KR101305621B1 - A pharmaceutical composition for allieviation, prevention or treatment of metabolic bone disease comprising an extract of fermented hizikia fusiforme and haelth fucntional food comprising the same - Google Patents

Abstract

The present invention provides a pharmaceutical composition for alleviating, preventing or treating metabolic bone diseases, particularly osteoporosis, comprising a fermented bovine extract as an active ingredient, and a health functional food for alleviating or preventing metabolic bone diseases, including a fermented bovine extract as an active ingredient. Fermented 톳 extract of the present invention is a natural-derived material used as a food has no side effects, has an effect on osteoblast activity and bone formation, is effective in inhibiting osteoclast differentiation, bone content and serum in the actual animal model Increasing the bone formation index, and reducing the bone absorption index can be useful for alleviating, preventing or treating metabolic bone diseases including osteoporosis.

Description

Pharmaceutical composition for alleviating, preventing or treating metabolic bone diseases comprising the extract of fermentation 톳 as an active ingredient, and a health functional food comprising the same FUCNTIONAL FOOD COMPRISING THE SAME}

The present invention relates to a pharmaceutical composition and a dietary supplement for the relief, prevention or treatment of metabolic bone diseases, in particular osteoporosis, comprising fermented bovine extract as an active ingredient.

With the development of science and civilization, the average life expectancy of human beings is gradually extended, increasing the elderly population and life after middle age, and in fact, one-third of the modern women's life is spent during menopause. Efforts have been made to improve the quality of life of women, and as part of this, many efforts are being made to treat osteoporosis in women caused by menopause.

Osteoporosis is a condition in which the bone matrix is pathologically diminished, resulting in reduced bone density and weakened bone strength, thereby increasing the risk of fracture (DJ L. Clinical guide for management of osteoporosis.The Korean Journal of Internal Medicine 1999; 57 (4)). : 801-4).

Skeletal strength is primarily formed and maintained by deposition of minerals in the bone matrix and bone matrix, which is clinically reflected in bone density. Physiologically, bone remodeling continues to replace bone matrix with new tissue (bone turnover), a process that balances bone resorption and bone formation. (Jerome CP. Hormonal therapies and osteoporosis. ILAR J. 2004; 45 (2): 170-8). Therefore, in healthy bones, bone density and strength of bones are maintained in a normal state according to balanced bone remodeling, but bone resorption occurs more than bone formation in osteoporosis-induced bones, resulting in imbalance of bone remodeling. As bone replacement decreases with bone quality and bone mass, histological bone atrophy and bone strength decreases, resulting in increased fracture rates (Belchetz PE. Hormonal treatment of postmenopausal women.N Engl J Med. 1994 Apr 14; 330 (15): 1062-71).

The primary clinical problem is postmenopausal osteoporosis, the first type of primary osteoporosis, and postmenopausal osteoporosis is the most common bone metabolic disease in older people that causes bone pain, fractures, and malformations (Lee WS PHBD.Prevalence). of Osteoporosis in Korean Women.The Journal of Korean Society of Menopause. 2003; 9 (4): 339-46).

Osteoporosis due to estrogen deficiency caused by ovarian extraction is a condition that can easily cause fractures. In this case, estrogen administration affects the composition of collagen fibers along with an increase in the mineral content of bone, thus preventing the fracture. (Clack AP, JA Schuttinga. Targeted estrogen / proestrogen replacement the graphics for osteoporosis. Calculation of health care cost savings. Osteoporos Int., 2, 195-200 (1992)). The functions of estrogens are diverse, which act directly on osteoblasts to maintain bone mass, inhibit new activation of new bone remodeling sites, and also stimulate osteoblasts and increase bone mineral TGF and IGF synthesis to act on osteoclasts By inhibiting interleukin-1 (II-1) and interleukin-1 (II-6). (Pfelschefter J., C. Chenu, A. Bird.Interleukin-1 and tumor necrosis factor stimulate the formation of human osteoclast-like cell in vitro. J. Bone Miner Res., 4, 113-118). 1989). Therefore, recently, drugs that block bone resorption by administering female hormone and Estrogen-like hormone to bone metabolic diseases (Recker RR, Saville PD, Heaney RP.Effect of estrogens and calcium carbonate on bone loss in postmenopausal) women.Annn Intern Med. 1977 Dec; 87 (6): 649-55). However, existing hormone preparations have limitations (SH C. Medical treatment of osteoporosis. HANYANG JOURNAL OF MEDICINE. 2002; 22 (1): 15-7).

