KR20190017704A - Composition for stimulation of bone formation comprising Curcuma xanthorrhiza extract or xanthorrhizol as effective component - Google Patents

Abstract

The present invention relates to a composition for promoting osteoanagenesis or osteogenesis containing an extract of Curcuma xanthorrhiza or xanthorrhizol separated from the same as an active component. The extract of Curcuma xanthorrhiza or xanthorrhizol can promote osteoanagenesis or osteogenesis by promoting differentiation of osteoblast or mineralization, and can be used as a medicine or food.

Description

Technical Field [0001] The present invention relates to a composition for promoting osteogenesis, which comprises as an active ingredient an extract of Javanese from Javanese or a nasal sol which is separated therefrom. [Technical Field] The present invention relates to a composition for promoting osteogenesis comprising Curcuma xanthorrhiza extract or xanthorrhizol as effective component,

The present invention relates to a composition for promoting bone regeneration or osteogenesis containing an extract of Java turmeric or a nansol sol isolated therefrom as an active ingredient.

Generally, osteoporosis occurring in bone is the most common skeletal disease among bone related diseases such as multiple myelosis and osteoarthritis, and is characterized by an increase in fracture and a decrease in bone strength. In the oral cavity, alveolar bone loss is caused by periodontal tissue inflammation caused by Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis , resulting in loss of teeth ( J. Immunol. Res. 2015: 1-10, 2015). For normal bone function, bone remodeling by bone homeostasis of bone formation by osteoclast and osteoblast is needed (for example, Rev. Endocrinol 8: 212227, 2011; J. Dent. Res. 91: 736-744, 2012).

However, excessive activity of osteoclasts and deactivation of osteoblasts cause imbalance in the remodeling process, leading to adult skeletal diseases such as osteoporosis. In order to alleviate this imbalance, methods for suppressing excessive activity of osteoclasts, promoting osteoclast activity, inhibiting osteoclast activity and promoting osteoclast activity are generally used, and they are used for treatment of osteoporosis (Nat. Rev. Rheumatol. 7: 631-638, 2011).

Osteoblasts are formed from mesenchymal stem cells. The mineralization, including calcium, formed by osteoblast differentiation not only maintains bone strength but also plays an important role in the homeostasis of calcium and hormone metabolism throughout the body. Calcium formation by osteoblast differentiation is regulated by vitamin D and parathyroid hormone, and it is known that bone morphogenetic protein (BMP), Wnt, calcineurin-calmurin kininase, (ALP) associated with the differentiation of osteoblasts by cross-talk of various signal transduction systems such as calmodulin kinase, nuclear factor kappa B (NF-κB) and activator protein The mineralization of bone hardness is synthesized by osteopontin (OPN), osteocalcin (OCN), type I collagen (COLA1) It is known that osteogenesis is induced by cell differentiation (Nat. Rev. Drug Discov. 11: 234-250, 2012).

A representative treatment for osteoporosis developed to date is bisphosphonates, which inhibits bone loss by weakening the function of osteoclasts (cells involved in bone resorption), and Merck's Fosamax, Procter Gamble And "Actonel" of the company. However, these treatments do not promote the formation of bones, and as a decomposition-inhibiting agent, it is necessary to substitute bone because it can induce additional fractures in continuous use, so it can not be a fundamental treatment. (Datamonitor Research Reports, 2007).

Java turmeric is a kind of Zingiberaceae, Curcuma xanthorrhiza Roxb. It is used as a spice in the tropical regions of Southeast Asia and has been widely used as a traditional medicine due to its strong antioxidant and anti-inflammatory action. Java turmeric is an antioxidant (J. Microbiol., 46: 228-232, 2008; Asian Pac. J. Trop. Biomed., 2: S637-S640, 2012), antioxidant (Evid. : 438356, 2012), anticancer (Phytother. Res. 22: 695-698, 2008), gastrointestinal protection (Biomed. Res. Int., 2014: 416409, 2014) ≪ / RTI > 2512-2517, 2010).

Xanthorrhizol isolated from Java turmeric is an antifungal agent (Fitoterapia, 71: 321-323, 2000; Planta Med., 66: 196-197, 2000), an antifungal (Phytother. Res., 21: 434-438, 2007 , Anti-inflammatory (J, Environ, Pathol, Toxicol, Oncol., 21: 141-148, 2002), anticancer (Biochem. Biophys. Res. Commun., 326: 210-217, 2005), anti-aging (Phytother. Res., 23: 1299-1302, 2009) and neuroprotection (J. Neurosci. Res., 82: 831-838, 2005).

However, there has been no report on the bone regeneration or the promotion of osteogenesis of Javanese extract or Jansori sol.

It is an object of the present invention to provide a technique capable of promoting osteogenesis and bone regeneration by inducing differentiation of osteoblasts without adverse effects.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

In order to achieve the above object, the present invention provides a pharmaceutical composition for promoting bone regeneration or bone formation comprising Curcuma xanthorrhiza extract or a fraction thereof as an active ingredient.

The present invention also provides a food composition for promoting bone regeneration or bone formation comprising Curcuma xanthorrhiza extract or a fraction thereof as an active ingredient.

In another aspect, the present invention provides a pharmaceutical composition for promoting bone regeneration or bone formation comprising xanthorrhizol represented by the following formula (1) or a salt thereof as an active ingredient:

[Chemical Formula 1]

In addition, the present invention provides a food composition for promoting bone regeneration or bone formation comprising xanthorrhizol or a salt thereof as an active ingredient.

The present invention relates to a composition for promoting osteogenesis comprising an extract of Java cornflakes or a jazzy sol as an active ingredient, and the Java corn oil extract or Janssorz extract is excellent in promoting osteoblast differentiation or promoting mineralization of osteoblasts Can promote bone regeneration or bone formation, and can be used for prevention or treatment of bone diseases.

