KR20160051658A - Composition for Effect of Protecting Liver Cells and Increasing Bone Density Using Germinated Fermented Soybean Extract Containing - Google Patents

Abstract

The present invention relates to a fermented germinated bean extract containing coumestrol, a manufacturing method thereof, and a composition comprising the fermented germinated bean extract. The fermented germinated bean extract of the present invention is confirmed to be effective to protect liver cells by measurement in vitro anti-oxidizing activity and cell survival rate for liver toxicity, implementation of a haematological test, and measurement of MDA, GST, GSH, SOD, CAT or the like on an animal model. Also, the fermented germinated bean extract effectively increases bone density by increasing mineral content of bone, increases thickness and the number of trabecular bones, and mitigates menopausal symptoms.

Description

[Technical Field] The present invention relates to a cermetosterol-containing germinated fermented soybean extract having a hepatocyte protective effect and a bone density increasing effect, and a composition containing the same. [0002]

The present invention relates to a cermetosterol-containing germinated fermented soybean extract having a hepatocyte protective effect and a bone density increasing effect, and a composition comprising the same. More specifically, the present invention relates to a method for improving the antioxidant activity and cell survival rate in vitro of hepatotoxicity caused by menopausal disorders using germinated fermented soybean extract as a component of improving natural lipid and menopausal disorders, (GST), GSH (reduced glutathione), SOD (superoxide dismutase), and catalase. In the present invention, the germinated fermented soybean extract is used as a sample in vivo In order to investigate the effect of bone loss on the improvement of bone loss during menopause, blood calcium and phosphorus levels, bone formation index, ALP activity and bone mineral content were examined. Various indexes of cancellous bone were examined by Micro-CT .

The fermented germinated soybean extract of the present invention significantly alleviates menopausal symptoms due to a significant increase in serum estrogen and testosterone concentrations and increases the protective effect of hepatocytes and the mineral content of bone in vitro and in vivo, The thickness and number of cancellous bone increases

The isozyme of didein in bean curd contains cousmenosterol. It regulates female hormone balance and content to prevent breast cancer and uterine cancer, slowing menopause and alleviating various symptoms associated with it.

Population aging is progressing rapidly throughout the world, especially in Korea, where the pace of aging is much faster than in other developed countries. As a result of the health promotion due to economic growth, the life expectancy of women in Korea has been extended and the intellectual level has been improved, so that the interest in improving the quality of life and health promotion of postmenopausal women after menopause has been increasing. Major symptoms of menopause include facial flushing, anxiety, depression, and nervousness. It is known that symptoms such as cardiovascular disease, osteoporosis and memory decay are typical, and the cause of these symptoms is the production and secretion of estrogen is stopped. Although estrogen replacement therapy has been proposed as a preventive measure, there are no results on usage, dose, or safety.

Phytoestrogen in natural foods has been studied and reported to be a reproductive hormone-like substance that can be used as a substitute for estrogen in postmenopausal women as the symptoms of postmenopause and the possibility of prevention of osteoporosis are attracting attention. Currently, synthetic estrogen replacement therapy is used in the treatment of menopausal symptoms, and most women experience side effects such as asymmetric menstruation, softening of the breasts and weight gain, and the use of foods with vegetable estrogen-like substances in natural foods Menopausal symptom relief will help to relieve side effects.

Vegetable estrogens include isoflavones and lignans and are similar to estrogen naturally occurring in plants such as beans, fruits, vegetables, peanuts, and grains. Isoflavones have attracted attention as a kind of phytoestrogen which binds weakly to the estrogen receptor in the body and has various physiological effects. Isoflavones extracted from soybeans are mainly present in glycosides in plants. They are absorbed into the human body and converted into aglycone-type genistein and daidzein by glycosidase and have estrogenic properties.

In relation to the health of women, consumption of isoflavones has been reported to reduce postmenopausal symptoms without side effects and to reduce cardiovascular risk and blood lipid and oxide levels in women with serum lipid levels and hypercholesterolemia, unlike conventional estrogen therapy There is a bar.