Seaweed has a high content of vitamins and dietary fiber compared to terrestrial organisms, and is known to contain many essential minerals such as magnesium, iron, iodine, calcium, and zinc (Lee JW, Sung NJ. of Minerals in Algae.J Food Sci Nutr. 1980; 9 (1): 51-7), Carbohydrates in Seaweeds are Effective in Preventing Vascular Cholesterol Deposition and Intestinal Exercise, Promoting Heavy Metal Release and Improving Hyperlipidemia Kim HS, Kim GJ.Effects of the Feeding Hijikia fusiforme (Harvey) Okamura on Lipid Composition of Serum in Dietary Hyperlipidemic Rats.J Food Sci Nutr. 1998; 27 (4): 718-23. There is a growing interest in the identification of substances and the development of functional foods.

 It is an object of the present invention to provide a composition for alleviating, preventing or treating metabolic bone diseases, in particular osteoporosis, comprising an extract obtained by fermenting 톳, a natural alga that is safer than synthetic chemicals.

The present invention provides a pharmaceutical composition for alleviating, preventing or treating metabolic bone diseases comprising the fermented bovine extract as an active ingredient.

In the present invention, 톳 ( Hijikia fusiformis = Hizikia fusiforme ) is a perennial seaweed that lives in the lower intertidal zone about 30cm above the average surface and has only a meteor generation. In Korea, it grows from the south of Jumunjin to Jangsan cape on the west coast, and is widely distributed in the south coast and Jeju region. The genus belongs to the taxonomic group, Heterokontophyta (Gold algae), Phaeophyceae (Galgae), Fucales (Horsewood), Sargassaceae (Hexaneaceae), and Hizikia . In addition to being a good source of dietary fiber, it contains a large amount of neutral polysaccharide laminaran and fucoidin, which are known to have functional properties such as blood coagulation and immune enhancing action. The high mineral content of is known to have excellent efficacy in the prevention and treatment of various adult diseases and chronic diseases. In addition, it has been confirmed that not only cholesterol deposition in blood vessels is prevented, but also intestinal motility is smoothly performed, so that it is effective not only for discharging heavy metals, but also has an anticancer effect.

In the present invention, the fermentation can be used strains isolated from doenjang and yeast, Bacillus sp. Strains and Aspergillus sp. Strains are preferred, for example Bacillus subtillis, Aspergillus oryzae, Aspergillus niger, Aspergillus preverse , Aspergillus fumigatus, Aspergillus ochraceus, Aspergillus sydowii, Aspergillus versicolor, Sepharodeca Sulfuria (Cephalotheca sulfurea), Cladorrhinum brunnescens, Cladosporium cladosporioides, Clavispora lusitaniae, Clitopilus passeckerianus ), Lasiosphaeria ovina, Neurospora terricola, Geomyces pannorum, Ciganus canarian Siganus canaliculatus, Trichomonascus ciferrii, Paecilomyces sp., Penicillium glandicola, Penicillium janthinellum, Penicill Penicillium spinulosum, Peniophora incarnata, Pesterootiopsis clavispora, Thermomyces lanuginosus and Trichoderma citrinobivir trinovidermarma It is preferable to include one or more selected from the group consisting of. Preference is given to fermentation by at least one selected. In the present invention, as a carbon source for fermentation, glucose, fructose, fructose, mannose, galactose, sucrose, maltose, and the like may be used. Bran, malt, or mixtures thereof, which are included substances, may be used. The mixing ratio of bran and malt may be various weight ratios (1: 3, 1: 7, 1:10), but a 1: 1 weight ratio is more preferable.

Fermented koji extract of the present invention may be prepared according to the conventional method for preparing algae extract. Fermentation 발효 extract in the present invention can be used after washing the 톳 1-5 times in the running water before fermentation, removing the impurities and salts on the surface and dried for 24 to 72 hours.

In the present invention, the extraction solvent is preferably a solvent selected from the group consisting of water, C ₁ -C ₄ alcohol, and water and C ₁ -C ₄ alcohol mixed solvent. Alcohols of C _{1 to} C ₄ include methanol, ethanol, isopropanol, n-propanol, n-butanol, isobutanol, t-butanol and the like, but ethanol is most preferred. Further, the extraction solvent is more preferably 70 to 90 (v / v)% ethanol aqueous solution.

