JP6560471B1 - Bone formation promoter and preventive and / or therapeutic agent for bone metabolic disease using the same - Google Patents

Abstract

The present invention provides a novel means capable of replacing a conventionally known preventive / therapeutic agent for bone metabolic diseases. An osteogenesis promoter comprising, as an active ingredient, an extract of one or more plants selected from the group consisting of sweet basil, holy basil, common time, Italian large leaf and fennel. This osteogenesis promoter is useful as a preventive and / or therapeutic agent for bone metabolic diseases accompanied by bone loss. [Selection] Figure 2

Description

  The present invention relates to an osteogenesis promoter and a preventive and / or therapeutic agent for bone metabolic diseases using the same.

  There are over 10 million osteoporosis patients in Japan, which is a big social problem. Moreover, it is speculated that the population over 65 years old will increase due to rapid aging, and the number of osteoporosis patients will continue to increase. In the human body, bones constantly repeat bone resorption and bone formation. At this time, osteoclasts are involved in bone resorption, and osteoblasts are involved in bone formation. Bone formation, maintenance, and repair depend on the balance between the formation and resorption of these cells. When this balance is lost, bone resorption exceeds bone formation, resulting in bone metabolic diseases such as osteoporosis. Are known. Therefore, it is thought that it is important for the maintenance and enhancement of bone mass that bone formation does not fall below bone resorption.

  Since bone resorption is performed by osteoclasts, the greater the differentiation and activation of osteoclasts, the higher the bone resorption rate. On the other hand, osteogenesis is performed by osteoblasts, and the more osteoblasts are differentiated and activated, the higher the bone formation rate. Therefore, if a composition having a bone resorption inhibitory action for reducing the bone resorption rate and a bone formation promoting action for increasing the bone formation rate is obtained, it is effective for prevention and treatment of osteoporosis. For example, a composition for improving bone metabolism using an extract of ginger containing 6-gingerol and 6-shogaol, which are pungent components of ginger, as an active ingredient is known. (For example, see Patent Document 1).

JP 2014-43416 A

  Conventionally, drugs such as estrogen, bisphosphonate, vitamin D, and calcitonin have been clinically applied to the treatment of bone metabolic diseases such as osteoporosis. However, there are cases where these treatment methods are not sufficient, and there is a need for new and better preventive / therapeutic agents.

  Accordingly, an object of the present invention is to provide a novel means that can be substituted for a conventionally known preventive / therapeutic agent for bone metabolic diseases.

  The present inventors have intensively studied to solve the above problems. As a result, the inventors have found that plant extracts such as sweet basil have an excellent bone formation promoting action and have completed the present invention.

  That is, according to one aspect of the present invention, it contains an extract of one or more plants selected from the group consisting of sweet basil, holy basil, common time, Italian large leaf and fennel as an active ingredient. An osteogenesis promoter is provided.

  Moreover, according to the other form of this invention, the extract of the 1 type, or 2 or more types of plant selected from the group which consists of sweet basil, holy basil, common time, Italian large leaf, and fennel is contained as an active ingredient. A food and drink composition for promoting bone formation is provided.

  Furthermore, according to still another aspect of the present invention, an extract of one or more kinds of plants selected from the group consisting of sweet basil, holy basil, common time, Italian large leaf and fennel is contained as an active ingredient. A food and drink composition for preventing and / or treating a bone metabolic disease accompanied by bone loss is provided.

  ADVANTAGE OF THE INVENTION According to this invention, the novel means which can substitute for the conventionally well-known preventive / therapeutic agent of a bone metabolic disease can be provided.

FIG. 1 shows the results of evaluating the bone formation promoting effect (osteoblast differentiation promoting action) of each sample using cultured cells (MC3T3-E1 cells) and staining with alkaline phosphatase (ALP) as an index in Examples. It is a photograph which shows. FIG. 2 shows the results of evaluating the osteogenesis promoting action (osteoblast differentiation promoting action) of each sample using cultured cells (MC3T3-E1 cells) in the Examples and using the activity of alkaline phosphatase (ALP) as an index. It is a graph which shows. FIG. 2 (A) is a graph comparing the measured values of ALP activity in the cells of the negative control (2) and the cells added with each sample as relative values when the measured value in the negative control (1) is 100%. It is. Moreover, FIG. 2 (B) compared the measured value of ALP activity in the cells to which each sample was added as a relative value (increase rate of ALP activity) when the measured value in the negative control (2) was taken as 100%. It is a graph. FIG. 3 shows the bone formation promoting action (osteoblast differentiation promoting action) of each sample using cultured cells (MC3T3-E1 cells) and staining of calcified sites using alizarin red S as an index in the examples. It is a photograph which shows the result of having evaluated.

  Embodiments of the present invention will be described below. In addition, this invention is not limited only to the following embodiment. In this specification, “X to Y” indicating a range means “X or more and Y or less”, “weight” and “mass”, “wt%” and “mass%”, and “part by weight” and “part by mass”. Is treated as a synonym. Unless otherwise specified, measurement of operation and physical properties is performed under conditions of room temperature (20 to 25 ° C.) / Relative humidity 40 to 50%.

