KR20180108471A - Calcium complex comprising mussel shell and oligosaccharide and uses thereof - Google Patents

Abstract

The present invention relates to a water-soluble calcium composite containing a nanocomposite composed of oligosaccharide and mussel shells as an active ingredient. More specifically, the calcium composite containing oligosaccharide and mussel shells as the active ingredient exhibits excellent water solubility, increases absorption rate, and shows outstanding osteogenic effects and bone tissue regeneration effects by containing substances safe to human body. Therefore, the calcium composite containing oligosaccharide and mussel shells as the active ingredient can be used as a composition effective in preventing or treating bone or joint disease.

Description

[0001] The present invention relates to a calcium complex comprising mussel shell and oligosaccharide as an active ingredient,

The present invention relates to a calcium complex agent containing as an active ingredient a nanocomposite comprising a mussel shell and an oligosaccharide, and a composition for preventing or treating bone or joint disease using the same.

Minerals such as calcium, iron, and zinc are regulators that are involved in the metabolism of various substances and calories in the human body, which are closely related to various diseases. Since the bioavailability of minerals varies depending on the intake level and chemical form of micronutrients, absorption promoting factors, interferences, and interactions with other nutrients, studies on proper intake and deficiency have been actively conducted.

Calcium is one of the most important minerals in the human body, and it shows a significant difference in the amount absorbed by the human body depending on the type, composition and dosage form ingested.

Since calcium is mostly used to make bones and teeth, a decrease in calcium absorption inevitably leads to osteoporosis. Osteoporosis is a degenerative disease caused by persistent loss of calcium in the bone, mainly in adults aged 35 to 40 years, and is more common in women than in men.

In the case of growing children, sufficient calcium must be supplemented in the body, as lack of calcium is likely to cause negative results such as limited key growth and malformations.

For this purpose, modern people mainly take calcium supplements as health supplement foods. Calcium preparations include calcium carbonate, calcium sulfate, calcium phosphate, calcium oxide, calcium hydroxide, calcium nitrate, calcium citrate and calcium gluconate, Calcium carbonate is inexpensive because it is fast in the body but is expensive. Calcium carbonate is most widely used because it is inexpensive. However, there is a problem that the absorption rate of calcium carbonate is substantially lower than that of human.

In recent years, as animal raw materials of egg shells and bones used as raw materials for natural calcium are not used due to infectious diseases of animals, there is a need to develop a product capable of promoting the calcium absorption rate at an inexpensive price.

Various shells and coral have been used as calcium carbonate and mineral fertilizer through plastic processing which is incinerated at high temperature by marine resources.

Among them, the mussel shell is rich in polypeptides such as conchiolin, functional amino acids and various minerals. Especially, the content of calcium is about 90%, which is higher than that of scallop or oyster shell. In addition, the phosphorus (P) content is low and the magnesium content is high as compared with other raw materials, which is advantageous for increasing the availability of calcium.

It is necessary to study a method for the development of a functional ingredient preserving inorganic substances such as calcium and water-soluble organic substances contained in the mussel shell.

Korean Patent Publication No. 2003-0006199 (published on Jan. 23, 2003)

As described above, according to the need to develop a product capable of promoting calcium absorption rate by using a safe and inexpensive substance in the human body, the present invention provides a method for producing a water-soluble organic substance and a calcium-rich mussel shell and an oligosaccharide And a calcium-containing compound.

The present invention provides a water-soluble calcium complex comprising a nanocomposite comprising a mussel shell and an oligosaccharide as an active ingredient.

The present invention provides a pharmaceutical composition for preventing or treating a bone or joint disease containing the water-soluble calcium complex as an active ingredient.

The present invention provides a health food for preventing or ameliorating a bone or joint disease containing the water-soluble calcium complex as an active ingredient.

In addition, the present invention provides a method for producing a mussel of the present invention, comprising: a first step of adding a mussel shell powder and an oligosaccharide to an acetic acid solution, mixing and stirring; And a second step of filtering the mixture. The present invention also provides a method for producing a water-soluble calcium complex.

According to the present invention, a calcium complex containing a nanocomposite composed of a mussel shell and an oligosaccharide as an active ingredient exhibits excellent water solubility and can exhibit an effect of improving the calcium absorption rate, and is a safe material for the human body, The calcium complex containing the mussel shell and oligosaccharide as an active ingredient can be effectively used as a composition for preventing or treating bone diseases and joint diseases.

Fig. 1 shows the results of confirming the heart rate of a zebrafish embryo administered with a mussel shell powder, a mussel shell / fructooligosaccharide mixture, and a mussel shell / fructooligosaccharide conjugate in a zebrafish embryo at 0.01 and 0.1 mg / ml, respectively.
FIG. 2 is a graph showing the effect of the combination of mussel shell powder, mussel shell / fructooligosaccharide mixture and mussel shell / fructooligosaccharide conjugate in zebrafish embryos at 0.01 and 0.1 mg / ml, respectively, And the degree of bone and cartilage formation.
FIG. 3 shows the results of comparing the solubilities of the water-soluble mussel shell and the oligosaccharide complex prepared from various oligosaccharides. As a result, it was confirmed that calcium carbonate (CaCO3), mussel shell calcium (MsCa), mussel shell / xylose oligosaccharide complex (XOS- (MOS), mussel shell / isomer maltooligosaccharide complex (IMOS-Ca), mussel shell / galactooligosaccharide complex (GOS-Ca) and mussel shell / fructooligosaccharide complex (FOS- For 30 minutes and then centrifuged at 12,000 × g for 15 minutes to obtain an insoluble residue. The residue was dried at a high temperature to measure the mass, and the solubility (w / v) in 100 mL of distilled water was determined.
FIG. 4 is a graph showing changes in blood calcium levels of mussel shell / galactooligosaccharide conjugate (GOS-Ca) and mussel shell / fructooligosaccharide conjugate (FOS-Ca) c mice were orally administered once daily for 5 days, and the blood was collected after 4 hours of the last administration and the blood calcium level was confirmed.
FIG. 5 shows the blood calcium level changes of the mussel shell calcium / fructooligosaccharide complex (MsCa + FOS) and the mussel shell / fructooligosaccharide complex (FOS-Ca) / c mice were orally administered once daily for 5 days, and the blood levels of 1, 2, and 4 hours after the last administration were collected to determine the calcium concentration in the blood.