In the present invention, the extraction may be powdered by extracting with the solvent and then concentrated and lyophilized, and in some cases, the solvent extract is then composed of hexane, methylene chloride, acetone, ethyl acetate, chloroform and mixtures thereof. The fractionation process can be further carried out with a solvent selected from. At least one solvent may be used in the fractionation. The extract preparation temperature may be 4 to 120 ° C, but is not limited thereto. The extraction time is not particularly limited, but may be at least 30 minutes to 7 days, and a conventional extraction device, an ultrasonic grinding extractor, or a fractionator may be used. The prepared extract can then be filtered under reduced pressure or lyophilized to remove the solvent, it can be carried out both under reduced pressure filtration and lyophilization.

In one embodiment of the present invention, the fermented 톳 extract is dried after removing the salt and impurities of the 제조 to prepare a dried sample, and fermented to the dry sample to obtain a fermented product, ethanol is added to obtain a crude extract, The extract may be concentrated under reduced pressure to prepare an extract. At this time, the removal of the salt and impurities of the 톳 may be performed by washing off with running water, and the dried sample may be used for fermentation by pulverizing the 제거한 from which the salt and the impurities have been removed. The drying may be preferably carried out by natural drying. The ethanol used as the solvent of the crude extract is preferably 50 to 99 (v / v)% ethanol aqueous solution, more preferably 70 to 90 (v / v)% ethanol aqueous solution, most preferably 70 (v / v) It may be a)% ethanol aqueous solution, the preparation of the crude extract may be carried out by a conventional extraction method such as cold extraction, hot extraction or heat extraction method, preferably may be carried out by a heat extraction method. The decompression concentration may use a decompression concentrate, the extract may be a crude extract concentrated under reduced pressure.

The obtained fermented Soybean extract can be stored in a deep freezer until use.

In addition, the fermented 톳 extract may be one obtained by completely removing the moisture through the concentration and lyophilization of the obtained extract, the 톳 extract is completely removed from the water used in powder form or by dissolving the powder in distilled water or a common solvent Can be used.

Accordingly, the present invention is directed to 1) Baillus sp. Strain and Aspergillus sp. Fermentation with; And 2) extracting the fermented product of step ₁ with an extraction solvent selected from the group consisting of water, C ₁ -C ₄ alcohol, and water and C ₁ -C ₄ alcohol mixed solvent and concentrating the extract. 톳 Provides an extract preparation method. In the present invention, the carbon source and the extraction solvent are as described above.

In the present invention, metabolic bone disease refers to bone atrophy, osteoporosis, rickets disease, osteomalacia, fibrous osteotrophy, etc., caused by degeneration of the bone due to the large intestine. In the present invention, osteoporosis refers to a metabolic bone disease whose primary lesion is a quantitative decrease in the constituents of the bone in a state in which the amount of bone is significantly reduced compared to normal people of the same age and sex. Osteoporosis in the present invention may include postmenopausal osteoporosis, senile osteoporosis and bone fragility, preferably may be postmenopausal osteoporosis. The osteoporosis is mainly associated with the thinning and weakening of the bone due to the reduction of bone tissue due to the puncture of the bone or the reduction of calcium salts, and the osteoporosis patients have been reported to develop bone fractures. .

The composition of the present invention may be in the form of a pharmaceutical composition comprising the fermented 톳 extract and a pharmaceutically acceptable carrier. The pharmaceutical composition may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, and the like, and sterile injectable solutions according to conventional methods, and include powders, tablets, capsules, injections, and A liquid agent is more preferable. Such formulations can be carried out by methods conventionally performed in the pharmaceutical art and can be formulated according to the respective diseases or according to the components using methods disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA.

The pharmaceutically acceptable carrier is lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline Cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil and the like.

Oral solid preparations include tablets, pills, powders, granules, capsules and the like, and such solid preparations include at least one excipient such as starch, calcium carbonate, sucrose or lactose. ), Gelatin, and the like, and may include a lubricant such as magnesium stearate, talc, and the like.

Oral liquid preparations include suspensions, solutions, emulsions, syrups, and the like, and may contain diluents such as water and liquid paraffin, wetting agents, sweetening agents, fragrances, preservatives and the like.

Examples of the non-aqueous solution include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions and freeze-dried preparations, and non-aqueous solvents and suspensions include vegetable oils such as propylene glycol, polyethylene glycol and olive oil, And the like.

The pharmaceutical composition of the present invention may include 0.01 to 99.9% by weight, preferably 0.1 to 99% by weight of the fermented extract, based on the total weight of the composition, and a composition for preventing or treating metabolic bone diseases including osteoporosis. Depending on the method of use and purpose of use, the content of the active ingredient can be properly adjusted.

