WO2017086608A1 - Composition for preventing or treating bone diseases and joint diseases, containing, as active ingredients, water-soluble ionized calcium and fructooligosaccharide complex - Google Patents

Abstract

The present invention relates to: a water-soluble ionized calcium and a fructooligosaccharide complex; a preparation method therefor; a pharmaceutical composition for preventing or treating bone diseases; and a food composition and a feed additive composition for preventing or alleviating bone diseases, wherein the pharmaceutical composition, the food composition, and the feed additive composition contain the complex as an active ingredient. The water-soluble ionized calcium and fructooligosaccharide complex of the present invention minimizes water-soluble organic material loss, has an excellent effect of improving calcium uptake, and can exhibit remarkable osteogenesis and bone tissue regeneration effects.

Description

Composition for the prevention or treatment of bone diseases and joint diseases comprising water-soluble ionized calcium and fructooligosaccharide complex as an active ingredient

The present invention is a water-soluble calcium ion and fructooligosaccharide complex and a method for preparing the same and a pharmaceutical composition for the prevention or treatment of bone diseases and joint diseases comprising the complex as an active ingredient, and foods for the prevention or improvement of bone diseases and joint diseases A composition and a feed additive composition.

Bone is a hard organ made up of a variety of nonprotein components, including various proteins and minerals like collagen. The bones serve to protect the soft tissue organs along with the mineral reservoir function of maintaining body shape and supporting the body and providing minerals to other soft tissues. The bone, like other organs, is a remodeled body that is created and destroyed repeatedly. Bone homeostasis is maintained with the elimination of old bone and new bone formation, which is essential for maintaining a healthy life.

The relationship between bone disease and joint disease and calcium is well known, and calcium intake is gaining attention as an important way to prevent bone disease and joint disease, but the actual intake is only 60% of the recommended amount. have. In particular, only about 30% of calcium is absorbed after ingestion, and the remaining 70% is one of the nutrients that are difficult to absorb. It is also important for bone health.

Pearl powder has a calcium content of about 90%, is rich in various minerals, and contains a large amount of magnesium to help the body absorb calcium, but it does not contain heavy metals such as lead. It is known as a material. In addition, unlike conventional shell calcium and seaweed calcium, freshwater pearl powder contains organic materials such as polypeptides, vitamins, amino acids, etc., and has attracted attention as a material having various functionalities derived from organic materials (Tong et al. The anti -aging effect of pearl oyster shell powder (POSP) .J Tradit Chin Med. 1988, 247-250). However, the pearl powder has a very low solubility in water, so it is difficult to expect the physiological activity by ingesting the pearl powder itself, and in particular, calcium or organic components are not soluble in water, so the pearl powder cannot be fully utilized in actual use. Accordingly, there is a demand for a method of preparing water-soluble pearl powders that are well soluble in water while preserving calcium and organic materials derived from pearl powders as much as possible, and ionizing calcium and minerals derived from pearl powders.

Under these circumstances, the present inventors have made diligent research efforts to develop a method of inducing calcium ionization and facilitating absorption of calcium while minimizing the loss of water-soluble organic substances contained in pearl powders. By reacting fructooligosaccharides, water-soluble complexes in which ionized calcium and fructooligosaccharides derived from pearl powder are combined can be produced, and the present invention has been confirmed that the complex agents exhibit an effect of bone formation or bone tissue regeneration.

An object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of bone diseases and joint diseases comprising a water-soluble calcium ion and fructooligosaccharide complex as an active ingredient.

Another object of the present invention is to provide a method for preparing a water-soluble calcium ion and fructooligosaccharide complex, and a raw material of water-soluble calcium ion and fructooligosaccharide complex prepared thereby.

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

In order to achieve the above object, the first aspect of the present invention provides a pharmaceutical composition for the prevention or treatment of bone diseases and joint diseases comprising a water-soluble ionized calcium and fructooligosaccharide complex as an active ingredient.

The second aspect of the present invention provides a method for preparing a water-soluble calcium ion and fructooligosaccharide complex comprising mixing and reacting pearl powder and fructooligosaccharide in an acid solution.