-catenin 신호전달계 유전자(LRP5, DVL2, β-catenin, CCND1)의 mRNA 발현량 증가효과를 확인한 결과이다.
도 4는 MC3T3-E1 조골세포에서 자바강황 초임계 추출물에 따른 골혈성 촉진 효과를 확인하기 위한 ALP 활성측정 결과이다.
도 5는 MC3T3-E1 조골세포에서 잔소리졸 처리에 따른 조골세포 분화 유전자(ALP) 및 무기질화 관련 유전자(COLA1, OPN, OCN)의 mRNA 발현량 증가효과를 확인한 결과이다.
도 6은 MC3T3-E1 조골세포에서 잔소리졸 처리에 따른 골형성 단백질 신호전달계 유전자(BMP2, RUNX2, OSX)의 mRNA 발현량 증가효과를 확인한 결과이다.
도 7은 MC3T3-E1 조골세포에서 잔소리졸 처리에 따른 Wnt/β-catenin 신호전달계 유전자(LRP5, DVL2, β-catenin, CCND1)의 mRNA 발현량 증가효과를 확인한 결과이다.
도 8은 MC3T3-E1 조골세포에서 잔소리졸에 따른 골혈성 촉진 효과를 확인하기 위한 ALP 활성측정 결과이다.
도 9는 치주염 유발 렛드 동물모델에서 자바강황 초임계 추출물 처리에 따른 치조골 골밀도 증가효과를 확인한 결과이다.FIG. 1 shows the results of confirming the effect of increasing osteoblast differentiation gene (ALP) and mineralization-related genes (COLA1, OPN, OCN) on MC3T3-E1 osteoblast by treatment with Java corn supercritical extract.
FIG. 2 shows the effect of increasing the mRNA expression level of osteogenic protein signaling pathway gene (BMP2, RUNX2, OSX) according to treatment with Java turmeric supercritical extract in MC3T3-E1 osteoblast.
FIG. 3 is a graph showing the effect of Wnt /

-catenin signaling pathway gene (LRP5, DVL2, β-catenin, CCND1).
Fig. 4 shows the result of measurement of ALP activity for confirming osteoclast stimulating effect on MC3T3-E1 osteoblast according to the extract of Java turmeric supercritical fluid.
FIG. 5 shows the results of confirming the effect of mRNA expression of osteoblast differentiation gene (ALP) and mineralization-related genes (COLA1, OPN, OCN) in MC3T3-E1 osteoblast following treatment with Janssol sol.
FIG. 6 shows the effect of increasing mRNA expression levels of osteogenic protein signaling pathway gene (BMP2, RUNX2, OSX) upon treatment with Janssolide in MC3T3-E1 osteoblasts.
FIG. 7 shows the effect of increasing the mRNA expression level of the Wnt / β-catenin signaling pathway gene (LRP5, DVL2, β-catenin, CCND1) upon treatment with Janssolide in MC3T3-E1 osteoblasts.
FIG. 8 shows the results of measurement of ALP activity for confirming osteoclast stimulating effect on the MC3T3-E1 osteoblast following zanthoxylase.
FIG. 9 shows the results of confirming the effect of increasing the bone density of alveolar bone according to the treatment with the extract of Java turmeric supercritical fluid in the periodontal model.

Hereinafter, the present invention will be described in detail.

According to one aspect of the present invention, there is provided a composition for promoting bone regeneration or bone formation comprising Curcuma xanthorrhiza extract or a fraction thereof as an active ingredient.

The present invention also provides a pharmaceutical composition for promoting bone regeneration or bone formation comprising xanthorrhizol or a salt thereof as an active ingredient.

The present inventors have made efforts to develop a natural substance that promotes bone formation. As a result, the present inventors have found that the extract of Javanese Turmeric, or Jansori sol isolated therefrom, has the effect of promoting bone regeneration or osteogenesis by increasing the expression and activity of genes involved in bone formation Respectively.

The composition of the present invention contains Java vulcanized extract as an active ingredient.

"Java turmeric (Curcuma xanthorrhiza)" to be used in the compositions of the present invention is native to tropical regions belonging to keokyu equine (Curcuma spp.) Of the "large kyuma nagging chair (Curcuma xanthorrhiza), ginger and (Zingiberaceae). It is used as a spice in Southeast Asia and is known to have antioxidant activity.

As used herein, the term " extract " means that a substance is dissolved in a solvent to separate the active ingredient or the characteristic ingredient. Specifically, the extract includes extracts obtained by extracting plants with an extracting solvent, and fractions obtained by fractionating the extracts prepared by extracting with an extracting solvent. Therefore, the Curcuma xanthorrhiza extract of the present invention is meant to include all of fractions obtained by extracting the extract obtained by adding a solvent to Java turmeric and a fraction obtained by adding fraction solvent thereto.

The Java turmeric extract can be prepared by mixing Java turmeric with water, a C1 to C4 lower alcohol or a mixture thereof, acetone, ether, benzene, chloroform, ethyl acetate, methylene chloride, hexane, cyclohexane, petroleum ether, And may be one selected from the group consisting of Java Persimmon Supernatant Extract or Java Persimmon Supercritical Extract, but is not limited thereto.

Preferably, the extract is dried by adding 3 to 10 times of water, alcohol, or a mixture thereof to the dried Java charcoal, and then extracted with 2 to 20 times the weight of Java , But is not limited thereto. The extraction temperature is preferably 30 ° C to 100 ° C, more preferably 60 ° C to 100 ° C, but is not limited thereto. The extraction time is preferably 1 hour to 10 hours, more preferably 2 hours to 5 hours, but is not limited thereto. The extraction method may be a cold extraction, an ultrasonic extraction, or a reflux cooling extraction method, and it is preferable to use a reflux cooling extraction method, but not limited thereto. The number of times of extraction is preferably 1 to 5 times, more preferably 2 to 3 times, but is not limited thereto.

In one embodiment of the present invention, the extract is prepared by subjecting the extract of Java corn ethanol to a silica gel column chromatography with a concentration gradient of a mixed solvent of hexane and ethyl acetate of 100: 1 (v / v) to 1: 1 (v / v) But the present invention is not limited thereto.

The fractions can be prepared by fractionating the active substance using techniques such as solvent fractionation, silica gel chromatography, pre-HPLC, and the like.

The extract or fraction may be extracted or fractionated, filtered under reduced pressure, or concentrated and / or lyophilized to concentrate or remove the solvent.

The dose of the above-described Java turmeric extract is preferably 1 μg / ml to 100 μg / ml, but is not limited thereto. At this time, when the extract is less than the above amount, the activity of promoting osteoblast differentiation or promoting mineralization of osteoblasts is lowered and it is difficult to exert the effect of promoting bone formation. , There may be concern about toxicity including cytotoxicity.

In addition, the composition of the present invention contains xanthorrhizol as an active ingredient.

&Quot; Xanthorrhizol " is a compound extracted and purified or synthesized from natural products including plants, animals and microorganisms and represented by the following formula (I), IUPAC name is 5 - [(1R) -1,5- 4-hexen-1-yl] -2-methyl-phenol.

[Chemical Formula 1]

The zanthoxylol may be derived from Curcuma xanthorrhiza . At this time, the leaves, stalks, flowers, roots, husks, fruits, or a mixture thereof of the Java turmeric can be extracted and are preferably roots, but are not limited thereto.

