JP2010030905A - Bmp-2 production promoter - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a medicinal composition effective for treating or preventing osteoporosis, using a new highly safe ingredient which is excellent in an osteoplasty-promoting action and a bone resorption-inhibiting action. <P>SOLUTION: The medicinal composition contains a water-soluble fraction of head-non-forming Brussels sprouts, obtained by mating Brassica oleracea var. acephala with Brassica oleracea var. gemmifera, for treating or preventing osteoporosis. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a BMP-2 production promoter and a pharmaceutical composition for preventing or treating osteoporosis.

Osteoporosis is a condition in which bones become brittle and tend to break. Fractures caused by osteoporosis are particularly common in elderly women, and are one of the main causes of QOL (quality of life) reduction and “bedridness”. In osteoporosis, the balance between bone resorption and bone formation is lost mainly with aging, and bone resorption is relatively dominant, resulting in a decrease in bone mass, and a change in bone microstructure reduces bone strength. It develops by.
The number of osteoporosis patients in Japan is currently estimated to be around 10 million. Furthermore, the aging rate in the total population is 19.5% in the 2005 report, and is increasing year by year. Therefore, the number of patients is expected to increase in the future. is there.
In the prevention of osteoporosis, it is important to increase the maximum bone mass as much as possible and maintain it for a long time, and to suppress even a slight decrease in bone mass that occurs after menopause and in old age. Therefore, intake of calcium, vitamin D, and vitamin K from meals and preparations is recommended, but it is difficult to suppress bone loss by itself.
In the treatment of osteoporosis, it is important to suppress bone resorption by osteoclasts while promoting bone formation by osteoblasts to increase overall bone mass.

Currently available osteoporosis treatments include bisphosphonates, estrogens, and calcitonin, but these treatments are bone resorption inhibitors and are effective in preventing and maintaining bone loss. However, the lost bone mass cannot be recovered significantly.
Furthermore, bisphosphonate preparations have side effects such as fever, esophageal ulcer, hypocalcemia, and rarely jaw osteonecrosis. Estrogen preparations have side effects that increase the risk of breast cancer, uterine cancer, myocardial infarction, and stroke. In addition, calcitonin preparations are not suitable for long-term treatment because they may diminish the effects when used continuously, and have side effects such as nausea and hot flashes, nasal inflammation, and rarely shock symptoms. .
In order to restore bone mass, in addition to inhibiting bone resorption, a therapeutic agent that can actively enhance bone formation to restore bone mass and bone structure and restore bone mechanical strength is desired. ing. Examples of drugs that are expected to enhance bone formation include statins used as therapeutic agents for hyperlipidemia. Statins promote osteogenesis by increasing the production of BMP-2 (Bone Morphogenic Protein-2 / Bone morphogenetic protein-2), a bone morphogenetic protein in osteoblasts. In non-patent literature 1). However, statins have side effects such as rhabdomyolysis, gastrointestinal dysfunction, liver dysfunction, rash and insomnia.

On the other hand, as a food-derived component, it has been reported that quercetin or a quercetin derivative has an effect of promoting OPG (Osteoprotegerin / osteoclast differentiation inhibitory factor) production. In addition, an extract of onion, spinach, kale, or parsley containing quercetin, or a hydrolyzate of extracts of buckwheat, tomato, green asparagus, azuki, potato, fig, apricot and lemon is an active ingredient for an OPG production promoter It is proposed to use as (patent document 1). However, only OPG production promoting action has been reported for these components.
Further, the present invention comprises a bone-derived promotion product or a bone-forming protein production enhancement characterized by containing, as an active ingredient, a plant-derived processed product selected from a processed product derived from a family of Apiaceae, a processed product derived from a plant belonging to the family Liliaceae, or a processed product derived from a family Asteraceae Has been proposed for the treatment or prevention of diseases requiring treatment (Patent Document 2).
. However, only an osteogenesis promoting action has been reported for these components. Moreover, the osteoporosis preventive agent which contains geraniol and / or perillyl alcohol as an active ingredient is proposed (patent document 3). These components have been reported to have both bone formation promoting action and bone resorption inhibiting action. However, these are fragrance components, the yield from the food material is small, and it is difficult to take an effective amount in terms of flavor.

