinated fermented soybeans provided by Hubio.

In vitro antioxidant activity (DPPH, ABTS, Polyphenol) measurement results
IC 50 Valued	ABTS (mg / ml)	DPPH (mg / ml)	Polyphenol (μg / ml)
GFS	4.2936 ± 0.03	2.4272 ± 0.04	324.09 ± 9.43
70% Ethanol GFS	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 Isolation and Purification of Experimental Materials (Germinated Fermented and Soybeans: GFS)

Referring to FIG. 8, 70% EtOH extract with high antioxidant activity was used to measure the antioxidant activity after separation of the material using Sep-pak. As a result, high polyphenol contents were shown at 30% and 70% of metanol. In addition, looking at the following Table 3 showed the highest antioxidant activity in 70% Methanol results of ABTS results of the antioxidant activity measurement method. Table 3 is a table showing the results of in vitro antioxidant activity (ABTS IC50value, Polyphenol) measurement.

70% Ethanol GFS Fraction	One	2	3	4	5	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. Determination of Protective Activity of Samples from Damage Caused by t-BHP Using HepG2 Cells

3.3.1. Cell survival rate

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

The absorbance measured at this wavelength reflects the concentration of living and metabolically active cells and is used as an indicator of mitochondrial activity. MTT is a test for measuring the mitochondrial activity to determine how toxic to a test substance by cytotoxicity test. If the test substance is toxic and the mitochondria are destroyed, the absorbance value is low.

 9A shows that when HepG2 cells were treated with only germinated fermented soybean samples, 0.0312 (98.52 ± 3.64), 0.0625 (97.08 ± 2.93) in the group treated with germinated fermented soybeans when compared with the untreated cells (100%). ), 0.125 (94.44 ± 1.72), 0.25 (96.46 ± 2.86), and 0.5 (96.19 ± 3.03) mg / mL concentrations showed significant toxicity.

In addition, as shown in Figure 9B the cell survival rate when the cells were treated with 70% EtoH extract germinated fermented soybean did not differ from the group not treated with the sample.

In addition, as shown in Figure 10A, when treated with germinated fermented soybeans and t-BHP using HepG2 cells, compared to cells that did not process anything, about 60% of cells treated with t-BHP only When compared with the cells treated with t-BHP alone, the germinated fermented soybean samples were not significantly different from those treated with t-BHP alone. However, referring to FIG. 10B, when the 70% EtoH extract germinated fermented soybean is compared with the cells treated with t-BHP only (64.67 ± 0.7 8%), the concentration of the cells is not significant but it can be seen that the survival rate of the cells 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. Reactive oxygen species (ROS) measurement using HepG2 cells

Dichlorofluorescin diacetate (DCFH-DA) reacts with free radicals (ROS) in the cell and converts it into a strong fluorescent substance, dichlorofluorescein (DCF). Therefore, the relative amount of ROS can be estimated by measuring the fluorescence by DCF.

In order to confirm the ROS effect on 70% EtoH germinated fermented soybean extract, 70% EtoH germinated fermented soybean extract was incubated for 12 hours in a non-cytotoxic concentration and t- BHP 1 mM was treated for 1 hour to induce oxidative stress. . After 25 μM of DCFH-DA was added for 60 minutes, the fluorescence of DCF was measured.

The result is shown in FIG. Referring to Figure 11, compared with Normal (100 ± 1.00%) fluorescence increased by (267.62 ± 2.68%) by t-BHP treatment, 0.5 (195.24 ± 1.95%) by 70% EtoH germinated fermented soybean extract pretreatment , 0.25mg / mL (195.24 ± 1.95), 0.125mg / mL (240 ± 2.41), 0.0625mg / mL (252.80 ± 2.53), significantly inhibited the concentration from the highest concentration to 70% EtoH germinated fermented soybean extract It was confirmed that the active oxygen reduction effect by.