In one specific embodiment, it exhibits excellent activity in the ALP activity of the extract of Java turmeric or the active ingredient of the present invention, zanthoxylate, mineralization activity of MC3T3-E1 osteoblasts, gene expression related to bone regeneration or osteogenesis Respectively.

In the present invention, " bone regeneration " includes promotion of osteogenesis by healing bone tissue lost due to promotion of osteoblast differentiation and activity. Herein, the promotion is not limited to this, but it can shorten the time for increasing the amount of bone tissue, osteocyte or bone matrix, or increasing the amount, and can be confirmed by the therapeutic effect of fracture and the like.

In this respect, bone regeneration or bone formation can be used interchangeably.

In the present invention, the term " bone " or " bone " includes the iliac bone (long bone), the staple bone (bone marrow), the flat bone (pelvic bone), and the alveolar bone.

In another embodiment of the present invention, the extract of the present invention was treated with the extract of the present invention in periodontitis-inducing group. As a result, the bone mineral density of the alveolar bone of the negative control group was lower than that of the normal group, but the bone mineral density of the alveolar bone of the group treated with the extract of the present invention Respectively. Accordingly, the composition of the present invention promotes bone regeneration or osteogenesis by promoting differentiation of osteoblasts, thereby preventing, treating, or ameliorating osteopathy caused by the inability of bone formation to occur smoothly.

Therefore, in this aspect, the composition of the present invention may be a pharmaceutical composition for promoting bone regeneration or bone formation.

As used herein, " prevention " refers to any action that inhibits or delays the onset of a disease or condition. In the present invention, it refers to inhibiting or delaying the symptoms of bone loss or osteoporosis through the action of promoting osteoblast differentiation.

In the present specification, the term " treatment " means any action that delays, halts, or reverses the progress of a disease or pathology. In the present invention, the term " treatment " means inducing regeneration or formation of bone or bone by promoting osteoblast differentiation it means.

In the present specification, 'improvement' means all actions that improve or improve disease or pathological conditions. In the present invention, it means improvement of bone loss or symptoms of osteoporosis by promoting osteoblast differentiation.

The composition of the present invention can be used for various diseases in which the risk of fracture is resolved due to low bone density and thus an increase in bone density is required.

Diseases that require bone regeneration and bone formation promotion in the present invention include, but are not limited to, osteoporosis, multiple myelopathy, osteoarthritis, osteoporotic fracture, diabetic fracture, nonunion fracture, bone defect, osteogenic insufficiency, osteomalacia, , Osteoarthritis, degenerative bone disease, and malocclusion, and it is difficult to restore natural bone due to such diseases, so that it can be used for the treatment or prevention of various diseases requiring surgical operations such as bone grafting.

More specifically, diseases for which the compositions of the present invention may be used include, but are not limited to, closed, open and non-fractured fractures; Prophylactic treatment of maximal bone density improvement with closed and open fracture reduction; Promoting bone healing in plastic surgery or plastic surgery; Maximum cemented orthopedic surgery and pre-menopausal women; Treatment of growth deficiency; Treatment of primary or secondary hyperparathyroidism; Treatment of osteolytic bone disease such as cancer; Increased bone formation during renal cartilage formation; Osteoporosis associated with aging, postmenopausal osteoporosis, glucocorticoid-induced osteoporosis, or occlusive osteoporosis.

The pharmaceutical composition of the present invention may include nasallyzol or a pharmaceutically acceptable salt thereof. The term " pharmaceutically acceptable " as used herein refers to those which are physiologically acceptable and do not normally cause an allergic reaction or a similar reaction when administered to humans, and the salts include pharmaceutically acceptable free acids acid is preferred.

The pharmaceutically acceptable salt may be an acid addition salt formed using an organic acid or an inorganic acid. The organic acid may be, for example, formic acid, acetic acid, propionic acid, lactic acid, butyric acid, isobutyric acid, trifluoroacetic acid, malic acid, But are not limited to, malonic acid, fumaric acid, succinic acid, succinic monoamide, glutamic acid, tartaric acid, oxalic acid, citric acid, glycolic acid, glucuronic acid, ascorbic acid, benzoic acid, phthalic acid, salicylic acid, anthranilic acid, dichloroacetic acid, aminooxyacetic acid, Toluenesulfonic acid or methanesulfonic acid. The inorganic acid includes, for example, hydrochloric acid, bromic acid, sulfuric acid, phosphoric acid, nitric acid, carbonic acid or boric acid. The acid addition salt may preferably be in the form of a hydrochloride or an acetate, more preferably in the form of a hydrochloride.

The above-mentioned acid addition salts may be prepared by a) directly mixing the azaSol sol and the acid, b) dissolving and mixing one of them in a solvent or a water solvent, or c) placing the yazosol sol in a solvent or an acid in an anhydrous solvent And mixing them.

Separately, additionally saltable forms include, but are not limited to, the salts of gabapentin, gabapentin, pregabalin, nicotinate, adipate, hemimarate, cysteine, acetylcysteine, methionine, arginine, lysine, Aspartate and the like.

The pharmaceutical composition for promoting bone regeneration or osteogenesis according to the present invention can be administered orally or rectally in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups and aerosols, external preparations, suppositories, And may contain suitable carriers, excipients or diluents conventionally used in the manufacture of pharmaceutical compositions for formulation.

The carrier or the excipient or diluent includes lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, Polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like.

In the case of formulation, a diluent or excipient such as a commonly used filler, a weight agent, a binder, a wetting agent, a disintegrant or a surfactant may be used.

Solid preparations for oral administration can be prepared by mixing at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin and the like in the above extract. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used.

Examples of the liquid preparation for oral administration include suspensions, solutions, emulsions, syrups and the like. In addition to water and liquid paraffin which are commonly used diluents, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included .

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous agents, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As a base for suppositories, witepsol, macrogol, tween 61, cacao paper, laurin, glycerol gelatin and the like can be used.

The preferable dosage of the pharmaceutical composition for promoting bone regeneration or osteogenesis according to the present invention may be appropriately selected by a person skilled in the art depending on the condition of the patient, body weight, degree of disease, drug form, administration route and period. However, for a desired effect, the dose may be 0.0001 to 2,000 mg / kg, preferably 0.001 to 2,000 mg / kg per day. The administration may be carried out once a day or divided into several doses. However, the scope of the present invention is not limited by the dosage.

The pharmaceutical composition for promoting bone regeneration or osteogenesis according to the present invention can be administered to mammals such as rats, mice, livestock, and humans in various routes. All modes of administration can be administered, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

The pharmaceutical composition for promoting bone regeneration or bone formation according to the present invention may be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy or biological response modifiers.