Mundy G, Garrett R, Harris S, Chan J, Chen D, Rossini G, Boyce B, Zhao M, Gutierrez G .: Stimulation of bone formation in vitro and in rodents by statins. Science. Dec 3; 286 (5446): 1825-6. (1999) JP 2007-137775 A International Publication No. 2004/030683 Pamphlet JP 2007-001894 A

  It is an object of the present invention to provide a novel component that has an excellent osteogenesis promoting action and is highly safe. Another object of the present invention is to provide a novel component having both an osteogenesis promoting action and a bone resorption suppressing action. And let it be a subject to provide a pharmaceutical composition effective in the treatment or prevention of osteoporosis using such a novel component. Moreover, this invention provides the technique of manufacturing such a novel component easily.

As a result of searching for a novel component having an excellent osteogenesis promoting action and high safety, the present inventors have conducted research on the effect, and as a result, kale ( Brassica oleracea var. Acephala ) and mecabbage ( Brassica oleracea var. Gemmifera ) It was found that the water-soluble fraction of non-tuberculous me cabbage obtained by crossing the BMP-2 has a BMP-2 production promoting action, and the present invention has been completed.

That is, the present invention is as follows.
(1) BMP-2 (Bone Morphogenetic Protein- containing as an active ingredient a water-soluble fraction of non- tuberculous me cabbage obtained by crossing kale ( Brassica oleracea var. Acephala ) and mecabe ( Brassica oleracea var. Gemmifera ) 2 / Osteogenic factor-2) Production promoter.
(2) The BMP-2 production promoter according to (1), which is further an OPG (Osteprotegerin / osteoclast differentiation inhibitory factor) production promoter.
(3) The BMP-2 production promoter according to (1) or (2), wherein the water-soluble fraction of the non-tuberculous mecha cabbage is a water extract of the non-tuberculous mecacab.
(4) The water-soluble fraction of the non-tuberculous me cabbage is an aqueous layer fraction obtained by further partitioning the water extract of the non-tuberculous me cabbage with water and an organic solvent, (1) or (2 ) BMP-2 production promoter described in the above.
(5) A pharmaceutical composition for preventing or treating osteoporosis, comprising the BMP-2 production promoter according to any one of (1) to (4).
(6) The water-soluble fraction of the non-tuberculous me cabbage is converted into the amount of 30-fold concentrated water extract, 0.001 mL / kg body weight to 100 mL / kg body weight per day, preferably 0.01 mL / kg body weight The pharmaceutical composition according to (5), which is administered in an amount of 10 mL / kg body weight.
(7) A step of distributing an aqueous extract of non- tuberculous me cabbage obtained by crossing kale ( Brassica oleracea var. Acephala ) and mecabbage ( Brassica oleracea var. Gemmifera ) with water and an organic solvent, and by partitioning The manufacturing method of a BMP-2 production promoter including the process of mix | blending an aqueous layer fraction as an active ingredient.

  By administering or ingesting the BMP-2 production promoter of the present invention, production of BMP-2 is promoted and bone formation is promoted. Furthermore, by administering or ingesting the BMP-2 production promoter of the present invention, OPG production is also promoted and bone resorption is suppressed. Since the BMP-2 production promoter of the present invention has both the effect of promoting the production of BMP-2 and the effect of promoting the production of OPG, and has high safety, the pharmaceutical composition for the treatment or prevention of osteoporosis It is suitable as a component.

BMP-2 production promoter of the present invention, kale (Brassica oleracea var. Acephala) and Brussels sprouts (Brassica oleracea var. Gemmifera) mated non-heading Brussels sprouts obtained (below, simply referred to as "non-heading Brussels sprouts" )) As an active ingredient.
Unlike kale, non-tuberculous mecha cabbage has a characteristic that bud leaves are not formed with bud leaves between petioles and stems.
There is a commercially available non-ballistic me cabbage, which can be used. As a commercial item, the brand name "petit veil" (trademark) made from Masuda Seed Co., Ltd. is mentioned, for example.