3.5. Hematological tests using animal models

Blood biochemical test results of the group administered with germinated fermented soybean and administered with t-BHP are shown in Table 4 below. Total Cholestrol, LDH, Total Bilirubin, ALP, ALT, and measurement results showed a significant difference (Table 4) when compared to the control group. LDH is a glycolytic enzyme and widely distributed in each tissue in the body. Especially, LDH in serum, liver, kidney, and muscle increases in heart disease, liver disease, malignancy, and leukemia, but has low specificity. In acute hepatitis, it escapes from hepatocytes like AST and ALT and increases in early acute hepatitis. In chronic hepatitis and cirrhosis, it increases to 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 used for hepatotoxicity studies because enzymes are released into the blood as hepatic necrosis and hepatic tissue destruction progress. 12 shows the results of measuring ALT and AST activity. The concentration of AST 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 the concentration of germinated fermented soybeans. Significantly lower compared to the group (94.25 ± 7.37 U / L). The ALT showed no significant change in the concentration of germinated fermented soybean at 0.5 g / kg (33.71 ± 5.59 U / L) and 1 g / kg (38.17 ± 4.49 U / L), but only the t-BHP was treated in the sample treatment group. Significantly lower compared to (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 0.5g / kg + t-BHP	66.29 ± 9.89 bc	127.0 ± 27.38 b	0.49 ± 0.07 bc
GFS 1g / kg + t-BHP	64.63 ± 7.89 c	102.20 ± 16.83 b	0.41 ± 0.04 c

3.6. MDA measurement results using animal models

Lipid peroxidation refers to a phenomenon in which polyunsaturated fatty acids in cell membranes are peroxidated by various free radicals derived from superoxide anion radicals that can occur naturally in a living body. Unsaturated fatty acids are degraded through peroxidation to produce MDA, which is an indicator of lipid peroxidation. 13, in the case of liver tissue, the group administered t- BHP after the administration of germinated fermented soybeans was significantly different between the groups at the sample concentration of 0.5 g / kg (34.25 ± 5.80) and 1 g / kg (32.68 ± 12.57). There was no difference, but it decreased as the concentration increased and decreased compared to the t- BHP-only group (44.66 ± 1.94).

3.7. GST measurement results using animal models

GST is an enzyme that exists in almost every cell of the body and is involved in many metabolism as well as detoxification of the liver. In particular, GST catalyzes the reaction of binding an electron-affinity substrate to GSH. Referring to FIG. 14, GST measurements germination fermentation after administration of the beans treated group, t -BHP 0.5g / kg, 1g / kg, only one group administered t -BHP (25.68 ± 4.28) Sample administration as compared to the concentration At 0.5 g / kg (33.65 ± 12.97) and 1 g / kg (48.43 ± 8.90), the sample concentration increased with increasing sample concentration, but significant difference with the t-BHP group was only observed at 1 g / kg.

3.8. GSH measurement result using animal model

GSH is a very important antidote and powerful antioxidant in the human body that plays a key role in detoxification, immune function, and antioxidant function in the body. Referring to FIG. 13, GSH measurements germination fermentation after administration of the beans treated group, t -BHP 0.5g / kg, 1g / kg, t -BHP only administered group 21.07 ± 8.33) Sample administration concentration of 0.5 as compared to the There was no significant increase as the sample concentration increased at g / kg (25.12 ± 2.44) and 1 g / kg (24.40 ± 2.52). In addition, there was no significant difference between the groups receiving the sample and the t- BHP group, but there was a slight increase in the group receiving the sample.

3.9. SOD measurement results using animal models

SOD is an enzyme that reduces superoxide anion (O ₂ ) to H ₂ O ₂ , and is present in cytosol and mitochondria. Only Cu / Zn-SOD exists in the cytosol, but Mn-SOD exists in the matrix of mitochondria, and a small amount of Cu / Zn-SOD exists between the inner and outer layers. SOD measurement results (Fig. 9) germination fermentation after administration of the beans when compared with the group (23.11 ± 1.71) of administration of only the group treated with t -BHP 0.5g / kg, 1g / kg, t -BHP administration sample concentration 0.5 There was no significant increase as the sample concentration increased at g / kg (27.35 ± 3.32) and 1 g / kg (25 .67 ± 0.02). There is also a significant difference between the group administered with the sample and the group administered with t- BHP. However, there was no concentration-dependent effect in the group administered the sample.

3.10. CAT measurement results using animal model

Catalase is an enzyme that protects the body from oxygen poison by reducing H ₂ O ₂ produced by the action of SOD or O ₂ reduction to non-toxic H ₂ O. Mostly, it is present in peroxisome and in small amounts in mitochondria. CAT measurement results (Fig. 10) after germination fermented beans treated with the group treated with t -BHP 0.5g / kg, 1g / kg, t -BHP only one dose group (182.24 ± 19.29) administration sample concentration of 0.5 as compared to the At g / kg (204.49 ± 41.51) and 1 g / kg (293.19 ± 61.02), the sample concentration increased significantly. In addition, there was a significant difference between the group administered with the sample and the t- BHP group, and the group administered with the sample showed a concentration-dependent effect.

