CN100548317C - Curative - Google Patents

Abstract

The present invention relates to need to promote skeletonization or be enhanced to treatment of diseases medicine that bone protein generates or preventive drug, skeletonization promoter or drBMP-2 generate reinforcing agent and be used to promote skeletonization or be enhanced to food, beverage or the feedstuff that bone protein generates, it is characterized in that containing handled thing from plant as effective ingredient.In addition, the present invention relates to use the drBMP-2 of specific cells generate potentiation assay method, have drBMP-2 generate potentiation material screening technique and have the preparation method that drBMP-2 generates the material of potentiation.

Description

Curative

Technical field

The present invention relates to needs to promote skeletonization or is enhanced to the disease that bone protein generates, for example useful medicine, food, beverage or feedstuff in the treatment of osteoporosis, fracture etc. or the prevention.

Background technology

In the osseous tissue, skeletonization and bone resorption are to keep certain equilibrated while repeatedly to carry out usually, and calcium concentration in bone strength and the blood is being regulated in this effect.It is believed that osteoblast and osteoclast play significant feature respectively in skeletonization and bone resorption process, the balance of ossification and broken bone effect causes osteoporosis because of certain reason when destroyed.Osteoporosis roughly is divided into: be reduced to the postmenopausal osteoporosis of main cause and be the senile osteoporosis of main cause with the age growth with estrogen secretion, known in addition also have incretion metabolism disease such as diabetes, hyperthyroidism and be the secondary osteoporosis of main cause with the disease of the medication of medicines such as steroid, digestive organs liver, hypovitaminosis C, immobility, oophorectomize, rheumarthritis etc.

Now, as the curative of osteoporosis, use estrogenic, calcitonin, bisphosphonate compound etc. mainly to be used for suppressing the medicine that the bone amount reduces by suppressing brokenly bone.But, in the treatment of using estrogenic, breast carcinoma, uterus carcinoma, cardiopathic dangerous rising etc., side effect strengthens, and when using calcitonin, easily develops immunity to drugs, can not be oral, bisphosphonate compound has absorbance difference, residual height, causes the shortcoming of the excessive passivation of bone metabolism.In addition, for making the bone metabolism activation use vitamin D ₃Preparation, to compare therapeutic effect lower with other drug, and the side effect of hypercalcemia etc. is big.The curative of these osteoporosis in the past can not make the bone that carried out run off and return to original state, is sufficient hardly as the curative of real osteoporosis.

With the closely-related cell of ossification be osteoblast.Osteoblast and chondrocyte, myocyte, adipose cell, tendon cell etc. originate from common mescenchymal stem cell, become sophisticated osteoblast via the preosteoblast strain in atomization.Osteoblast produces in maturation process with a large amount of extracellular matrixs headed by the collagen protein of bone constituent, in addition, expresses alkali phosphatase and causes that calcium phosphate deposits in the substrate.Part osteoblast is imbedded in the substrate of calcification like this, and then is divided into osteocyte.

Human bone amount was a maximum in the time of 20～30 years old, reduced gradually afterwards.Osteoblast in the osseous tissue reduces with age, is divided into osteoblastic ability by mescenchymal stem cell in addition and also reduces.In contrast, it is said that the adipose cell quantity from identical mescenchymal stem cell increases with age.Therefore in the osteogenic phase, improve osteoblast, osteocyte amount, further optionally promote to be divided into osteoblast by mescenchymal stem cell after geratic period, menopause, improve ossification, it is effective being considered to osteoporosis prevention, treatment.

Come in to carry out to promote the exploitation of the medicine of skeletonization always, disclose the skeletonization facilitation that 1-benzopyran derivatives (for example opening flat 7-291983 communique with reference to the spy), phenyl substituted hydroxy cyclopentenone analog (for example opening flat 11-43460 communique with reference to the spy), PGA1 analog (for example opening flat 11-43461 communique with reference to the spy), benzo are considered flat (ペ Application ソ チ エ ピ Application) derivant (for example opening the 2000-109480 communique with reference to the spy), chromone derivative (for example opening the 2001-139571 communique with reference to the spy).But effectiveness, safety evaluatio are still not enough, also do not arrive the practical stage.

Found to contain in the bone matrix albumen sex factor of induced osteogenesis effect, called after drBMP-2 (bone morphogenetic protein is hereinafter referred to as BMP).To its essence and unclear, be opportunity to clone 4 kinds of BMP genes in for a long time, progressively known now tens kinds of molecular speciess surmount kind and extensively are present in the animal.BMP acts on the preosteoblast strain, improves alkaline phosphatase activities, responsiveness, the bone calcium of parathyroid hormone is generated the synthetic etc. of plain, collagen protein, induces to osteoblastic differentiation.The BMP basis has the differential period of the undifferentiated mesenchymal cell of multiple differentiation capability, differentiating cartilage-forming cell, osteoblast, adipose cell respectively.BMP is suppressed to the myocyte and is divided into muscle, makes it to become to be divided into osteocyte.In addition, known BMP has the induced activity from secondary multiplicaiton factor such as osteoblastic Insulin-Like multiplicaiton factor.By with the subcutaneous or muscle administration simultaneously of BMP and carrier, induced osteogenesis effect.Confirmed that among recombinant human B MP BMP-2 ,-4 ,-5 ,-6 ,-7 has the activity of induced osteogenesis effect separately.Wherein known recombined human BMP-2 has strong osteogenic activity, in the damaged model of bone of rat, sheep, Canis familiaris L., monkey etc., recovers defective tissue.In addition, BMP-4 ,-5 relevant with the fracture healing process is arranged, the report that BMP-6 is relevant with endochondral ossification, known BMP-12 have into ligament, become the tendon activity, and BMP-13 has into the cartilage activity.By having confirmed among animal and the old people that at advanced age the BMP amount in the bone matrix reduces, osteoblast reduces the sensitivity of BMP, pointed out that BMP is relevant with senile osteoporosis.

BMP not only has ossification, even also play an important role in growth course, BMP-2 ,-4 ,-7 ,-8 ,-11 etc. is with to form dorsoventral axis, mesoderm, heart, kidney, eyes, sperm etc. relevant.The animal of rejecting BMP can cause death or have serious obstacle.Therefore known BMP is essential to body and has a lot of physiologically actives.

Because BMP shows above-mentioned various effect, has also attempted BMP self directly is used for osteoporosis and treatment of fractures as protein formulation.But, therefore have the restriction of medication and problem such as Drug resistance occurs because BMP is protein.In addition, the receptor wide expression of BMP is in a lot of tissues, so worry can exert an influence to the tissue beyond the bone during whole body administration.Because these shortcomings, can be during as the actual use of curative with BMP self with all restrictions.But can consider not BMP from outside administration,, just can need to strengthen in treatment of diseases that BMP generates, the prevention effective in osteoporosis, fracture etc. if the generation of BMP is strengthened.In recent years, disclose the specific compound (for example with reference to No. 97/15308 open pamphlet of WO) that contains two fragrant families, statin compounds such as mevastatin, lovastatin, pravastatin and simvastatin (for example with reference to No. 98/25460 open pamphlet of WO) have the activity that strengthens the BMP-2 generation.In addition, helioxanthin (helioxanthin) (for example opening flat 8-245386 communique with reference to the spy), the activity with enhancing BMP effect is disclosed in the condensation thiophene derivant (for example opening flat 9-151132 communique with reference to the spy).But the evaluation of safety, effectiveness about them is still not enough, does not also arrive the practical stage.

In recent years, carried out BMP is used for the exploitation of osteanagenesis medical treatment, carried out following trial: with the complex and the embedded material combination of BMP and carrier, by it is implanted fracture site, effect obtains medical treatment.But,, also producing problem aspect safety, the economy because a large amount of BMP is brought in the body.Can consider to avoid this shortcoming, but not have enough methods in actual use by using the material that strengthens the BMP generation, promotes skeletonization to replace BMP.

Like this, can consider by promoting skeletonization, strengthening the BMP generation, make treatment or prevent associated various diseases to become possibility, and do not see toxicity and side effect, can promote suitably that as required skeletonization, the material that strengthens the BMP generation, method etc. are still not known.

Angelica keiskei koidzumi is umbelliferous large-scale herbaceos perennial, and known have various sanatory effects.For example the physiologically active that known angelica keiskei koidzumi had has: the effect of prophylaxis of hypertension, antibacterial action, antitumor action, gastric acid inhibitory secretory action, anticancer effect, nerve growth factor generate reinforced effects, hepatocyte proliferation factor generates potentiation (for example with reference to WO01/76614 number open pamphlet), but, for promote skeletonization, that BMP generates potentiation is then not known so far.

Herba Apii graveolentis is to belong to umbellate form section, known plant with various physiological actions.The physiological action of Herba Apii graveolentis is known blood coagulation resisting function, cancer suppressing action etc.But, for promote skeletonization, that BMP generates potentiation is then not known so far.

Bulbus Lilii is to belong to Liliaceae, known plant with various physiological actions.The physiological action of Bulbus Lilii is known antiinflammatory, diuresis, antitussive effect, sedation etc.But, for promote skeletonization, that BMP generates potentiation is then not known so far.

Aloe is to belong to Liliaceae, known plant with various physiological actions.Aloetic physiological action is known antitumor action, mitogenic activity, enhance immunity activity, cure the cold injury effect, the effect of antibacterial action, anti-allergic effects, anti-inflammatory effect, healing burn, blood sugar lowering effect, moisture-keeping function etc.But, for promote skeletonization, that BMP generates potentiation is then not known so far.

Folium Artemisiae Argyi is to belong to Compositae, known plant with various physiological actions.The physiological action of Folium Artemisiae Argyi is known antibacterial action, sedation, the effect of digestion active oxygen, anastalsis, antihistamine effect etc.But, for promote skeletonization, that BMP generates potentiation is then not known so far.

Summary of the invention

The objective of the invention is to develop from natural goods, can absorb safe and simplely, be suitable as food material, medicine material, have the compositions that promotes skeletonization, drBMP-2 generation potentiation, and medicine, food, beverage or the feedstuff that uses said composition is provided.

Below the present invention is summarized.The 1st～3 invention of the present invention relates to that needs promote skeletonization, are enhanced to treatment of diseases medicine that bone protein generates or preventive drug, ossification promotes medicine or BMP to generate food, beverage or the feedstuff that strengthens medicine, is used to promote skeletonization or strengthens the BMP generation, it is characterized in that containing be selected from following (a)～(c) from the handled thing of plant as effective ingredient.