In another aspect, the pharmaceutical composition of the present invention may be provided as a formulation of a topical agent containing Java panacea extract or jazzarizol as an active ingredient.

When the pharmaceutical composition for promoting bone regeneration or bone formation according to the present invention is used as an external preparation for skin, it may further contain a fatty substance, an organic solvent, a solubilizing agent, a thickening agent and a gelling agent, a softening agent, an antioxidant, a suspending agent, a stabilizing agent, a surfactant, a water, an ionic emulsifier, a nonionic emulsifier, a filler, a sequestering agent, a chelating agent, a preservative, a vitamin, a blocking agent, a wetting agent, an essential oil, a dye, a pigment, a hydrophilic active agent, An adjuvant commonly used in the field of dermatology, such as an active agent or any other ingredient commonly used in skin external preparations such as lipid vesicles. The components can also be introduced in amounts commonly used in the field of dermatology.

When the pharmaceutical composition for promoting bone regeneration or bone formation of the present invention is provided as an external preparation for skin, it may be a formulation such as an ointment, a patch, a gel, a cream or a spray.

In another aspect, the composition of the present invention can be prepared from a food composition for promoting bone regeneration or bone formation.

The food composition of the present invention includes all forms of functional foods, nutritional supplements, health foods, food additives and feeds, It is targeted for eating.

Food compositions of this type may be prepared in a variety of forms according to conventional methods known in the art. There is no particular limitation on the type of the food, and examples of the food to which the above substance can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, , Various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and may include foods in a conventional sense.

Generally, the above Java potash extract or fraction may be added in an amount of not more than 15 parts by weight, preferably not more than 10 parts by weight, based on 100 parts by weight of the raw material. However, in the case of long-term consumption intended for health and hygiene purposes or for health control purposes, the amount may be less than the above range. Further, since the present invention uses fractions from natural products, there is no problem in terms of safety, Or more.

The beverage in the food according to the present invention may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. The above-mentioned natural carbohydrates may be monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like. The ratio of the natural carbohydrate may be about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 mL of the beverage according to the present invention.

In addition to the above, the food for improving bone diseases according to the present invention may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickening agents, pH adjusting agents, A preservative, a glycerin, an alcohol, and a carbonating agent used in a carbonated drink. In addition, the composition for improving sleep of the present invention may contain flesh for the production of natural fruit juice, fruit juice drink and vegetable drink. These components may be used independently or in combination. The ratio of such additives is not limited, but is generally selected in the range of 0.01 to 0.1 parts by weight based on 100 parts by weight of the food of the present invention.

The food composition of the present invention may be added to a feed additive or feed composition for the purpose of promoting bone regeneration or promoting bone formation.

The term " feed additive " in the present invention means a substance added to feed for various purposes such as nutrient supplementation and weight loss prevention, promotion of digestive utilization of fibrin in feed, improvement of oil quality, prevention of reproductive disorder, improvement of conception rate, . The feed additive of the present invention corresponds to an auxiliary feed in the feed control method and is a mineral preparation such as sodium hydrogencarbonate, bentonite, magnesium oxide and complex mineral, a mineral preparation such as zinc, copper, cobalt and selenium, , Vitamins such as vitamin AD, E, nicotinic acid and vitamin B complex, protective amino acids such as methionine and lysine, protective fatty acids such as calcium salt of fatty acid, live bacteria such as probiotics (lactic acid bacteria), yeast cultures, A yeast agent, and the like may be further included.

The term " feed " in the present invention refers to any composition for promoting osteogenesis according to the present invention, which is any natural or artificial diet, single meal or the like component for feeding, ingesting, digesting, As an active ingredient can be produced in various types of feeds known in the art, preferably, but not exclusively, concentrated feed, forage and / or special feed.

Concentrated feeds include seeds containing cereals such as wheat, oats, and corn, and by-products such as rice bran, wheat bran, barley, etc., Which is the by-product of the starch residue, which is the main component of the starch residue, which is the remaining product of starch minerals, sweet potatoes, potatoes, etc., which is a byproduct obtained by concentrating the fresh liquid product obtained from fish meal, fish meal, fish residue, The animal feed such as fish soluble, meat powder, blood meal, wheat meal, skim milk powder, cheese in milk, dried whey in the case of casein production in skim milk, dry yeast, yeast, Chlorella, algae, but not limited thereto.

Roughage includes root vegetables such as wild grasses, grasses and fodder, raw turnip for feed, turnip for feed, feed bit, turnip root, Silage, which is filled with lactic acid and fermented by lactic acid, hay, dried hay, herbage straw, straw of leguminous crops, leaves of leguminous plants, and the like. Special feeds include mineral feeds such as oyster shells and rock salt, urea feeds such as urea and its derivatives diuret isobutane, supplemented with ingredients that are insufficient when mixed with natural feed ingredients, or mixed feeds to improve the storage stability of the feed. Feed additives, dietary supplements, which are substances added in small quantities, but are not limited thereto.

The feed additive for promoting osteogenesis according to the present invention can be prepared by adding Janssorzol or Java Turmeric Extract in an appropriate effective concentration range according to various feed production methods known in the art.

The feed additive according to the present invention can be applied to any object for promoting bone formation without limitation. For example, it can be applied to any non-human animal such as a monkey, a dog, a cat, a rabbit, a guinea pig, a rat, a mouse, a cattle, a sheep, a pig,

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

[Example]

Example 1. Preparation of Java turmeric extract

1-1. Manufacture of Java Turmeric Methanol Extract

The dried Java crumbs were pulverized with a mixer, and then 100 g of the crushed Java turbidity sample was added to 1 L of 100% methanol and extracted for 3 hours at room temperature for 24 hours. The extracted sample was filtered under reduced pressure with Whatman No. 2 filter paper, and the filtered extract was concentrated with a vacuum rotary condenser to remove the solvent components, and then a methanol extract of Java was obtained.

1-2. Preparation of Java Turmeric Ethanol Extract

The procedure of Example 1-1 was repeated except that 100% ethanol was used to obtain a Java ethanol extract of turmeric.

1-3. Preparation of Java Turmeric Ethyl Acetate Extract

The procedure of Example 1-1 was repeated except that 100% ethyl acetate was used to obtain an extract of Java turmeric ethyl acetate.

1-4. Preparation of Java Turmeric Hexane Extract

The procedure of Example 1-1 was repeated except that 100% hexane was used to obtain the extract of Java turmeric hexane.