Korean Patent Laid-Open Publication No. 10-2014-0053471 (a composition for preventing and treating the symptoms of menopausal symptoms containing soybean extract as an active ingredient including quimestrol) is used as an effective ingredient for a menopausal In order to confirm the above-described cell experiments, the present invention was conducted in order to confirm the effects of improving the menopausal symptoms and protecting the hepatocytes through in vivo experiments. And improved germination fermented soybean extract to obtain advanced methods and results.

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 in 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., 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., et 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 aims at alleviating menopausal symptoms by using fermented germinated soybean extract, increasing hepatocyte protective effect and increasing bone mineral content.

As a result of the health promotion due to economic growth, the life expectancy of women in Korea has been extended and the intellectual level has been improved, so that the interest in improving the quality of life and health promotion of postmenopausal women after menopause has been increasing. Major symptoms of menopause include facial flushing, anxiety, depression, and nervousness. It is known that symptoms such as cardiovascular disease, osteoporosis and memory decay are typical, and the cause of these symptoms is the production and secretion of estrogen is stopped. Although estrogen replacement therapy has been proposed as a preventive measure, there are no results on usage, dose, or safety. Furthermore, women who are menopausal are lacking adequate medicines and health foods that have the effect of protecting hepatocytes and increasing bone density.

The present invention relates to a cermetosterol-containing germinated fermented soybean extract having an effect of protecting hepatocytes and increasing bone density in women of menopause, and a composition containing the same. The above-mentioned germinated fermented soybean extract containing cucumber sterol is used to examine the effect of protecting the hepatocyte and increasing the bone density in postmenopausal women. In the present invention, the germinated fermented soybean extract is used as a sample in vivo To investigate the effect of calcium and phosphorus concentration, bone formation index, ALP activity and bone mineral content in the menopausal women who may develop during the menopausal period, Various indicators were measured using Micro-CT.

It is expected that isoflavone of germinated fermented soybean can replace the role of estrogen by the significant increase of blood estrogen concentration and it will alleviate menopausal symptoms. Testosterone concentration was significantly increased (p <0.05) compared to ovariectomized control group.

The present invention was found to be effective for the hepatocyte protection through in vitro antioxidant activity, cell viability and hematological test, MDA, GST, GSH, SOD, CAT and the like in the hepatotoxicity observed in menopausal disorders. It was also confirmed that the administration of fermented soybean germ increased the mineral content of the bone. The bone density was significantly increased in rats fed with fermented soybeans and the thickness and number of cancellous bone increased. It is expected that isoflavone of germinated fermented soybean can replace the role of estrogen by the significant increase of blood estrogen concentration and it will alleviate menopausal symptoms. In particular, the concentration of triglyceride in blood and the concentration of glucose tend to decrease, so that administration of germinated fermented soybeans is expected to help improve menopausal obesity, and the administration of high-concentration germinated fermented soybeans is similar to estrogen administration To prevent osteoporosis. Antioxidant enzymes such as catalase, SOD, GSH and GST are required for the oxidative damage of the body to oxidative stress, which is regarded as a cause of various human diseases. As a result, the antioxidative activities of liver enzymes were significantly decreased and increased compared with those of t- BHP alone, such as SOD, GSH, GST, and MDA, in an animal model with artificial liver damage using germinated fermented soybeans Respectively. Thus, the germinated fermented soybeans showed a liver protective effect against oxidative stress.

Figure 1 shows the weight gain of the group to which estrogen was administered.
Figure 2 shows serum lipid, cholesterol and glucose concentrations
Figure 3 shows changes in hormones.
Fig. 4 shows the measurement result of the bone index indicator.
Fig. 5 shows the result of bone mineral content measurement.
Fig. 6 shows the bone microstructure index.
Fig. 7 shows a 2D image photographed using Micro-CT.
Fig. 8 shows a sep-pak separation process of 70% ethanol germinated fermented soybean.
9 shows cell viability of germinated fermented soybean and 70% ethanol-extracted germinated fermented soybean.
10 shows the survival rate of t- BHP cells in germinated fermented soybean and 70% ethanol-extracted germinated fermented soybean.
FIG. 11 shows ROS measurement results of germinated fermented soybean and 70% ethanol-extracted germinated fermented soybean.
Figure 12 shows the results of the animal model hematological test AST (GOT) and ALT (GPT) measurements.
13 shows the result of MDA measurement in the liver of the group administered t- BHP after administration of fermented soybeans.
14 shows the result of GST measurement in liver of the group administered t- BHP after administration of fermented soybeans.
FIG. 15 shows the results of GSH measurement in the liver of the group administered t- BHP after administration of fermented soybeans.
FIG. 16 shows SOD measurement results in the liver of the group administered t- BHP after administration of fermented soybeans.
FIG. 17 shows the result of CAT measurement in the liver of the group administered t- BHP after administration of fermented soybeans.

