JP2012232916A - Composition for prevention or treatment of bone disease - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a composition for prevention or treatment of bone disease, which has a preventive or curative effect.SOLUTION: The composition contains heptamethoxyflavone as an effective ingredient, or optimally contains the heptamethoxyflavone and polymethoxyflavone combined with at least one chosen from a group of nobiletin, tangeretin, and tetramethoxyflavone.

Description

  The present invention relates to a composition for preventing or treating bone diseases.

Today, as Japan faces an aging society, the number of osteoporosis patients in Japan exceeds 10 million, one in three menopause women, and the majority of the elderly are said to suffer from osteoporosis. The increase is a serious social and economic problem. In osteoporosis, in the process of bone remodeling in which old bones are replaced with new bones, the amount of bone resorption exceeds the amount of bone formation, resulting in a decrease in bone mass. In osteoporosis, in addition to a decrease in the amount of bone mineral, the fine structure of the bone collapses, so that many elderly patients become fragile and become bedridden due to a fracture.
Conventionally, the treatment for osteoporosis has mainly been oral drugs that suppress bone resorption or promote bone formation. At present, the most widely used treatment method is to take a bisphosphonate preparation, a hormonal agent (estrogen), a calcium preparation, vitamin D or a derivative thereof, and try to improve bone metabolism systemically (for example, Patent Document 1). ).

On the other hand, it is known that naturally derived flavonoids have various functions. For example, Patent Document 2 discloses that polymethoxyflavonoids contained in seeker extract and the like are effective in preventing and treating periodontal disease. Patent Document 3 discloses tangeretin, nobiletin, and the like as flavonoids that suppress an increase in blood pressure or an increase in blood sugar level. Non-Patent Document 1 discloses that naringin, a citrus-derived flavonoid, inhibits osteoclast formation and increases bone mass. Patent Document 4 discloses that heptamethoxyflavone, hexamethoxyflavone, pentamethoxyflavone and the like have an effect of inhibiting fat accumulation.

  In particular, nobiletin improves inflammatory bone resorption by suppressing PGE production and NF-κB activation, and prevents bone loss in ovariectomized mice (Non-Patent Document 2), and osteoporosis model. It is known to partially improve bone destruction (Non-patent Document 3).

JP 2010-106042 A JP 2006-105969 A JP 2001-240539 A JP 2008-273935 A

J. Health Sci., (2009) Vol.55, pp.463-467 J. Pharmacol. Sci., (2011) Vol.115, pp.89-93 BioFactors, (2007) Vol.30, pp.179-192

  However, the effect of prevention or treatment of bone disease varies among individuals, and there is still room for improvement in order to obtain a more reliable prevention or treatment effect.

  Accordingly, an object of the present invention is to provide a composition for the prevention or treatment of bone diseases having a high prevention or treatment effect.

The present invention is as follows.
[1] A composition for preventing or treating bone disease comprising heptamethoxyflavone as an active ingredient.
[2] The composition for preventing or treating bone disease according to [1], comprising a polymethoxyflavone blend containing the heptamethoxyflavone and at least one selected from the group consisting of nobiletin, tangeretin and tetramethoxyflavone. [3] The composition for preventing or treating bone disease according to [2], wherein the polymethoxyflavone compound contains heptamethoxyflavone and nobiletin, and the mass ratio of heptamethoxyflavone to nobiletin is 0.8 to 1.5. object.
[4] The composition for preventing or treating bone disease according to [2] or [3], wherein the polymethoxyflavone blend contains at least 15% by mass of the heptamethoxyflavone.
[5] The heptamethoxyflavone or the polymethoxyflavone blend is obtained by extracting at least one of citrus peel oil, essential oil extract and squeeze cake with an organic acid aqueous solution [1]. -The composition for bone disease prevention or treatment according to any one of [4].

  ADVANTAGE OF THE INVENTION According to this invention, the composition for bone disease prevention or treatment which has a high prevention or treatment effect can be provided.