The pharmaceutical composition of the present invention can be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically) according to the desired method, and the dosage is based on the weight, age, sex and health of the patient. The range varies depending on the diet, the time of administration, the method of administration, the rate of excretion and the severity of the disease. The daily dosage of the fermented bovine extract of the present invention is about 0.1 to 1,000 mg / kg, preferably 200 to 300 mg / kg.

The pharmaceutical composition of the present invention may contain one or more active ingredients exhibiting the same or similar function as the alleviation, prevention or treatment of metabolic bone diseases including osteoporosis, in addition to the fermentation extract.

The pharmaceutical composition of the present invention may be used alone or in combination with methods using surgery, hormone therapy, drug therapy and biological response modifiers for the prevention and treatment of metabolic bone diseases including osteoporosis.

The present invention also provides a method for alleviating, preventing or treating osteoporosis by administering the pharmaceutical composition of the present invention to a mammal including a human in need of alleviating, preventing or treating osteoporosis.

The present invention provides a health functional food for alleviating or preventing osteoporosis or bone diseases comprising the fermented bovine extract as an active ingredient. In the health functional food of the present invention, fermentation, extraction solvent, extraction method and the like are as described above in the pharmaceutical composition. Health functional food of the present invention, the total weight of the composition may comprise 0.01 to 99.9% by weight of the fermented extract.

The health functional food of the present invention may be a formulation selected from powders, tablets, capsules, injections, solutions, and dairy products, and their formulation may be the same as the preparation method of a pharmaceutical composition, or may be prepared according to a general health functional food manufacturing method. have.

The health functional food of the present invention includes the food added fermented 톳 extract of the present invention as a food additive for producing a health functional food.

Fermented Soybean Extract of the present invention not only increases the expression of Osteocalcin, a bone-forming biomarker, and reduces the expression of CTx, a bone-absorbing biomarker, even when estrogen secretion is decreased, and bone density in mouse osteoporosis rat model induced by ovarian extraction. It has a good effect on alleviating, preventing or treating bone diseases including osteoporosis by improving the. Furthermore, since the fermented bovine extract is a natural product, it is expected that there will be no problem with the side effects of the conventional osteoporosis therapeutic agent, and thus it can be used as a preventive or therapeutic agent for metabolic bone diseases including osteoporosis or as a health functional food.

1 is a graph showing the effect of fermented 톳 extract on the expression of Osteocalcin, a bone-forming biomarker.
Figure 2 is a graph showing the effect of fermented 톳 extract on the expression of bone absorption biomarker CTx (C-Telepeptide).
Figure 3 is a graph showing the effect of fermented 톳 extract on bone density.
Figure 4 is a photograph showing the effect of fermented 톳 extract on bone tissue formation.
Figure 5 is a graph showing the effect of fermented 톳 extract on the activity of Alkaline phosphatase.
Figure 6 is a graph showing the effect of fermentation 톳 extract on the activity of Minerailization.
Figure 7 is a graph showing the effect of fermented 톳 extract on the inhibition of osteoclast differentiation.

Hereinafter, the structure of the present invention will be described in more detail with reference to examples, but the scope of the present invention is not limited to the following examples. In addition, the significance between the control group, the comparison group and the experimental group according to the experimental results described below was verified by ANOVA, and then compared with the Scheffe method, Bonferroni method at a = 0.05 level.

< Example  1> Preparation of Soybean Extract

Naturally dried 톳 (Honbok Country, Korea, Hijikia fusiformis is synonymous with Hizikia fusiforme) Wash 1 kg three times in running water to remove surface impurities and salt, and then dry and grind for 48 hours to prepare a dry sample. It was.

500 g of the dried sample was extracted with hot water at 100 ° C., extracted for 2 hours, and then obtained a crude extract by filtration, and then concentrated under reduced pressure at 55 ° C. using a rotary evaporator N-1000, EYELA, Japan. After lyophilization to prepare the extract of the title in powder form.

