JP2014076972A - Bone density decrease inhibition pharmaceutical composition and food additive including dioscin as active ingredient - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide novel means which can inhibit decrease of bone density.SOLUTION: A bone density decrease inhibition pharmaceutical composition contains dioscin.

Description

  The present invention relates to a bone mineral density reduction-suppressing pharmaceutical composition and a food additive effective for preventing, treating, and / or improving a disease caused by a decrease in bone density such as osteoporosis.

  Osteoporosis is caused by various factors such as aging, menopause, extreme dieting, lack of exercise, and smoking. Bone resorption by osteoclasts significantly exceeds bone formation by osteoblasts, resulting in decreased bone density and bone loss. It is a disease that tends to break.

  So far, several substances having an effect of suppressing the decrease in bone density have been found as having an effect of preventing osteoporosis, and examples of such substances include diosgenin and methyl saponin derived from plants. Examples include protodioscin and fenugreek extract (Non-patent Documents 1 and 2 and Patent Document 1).

  In addition, the present inventors have previously shown that fenugreek (Trigonella foenum-graecum L.) seeds and bitterness-reduced fenugreek seeds treated with a saponin, which is a bitter component, have an effect of suppressing a decrease in bone density. (Non-Patent Document 3).

  However, it cannot be said that the effect which these substances have is sufficient, and development of the new means which can suppress the reduction | decrease in a bone density is still anxious in the said field | area.

JP 2005-112832 A

Kent Higdon et al., Biomed Sci Instrum. 2001; 37: 281-6. Jun Yin et al., Planta Med. 2004 Mar; 70 (3): 220-6. Chiba Taisei et al., 3E-05p “Effect of preventing osteoporosis focusing on fenugreek seed with reduced bitterness”, Annual Meeting of the Japan Society of Nutrition and Food Science, Section 195, May 1, 2009

  Then, an object of this invention is to provide the novel means which can suppress the reduction | decrease in bone density.

As a result of intensive studies in order to solve the above-mentioned problems, the present inventors have found that dioscin has an effect of suppressing a decrease in bone density, and have completed the present invention. The present invention includes the following inventions.
[1] A bone mineral density-inhibiting pharmaceutical composition comprising dioscin.
[2] The pharmaceutical composition according to [1], wherein the dioscin is obtained by treating β-glucosidase with an extract from fenugreek (Trigonella foenum-graecum L.) seeds.
[3] A food and beverage additive having a bone density decrease inhibitory effect, comprising dioscin.
[4] The food and beverage additive according to [3], wherein the dioscin is obtained by treating a fenugreek (Trigonella foenum-graecum L.) seed extract with β-glucosidase.
[5] A food or drink comprising the food or drink additive of [3] or [4].
[6] A method for producing a food or drink product having a bone density reduction inhibitory action, comprising adding dioscin to the material of the food or drink product.
[7] The method according to [6], wherein the dioscin is obtained by treating a fenugreek (Trigonella foenum-graecum L.) seed extract with β-glucosidase.

  ADVANTAGE OF THE INVENTION According to this invention, the novel pharmaceutical composition etc. which can suppress the reduction | decrease in bone density are provided. By ingesting the pharmaceutical composition of the present invention, a disease caused by a decrease in bone density such as osteoporosis can be prevented, treated and / or improved.