It is a graph which shows the osteoclast formation suppression rate of each polymethoxyflavone concerning Example 1 of this invention. It is a graph which shows the bone resorption activity inhibitory effect of the heptamethoxyflavone concerning Example 2 of this invention. It is a graph which shows the bone resorption activity inhibitory effect of the PMF compound concerning Example 2 of the present invention. It is a graph which shows the change of the bone density of the femur distal by ingestion of the PMF compound concerning Example 3 of the present invention. It is a micro CT photograph image which shows the change of the amount of distal femoral cancellous bone by ingestion of the PMF compound concerning Example 3 of the present invention.

The composition for preventing or treating bone disease of the present invention contains heptamethoxyflavone as an active ingredient.
According to the present invention, since heptamethoxyflavone is included as an active ingredient, a bone disease prevention or treatment composition having a high bone disease prevention or treatment effect can be provided.
In this specification, the term “process” is not limited to an independent process, and is included in this term if the intended action of this process is achieved even when it cannot be clearly distinguished from other processes. .
Moreover, the numerical value range shown using "to" in this specification shows the range which includes the numerical value described before and behind "to" as a minimum value and a maximum value, respectively.
Further, in the present invention, when referring to the amount of each component in the composition, when there are a plurality of substances corresponding to each component in the composition, the plurality present in the composition unless otherwise specified. Means the total amount of substances.
The present invention will be described below.

  The composition for preventing or treating bone disease of the present invention contains heptamethoxyflavone as an active ingredient. Heptamethoxyflavone is a compound having seven methoxy groups in the flavone skeleton, and the position of the methoxy group is not particularly limited. Examples of the heptamethoxyflavone in the present invention include those having the following structures. The heptamethoxyflavone in the present invention may be composed of a single heptamethoxyflavone having the same structure, or a combination with a plurality of heptamethoxyflavones having different methoxy group positions.

  Further, the composition for preventing or treating bone disease according to the present invention may use heptamethoxyflavone alone, and includes a polymethoxyflavone blend containing heptamethoxyflavone and other methoxyflavones. Also good. As such a polymethoxyflavone blend, it is a polymethoxyflavone blend containing heptamethoxyflavone and at least one selected from the group consisting of nobiletin, tangeretin and tetramethoxyflavone, to prevent bone disease or It is preferable from the viewpoint of therapeutic effect. Tetramethoxyflavone is a flavone derivative having four methoxy groups, and examples thereof include the following structures, but the position of the methoxy group is not limited to the following. Also. Tetramethoxyflavone may be used alone, or a combination of plural kinds of tetramethoxyflavones having different methoxy group positions may be used.

  Although there is no restriction | limiting in particular as content in the said polymethoxyflavone compound of heptamethoxyflavone, For example, it contains at least 15 mass%, and is 15 mass%-60 mass% from a viewpoint of a bone disease prevention or therapeutic effect. It is preferably 25% by mass to 45% by mass.

When nobiletin is contained in the polymethoxyflavone compound, nobiletin may be, for example, 20% to 50% by mass, or 30% to 40% by mass of the total mass of the polymethoxyflavone compound. Is preferred.
When the polymethoxyflavone compound contains tangeretin, the tangeretin may be, for example, 5% to 25% by mass, or 8% to 18% by mass of the total mass of the polymethoxyflavone compound. Is preferred.
When tetramethoxyflavone is contained in the polymethoxyflavone compound, the tetramethoxyflavone may be, for example, 1% by mass to 17% by mass, or 2% by mass to 13% by mass of the total mass of the polymethoxyflavone compound. % Is preferred.

  As an example of the polymethoxyflavone compound, it is preferable that it contains heptamethoxyflavone and nobiletin from the viewpoint of bone disease prevention or treatment effect, and is a polymethoxyflavone compound containing heptamethoxyflavone, nobiletin, and tangeretin. It is more preferable.

  In the case of a polymethoxyflavone compound containing heptamethoxyflavone and nobiletin, the amount of heptamethoxyflavone and nobiletin in the polymethoxyflavone compound is not particularly limited, but from the viewpoint of bone disease prevention or treatment effect, hepta It is preferably a polymethoxyflavone blend containing 15% to 60% by weight of methoxyflavone and 20% to 50% by weight of nobiletin, 25% to 45% by weight of heptamethoxyflavone and 30% to 40% by weight of nobiletin. It is more preferable that it is a polymethoxyflavone compound containing%.