< Example  2> Preparation of Fermented Soybean Extract

1 kg of naturally dried 톳 (Honbok Country, Korea, Hijikia fusiforme) is washed three times in running water to remove the impurities and salts on the surface, and then dried and ground for 48 hours to prepare a dry sample. It was. A mixture of bran and malt (bran: malt = 1: 1 (weight ratio)) was used as a carbon source to help the growth of the strain in the pulverized dried pan, and 500 g of the pan and carbon source were mixed with each other and then separated from miso and yeast. Bacillus Subtillis and Aspergillus oryzae strains were inoculated at 4% of the total volume of the mixture of murine and carbon sources and fermented in a 37 ℃ incubator for 3 weeks. The end of fermentation was determined by extracting a certain amount at a weekly interval and then concentrated by cell proliferation assay. 500 g of the fermented sample was extracted for 24 hours using an aqueous 70% (v / v) ethanol solution, and after extraction, a crude extract was obtained by filtration and then, using a rotary evaporator N-1000, EYELA, Japan. Concentrated under reduced pressure at 55 ℃ lyophilized to prepare a powder form.

< Experimental Example  1> Measurement of breeding conditions and weight change of experimental animals

1-1. Breeding conditions of experimental animals

In order to measure the bone formation promoting effect of the fermentation 톳 extracts of Examples 1 and 2, 48 rats of 6-week-old Sparaque-dawley-based female rats (Dae Biolink, South Korea) having an average weight of 160 g were used at 22 ° C to 24 ° C. Breeding was conducted under conditions of temperature and relative humidity of 55% to 60%. The feed for the experimental animals was used solid feed (Dae Biolink, Korea), and the feed and water were always available.

The experimental animals were eight Sham control group (Sham), control group 8 ovary removal group (OVX), after removing the ovary, 100 mg / kg (체) extract, 톳 25, 50, 100 mg / kg (발효) fermentation The experiment was divided into eight experimental groups each administered the body weight, which is shown in [Table 1]. 톳 extract and fermentation 톳 extract used in the experimental group was prepared in Examples 1 and 2. The removal of the ovary was performed by dividing the group by the ovarian method for adaptation to the surrounding environment for one week and performing ovarian resection. In ovarian resection surgery, Ketamin and Rumpun are mixed at a volume ratio of 4: 1 and injected at 0.002ml per g to anesthetize the experimental animal. It was carried out by the method.

The experimental group was bred under the same conditions as other experimental animals except that the fermentation extract was administered to determine the effect of the fermentation extract. The fermentation 톳 extract was administered by the method of orally administering the test sample prepared by dissolving the powder of Example 1 in 1 ml of distilled water at the above dosage from 15 weeks after the ovarian resection procedure. The control group and the control group were administered the same dose of 0.5 (v / v)% CMC (Carboxy methyl Cellulose) as the experimental group.

[Table 1]

1-2. Confirmation of Changes in Weight of Laboratory Animals

The experimental animals measured the weight of the experimental animals at a certain time every week after the first one week of the total 21 weeks breeding period including the period in which the sample was administered, the results are summarized in [Table 2].

[Table 2]

Similar to the previous reports that the reduction of estrogen secretion by ovarian removal leads to weight gain, in this experiment, the ovary removal group (OVX) gained weight compared to the control group without ovarian removal (Sham).

On the other hand, the experimental group from which the ovary was removed also showed a high weight gain compared to the control group, it was confirmed that the extract did not significantly affect the weight loss.

< Experimental Example  2>: Bone Formation and Bone Resorption of Fermented Soybean Extract biomarker  Expression measurement

2-1. Blood collection

In order to collect blood of the experimental animals, the experimental animals of Experimental Example 1-1 were fasted 24 hours before dissection. The experimental animals fasted for 24 hours were anesthetized using ether, and then opened, and blood was collected from the abdominal vein.

2-2. Bone Formation and Bone Resorption biomarker  Expression measurement

The blood collected above was left at room temperature for 30 minutes to measure enzyme activity and lipid concentration, and then centrifuged for 10 minutes at 3,000 rpm at 4 ° C. Serum was separated from the blood separated by the centrifugation. The isolated serum was placed in a heparinized tube, and then, bone formation and bone resorption biomarkers were obtained using Rat-MID ™ Osteocalcin ELA (Nordic bio Sci., USA) and Rat-Laps ™ ELA (Nordic bio Sci., USA). Osteocalcin (called osteoblastic hormone, which acts to hold calcium in the bone) and C-telopeptide (CTx, produced more as bone breaks occur) were measured and the results are shown in [FIG. 1] and [FIG. 2]. (In FIG. 1 and FIG. 2, * indicates p-value <0.05, ** indicates p-value <0.01, *** indicates p-value <0.001).