A third aspect of the present invention provides a water-soluble calcium ionized and fructooligosaccharide complex prepared by mixing and reacting pearl powder and fructooligosaccharide in an acid solution.

The water-soluble ionized calcium and fructooligosaccharide complex of the present invention is characterized by a novel ionized calcium and fructooligosaccharide complex that promotes bone tissue regeneration and osteogenic differentiation.

Throughout this specification, when a part is said to "include" a certain component, it means that it can further include other components, without excluding the other components unless specifically stated otherwise.

As used throughout this specification, the terms "about", "substantially" and the like are used at, or in the sense of, numerical values when a manufacturing and material tolerance inherent in the stated meanings is indicated, Accurate or absolute figures are used to assist in the prevention of unfair use by unscrupulous infringers. As used throughout this specification, the term "step to" or "step of" does not mean "step for."

Throughout this specification, the term “A and / or B” means “A or B, or A and B”.

Throughout this specification, the term "combination of these" included in the expression of the makushi form means one or more mixtures or combinations selected from the group consisting of constituents described in the expression of the makushi form, wherein the constituents It means to include one or more selected from the group consisting of.

Hereinafter, the present invention will be described in detail.

According to one embodiment of the present invention, a first step of mixing and stirring pearl powder and fructooligosaccharide in an acidic solution; And it can provide a method for producing a calcium ion and fructooligosaccharide complex comprising a second step of filtering the mixture.

According to one embodiment of the present application, the pearl powder may be a freshwater pearl powder, but may not be limited thereto.

According to one embodiment of the present application, the average particle diameter of the pearl powder may be about 10 nm to about 500 nm, but may not be limited thereto. For example, the average particle diameter of the pearl powder is about 50 nm to about 500 nm, about 100 nm to about 500 nm, about 300 nm to about 500 nm, about 10 nm to about 300 nm, about 10 nm to about 100 nm , About 10 nm to about 50 nm, about 90 nm to about 110 nm, or about 100 nm, but may not be limited thereto.

According to one embodiment of the present application, the acidic solution may be about 10% to about 30% acetic acid solution, but may not be limited thereto. For example, the acidic solution may be about 10% to about 30%, about 20% to about 30%, about 10% to about 20%, about 10% to about 15%, or about 10% acetic acid solution. However, this may not be limited.

According to one embodiment of the present application, the pearl powder and fructooligosaccharide may be mixed in a weight ratio of about 10: 1 to about 1:10, but may not be limited thereto. For example, the pearl powder and fructooligosaccharide may be mixed in a weight ratio of about 1: 1, but may not be limited thereto.

According to one embodiment of the present application, the first step of mixing and stirring the pearl powder and fructooligosaccharide in the acid solution, the process of adding the pearl powder and fructooligosaccharide to the acid solution and stirring once to 20 times It may include repeating, but may not be limited thereto.

According to one embodiment of the present application, the mixing and stirring may be performed at a temperature of about 30 ℃ to about 80 ℃, but may not be limited thereto. For example, the mixing and stirring may be performed at a temperature of about 30 ° C. to about 60 ° C., about 30 ° C. to about 50 ° C., about 50 ° C. to about 80 ° C., about 60 ° C. to about 80 ° C., or about 60 ° C. It may be, but may not be limited thereto.

According to one embodiment of the present application, the method may further include, but not limited to, drying the mixture after filtration.

According to one embodiment of the present application, comprising an ionized calcium and fructooligosaccharide complex prepared by the method for producing an ionized calcium and fructooligosaccharide complex of the present application as an active ingredient, for the prevention or treatment of bone diseases and joint diseases A composition can be provided.

According to one embodiment of the present disclosure, the bone disease and joint disease may be selected from the group consisting of degenerative arthritis, rheumatoid arthritis, deformable arthritis, osteomalacia, osteoporosis, rickets and Paget's disease, but is not limited thereto. Can be.