<Bone formation promoter>
One embodiment of the present invention is an extract of one or more plants selected from the group consisting of sweet basil, holy basil, common time, Italian large leaf and fennel (hereinafter collectively referred to as “plant extract”). It is a bone formation promoter containing as an active ingredient. The scientific name of sweet basil is Ocium basilicum, which belongs to the genus Meboki (Japanese name is Mebuki). Holy Basil's scientific name is Ocimum tenuiflorum, Syn. O. sanctum, which belongs to the family Lamiaceae (Japanese name is Kamebouki). The common time scientific name is Thymus vulgaris L. This belongs to the family Lamiaceae, which belongs to the genus Ichikisou. Italian large leaf has the same scientific name as sweet basil and belongs to the same family and genus, but is a large leaf cultivar with larger leaves. The scientific name of fennel is Foenicum vulgare, which belongs to the genus Felioaceae (Japanese name is Fennel).

  The extract which is an active ingredient of the osteogenesis promoter according to the present invention can be obtained by extracting all or part of the various plants described above with a suitable solvent.

  Here, the part to be extracted in the plant is not particularly limited, and may be the whole plant or a part (leaf, seed, root, etc.). Of these, it is preferable to extract the leaves in consideration of the bone formation promoting effect.

  In addition, the whole plant to be extracted or a part thereof (hereinafter, also simply referred to as “extraction raw material”) may be subjected to extraction in the form as it is, but before being subjected to extraction, it is dried and dried in advance. It is preferable to be pulverized. Thereby, a desired active ingredient can be more efficiently extracted from said plant.

  Here, the drying conditions for drying the extraction raw material in advance are not particularly limited. Considering ease of pulverization and the like, the drying temperature is preferably 20 to 60 ° C, more preferably 20 to 40 ° C. The drying time is preferably 24 to 120 hours, more preferably 48 to 96 hours. Or you may dry an extraction raw material by methods, such as a freeze-drying powdering method, a high pressure method, and an ultrahigh pressure method. Among these, the high pressure method is a method of crushing cells in the extraction raw material under pressure by, for example, high pressure heat treatment at 100 to 150 ° C. for 2 to 10 hours (for example, 120 ° C. for 4 hours).

  Moreover, the pulverization conditions when the extraction raw material is pulverized in advance are not particularly limited. The size of the extraction raw material after pulverization is preferably about 0.5 to 10 mm, more preferably about 1 to 5 mm in consideration of extraction efficiency and the like. Examples of the pulverization method include a method of chopping with a cutting machine, a slicer, a cutter, a peeler, a jaw crusher, a jet mill, a blender, and the like.

  Next, the extraction raw material previously dried and / or pulverized if necessary is extracted using an appropriate solvent. The solvent that can be used here is not particularly limited as long as it can extract the active ingredient from the extraction raw material, and is appropriately selected. Specifically, water (including tap water, industrial water, distilled water, reverse osmosis membrane water, filtered water, sterilized water, purified water, etc.); methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, Examples include alcohols such as 2-butanol, propylene glycol, and 1,3-butylene glycol; esters such as methyl acetate and ethyl acetate; ketones such as acetone; diethyl ether, formaldehyde, and dimethyl sulfoxide. The said solvent may be used independently or may be used with the form of 2 or more types of mixed solvents. Of these, the solvent is preferably water or a mixed solution of water and at least one alcohol selected from the group consisting of methanol, ethanol, and isopropanol in consideration of extraction efficiency, safety, and the like. That is, in a preferred embodiment of the present invention, the plant extract is an extract from water, alcohol (preferably ethanol) or a mixture of water and alcohol. In addition, as adopted in the examples described later, a mixture of water and alcohol (for example, ethanol) is used as an extraction solvent, and extraction is sequentially performed using a plurality of such mixtures whose alcohol concentration is sequentially increased. This is also a preferred embodiment.

  If necessary, the extraction raw material may be extracted under acidic or alkaline conditions. That is, an acidic or alkaline solvent may be used. Here, the acid that can be used when preparing the acidic solvent is not particularly limited, but inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, citric acid, oxalic acid, succinic acid, formic acid, propionic acid, etc. Organic acids and the like. Moreover, as alkalinity, although it does not restrict | limit especially as an alkali which can be used when preparing a solvent, Potassium hydroxide, sodium hydroxide, sodium carbonate etc. are mentioned. The pH of the solvent when extracting the plant material under acidic or alkaline conditions is appropriately selected in consideration of the extraction conditions and the like. Moreover, when an acidic solvent or an alkaline solvent is used, it is preferable to neutralize the extract so that it becomes neutral (pH = about 7 ± 1) after extraction.

  The amount of the solvent added is not particularly limited as long as the active ingredient can be extracted from the extraction raw material. Specifically, it is preferable to add the solvent in an amount of 1 to 100 mL, more preferably 5 to 50 mL, with respect to 1 g of the extraction raw material.

  The extraction conditions are not particularly limited as long as the active ingredient can be extracted from the extraction raw material. Specifically, the extraction temperature is preferably 15 to 200 ° C, more preferably 20 to 150 ° C. The extraction time is preferably 12 hours to 6 days, more preferably 12 hours to 3 days. Under such conditions, the active ingredient can be efficiently extracted from the extraction raw material.