&Lt; Example 1 > Materials and methods

1.1. Experimental material

Germinated fermented soybeans used in this experiment were supplied from Hibio. Soybeans obtained from Jeju Agricultural Research and Extension Services are germinated according to previous studies and fermented by inoculation with Aspergillus oryzae. After washing with water, purified water and ultrasonic, it was dried and used as an experimental sample.

1.2. Preparation of sample

Soybeans were washed, washed, germinated and Aspergillus After filtration through fermentation process using oryzae, it was dried at 60 ° C. 80 g of ethanol was added to 10 g of the dried sample at a rate of 7 mL / g, followed by hydrothermal extraction using ultrasound. Thereafter, the germinated fermented soybean extract was obtained by centrifugation, which was then filtered through a PTFE membrane and dried to obtain a sample. The above samples were dissolved in tertiary distilled water and used in animal experiments

1.3. Experimental animal

Sprague-Dawley female rats, 8 weeks old, were purchased from BioLink (Daejeon) and reared in a laboratory (Purina). The animals were adapted to the control diet for 2 weeks before starting the experiment. The ovariectomized control group (sham), the estrogen-treated group (OVX-estrogen), the germinated fermented soybean extract (0.5 g / kg body weight) (OVX-0.5), 1.0 g / kg of germinated fermented soybean extract (OVX-1.0) and 2.0 g / kg of germinated fermented soybean extract (OVX-2.0) Experimental animals were maintained in a laboratory cage (20 × 26 × 13 cm) at a laboratory temperature of 22-24 ° C and a humidity of 60 ± 5%, and day and night cycles (12 hours light / 12 hours dark) After receiving approval from Korea University Animal Experimentation Ethics Committee, it was conducted in accordance with 'Guide for the Care and Use of Laboratory Animals (NRC)'. Water and diet were fed ad libitum. Experimental samples were dissolved in tertiary distilled water and administered orally in 3 ml daily. Control group (Negative-control, sham) was orally administered with the same volume of tertiary distilled water daily.

1.4. Ovariectomy

For 2 weeks, the patient was adjusted to the surrounding environment and divided into groups according to body weight by randomized complete block design. Surgery was performed with an isoflurane anesthesia. After reaching the deep anesthesia, the dorsal flank was opened to remove the ovaries and the incision was closed. In the non-ovariectomized control group (Sham), the fats at the side of the ovary were removed in the same manner and the incision site was closed. Samples were given orally after 2 weeks of recovery after surgery.

1.5. Analysis of Serum Lipid Concentration and Enzyme Activity

(TG), total cholesterol (TCHO), high density lipoprotein cholesterol (HDL), low density lipoprotein cholesterol (LDL) and glucose (GLU) were measured using FUJI DRI-CHEM 3500. The activity of glutamate oxaloacetate transaminase (GOT) and glutamate pyruvate transmaminase (GPT) was also measured by the same method.

1.6. Hormone measurement

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

1.7. Measurement of bone metabolism index

Plasma calcium and phosphorus concentrations and plasma alkaline phosphatase (ALP) concentrations were measured by FUJI DRI-CHEM 3500 as a bone formation index. BONE-alkaline phosphatase (BONE-ALP) was measured by previous studies. The femur was dried, and the femur bone mass was measured.

