JP2000191526A - Composition for stimulating formation of bone and preventing and treating bone osteoporosis - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a composition capable of being safely and continuously ingested, having effects for stimulating the growth of bones and the formation of bones, and capable of being used for foods and medicines effective for the prevention and treatments of bone osteoporosis, bone fracture, etc. SOLUTION: This composition (soybean soup) for stimulating the formation of bones and preventing and treating bone osteoporosis contains saponins, daizin, daidzein, genistin and genistein as main active ingredients, and can give stronger effects than those singly using the active ingredients.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a bone growth and metabolic bone disease such as osteoporosis and a bone fracture which contain both saponin, daidzin, daidzein, genistin and genistein as bone active ingredients. The present invention relates to an osteogenesis promoting and anti-osteoporotic composition.

[0002]

2. Description of the Related Art Bone mass decreases with aging, and osteoporosis tends to occur. Osteoporosis is a bone disease that is prone to fracture due to a decrease in bone mass. In recent years, osteoporosis has received much attention throughout Japan and overseas. Therefore, osteoporosis is positioned as a more important disease to prevent than to treat.

[0003] Therapeutic agents for osteoporosis include calcium,
Active vitamin D _3, estrogen, calcitonin, ipriflavone, vitamin K ₂ and bisphosphonate related compounds have been used. The main effect of these drugs is to suppress the action of osteoclasts, which function when bone mineral is dissolved from bone tissue. Currently, osteoporosis therapeutics under development are derivatives of the above related compounds. Since the use of these drugs becomes expensive and can be used after being diagnosed with osteoporosis, it is impossible to prevent osteoporosis, and a food material capable of preventing osteoporosis has been demanded.

In order to prevent osteoporosis, it is essential to increase bone mass from a young age, and to actively consume nutrients necessary for bone growth and bone formation and foods that promote bone formation on a daily basis. Has become recognized as very important. Currently, calcium and vitamin D are mainly used as foods for strengthening bones. Casein phosphopeptides that promote the absorption of calcium from the intestinal tract have also been used. Furthermore, it has been considered to utilize vitamin K ₂ in foods known as menaquinones (MK) -1 to 14 depending on the difference in side chain length. recently,
Isoflavonoids such as genistin are being used. However, in order to utilize these substances for the prevention of osteoporosis, it is necessary to add a large amount to food alone and fortify it, and it cannot be used as a food material,
Even if the functionality of food is enhanced, there are problems such as a decrease in taste. At present, no suitable food material for preventing osteoporosis has been developed.

[0005] Soybean seeds are rich in isoflavone components. The isoflavones include daidzin, genistin, glycine and those in which a malonyl group is bonded thereto, and daidzein or genistein from which sugar is removed. In recent years, biological effects such as anti-osteoporosis and female hormone activity have been reported on some of these components and have attracted attention. On the other hand, there are some reports that isoflavone has anti-osteoporosis activity, but it mainly uses daidzein or genistein as a single product (Yama).
guchi M, Gao YH: Res. Exp.
Med. , 197: 101-107, 1997;
Aguchi M, Gao YH: Mol. Cel
l. Biochem. 178: 377-382,19.
98; Yamaguchi M, Gao YH: Bi
ochem. Pharmacol. , 55: 71-7
6, 1998). On the other hand, soybeans and their hypocotyls contain high levels of saponin, daidzin, daidzein, genistin, and genistein. In addition, the above components are contained in a high concentration in the broth produced in a large amount during the process of producing natto from soybeans, and this composition is considered to have more effective and higher functionality than each single component. No findings were reported. On the other hand, soy broth and soybean hypocotyl are not used as food and are mainly discarded. If functionalities such as anti-osteoporosis effects can be found in these, they can be reused as functional foods.

[0006]

In the prevention of osteoporosis, the ingestion of foods for the prevention of osteoporosis for a long period of time requires absolutely excellent safety. Calcium conventionally used, such as isoflavones and vitamin K ₂ for adding and enhanced foods, today, it is not obtained findings in terms of their safety, a strong possibility that many problems in the future arises . However, broth and hypocotyls produced from soy foods are natural food materials, and their safety is guaranteed from the experience of traditional eating habits. Therefore, long-term intake is extremely effective for increasing bone mass and preventing osteoporosis. Functionality such as a bone mass increasing effect and an anti-osteoporosis effect of this broth has not been known so far, and no functional food using a broth material has been developed. An object of the present invention is to obtain a food composition for promoting bone growth and bone formation of soybean broth and for preventing and treating anti-osteoporosis.