According to one embodiment of the present application, the pharmaceutical composition of the present application may be one having one or more effects of bone formation and bone tissue regeneration, but may not be limited thereto.

According to one embodiment of the present application, comprising the ionized calcium and fructooligosaccharide complex prepared by the method for producing an ionized calcium and fructooligosaccharide complex of the present application as an active ingredient, food composition for the prevention or improvement of bone diseases and joint diseases Can be provided.

According to one embodiment of the present application, comprising an ionized calcium and fructooligosaccharide complex prepared by the method for producing an ionized calcium and fructooligosaccharide complex of the present application as an active ingredient, feed additive for the prevention or improvement of bone diseases and joint diseases A composition can be provided.

Pearl powder may be prepared by grinding through a grinding process commonly used in the art, etc., may be further performed sterilization process, pretreatment process and / or classification process, if necessary. For example, the pearl powder used in the present invention is an edible pearl powder obtained by crushing freshwater pearls into particles of 10 to 500 nm in size. More than 99% is calcium carbonate, rich in various minerals, and helps the body absorb calcium. Although it contains a large amount of magnesium, it does not contain heavy metals such as lead, so it is known as a material for excellent calcium development (Table 1; calcium content in freshwater pearls by ICP atomic emission spectroscopy). Unlike conventional shell calcium or seaweed calcium, freshwater pearl powder is rich in polypeptides such as conchiolin, which makes it easier to manufacture water-soluble calcium than other calcium sources. Can be. However, the pearl powder itself is very low solubility in water (0.03 mg / mL, 0.003%) to be prepared as an aqueous solution, it is difficult to expect the physiological activity by ingesting it.

In a preferred embodiment of the present application, by mixing and reacting the pearl powder and fructooligosaccharide in an acid solution, a complex agent exhibiting water solubility in the form of a combination of ionized calcium and fructooligosaccharide derived from pearl powder can be prepared, thereby It has been found that not only the absorption of large amounts of calcium contained in the powder is easy, but also the combination shows an effect of promoting bone formation and bone tissue regeneration. The present invention is based on this.

The present invention provides a pharmaceutical composition for preventing or treating bone diseases and joint diseases comprising a water-soluble ionized calcium and fructooligosaccharide complex as an active ingredient.

The water-soluble ionized calcium and fructooligosaccharide complex of the present invention may be formed by combining an ionized calcium and fructooligosaccharide derived from pearl powder, and the ionized calcium may be produced by acid treatment of the pearl powder. For example, the acid treatment may be performed using an organic acid solution, for example, one or more organic acids selected from edible glacial acetic acid, lactic acid, tartaric acid and citric acid may be used, but is not limited thereto.

The ionized calcium and fructooligosaccharide complexes of the present invention exhibit water solubility, thereby facilitating absorption of large amounts of calcium due to improved bioavailability. In addition, the water-soluble ionized calcium and fructooligosaccharide complex of the present invention promotes ossification (deposition of calcium) and formation of cartilage tissue, and can activate the function of osteoblasts (FIGS. 5 to 7). Therefore, the water-soluble calcium ion and fructooligosaccharide complex according to the present invention can be usefully used for pharmaceuticals and quasi-drugs as a pharmaceutical composition for the prevention or treatment of bone diseases and joint diseases.

The term "prevention" of the present invention means any action that inhibits or delays the development and spread of bone disease and joint disease by administration of the composition of the present invention, and "treatment" means bone disease and the administration of the composition of the present invention. Means any behavior that improves or beneficially changes the symptoms of joint disease.

Bone diseases and joint diseases that can be prevented or treated by the pharmaceutical compositions of the present invention include degenerative arthritis, rheumatoid arthritis, deformable arthritis, osteomalacia, osteoporosis, rickets and Paget's disease, etc. And / or bone diseases and joint diseases known to those skilled in the art that can be treated or ameliorated due to the ability to promote cartilage production. In particular, the pharmaceutical composition of the present invention can be effectively used for the prevention or treatment of bone diseases and joint diseases by promoting bone formation and regeneration of cartilage tissue.