1.8. Bone morphometry: Micro-CT

The 2-mm-high portion of the tibia 1 mm from the growth plate of the sacrificed rats was photographed using Micro-CT (Skyscan 1072, SKYSCAN, Antwerpen, Belgium). The tube voltage was 50 kVp, the tube current was 500 μA, and 0.5 mm aluminum filtration was used. The imaging time was 210 ms and the pixel size was 18 μm. The threshold value was fixed at 58 and the 3D image was reconstructed in order to separate the osteotomy and osteotomy from the individual images. Skyscan ^™ CT-analyzers software was used for images taken by micro-CT to analyze bone microstructure parameters. The trabecular thickness (Tb.Th (μm)), the trabecular separation (Tb.Sp (μm)), the trabecular number (Tb.N) pattern factor (Tb.pf ()), and structure model index (SMI).

1.9. Statistical processing

The data from the study were processed using statistical package for statistical package for social science, version 12.0. T-tests were performed on the negative-control group to identify significant differences between groups. P <0.05, p <0.01, p <0.001 were indicated as ^{*, **, ***} , respectively.

&Lt; Example 1 >

2.1. Weight change and dietary intake

During the 6-week experiment, we found a tendency for body weight to increase in all the groups that induced menopause by ovariectomy compared to non-ovariectomized controls (Fig. 1). The body weight gain of the non-ovariectomized control group was 20.4 ± 3.3 g, compared with the control group, 0.5 g / kg, 1.0 g / kg, and 2.0 g / kg of ovariectomized control group and 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-treated group was 12.1 ± 8.7g, which showed a tendency to decrease, but there was no significant difference in the germinated fermented soybean group. There was no significant difference in dietary intake. The concentrations of 0.5, 1.0, and 2.0 mg / kg, 0.5 g / kg, 1.0 g / kg, and 2.0 g / kg of germinated fermented soybeans were 0.5, 1.0, and 2.0, respectively, in the ovariectomized control group, non-ovariectomized control group and estrogen treated group Respectively. Values are expressed as mean ± standard error.

2.2. Analysis of Serum Lipid Concentration and Enzyme Activity

Like women's menopause, estrogen secretion decreases sharply in the ovariectomized group. At this time, the body will accumulate a lot of fat cells that can produce estrogen to supplement the decreased secretion of estrogen, which leads to menopausal obesity. The lipid, cholesterol, and glucose concentrations in the blood were determined as an index of menopausal obesity, and the results are shown in FIG.

The triglyceride levels of the germinated fermented soybeans fed 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 and 55.8 ± 8.8 mg / dl respectively, Which was similar to that of the non-ovariectomized control group (66.8 ± 12.6 mg / dl).

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

2, the concentration of glucose in the blood was also 98.3 ± 16.1 mg / dl, 123.3 ± 14.4 mg / dl and 112.8 ± 6.6 mg / dl in all groups administered with germinated fermented soybeans, and the blood glucose concentration of the ovariectomized control group was 162.7 ± And 18.6 mg / dl, respectively.

The concentration of triglyceride and glucose concentration in the blood tended to decrease, suggesting that administration of fermented soybean germs would help improve menopausal obesity.

GOT, GPT is a normal enzyme made in the liver. These liver enzymes are used as an indicator of representative hepatotoxicity since liver enzymes are liberated into the blood and increase more than normal when hepatic cells are broken when the liver is damaged. As shown in the following Table 1, serum GOT and GPT activity analyzes do not show significant differences among 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 causes of various menopausal symptoms are ovarian senescence and estrogen production and secretion reduction. Therefore, it is important to confirm the increase of serum estrogen concentration in order to see the effect of alleviating menopausal symptoms of germinated fermented soybean.

2.3.1. Estrogen

The estrogen level of the ovariectomized control group was 500.3 ± 27.8 pg / ml compared with the estrogen concentration of 1020. ± 63.6 pg / ml in the non-ovariectomized control group. The estrogen level was significantly decreased (p <0.001) . In the estrogen-treated group, the serum estrogen level was elevated to 1134.1 ± 205.8 pg / ml, which was similar to that of the non-ovariectomized control group (FIG. 3). In the germinated fermented soybean group, blood estrogen levels were significantly increased (1577.1 ± 341.6 pg / ml, 2358.6 ± 191.8 pg / ml, and 938.5 ± 51.2 pg / ml, respectively) in comparison with the ovariectomized control group (FIG. The significant increase in serum estrogen concentration can be expected to replace the role of estrogen in the germinated fermented soybean extract containing the cercerosterol, thereby alleviating the symptoms of menopausal symptoms.