  After the extraction step, the solid (residue of the extraction raw material) is removed from the liquid mixture of the extraction raw material and the solvent, and the extraction liquid is separated. Here, the separation method is not particularly limited, and examples thereof include filtration and centrifugation. Furthermore, this extract may be used as it is, but if necessary, diluted form by dilution, extract by concentration, paste or solid form, frozen form by freezing, dry powder form by freeze drying, etc. You may convert into various forms (extract). Here, the conversion method may be applied alone or in combination of two or more. Preferably, a method of concentrating the extract solution to an appropriate concentration (for example, concentration under reduced pressure) is preferably used.

  The extract or extract may be further purified. Here, the purification method is not particularly limited, and a known purification method can be used. Specifically, a quaternary ammonium salt such as cetylpyridinium chloride is added to obtain a precipitate, and this precipitate is washed with an appropriate solvent (eg, water, alcohol), anion exchange ion exchange chromatography, Examples include cation exchange ion exchange chromatography, phosphocellulose chromatography, hydrophobic reaction chromatography, affinity chromatography, methods using chromatography using hydroxyapatite chromatography, lectin chromatography, and the like, and recrystallization methods.

  The plant extract according to the present invention obtained above has an effect of promoting bone formation. Therefore, the plant extract according to the present invention can be suitably used as an osteogenesis promoter. The osteogenesis promoting action of the plant extract according to the present invention is due to the fact that any active ingredient contained in the plant extract promotes osteoblast differentiation (that is, the extract is osteoblast differentiation. It is estimated that bone formation is promoted through the promotion of Here, it can be confirmed by the following method that the plant extract according to the present invention has an activity of promoting bone formation. MC3T3-E1 cells may be brought into contact with plant extracts, and the degree of differentiation of MC3T3-E1 cells may be confirmed using the activity of alkaline phosphatase (ALP), which is a differentiation marker, as an index. When ALP activity increases, it can be evaluated that osteoblast differentiation is promoted. In addition, osteoblast differentiation may be evaluated by, for example, the amount of calcium accumulated by the alizarin red S staining method.

  Here, the use target of the osteogenesis promoter is not particularly limited, but mammals and birds, preferably mammals and birds suffering from a bone metabolic disease accompanied by bone loss. Here, mammals include primates such as humans, monkeys, gorillas, chimpanzees, orangutans, and non-humans such as mice, rats, hamsters, guinea pigs, rabbits, dogs, cats, pigs, cows, horses, sheep, camels, and goats. Includes both human mammals. Examples of birds include chickens, quails and pigeons. Of these, humans, hamsters, guinea pigs, rabbits, dogs, cats and pigs are preferred. The target of use is more preferably a pet animal such as a rabbit, dog or cat, except for humans.

  The osteogenesis promoter according to the present invention is effective for the prevention and / or treatment of bone metabolic diseases accompanied by bone loss. Therefore, the osteogenesis promoter according to the present invention can be suitably used as a preventive and / or therapeutic agent for bone metabolic diseases accompanied by bone loss. As used herein, “prevention and / or treatment of a bone metabolic disease associated with bone loss” refers to suppressing the progression of the disease in a patient suffering from a bone metabolic disease associated with bone loss, and bone loss. Eliminate bone metabolic diseases that accompany it, suppress progression of bone metabolic diseases that accompany bone loss, prevent the development of bone metabolic diseases that accompany bone loss, and recurrence of bone metabolic diseases that accompany bone loss It means satisfying at least one of prevention. Therefore, in the present specification, the “prophylactic and / or therapeutic agent for bone metabolic disease accompanied by bone loss” means the above-mentioned preventive and / or therapeutic effect when used for the treatment of bone metabolic disease accompanied by bone loss. Means the indicated drug.

  Here, the kind of the disease which becomes the object of the preventive and / or therapeutic agent for bone metabolic disease accompanied by bone loss is not particularly limited. For example, osteoporosis, juvenile osteoporosis, bone dysplasia, hypercalcemia, hyperparathyroidism, osteomalacia, osteocalcinosis, osteolytic bone disease, osteonecrosis, Paget's disease, rheumatoid arthritis, deformity Bone loss due to osteoarthritis, inflammatory arthritis, osteomyelitis, glucocorticoid treatment, metastatic bone disease, periodontal bone loss, bone loss due to cancer, and bone loss due to aging, and the like.

  The preventive and / or therapeutic agent for bone metabolic diseases associated with bone loss according to the present invention can be used in the same dosage form as in the prior art, except that it contains the plant extract according to the present invention as an active ingredient. That is, the preventive and / or therapeutic agent for bone metabolic disease accompanied by bone loss according to the present invention is suitable for parenteral administration, oral administration or external administration by mixing with pharmaceutically acceptable additives such as excipients. It can be used in the form of pharmaceuticals, quasi drugs, foods and cosmetics. In food, it can be added to oil and fat products, emulsified products, soft drinks and the like. In pharmaceuticals, it can be added to oral preparations, external preparations, injections, inhalants, nasal drops, eye drops, etc., depending on the method of use, tablets, solutions, injections, ointments, creams, lotions, aerosols And a desired dosage form such as a suppository. Moreover, an excipient | filler, a base, an emulsifier, a stabilizer, a solubilizing agent, a corrigent, a preservative, a fragrance | flavor, a coloring agent, a coating agent, etc. can be suitably mix | blended as needed. For quasi-drugs and cosmetics, it can be added to lotions, emulsions, creams, etc., if necessary, oil, moisturizer, UV absorber, water-soluble polymer, antioxidant, surfactant, metal An ion sequestering agent, an antibacterial preservative and the like can be blended.