FIG. 1 shows the measurement results of the body weight, feed intake, uterine weight, and white fat weight of test animals administered with each experimental diet. Results were expressed as mean ± standard error (n = 5), and different alphabets were considered significant (P <0.05). FIG. 2-1 shows the measurement results of the total bone density of the femurs of the test animals administered with each experimental meal. Results were expressed as mean ± standard error (n = 5), and different alphabets were considered significant (P <0.05). FIG. 2-2 shows the measurement results of the cortical bone density (A) and cancellous bone density (B) of the femur of each test animal administered with each experimental diet. Results were expressed as mean ± standard error (n = 5), and different alphabets were considered significant (P <0.05). FIG. 3 shows the measurement results of the maximum point load in a three-point bending test using the femurs of test animals administered with each experimental meal. Results were expressed as mean ± standard error (n = 5), and different alphabets were considered significant (P <0.05). FIG. 4 shows the measurement results of the bone mass (A) of the distal cancellous cancellous bone and the bone resorption rate (B) of the cancellous bone of the test animals administered with each experimental diet. Results were expressed as mean ± standard error (n = 5), and different alphabets were considered significant (P <0.05). FIG. 5 shows the measurement results of type I collagen C-terminal telopeptide (CTx) concentration (A) and osteocalcin (OC) concentration (B) in the serum of test animals administered with each experimental diet. Results were expressed as mean ± standard error (n = 5), and different alphabets were considered significant (P <0.05). FIG. 6 shows the measurement results of the sRANKL concentration (A) in the serum and the ratio of the sRANKL concentration to the osteoprotegerin (OPG) concentration (B) in the test animals administered with each experimental diet. Results were expressed as mean ± standard error (n = 5), and different alphabets were considered significant (P <0.05). FIG. 7 shows the measurement results of TNF-α concentration (A) and IL-1 concentration (B) in the serum of test animals administered with each experimental diet. Results were expressed as mean ± standard error (n = 5), and different alphabets were considered significant (P <0.05).

  Dioscine is a kind of glycoside of diosgenin, and is a steroid saponin known to be contained in plants such as the genus Dioscorea.

  In the present invention, dioscin used as an active ingredient for inhibiting bone density reduction may be chemically synthesized or extracted from a plant known to contain dioscin (for example, a plant of the genus Yamaceae). And / or purified.

  Alternatively, in the present invention, the dioscin may be obtained by treating an extract of fenugreek (Trigonella foenum-graecum L.) seeds with β-glucosidase. More specifically, dioscin can be produced by adding an extraction solvent to fenugreek seeds to elute the seed components (saponin and the like) and treating the resulting extract with β-glucosidase.

  Here, the “fenugreek seed” may be not only a seed before germination but also a seed after germination. The seed may be in the form of powder or crushed material.

  Examples of the “extraction solvent” include hot water or water, lower alcohol (methanol, ethanol, etc.), or a mixture thereof. Particularly preferred is hot water or a mixture of water and ethanol. The amount of extraction solvent added to fenugreek seeds is 30 to 100 parts by weight, preferably 30 to 600 parts by weight, more preferably 60 to 400 parts by weight, and still more preferably 200 to 300 parts by weight with respect to 100 parts by weight of fenugreek seeds. is there. In the case of an alcohol extract, it is necessary to evaporate the alcohol by heating or the like before the enzyme reaction.

  “Β-Glucosidase” is not particularly limited, such as microorganism-derived or plant-derived, but it is preferable to use a microorganism-derived one from the viewpoint of strength of enzyme activity and substrate compatibility. Examples thereof include Trichoderma reesei (Trichoderma reesei RUT-C30 (ATCC No. 56765), Trichoderma reesei QM9414 (ATCC No. 26721)). Examples of plant-derived substances include almond-derived β-glucosidase. In addition to purified β-glucosidase, an enzyme preparation containing β-glucosidase can also be used as β-glucosidase. Examples of the enzyme preparation include microorganism-derived Multifect BGL, SPEZYME CP (Genencor Kyowa), naringinase (Tanabe Seiyaku), cellulase SS (Nagase ChemteX) and the like. Multifect BGL and SPEZYME CP (Genencor Kyowa) are liquid enzyme preparations, and naringinase is a powdery enzyme preparation. The amount of β-glucosidase added may vary depending on the form of fenugreek seed used and the type of β-glucosidase. SPEZYME CP may be added from 0.001 ml to 20 ml to 20 g of fenugreek seed powder. β-glucosidase may be added in advance to an extraction solvent before being added to fenugreek seeds, or may be added to an extract obtained by adding an extraction solvent to elute the seed components.

  The “β-glucosidase treatment” can be performed, for example, at 20 ° C. or higher, preferably 40 ° C. to 60 ° C. for 48 hours or less, preferably about 2 hours to 24 hours. By this treatment, glucose at position 26 of protodioscin contained in the extract can be cleaved to produce dioscin. After the treatment, in order to heat-inactivate β-glucosidase, for example, heat treatment can be performed at 80 ° C. to 100 ° C. for 5 minutes to 10 minutes.