2.3.2. Testosterone

Testosterone is a representative anabolic hormone, involved in muscle metabolism and fat metabolism. When the level of this hormone is lowered, the amount of muscle in the body is lowered, lowering the basal metabolic rate and causing obesity easily. It also induces skin keratinization which is one of the typical symptoms of menopausal period. As shown in FIG. 3, the concentration of testosterone in blood was lower in all the ovariectomized group than in the non-ovariectomized control group. The testosterone concentrations of 2797.8 ± 517.3 pg / ml and 1969.8 ± 164.2 pg / ml in the estrogen-treated group and 1.0 g / kg of fermented soybean group were confirmed, respectively. (P <0.05) than the control group.

2.4. Measurement of bone metabolism index

FIG. 4 shows that there was no significant difference in serum calcium concentration between the groups, and in the case of phosphorus, the difference was significant in the group administered with 1.0 g / kg of germinated fermented soybeans (p <0.05).

Alkaline phosphatase (ALP) is a biomarker of bone cells involved in bone metabolism. When there is a rapidly increasing disease of bone metabolism, such as osteoporosis, the level of ALP also increases. Serum ALP levels in the ovariectomized controls were 454.0 ± 69.9 U / L, which was higher than the serum ALP levels in the non - ovariectomized controls (340.2 ± 33.31 U / L). On the other hand, in the 0.5 g / kg germinated fermented soybean group, 260.0 ± 646.9 U / L was significantly lower than the control group (p <0.05). In addition, the Bone-ALP level was 140.8 ± 58.5 U / L, which was significantly lower (p <0.05) in the 0.5 g / kg germinated fermented soybean group than the ovariectomized control group .

As shown in FIG. 5, after oral administration for 6 weeks, bone mass was significantly lowered in the ovariectomized group compared to the non-ovariectomized group (p <0.05). In the group treated with 1.0 g / kg and 2.0 g / kg germinated fermented soybeans, the bone mineral content was 61.3 ± 0.36% and 60.8 ± 0.31%, respectively. This was significantly increased (p <0.05) compared to ovariectomized controls. It was confirmed that the administration of fermented soybeans increased the mineral content of bone.

2.5. Bone morphometry: Micro-CT

The values of percent bone volume (BV / TV (%)), trabecular number (Tb.N mm ^-1 ) and trabecular thickness (Tb.Th (μm)) in all ovariectomized group Of the total number of workers. The values of estrogen - treated group and germinated fermented soybean group (2.0 g / kg) were significantly higher than those of ovariectomized group. In the case of BV / TV, the ovariectomized control group decreased to 13.7 ± 1.3%, and the BT / TV of the germinated fermented soybean group 2.0 g / kg was 23.3 ± 1.3% (p <0.01 ) (Fig. 6). Tb.N was 1.17 ± 0.09 mm ^-1 in the ovariectomized control group but increased to 1.69 ± 0.15 mm ^-1 in the group treated with 2.0 g / kg germinated fermented soybeans, similar to that in the estrogen treated group (P < 0.05). Tb.Th was significantly lower in the ovariectomized control group (0.12 ± 0.00 mm ^-1 ), but Tb.Th was significantly increased at all concentrations of estrogen and germinated fermented soybean (FIG. 6). This indicates that bone density was significantly increased in rats fed with germinated fermented soybeans, and the thickness and number of cancellous bone increased.

6, trabecular separation (Tb.Sp), trabecular pattern factor (Tb.pf (mm- ¹ )) and structure model index (SMI) were significantly higher in all ovariectomized non-ovariectomized controls The figures have increased compared to. The Tb.Sp value was 0.70 ± 0.10 in the group treated with 2.0 g / kg of germinated fermented soybeans (p <0.05). These results indicate that the dispersion of cancellous bone, which is closely related to osteoporosis, is reduced in the group treated with germinated fermented soybeans compared to the ovariectomized control group. Tb.pf was significantly (p <0.01, p <0.05) lower in estrogen treated group and 2.0 g / kg germinated fermented soybean group compared to ovariectomized 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 inde x (SMI), 1.54 ± 0.07 and 1.53 ± 0.10 in the estrogen-treated group and 2.0 g / kg of the germinated fermented soybean group were significantly (p <0.01) Respectively. Therefore, 2.0 g / kg of germinated fermented soybeans is expected to prevent osteoporosis at a level similar to estrogen administration.