(a) from the handled thing of umbellate form section plant

(b) from the handled thing of liliaceous plant

(c) from the handled thing of feverfew

In the 1st～3 invention of the present invention, use angelica keiskei koidzumi, Herba Apii graveolentis, Bulbus Lilii, Aloe or Folium Artemisiae Argyi as plant optimization.

The 4th～6 invention of the present invention relates to needs and promotes the treatment of diseases medicine of skeletonization, enhancing BMP generation or preventive drug, ossification promotion medicine or BMP to generate food, beverage or the feedstuff that strengthens medicine, is used to promote skeletonization or enhancing BMP generation, it is characterized in that containing the chemical compound shown in the following formula (A), its derivant or their salt as effective ingredient.

The 7th invention of the present invention relates to the assay method that strengthens the BMP nucleus formation, it is characterized in that comprising following step:

(a) with condition that test sample contacts under, cultivate the step of using HuO9 cell or its substrain or the hybridoma that any one cell strain obtains among this and

(b) the BMP amount in the culture fluid that obtains of determination step (a) generates the step of potentiation index as the BMP of test sample.

The 8th invention of the present invention relates to the screening technique of the material with BMP generation potentiation, it is characterized in that comprising following step:

(a) with condition that test sample contacts under, cultivate the step of using HuO9 cell or its substrain or the hybridoma that any one cell strain obtains among this and

(b) step of BMP amount in the culture fluid that obtains of determination step (a), at this, with not with condition that test sample contacts under or generate and compare when cultivating above-mentioned cell under the condition that the contrast material of potentiation contact with having BMP, BMP measures for a long time, is judged as test sample and has BMP generation potentiation.

The 9th invention of the present invention relates to the preparation method of the material with BMP generation potentiation, it is characterized in that comprising following step:

(a) obtain having BMP generate potentiation material step and

(b) use the of the present invention the 7th assay method of inventing, the BMP that is determined at gained material in the step (a) generates the step of potentiation.

In addition, the present invention relates to have the preparation method that BMP generates the material of potentiation, it is characterized in that comprising following step:

(a) with condition that test sample contacts under, cultivate the step of using HuO9 cell or its substrain or the hybridoma that any one cell strain obtains among this and

(b) step of the BMP amount in the culture fluid that obtains of determination step (a), at this, with not with condition that test sample contacts under or compare when BMP generates under the condition that the contrast material of potentiation contact the above-mentioned cell of cultivation with having, the BMP amount for a long time, be judged as test sample and have BMP generation potentiation, generate the material of potentiation and obtain this test sample as having BMP.

The accompanying drawing summary

Fig. 1 represents the mass spectrum of compound a.

Fig. 2 represents compound a ¹The H-NMR spectrum.

Fig. 3 represents compound a ¹³The C-NMR spectrum.

The specific embodiment

In this description, " BMP generates potentiation " and " BMP generates enhanced activity " instructed the enhancing that causes the BMP generation respectively and strengthened the function that BMP generates, but their not differences of strictness especially on this meaning." enhancing " comprise with effective ingredient effect of the present invention before compare the situation that the amount of effect back target substance increases; Also comprise the effective ingredient effect that the present invention relates to by making simultaneously, generated the situation (inducing) of target substance.In addition in this manual, any material of enumerating as effective ingredient can be applied among the present invention separately or with mixing more than 2 kinds or 2 kinds.

As effective ingredient of the present invention, use handled thing from plant, it is selected from from the handled thing of umbellate form section plant, from the handled thing of liliaceous plant with from the handled thing of feverfew.

Among the present invention, so-called umbellate form section plant is the plant that belongs to angiosperm umbellate form section, for example angelica keiskei koidzumi, Herba Apii graveolentis, Herba Oenanthes Javanicae, Caulis et folium cryptotaeniae japonicae, Radix Angelicae Pubescentis, Radix Ginseng etc.Among the present invention, especially preferably use large-scale herbaceos perennial angelica keiskei koidzumi, the Herba Apii graveolentis of umbellate form section.

Among the present invention, can exemplify Aloe as liliaceous plant, Bulbus Lilii, Cuculus polioephalus, savatier monochasma, the rapids grass of tail, シ ヨ ウ ジ ヨ ウ バ カ マ (Heloniopsis orientalis), yellow villous themeda (キ ス ゲ), Herba Plantaginis (ギ ボ シ), Herba Alii fistulosi, Bulbus Allii, Folium Allii tuberosi, black Bulbus Lilii (Network ロ ユ リ), Flos Tulipae Gesnerianae, Rhizoma Dioscoreae head (カ Network リ), Pseudobulbus cremastrae seu pleiones (ア マ Na), Rhizoma Polygonati Odorati (ア マ De コ ロ), ケ シ マ ラ Application, seven muscle aunts (Star バ メ オ モ ト), Radix et rhizoma smilacinae japonicae (ユ キ ザ サ), dance crane grass (マ イ ズ Le ソ ウ), チ go ユ リ, Trillium tschonoskii Maxim (エ Application レ イ ソ ウ), Rhizoma Paridis (Star Network バ ネ ソ ウ), Rohdea japonica Roth (オ モ ト), Radix Ophiopogonis (ジ ヤ ノ ヒ ゲ), Radix Ophiopogonis seu Liriopis (ヤ Block ラ Application), alertris spicata (ソ Network シ Application ラ Application), Autumn Crocus (イ ヌ サ Off ラ Application), Bulbus scillae scilloidis (Star Le ボ), narcissus (ス イ セ Application), kafir lily (Network Application シ ラ Application), Bulbus Lycoridis Radiatae (ヒ ガ Application バ Na), Bulbus et Radix Crini Sinici (Ha マ オ モ ト) etc.Especially preferably use Aloe and Bulbus Lilii among the present invention.

In addition, so-called Aloe can be used to classify and be belonged to any plant of Aloe, for example Aloe ferox Miller (ケ-プ ア ロ エ), big Aloe (キ ダ チ ア ロ エ), Ji Lasuo Aloe (ア ロ エ ベ ラ) etc.Big Aloe can be called as Aloe arborescens var. natalensis (ギ チ ロ カ イ) on the Plant Taxonomy, this also is contained among the Aloe described in this description.In addition, so-called Bulbus Lilii can be used to classify and be belonged to any plant of lilium, for example Lilium longiflorum (テ Star Port ウ ユ リ), deer Bulbus Lilii (カ ノ コ ユ リ), speckle Bulbus Lilii (ス カ シ ユ リ), goldband lily (ヤ マ ユ リ), tiger lily (オ ニ ユ リ) etc.

Among the present invention, can exemplify Folium Artemisiae Argyi as feverfew, lion foot Folium Artemisiae Argyi (サ ザ バ Le ヨ モ ギ), Ji (ア ザ ミ), Fructus Arctii (go ボ ウ), Caulis et Folium Chrysanthemi segeti (シ ユ Application ギ Network), spring Herba Erigerontis aceris (Ha Le ジ オ Le), Radix Asteris (シ オ Application), Herba Kalimeridis (ヨ メ Na), Herba Lycopi (Off ジ バ カ マ), artemisiifolia (Block Network サ), Herba Xanthii (オ Na モ ミ), Dahlia Pinnata Cav. (ダ リ ア), coreopsis (コ ス モ ス), Helianthi (ヒ マ ワ リ), Herba Gnaphalii Affinis (Ha Ha コ グ サ), Leontopodium leontopodioides (Willd.) Beauv. (ウ ス ユ キ ソ ウ), African Chrysanthemum (ガ-ベ ラ), Petasites japonicus (Off キ), ウ サ ギ ギ Network, Huang Yuan (キ オ Application), wild chrysanthemum (シ ヨ ウ ジ ヨ ウ Ha グ マ), Herba Ixeritis Denticulatae (ニ ガ Na), Herba Lactucae Indicae (ア キ ノ ノ ゲ シ), Herba Taraxaci ( Application Port Port), Hibisci Mutabilis chrysanthemum (Off マ Application) etc.Especially preferably use Folium Artemisiae Argyi and lion foot Folium Artemisiae Argyi among the present invention.

The plant of using among the present invention is not particularly limited, but can use its fruit, seed, kind skin, flower, leaf, stem, root, rhizome and/or directly use plant integral body.

Conduct is from the handled thing of plant among the present invention, generate potentiation as long as have the skeletonization of promotion or BMP, being not particularly limited, for example can be extract, ground product, squeezeding juice, broken thing, chemical treatments, enzyme handled thing, preferred especially extract, ground product and squeezeding juice.So long as use, the form from the handled thing of plant is not particularly limited as effective ingredient of the present invention.

So-called extract is to use and extracts the material that solvent obtains through extracting the step of operation among the present invention.Extraction can be undertaken by known extracting method is following.Raw material, extraction solvent, the following suitable setting of extraction temperature: after for example can or chopping up, use solvent to extract with batch process or continuation mode with raw material pulverizing.Extraction solvent when obtaining extract, be not particularly limited, can enumerate alcohols such as water, hexane, chloroform, ethanol, methanol, isopropyl alcohol, ketone such as acetone, butanone, hydrophilic such as methyl acetate, ethyl acetate or oil loving solvent can use or use suitable mixed liquor separately as required.The amount of extracting solvent can suitably determine, usually, the raw material weight when extracting operation can preferably use the extraction solvent of 0.1～100 times of amount.Extracting temperature can be according to the suitable decision of purpose, and during water extraction preferred 4～130 ℃ usually, more preferably 25～100 ℃.In addition, when containing ethanol in the solvent, preferred 4～60 ℃ scope.Extraction time can consider that also extraction efficiency decides, usually preferred several seconds～several days, more preferably 5 minutes～24 hours scope.Pressure during extraction is not particularly limited, and decision as required can suit.Extract operation as required alternative condition for example can be under normal pressure, add and depress, carry out under the decompression of filtered off with suction etc.Extract operational example and carry out, can extract for several times repeatedly as required as can under agitation or leaving standstill down.By above operation, the plant extract that obtains using among the present invention (below be sometimes referred to as extract of the present invention).Extract can filter as required, centrifugalize, concentrate, the processing of ultrafiltration, molecular sieve etc., can prepare to have promotes skeletonization or BMP to generate the concentrated extract of the purpose composition of potentiation (below, be called ossification and promote composition or BMP to generate enhancing ingredients).The skeletonization facilitation of extract and concentrated extract or BMP generate potentiation and can measure easily according to following embodiment 2 or 4 described methods.In addition, the plant that the present invention uses is made the Folium Camelliae sinensis shape with known method,, also can be used as extract of the present invention and use as long as the extract that uses this Folium Camelliae sinensis shape material to obtain has the skeletonization facilitation or BMP generates potentiation.In addition, can be among the present invention with 2 kinds or the multiple extract merging use that obtains by different extracting method.