  The dosage of the preventive and / or therapeutic agent for bone metabolic disease accompanied by bone loss according to the present invention includes the route of administration; the nature of the patient's disease; the size, weight, surface area, age and sex of the patient; It depends on the drug; and the judgment of the attending physician. A suitable dose (in terms of dry weight of the plant extract) is, for example, 10 to 100 mg / kg body weight per day. Given the different available compositions and the different effectiveness of the different routes of administration, it is expected that the required dosage can vary widely. These dosage level variations can be adjusted using standard empirical procedures for optimality known in the art. In particular for oral delivery, encapsulation of the composition in a suitable delivery vehicle (eg, polymer microparticles or implantable devices) increases delivery efficiency. The dose may be divided once or multiple times a day. Or, in some cases, it may be administered less frequently (eg, weekly or monthly). In addition, even in the same patient, the dose may vary depending on the patient's symptoms and severity.

  A therapeutically effective amount of a plant extract is formulated with one or more pharmaceutically acceptable carriers (additives) and / or diluents in prophylactic and / or therapeutic agents for bone metabolic disorders with bone loss Can be done. That is, the preventive and / or therapeutic agent for bone metabolic diseases accompanied by bone loss according to the present invention can be provided in the form of a pharmaceutical composition further containing a pharmaceutically acceptable additive (eg, excipient, carrier, etc.). Agents for the prevention and / or treatment of bone metabolic disorders associated with this form of bone loss can be specifically formulated for solid or liquid administration, as described in detail below. As oral administration, for example, liquid medicine (aqueous solution or non-aqueous solution or suspension), tablet, bolus, powder medicine, granule, paste for application to the tongue can be exemplified. For parenteral administration, for example, as a sterile solution or suspension, for example, a formulation for subcutaneous, intramuscular or intravenous injection, or as a topical, for example as a cream, ointment or spray applied to the skin, or It can be formulated in the vagina or rectum, for example, as a vaginal suppository, cream or foam.

  As used herein, “therapeutically effective amount” as used herein is effective to produce any desired therapeutic effect at a reasonable benefit / risk ratio applicable to any medical treatment. Means the amount of the active agent or composition. For example, although the dose of the preventive and / or therapeutic agent for bone metabolic disease associated with bone loss according to the present invention varies depending on the target disease, administration subject, administration route, etc., the present invention is used for the purpose of prevention and / or treatment. When a prophylactic and / or therapeutic agent for bone metabolic diseases associated with bone loss associated with the above is orally administered, the volume can be easily varied depending on conditions such as the body weight of the subject person, and thus can be appropriately selected by those skilled in the art. In the end, the attending physician will make an appropriate selection in consideration of the patient's symptoms and severity.

  As used herein, “pharmaceutically acceptable” means that within a reasonable medical judgment, commensurate with a reasonable benefit / risk ratio, without problems or complications such as excessive toxicity, irritation, and allergic reaction, Used to refer to compounds, materials, compositions, and / or dosage forms suitable for use in contact with tissue of a subject to be treated (human, mammal, etc.).

  The pharmaceutically acceptable carrier means to transport or transport the preventive and / or therapeutic agent for bone metabolic diseases accompanying bone loss of the present invention from one organ or part of the body to another organ or part of the body. Means a pharmaceutically acceptable material, composition or excipient, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material involved. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the dosage form and not injurious to the patient. Pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; Powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository wax; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols such as propylene glycol; Polyols such as glycerin, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; magnesium hydroxide and water Buffers such as aluminum; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solution; and other adaptations non-toxic substance which is used in the drug formulation and the like. In some embodiments, the drug formulation is non-pyrogenic. That is, the thing which does not raise a patient's body temperature is desirable. Other wetting agents such as sodium lauryl sulfate and magnesium stearate, emulsifiers and lubricants, and colorants, release agents, coatings, sweeteners, flavors and fragrances, preservatives and antioxidants with bone loss It may be contained in an agent for preventing and / or treating bone metabolic diseases.

  Pharmaceutically acceptable antioxidants include, but are not limited to, water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium disulfite, sodium sulfite and the like; ascorbyl palmitate, butylhydroxyanisole ( Oil-soluble antioxidants such as BHA), butylhydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol and the like; and citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid and the like A metal chelating agent is mentioned.

  The agent for preventing and / or treating bone metabolic diseases accompanied by bone loss according to the present invention includes various agents such as oral, nasal, topical (including oral and sublingual), rectal, vaginal and / or parenteral administration. Can be used in form. The dosage form may be conveniently presented in unit dosage form and may be prepared by any method well known in the pharmaceutical art. The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated, the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form is generally the amount of compound that produces a therapeutic effect, but in general, the amount of active ingredient (plant extract) reduces bone loss. The amount is about 0.1 to about 99 parts by weight, preferably about 1 part to about 70 parts by weight, more preferably about 5 parts by weight with respect to 100 parts by weight of the preventive and / or therapeutic agent for the accompanying bone metabolic disease. Part to about 50 parts by weight.

  Methods for preparing these dosage forms or compositions comprise the step of combining the plant extract according to the present invention and optionally one or more accessory ingredients. In general, the dosage forms are prepared by uniformly and intimately bringing together the plant extract according to the invention with a liquid carrier, a finely divided solid carrier or both, and if necessary shaping the product.