1-5. Preparation of hot water extract of Java

The dried Java crumbs were pulverized with a mixer, and 100 g of the crushed Java turbidity sample was added to 1 L of water and extracted with stirring at 80 ° C for 2 hours. The extracted sample was filtered under vacuum with Whatman No. 2 filter paper, and the filtered extract was concentrated with a vacuum rotary condenser to remove solvent components, and then a Java hot water extract was obtained.

1-6. Manufacture of Ultra High Pressure Extracts from Java

The dried Java crushed sulfur was pulverized with a mixer and 1 g of crushed Java turcyan and 76 mL of 18% ethanol were placed in a polyethylene pack, sealed and then extracted with an ultra high pressure extraction apparatus (Frescal MFP-7000; Mitsubishi Heavy Industries) Respectively. Extraction pressure was 320 MPa and extraction time was 5 min. The extracted sample was filtered with Whatman No. 2 filter paper, and the filtered extract was concentrated with a vacuum rotary condenser to remove the solvent components, thereby obtaining a Java vulcanization ultra-high pressure extract.

1-7. Manufacture of Supercritical Extracts of Java Turmeric

The dried Java crushed sulfur was pulverized with a mixer, and 1 g of the ground Java crushed sulfur sample was charged into a sample cartridge and extracted with a supercritical fluid extraction apparatus (SCFE-P400, Ilshin autoclave Co., Ltd., Korea). Supercritical fluid extraction conditions were extraction pressure 40 MPa, extraction temperature 50 ℃ and extraction time 240 min. Upon completion of the supercritical fluid extraction, the supercritical carbon dioxide fluid extract of Java turmeric supercritical carbon dioxide was obtained by releasing the supercritical fluid state by lowering the pressure of the extraction apparatus.

1-8, Manufacture of Java Turmeric Subcritical Fluid Extract

The dried Java crushed sulfur was pulverized with a mixer, and then 50 g of the ground Java coal tar was put in a subcritical extraction device (Biovan, Gyeonggi, Korea) with 1 L water and sealed. After sealing, the temperature of the reactor was raised to 200 DEG C, and when the temperature of the reactor reached 200 DEG C, the temperature was maintained for 20 minutes to extract. Then, the extract was transferred to a storage tank to which cooling water was supplied, cooled rapidly to 30 ° C, and centrifuged at 3,600 rpm for 30 minutes to separate the floating residue. The solvent was completely removed by using a freeze dryer (ilShin Lab Co. Ltd., Seoul, Korea) to obtain a subtropical extract of Javanese Persimmon.

Example 2. Separation of Nazorizole

Using a solvent system in which the concentrated Java CaO ethanol extract (11.1 g) obtained in Example 1-2 was loaded on a column packed with silica gel and hexane and ethyl acetate were mixed at a ratio of 10: 1 (v / v) Lt; / RTI > The fractions were divided into 6 fractions according to the sorting procedure, and the fractions were concentrated and dried. Five fractions (fraction 5) of the six fractions were fractionated using 80% methanol eluting with Rp-18 reverse phase column chromatography (Lichroprep Rp-18 25-40 um, Merck & Co., USA). The fractions were divided into three fractions according to the above sorting order and concentrated and dried. The third fraction (fraction 3-3) of the three fractions was concentrated to dryness to isolate the pure single active substance zanthoxylate (0.52 g).

[Experimental Example]

Experimental Example 1. Confirmation of osteogenesis promoting effect of Java turmeric extract

1-1. Confirmation of the effect of increasing the amount of expression promoting gene of osteogenesis by the extract of Java turmeric (Fig. 1)

-glycerol phosphate disodium salt hydrate (Sigma-adrich)이든 배양액에 상기 실시예 1-7에서 제조한 자바강황 초임계 이산화탄소 유체 추출물을 1, 10 ㎍/㎖ 농도로 조골세포에 처리하여 3일 동안 조골세포 분화를 유도하였다. 분화한 세포로부터 TRIzol시약(Invitrogen, Carlsbad, CA, USA)을 사용하여 총 RNA를 분리하였다. 분리한 총 RNA는 나노드랍(NanoDrop 1000; Thermo Fisher Scientific Inc., MA, USA)을 이용하여 정량하였다. 정량된 16 μL의 RNA를 Reverse Transcriptase Premix (ELPIS-Biotech, Daejeon, Korea)와 PCR 기계(Gene Amp PCR System 2700; Applied Biosystems, MA, USA)를 이용하여 42℃ 55분, 70℃ 15분의 조건에서 cDNA로 합성하였다. 16 μL의 생성된 cDNA 중 4 μL의 cDNA, 하기 [표 1]의 특정 프라이머(Bioneer, Daejeon, Korea)를 이용하여 PCR premix(ELPIS-Biotech)로 95℃에서 30초, 60℃에서 1분, 72℃에서 1분을 30번 반복하여 PCR을 수행하였다. PCR 결과 증폭된 cDNA를 1.5% agarose gel로 전기영동하여 분리하였으며, G;BOX EF imaging system (Syngene, Cambridge, UK)을 이용하여 결과를 확인하였다.MC3T3-E1 osteoblast (American Type Culture Collection, Manassas, Va., USA) was inoculated with alpha-modified Eagle's Medium (α-MEM) containing 10% fetal bovine serum (FBS; Hyclone Laboratories, Inc., (Gibco, Grand Island, NY, USA) and placed in a 6-well microtiter plate at 1 × 10 ⁵ cells / well. L-ascorbic acid (Sigma-adrich, St. Louis, Mo., USA) was added to the osteoblast at a concentration of 100%

-glycerol phosphate disodium salt hydrate (Sigma-adrich) was added to the osteoblast cells at a concentration of 1, 10 / / ㎖, and the osteoblast cells were cultured for 3 days. Lt; / RTI > Total RNA was isolated from differentiated cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The isolated total RNA was quantitated using NanoDrop 1000 (Thermo Fisher Scientific Inc., MA, USA). The quantified 16 μL of RNA was amplified by PCR using a Reverse Transcriptase Premix (ELPIS-Biotech, Daejeon, Korea) and a PCR machine (Gene Amp PCR System 2700; Applied Biosystems, MA, USA) at 42 ° C for 55 minutes and 70 ° C for 15 minutes . 4 μL of cDNA of 16 μL of the resulting cDNA was amplified with PCR primer (ELPIS-Biotech) at 95 ° C. for 30 seconds, at 60 ° C. for 1 minute, using specific primer (Bioneer, Daejeon, Korea) PCR was performed by repeating the PCR reaction at 72 ° C for 1 minute 30 times. The amplified cDNA was separated by electrophoresis on 1.5% agarose gel, and the results were confirmed using G; BOX EF imaging system (Syngene, Cambridge, UK).