Among the present invention, component that obtains with known method separating plant extract or the component that obtains by the operation of repeated isolation repeatedly are also included within the extract of the present invention.Above-mentioned separation method has extraction, fractional precipitation, column chromatography, thin layer chromatography etc.To promote skeletonization or BMP to generate potentiation is index, by further refining to obtained component, can isolate ossification and promote composition or BMP to generate enhancing ingredients.

In addition, as the handled thing outside the plant extract, can enumerate the plant ground product from plant.As the preparation method of ground product, for example can be by plant drying, pulverizing are obtained pulverous plant ground product.

Can enumerate known plant expressed juice method as the preparation method of plant expressed juice liquid, be not particularly limited, for example can use squeezers such as screw, gear type, rotor formula or fruit juice mixer to squeeze the juice.As pre-treatment, after raw material can being chopped up or grinds,, obtain squeezeding juice with squeezings such as above-mentioned fruit juice mixer or cloth.

Broken thing is meant the material that the plant fragmentation as active ingredient raw materials is obtained, and is bigger than the tissue of ground product usually, for example can use disintegrating machine to prepare.In addition, chemical treatments is not particularly limited, the material that for example this plant can be carried out acid treatment, alkali treatment, oxidation processes, reduction processing etc. and obtain, for example this plant can be soaked in mineral acid or organic acid such as containing hydrochloric acid, sulphuric acid, nitric acid, citric acid, acetic acid, perhaps prepare in the aqueous solution of inorganic base such as sodium hydroxide, potassium hydroxide, ammonia or organic base.Chemical treatments comprises from the total material that has passed through above-mentioned chemically treated plant.The enzyme handled thing is meant the enzyme handled thing of for example handling through pectase, cellulase, xylanase, amylase, mannase, glucosidase etc., the enzymatic reaction product of microbial treatments (for example tunning) for example can be used for preparing with this plant in suitable buffer by making above-mentioned enzyme.The enzyme handled thing comprises the total material of the plant that the above-mentioned enzyme of hanging oneself is handled.And, as the handled thing from plant of the present invention, for example also comprise the stem of above-mentioned plant cut off, the juice that obtains by its section.

Among the present invention, being not particularly limited for the shape from the handled thing of plant, as long as have the skeletonization of promotion or BMP generates potentiation, can be Powdered, solid, shaped, the arbitrary shape in liquid.Also this handled thing can be used the known method pelletize in addition, use as the handled thing from plant of the present invention with the granular solid matter that obtains.Prilling process is not particularly limited, and the pelletize of rotation, stirring-granulating, fluidized bed prilling, air-flow pelletize, extruder grain, compacting pelletize, size degradation pelletize, air prilling or mist projection granulating etc. are for example arranged.With pulverous should from handled thing of plant be dissolved in liquid for example water, alcohol etc. make liquidly, this also can be used as the handled thing from plant of the present invention and uses.

Handled thing from plant of the present invention is compared with the plant self as raw material, and preferred especially high concentration and/or high-purity contain skeletonization and promote composition or BMP to generate enhancing ingredients.Here so-called high concentration is meant that skeletonization promotes that the weight of composition or BMP generation enhancing ingredients is many in the material plant body of the weight ratio Unit Weight that the handled thing skeletonization promotion composition or the BMP from plant of Unit Weight generates enhancing ingredients.In addition, so-called high-purity is meant, the skeletonization that likens to the plant of raw material from the handled thing of plant promotes composition or BMP to generate the containing ratio height of enhancing ingredients.

High concentration and/or high-purity also are provided among the present invention contain food, beverage or feedstuff from the handled thing of plant, this is meant with in the past food, beverage or feedstuff and compares that food of the present invention, beverage or feedstuff middle and high concentration and/or high-purity ground contain skeletonization and promotes composition or BMP to generate enhancing ingredients.

Among the present invention, can will use as the effective ingredient that curative of the present invention, preventive drug, ossification promoter, BMP generate reinforcing agent, food, beverage or feedstuff from represented chemical compound, its derivant or their salt (hereinafter referred to as chemical compound of the present invention) of above-mentioned formula (A) that the handled thing of plant obtains by umbellate form section.Chemical compound of the present invention can be distinguished use separately or two or more is mixed use.This chemical compound can be to make with extra care the material that obtains by above-mentioned known separation method by umbellate form section plant, maybe can be the semi-synthetic or synthetic material of chemistry.

Concrete preparation method as the chemical compound shown in the formula (A) for example has the method described in the following embodiment 1.In addition, for example obtain semi-synthetic as raw material by organic synthesis, obtain composite through organic synthesis fully with chalcone chemical compound from natural goods.The method of organic synthesis for example can be with reference to Alessandra Lattanzi etc., Synlett.2002, No.6, p942-946; L.Claisen A. etc., Ber.1881, No.14, p2460 etc.

The derivant of chemical compound shown in the above-mentioned formula (A) is meant in this description, this chemical compound is synthetic as initial compounds, have and (A) shown in the equal effect of chemical compound, that is, skeletonization facilitation or BMP generate the chemical compound of potentiation.As this derivant, for example can enumerate the carboxylate, etherate, glucosides of chemical compound shown in the above-mentioned formula (A) etc., hydrolysis easily in vivo can be brought into play the chemical compound (prodrug) of required effect.This prodrug can prepare according to known method.In addition, this derivant can be its salt.

In the chemical compound of the present invention, the preferred officinal salt of salt.The salt that the salt that uses among the present invention for example has alkali metal salt, alkali earth metal salt, form with organic base etc.The preferred officinal salt of this salt.In addition, the officinal salt that uses among so-called the present invention is meant the salt nontoxic substantially to biology.For example have and sodium, potassium, calcium, magnesium, ammonium or protonated benzyl star (benza thine) (N as this salt, N '-two-benzyl ethylenediamine), the salt of choline, ethanolamine, diethanolamine, ethylenediamine, meglumine (メ グ ラ ミ Application) (N-methylglucosamine), benethamine (N-benzyl-1-phenylethylamine), piperazine or trihydroxy methyl aminopropane (ト ロ メ ミ Application) (2-amino-2-methylol-1, ammediol) formation.

In addition, in the present invention, handled thing and/or chemical compound of the present invention from plant used in the present invention is called effective ingredient of the present invention, and the needs that will contain effective ingredient of the present invention promote the treatment of diseases medicine or the preventive drug of skeletonization or enhancing BMP generation to be called curative of the present invention or preventive drug.

In the effective ingredient of the present invention, do not see toxicity as described later.In addition, do not worry having side effects.That is because as long as use this effective ingredient, just can securely and effectively carry out treatment of diseases or prevention.Therefore, contain curative of the present invention, preventive drug, ossification promoter, BMP generation reinforcing agent, food, beverage or the feedstuff of this effective ingredient, effective to treatment of diseases or prevention that needs promote skeletonization or enhancing BMP to generate.

The so-called skeletonization that promotes among the present invention, so long as promote the ossification of bone, cartilage to get final product, be not particularly limited, for example can exemplify the promotion mescenchymal stem cell and be divided into osteoblastic effect, promote undifferentiated mesenchymal cell to be divided into osteoblastic effect, promote the preosteoblast strain to be divided into the calcification of osteoblastic effect, bone matrix formation facilitation, bone matrix, to promote the osteoblastic effect of osteoblast differentiation, endochondral ossification inducing action etc.In addition,, be not particularly limited, can for example, can measure the promotion mesenchymal cell easily and be divided into osteoblastic effect according to following embodiment 2 described methods about promoting having or not of skeletonization.Here, comprise " inducing " in " promotion ".

Among the present invention, can exemplify BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, BMP-10, BMP-11, BMP-12, BMP-13, BMP-14, BMP-15 etc. as BMP, preferred especially BMP-2, BMP-4 or BMP-7.Generate having or not of potentiation about BMP in addition, be not particularly limited, can measure easily according to following embodiment 4 described methods.

BMP is the factor that promotes that strongly bone, cartilage, ligament, tendon etc. form, and acts on the preosteoblast strain and promotes it to be divided into osteoblast, is considered to relevant with the healing process of the generation of bone, growth, bony remodeling (リ モ テ リ Application グ), fracture.In addition, BMP acts on undifferentiated mesenchymal cell, and is by being divided into chondroblast, osteoblast, adipose cell respectively according to differential period, extensively relevant with maturation and propagation from mesochymal cell.And then, even BMP in the generating process of individuality, also plays an important role to dorsoventral axis formation, mesoderm formation etc.In BMP, BMP-2 ,-4 ,-7 osteogenic activities are strong especially, and recombinant human B MP-2 is acted on the damaged animal of bone, and it is damaged to treat bone.Can reference example such as J.M.Wozney etc. about the detailed description of recombinant human B MP-2., Science Vol.242p1528-1534 (1988).In addition, in dental field, can confirm that BMP-2 can make paradenlal tissue regenerations such as alveolar bone sclerotin, periodontal membrane tissue.

Among the present invention, promote skeletonization or strengthen the disease that BMP generates as needs, can exemplify osteoporosis (for example, chronic osteoporosis, unusually the osteoporosis that causes by hormonal balance after the menopause, with diabetes, the secondary osteoporosis that the side effect of steroid medicine etc. is concurrent etc.), fracture, refracture, bone is damaged, osteogenesis imperfecta, osteomalacia, the bone Behcet disease (Bechet ' s disease), tetanic property myelitis, rheumatoid arthritis, osteoarthrisis deformans knee, the osteoarthrisis deformans knee relevant with cartilage, periodontal disease, periodontal tissue in the periodontal disease is damaged, the root of the tooth alveolus is damaged, form alveolar ridge, cleft palate.In addition, the bone tissue restoration agent that can be used as behind the surgical operation of multiple myeloma, pulmonary carcinoma, breast carcinoma etc. of curative of the present invention or preventive drug is used.And then curative of the present invention or preventive drug can be used for the osteanagenesis in regenerative medicine field.Particularly, curative of the present invention or preventive drug can be used for the activation stabilisation of artificial bone, artificial root of the tooth, maybe can from fall ill preceding or patient's body of falling ill in gather cell, make curative of the present invention or preventive drug effect external, after forming the regeneration osseous tissue, replant in patient's the body.

As curative of the present invention or preventive drug, can enumerate the preparation that to make with above-mentioned effective ingredient that the present invention relates to and known drug regimen.Effective ingredient of the present invention can with the medicine that for example suppresses bone resorption, for example estrogenic, calcitonin, activated vitamin D ₃, diphosphate etc. is common uses.In addition, effective ingredient of the present invention can use jointly with BMP.As described in the embodiment 13, can expect to obtain and the synergy that promotes that skeletonization is relevant thus.At this, preferably use above-mentioned recombinant human B MP-2 as BMP.Therefore, as curative of the present invention or the preferred form of preventive drug, can enumerate the form that contains above-mentioned handled thing and/or chemical compound of the present invention and recombinant human B MP-2 from plant.This composition also is the preferred configuration that skeletonization promoter of the present invention, BMP generate reinforcing agent, food, beverage, feedstuff.