  The extract treated with β-glucosidase can be fractionated or purified to obtain dioscin. The fractionation or purification treatment may be any method that fractionates dioscin contained in the extract and increases the degree of purification thereof. Filtration treatment or adsorption / desorption treatment using an ion exchange resin or activated carbon column according to a conventional method. Etc. can be performed. The obtained dioscin may be dried (hot air drying, freeze drying, spray drying, etc.).

  The dioscin used in the present invention may be purified or not purified, but is preferably in a purified state. “Purified state” means a state in which the content of substances other than dioscin is 10% or less, 5% or less, 1% or more, 0.5% or less, 0.1% or less, or less. .

  The present invention relates to a bone mineral density reduction-suppressing pharmaceutical composition (hereinafter referred to as the pharmaceutical composition of the present invention) containing dioscin as an active ingredient.

  The pharmaceutical composition of the present invention has an effect of suppressing a decrease in bone density, more specifically, an effect of suppressing bone resorption. Therefore, by administering the pharmaceutical composition of the present invention, for example, prevention and treatment of diseases caused by bone density decrease such as osteoporosis due to various factors such as aging, menopause, extreme diet, lack of exercise, smoking, And / or can be improved.

  The pharmaceutical composition of the present invention can be formulated for various dosage forms. The formulation form of the pharmaceutical composition is not particularly limited, and includes normal formulation forms such as oral preparations such as liquids, tablets, granules, fine granules, powders, chewable preparations, and parenteral preparations such as injections. It is preferable that The above preparation contains dioscin and other pharmaceutically acceptable ingredients such as excipients (eg, pharmaceutically acceptable excipients, binders, disintegrants, surfactants, absorption enhancers, adsorbents, fillings) And the like, preservatives, stabilizers, emulsifiers, etc.).

  The pharmaceutical composition of the present invention preferably contains dioscin, which is an active ingredient, so that it can be ingested in an amount of 0.01 to 1 mg / kg body weight per day.

  This invention also relates to the food-drinks additive which has a bone density reduction inhibitory effect (it is hereafter described as the food-drinks additive of this invention) which contains a dioscin as an active ingredient.

  The food / beverage food additive of the present invention can give the food / beverage products the effect of suppressing a decrease in bone density, more specifically, the effect of suppressing bone resorption, by adding the food / beverage products. Therefore, by ingesting a food or drink to which the food or drink additive of the present invention is added, a disease caused by a decrease in bone density such as osteoporosis can be prevented and / or improved.

  The food / beverage product additive of the present invention can be prepared by a conventional method using dioscin and various additives that are usually added and blended in the food / beverage product as long as the desired effects of the present invention are not impaired. Examples of various additives that are added and blended with dioscin include, but are not limited to, various vitamins, minerals, fragrances, sweeteners, colorants, flavor substances, salts, and the like.

  The form of food or drink to which the food or drink additive of the present invention can be added is not particularly limited, and examples thereof include drinks, solid foods, and semisolid foods. Specific examples of the beverage include fruit juice beverages, soft drinks, and alcoholic beverages. Examples of solid food include tablets (tablets) and sugar-coated tablets including candy, troches, etc., granules, powdered beverages, powdered soups, powdered blocks, confectionery such as biscuits, capsules, jelly, etc. There are various forms such as As a semi-solid food, the form of paste, such as curry roux, is mentioned, for example.

  The present invention further relates to a method for producing a food / beverage product having a bone density reduction-inhibiting action (hereinafter referred to as “the method of the present invention”), which comprises adding dioscin to the material of the food / beverage product. In the method of the present invention, by adding dioscin to the material of the food or drink, the obtained food or drink can be imparted with an action of suppressing a decrease in bone density, more specifically, an action of suppressing bone resorption. Therefore, by ingesting the food or drink, a disease caused by a decrease in bone density such as osteoporosis can be prevented and / or improved. The above-mentioned thing is mentioned as a form of food / beverage products.

In the method of the present invention, the amount of dioscin blended in the material of the food or drink may be an amount that can ingest 0.01 to 1 mg / kg body weight of dioscin per day by ingesting the food or drink.
Hereinafter, the present invention will be described more specifically with reference to examples.