gene Type of primer The base sequence (5 '-> 3') SEQ ID NO: ALP Forward primer 5'-TGACAATCACATCTGAAGGCTCTC-3 ' One Reverse primer 5'-GGAACATGATGACATTCTTGGCTAC-3 ' 2 COLA1 Forward primer 5'-CTAGGGTCTAGACATGTTCAGCTTT-3 ' 3 Reverse primer 5'-GACTCCTACATCTTCTGAGTTTGGT-3 ' 4 OPN Forward primer 5'-GACCACATGGACGACGATG-3 ' 5 Reverse primer 5'-TGGAACTTGCTTGACTATCGA-3 ' 6 OCN Forward primer 5'-GAACAGACAAGTCCCACACAGC-3 ' 7 Reverse primer 5'-GTCAGCAGAGTGAGCAGAAAGAT-3 ' 8 β-Actin Forward primer 5'-GAAGGAGATTACTGCTCTGGCTC-3 ' 9 Reverse primer 5'-CTCAGTAACAGTCCGCCTAGAA-3 ' 10

As a result, as shown in Fig. 1, the expression of the osteoblast differentiation gene (ALP), which is known to be expressed at the early stage of differentiation, was increased by the treatment of the extract, and the expression of the mRNA of the mineralization-related genes (COLA1, OPN, OCN) And the expression level was also increased. This means that the Java turmeric supercritical carbon dioxide fluid extract of the present invention is excellent in the ability to promote osteoblast differentiation and mineralization.

1-2. Confirmation of activation of osteogenic protein signal transduction system by Java turmeric extract (Fig. 2)

Except that the primer (Bioneer, Daejeon, Korea) of the following [Table 2] was used.

gene Type of primer The nucleotide sequence (5 '- > 3) SEQ ID NO: BMP2 Forward primer 5'-CCAAGACACAGTTCCCTACA-3 ' 11 Reverse primer 5'-CACGGCTTCTAGTTGATGGA-3 ' 12 RUNX2 Forward primer 5'-ACAAACAACCACAGAACCACAAG-3 ' 13 Reverse primer 5'-AGTTGTACAGTATGCCTGGAGT-3 ' 14 OSX Forward primer 5'-CTTCCCAATCCTATTTGCCGTTT-3 ' 15 Reverse primer 5'-CGGCCAGGTTACTAACACCAATCT-3 ' 16 β-Actin Forward primer 5'-GAAGGAGATTACTGCTCTGGCTC-3 ' 9 Reverse primer 5'-CTCAGTAACAGTCCGCCTAGAA-3 ' 10

As a result, as shown in Fig. 2, it was confirmed that the extract of Java corpuscle supercritical carbon dioxide increased mRNA expression levels of BMP2, RUNX2 and OSX genes related to osteogenic protein signal transduction system. This means that the Java turmeric supercritical carbon dioxide fluid extract of the present invention is excellent in the ability to activate the bone morphogenetic protein signaling system.

1-3. Confirmation of Wnt / β-catenin signal transduction activation by Java turmeric extract (FIG. 3)

Except that the primer (Bioneer, Daejeon, Korea) of the following [Table 3] was used.

gene Type of primer The base sequence (5 '-> 3') SEQ ID NO: LRP5 Forward primer 5'-CTTCATGGATGGGACCAATTGTATG-3 ' 17 Reverse primer 5'-TAATCGCTATATTGAGTCAGGCCAA-3 ' 18 DVL2 Forward primer 5'-GATGTTATGTAGGGACCCATCTTGA-3 ' 19 Reverse primer 5'-ATAGAGTAACAAAGGCAGCTACACA-3 ' 20 β-Catenin Forward primer 5'-AGTGTTATGTTCTAGTGAACCTGCT-3 ' 21 Reverse primer 5'-CTATAACCGCATCTGTTGAAGCATT-3 ' 22 CCND1 Forward primer 5'-TTGTGCATCTACACTGACAACTCTA-3 ' 23 Reverse primer 5'-CAGTCCCGGGTCACACTTGAT-3 ' 24 β-Actin Forward primer 5'-GAAGGAGATTACTGCTCTGGCTC-3 ' 9 Reverse primer 5'-CTCAGTAACAGTCCGCCTAGAA-3 ' 10

As a result, it was confirmed that the Java turmeric supercritical carbon dioxide fluid extract increased the amount of mRNA expression of LRP5, DVL2, β-catenin, and CCND1, which are major genes of the Wnt / β-catenin signal transduction system there was. This means that the extract of the Java turmeric supercritical carbon dioxide fluid extract of the present invention has an excellent ability to activate the Wnt /? - catenin signal transduction system.

1-4. Confirmation of ALP activity increasing effect by Java turmeric extract (FIG. 4 )

MC3T3-E1 osteoblast differentiated in the same manner as in Example 3 was dissolved in 0.2% Triton X-100. The supernatant was centrifuged at 2,500 g for 15 minutes at 4 ° C to obtain a Bio-Rad protein Proteins in the supernatant were quantified by Bradford method using an assay kit (Bio-Rad Laboratories, Hercules, Calif., USA). ALP activity was measured using a SensoLyte p-nitrophenyl phosphate (p-NPP) ALP activity assay kit (AnaSpec, Inc., Fremont, CA, USA).

As a result, as shown in Fig. 4, the extract of Java turmeric supercritical carbon dioxide increased ALP activity in a concentration-dependent manner. This means that the Java turmeric supercritical carbon dioxide fluid extract of the present invention has an excellent ability to promote osteoblast differentiation by increasing ALP activity as an early differentiation marker of osteoblast.

Experimental Example 2. Confirmation of the effect of accelerating the osteogenesis of Jansori sol

2-1. Confirming the effect of increasing the amount of expression of the osseigenesis promoting gene by Jansorizol (Fig. 5)

The same procedure as in Experimental Example 1-1 was carried out except that the Nansorizol isolated and purified in Example 2 was treated at a concentration of 1 and 10 μM.

As a result, as shown in Fig. 5, it was confirmed that Jansorizol increases mRNA expression level of osteoblast differentiation gene (ALP) and mineralization-related genes (COLA1, OPN, OCN). This means that the zansori sol of the present invention is excellent in the ability to promote the differentiation and mineralization of osteoblasts.

2-2. Confirmation of activation of osteogenic protein signal transduction system by zanthoxylose (Fig. 6)

The same procedure as in Experimental Example 1-2 was carried out except that the zanthoxyl sols isolated and purified in Example 2 were treated at a concentration of 1 and 10 μM.