The preparation of curative of the present invention or preventive drug, usually above-mentioned effective ingredient and pharmaceutically acceptable liquid state or solid-state carrier are cooperated and to carry out, add solvent, dispersant, emulsifying agent, buffer agent, stabilizing agent, excipient, binding agent, disintegrating agent, lubricant etc. as required, can make liquid preparations such as solid preparations such as tablet, granule, powder, powder agent, capsule, regular solution agent, suspensoid, Emulsion.Also can make before use by adding appropriate carriers and can form the dry product of liquid state, in addition, also can make external preparation

Medicine can be selected according to the administering mode and the dosage form of curative or preventive drug with carrier.When making the oral formulations that contains solid composite, for example starch, lactose, white sugar, mannitol, carboxymethyl cellulose, corn starch, inorganic salt etc. can be utilized, tablet, pill, capsule, powder, granula subtilis, granule etc. can be made.When making oral formulations in addition, can cooperate binding agent, disintegrating agent, surfactant, lubricant, fluidizer, correctives, coloring agent, spice etc.When for example making tablet or pill, also can be coated with the sugar-coat of sucrose, gelatin, hydroxypropyl cellulose etc. or the thin film of gastric solubility or enteric solubility matters as required.Make the oral formulations when containing fluid composition, for example can utilize distilled water, ethanol etc., make pharmaceutically useful opacifiers, solution, suspensoid, syrup etc. as carrier.In addition, can add the auxiliary agent, sweeting agent, correctives, antiseptic etc. of wetting agent, suspending agent and so on as required.

On the other hand, when making non-oral formulation,, add antibacterial, stabilizing agent, isoosmotic adjusting agent, analgesic as required and wait and prepare by making above-mentioned effective ingredient dissolving of the present invention or being suspended in distilled water for injection as diluent, normal saline, D/W, injection vegetable oil, Oleum sesami, Oleum Arachidis hypogaeae semen, soybean oil, Semen Maydis oil, propylene glycol, Polyethylene Glycol etc. according to well-established law.In addition, also solid composite be can prepare, sterilized water or aseptic injection are dissolved in then before use with using in the solvent.

As external preparation, comprise percutaneous dosing with or solid, semisolid or the liquid preparation of through mucous membrane (in the oral cavity, nasal cavity) administrable.Also comprise suppository etc. in addition.For example, can make opacifiers such as Emulsion, lotion, the liquid preparations such as liquid preparation of external-use tincture, transmucosal administration, ointments such as oiliness ointment, hydrophilic ointment, the patch of percutaneous dosings such as membrane, patch (テ one プ drug), paste usefulness or transmucosal administration etc.

Above various preparations use known medicine with carrier etc. respectively, can be by the suitable preparation of well-established law.In addition, the content of effective of said preparation is considered its administering mode, medication etc., preferably in following dosage scope, so long as can just be not particularly limited to the amount with this effective ingredient.For example, by using 20mL water as the extraction operation of extracting solvent, the plant drying thing that is used as raw material from 1g in the present invention obtains extract, the extract that obtains is concentrated into 5mL, with this during as effective ingredient, in curative or preventive drug 100 weight %, 0.001～100 weight % normally, preferred 0.01～90 weight %, more preferably 0.1～80 weight %.In addition, when chemical compound of the present invention is used as effective ingredient, be not particularly limited, in curative or preventive drug 100 weight %, 0.000001～100 weight % normally, preferred 0.00001～90 weight %, more preferably 0.0001～80 weight %.

Effective ingredient of the present invention and above-mentioned reorganization BMP-2 and time spent, with respect to 100 weight portion effective ingredient, reorganization BMP-2 is preferred usually to be used about 0.1～100000 weight portion.This effective ingredient and reorganization BMP-2 and the ratio of time spent are same for following skeletonization promoter, BMP generation reinforcing agent, food, beverage, feedstuff.

Curative of the present invention or preventive drug are according to the suitable route of administration administration that adapts with dosage form.Medication is not particularly limited, can be take orally, external and injection.Injection

For example can vein, administration such as intramuscular, subcutaneous, Intradermal, as the external agent, suppository for example then can be by the medication administration that adapts with suppository.

The dosage of curative of the present invention or preventive drug was suitably set according to age of the administration subject patient of said preparation form, medication, application target and this curative or preventive drug, body weight, symptom, was not unalterable.Common dosage according to the above-mentioned effective ingredient that contains in the preparation, for example, by using 20mL water as the extraction operation of extracting solvent, the plant drying thing that is used as raw material from 1g in the present invention obtains extract, the extract that obtains is concentrated into 5mL, and during as effective ingredient, people (for example adult) is 0.0001mg～2000mg/kg body weight every day with this, preferred 0.001mg～1000mg/kg body weight, more preferably 0.01mg～100mg/kg body weight.When chemical compound of the present invention is used as effective ingredient, be not particularly limited, people (for example adult) is 0.0001 μ g～2000mg/kg body weight every day, preferred 0.001 μ g～1000mg/kg body weight, more preferably 0.01 μ g～100mg/kg body weight.Certainly dosage is because of various conditions, for example extracts solvent types, uses the consumption etc. of solvent and change, and has therefore that to lack than above-mentioned dosage be enough situations, and the situation that need exceed above-mentioned scope is also arranged.Can be in the dosage scope of needs, single-dose in a day, or administration several times, also can be in accordance with regulations time administration.In addition, curative of the present invention or preventive drug can be added to arbitrarily and carry out daily picked-up in the beverage/food except direct oral administration.

In addition, the present invention can also provide the skeletonization promoter and the BMP that contain above-mentioned effective ingredient to generate reinforcing agent.Generate reinforcing agent as this skeletonization promoter and BMP, can be above-mentioned effective ingredient self, or also can be the compositions that contains above-mentioned effective ingredient.Skeletonization promoter and BMP generate reinforcing agent, for example with above-mentioned effective ingredient and other cooperations that become to grade that can be used for this effective ingredient same use, according to the preparation method of above-mentioned curative or preventive drug, can be prepared as the form of normally used reagent.This skeletonization promoter or BMP generate above-mentioned content of effective in the reinforcing agent, consider that this skeletonization promoter or BMP generate the medication of reinforcing agent, application target etc., get final product so long as can reach the amount of effect of the presently claimed invention, are not particularly limited.For example, by using 20mL water as the extraction operation of extracting solvent, the plant drying thing that is used as raw material from 1g in the present invention obtains extract, the extract that obtains is concentrated into 5mL,, generate among the reinforcing agent 100 weight % skeletonization promoter or BMP, normally 0.001～100 weight % during with this as effective ingredient, preferred 0.01～90 weight %, more preferably 0.1～80 weight %.In addition, when chemical compound of the present invention is used as effective ingredient, be not particularly limited, generate among the reinforcing agent 100 weight % skeletonization promoter or BMP, 0.000001～100 weight % normally, preferred 0.00001～90 weight %, more preferably 0.0001～80 weight %.

This skeletonization promoter or BMP generate the consumption of reinforcing agent, get final product so long as can reach the amount of effect of the presently claimed invention, are not particularly limited.Especially for when the body administration, preferably in the dosage scope of curative or prevention the effective elements of the medicine, so long as the amount with this effective ingredient of can giving gets final product.It is being useful with promote skeletonization or BMP to generate strengthening in the relevant disease that skeletonization promoter of the present invention or BMP generate reinforcing agent.

Skeletonization promoter of the present invention or BMP generation reinforcing agent can be contained in the implant and use.Thus, for example when fracture,, promote bone to engage, the integration of symphysis or implant and osseous tissue is quickened by using this implant.At this, so-called implant is meant and is imported into intravital utensil at least in part in operating process, uses at the fracture or the damage of joint, bone, tooth, ligament or tendon etc.Implant can stay in the body forever, or is absorbed by biodegradation.Skeletonization promoter of the present invention or BMP generate reinforcing agent can be contained in the inside of implant, or also can make it to be contained in the implant surface coatings.As long as obtaining desired effect, the content of skeletonization promoter of the present invention or BMP generation reinforcing agent can make when setting.Implant can use effective ingredient of the present invention to prepare by known method.

Skeletonization promoter of the present invention or BMP generation reinforcing agent can be contained in the toothpaste and use.This toothpaste generates potentiation by skeletonization facilitation of the present invention or BMP, can carry out periodontal tissue regeneration, periodontitis prevention and promote calcification again.As long as the content of skeletonization promoter of the present invention or BMP generation reinforcing agent can obtain desired effect and can suitably set.Toothpaste can use effective ingredient of the present invention to prepare by known method.

It is also useful in the drug screening of the disease of closing with bone photo that this skeletonization promoter or BMP generate reinforcing agent.Further, this skeletonization promoter or BMP generate reinforcing agent with about the functional study of bone physical change in also useful.

Toxicity is not seen by effective ingredient of the present invention such as following institute.In addition, do not worry having side effects.That is because as long as use this effective ingredient, just can bring into play promotion skeletonization or BMP and generate potentiation safe and suitablely.Therefore, contain medicine of the present invention, food, beverage or the feedstuff of this effective ingredient, effective to treatment of diseases or prevention that needs promote skeletonization or enhancing BMP to generate.

The invention provides the food, beverage or the feedstuff that are used to promote skeletonization or enhancing BMP generation that contain above-mentioned effective ingredient.At this, so-called " containing " is meant the meaning that contains, adds and/or dilute.Food of the present invention, beverage or feedstuff promote skeletonization or BMP to generate potentiation by it, and needs are promoted that improvement, the prevention of the disease symptoms that skeletonization or enhancing BMP generate are extremely useful.

In this manual, above-mentioned what is called " contains " form that contains effective ingredient used in the present invention in food, beverage or feedstuff that is meant; Above-mentioned what is called " interpolation " is meant the form of adding effective ingredient used in the present invention in food, beverage or feedstuff; Above-mentioned what is called " dilution " is meant in effective ingredient used in the present invention, adds the form of the raw material of food, beverage or feedstuff.

The preparation method of food of the present invention, beverage or feedstuff is not particularly limited.For example, cooperation, the cooking, processing etc. can be carried out according to general food, beverage or feedstuff, preparation method according to them is made, and promotes skeletonization or BMP to generate the of the present invention above-mentioned effective ingredient of potentiation as long as contain in the food that obtains, beverage or the feedstuff to have.