1: Experimental Method As test animals, 20 8-week-old ddY female mice were used, and sham operation (Sham) or ovariectomy (OVX) was performed. Ovariectomized mice are generally used as postmenopausal osteoporosis model mice. Thereafter, preliminary breeding was performed with the AIN93G composition (Table 1), which is a reference diet, and divided into the following 6 groups according to the experimental diet to be administered. That is, (i) Sham group administered with a reference diet (hereinafter sometimes referred to as “Sham group”), (ii) OVX group administered with a reference diet (hereinafter referred to as “OVX group”) ), (Iii) OVX group (hereinafter sometimes referred to as “OVX + DC group”) administered with a standard diet containing 0.0028% by weight of dioscin (Table 1), (iv) 0.0034% by weight of protodioscin OVX group (hereinafter sometimes referred to as “OVX + PDC group”) administered with a standard diet (Table 1) containing, and (v) a standard diet containing 0.0013 wt% diosgenin as a positive control (Table 1) OVX group (hereinafter sometimes referred to as “OVX + DG group”), (vi) OVX group (hereinafter, referred to as “OVX + DG group”) administered with a standard diet (Table 1) containing a crude saponin fraction of 0.006% by weight. OVX + SP group ”, which was sometimes described as“ OVX + SP group ”and reared for 8 weeks.

  Dioscin, protodioscin, and diosgenin were commercially available. Dioscine was purchased from Canfo chemicals (China). Protodioscin was purchased from Aktin chemicals (China) with a 40% powder and purified by medium pressure chromatography using Lichroprep RP-18 resin. Diosgenin was purchased from Wako Pure Chemical.

  The crude saponin fraction is a mixture of fenugreek saponins based on protodioscin. Specifically, fenugreek powder was extracted with hydrous ethanol, concentrated, purified by medium pressure chromatography using ODP-400 resin, purified by preparative ODS column, and lyophilized.

  In addition, dioscin, diosgenin, and protodioscin were added to the standard diet with the same number of moles. The crude saponin fraction is set as a positive control that suppresses bone loss and is not matched with other groups.

  After collecting the blood of the mice raised for 8 weeks, the mice were sacrificed and dissected, and femur bone density (BMD) measurement, femur bone quality evaluation, and femur bone morphometry were performed. The femur bone density (BMD) is measured by qCT (LaTheta LCT-100, ALOKA), the femur bone quality is evaluated by a three-point bending test, and the femur bone morphometry is measured This was carried out by measuring the bone mass and bone resorption rate of the distal cancellous bone.

  Furthermore, bone metabolism markers in the collected blood (bone resorption markers: type I collagen C-terminal telopeptide (CTx), bone formation markers: osteocalcin (OC)), bone metabolism regulators (sRANKL, osteoprotegerin (OPG)) The concentrations of inflammatory cytokines (TNF-α, IL-1) were measured by ELISA, respectively.

2: Results (1) Influence of Administration of Experimental Food As shown in FIG. 1, no significant influence on body weight, sample intake, and white fat weight by administration of experimental food was observed in the test animals. Uterine weight was reduced by OVX treatment, but no effect of experimental diet was observed.

(2) BMD measurement results
As is clear from the comparison between the Sham group and the OVX group, the total bone density (Fig. 2-1), cortical bone density (Fig. 2-2 (A)) and cancellous bone density (Fig. 2-2) of the femur (B)) showed a significant decrease with OVX treatment.

  These reductions in bone density could be suppressed by administering dioscin, diosgenin, or crude saponin fraction (“OVX + DC group”, “OVX + DG group” and “OVX + Sp” in each figure). group"). On the other hand, it was shown that administration of protodioscin cannot suppress such a decrease in bone density (“OVX + PDC group” in each figure).

(3) Bone quality evaluation results As shown in FIG. 3, the maximum point load in the three-point bending test was reduced by the OVX treatment, as is clear from the comparison between the Sham group and the OVX group. This decrease in the maximum point load could be suppressed by administering dioscin, diosgenin, or crude saponin fraction (“OVX + DC group”, “OVX + DG group” and “OVX +” in each figure). Sp group "). On the other hand, it was shown that administration of protodioscin cannot suppress the decrease in the maximum point load (“OVX + PDC group” in each figure).