3. Antioxidant effect of liver toxicity model

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

The antioxidative activity and the polyphenol content of the germinated fermented soybeans extracted from 70% EtOH were higher than those of the fermented soybeans provided by Hibio, as shown in Table 2 below.

In vitro antioxidant activity (DPPH, ABTS, Polyphenol) 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
(IC 50 μg / ml) 68.2545 0.95 30.6857 + 1.87 -

3.2 Separation and Purification of Germinated Fermented and Soybeans (GFS)

As shown in FIG. 8, the antioxidative activity of the 70% EtOH extract was higher than that of methanol (30% and 70%) after Separation using Sep-pak. As shown in Table 3 below, the antioxidative activity of ABTS showed the highest antioxidative activity in 70% methanol. Table 3 shows the results of measurement of in vitro antioxidant activity (ABTS IC50 value, Polyphenol).

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 μg / 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 survival rate

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

The absorbance measured at this wavelength reflects the concentration of living, metabolically vigorous cells and is used as an indicator of mitochondrial activity. MTT is a test to measure the activity of mitochondria, which shows some degree of toxicity to the test substance by cytotoxicity. As the test substance is toxic, the absorbance value is low when the mitochondria are destroyed.

 FIG. 9A shows that when the germinated fermented soybean samples were treated with only HepG2 cells, 0.0312 (98.52. + -. 3.64), 0.0625 (97.08. 2.93 ), 0.125 (94.44 ± 1.72), 0.25 (96.46 ± 2.86), and 0.5 (96.19 ± 3.03) mg / mL, respectively.

9B, the cell viability of the cells treated with 70% EtoH extract germinated fermented soybeans was not different from that of the untreated cells.

In addition, FIG. 10A shows that when treated with germinated fermented soybeans and t-BHP using HepG2 cells, about 60% of cells treated with t-BHP alone BHP-treated cells were not significantly different from those treated with t-BHP only when treated with germinated fermented soybeans. However, as shown in FIG. 10B, when the 70% EtoH extract germinated fermented soybean was compared with the cells treated with t-BHP only (64.67 ± 0.7 8%), the survival rate of the cells was increased although the concentration was not significant. 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) reacts with reactive oxygen species (ROS) in the cell and is converted to DCF (dichlorofluorescein), a strong fluorescent substance. Therefore, the relative amount of ROS can be estimated by measuring the fluorescence intensity by DCF.

70% EtoH germination To confirm the ROS effect on the fermented soybean extract, 70% EtoH germinated fermented soybean extract was cultured for 12 hours at a concentration without cytotoxicity and treated with 1 mM t- BHP for 1 hour to induce 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, fluorescence was increased by t-BHP treatment (267.62 ± 2.68%) compared with Normal (100 ± 1.00%), and 0.5 (195.24 ± 1.95%) by pretreatment of 70% EtoH germinated fermented soybean extract. (252.80 ± 2.53), the highest concentrations were inhibited by the concentration of 0.25 mg / mL (195.24 ± 1.95), 0.125 mg / mL (240 ± 2.41) and 0.0625 mg / And the effect of reducing the free radicals was confirmed.

3.5. Hematological tests using animal models

Blood biochemical test results of the group treated with germinated fermented soybeans and administered with t-BHP are shown in Table 4 below. Total cholesterol, LDH, total bilirubin, ALP, and ALT were measured and compared with the control group (Table 4). LDH is widely distributed in various tissues of the body, and especially in the heart, liver, kidney and muscle, LDH in the blood is increased in heart disease, liver disease, malignant tumor and leukemia but low specificity. In acute hepatitis, as in AST and ALT, it exits the hepatocyte and increases in the early stage of acute hepatitis. In chronic hepatitis and liver cirrhosis, it is normal or increases with hardness. Significant differences were found in AST and ALT, one of the indicators of liver failure.