  For example, dosage forms of the invention suitable for oral administration are in the form of capsules, sachets, pills, tablets, lozenges (with seasoned active ingredients, usually sucrose and gum arabic or tragacanth), powders, granules Or as solutions or suspensions in aqueous or non-aqueous liquids, or as oil-in-water or water-in-oil liquid emulsions, or as elixirs or syrups, or pastilles (such as gelatin and glycerin, or sucrose and gum arabic) And / or a gargle, etc., each containing a predetermined amount of the plant extract according to the present invention as an active ingredient. The agent for preventing and / or treating a bone metabolic disease accompanied by bone loss according to the present invention may be administered as a bolus, electuary or paste.

  In solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules, etc.), the active ingredient (plant extract) is one or more such as sodium citrate or dicalcium phosphate A pharmaceutically acceptable carrier and / or any of the following: a filler or bulking agent such as starch, lactose, sucrose, glucose, mannitol, and / or silicic acid; for example, carboxymethylcellulose, alginate, Binders such as gelatin, polyvinylpyrrolidone, sucrose and / or gum arabic; humectants such as glycerol; such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate Disintegrants; dissolution retardants such as paraffin Absorption enhancers such as quaternary ammonium compounds; wetting agents such as cetyl alcohol and glycerol monostearate; absorbents such as kaolin and bentonite clay; talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate And lubricants such as mixtures thereof; and colorants. In the case of capsules, tablets and pills, the preventive and / or therapeutic agent for bone metabolic diseases accompanied by bone loss according to the present invention may contain a buffer. Similar types of solid compositions can also be used as fillers in soft and hard-filled gelatin capsules with excipients such as lactose or lactose and high molecular weight polyethylene glycols and the like.

  A tablet may also be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets can be binders (eg, gelatin or hydroxypropylmethylcellulose), lubricants, inert diluents, preservatives, disintegrants (eg, sodium glycolate starch or crosslinked sodium carboxymethylcellulose), surfactants Alternatively, it can be prepared using a dispersant. Molded tablets can be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

  Solid dosage forms such as tablets, such as dragees, capsules, pills and granules, can be optionally scored or prepared using coatings and shells such as enteric coatings well known in the pharmaceutical formulating art. Good. They are controlled or controlled release of the internal active ingredient using, for example, hydroxypropyl methylcellulose, other polymer matrices, liposomes and / or microspheres in various ratios to provide the desired release profile. It may be formulated to provide release. They may be sterilized, for example, by filtration through a bacteria retaining filter, or by incorporating a sterilant in the form of a sterile solid composition that can be dissolved in a sterile injectable medium such as sterile water immediately before use. . These compositions may optionally contain opacifiers, in compositions that release one or more active ingredients only in certain parts of the gastrointestinal tract or preferentially there, optionally in a delayed manner. There may be. Examples of embedding compositions that can be used are polymeric substances and waxes. The active ingredient may be in microencapsulated form, if appropriate, with one or more of the above-described excipients.

  Liquid dosage forms for oral administration of the preventive and / or therapeutic agent for bone metabolic diseases with bone loss according to the present invention include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. is there. Liquid dosage forms include, in addition to the active ingredient, inert diluents commonly used in the art, such as water and other solvents, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzoic acid Like fatty acids esters of benzyl, propylene glycol, 1,3-butadiene glycol, oils (especially cottonseed oil, peanut oil, corn oil, embryo oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofuryl alcohol, polyethylene glycol and sorbitan Solubilizers and emulsifiers, and mixtures thereof. In addition to inert diluents, the oral compositions may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, flavoring and preserving agents. Suspensions may be suspended in addition to the active compound, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar and tragacanth, and mixtures thereof. A turbidity agent may be included.

  The dosage form of the present invention for preventing and / or treating bone metabolic diseases with bone loss according to the present invention for rectal or vaginal administration may be presented as a suppository. This suppository comprises mixing one or more agents of the present invention with one or more suitable nonirritating excipients or carriers including, for example, cocoa butter, polyethylene glycol, suppository waxes or salicylates. Which is solid at room temperature but liquid at body temperature, it will melt in the rectum or vaginal cavity and release the active compound. Dosage forms suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray dosage forms containing carriers as known to be suitable in the art.

  Dosage forms for topical or transdermal administration of agents for the prevention and / or treatment of bone metabolic disorders with bone loss according to the present invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and Contains inhaled drugs. The active ingredient (plant extract) may be mixed under sterile conditions with a pharmaceutically acceptable base material and, if necessary, preservatives, buffers, or propellants. Ointments, pastes, creams and gels contain, in addition to active ingredients (plant extracts), animal or vegetable fats, oils, waxes, paraffins, starches, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acids, talc And may include excipients such as zinc oxide, or mixtures thereof.

  Powders and sprays may contain, in addition to the active ingredient (plant extract), excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicate and polyamide powder, or mixtures of these substances . The spray may further comprise customary high pressure gases such as chlorofluorinated hydrocarbons and volatile unsubstituted hydrocarbons such as butane and propane.