Foods or drinks of the present invention is not particularly limited, and for example has: corn fabricated product (the wheat flour fabricated product that contains above-mentioned effective ingredient of the present invention, the starch based fabricated product, the premix fabricated product, Noodles, the macaroni class, Bread and Pastries, bean filling bag class, the Semen Fagopyri Esculenti Noodles, Testa Tritici, rice flour, vermicelli, packing Oryza glutinosa cake etc.), oils and fats fabricated product (plasticity oils and fats, frying oil, salad oil, mayonnaise, tartar sauce etc.), soybean processing goods (Tofu, bean sauce, natto etc.), meat fabricated product (Petaso, bacon, the compacting Petaso, bologna sausage etc.), aquatic products (freezing minced fish, breaded fish stick, cylindric breaded fish stick, the breaded fish stick sheet, fried Radix Dauci Sativae fish meat sheet, quick-boil fish pill, muscle, fish ham, intestinal, the Skipjack wood shavings, the fish roe fabricated product, canned aquatic products, the sweet seafood etc. of cooking), milk product (raw milk, butter, Yoghurt, butter, cheese, practice breast, milk powder, ice cream etc.), vegetable fruit fabricated product (is stuck with paste class, jam, the brined vegetable class, fruit nectar, vegetable beverage, bland etc.), cake class (chewing gum, maltose, chocolate, Biscuits, the cake Bread and Pastries, cake, Oryza glutinosa cake (Japonica type), Oryza glutinosa Biscuits etc.), alcoholic beverage (Janpanese sake, Chinese wine, wine, whiskey, liquor, vodka, brandy, gin, rum, medicated beer, refrigerant alcoholic beverage, fruit wine, liqueur etc.), hobby beverage (green tea, black tea, oolong tea, coffee, refreshment drink, lactic acid beverage etc.), flavoring agent (soy sauce, dip, vinegar, sweetener wine etc.), canned bottle pack dress food (beef rice, portioned rice bowl, RED BEAN RICE, curry, other various processed foods etc.), partial desiccation or reserve ration (liver pat, other tartar sauce, Fagopyrum esculentum Moench noodles juice, condensed soup class etc.), dried foods (disposable noodles, the fast food curry, instant coffee, juice powder, the powder end, the fast food Miso Soup, processed food, processing beverage, processing soup etc.), frozen food (Sukiyaki, Steamed Egg Custard, Broiled River Eel, Hamburg steak, steamed dumplings, dumpling, various soup stocks, fruit juice cocktail etc.), solid-state food, liquid food (soup etc.), agricultural production forest products fabricated products such as fragrant peppery flavoring agent class, the livestock products fabricated product, aquatic products processing goods etc.As food of the present invention, preferred especially chewing gum, maltose class etc.This food if contain effective ingredient of the present invention therein, just can prove effective by effective ingredient of the present invention owing to chew the regular hour in mouth, for example, the effect of calcification of regeneration effect, the promotion tooth of periodontal tissue is more effectively brought into play.

Foods or drinks of the present invention contains, adds and/or dilutes one or more above-mentioned effective ingredient, its content promotes skeletonization or the necessary amount of BMP generation potentiation to get final product so long as bring into play it, there is no particular limitation to its shape, also comprise can orally ingestible shape such as lamellar, graininess, capsule shape.

Beverage of the present invention can with the plant beyond effective ingredient of the present invention and Umbelliferae, Liliaceae and the Compositae for example the squeezeding juice of vegetable beyond them or fruit etc. mix or squeeze the juice simultaneously with them, make healthy beverage.For example with the squeezeding juice dilute with water of angelica keiskei koidzumi, Herba Apii graveolentis, Bulbus Lilii, Aloe or Folium Artemisiae Argyi, squeezeding juice with Xiao Song dish, Radix Brassicae rapae, blue or green stalk dish, Fructus Lycopersici esculenti, mandarin orange, Fructus Citri Limoniae, grapefruit, kiwi fruit, Herba Spinaciae, Radix Raphani, Radix Raphani, Chinese cabbage, Brassica oleracea L.var.capitata L., the Caulis et Folium Lactucae Sativae of cooking salad, Caulis et Folium Lactucae Sativae, Flos abelmoschi manihot, CAIJIAO, Fructus Cucumidis sativi, Kidney bean, green soyabeans, Semen Pisi sativi, corn, rocket salad, Folium Eriobotryae, Fructus Citri tangerinae, sweet summer Fructus Citri tangerinae etc., milk, the healthy beverage with promotion skeletonization or BMP generation potentiation is made in mixing such as bean milk.

Alcoholic beverage as a kind of embodiment of beverage of the present invention, Umbelliferae, Liliaceae and/or feverfew that the present invention uses, for example angelica keiskei koidzumi, Herba Apii graveolentis, Bulbus Lilii, Aloe and/or Folium Artemisiae Argyi be impregnated in the alcohol type of drinking, it directly or according to the manufacture method of known alcoholic beverage is obtained alcoholic beverage, and this alcoholic beverage can be for drinking.

The handled thing from plant that uses as effective ingredient in the present invention, for example, the form that ground product, broken thing are made tablet etc. according to known method also is contained in of the present invention making in the product.

Above-mentioned content of effective among the present invention in the Foods or drinks is not particularly limited, consider and suitably to select from bringing into play its function and active viewpoint, for example in the present invention by using 20mL water as the extraction operation of extracting solvent, the plant drying thing that is used as raw material from 1g in the present invention obtains extract, the extract that obtains is concentrated into 5mL, with this during as effective ingredient, in food 100 weight %, content is more than the 0.1 weight %, preferred 1～100 weight %, more preferably 6～100 weight %; In beverage 100 weight %, content is more than the 0.1 weight %, preferred 1～100 weight %, more preferably 6～100 weight %.In addition, when chemical compound of the present invention is used as effective ingredient, be not particularly limited, in food 100 weight %, content is more than the 0.0001 weight %, preferred 0.001～50 weight %, more preferably 0.006～10 weight %; In beverage 100 weight %, content is more than the 0.0001 weight %, preferred 0.001～50 weight %, more preferably 0.006～10 weight %.In addition, Foods or drinks of the present invention, when the effective ingredient that wherein preferably contains is, for example, operate as the extraction of extracting solvent by using 20mL water, the plant drying thing that is used as raw material from 1g in the present invention obtains extract, the extract that obtains is concentrated into 5mL, and during as effective ingredient, people (for example adult) can absorb 0.0001 μ g～2000mg/kg body weight every day with this, preferred 0.001 μ g～1000mg/kg body weight, more preferably 0.01 μ g～100mg/kg body weight.When chemical compound of the present invention is used as effective ingredient, be not particularly limited, people (for example adult) is 0.0001 μ g～2000mg/kg body weight every day, preferred 0.001 μ g～1000mg/kg body weight, more preferably 0.01 μ g～100mg/kg body weight.Certainly intake is because of various conditions, for example extracts solvent types, uses the consumption etc. of solvent and change, and has therefore that to lack than above-mentioned intake be enough situations, and the situation that need exceed above-mentioned scope is also arranged.

The invention provides and contain biology feedstuff above-mentioned effective ingredient, that have promotion skeletonization or BMP generation potentiation, and then, as another embodiment, also provide biological method for breeding, it is characterized in that using above-mentioned effective ingredient to biology.As another embodiment of the present invention, provide the biological agent of raising in addition, it is characterized in that containing above-mentioned effective ingredient.At this, so-called " containing " is meant the above-mentioned meaning that contains, adds and/or dilute.

In these inventions, so-called biology is for example cultivated animals, pet animals etc., has exemplified domestic animal, laboratory animal, poultry, Fish, shell-fish or shellfish as cultivated animals.Feedstuff for example has keeping and/or improves the feedstuff of health.Biological raising agent for example has impregnating agent, feed additive, beverage additive.

According to these inventions, in the biology that exemplifies more than this invention of use, promotion skeletonization or BMP generation potentiation based on the above-mentioned effective ingredient that uses among the present invention are expected to bring into play and above-mentioned curative of the present invention or the same effect of preventive drug.That is, can bring into play the needs promotion skeletonization of this biology or treatment of diseases or the preventive effect that enhancing BMP generates.

The above-mentioned effective ingredient that uses among the present invention, usually for example in the present invention by using 20mL water as the extraction operation of extracting solvent, the plant drying thing that is used as raw material from 1g in the present invention obtains extract, the extract that obtains is concentrated into 5mL, with this during as effective ingredient, object organisms is used 0.0001 μ g～2000mg/kg body weight, preferred 0.001 μ g～1000mg/kg body weight, more preferably 0.017 μ g～100mg/kg body weight every day.When chemical compound of the present invention is used as effective ingredient, be not particularly limited, object organisms is used 0.0001 μ g～2000mg/kg body weight, preferred 0.001 μ g～1000mg/kg body weight, more preferably 0.01 μ g～100mg/kg body weight every day.Certainly throw with amount because of various conditions, for example extract solvent types, use the consumption etc. of solvent and change, have therefore that to lack than above-mentioned throwing and amount be enough situations, the situation that need exceed above-mentioned scope is also arranged.Using can be by for example adding this effective ingredient, is mixed in the raw material to the artifical compound feed of object organisms in advance, or with after the powder stock of artifical compound feed mixes, adds other raw material again, mixes.In addition, the content of above-mentioned effective ingredient in feedstuff is not particularly limited, can suitably determine that for example in the present invention by using 20mL water as the extraction operation of extracting solvent, the plant drying thing that is used as raw material from 1g in the present invention obtains extract according to purpose, the extract that obtains is concentrated into 5mL, during as effective ingredient, in feedstuff 100 weight %, content is more than the 0.1 weight % with this, preferred 1～100 weight %, more preferably 6～100 weight %.When chemical compound of the present invention is used as effective ingredient, be not particularly limited, in feedstuff 100 weight %, content is more than the 0.0001 weight %, preferred 0.001～50 weight %, more preferably 0.006～10 weight %.

The preparation method of feedstuff of the present invention is not particularly limited, and its mixing can be carried out according to common feedstuff, promotes skeletonization or BMP to generate the of the present invention above-mentioned effective ingredient of potentiation as long as contain in the prepared feedstuff to have.

Biological raising agent can be according to situation preparation, the use of above-mentioned feedstuff.

Applicable biology of the present invention is not particularly limited, and is widely used in the domestic animal such as horse, cattle, pig, sheep, goat, camel, llama in the cultivated animals; Laboratory animals such as mice, rat, Cavia porcellus, rabbit; Poultry such as chicken, duck, turkey, Ostriches; And as the Canis familiaris L. of pet animals, cat etc.