<Application example>

The present invention may include one or more selected from the group consisting of excipients (disintegrants, binders, lubricants), stabilizers, emulsifiers, additives, etc., which are acceptable under the pharmacological, health functional food method, food hygiene method, in addition to the extract of germinated fermented soybean. Can be. In addition, the composition may be made and used for oral use such as warnings, granules, powders, tablets, or capsules. The dosage of the composition should be adjusted according to the criteria set forth in the relevant laws (use, purpose of use, patient's condition, age, sex, weight, prescription drug, disease).

<Application Example 1>; Formulation

The formulation was prepared by mixing the germinated fermented soybean extract with excipients or carriers acceptable in the pharmacological method to prepare powders, tablets, capsules, pills, and granules as preparations.

<Product>

2 g of germinated fermented soybean extract and 1 g of lactose were mixed and filled into an airtight cloth to prepare a powder.

<Tablet>

Germinated fermented soybean extract 100 mg, corn starch 100 mg, lactose 100 mg and stearic acid magnesium 2 mg were mixed and then compressed into tablets.

<Capsules>

Germinated fermented soybean extract 100 mg, corn starch 100 mg, lactose 100 mg, magnesium stearate 2 mg was mixed, and then prepared by filling in gelatin capsules.

<Hwan>

After germination of fermented soybean extract 1 g, lactose 1.5 g, glycerin 1 g, and xylitol 0.5 g, a ring was prepared in a size of 4 g.

<Granule>

After germination of fermented soybean extract 150 mg, soy extract 50 mg, glucose 200 mg, and starch 600 mg, 100 mg of 30% ethanol solution was added and dried at 60 ° C. to form granules, which were then filled into fabric.

<Application Example 2>; food

Foods were prepared by mixing the germinated fermented soybean extract with excipients or carriers allowed by the food hygiene or dietary supplement method.

<Health food>

Germinated fermented soybean extract 1g, Vitamin A 70 μg, Vitamin E 1.0 mg, Vitamin 0.13 mg, Vitamin B2 0.15 mg, Vitamin B6 0.5 mg, Vitamin B12 0.2 μg, Vitamin C 10 mg, Biotin 10 μg, Nicotinamide 1.7 mg, Folic acid 50 ㎍, calcium pantothenate 0.5 mg, mineral mixture appropriate amount, ferrous sulfate 1.75 mg, zinc oxide 0.82 mg, magnesium carbonate 25.3 mg, potassium monophosphate 15 mg, calcium diphosphate 55 mg, potassium citrate 90 mg, carbonate Health food was prepared by mixing 100 mg of calcium and 24.8 mg of magnesium chloride.

The proportion of the vitamin and minerals mixed with the germinated fermented soybean extract may be variously modified according to the formulation or the consumer layer.

<Beverage>

Germinated fermented soybean extract 1%, liquid fructose 0.5%, oligosaccharide 2%, sugar 2%, salt 0.5% and the balance of the purified water and subsidiary materials were combined and sterilized and then packaged to prepare a beverage.

The ratio of sugars, dietary fiber, vitamins and minerals to the germinated fermented soybean extract may be variously modified depending on the dosage form or consumer layer.

Germinated fermented soybean extract of the present invention significantly increases blood estrogen and testosterone concentrations, thereby alleviating menopausal symptoms, increasing bone mineral content, significantly increasing bone density, and the thickness and number of sponges Increases. Therefore, there is industrial applicability.

Claims (10)

1. Germinated fermented soybean extract containing cumesterol.
2. The germinated fermented soybean extract of claim 1, wherein the extract is for protecting hepatocytes.
3. A composition for protecting hepatocytes comprising the germinated fermented soybean extract of claim 1.
4. The germinated fermented soybean extract according to claim 1, wherein the extract is for increasing bone density.
5. Composition for increasing bone density comprising the germinated fermented soybean extract of claim 1.
6. The composition according to claim 3 or 5, which is a pharmaceutical composition further comprising an excipient acceptable in the pharmacological method.
7. The nutraceutical composition according to claim 3 or 5, further comprising an excipient which is allowed by the nutraceutical method.
8. The composition according to claim 3 or 5, which is a food composition further comprising an excipient acceptable in the food hygiene method.
9. The composition of any one of claims 3 to 5, wherein the formulation of the composition is a liquid, granule, pill or capsule.
10. Germinating soybeans to inoculate and ferment the Rheum bacteria, and filtering and drying to obtain germinated fermented soybeans;

    Decomposing by adding ethanol solution to the dried germinated fermented soybeans, and extracting hot water by ultrasonication to obtain a hot water extract; And

    Filtrating the germinated fermented soybean extract obtained by centrifuging the hot water extract, and then drying to obtain a germinated fermented soybean extract.

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