The pharmaceutical composition may include, but is not limited to, oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations such as powders, gels, ointments, creams, or sterilizations. It can be formulated and used in the form of injectable solutions. The composition of the present invention may further comprise suitable antibiotics, carriers, excipients and / or diluents commonly used in the manufacture of a medicament.

The composition of the present invention may be formulated in various forms, such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, oral formulations such as aerosols, and injections of sterile injectable solutions, according to conventional methods, for the purpose of each use. It may be used orally or may be administered through various routes including intravenous, intraperitoneal, subcutaneous, rectal, topical, and the like. Examples of suitable carriers, excipients or diluents that may be included in such compositions include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil and the like. In addition, the compositions of the present invention may further include fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives and the like.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, which may comprise at least one excipient in the composition, for example starch, calcium carbonate, sucrose, lactose, gelatin, etc. Are formulated by mixing. In addition to the simple excipients, lubricants such as magnesium stearate, talc may be used.

Oral liquid preparations include suspensions, solvents, emulsions, syrups, and the like, and include various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. Can be.

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. The suppository base includes Uittepsol, Macrogol, and Twin 61. Cacao butter, laurin butter, glycerogelatin and the like can be used. Injectables may include conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, preservatives, and the like.

The composition of the present invention is administered in a pharmaceutically effective amount.

As used herein, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment and that does not cause side effects. Well-known factors in the field of bone and joint diseases, including the severity, activity of the drug, sensitivity to the drug, method of administration, time of administration, route of administration and rate of release, duration of treatment, combination or simultaneous use of drugs and other medical fields It can be determined according to the factor. The compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered in single or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects, which can be easily determined by those skilled in the art.

Specifically, the effective amount of the combination in the composition of the present invention may vary depending on the age, sex and weight of the subject, and generally 1 to 10 mg, preferably 1 to 5 mg per kg body weight daily or every other day or 1 It can be administered in 1 to 3 times a day. However, the dosage may be increased or decreased depending on the route of administration, the severity of the disease, sex, weight, age, etc., and the above dosage does not limit the scope of the present invention in any way.

In addition, the present invention provides a method for preventing or treating bone diseases and joint diseases, comprising administering to a subject in need thereof a pharmaceutical composition comprising a water-soluble ionized calcium and fructooligosaccharide complex as an active ingredient. .

The term "individual" of the present invention refers to monkeys, cows, horses, sheep, pigs, chickens, cats, dogs, mice, rats, rabbits or guinea pigs, including humans who may or may develop the bone disease and joint disease. By means of any animal, including, and administering a pharmaceutical composition of the present invention to a subject can effectively prevent or treat the disease. The pharmaceutical composition of the present invention can be administered in parallel with existing therapeutic agents.

As used herein, the term "administration" means providing a subject with any suitable method, and the route of administration of the composition of the present invention may be administered via any general route as long as it can reach the desired tissue. have. For example, the route of administration may be selected from, but is not limited to, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration and intranasal administration. In addition, the pharmaceutical compositions of the present invention may be administered by any device in which the active substance may migrate to target cells. Preferred modes of administration and preparations may include intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, instillation injections. Injections include non-aqueous solvents such as aqueous solvents such as physiological saline solution and ring gel solution, vegetable oils, higher fatty acid esters (e.g., oleic acid, etc.), and alcohols (e.g., ethanol, benzyl alcohol, propylene glycol, glycerin, etc.). Stabilizers (e.g. ascorbic acid, sodium bisulfite, sodium pyrosulfite, BHA, tocopherol, EDTA, etc.), emulsifiers, buffers for pH control, to prevent microbial growth Preservatives (eg, mercury nitrate, chimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol, etc.) may be included.

In another aspect, the present invention is a first step of mixing and stirring the pearl powder and fructooligosaccharide in an acidic solution; And it provides a method for producing a water-soluble calcium ion and fructooligosaccharide complex comprising a second step of filtering the mixture.