  The transdermal patch has the further advantage that the preventive and / or therapeutic agent for bone metabolic diseases associated with bone loss according to the present invention is controlled and delivered to the body. Such dosage forms can be made by dissolving or dispersing the cytostatic agent of the present invention in an appropriate medium. Absorption enhancers can also be used to increase the flux of substances containing preventive and / or therapeutic agents for bone metabolic disorders with bone loss according to the invention across the skin. The speed of such flux can be controlled by either providing a speed control membrane or by dispersing the compound in a polymer matrix or gel.

  The preventive and / or therapeutic agent for bone metabolic diseases accompanied by bone loss according to the present invention suitable for parenteral administration, together with the plant extract according to the present invention, one or more pharmaceutically acceptable sterile isotonic aqueous solutions or non- Includes aqueous solutions, dispersions, suspensions or emulsions, or sterile powders that can be reconstituted in sterile injectable solutions or dispersions just before use, for antioxidants, buffers, bacteriostats, preparations Solutes that are isotonic with the blood of the recipient, or suspensions or concentrates may be included.

  Examples of suitable aqueous and non-aqueous carriers that can be used in the preventive and / or therapeutic agent for bone metabolic diseases associated with bone loss according to the present invention include water, ethanol, polyol (eg, glycerol, propylene glycol, polyethylene glycol). Etc.), and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. The inherent fluidity can be maintained, for example, by the use of a coating material such as lecithin, by the maintenance of the required particle size in the case of dispersants and by the use of surfactants.

  The preventive and / or therapeutic agent for bone metabolic disease accompanied by bone loss according to the present invention may include adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the activity of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents such as parabens, chlorobutanol and phenol sorbate. It may be desirable to include isotonic agents such as sugars, sodium chloride in the composition. In addition, prolonged absorption of the injectable drug form can be brought about by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin.

  Another aspect of the present invention relates to a method for promoting osteogenesis in a patient, comprising administering the osteogenesis promoter according to the present invention to a patient in need thereof. Furthermore, still another aspect of the present invention involves bone loss including administration of a preventive and / or therapeutic agent for bone metabolic disease associated with bone loss according to the present invention to a patient in need thereof. The present invention relates to a method for preventing and / or treating bone metabolic diseases.

<Eating and drinking composition for promoting bone formation>
According to still another aspect of the present invention, an active ingredient contains an extract of one or more kinds of plants selected from the group consisting of sweet basil, holy basil, common time, Italian large leaf and fennel. A food and drink composition for promoting bone formation is provided.

  The form of the bone formation promoting food and drink composition is not particularly limited, and is a liquid, gel or solid food (eg juice, soft drink, tea, soup, soy milk, salad oil, dressing, yogurt, jelly, pudding, sprinkle , Infant formula, cake mix, powdered or liquid dairy products, bread, cookies, etc.) or pellets, tablets, excipients such as dextrin, lactose, starch, flavorings, pigments, etc. It may be processed into granules. In addition, the plant extract is coated with gelatin or the like and molded into a capsule, which can be used as a health food or a nutritional supplement. The amount of the plant extract contained in the bone formation promoting food and beverage composition is difficult to define uniformly depending on the type and state of the food or composition, but 0.01 to 90% by weight, more preferably 0.1 to 80% by weight. If it is such a compounding quantity, the effect by a plant extract can fully be exhibited, without impairing flavor. Moreover, food can be easily prepared.

  Here, the “food / beverage product composition” means a composition intended to be ingested by mammals (including pets) such as humans. For example, a pet food composition is a food or drink composition intended for consumption by pets. Food / beverage composition is widely known in the art. A pet food composition may or may not be nutritionally balanced, and in addition to supplements (eg, food), it is a nutritionally balanced composition suitable for a daily diet. May be. Here, “nutritionally balanced” means that the composition of the present invention has known nutrients necessary to maintain life in an appropriate amount and proportion in the pet nutrition field. To do.

  In a preferred embodiment of the present invention, the bone formation promoting food or drink composition is a human health food or pet food composition.

  Here, when the plant extract according to the present invention is added to and used in human health food (health food for humans) and pet food, the amount of plant extract added (content) is not particularly limited, The amount is preferably about 0.01 to 30% by weight, more preferably about 0.05 to 20% by weight, based on health food and pet food (in terms of solid content). Alternatively, the amount is such that the plant extract is administered at a total daily weight of preferably about 0.1 to 1000 mg / kg body weight, more preferably about 1 to 500 mg / kg body weight. If it is such an amount, the preventive effect of the bone metabolic disease accompanied by sufficient bone loss can be achieved. In addition, if the amount is as described above, the pet will not disturb the pet's taste and the pet will consume the entire amount of the given pet food.