As a result, as shown in Fig. 6, it was confirmed that Janssorzol increases the mRNA expression level of BMP2 and RUNX2 OSX genes related to osteogenic protein signal transduction system. This means that the zansori sol of the present invention is excellent in the ability to activate the osteogenic protein signal transduction system.

2-3. Activation of the Wnt / β-catenin signal transduction system by xanthorrhizol (FIG. 7)

The same procedure as in Experimental Example 1-3 was carried out except that the Nansorizol isolated and purified in Example 2 was treated at a concentration of 1 and 10 μM.

As a result, as shown in Fig. 7, it was confirmed that Jansorizol increased mRNA expression of LRP5, DVL2, β-catenin and CCND1, which are major genes of the Wnt / β-catenin signal transduction system. This means that the extract of Jansorizole of the present invention has an excellent ability to activate the Wnt /? - catenin signal transduction system.

2-4. Confirming the effect of increasing the ALP activity by zanthoxylase (Fig. 8)

The same procedure as in Experimental Example 1-4 was carried out except that the Nansorizol isolated and purified in Example 2 was treated at a concentration of 1 and 10 μM.

As a result, as shown in Fig. 8, the Nansorizol has ALP activity in a concentration-dependent manner. This means that the zansori sol of the present invention has an excellent ability to promote osteoblast differentiation by increasing the ALP activity as an early differentiation marker of osteoblasts.

Experimental Example 3. Confirmation of alveolar bone formation promoting effect of Java turmeric extract on experimental animals

3-1. Experimental animal breeding and Java turmeric extract treatment

Male Sprague Dawley rats (Orient Bio, Seongnam, Korea), 9 weeks of age, were purchased as experimental animals. All animals were maintained at Chung Ang University (Chung-Ang University, Seoul, Korea), and the breeding environment was maintained at a temperature of 23 ± 2 ° C and a relative humidity of 55 ± 10%. Before starting the experiment, 32 rats were randomly divided into 8 normal rats, periodontitis induction group, Java turmeric 30 group, and Java turmeric 100 group. The animals were anesthetized with ether and 10 ㎎ / ㎖ lipopolisaccharide (Sigma-aldrich) was injected bilaterally between the mandibular first and second molars A total of 10 injections were made at intervals of 2 days to induce periodontitis. After the 6th injection, the extract of Jakuzan supercritical carbon dioxide fluid extract prepared in Example 1-7 at 30 mg / kg / day and the extract of Jakuzan supercritical carbon dioxide fluid at 100 mg / / Kg / day. At this time, the normal group and periodontitis induction group were orally administered with corn oil instead of Java corn supercritical carbon dioxide fluid extract. The animals were sacrificed by collecting blood after anesthesia with respiration using ether. The alveolar bone was separated from the extracted mandibular gingiva, confirming that the heart beat was stopped.

3-2. Increase in Bone Mineral Density by Treatment of Java Cranberry Extract (FIG. 9)

The bone mineral density in the alveolar bone isolated in Experimental Example 3-1 was measured using Micro-CT (Skyscan 1076, Skyscan, Kontich, Belgium). The measurement conditions were Total rotation 360 °, Rotation step 0.5 °, Pixel size 18um, Voltage 100kV, Current 100uA and Exposure time 1475 ms. Bone mineral density (BMD) was analyzed based on the images taken.

As a result, as shown in Fig. 9, the bone mineral density of the alveolar bone of the experimental animals in the periodontitis induction group was lower than that of the normal group, but it was confirmed that the administration of the Java corn oil supercritical carbon dioxide fluid extract was increased. This means that the Java turmeric supercritical carbon dioxide fluid extract of the present invention is excellent in bone formation promoting ability.

Hereinafter, examples of the production of medicines and foods containing the extract of Javanese Turmeric or Jansori sol as the active ingredient in Examples 1 and 2 according to the present invention will be described, but the present invention is not intended to be limited thereto but is specifically described below to be. Based on the following composition components and composition ratios, the medicines or foods of Preparation Examples 1 to 2 were prepared according to a conventional method, with the extracts of Javanese Calcium Sulfate having excellent bone formation promoting effect or the Nansorizol.

[Production Example 1]

1-1. Powder

50 mg of Javanese Turmeric extract or 2 g of crystalline cellulose were mixed in the above Examples 1 and 2 and filled in an airtight container according to a conventional acid preparation method to prepare a powder.

1-2. refine

The tablets were prepared by mixing 50 mg of Javanese Talc extract or 400 mg of crystalline cellulose and 5 mg of magnesium stearate in the above Examples 1 and 2 or tablets according to a conventional tablet preparation method.

1-3. Capsule

After mixing 30 mg of Javanese Turmeric extract or 100 mg of whey protein, 400 mg of crystalline cellulose and 6 mg of magnesium stearate in the above Examples 1 and 2, the mixture was filled in a gelatin capsule according to a conventional method for preparing a capsule, .

1-4. Injection

The active ingredient was dissolved in distilled water for injection and the pH was adjusted to about 7.5. Then, 100 mg of Zazorizol in Example 2, distilled water for injection, and pH adjusting agent were mixed and filled into a 2 mL volume ampoule And sterilized to prepare an injection.

[Production Example 2]

2-1. Manufacture of health food

The extracts of Examples 1 and 2 or Jansori sol 1000 mg, vitamin A acetate 70 ug, vitamin E 1.0 mg, vitamin B1 0.13 mg, vitamin B2 0.15 mg, vitamin B6 0.5 mg, vitamin B12 0.2 ug, vitamin C 10 10 mg of biotin, 1.7 mg of nicotinic acid amide, 50 mg of folic acid, 0.5 mg of calcium pantothenate, 1.75 mg of ferrous sulfate, 0.82 mg of zinc oxide, 25.3 mg of magnesium carbonate, 15 mg of potassium phosphate monobasic, 55 mg of calcium dihydrogenphosphate , Potassium citrate (90 mg), calcium carbonate (100 mg) and magnesium chloride (24.8 mg). The compounding ratio may be arbitrarily changed. The above ingredients are mixed according to a conventional method for preparing healthy foods, Granules can be prepared and used in the manufacture of a health food composition according to conventional methods.

2-2. Manufacture of health drinks

Purified water was added to 1000 mg of Javanese Turmeric extract or 1000 mg of Jansori sol, 100 g of citric acid, 100 g of oligosaccharide, 2 g of plum concentrate and 1 g of taurine in the above Examples 1 and 2 to prepare the above components And the mixture is stirred and heated at 85 for about 1 hour. The resulting solution is then filtered and sterilized in a sterilized 2 L container. The resulting solution can be stored in a refrigerator for use in the manufacture of a health beverage composition.