Specifically, according to a preferred embodiment of the present application, the reaction of the pearl powder and fructooligosaccharide is carried out by mixing the reactants in an aqueous solution containing an acid in a weight ratio of 10: 1 to 1:10 and stirring at 30 to 80 ℃ can do. In the first step, a process of adding pearl powder and fructooligosaccharide to an acidic solution and stirring may be repeated 1 to 20 times.

In addition, the pearl powder may be a pearl unit of the nano unit pulverized into particles of 10 to 500 nm size, if the size of the powder is large, the ionization reaction by acetic acid is too weak, it may be difficult to ionize. As the acid, glacial acetic acid may be used, but is not limited thereto, and may be prepared by using 10% to 30% acetic acid aqueous solution. Using the 10% to 30% acetic acid solution, the calcium carbonate in the used pearl powder is dissolved in water with calcium ions, and the water-soluble organic substances bound to the pearl powder in the process are added to the ionized pearl powder solution. It can be melted and the functionalities of these organic components can be obtained. In addition, the ionized calcium and fructooligosaccharide produced by the acid solution may be combined to form a complex agent, and the ionized calcium and fructooligosaccharide complex dissolved in the aqueous solution may be filtered and dried and then powdered. It can be made into a powder.

The fructooligosaccharide used in the present invention is a mixture of sugars in which 1-3 fructose is bound to a fructose residue of sugar, which helps the growth of the intestinal beneficial bacteria Bifidus and inhibits intestinal harmful bacteria. It is known to exhibit efficacy in promoting absorption. Therefore, the water-soluble ionized calcium and fructooligosaccharide complexes prepared by the preparation method of the present invention may improve bioavailability due to the water-soluble properties, and also exhibit an effect of enhancing calcium absorption and promoting bone formation and bone tissue regeneration. Can be. In one embodiment of the present invention, it was confirmed that the water-soluble calcium ion and fructooligosaccharide complexes exhibited more osteogenic and cartilage-forming effects compared to the simple mixture of calcium ion and fructooligosaccharide, through which ionized calcium and fructo It was confirmed that oligosaccharides can exhibit superior bone formation and bone tissue production efficacy by forming a complex (FIG. 6).

As another aspect, the present invention provides a water-soluble ionized calcium and fructooligosaccharide complex prepared by mixing and reacting pearl powder and fructooligosaccharide in an acid solution. Since the water-soluble ionized calcium and fructooligosaccharide complexes exhibit excellent bone formation and bone tissue regeneration effects, they may be included as an active ingredient in a food composition for preventing or improving bone diseases and joint diseases.

The food composition may include a food supplement acceptable food additive in addition to the active ingredient. The term "food supplement" used in the present invention means a component that can be added to food supplements, and can be appropriately selected and used by those skilled in the art as being added to prepare a health functional food of each formulation. Examples of food additives include flavors such as various nutrients, vitamins, minerals (electrolytes), synthetic and natural flavors, colorants and fillers, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners. , pH regulators, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like, but is not limited to the kind of food additives of the present invention by the above examples.

The food composition of the present invention may include a health functional food. As used herein, the term "health functional food" refers to a food prepared and processed in the form of tablets, capsules, powders, granules, liquids and pills using raw materials or ingredients having useful functions for the human body. Here, 'functional' means to obtain a useful effect for health purposes such as nutrient control or physiological action on the structure and function of the human body. The health functional food of the present invention can be prepared by a method commonly used in the art, and the preparation can be prepared by adding raw materials and ingredients commonly added in the art. In addition, the formulation of the health functional food can also be prepared without limitation as long as the formulation is recognized as a health functional food. Food composition of the present invention can be prepared in various forms of formulation, unlike the general medicine has the advantage that there is no side effect that can occur when taking a long-term use of the drug as a raw material, and excellent portability, the present invention Health functional foods can be taken as a supplement to promote the improvement effect of bone diseases and joint diseases.