  When the polymethoxyflavone compound contains heptamethoxyflavone and nobiletin, the mixing ratio of heptamethoxyflavone and nobiletin is not particularly limited, but from the viewpoint of bone disease prevention or therapeutic effect, the mass of heptamethoxyflavone relative to nobiletin The ratio may be, for example, 0.8 to 1.5, preferably 0.9 to 1.5, and more preferably 1.0 to 1.3.

  Heptamethoxyflavone may be a synthesized product or a commercially available product, but it is preferable to obtain it as a component of an extract from a natural product from the viewpoint of availability and bone disease prevention or treatment effect. The natural products that supply heptamethoxyflavone can be obtained from citrus fruits such as orange, group fruit, lemon, Wenzhou orange, summer orange, Yawata, Iyokan, winter orange, Sikhwasa, Navel, Yuzu, etc. From the viewpoint of availability, orange, grapefruit, seeker, lemon, Unshu mandarin orange and the like are particularly preferable. In addition, examples of the extraction site include fruit skin oil, essential oil extract or squeezed lees, and fruit oil or essential oil extract is preferable from the viewpoint of the content of heptamethoxyflavone.

The polymethoxyflavone blend containing heptamethoxyflavone or heptamethoxyflavone can be obtained as an extract of citrus fruits or a purified product from the extract.
The polymethoxyflavone compound as a citrus extract is not particularly limited, but from the viewpoint of productivity and purity of heptamethoxyflavone, at least one of citrus peel oil, essential oil extract and squeezed koji is used as an organic acid aqueous solution. It is preferable that it is the organic acid aqueous solution extract obtained by extracting by (3). The organic acid aqueous solution extract contains heptamethoxyflavone in the predetermined ratio described above, and contains nobiletin, tangeretin and tetramethoxyflavone in a predetermined ratio in addition to heptamethoxyflavone, and has high bone disease It is preferable because it has a preventive or therapeutic effect.

The organic acid aqueous solution extract is preferably obtained, for example, by the production method described in JP-A-2009-51738. The production method includes extracting polymethoxyflavones using an aqueous organic acid solution as an extraction solvent for at least one of citrus peel oil, essential oil extract, and juice extract (hereinafter referred to as an extraction step).
As for the citrus fruits as the source, as in the case of heptamethoxyflavone, for example, oranges, group fruits, lemons, Unshu oranges, summer oranges, Yawata, Iyokan, winter oranges, shikuwasa, navel, yuzu, etc. In view of the blending ratio, purity, and availability of each polymethoxyflavone, orange, grapefruit, seeker, lemon, Unshu mandarin orange and the like are particularly preferable. The extraction site is preferably peel oil or essential oil extract from the viewpoint of the content of heptamethoxyflavone, as in the case of heptamethoxyflavone.

Examples of the organic acid aqueous solution used in the extraction step include formic acid, acetic acid, propionic acid, butyric acid, lactic acid, pyruvic acid, citric acid and the like.
The content rate of the polymethoxyflavone in the organic acid aqueous solution extract obtained by the said manufacturing method is 0.3 mass%-3.5 mass%, for example.
When only heptamethoxyflavone is obtained, a step of purifying heptamethoxyflavone may be added after the extraction step in the production method. There is no restriction | limiting in particular about the refinement | purification means used for a refinement | purification process, The well-known refinement | purification means which can be refine | purified based on the structure of heptamethoxyflavone can be used.

The composition for preventing or treating bone disease may be in any form such as liquid, solid, powder, or gel, and as a known solvent or excipient that can be used based on the applied form. It may further contain an acceptable additive component or carrier.
The composition for preventing or treating bone disease of the present invention can be administered at an appropriate dose depending on age, weight and symptoms. For example, in the case of intravenous administration, the composition for preventing or treating bone disease can be administered as an active ingredient amount per day for an adult, for example, 1 to 100 mg / kg. The composition for preventing or treating bone disease can be administered as an active ingredient amount per day for an adult, for example, 2 to 200 mg / kg. The administration period of the composition for preventing or treating bone disease can be arbitrarily determined according to age and symptoms.