Containing having of using among the present invention by picked-up promotes skeletonization or BMP to generate the feedstuff of the above-mentioned effective ingredient of potentiation, or object organisms is soaked in the liquid of the above-mentioned effective ingredient that uses among the present invention with promotion skeletonization or BMP generation potentiation, can keep or improve the health of domestic animal, experimental animal, poultry, animal pets etc. well, in addition, these embodiments also become a kind of scheme of biological method for breeding provided by the invention.

Next, to BMP of the present invention generate potentiation assay method, have that BMP generates the screening technique of material of potentiation and the preparation method of this material describes.These methods can be preferably used for: the plant of selecting to use as raw material among the present invention, the BMP that compositions had such as curative, food that obtain effective ingredient of the present invention, estimate effective ingredient of the present invention and comprise this effective ingredient generate potentiation.

As other embodiments of the present invention, the assay method (being called assay method of the present invention) of the BMP generation potentiation of test sample is provided, it is characterized in that comprising (a) with condition that test sample contacts under, cultivate to use HuO9 cell or its substrain or any one cell strain obtains among this hybridoma (below, these cells are referred to as cell strain of the present invention) step and the BMP amount in the culture fluid that (b) obtains with step (a) generate the step that the index of potentiation is measured as BMP.

Assay method of the present invention in various BMP cellulations, uses cell strain of the present invention, has stably measured the BMP growing amount of cell first, has finished this assay method.It is in addition, described later that BMP generates the screening technique of material of potentiation and the preparation method of this material also is same to having.

Above-mentioned HuO9 cell (human osteosarcoma cell) has commercially available, can obtain.As the substrain of HuO9 cell, variants such as natural mutant, artificial variation's strain are for example arranged.Above-mentioned artificial variation's strain can for example be handled by known variation, and mutagenic agent and ultraviolet radiation prepare.Prepare as using the hybridoma that any one cell strain obtains among this, can operating, for example the cell strain that obtains by methods such as cell fusion with the myeloma cell by known cell fusion.

About assay method of the present invention, the cell strain that uses among the present invention is cultivated the culture medium of usefulness, be not particularly limited, so long as select the culture medium of this cell growth promoter is got final product, can use known commercially available culture medium.Incubation time as the cell strain that uses among the present invention, as long as longer than the needed time of BMP growing amount of improving cell because of the effect of test sample, be not particularly limited, can exemplify a few minutes～10 day, preferred 1 hour～5 days, more preferably 12 hours～3 days.About cultivation temperature, be not particularly limited, cultivate under the temperature that the growth promoter of the cell strain that can use in the present invention suits, for example 0～100 ℃, preferred 10～60 ℃, more preferably 20～50 ℃.

In the step (a) with condition that test sample contacts under the cultivation of cell strain of the present invention, can be by for example when the cultivation with cell strain of the present invention begins or in the incubation, to adding in the culture fluid that contains this cell strain, mixing test sample, begin or proceed to cultivate.The addition of test sample is not particularly limited, and can suitably set for obtaining desired effect.The amount of the cell strain of the present invention during as the interpolation test sample in the culture fluid, preferred 1 * 10 ³～1 * 10 ⁶About cell/mL.

After cultivating certain hour, in step (b), measure the BMP amount that generates by cell strain of the present invention.The mensuration of the BMP amount in the culture fluid, be not particularly limited, can carry out by the following method, for example enzymoimmunoassay, radioimmunoassay, western blotting, or with the biological action of BMP, for example bioassay (for example reference of carrying out as index to osteoblastic induction ability, Katagiri etc., BBRC Vol.172, No.1, p295-299 (1990); Yamaguchi etc., BBRC Vol.120, No.2, p366-371 (1996); Takuwa etc., BBRC Vol.174, No.1, p96-101 (1991)).

In the assay method of the present invention, will generate potentiation as index determining BMP by the BMP amount that cell strain of the present invention generates.For example, embodiment 4 as described later, and the BMP amount that is generated by cell strain of the present invention when not contacting with test sample is as 100%, and the BMP when relatively expression contact with test sample measures, and can measure the BMP generation potentiation of (evaluation) test sample quantitatively.That is, the BMP amount surpasses at 100% o'clock, can judge that test sample has BMP and generates potentiation, and the BMP amount is represented with relative quantity with numerical value, can estimate its effect quantitatively with this.

As other embodiments of the present invention, provide and have the screening technique (hereinafter referred to as screening technique of the present invention) that BMP generates the material of potentiation, it is characterized in that, comprise (a) with condition that test sample contacts under cultivate cell strain of the present invention step, (b) step of the BMP amount in the culture fluid that obtains of determination step (a), at this, with not with condition that test sample contacts under or compare when BMP generates under the condition that the contrast material of potentiation contact the above-mentioned cell strain of cultivation with having, the BMP amount judges that test sample has BMP and generates potentiation for a long time.

Screening technique of the present invention can similarly be implemented with the assay method of the invention described above.

For example, as mentioned above, the BMP amount that will the cell strain of the present invention when not contacting with test sample generates is as 100%, BMP when relatively expression contacts with test sample measures, the BMP that represents test sample quantitatively generates potentiation, the BMP amount surpasses at 100% o'clock, can judge that test sample has BMP and generates potentiation.In addition, for example measure test sample and (for example have contrast material that certain BMP generates potentiation, chemical compound shown in the above-mentioned formula (A)) BMP separately generates potentiation, the BMP of contrast material is measured as 100%, the BMP amount of representing test sample according to said circumstances quantitatively, by relatively, can screen material with more excellent BMP generation potentiation with both.Generate in the mensuration of potentiation at this BMP, can use the assay method of the invention described above.

As other embodiments of the present invention, provide and have the preparation method (hereinafter referred to as preparation method 1 of the present invention) that BMP generates the material of potentiation, it is characterized in that, comprise (a) obtain having BMP generate potentiation material step and (b) use assay method of the present invention, the BMP of the material that obtains in the determination step (a) generates the step of potentiation.Preparation method 1 of the present invention is useful in the preparation of the BMP generation enhancing substance of having examined and determine BMP generation potentiation.

Step (a) can be undertaken by for example screening technique acquisition BMP generation enhancing substance according to the invention described above.

In the step (b), for example, can generate enhancing substance separation and purification BMP by the BMP that obtains and generate enhancing ingredients.Promptly, the BMP of the material that obtains in the assay method determination step (a) of the application of the invention in step (b) generates potentiation, is used as index with this, and the limit confirms that BMP generates the refining degree of enhancing ingredients, the separation and purification operation is carried out on the limit, can obtain required composition.BMP generates the separation and purification of enhancing ingredients, can be applicable to that effective ingredient of the present invention carries out from the above-mentioned known method of the method for the handled thing of plant according to conduct.

A kind of form of implementation as preparation method of the present invention, provide and have the preparation method (hereinafter referred to as preparation method 2 of the present invention) that BMP generates the material of potentiation, it is characterized in that, comprise (a) with condition that test sample contacts under cultivate cell strain of the present invention step, (b) step of the BMP amount in the culture fluid that obtains of determination step (a), at this, with not with condition that test sample contacts under or compare when BMP generates under the condition that the contrast material of potentiation contact the above-mentioned cell strain of cultivation with having, the BMP amount for a long time, judge that test sample has BMP and generates potentiation, obtain this test sample as material with BMP generation potentiation.

This preparation method 2 of the present invention can similarly be implemented with the screening technique of the invention described above.In preparation method 2 of the present invention, judge when test sample has BMP generation potentiation, obtain this test sample as material with BMP generation potentiation.The material with BMP generation potentiation that obtains can be used for preparation method 1 of the present invention, carry out the separation and purification that BMP generates enhancing ingredients.

The above-mentioned effective ingredient that uses among the present invention even with the effective dose of bringing into play its effect biology is carried out administration, is not seen toxicity yet.For example, during oral administration, even with the water extract of angelica keiskei koidzumi, Herba Apii graveolentis, Bulbus Lilii, Aloe or Folium Artemisiae Argyi with the 1g/kg body weight to the mice single-dose, do not see dead example yet.In addition, though above-mentioned effective ingredient to rat with 1g/kg body weight oral administration, do not see dead example yet.

Embodiment

Below, enumerate embodiment and illustrate in greater detail the present invention, but the present invention is not subjected to any qualification of these records.In addition, do not specify that the % among the embodiment is meant volume % as long as have.

The preparation of embodiment 1 (E)-1-(5,6,7,8,8a, 10a-six hydrogen-1,7-dihydroxy-8,8,10a-trimethyl-9H-xanthene-4-yl)-3-(4-hydroxyphenyl)-2-propylene-1-ketone

(1) in the dried powder 5.8kg of angelica keiskei koidzumi (Angelica keiskei Koidz.) root, add the 24L ethyl acetate, stir under the room temperature, carry out extracting in 3 hours, after the filtered off with suction, separating ethyl acetate extracting solution and residue.Behind acetic acid ethyl acetate extract usefulness Rotary Evaporators concentrating under reduced pressure, be dissolved in the chloroform, and make it all be adsorbed in silica gel BW-300SP (シ リ シ ア chemical company of Fuji system: 750mL).Then use hexane successively: chloroform=2: 5 (750mL), chloroform (1000mL), chloroform: methanol=10: 1 (1100mL) is with the adsorbate gradient elution.

(2) with the chloroform that obtains among the embodiment 1-(1): methanol=10: 1 elution fraction is dissolved in the 30mL chloroform after concentrating and solidifying, make wherein half be adsorbed in silica gel (BW-300SP, 300mL).Then use chloroform (1800mL), chloroform successively: methanol=500: 7 (300mL), ethyl acetate (300mL) is with the adsorbate gradient elution.

(3) the eluent ethyl acetate component that obtains among the embodiment 1-(2) is concentrated solidify after, be dissolved in chloroform: in the methanol=50: 1 (20mL), be adsorbed in silica gel (BW-300SP, 300mL).Then use chloroform successively: methanol=500: 10 (1200mL), chloroform: methanol=500: 13 (500mL), chloroform: methanol=500: 19 (500mL), chloroform: methanol=500: 22 (800mL), ethyl acetate (500mL) is got 18mL with the adsorbate gradient elution in per 1 component.

(4) the silica gel column chromatography component that obtains among the collection embodiment 1-(3) is No. 115 to No. 155, and concentrating under reduced pressure is dissolved in the dimethyl sulfoxide of 7mL.With wherein half uses reversed phase chromatography separation.Resin adopts COSMOS IL 140C18-OPN, and (Na カ ラ イ テ ス Network company makes: amount of resin 50mL).Then use distilled water (40mL) successively, 20% ethanol water (50mL), 30% ethanol water (50mL), 50% ethanol water (the 1st eluting 50mL, the 2nd eluting 50mL afterwards), ethanol (50mL) carries out eluting.