As shown in FIG. 1 and FIG. 2, the results of Osteocalcin (314.19 ± 143.2 ng / ml) and CTx (118.88 ± 21.5 ng / ml) of the control group (OVX) were compared to Osteocalcin (565.60) of the control group (Sham). Comparing to the results of ± 90.5 ng / ml) and CTx (58.22 ± 39.6 ng / ml), Osteocalcin was less and CTx was more expressed. On the other hand, in the experimental group to which 톳 and fermented 톳 extracts were administered after ovarian resection, Osteocalcin fermented 톳 100 mg / kg (367.47 ± 105.5 ng / ml) and fermented 톳 25 mg / kg (601.55 ± 110.4 ng / ml) 톳 50mg / kg (557.81 ± 171.1 ng / ml) fermentation 톳 100mg / kg was (822.61 ± 137.8 ng / ml) fermentation 톳 100 ㎎ / kg showed more 145% expression than Sham group Compared with the OVX control, 262% of the expression showed that the fermentation process increased the expression of Osteocalcin, a biomarker involved in bone formation. In the case of CTx, 100 mg / kg (85.48 ± 40.9 ng / ml), 25 mg / kg of fermentation (61.48 ± 30.6 ng / ml), 50 mg / kg of fermentation (57.91 ± 3.7 ng / ml), 100mg / kg of fermentation showed (47.26 ± 6.5 ng / ml), and the expression of CTx in fermentation 톳 100mg / kg was 81% in sham and 40% in OVX control. Inhibition of bone absorption was confirmed by reducing the expression of CTx, a biomarker involved in

As described above, since the composition of the present invention increases Osteocalcin and decreases CTx compared to the comparison group (ovx), the experimental group is expected to have a beneficial effect on bone metabolism after menopause, that is, to treat osteoporosis.

< Experimental Example  3> Determination of the Effect on Fermentation Bone Extracts on Bone Mineral Density

Bone density is the most widely used indicator of osteoporosis and according to the KFDA Osteoporosis Guidelines (2008), it is the most important indicator of osteoporosis. In other words, if the bone density increases compared to the comparison group, it can be considered that there is a therapeutic effect of osteoporosis. Accordingly, the present inventors have examined the effect of fermented Soybean extract on bone density in order to investigate the effect of fermented Soybean extract to treat or prevent osteoporosis.

3-1. Organ extraction

The long-term extraction and storage of the experimental animals were removed at the left and right femurs of the experimental animals from which the blood was collected in Experimental Example 2-1. All muscle tissues and ligaments except bone tissue were removed and stored at -70 ° C until the experiment.

3-2. Bone density measurement

Measurement of bone density was measured using a bone mineral density meter (PIXImus, Lunar, USA) at Hallym University, the results are shown in Figure 3 and Table 3 (Fig. 3 and Table 3). Where * denotes p-value <0.05, ** denotes p-value <0.01, *** denotes p-value <0.001).

[Table 3]

< Experimental Example  4> Effect of Fermented Soybean Extract on Bone Tissue Change

After confirming the bone density of the experimental animal and then paraffin embedding in order to confirm histologically, the tissue was thinned to make a slide and then confirmed the formation of bone using a dye, the result is [Fig. 4] Written on.

Using FIG. 4, the amount of new bone for all bones was confirmed through image analysis (BV / TV), and the results were prepared in [Table 4].

[Table 4]

Experimental Example 5 Effect of Fermented Soybean Extract on the Activity of Osteoblasts and Osteoclasts in Vitro