In the present invention, the bone disease may be any disease that develops in relation to bone metabolism failure, increased bone destruction, and the like. Osteoporosis, periodontal disease, bone metastasis of cancer, rheumatoid arthritis, deformability Examples include arthropathy, osteomalacia, hyperparathyroidism, and Paget's disease.
The “prevention or treatment” in the present invention is not limited to the distinction between prevention and treatment, as long as it can suppress or maintain or inhibit the development of symptoms related to bone metabolism failure and bone destruction. May be.

  The composition for preventing or treating bone disease of the present invention is not limited in its use as a composition as long as it contains the above-mentioned components. For example, food additives, nutritional supplements, health supplements for oral care, It may be used as a functional food of any type and form such as an additive for oral care, a nutrient, a therapeutic food, a nutritional supplement, a health food, or a material thereof.

  Hereinafter, the present invention will be described in detail with reference to examples. However, the present invention is not limited to them. Unless otherwise specified, “%” is based on mass.

[Example 1]
(1) Preparation of heptamethoxyflavone and polymethoxyflavone blend Heptamethoxyflavone and nobiletin were obtained from Wako Pure Chemicals.
The polymethoxyflavone formulation was prepared from orange peel oil according to the method described in JP 2009-51738. The composition of the obtained polymethoxyflavone blend (PMF1: purity 87.8%) is as follows. Further, by using 40% formic acid, 40% propionic acid, or 40% butyric acid instead of 40% acetic acid as the extraction solvent, a plurality of PMF blends having different heptamethoxyflavone contents can be obtained ( PMF2-PMF5), respectively.
Hereinafter, PMF1 was used for evaluation of the polymethoxyflavone blend. Hereinafter, PMF1 is referred to as “PMF formulation” or simply “PMF”.

(2) Inhibitory evaluation on osteoclastogenic differentiation Bone marrow cells were collected from 6-week-old ddy mice by a conventional method, and 2 × 10 ⁶ cells / well and 1 × 10 ^{4 of} primary osteoblasts were collected in a 24-well plate. 5% CO ₂ -95% using 0.5 mL of 10% (v / v) FBS / PR (−) αMEM seeded at a cell concentration of 1 cell / well and containing IL-1 at a final concentration of 2 ng / mL. The cells were cocultured at 37 ° C. for 7 days in the air phase. Every 3 days, 0.4 ml of the culture solution was changed. Heptamethoxyflavone, nobiletin, and the PMF formulation obtained above were added at the concentrations shown in Table 2 at the start of culture and at the time of culture medium exchange.
After the culture, the cells were stained with a tartrate-resistant acid phosphatase (hereinafter, TRAP) solution, which is an osteoclast marker enzyme, and then dried at room temperature. Three or more TRAP-positive multinucleated cells were counted as osteoclasts and evaluated as osteoclast formation rate. The results are shown in Table 2 and FIG. In addition, in FIG. 1, the black rhombus shows the case of a PMF compound, the black square shows heptamethoxyflavone alone, and the black triangle shows nobiletin alone. The numerical values in Table 2 indicate the number of osteoclasts when each component was added and cultured when the number of osteoclasts induced by IL-1 (2 ng / mL) was 100% (mean) ± SEM).

  As shown in Table 2 and FIG. 1, the formation of osteoclasts is stronger at a lower concentration when heptamethoxyflavone alone and a PMF formulation containing heptamethoxyflavone are added than when only nobiletin is added. Suppressed. It was also found that a PMF formulation containing heptamethoxyflavone in a predetermined amount was particularly strong and inhibited osteoclast formation.