(5) with behind the 1st eluting part 50mL concentrating under reduced pressure among the 50% ethanol water eluting component of embodiment 1-(4), be dissolved in 50% ethanol water of 3mL, use reversed phase chromatography separation.Below narrate its condition.Chromatographic column is used TSK gel ODS-80Ts (21.5mm * 30cm: eastern ソ-company makes).Use distilled water and acetonitrile to mix the gained solvent as solvent with volume ratio 1 to 1, elution speed is 5mL/ minute, detects at 215nm.Uv absorption with eluent is that index is collected reversed phase chromatography component 1～5.

(6), measure the mass spectrum (MS) of the reversed phase chromatography component 2 (containing the component that retention time is 24.1 minutes detected peaks) that obtains among the embodiment 1-(5) by the means of FAB-MS by mass spectrometer (DX302: NEC company makes).Substrate is used m-nitrobenzyl alcohol.The result detect m/z 407 (M-H)-the peak.Fig. 1 represents this mass spectrum.Transverse axis is represented the m/z value among Fig. 1, and the longitudinal axis is represented relative intensity.Then, use nuclear magnetic resonance, NMR (NMR) spectral apparatus (the AVANCE600 type: Bruker BIOSPIN company makes), measure the various NMR spectrums of reversed phase chromatography component 2, carry out structure elucidation.Below the signal of expression NMR belongs to.In addition, the ownership number at peak is as with shown in the following formula (B).

¹H-NMR: δ 0.81 (3H, s, 7 "-CH ₃), 1.03 (3H, s, 7 "-CH ₃), 1.25 (3H, s, 3 "-CH ₃), 1.54 (1H, m, 5 "-H), 1.61 (1H, dd; J=4.8,13.2Hz, 2 "-H), 1.71 (1H, m, 5 "-H); 1.75 (1H, m, 4 "-H), 1.87 (1H, m, 4 "-H); 2.34 (1H, dd, J=13.2,16.8Hz, 1 "-H), 2.67 (1H, dd, J=4.8,16.8Hz, 1 "-H), 3.27 (1H, m; 6 "-H), 4.65 (1H, d, J=4.8Hz, 6 "-OH); 6.47 (1H, d, J=8.4Hz, 5 '-H), 6.83 (2H; d, J=8.4Hz, 3-H and 5-H), 7.39 (1H, d; J=8.4Hz, 6 '-H), 7.42 (1H, d, J=15.6Hz; β-H), 7.48 (1H, d, J=15.6Hz, α-H); 7.51 (2H, d, J=8.4Hz, 2-H and 6-H), 9.97 (1H, br-s, 4-OH), 10.22 (1H, br-s, 4 '-OH)

¹Among the H-NMR, sample dissolution is in deuterated dimethyl sulfoxide, and the remaining proton chemical shifts value representation of deuterated dimethyl sulfoxide is 2.51ppm.Fig. 2 represents ¹The H-NMR spectrum.Among Fig. 2, transverse axis is represented chemical displacement value, and the longitudinal axis is represented the intensity of signal.

¹³C-NMR: δ 15.3 (7 "-CH ₃), 18.8 (1 " C), 20.7 (3 "-CH ₃), 28.1 (7 "-CH ₃), 28.9 (5 "-C), 38.3 (4 "-C), 38.9 (7 "-C); 46.4 (2 "-C), 76.8 (6 "-C), 77.9 (3 "-C), 107.7 (5 '-C), 110.4 (3 '-C), 116.8 (3-C and 5-C), 120.8 (1 '-C), 125.2 (α-C), 127.1 (1-C), 130.2 (6 '-C), (130.8 2-C and 6-C), 141.2 (β-C), 154.9 (2 '-C), 160.3 (4-C), 160.6 (4 '-C), 189.8 (C=O)

¹³Among the C-NMR, sample dissolution is in deuterated dimethyl sulfoxide, and the chemical displacement value of deuterated dimethyl sulfoxide is expressed as 40.2ppm.Fig. 3 represents ¹³The C-NMR spectrum.Among Fig. 3, transverse axis is represented chemical displacement value, and the longitudinal axis is represented the intensity of signal.

More than, by reversed phase chromatography component 2 being carried out the result of mass spectrum, NMR analysis of spectrum, determined that reversed phase chromatography component 2 is (E)-1-(5,6,7,8,8a, 10a-six hydrogen-1,7-dihydroxy-8,8,10a-trimethyl-9H-xanthene-4-yl)-3-(4-hydroxyphenyl)-2-propylene-1-ketone (molecular weight 408 is hereinafter referred to as compound a).

It is osteoblastic effect that embodiment 2 compound as are induced the ST-2 cell differentiation

(1) the mesenchymal cell strain ST-2 with mice is suspended in the DMEM culture medium (manufacturing of Bio Whittaker company) that contains 10% hyclone (manufacturing of BioWhittaker company), makes to reach 3 * 10 ⁴Cell/mL respectively inoculates 0.1mL in the flat board in 96 holes, cultivate under aseptic condition.Cultivate after 2 days, more renew culture medium.The compound a from blade root tomorrow that obtains in the embodiment 1 that wherein adds as sample was cultivated 3.Then, be index with the alkali phosphatase, measure the ST-2 cell to osteoblastic differentiation.With the PBS washed cell once, add response matrix liquid (100mM diethanolamine buffer pH10.0,2mM magnesium chloride, 1mM p-nitrophenyl phosphate ester) 100 μ L, 37 ℃ of reactions 30 minutes.Then, the sodium hydroxide 100 μ L that add 0.2N stop reaction, measure free paranitrophenol amount at 405nm.Not add sample in contrast, be that 100% expression is induced to differentiate into osteoblastic activity with the alkaline phosphatase activities that contrasts.The addition of sample is as shown in table 1.This experiment carries out twice continuously, gets its meansigma methods.Its result induces to osteoblastic differentiation with showing the compound a concentration dependent.Its result of table 1 expression.

It is osteoblastic activity that table 1 compound a is induced the ST-2 cell differentiation

Embodiment 3 compound as induce the MC3T3-E1 cell differentiation to become osteoblastic effect

The preosteoblast strain MC3T3-E1 of mice is suspended in the DMEM culture medium that contains 10% hyclone, makes to reach 3 * 10 ⁴Cell/mL respectively inoculates 0.1mL in the flat board in 96 holes, cultivate under aseptic condition.Cultivate after 2 days, more renew culture medium.The compound a from blade root tomorrow that obtains in the embodiment 1 that wherein adds as sample was cultivated 5.Then, be index with the alkali phosphatase, use the method identical to measure to osteoblastic differentiation with embodiment 2.Its result induces to osteoblastic differentiation with showing the compound a concentration dependent.Its result of table 2 expression.

It is osteoblastic activity that table 2 compound a is induced the MC3T3-E1 cell differentiation

The BMP-2 of embodiment 4 angelica keiskei koidzumis generates potentiation

(1) with respect to the dry thing 2g of blade root tomorrow that pulverizes, adds water 40mL, stir down and extracted 30 minutes at 60 ℃.Then carry out centrifugalize and make supernatant and precipitate and separate.For precipitation, repeat 2 same operations.The supernatant that collection obtains is concentrated into 10mL, preparation blade root water extract tomorrow.

(2) human osteosarcoma cell HuO9 is suspended in the DMEM culture medium that contains 10% hyclone, makes to reach 1 * 10 ⁵Cell/mL respectively inoculates 0.1mL in the flat board in 96 holes, cultivate under aseptic condition.Cultivate after 2 days, more renew culture medium.Blade root water extract tomorrow that obtains in the embodiment 4-(1) that wherein adds as sample was cultivated 48 hours.Then, measure the concentration of drBMP-2-2 (BMP-2) in the culture fluid by enzymoimmunoassay (manufacturing of BMP-2Immunoassay:GT company).Not add sample in contrast, be that 100% expression BMP-2 generates enhanced activity with the BMP-2 concentration in this cell culture fluid.The addition of sample is as shown in table 3.This experiment carries out twice continuously, gets its meansigma methods.Its result shows the generation of the blade root water extract concentration dependent ground enhancing tomorrow BMP-2 that embodiment 4-(1) obtains.Its result of table 3 expression.

The BMP-2 of table 3 blade root tomorrow water extract generates potentiation

The BMP-2 growing amount of contrast is 0.270ng/ml.

Embodiment 5 angelica keiskei koidzumis are induced the osteoblastic effect that is divided into

C3H10T1/2 is suspended in the DMEM culture medium that contains 10% hyclone with the strain of mice blastocyte, makes to reach 3 * 10 ⁴Cell/mL respectively inoculates 0.1mL in the flat board in 96 holes, cultivate under aseptic condition.Cultivate after 3 days, more renew culture medium.Toward wherein adding, cultivated 6 as blade root water extract tomorrow that obtains among the sample embodiment 4-(1).Then, be index with the alkali phosphatase, use the method identical to measure to osteoblastic differentiation with embodiment 2.Its result shows that embodiment 4-(1) obtains tomorrow blade root water extract concentration dependent induce to osteoblastic differentiation.Its result of table 4 expression.

Table 4 blade root tomorrow water extract is induced to differentiate into osteoblastic effect

Embodiment 6 generates potentiation from the BMP-2 of COSMOSIL segmentation thing of blade root water extract tomorrow

(1) with respect to the dry thing 3kg of blade root tomorrow that pulverizes, adds water 30L, stir down and extracted 60 minutes at 60 ℃.Then carry out centrifugalize and make supernatant and precipitate and separate.For precipitation, add water 20L, stir, extracted 60 minutes under the room temperature.Then carry out centrifugalize and make supernatant and precipitate and separate.Merge supernatant, preparation blade root aqueous extract 45L tomorrow.

(2) use reversed phase chromatography that blade root aqueous extract tomorrow that obtains among the embodiment 6-(1) is separated.Its condition is as follows.Resin uses COSMOSIL 140 C18-OPN (amount of resin 700mL).Provide tomorrow blade root aqueous extract 1.5L, water (2.1L) successively, 10% ethanol (2L), 20% ethanol (2.3L), 40% ethanol (2L), 60% ethanol (3.5L) carries out eluting as launching solvent, obtains each COSMOSIL segmentation thing.