2-3. Chewing gum

Chewing gum was prepared in a conventional manner by mixing 20% by weight of a gum base, 76.9% by weight of sugar, 1% by weight of a flavor and 2% by weight of water and 0.1% by weight of a Java turmeric extract or 0.1%

2-4. candy

A candy was prepared by blending 60% by weight of sugar, 39.8% by weight of starch syrup, 0.1% by weight of fragrance and 0.1% by weight of Javanese Turmeric extract or Jansori sol of Examples 1 and 2.

2-5. Biscuit

A mixture of 0.75% by weight of sodium chloride, 0.78% by weight of glucose, 11.78% by weight of palm shortening, 1.54% by weight of ammonium, 0.17% by weight of sodium bicarbonate and 0.16% by weight of sodium bisulfite, 25.59% by weight of Grade I, 22.22% 1.45 wt% of rice flour, 0.0001 wt% of vitamin B, 0.04 wt% of milk fractions, 20.6998 wt% of water, 1.16 wt% of whole milk powder, 0.29 wt% of replacement milk powder, 0.03 wt% of calcium phosphate, 0.29 wt% 7.27% by weight of milk and the Java turmeric extract or the nasal decaned sol of% by weight of the above Examples 1 and 2 were mixed to prepare biscuits by a conventional method.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

<110> Industry-Academic Cooperation Foundation, Yonsei University <120> Composition for stimulation of bone formation          xanthorrhiza extract or xanthorrhizol as effective component <130> 1064441 <150> KR 10-2017-0101489 <151> 2017-08-10 <160> 24 <170> Kopatentin 2.0 <210> 1 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for ALP <400> 1 tgacaatcac atctgaaggc tctc 24 <210> 2 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for ALP <400> 2 ggaacatgat gacattcttg gctac 25 <210> 3 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for COLA1 <400> 3 ctagggtcta gacatgttca gcttt 25 <210> 4 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for COLA1 <400> 4 gactcctaca tcttctgagt ttggt 25 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for OPN <400> 5 gaccacatgg acgacgatg 19 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for OPN <400> 6 tggaacttgc ttgactatcg a 21 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for OCN <400> 7 gaacagacaa gtcccacaca gc 22 <210> 8 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for OCN <400> 8 gtcagcagag tgagcagaaa gat 23 <210> 9 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for bata-Actin <400> 9 gaaggagatt actgctctgg ctc 23 <210> 10 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for beta-Actin <400> 10 ctcagtaaca gtccgcctag aa 22 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for BMP2 <400> 11 ccaagacaca gttccctaca 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for BMP2 <400> 12 cacggcttct agttgatgga 20 <210> 13 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Runx2 <400> 13 acaaacaacc acagaaccac aag 23 <210> 14 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Runx2 <400> 14 agttgtacag tatgcctgga gt 22 <210> 15 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for OSX <400> 15 cttcccaatc ctatttgccg ttt 23 <210> 16 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for OSX <400> 16 cggccaggtt actaacacca atct 24 <210> 17 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for LRP5 <400> 17 cttcatggat gggaccaatt gtatg 25 <210> 18 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for LRP5 <400> 18 taatcgctat attgagtcag gccaa 25 <210> 19 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for DVL2 <400> 19 gatgttatgt agggacccat cttga 25 <210> 20 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for DVL2 <400> 20 atagagtaac aaaggcagct acaca 25 <210> 21 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for beta-Catenin <400> 21 agtgttatgt tctagtgaac ctgct 25 <210> 22 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for beta-Catenin <400> 22 ctataaccgc atctgttgaa gcatt 25 <210> 23 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for CCND1 <400> 23 ttgtgcatct acactgacaa ctcta 25 <210> 24 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for CCND1 <400> 24 cagtcccggg tcacacttga t 21

Claims (14)

A pharmaceutical composition for promoting bone regeneration or bone formation comprising Curcuma xanthorrhiza extract or fractions thereof as an active ingredient.
The method according to claim 1,
Wherein the extract is selected from the group consisting of water, C1 to C4 lower alcohols or mixtures thereof, acetone, ether, benzene, chloroform, ethyl acetate, methylene chloride, hexane, cyclohexane, petroleum ether, subcritical fluid and supercritical fluid Wherein the composition is one or more solvent extracts.
The method according to claim 1,
The fractions were subjected to silica gel column chromatography using a mixture of hexane and ethyl acetate in a concentration gradient of 100: 1 (v / v) to 1: 1 (v / v) &Lt; / RTI &gt;
The method according to claim 1,
Wherein the extract or fraction is an extract or fraction of leaf, stem, flower, roots, husks, fruit or mixture thereof of Java turmeric.
The method according to claim 1,
Wherein the Jaszorz extract promotes osteoblast differentiation or promotes mineralization.
The method according to claim 1,
Wherein the extract or fraction thereof comprises a xanthorrhizol compound of formula 1:
[Chemical Formula 1]

A pharmaceutical preparation for the treatment of diseases requiring bone regeneration or promotion of osteogenesis, comprising the composition according to any one of claims 1 to 6.
The method according to claim 7, wherein the diseases requiring bone regeneration or promotion of osteogenesis are osteoporosis, multiple myelopathy, osteoarthritis, osteoporotic fracture, diabetic fracture, nonunion fracture, bone defect, osteoporosis, A dysmorphic disorder, a degenerative bone disease, and a malocclusion.
A composition for promoting bone regeneration or bone formation comprising Curcuma xanthorrhiza extract or a fraction thereof as an active ingredient.
A pharmaceutical composition for promoting bone regeneration or osteogenesis comprising xanthorrhizol or a salt thereof represented by the following formula (1) as an active ingredient:
[Chemical Formula 1]

11. The method of claim 10,
Wherein the jazorsol is derived from Curcuma xanthorrhiza .
11. A pharmaceutical preparation for the treatment of diseases requiring bone regeneration or promotion of osteogenesis, comprising the composition according to claim 10 or 11.
13. The method according to claim 12, wherein the diseases requiring bone regeneration or promotion of osteogenesis are osteoporosis, multiple myelopathy, osteoarthritis, osteoporotic fracture, diabetic fracture, nonunion fracture, bone defect, osteogenesis imperfecta, A dysmorphic disorder, a degenerative bone disease, and a malocclusion.
A food composition for promoting bone regeneration or bone formation comprising xanthorrhizol or a salt thereof as an active ingredient.

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