Water is preferable as a solvent used for extraction to obtain a water-soluble fraction of non-tuberculous me cabbage.
The site of the non-heading mecha cabbage for extraction is not particularly limited, but leaves are preferred. Usually, after leaves are dried or dried and cut or crushed, extraction is performed by adding a solvent. The amount of the solvent to be added is not particularly limited, but is usually 1 mL to 100 mL of the solvent with respect to 1 g of the dried product of non-ballistic mecabe. The temperature of the solvent used for extraction is not particularly limited, but is preferably 10 to 30 ° C. The extraction time may be adjusted depending on the temperature of the solvent, but when extracting at 10 to 30 ° C., it is preferably 10 to 15 minutes.
The extract thus obtained is preferably concentrated by a conventional method, more preferably freeze-dried.

The obtained extract can be used as an active ingredient of the BMP-2 production promoter of the present invention. Preferably, the extract is purified and then used as an active ingredient of a BMP-2 production promoter. For example, the extract obtained as described above is distributed with a hydrophilic solvent and a hydrophobic solvent such as ethyl acetate, ethyl ether, chloroform, and the hydrophilic solvent fraction is separated.
The water-soluble fraction of non-ballistic me cabbage, which is an active ingredient of the BMP-2 production promoter of the present invention, is a non-ballistic mecha cabbage water extract, or water, and hydrophobicity such as ethyl acetate. The aqueous layer fraction obtained by partitioning with a solvent is preferred.

  The BMP-2 production promoter of the present invention is preferably also an OPG production promoter.

  The BMP-2 production promoter of the present invention may contain other optional components in addition to the water-soluble fraction of non-tuberculous mecabe. As such an optional component, a substance that is expected to have a BMP-2 production promoting action, an OPG production promoting action and the like and has been confirmed to be safe can be used without particular limitation. In addition, excipients, binders, disintegrants, lubricants, stabilizers, preservatives, flavoring agents, diluents and the like may be added.

  The content of the water-soluble fraction of the non-tuberculous me cabbage when an optional component is blended with the BMP-2 production promoter of the present invention is the amount of the water-soluble fraction of the non-tuberculous me cabbage effective for promoting the production of BMP-2. It is not particularly limited as long as it contains a minute amount, and can be appropriately adjusted depending on the dosage form, use form, use object, use method and the like.

The BMP-2 production promoter of the present invention is a pharmaceutical composition for treatment or prevention of a disease requiring promotion of bone formation by formulation as it is or in combination with components usually used in a pharmaceutical composition. (Hereinafter also referred to as “the pharmaceutical composition of the present invention”). Examples of the disease requiring the promotion of bone formation include osteoporosis, fracture, osteogenesis imperfecta, osteomalacia, bone Pechet disease, rheumatoid arthritis and the like.
Since the BMP-2 production promoter of the present invention has both the effect of promoting the production of BMP-2 and the effect of promoting the production of OPG, the active ingredient of the pharmaceutical composition for the treatment or prevention of osteoporosis in particular. It is suitable as.

The optional components that can be used in the pharmaceutical composition of the present invention are not particularly limited as long as safety is confirmed.
The dosage form of the pharmaceutical composition of the present invention is not particularly limited, and is generally mixed with one or more pharmaceutically acceptable carriers, excipients, integrating agents, preservatives, stabilizers, flavoring agents and the like. , Tablets, granules, capsules, liquid medicines, drinks, etc. Such formulation can be carried out according to a conventional method used for the production of a medicine.