(3) 1/50 amount with each COSMOSIL segmentation thing of obtaining among the embodiment 6-(2) is concentrated into 10mL.The BMP-2 that measures each concentrate with the method identical with embodiment 4-(2) generates enhanced activity.Its result shows the water elution component, and 10% ethanol elution component, 20% ethanol elution component have BMP-2 and generate enhanced activity.Its result of table 5 expression.

Table 5

The BMP-2 growing amount of contrast is 0.126ng/ml.

Embodiment 7 generates potentiation from the BMP-2 of silicagel column segmentation thing of blade root water extract tomorrow

(1) embodiment 6-(1) will be concentrated into 19L with 41L, preparation blade root water extraction concentrated solution tomorrow among the blade root water extract at tomorrow of obtaining.

(2) add 200mL ethanol toward the tomorrow that embodiment 7-(1) obtains among the blade root water extraction concentrated solution 300mL, left standstill under the room temperature 1 hour, carry out centrifugalize then and make supernatant and precipitate and separate.In precipitation, add the 300mL distilled water, carry out same operation repeatedly.Merge the supernatant that obtains, behind concentrated the curing, be dissolved in the 108mL distilled water, preparation is from the ethanol precipitation handled thing of blade root water extract tomorrow.

(3) use silica gel column chromatography to embodiment 7-(2) obtain from tomorrow the blade root water extract the ethanol precipitation handled thing separate.Its condition is as follows.With 25mL from tomorrow the blade root water extract the ethanol precipitation handled thing concentrate to solidify after, be dissolved in volume ratio and be in 6 to 3 the chloroform and alcoholic acid mixed solution, it is adsorbed on the silica gel BW-300SP (amount of resin 300mL).Then, use chloroform successively: ethanol=6: 3 (600mL), chloroform: ethanol: water=30: 15: 1 (900mL), chloroform: ethanol: water=12: 8: 1 (500mL), ethanol: water=10: 1 (600mL), ethanol: water=20: 3 (200mL), ethanol: water=4: 1 (250mL), ethanol: the gradient elution adsorbate of water=1: 1 (300mL).

Collect chloroform: ethanol: back half eluting part (200mL) and ethanol of water=12: 8: 1: preceding half eluting of water=10: 1 is (200mL) partly, obtains silicagel column elution fraction 1.Collect ethanol: back half eluting part (400mL) and ethanol of water=10: 1: water=20: 3 eluting part, ethanol: water=4: 1 eluting parts obtains silicagel column elution fraction 2.With ethanol: water=the eluting part obtained as silicagel column elution fraction 3 in 1: 1.

(4) 1/5 amount with each silicagel column segmentation thing of obtaining among the embodiment 7-(3) is concentrated into 1.25mL.The BMP-2 that measures each concentrate with the method identical with embodiment 4-(2) generates enhanced activity.Its result shows that component shown in the table 6 has BMP-2 and generates enhanced activity.

Table 6

The BMP-2 growing amount of contrast is 0.096ng/ml.

The BMP-2 of embodiment 8 leaf of Herba Apii graveolentis portion water extract generates potentiation

(1) with respect to the dry thing 2g of the leaf of Herba Apii graveolentis portion that pulverizes, adds water 40mL, stir down and extracted 30 minutes at 60 ℃.Then carry out centrifugalize and make supernatant and precipitate and separate.For precipitation, repeat 2 same operations.The supernatant that collection obtains is concentrated into 10mL, preparation leaf of Herba Apii graveolentis portion water extract.

(2) BMP-2 that uses the method identical with embodiment 4-(2) to measure the leaf of Herba Apii graveolentis portion water extract of embodiment 8-(1) preparation generates enhanced activity.Its result shows that leaf of Herba Apii graveolentis portion water extract has BMP-2 and generates enhanced activity.

The BMP-2 of table 7 leaf of Herba Apii graveolentis portion water extract generates potentiation

The BMP-2 growing amount of contrast is 0.119ng/ml.

The BMP-2 of embodiment 9 aloe water extracts generates potentiation

(1) with respect to the dry thing 2g of the Aloe of pulverizing, adds water 40mL, stir down and extracted 30 minutes at 60 ℃.Then carry out centrifugalize and make supernatant and precipitate and separate.For precipitation, repeat 2 same operations.The supernatant that collection obtains is concentrated into 10mL, preparation aloe water extract.

2) BMP-2 that uses the method identical with embodiment 4-(2) to measure the aloe water extract of embodiment 9-(1) preparation generates enhanced activity.Its result shows that the aloe water extract has BMP-2 and generates enhanced activity.

The BMP-2 of table 8 aloe water extract generates potentiation

The BMP-2 growing amount of contrast is 0.274ng/ml.

The BMP-2 of embodiment 10 Folium Artemisiae Argyi water extract generates potentiation

(1) with respect to the dry thing 2g of the Folium Artemisiae Argyi of pulverizing, adds water 40mL, stir down and extracted 30 minutes at 60 ℃.Then carry out centrifugalize and make supernatant and precipitate and separate.For precipitation, repeat 2 same operations.The supernatant that collection obtains is concentrated into 10mL, preparation Folium Artemisiae Argyi water extract.

(2) BMP-2 that uses the method identical with embodiment 4-(2) to measure the Folium Artemisiae Argyi water extract of embodiment 10-(1) preparation generates enhanced activity.Its result shows that the Folium Artemisiae Argyi water extract has BMP-2 and generates enhanced activity.

The BMP-2 of table 9 Folium Artemisiae Argyi water extract generates potentiation

The BMP-2 growing amount of contrast is 0.119ng/ml.

Embodiment 11 aloe water extracts are induced the osteoblastic effect that is divided into

Measure the aloe water extract of preparation among the embodiment 9-(1) with the method identical and induce the osteoblastic effect that is divided into embodiment 5.Its result induces to osteoblastic differentiation with showing the aloe water extract concentrations dependency that obtains among the embodiment 9-(1).Its result of table 10 expression.

Table 10 aloe water extract is induced to differentiate into osteoblastic effect

Embodiment 12 Flos Lilii viriduli water extract are induced the osteoblastic effect that is divided into

(1) with respect to the dry thing 2g of the Flos Lilii viriduli portion that pulverizes, adds water 40mL, stir down and carry 30 minutes at 60 ℃.Then carry out centrifugalize and make supernatant and precipitate and separate.For precipitation, repeat 2 same operations.The supernatant that collection obtains is concentrated into 10mL, preparation Flos Lilii viriduli water extract.

(2) the Flos Lilii viriduli water extract of using the method identical with embodiment 5 to measure preparation among the embodiment 12-(1) is induced the osteoblastic effect that is divided into.Its result induces to osteoblastic differentiation with showing the Flos Lilii viriduli portion water extract concentration dependent that obtains among the embodiment 12-(1).Its result of table 11 expression.

Table 11 Flos Lilii viriduli water extract is induced to differentiate into osteoblastic effect

Embodiment blade root water extract 13 tomorrow and recombinant human B MP-2 induce the synergism that is divided into osteoblastic effect

C3H10T1/2 is suspended in the DMEM culture medium that contains 10% hyclone with the strain of mice blastocyte, makes to reach 3 * 10 ⁴Cell/mL respectively inoculates 0.1mL in the flat board in 96 holes, cultivate under aseptic condition.Cultivate after 4 days, more renew culture medium.The blade root water extract and reorganization BMP-2 (manufacturing of GT company) tomorrow that obtains in the embodiment 4-(1) that wherein adds as sample was cultivated 11.Then, be index with the alkali phosphatase, use the method identical to measure to osteoblastic differentiation with embodiment 5.Its result induces to osteoblastic differentiation with showing recombinant human B MP-2 concentration dependent, but by and with blade root water extract tomorrow, strengthened to osteoblastic differentiation-inducing action.Its result of table 12 expression.

Table 12 blade root tomorrow water extract and BMP-2 are induced to differentiate into osteoblastic effect

Embodiment blade root water extract 14 tomorrow inducing mesenchymal stem cell is divided into osteoblastic effect

Human mesenchymal stem cell (manufacturing of TAKARA BIO company) is suspended in the DMEM culture medium (manufacturing of TAKARA BIO company) that contains 10% hyclone, makes to reach 1.2 * 10 ⁴Cell/mL respectively inoculates 0.5mL in the flat board in 48 holes, cultivate under aseptic condition.Cultivate after 5 days, be replaced by the new culture medium that contains 5mM β-phosphoglycerol, 100nM dexamethasone.Adding blade root water extract tomorrow that obtains among the embodiment 4-(1) in this culture medium cultivates as sample.Changed culture medium in per 3,4 days.Human mesenchymal stem cell to osteoblastic differentiation with the calcium accumulation of alkaline phosphatase activities and cell as index.Measure alkaline phosphatase activities with the method identical, be expressed as 1 minute free paranitrophenol amount in every hole with embodiment 2.The calcium amount of accumulating in the cell is following mensuration, toward with adding 6N hydrochloric acid in the washed cell of PBS, for the solution that carries out ultrasonic disruption, uses the quantitative box of calcium (the pure pharmaceutical worker's industry of Calucium E-testWako and light company makes) to measure.

Its result, the blade root water extract will promote alkaline phosphatase activities of following when mescenchymal stem cell is divided into osteoblast and the calcium of following in the osteoblast maturation to accumulate in time powerfully tomorrow.The measurement result of table 13 expression alkaline phosphatase activities, the measurement result that the calcium of table 14 expression cell is accumulated.

Table 13

Table 14

Industrial applicibility

The invention provides the handled thing that contains from Umbelliferae, Liliaceae or feverfew, be used for Treatment or prevention need the promotion skeletonization or are enhanced to medicine, the skeletonization of the disease of bone protein generation Effect promoter or BMP generate reinforcing agent, food, beverage or feed. This medicine can be used as Curative or the preventive medicine of osteoporosis, fracture etc. and bone photo related disorders. In addition, skeletonization is done Generate reinforcing agent with promoter or BMP, can be used as planting of using in fractures, the tooth treatment Enter agent, toothpaste. In addition, said preparation generates the screening that strengthens medicine at bone functional study, BMP In also useful. In addition, this Foods or drinks, by absorbing as daily beverage/food, can To improve the symptom that needs the promotion skeletonization or strengthen the disease of BMP generation. In addition, by making Thing absorbs this feed also can expect same effect.

Claims (3)

1. the handled thing from angelica keiskei koidzumi needs to promote skeletonization or be enhanced to the disease treatment medicine of bone protein generation or the application in the preventive drug in preparation.

2. generate application in the reinforcing agent from the handled thing of angelica keiskei koidzumi at preparation skeletonization promoter or drBMP-2.

3. be used for promoting skeletonization or be enhanced to the application of food, beverage or feedstuff that bone protein generates in preparation from the handled thing of angelica keiskei koidzumi.