 ALT and AST are hepatocyte leukocyte enzymes that are used for hepatotoxicity studies because the enzymes are released into the blood as necrosis of hepatocytes and destruction of liver tissues progresses. Figure 12 shows the results of ALT and AST activity measurements. In AST, there was no significant change in the concentration of germinated fermented beans at 0.5 g / kg (83.20 ± 2.86 U / L) and 1 g / kg (81.80 ± 6.72 U / L) (94.25 ± 7.37 U / L), respectively. In ALT, the germinated fermented soybean concentration was not changed to 0.5 g / kg (33.71 ± 5.59 U / L) and 1 g / kg (38.17 ± 4.49 U / L) (48.25 ± 16.19 U / L), respectively.

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 0.5 g / kg + t-BHP 66.29 ± 9.89 ^bc 127.0 ± 27.38 ^b 0.49 ± 0.07 ^bc GFS 1 g / kg + t-BHP 64.63 + - 7.89 ^c 102.20 ± 16.83 ^b 0.41 0.04 ^c

3.6. Results of MDA measurements using animal models

Lipid peroxidation refers to a phenomenon in which polyvalent unsaturated fatty acids in the cell membrane are peroxidized by various free radicals derived from superoxide anion radicals, which can occur naturally in the body. Unsaturated fatty acids are degraded through peroxidation process to produce MDA, which is used as an index to measure lipid peroxidation. 13, in the case of liver tissue, the group administered t- BHP after administration of fermented soybeans showed a significant difference between the groups at 0.5 g / kg (34.25 ± 5.80) and 1 g / kg (32.68 ± 12.57) There was no significant difference, but it decreased with increasing concentration and decreased when compared with t- BHP alone group (44.66 ± 1.94).

3.7. Results of GST measurement using animal model

GST exists in almost all cells of the body and is an enzyme involved in many metabolism as well as liver detoxification. 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 The increase in sample concentration was observed at 0.5 g / kg (33.65 ± 12.97) and 1 g / kg (48.43 ± 8.90), respectively, but the significant difference was only 1 g / kg with t-BHP.

3.8. GSH measurement results using dynamic model

GSH is a very important antioxidant and a very important antioxidant in human body which plays a key role in detoxification, immune and antioxidant functions in the body. FIG. 13 shows that when GSH measurement was compared with 0.5 g / kg, 1 g / kg, and t- BHP only group 21.07 ± 8.33 after administration of t -BHP after administration of fermented soybeans, sample concentration 0.5 g / kg (25.12 ± 2.44) and 1 g / kg (24.40 ± 2.52), respectively. In addition, there was no significant difference between the groups administered with t- BHP and the groups administered with t- BHP, but it showed a slight increase in the group administered with the sample.

3.9. SOD measurement using animal model

SOD is an enzyme that acts to reduce superoxide anion (O ₂ ) to H ₂ O ₂ , which is present in cytosol and mitochondria. There is only Cu / Zn-SOD in cytosol, but Mn-SOD in mitochondria matrix and Cu / Zn-SOD between inner membrane and outer membrane. 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 g / kg (27.35 ± 3.32) and 1 g / kg (25.67 ± 0.02), respectively. There is also a significant difference between the groups administered t- BHP and the group administered t- BHP. However, the concentration-dependent effect was not shown in the group administered with the sample.

3.10. Results of CAT measurements 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. 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 g / kg (204.49 ± 41.51) and 1 g / kg (293.19 ± 61.02), respectively. In addition, there was a significant difference between the groups administered with t- BHP and the groups administered with t- BHP, showing a concentration-dependent effect in the group administered with the sample.

<Application example>

The present invention includes at least one selected from the group consisting of excipients (disintegrants, binders, lubricants), stabilizers, emulsifiers, additives and the like which are acceptable under the pharmacological, health functional foods and food hygiene laws in addition to the extracts of fermented soybeans . In addition, the composition may be used for oral administration such as an agent, granule, powder, tablet, or capsule. The dosage of the composition should be adjusted according to the criteria set out in the relevant legislation (purpose of use, purpose of use, patient's condition, age, sex, weight, prescription drug, disease).