In addition, the content of the BMP-2 production promoter in the pharmaceutical composition of the present invention is not particularly limited as long as it is in a range suitable for continuously administering an effective amount of the water-soluble fraction of non-tuberculous me cabbage. That is, a pharmaceutical composition can be produced by selecting an appropriate dose according to lifestyle, such as symptom, age, sex, and dietary habit of the subject to be administered. Usually, the amount of 0.001 mL / kg body weight to 100 mL / kg body weight, preferably 0.01 mL / kg body weight to 10 mL / kg body weight, converted into the amount of 30-fold concentrated water extract per day per adult The content of the BMP-2 production promoter of the present invention in the pharmaceutical composition is adjusted so as to be in a range suitable for administration of the water-soluble fraction of non-tuberculous me cabbage. In the present invention, the “30-fold concentrated water extract” means a water extract obtained by the method of Example 1 described later. "Means an amount corresponding to the amount of the water-soluble fraction in the water extract obtained by the method of Example 1 described later.
Moreover, it is preferable to administer the pharmaceutical composition of the present invention by oral administration. The dose is not limited as long as an effective amount of the water-soluble fraction of non-tuberculous me cabbage can be administered, and can be appropriately adjusted according to the subject to be administered in the same manner as described above. Usually, the amount of 0.001 mL / kg body weight to 100 mL / kg body weight, preferably 0.01 mL / kg body weight to 10 mL / kg body weight, converted into the amount of 30-fold concentrated water extract per day per adult It is preferable to administer the water-soluble fraction of non-tuberculous me cabbage. The administration method is not particularly limited, but it is preferably administered continuously over a period of 3 months or more.

1. Preparation of water-soluble fraction of non-tuberculous mecha cabbage Using the outer leaves harvested in the winter season when the brand name “Petitvale” (registered trademark) (manufactured by Masuda Seed Co., Ltd.) is reached, freeze-dried and powdered . Add 100 mL of distilled water to 6.25 g of petit veil lyophilized powder, shake with a shaker (Iwaki Co., Ltd.) for 10 minutes, and centrifuge at 14,000 rpm for 10 minutes (Hitachi Koki Co., Ltd.). Qing was recovered.
The same operation was performed twice on the residue, and the collected supernatant, a total of 300 mL, was concentrated to 10 mL with a rotary evaporator (Asahi Glass Co., Ltd.) to obtain a water extract (pH 5.7).
30 mL of distilled water was added to 10 mL of the water extract, adjusted to pH 3.0 with 0.5 M hydrochloric acid, and extracted four times with 100 mL of ethyl acetate. The collected supernatant (400 mL in total) was concentrated to dryness and dissolved in 10 mL of DMSO as the solvent layer fraction.
The lower layer 40 mL after extraction with ethyl acetate was adjusted to pH 5.7 with 0.5 M sodium hydroxide and concentrated under reduced pressure to 10 mL. This was made into the water layer fraction.
The water extract, solvent layer fraction, and water layer fraction were each filtered through a 0.22 μm filter and sterilized for use in the test.

2. Test of mRNA expression promoting effect of osteoblast-related factor For each fraction, human strain osteoblast MG-63 was used to promote the mRNA expression of osteoblast-related factor in the water-soluble fraction of petitvale. Examined. As osteoblast-related factors, BMP-2 involved in bone formation and OPG involved in bone resorption were targeted. MG-63 cells were cultured in MEM medium containing 10% fetal bovine serum (Earle's Minimum Essential Medium, Gibco) at 37 ° C. in the presence of 5% by volume of carbon dioxide. A 60 mm dish was seeded with a predetermined amount of MG-63 cells in three groups, cultured to a subconfluent (about 60%) state, then replaced with serum-free MEM medium and cultured for 24 hours.
Thereafter, each fraction, and as a control, sterile water (pH 5.7) and DMSO were replaced with serum-free MEM medium supplemented with 1% by volume of each medium, cultured for 48 hours, and then RNeasy Mini kit ( Total RNA was extracted using QIAGEN).
Using 0.5 μg of total RNA as a template, cDNA was prepared by PCR using a commercially available kit (SuperScript First-Strand Synthesis System, Invitrogen).
PCR reaction was performed using 2 μL of 20 μL of the prepared cDNA solution as a template, BMP-2 primer (SEQ ID NO: 1 and 2), OPG primer (SEQ ID NO: 3 and 4), and internal standard substance glyceraldehyde-phosphate dehydrogenase (GAPDH) primer. (SEQ ID NOS: 5 and 6) were added so that both the Sense primer and the Antisense primer were 10 μM, respectively, and a cycle of 94 ° C. for 2 minutes, 94 ° C. for 1 minute, 58 ° C. for 1 minute, and 72 ° C. for 1 minute was performed with BMP-2. 25 cycles, 19 cycles of OPG, 15 cycles of GAPDH.
The PCR product was electrophoresed with 5% polyacrylamide gel, stained with Vistra Green (Amarsham Pharmacia Biotech), and then measured with Lumino Image Analyzer LAS-3000 (Fuji Film Co., Ltd.).