In addition, the water-soluble ionized calcium and fructooligosaccharide complexes of the present invention are monkeys, cows, horses, sheep, pigs, chickens, cats, dogs, mice, rats, rabbits or guinea pigs that may or may develop bone diseases and joint diseases. It may be included as an active ingredient of the feed additive composition for the prevention or improvement of bone diseases and joint diseases supplied to all animals, including. The feed may be prepared by adding the feed additive of the present invention to the feed, or may be prepared by directly adding a composition comprising the water-soluble ionized calcium and fructooligosaccharide complex. The type of the feed is not particularly limited in the present invention, it is possible to use a feed commonly used in the art. Non-limiting examples of the feed may include plant feeds such as cereals, fruits, food processing by-products, algae, fibres, pharmaceutical by-products, oils, starches, gourds or grain by-products; And animal feeds such as proteins, minerals, fats and oils, minerals, fats and oils, single cell proteins, zooplankton or foods. These may be used alone or in combination of two or more thereof.

The water-soluble ionized calcium and fructooligosaccharide complexes of the present invention minimize the loss of water-soluble organic substances and have an excellent effect of improving calcium absorption, and can exhibit superior bone formation and bone tissue regeneration efficacy. Accordingly, the water-soluble ionized calcium and fructooligosaccharide complex may be included as an active ingredient of the composition for preventing or treating bone diseases and joint diseases.

1 is a view showing a dry powder of the water-soluble ionized calcium and fructooligosaccharide complex of the present invention.

Figure 2 is a graph showing the heart rate of zebrafish embryos according to the concentration of the water-soluble calcium ion and fructooligosaccharide complex of the present invention.

Figure 3 is a view of the morphological changes of zebrafish embryos according to the water-soluble calcium ion and fructooligosaccharide complex of the present invention.

Figure 4 is a diagram confirming the degree of absorption of zebrafish absorption of the water-soluble calcium ion and fructooligosaccharide complex of the invention with a fluorescence microscope.

Figure 5 is a view of the degree of bone and cartilage formation by the water-soluble calcium ion and fructooligosaccharide complex of the present invention. The degree of staining of alcian blue (bone) and alizarin red (cartilage) is increased by the water-soluble ionized calcium and fructooligosaccharide combination.

6 is a view illustrating the efficacy of bone and cartilage formation by the water-soluble calcium ion and fructooligosaccharide complex of the present invention, and a simple mixture of ionized calcium and fructooligosaccharide.

Figure 7 is a diagram showing the expression level of the bone related genes by the water-soluble calcium ion and fructooligosaccharide complex of the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are intended to illustrate the present invention more specifically, but the scope of the present invention is not limited by these examples.

Example  1: calcium ionization and Fructooligosaccharide  Preparation of the Composite

20 mL of glacial acetic acid was mixed with 180 mL of distilled water to prepare a 10% acetic acid solution, and 2 g of freshwater pearl powder and 2 g of fructooligosaccharide were added to 200 mL of the 10% acetic acid solution. Thereafter, the mixture was dissolved for 10 minutes at 60 ° C. until all the bubbles were removed. Every 10 minutes, fresh water pearl powder and fructooligosaccharide were added 2 g each, and then the pearl calcium powder and fructooligosaccharide powder were put into the foam so that the pearl calcium powder placed on the foam was mixed well with the calcium acetate solution. This process was repeated nine times, and finally 20 g of pearl calcium and fructooligosaccharide were dissolved in total. The obtained calcium ion and fructooligosaccharide complex aqueous solution was filtered and dried in a drying oven at 80 ° C. to obtain a dry powder of calcium ion and fructooligosaccharide complex (FIG. 1).

Example  2: calcium ionization and Fructooligosaccharide  Acute Toxicity Assessment of Combinations

To analyze the acute toxicity of the calcium ion and fructooligosaccharide combinations, water-soluble calcium ion and fructooligosaccharide complexes were concentrated in concentrations (37.5 to 2400 μg / mL) in cultures of zebrafish embryos 24 h after fertilization (24 hpf). Treated. To determine the toxicity at 48 hpf, the embryos were removed and the heart rate was measured and phenotype was confirmed. Heart rate was measured three times and statistically processed. Morphological changes were observed based on the presence of bleeding and deformation of the trunk.