[Example 2]
(Inhibition evaluation for increased bone resorption activity)
A parietal bone was collected from a 5-day-old ddy mouse and used in a 24-well plate with 1 mg / ml BSA / BGJb medium (containing penicillin (50 mg / L) and streptomycin (100 mg / L)) at 37 ° C. for 24 hours. Cultured. Thereafter, the medium was changed to a medium supplemented with IL-1 (IL-1 having a final concentration of 4 ng / mL) and heptamethoxyflavone or PMF formulation was added at 13 μg / mL or 26 μg / mL and cultured for 5 days. After the culture, the calcium concentration in the culture supernatant was measured. The calcium concentration in the case of only the culture solution was subtracted, and the increase in the calcium concentration was determined as calcium released from the parietal bone and used as an index of bone resorption activity. The results are shown in FIGS.

  As shown in FIG. 2 and FIG. 3, on the fifth day after treatment, destruction of the parietal bone was promoted by the addition of IL-1, and an increase in bone resorption activity was observed, but it was significantly suppressed by heptamethoxyflavone ( Figure 2). Moreover, even when the PMF compound was used, the bone resorption activity by IL-1 was remarkably suppressed (FIG. 3). That is, it was found that the heptamethoxyflavone and PMF compound has an inhibitory effect on the bone resorption activity enhancement by the inflammatory bone resorption factor, and can prevent bone destruction.

[Example 3]
Five week old female mice were subjected to ovariectomy (OVX) or sham operation (Sham) by a conventional method. From the next day of OVX, the PMF formulation was orally administered daily by a sonde in an amount of 5 mg per mouse once a day. At 4 weeks after the operation, the body weight and uterine mass were measured, the femur was removed, and the bone density was measured with a dual energy X-ray absorption measuring device (DCS-600R; Aroka). In the control group, a solvent (gum arabic) was orally administered daily by a sonde instead of the PMF formulation. The results are shown in FIG. In addition, the amount of trabecular bone distal to the femur was three-dimensionally observed using micro CT (Inspeio SMX-90CT, Shimadzu Corporation). The results are shown in FIG.

As shown in FIG. 4, the distal bone density of the femur is reduced in the OVX group as compared to the sham group, but the OVX group ingests the PMF formulation to allow the distal femur Bone density was significantly increased (FIG. 4, * p <0.05, ** p <0.001). On the other hand, in the Sham mice, the bone density of the femur was not changed by ingestion of the PMF formulation. Further, as shown in FIG. 5, when the amount of cancellous bone distal to the femur was three-dimensionally observed by micro CT, the cancellous bone significantly decreased by OVX was improved by oral intake of the PMF formulation. It should be noted that no change in body weight was observed in the Sham group and the OVX group due to ingestion of the PMF formulation. Moreover, although the uterus mass of the OVX group was significantly lower than that of the Sham group, the uterine mass was not changed in both the Sham group and the OVX group by ingestion of the PMF formulation.
This indicates that in postmenopausal osteoporosis models, PMF formulations can improve bone destruction by a mechanism that is not similar to female hormones.

Thus, it has been found that taking a heptamethoxyflavone and a PMF formulation containing heptamethoxyflavone exerts a preventive and therapeutic effect on osteoporosis.
Therefore, the composition of the present invention containing heptamethoxyflavone is effective in maintaining bone health and has the effect of preventing and treating bone diseases.

Claims (5)

  A composition for preventing or treating bone disease comprising heptamethoxyflavone as an active ingredient.   The bone disease prevention or treatment composition according to claim 1, comprising a polymethoxyflavone blend containing the heptamethoxyflavone and at least one selected from the group consisting of nobiletin, tangeretin and tetramethoxyflavone.   The composition for preventing or treating a bone disease according to claim 2, wherein the polymethoxyflavone compound contains heptamethoxyflavone and nobiletin, and the mass ratio of heptamethoxyflavone to nobiletin is 0.8 to 1.5.   The composition for preventing or treating a bone disease according to claim 2 or 3, wherein the polymethoxyflavone compound contains at least 15% by mass of the heptamethoxyflavone.   The said heptamethoxyflavone or the said polymethoxyflavone compound is obtained by extracting at least 1 sort (s) of citrus peel oil, an essential oil extract, and a squeezed rice cake with an organic acid aqueous solution. 5. The composition for preventing or treating bone disease according to any one of 4 above.