5-1. Alkaline 포스화제 activity  Confirm

Osteoblasts contain alkaline phosphatase (ALP) in the cell membrane, and ALP is an enzyme involved in calcium and phosphorus metabolism. It is found in high concentrations in the stromal vesicles of the extracellular membrane and calcified tissues. As ALP hydrolyzes organic phosphate, it increases the concentration of phosphate ions locally at the site of calcification and induces calcification by depositing calcium phosphate on extracellular matrix. It was. The ALP activity test was performed by measuring the amount of p-nitrophenol, a product of hydrolysis reaction, by using ALP as a catalyst for the hydrolysis reaction of p-nitrophenyl phosphate (pNPP, Sigma, St. Louis, MO, USA). Indirectly calculating the concentration of ALP. The cultured MC3T3-E1 cells were adjusted to 1x10 ⁴ cells / well and dispensed into 96well plates. After 24 hours, differentiation-inducing media (50 ㎍ / ml Vit. C (ascorbic acid) and 10 mM β-glycerophosphate (β-GP) Exchanged with the included α-MEM medium) and cultured for 24 hours for sample treatment. 톳, fermented 톳 extract and dexamethron (sample) were dissolved in α-MEM medium and treated at 1, 10, 100 ㎍ / ml, and the control (treatment) treated only α-MEM medium at 200 μl per well without sample. After 3 days, the cells were washed with PBS, and 20 μl of 0.1% Triton X-100 was added thereto, followed by lysis at 37 ° C. for 30 minutes. Then, 20 μl 0.1 M glycine-NaOH buffer (pH 10.4) and 20 μl of p-nitrophenyl phosphate (p-NPP) were added, followed by reaction at 37 ° C. for 30 minutes. After the reaction, the reaction was stopped with 100 μl of 0.1N NaOH, and the absorbance was measured at 405 nm. ALP activity was determined by measuring p-nitrophenol (p-NP) generated from p-NPP, preparing a standard curve for p-NP, and then comparing the activity with the control group. The results were prepared in [FIG. 5] and [Table 5]. Comparison of the experimental group used Dexamethasone having bone formation efficacy as a positive control (in Fig. 5 and Table 5, * indicates p-value <0.05, ** indicates p-value <0.01, ** * Indicates p-value <0.001).

[Table 5]

5-2. Mineralization activity  Confirm

Fermentation Mineral Mineralization activity was confirmed to determine whether osteoblasts are differentiated by the extract to form bone. MC3T3-E1 osteoblasts were attached to a polystyrene cell culture dish and 5% using α-MEM (PAA) containing 1% antibacterial-antifungal solution (PAA) containing penicillin and streptomycin and 10% FBS (PAA). Basic incubation in a 37 ° C. incubator with CO ₂ and 95% humidity.

Differentiation of MC3T3-E1 cells into osteocytes was treated with 3 × 10 ⁴ cells / well in a ²⁴ well plate to form monolayers of 50 μg / ml Vit. It was induced by C (ascorbic acid) and 10 mM β-glycerophosphate (β-GP) treatment. After incubation for one day, the mineralization degree of fermentation extract was measured using Vit C and β-GP, which mineralize MC3T3-E1 cells in 24well plates. Cells treated with Vit C and β-GP were dissolved in 톳 and fermented 톳 extracts and positive control Dexamethason at 1, 10, 100 ㎍ / ml in α-MEM medium, and treated with α-MEM medium except Sample. As a control, each well was treated with 1 ml and incubated in a 37 ° C. CO ₂ incubator for 3 weeks, the medium was removed from the mineralized 24 well plate, the plate was washed with PBS, and then washed with 10% formalin solution. Fixed at 4 ° C. for 12 hours.

Alizarin red solution (ARS, Sigma, USA) was prepared at a concentration of 40 mM during cell fixation to adjust the pH to 4.2. After staining the fixed cells with ARS for 5 minutes, wash the remaining dye using DW (distilled water), and then dry the stained area using PBS. After confirming nodule formation under a microscope, 10% cetylpyridinium chloride (Sigma, USA) was reacted for 15 minutes, and then the solution was transferred to a 96well plate to confirm absorbance at 562 nm. The results were prepared in [FIG. 6] and [Table 6]. Dexamethasone was also used as a positive control in the above experiments (in FIG. 6 and Table 6, * indicates p-value <0.05 and ** indicates p-value <0.01).

TABLE 6

5-3. Confirmation of inhibition of osteoclast differentiation

Raw 264.7 cells differentiated into osteoclasts by RankL were subjected to TRAP staining to confirm whether the extract inhibited differentiation. Raw 264.7 osteoclasts were attached to a polystyrene cell culture dish and α-MEM (PAA) containing 1% antibacterial-antifungal solution (PAA) containing penicillin and streptomycin and 10 (v / v)% FBS (PAA). Were incubated in a 37 ° C. incubator maintained at 5% CO ₂ and 95% humidity. The degree of differentiation of osteostelasts was confirmed by tartrate-resistant acid phosphatase (TRAP, sigma, USA) staining. RAW264.7 osteoclast progenitor cells were treated with 1 × 10 ⁴ cell / well on a 24well plate, and 50 ng / mL recombinant murine RANKL and 톳, fermented 톳 extract and positive control Dexamethason were added to α-MEM medium in 1, 10, 100 ㎍. It was dissolved in / ml, and treated only with α-MEM medium except for the sample as a control (control), 1ml each well was incubated for 3 days. Then, remove the medium, wash with 1X PBS, fixative solution (25ml citrate solution, 65ml acetone, 8ml 37% formaldehyde) 0.5ml per well to fix, wash with DW and staining solution (45ml DW-prewarmed to 37 ℃, 0.5ml Fast Garnet GBC base solution, 0.5ml Sodium Nitrate solution, 0.5ml Naphthol AS-BI phosphate solution, 2ml acetate, 1ml Tartrate solution) was treated with 300µl per well. After blocking the light for 1 hour in a 37 ℃ incubator, washed with DW and counterstained with H & E solution for 2 minutes, residual dye was washed with DW and dried to confirm the number of mature Osteoclast under a microscope. The confirmed Osteoclast was compared with the control group by measuring the intensity using an image analysis program (Leica application suite, Leica, Germany). The results were prepared in [Figure 7] and [Table 7]. The experiment also used Dexamethasone as a positive control. (In FIG. 7 and Table 7, ** indicates p-value <0.01 and *** indicates p-value <0.001).