As a result, when the calcium ionized fructooligosaccharide complex was treated in zebrafish embryos at a concentration of 37.5 to 2400 μg / mL, there was no significant difference in heart rate (FIG. 2). In addition, typical morphological toxic effects such as bleeding or abnormal formation of the head, embryo, and tail were not observed when the calcium ion and fructooligosaccharide combinations were treated in zebrafish embryos at a concentration of 10-100 μg / mL ( 3). From the above results, it was confirmed that the calcium ion and fructooligosaccharide complex prepared in Example 1 did not show toxicity based on heart rate, bleeding, and morphological changes.

Example  3: calcium ionization and Fructooligosaccharide  Complex Bone formation  Active evaluation

3-1. Calcium ion and Fructooligosaccharide  Containing complex Zebrafish  Embryo Diet and Confirmation of Absorption

When the egg yolk of the zebrafish embryo disappeared and the reaction to the food began to be fed, a diet containing a combination of ionized calcium and fructooligosaccharide was fed. It was prepared with gluten granules and supplemented with calcium as a supplement while feeding normally. Based on the administration of calcium as an adjuvant in the adult range (0.4 g-2 g (60 kg basis)), the doses of calcium ionized and fructooligosaccharide combinations were set for zebrafish. To make sure that the embryos are properly fed the zebrafish diet, mix the gluten powder with 10 mg / mL of Neil Red (dye reagent), add gluten powder to the mortar and add 1 mL of distilled water. Kneading was added slowly. The homogeneously kneaded gluten was lyophilized. The gluten after drying was passed through a sieve of 1000, 800, and 700 μm in size to obtain granules of 700 μm and fed to zebrafish.

After 4, 8, 12, and 24 hours of the administration of the diet prepared above, the degree of absorption of the zebrafish anesthetized with tricaine (Tricaine) under fluorescence microscopy was confirmed. It was confirmed that the red fluorescence continuously appeared for 4 hours to 24 hours in the intestine (FIG. 4).

3-2. Calcium ion and Fructooligosaccharide  Complex Bone formation  Active evaluation

When the yolk disappearance of zebrafish embryos and the response to food began to appear, a diet containing calcium ion and fructooligosaccharide complex concentrations was administered once daily. At 21 days after the administration, staining with Alcian blue and Alizarin red was performed to compare the bone formation differences. The method for staining was as follows: zebrafish was anesthetized with 0.016% tricaine and fixed with 3.7% neutral buffered formaldehyde. The zebrafish was washed three times for 5 minutes with 1 × PBS-T, and zebrafish was transferred to 0.1% Alcian blue solution and stained for 4 hours to overnight. After staining, dehydration was performed in 70% ethanol and 30% PBST, 50% ethanol and 50% PBST, 30% ethanol and 70% PBST. The zebrafish was transferred to 0.05% trypsin, reacted for 1 hour, and washed three times with 1 × PBS-T for 5 minutes. The zebrafish was transferred to 0.05% alizarin red solution, stained for 4 hours or more, and observed to the degree of staining under a microscope.

As a result of comparative analysis of staining of Alcian blue (bone) and alizarin red (cartilage), cartilage and bone were treated in the water-soluble ionized calcium and fructooligosaccharide combination group compared to the control group (untreated group of calcium ion and fructooligosaccharide combination). It was confirmed that the degree of staining for all increased (Fig. 5).

3-3. Calcium ion and Fructooligosaccharide  Combinations; And simple mixtures Bone formation  Activity comparison

Once the yolk disappears from the zebrafish embryos and begins to respond to food, a diet containing 0.1 mg / mL of calcium ion and fructooligosaccharide mixture (B), or an ionized calcium and fructooligosaccharide combination (C), is taken daily. Administration was once. At 21 days after administration, the difference in bone formation was analyzed by staining with alcian blue and alizarin red. The staining method was carried out in the same manner as the staining method used in the evaluation of bone formation activity of the ionized calcium and fructooligosaccharide complex of Example 3-2.