(4) Measurement results of femur bone morphology (4-1) Bone mass of distal cancellous cancellous bone (FIG. 4 (A))
As is clear from the comparison between the Sham group and the OVX group, the bone mass showed a marked decrease by the OVX treatment.

  The decrease in bone mass could be suppressed by administering dioscin, diosgenin or crude saponin fraction (“OVX + DC group”, “OVX + DG group” and “OVX + Sp” in each figure). group"). On the other hand, it was shown that administration of protodioscin cannot suppress such a decrease in bone mass.

(4-2) Bone resorption rate of the trabecular bone of the distal femur (FIG. 4B)
As is clear from the comparison between the Sham group and the OVX group, the bone resorption rate showed a significant increase by the OVX treatment.

  The increase in the bone resorption rate could be remarkably suppressed by administering dioscin, diosgenin or crude saponin fraction (“OVX + DC group”, “OVX + DG group” and “ OVX + Sp group ").

(5) Measurement results of bone metabolism markers
As is clear from the comparison between the Sham group and the OVX group, the concentrations of CTx (FIG. 5 (A)) and OC (FIG. 5 (B)) in the serum showed a significant increase by the OVX treatment. On the other hand, these increases in serum concentration could be remarkably suppressed by administering dioscin or the like (“OVX + DC group”, “OVX + PDC group”, “OVX + DG group” in each figure) And “OVX + Sp group”).

(6) Measurement results of bone metabolism regulator
As is clear from comparison between the Sham group and the OVX group, the serum concentration of sRANKL (FIG. 6 (A)) showed a marked increase with OVX treatment. In addition, the ratio of sRANKL to the serum OPG concentration (FIG. 6 (B)) showed a marked increase by OVX treatment.

  The increase in these values could be significantly suppressed by administering dioscin. On the other hand, it was shown that administration of other than dioscin could not remarkably suppress the increase in these values (“OVX + PDC group”, “OVX + DG group” and “OVX + Sp group” in each figure)).

(7) Measurement results of inflammatory cytokines
As is clear from the comparison between the Sham group and the OVX group, the concentrations of TNF-α (FIG. 7A) and IL-1 (FIG. 7B) in the serum increased with OVX treatment. On the other hand, these increases in serum concentration could be suppressed by administering dioscin or the like (“OVX + DC group”, “OVX + PDC group”, “OVX + DG group” and “ OVX + Sp group ").

3: Effect of dioscin From the above results, administration of dioscin shows a bone density decrease inhibitory effect that can suppress a decrease in bone density, bone quality, bone mass, and bone resorption rate in osteoporosis. It was. In addition, it has been shown that administration of dioscin can suppress increases in serum bone metabolism markers CTx, bone metabolism regulator sRANKL, and inflammatory cytokines TNF-α and IL-1. It was done. These factors are generally known to be involved in bone resorption by osteoclasts. Therefore, it was suggested that the bone density reduction inhibitory effect by administration of dioscin is due to suppression of bone resorption.

  On the other hand, administration of protodioscin, which is a glycoside of diosgenin, did not provide the above-described bone density reduction inhibitory effect. This shows that the glycoside of diosgenin, which is known to have a bone density reduction inhibitory effect, does not uniformly have a bone density reduction inhibitory effect.

Claims (7)

  A pharmaceutical composition for inhibiting bone density reduction, comprising dioscin.   The pharmaceutical composition according to claim 1, wherein the dioscin is obtained by treating β-glucosidase with an extract from fenugreek (Trigonella foenum-graecum L.) seeds.   A food additive having an inhibitory action on bone density reduction, comprising dioscin.   The food additive according to claim 3, wherein the dioscin is obtained by treating a fenugreek (Trigonella foenum-graecum L.) seed extract with β-glucosidase.   Food-drinks containing the food additive of Claim 3 or 4.   The manufacturing method of the food-drinks composition which has mix | blending dioscin with the material of food-drinks composition, and has the bone density reduction inhibitory effect.   The method according to claim 6, wherein the dioscin is obtained by treating β-glucosidase with an extract of fenugreek (Trigonella foenum-graecum L.) seeds.

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