[0007]

Means for Solving the Problems The composition of soybean soup having the effects of promoting bone growth and bone formation and preventing bone loss according to the present invention contains saponin, daidzin, daidzein, genistin, and genistein as active ingredients. It is characterized by.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The present inventors have conducted intensive studies on food components that affect bone metabolism, and have found that a soybean soup composition enhances bone components, thereby completing the present invention. The effect and effect of the composition of the present invention is to orally administer the boiled liquid of the present invention to 4-week-old young rats, and to examine changes in bone components of the femoral shaft and metaphyseal tissue of the rat. Revealed by That is, when a broth suspension is orally administered to rats, the amount of calcium as bone mineral in bone tissue, the activity of alkaline phosphatase, an enzyme promoting bone mineralization, and the number of cells as an indicator of the number of cells in bone tissue are increased.
As a result of measuring the amount of A, it was found that the amount of all these bone components significantly increased. From these results, the broth composition of the present invention has an anti-osteoporosis effect based on a bone growth and bone formation promoting effect, has an effect of preventing a decrease in the amount of bone mineral, and is useful for prevention of osteoporosis and fracture. Expected to be useful.

<Experimental Examples> The present invention will be described in more detail with reference to the following experimental examples. <Explanation of Experimental Method> Six young male Wistar rats were used as one group. The dried broth powder produced during the natto manufacturing process was suspended in purified distilled water to prepare a 100 mg / ml test solution. Saponin (66m
g), daidzin (77 mg), daidzein (0.6 m)
g), genistin (58 mg) and genistein (0.
6 mg). The test solution was orally administered at a dose of 100 mg / ml per 100 g of body weight of a rat using a gastric tube. The administration was performed twice a day (10 am and 4 pm) for 14 days. Twenty-four hours after the last dose, the rats are sacrificed,
The femur was removed. For the control group, purified distilled water was added at 1 ml /
100 g body weight was administered orally. The extracted femur was divided into a diaphyseal part (cortical bone) and a metaphyseal part (cancellous bone) and used for the measurement of bone components.

Measurement of Calcium Content in Bone Tissue The diaphyseal tissue and metaphyseal tissue of the femur were lightly washed in a 0.25 M sucrose solution and dried in a dryer at 100 ° C. for about 6 hours. After measuring the dry weight, the mixture was placed in a test tube, 3 ml of concentrated nitric acid was added, and the mixture was decomposed at 120 ° C. for 24 hours.
Using this as a sample solution, the amount of calcium was quantified with an atomic absorption spectrophotometer. The amount of calcium is mg per 1 g of bone dry weight.
Displayed as

Measurement of Bone Alkaline Phosphatase Activity The diaphyseal tissue and metaphyseal tissue of the femur were washed with a cold 0.25 M sucrose solution and then cooled with a cold 6.5 mM barbital buffer (p
H7.4) Crushed in 3 ml and sonicated for 60 seconds. The mixture was further centrifuged at 3,000 rpm for 5 minutes, and the supernatant fraction was used as a crude enzyme solution. Bone alkaline phosphatase activity is determined by the method of Walter and Schutt (Bergmeyer HU (ed). Method).
s of Enzymatic analysis, Vo
l. 1-2, AcademicPress, New Y
ork, pp. 856-860, 1965). The enzymatic reaction is performed by adding 0.05 ml of the enzyme solution to 2 ml of 0.1 M diethanolamine hydrochloride buffer (pH 9.8) containing disodium p-nitrophenylphosphate as a substrate.
The reaction was started by adding. The reaction is 37
Incubation was performed at 30 ° C. for 30 minutes. The reaction was stopped by adding 10 ml of 0.05N NaOH, and the amount (nmol) of released p-nitrophenol was measured with a spectrophotometer (405 nm).
The enzyme activity was expressed in terms of the amount (nmol) of p-nitrophenol produced by the reaction for 1 minute of incubation per amount of enzyme protein used (mg). The protein concentration was determined by the method of Lowry et al. (J. Biol. Chem., 19).
3: 265-273, 1951).

Measurement of Deoxyribonucleic Acid (DNA) Content in Bone Tissue The diaphyseal tissue and metaphyseal tissue of the femur were washed with a cold 0.25 M sucrose solution, and after removing water, the wet weight was measured. This was crushed in 4.0 ml of 0.1 N NaOH,
Shake for 4 hours and extract. Thereafter, centrifugation was performed at 3000 rpm for 5 minutes, and the supernatant fraction was used as a sample for DNA measurement. The amount of DNA was measured according to the method of Ceriotti (J. Biol. Chem., 214: 39-77, 1).
955). 1.0 ml of concentrated hydrochloric acid and 1.0 ml of a 0.04% indole solution were added to 2.0 ml of the sample, and the tester was covered with an aluminum cap.
After heating for 0 minutes, the reaction was stopped by quenching in ice. Extraction with 4.0 ml of chloroform for 3 to 4 minutes was repeated several times, and the amount of DNA was measured with a spectrophotometer (490 nm). The amount of DNA (mg) is the bone tissue wet weight (g)
Calculated per hit.

Statistical processing method The significance test of each measured value was performed by Student's
Performed using -test. Those with a risk factor of 5% or less were determined to have a significant difference.