  The pet food contains the same components as the conventional one except that it contains the plant extract according to the present invention. For example, in addition to the plant extract according to the present invention, a pet food is composed of monosaccharides, oligosaccharides, polysaccharides, dietary fibers, starches (for example, waxy corn starch, corn starch, wheat starch, rice starch, sticky rice starch, Potato starch, honeydew starch, tapioca starch, sago starch, chemically modified or chemically modified starch) and cereals (eg, corn, barley, wheat, rye, sorghum, rice, leeches, sweet potato, amaranthus) Carbohydrate sources such as cattle, pigs, sheep, rabbits, kangaroos, by-products and processed products, chickens, turkeys, poultry and other meats, by-products and household goods, fish , Fish meat such as white fish, its by-products and processed products, meat meal, meat bones, chicken meal, porter meal, Animal protein sources such as lettering of the above-mentioned raw materials such as schmir; vegetable protein sources such as soy protein, wheat protein, wheat gluten, corn gluten; α-sitosterol, β-sitosterol, stigmasterol, campesterol, Plant sterols such as α-sitostanol, β-sitostanol, stigmasteranol, campestanol, cycloartenol, and other free forms, and fatty acid esters, ferulic acid esters, cinnamic acid esters, and the like; rice bran, bran, etc. Examples include rice bran, soybeans such as soybean meal, vegetables such as vegetable extract, vitamins A, B1, B2, D, E, vitamins such as niacin, pantothenic acid, and carotene. In addition to the above, other additives such as a gelling agent, a shape-retaining agent, a pH adjuster, a seasoning, an antiseptic, and a nutrient reinforcing agent that are generally used in pet foods may be contained. In addition to or in place of the other additives, an antioxidant such as butylhydroxyanisole (BHA), dibutylhydroxytoluene (BHT), ethoxyquin, tert-butylhydroquinone (TBHQ), propyl gallate, etc. It can also be used in combination. The addition amount (content) of each component described above is not particularly limited, and the same amount as conventional can be applied.

  Pet food is manufactured by a conventionally well-known method. For example, it can be produced by mixing the plant extract according to the present invention and the necessary components described above into a desired form. For example, plant extract, cereal, meat meal, and mineral components such as iron and copper are mixed with citric acid or citrate if necessary. Extrude with a ruder. Thereafter, it is preferably dried with hot air until the water content becomes 10% or less, to produce a pet food. In addition, when pet food contains the fats and oils which have the fatty acid which has two or more double bonds, it is desirable to coat after drying with hot air.

The pet food may be in any form such as dry type, wet type, semi-moist type, jerky type, biscuits type, gum type, granular, powdery, soup, etc. It is preferable from the property. Examples of dry-type pet foods include kibble shapes, flat plate shapes, and bone shapes. From the standpoint of obtaining pets that are easy to chew and handle, the bulk density is 100 kg / m ³ or more, preferably 300 kg / m ³ or more, and 900 kg / m ³ or less, preferably 700 kg / m ³ or less. Or 100 to 900 kg / m ³ , particularly 300 to 700 kg / m ³ is preferable. In addition, the pet food can be provided in the form of bagging, boxing, packing, canning, and retort pouch.

  The effects of the present invention will be described using the following examples and comparative examples. However, the technical scope of the present invention is not limited only to the following examples. In the following examples, the operation was performed at room temperature (25 ° C.) unless otherwise specified.

[Preparation of various plant-derived extracts]
As plants according to Examples, sweet basil (1) (sample number 2), holy basil (sample number 3), common time (sample number 6), sweet basil (2) (sample number 8), Italian large leaf (sample) Number 9) and fennel (sample number 10) (both manufactured by BS East Japan Tech Co., Ltd.) were prepared. Similarly, as a plant according to a comparative example, estragon (sample number 1), baigapao (sample number 4), italian parsley (sample number 5), arugula (sample number 7), rosemary (sample number 11), selbatica (sample) No. 12) and Chervil (Sample No. 13) (both manufactured by BS East Japan Tech Co., Ltd.) were prepared.

  The various plants prepared above were thoroughly washed with water and dried, and then the whole plant was placed in an extraction container (bottle) and extracted at 20 ° C. in the dark using an extraction solvent. Here, as the extraction solvent, four kinds of solvents (the balance is water) having an alcohol concentration of 40% by volume, 60% by volume, 80% by volume or 100% by volume were prepared. And using the said solvent so that alcohol concentration might become high in order, the solvent was put to the extent that the plant which is an extraction raw material was immersed in a solvent, and it extracted for 3 to 4 days at a time. When the extraction raw material did not soak in the solvent, the same concentration of solvent was added. In addition, the extraction container was shaken well once or twice a day for the purpose of promoting the leaching of the active ingredient.

  After completing the extraction operation as described above, the supernatant was collected by filtration and dried to obtain a residue as a plant extract.

[Evaluation of extract activity]
(Cell culture)
Mouse skull-derived progenitor osteoblast MC3T3-E1 cells were obtained from RIKEN (Tsukuba City). MC3T3-E1 cells were cultured in MEM-α (above Invitrogen, MD, USA) medium containing 10% fetal bovine serum (FBS), 100 U / mL penicillin, and 100 μg / mL streptomycin at 37 ° C.-CO. _The cells were cultured in an incubator (air: CO ₂ = 95: 5 (volume%)).

(Alkaline phosphatase (ALP) staining)
100 μL of MC3T3-E1 cells (1 × 10 ⁵ cells / mL) were seeded in a 96-well multiplate and pre-cultured for 12 hours. Thereafter, each sample was added to a final concentration of 20 μg / mL and cultured for 72 hours. After 72 hours, the cells were washed twice with PBS, and the cells were fixed with 70 w / v% ethanol for 10 minutes. Thereafter, an ALP staining (TRACP & ALP double-stain Kit (MK300), Takara Bio) solution was added in an amount of 50 μL / well and stained at 37 ° C. for 1 hour. As a negative control, a system in which culture was performed without adding a sample was performed. As a positive control, BMP-2 (R & D, CA, USA), which is an osteoblast differentiation inducer, was added to a final concentration of 100 ng / mL and cultured.