[Table 7]

< Manufacturing example > Manufacture of Milk Containing Fermented Soybean Extract

Milk containing the components shown in [Table 8] and the weight% thereof can be prepared according to a conventional milk production process.

[Table 8]

<Manufacturing Method>

The method for preparing milk containing fermented Soybean extract according to the present invention can be prepared simply by the following conventional method. Bacterial number 1 grade crude milk and ingredients other than the milk shown in Table 8 may be mixed and manufactured through a process consisting of dissolution step, homogeneous step, present sterilization step, storage and the like.

Preparation Example 2 Preparation of Pharmaceutical Composition Containing Fermented Soybean Extract

A. Preparation of Powders

Fermented Soybean Extract 200 mg

Lactose 100 mg

Talc 10 mg

The above components are mixed and filled in airtight bags to prepare powders.

B. Preparation of Tablets

Fermented Soybean Extract 200 mg

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

After mixing the above components, tablets are prepared by tableting according to the usual preparation method of tablets.

C. Preparation of Capsule

Fermented Soybean Extract 200 mg

Crystalline cellulose 3 mg

Lactose 14.8 mg

Magnesium stearate 0.2 mg

The above components are mixed according to a conventional capsule preparation method and filled in gelatin capsules to prepare capsules.

D. Preparation of Injection

Fermented Soybean Extract 200 mg

180 mg mannitol

Sterile sterilized water for injection 2974 mg

Na ₂ HPO ₄ 12H ₂ O 26 mg

(2 ml) per ampoule in accordance with the usual injection method.

E. Preparation of Liquids

Fermented Soybean Extract 200 mg

10 g per isomer

5 g mannitol

Lemon incense quantity

Purified water quantity

Each component was added to purified water in accordance with the usual preparation method of the liquid preparation and dissolved, and the lemon flavor was added in an appropriate amount. Then, the above components were mixed and then purified water was added thereto. The whole was adjusted to 100 ml with purified water, And sterilized to prepare a liquid preparation.

It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims (15)

A pharmaceutical composition for alleviating, preventing or treating osteoporosis comprising as an active ingredient a fermented Soybean extract fermented by at least one selected from Bacillus sp. Strain and Aspergillus sp. Strain. delete The pharmaceutical composition according to claim 1, wherein the extraction is performed using a solvent selected from the group consisting of water, C ₁ -C ₄ alcohols, and a mixed solvent of water and C ₁ -C ₄ . The pharmaceutical composition according to claim 3, wherein the extraction is 70-90 (v / v)% aqueous ethanol solution. The pharmaceutical composition according to claim 1, wherein the composition is a formulation selected from powders, tablets, capsules, injections and solutions. The pharmaceutical composition according to claim 1 or 3 to 5, wherein the osteoporosis is postmenopausal osteoporosis.  A health functional food for alleviating or preventing osteoporosis, comprising as an active ingredient a fermented bovine extract fermented by at least one selected from Bacillus sp. Strain and Aspergillus sp. Strain. delete The dietary supplement according to claim 7, wherein the extraction is performed using a solvent selected from the group consisting of water, C ₁ -C ₄ alcohols, and a mixed solvent of water and C ₁ -C ₄ . 10. The dietary supplement of claim 9, wherein the extract is 70-90 (v / v)% aqueous ethanol solution. The health functional food according to any one of claims 7 or 9 to 10, wherein the osteoporosis is postmenopausal osteoporosis. The dietary supplement of claim 11 which is a formulation selected from powders, tablets, capsules, solutions and dairy products. delete delete delete

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