Description of experimental results [0014] The diaphyseal tissue of the femur when the rat was orally administered with a sample solution (100 mg of broth / ml) in which dried broth powder produced in the process of natto production was suspended orally at a dose of 1 ml per 100 g of body weight for 2 weeks. The change of the bone component of the metaphyseal tissue was examined. Table 1 shows the results. Calcium content and DNA in diaphyseal tissue
The amount was significantly increased by the administration of broth. Furthermore, the amount of calcium, alkaline phosphatase activity and the amount of DNA in the metaphyseal tissue were significantly increased by the administration of broth. From the results, saponin (66 μg / ml), soybean (77 μg / ml), daidzein (0.6 μg / m
l), a broth composition containing genistin (58 μg / ml) and genistein (0.6 μg / ml) can prevent bone growth due to promotion of bone formation and decrease in bone mass due to decrease in bone metabolic activity due to aging. It was found that the food material had an effective composition. In addition, saponin (66 μg / ml) and soybean (77 μg / m
l), daidzein (0.6 μg / ml) and genistein (0.6 μg / ml) were orally administered alone, but no significant increase in the bone component was observed. From this, it was found that the effect of increasing the bone component by oral administration of the broth was based on the combined synergistic effect of the above components. This opens up the possibility that the onset of aging osteoporosis can be prevented by ingesting the broth composition.

[0015]

[Table 1] Bone components in rat femur bone tissue were increased by oral administration of broth Each value represents the mean ± standard error of bone tissue of 6 rats.

The food material containing the composition of the present invention has an anti-osteoporosis effect without any side effects, and can be taken on a daily basis for the purpose of preventing osteoporosis from a young age. In addition to protecting the quality of life, it can be expected to contribute to reducing medical costs in an aging society. At the same time, food materials with the composition provided by the present invention can also enhance nutritional components.

Safety Test The acute toxicity test of the powder sample solution prepared in the examples was performed by the following method. <Test Method> One 4-week-old male Wistar rat was grouped 1
0 animals were used. Administration of test substance is 1 g per individual body weight
/ 100g body weight and 10g / 100g
After an 18-hour fast, gavage administration was performed using a gastric tube.
The rats were bred under constant temperature and humidity conditions, and the body weight of each individual was measured with the administration day being 0, and the general symptoms and the state of life and death were observed for 14 days. During this time, water and food were provided ad libitum.

<Test Results> In the administration of the powder sample solution, no fatal cases were observed, and the body weight increased steadily. The appearance was not different from that of a normal rat. Therefore, the LD50 of each test substance was greater than 10 g / 100 g, indicating that the composition was an extremely safe composition. Further, since this sample liquid is a material having a natural food component, it is determined that the sample liquid is extremely safe with respect to chronic toxicity.

[0019]

EXAMPLES Example 1 Powder Material Soybean Boiled Dry Powder (Saponin 66mg / 100g, Daizin 77mg / 100g, Daidzein 0.6mg / 100g, Genistin 58mg / 100g, Genistin 0.6mg / 100g) 700 g corn starch 280 g extracted tocopherol 20 g The above raw materials were mixed by a high-speed mixer to obtain a uniform powder. This powder can be mixed with various foods.

Example 2 Tablets Soybean broth dried powder (containing the same ingredients as in Example 1) 100 g corn starch 125 g crystalline cellulose 25 g The above ingredients were mixed uniformly. 200 ml of a 7.5% aqueous hydroxypropyl cellulose solution was added, granulated by an extrusion granulator using a screen with a diameter of 0.5 mm, immediately rounded by an elmerizer, and dried to obtain granules.

Method of drying soybean broth and hypocotyl and range of application Hypocotyl and broth generated during processing of soybean food must be dried immediately to remove water, and spoilage microorganisms and the like will proliferate and the composition of components will change. I will. As a drying method, all the methods usually used for drying foods can be applied.

[0022]

EFFECTS OF THE INVENTION A composition using a hypocotyl or broth (containing saponin, soybean, daidzein, genistin and genistein) produced during the processing of soybean food as a food material has a strong bone growth and bone formation promoting effect and bone mineral content. Especially useful for the treatment and prevention of osteoporosis in the elderly, as it has an increasing effect and can be taken not only as granules that are easy to be taken as health foods, but also as a food to eat on a daily basis without discomfort Therefore, even for young people, it is highly effective in preventing a delay in bone growth and bone formation caused by an overweight diet. Further, it is useful as a preventive food or pharmaceutical for fractures, osteomalacia, osteoporosis, rheumatic osteopenia and low back pain caused by abnormal bone formation and bone metabolism.

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Claims (3)

[Claims] 1. An osteogenesis-promoting and anti-osteoporosis composition comprising saponin, daidzin, daidzein, genistin and genistein as main active ingredients. 2. The composition according to claim 1, wherein the saponin, daidzin, daidzein, genistin and genistin are derived from soybean broth or soybean or soybean hypocotyl. 3. Saponin 50 mg or more, daidzin 50 mg or more, daidzein 0.4 mg or more, genistin 40 mg or more, and genistein 0.4 m in 100 g of solid matter
The composition according to any one of claims 1 to 3, which is not less than g.