  The results are shown in FIG. As shown in FIG. 1, sweet basil (sample numbers 2 and 8), holy basil (sample number 3), common time (sample number 6), Italian large leaf (sample number 9) and fennel (which are plants of the examples) It can be seen that in the cells to which the extract of sample number 10) was added, the ALP staining was significantly increased compared to the negative control. In contrast, such a significant increase in ALP staining was not observed in the cells to which the plant extract of the comparative example was added.

(Evaluation of ALP activity)
500 μL of MC3T3-E1 cells adjusted to 1 × 10 ⁵ cells / mL were seeded in a 24-well multiplate and pre-cultured in DMEM (Dulbecco's modified Eagle's medium) medium for 12 hours. Thereafter, the culture was performed using a differentiation induction medium (OSTM) supplemented with each sample of the example in which the ALP staining was confirmed as described above to a final concentration of 50 μg / mL. The differentiation induction medium (ODM) is 10 w / v% FBS, 10 mM β-glycerophosphate (Sigma, St. Louis, USA), 10 mM HEPES (pH 6.8), 0.28 mM (or 50 μg / mL). ) Α-MEM medium containing L-ascorbic acid and no phenol red was used. After culturing for 72 hours, the cells were washed twice with PBS (−), and 50 μL of cell lysate (10M Tris-HCl, 1 mM MgCl ₂ , 0.5% Triton X-100, pH 8.0) was added to each well. In addition, the cells were lysed. Each cell lysate was transferred to a 0.5 mL tube, subjected to ultrasonic disruption, and centrifuged at 6,200 × g to obtain a supernatant. The ALP activity in the supernatant was developed with LabAssay ^™ ALP kit (Fujifilm Wako Pure Chemical Industries, Ltd.), and the absorbance at a wavelength of 405 nm was measured using a spectrophotometer Varioskan LUX (Thermo Fisher Scientific, MA, USA). And from this measured value, ALP activity was calculated | required with the following formula.

Activity (units / μL) = [{p-nitrophenol concentration (nmol / μL) relative to (absorbance of treated sample−absorbance of blank))} / 15 (reaction time)] × 2 (dilution factor)
ALP activity (units / μg protein) = activity (units / μL) / protein concentration (μg / μL)
The protein concentration was measured with a Bio Rad DC protein assay kit (Bio Rad, Hercules, CA, USA). As a negative control, first, a system (negative control (1)) in which no sample was added and cultured in a DMEM medium instead of a differentiation induction medium was performed. As another negative control, a system (negative control (2)) cultured in ODM medium without adding a sample was also carried out.

  The measurement result of ALP activity is shown in FIG. Here, FIG. 2 (A) shows the measured value of ALP activity in the cells of the negative control (2) and the cells to which each sample was added, as a relative value when the measured value in the negative control (1) is 100%. It is the graph compared. Moreover, FIG. 2 (B) compared the measured value of ALP activity in the cells to which each sample was added as a relative value (increase rate of ALP activity) when the measured value in the negative control (2) was taken as 100%. It is a graph. As shown in FIG. 2, a very significant increase in ALP activity was observed in all of the samples of Examples.

(Alizarin Red S staining)
MC3T3-E1 cells adjusted to 1 × 10 ⁵ cells / mL were seeded in 500 μL each in a 24-well multiplate and pre-cultured in DMEM medium for 12 hours. Then, the culture | cultivation was performed using the differentiation induction culture medium (osteoblast differentiation medium (ODM)) which added each sample of the Example by which ALP dyeing | staining was confirmed above so that final concentration might be set to 40 micrograms / mL. After culturing for 28 days, the cells were washed twice with chilled PBS and then with an appropriate amount of ethanol. Then, the alizarin red S aqueous solution which can dye | stain calcification by couple | bonding with calcium was added, and the calcification site | part in a cultured cell was dye | stained. As a negative control, first, a system (negative control (1)) in which no sample was added and cultured in a DMEM medium instead of a differentiation-inducing medium was performed. As another negative control, a system (negative control (2)) cultured in ODM medium without adding a sample was also carried out. On the other hand, as a positive control, a system in which daidzein, which is known to have an osteogenesis promoting action, was added at the same concentration as each sample and cultured in an ODM medium was performed.

  The results are shown in FIG. As shown in FIG. 2, it was observed that the calcification of cultured cells was promoted in all of the samples of the Examples as compared with the negative control.

  From the above, it was shown that the plant extract according to the present invention exhibits an excellent osteogenesis promoting action through the promotion of osteoblast differentiation.

Claims (4)

A progenitor osteoblast differentiation inducer containing, as an active ingredient, one or more plant extracts selected from the group consisting of sweet basil, holy basil, common time, Italian large leaf and fennel. The progenitor osteoblast differentiation inducer according to claim 1 , wherein the extract comprises an extract of the leaves of the plant. A food and beverage composition for inducing differentiation of progenitor osteoblasts , comprising as an active ingredient an extract of one or more kinds of plants selected from the group consisting of sweet basil, holy basil, common time, Italian large leaf and fennel . The food / beverage product composition for progenitor osteoblast differentiation induction according to claim 3 , which is a human health food or pet food composition.