As a result, it was confirmed that the degree of staining for cartilage and bone is increased in the water-soluble calcium ion and fructooligosaccharide mixed agent treated group compared to the control group (calcium ionized fructooligosaccharide complex, and the mixture untreated group). However, as a result of comparing the ionized calcium and fructooligosaccharide mixture treatment group with the ionized calcium and fructooligosaccharide composite treatment group, the degree of ossification (calcium deposition) was more improved in the treatment group of the composite despite the same concentration. Observation of the cartilage tissue was also greatly increased (FIG. 6).

From the above results, the water-soluble ionized calcium and fructooligosaccharide complexes formed better ossification (deposition of calcium) than conventional ionized calcium or ionized calcium and fructooligosaccharide mixtures prepared by simply combining water-soluble calcium and fructooligosaccharides. It was confirmed that the production of cartilage tissue.

3-4. Calcium ion and Fructooligosaccharide  According to the combination Bone formation  Related gene expression level analysis

When the egg yolk of the zebrafish embryo disappeared and the response to the food began to be seen, the diet containing the water-soluble calcium ion and fructooligosaccharide complex was administered once daily, and then RNA was separated from the zebrafish after 21 days. After cDNA synthesis from the isolated RNA, the expression level of the gene was analyzed by amplification by PCR using primers of Dlx5, collagen type I (Type I collagen), osteocalcin, and β-actin gene.

As a result, the water-soluble calcium ion and fructooligosaccharide complex of the present invention increases the expression level of Dlx5 and collagen type I genes, which are used as markers for bone / joint formation, while the osteocalcin genes that cause bone destruction are increased. It did not appear to affect (FIG. 7). From the above results, it was confirmed that osteoblast function is enhanced by the water-soluble ionized calcium and fructooligosaccharide complex prepared in Example 1.

So far I looked at the center of the preferred embodiment for the present invention. Those skilled in the art will appreciate that the present invention can be implemented in a modified form without departing from the essential features of the present invention. Therefore, the disclosed embodiments should be considered in descriptive sense only and not for purposes of limitation. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the scope will be construed as being included in the present invention.

Claims (12)

1. A first step of mixing and stirring pearl powder and fructooligosaccharide in an acidic solution; And

   A second step of filtering the mixture;

   A method of producing a calcium ion and fructooligosaccharide complex comprising a.
2. The method of claim 1, wherein the pearl powder is a freshwater pearl powder.
3. The method of claim 1, wherein the pearl particle has an average particle diameter of 10 nm to 500 nm.
4. The method of claim 1, wherein the acidic solution is a 10% to 30% acetic acid solution.
5. The method of claim 1, wherein the pearl powder and fructooligosaccharide are mixed in a weight ratio of 10: 1 to 1:10.
6. According to claim 1, wherein the first step of mixing and stirring the pearl powder and fructooligosaccharide in the acid solution, the process of adding the pearl powder and fructooligosaccharide to the acid solution and stirring 1 to 20 times Wherein the mixing and stirring is performed at a temperature between 30 ° C. and 80 ° C. 6.
7. The method of claim 1, further comprising drying the mixture after filtration.
8. A pharmaceutical composition for the prevention or treatment of bone diseases and joint diseases, comprising an ionized calcium and fructooligosaccharide complex prepared by the method of any one of claims 1 to 7 as an active ingredient.
9. The pharmaceutical composition of claim 8, wherein the bone disease and joint disease are selected from the group consisting of degenerative arthritis, rheumatoid arthritis, deformable arthritis, osteomalacia, osteoporosis, rickets and Paget's disease.
10. The pharmaceutical composition of claim 8, having one or more effects of bone formation and bone tissue regeneration.
11. Claims 1 to 7 comprising the ionized calcium and fructooligosaccharide complex prepared by the method of any one of the active ingredient, a food composition for the prevention or improvement of bone diseases and joint diseases.
12. Claims 1 to 7 comprising the ionized calcium and fructooligosaccharide complex prepared by the method of any one of the active ingredient, a feed additive composition for the prevention or improvement of bone diseases and joint diseases.

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