&Lt; Application Example 1 > Formulation

The preparation was prepared by blending the germinated fermented soybean extract with excipients or carriers allowed by pharmacopoeia to prepare powders, tablets, capsules, rings and granules.

<Sanju>

2 g of fermented soybean germ extract and 1 g of lactose were mixed and packed in airtight bags to prepare powders.

<Refining>

100 mg of germinated fermented soybean extract, 100 mg of corn starch, 100 mg of lactose and 2 mg of magnesium stearate were mixed and then tableted to prepare tablets.

&Lt; Capsule >

100 mg of germinated fermented soybean extract, 100 mg of corn starch, 100 mg of lactose and 2 mg of magnesium stearate were mixed and filled in a gelatin capsule.

<Exchange>

1 g of germinated fermented soybean extract, 1.5 g of lactose, 1 g of glycerin and 0.5 g of xylitol were mixed to prepare a ring of 4 g size.

<Granule>

150 mg of germinated fermented soybean extract, 50 mg of soybean extract, 200 mg of glucose, and 600 mg of starch were premixed, and then 100 mg of 30% ethanol solution was added and dried at 60 ° C to form granules.

&Lt; Application Example 2 > food

Foods were prepared by mixing the germinated fermented soybean extract with excipients or carriers permitted by the Food Sanitation Act or the Health Functional Food Act to produce health foods and beverages.

<Health food>

1 g of germinated fermented soybean extract, 70 g of acetic acid vitamin A, 1.0 mg of vitamin E, 0.13 mg of vitamin, 0.15 mg of vitamin B2, 0.2 mg of vitamin B12, 10 mg of vitamin B, 10 mg of biotin, 1.7 mg of nicotinic acid amide, 50 mg of folic acid, 0.5 mg of calcium pantothenate, 1.75 mg of ferrous sulfate, 0.82 mg of zinc oxide, 25.3 mg of magnesium carbonate, 15 mg of potassium phosphate monobasic, 55 mg of dibasic calcium phosphate, 90 mg of potassium citrate, 100 mg of calcium, and 24.8 mg of magnesium chloride were mixed to prepare a health food.

The ratio of the mixture of vitamins and minerals to the germinated fermented soybean extract can be variously modified depending on the formulations and the consumer layer.

<Drink>

The beverage was sterilized by mixing 1% of the germinated fermented soybean extract, 0.5% of the liquid fructose, 2% of the oligosaccharide, 2% of the sugar, 0.5% of the salt, and the remaining part.

The ratio of the saccharide, dietary fiber, vitamins and minerals to the germinated fermented soybean extract may be varied depending on the formulation and the consumer layer.

The germinated fermented soybean extract of the present invention significantly increases serum estrogen and testosterone concentrations, thereby alleviating menopausal symptoms, increasing the bone mineral content, significantly increasing bone density, and reducing the thickness and number of cancellous bone . Therefore, there is an industrial possibility.

Claims (10)

Germinated fermented soybean extract containing quercetesterol. The fermented soybean extract of claim 1, wherein the extract is for hepatocyte protection. A composition for protecting hepatocytes comprising the germinated fermented soybean extract of claim 1. The fermented soybean extract of claim 1, wherein the extract is for increasing bone density. A composition for increasing bone density comprising the germinated fermented soybean extract of claim 1. The composition according to claim 3 or 5, further comprising an excipient which is acceptable in pharmacology. The composition according to claim 3 or 5, which is a health functional food composition further comprising an excipient acceptable in the Health Functional Food Act. The composition according to claim 3 or 5, further comprising an excipient acceptable in the Food Sanitation Act. 6. The composition according to any one of claims 3 to 5, wherein the formulation of the composition is a liquid, granule, pill or capsule. Fermenting the soybean germinated by inoculation with Hwanggukjun, filtering and drying to obtain germinated fermented soybean;
Extracting the dried germinated fermented soybeans with an ethanol solution to obtain a hydrothermal extract by hydrothermal extraction with ultrasonic waves; And
Fermenting soybean extract obtained by centrifuging the hot-water extract, filtering and then drying to obtain a germinated fermented soybean extract.

Images