3. Using statistical processing statistical analysis software SPSS13.0J (S. P. S. S Co., Ltd.), after the average value ANOVA to test for differences in (ANOVA), using multiple comparison Dunnett method p < A significant difference was determined at 0.05.

4). Results The effect of BMP-2 on mRNA expression is shown in FIG. 1, and the effect of OPG on mRNA expression is shown in FIG.
BMP-2 and OPG mRNA expression levels were expressed as relative values to GAPDH mRNA expression levels. In addition, the mRNA expression level when adding the water extract and the aqueous layer fraction is shown as a ratio to the mRNA expression level when adding sterilized water (pH 5.7), and the mRNA expression level when adding the solvent layer fraction is DMSO added. It was shown as a ratio to the amount of mRNA expression at the time.

The water extract and the water layer fraction increased BMP-2 and OPG mRNA expression levels.
However, the solvent layer fraction did not increase both BMP-2 and OPG mRNA expression levels.
As mentioned above, it turned out that the component effective for BMP-2 production promotion and OPG production promotion is a water-soluble fraction of non-tuberculous mecabe. In addition, an aqueous extract of non-tuberculous me cabbage and an aqueous fraction obtained by removing hydrophobic components from the aqueous extract with ethyl acetate increase BMP-2 production, promote bone formation, and It was suggested that the production amount of OPG is increased and osteoclast differentiation is suppressed to suppress bone resorption.
In addition, although the content of flavonoids such as quercetin contained in non-tuberculous mecha cabbage is not grasped, it is estimated that these substances are contained in the solvent layer fraction.

It is a figure which shows the influence which each fraction has on the expression level of BMP-2 mRNA of MG-63. It is a figure which shows the influence which each fraction has on the expression level of OPG mRNA of MG-63.

Claims (7)

BMP-2 (Bone Morphogenetic Protein-2 / bone containing, as an active ingredient, a water-soluble fraction of non- tuberculous me cabbage obtained by mating kale ( Brassica oleracea var. Acephala ) and mecabe ( Brassica oleracea var. Gemmifera ) Forming factor-2) production promoter.   Furthermore, the BMP-2 production promoter of Claim 1 which is an OPG (Osteoprotegerin / osteoclast differentiation inhibitory factor) production promoter.   The BMP-2 production promoter according to claim 1 or 2, wherein the water-soluble fraction of the non-tuberculous me cabbage is an aqueous extract of the non-tuberculous mecabbage.   3. The BMP according to claim 1, wherein the water-soluble fraction of the non-ballistic me cabbage is an aqueous layer fraction obtained by further partitioning the water extract of the non-ballistic me cabbage with water and an organic solvent. -2 production promoter.   The pharmaceutical composition for the prevention or treatment of osteoporosis containing the BMP-2 production promoter as described in any one of Claims 1-4.   The water-soluble fraction of the non-tuberculous mecabbage is administered in an amount of 0.001 mL / kg body weight to 100 mL / kg body weight per day in terms of the amount of 30-fold concentrated water extract. The pharmaceutical composition as described. A step of partitioning a water extract of non- tuberculous me cabbage obtained by mating kale ( Brassica oleracea var. Acephala ) and mecabe ( Brassica oleracea var. Gemmifera ) with water and an organic solvent, and an aqueous layer obtained by the partitioning The manufacturing method of a BMP-2 production promoter including the process of mix | blending a fraction as an active ingredient.

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