The present invention can provide a water-soluble calcium complex comprising a nanocomposite comprising a mussel shell and an oligosaccharide as an active ingredient.

More specifically, the nanocomposite may be prepared by adding a mussel shell powder and an oligosaccharide to an acetic acid solution, mixing and stirring the nanocomposite, and the size of the nanocomposite may be 100 to 500 nm.

The mussel shell powder may be a fine powder having an average diameter of 30 nm to 400 nm.

More specifically, the mussel shell powder may be prepared by pulverizing through a pulverization process commonly used in the art, and may be further subjected to a sterilization process, a pretreatment process, and / or a classification process, if necessary. The mussel shell contains water-soluble organic substances such as conchiolin and has a higher calcium content and lower phosphorus content than other shells.

Minerals Scallop shell powder Oyster shell powder Mussel shell powder Iron (Fe) - - - Calcium (Ca) 26,104.8 32,516.5 34,849.7 Zinc (Zn) - - - In (P) 39.2 40.0 2.6

The oligosaccharide may be any one selected from the group consisting of mannan oligosaccharide, fructooligosaccharide, soy oligosaccharide, galactooligosaccharide, isomaltooligosaccharide, xylooligosaccharide, agarooligosaccharide and maltooligosaccharide.

The calcium complex may comprise 10 to 90% by weight of a mussel shell and 10 to 90% by weight of an oligosaccharide.

The present invention can provide a pharmaceutical composition for preventing or treating a bone or joint disease containing the water-soluble calcium complex as an active ingredient.

The bone or joint disease may be selected from the group consisting of degenerative arthritis, rheumatoid arthritis, deformed arthritis, osteomalacia, osteoporosis, rickets and Paget's disease.

The water-soluble calcium complex can exhibit bone formation or bone tissue regeneration effect.

In one embodiment of the present invention, the pharmaceutical composition for preventing or treating bone diseases and joint diseases containing the water-soluble calcium complex as an active ingredient may be administered orally or parenterally in the form of injections, granules, powders, tablets, pills, capsules, suppositories, Gels, suspensions, emulsions, drops, or liquid preparations may be used.

In another embodiment of the present invention, the pharmaceutical composition for preventing or treating bone diseases and joint diseases containing a water-soluble calcium complex as an active ingredient may be formulated with a suitable carrier, excipient, disintegrant, sweetener, A lubricant, a lubricant, a flavoring agent, an antioxidant, a buffer, a bacteriostatic agent, a diluent, a dispersant, a surfactant, a binder and a lubricant.

Specific examples of carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. Solid formulations for oral administration may be in the form of tablets, pills, powders, granules, capsules These solid preparations can be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc., into the composition. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, syrups and the like, and various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin which are commonly used simple diluents. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, and the like. 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 the suppository base, witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like can be used.

According to one embodiment of the present invention, the pharmaceutical composition may be administered orally, intraarterally, intraperitoneally, intramuscularly, intraarterally, intraperitoneally, intrasternally, transdermally, nasally, inhaled, topically, rectally, ≪ / RTI > can be administered to the subject in a conventional manner.

The preferred dosage of the pharmaceutical composition may vary depending on the condition and body weight of the subject, the type and degree of disease, the drug form, the administration route and the period, and may be appropriately selected by those skilled in the art. According to one embodiment of the present invention, the daily dose may be 0.01 to 200 mg / kg, specifically 0.1 to 200 mg / kg, more specifically 0.1 to 100 mg / kg, though it is not limited thereto. The administration may be performed once a day or divided into several times, and thus the scope of the present invention is not limited thereto.

In the present invention, the 'subject' may be a mammal including a human, but is not limited thereto.

The present invention can provide a health food for preventing or ameliorating a bone or joint disease containing the water-soluble calcium complex as an active ingredient.

The health food is used together with other food or food additives in addition to the water-soluble calcium complex, and can be suitably used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to its use purpose, for example, prevention, health or therapeutic treatment.

The effective dose of the compound contained in the above-mentioned health food may be used in accordance with the effective dose of the therapeutic agent, but may be less than the above range for health and hygiene purposes or for long-term intake for health control purposes, It is clear that the component can be used in an amount of more than the above range since there is no problem in terms of safety.

There is no particular limitation on the type of the health food, and examples thereof include meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, Drinks, alcoholic beverages and vitamin complexes.

In addition, the present invention provides a method for producing a mussel of the present invention, comprising: a first step of adding a mussel shell powder and an oligosaccharide to an acetic acid solution, mixing and stirring; And a second step of filtering the mixture.

The acetic acid solution may be 1 to 30% by weight, but is not limited thereto.

The water-soluble calcium complex can be mixed with 10 to 90% by weight of a mussel shell and 10 to 90% by weight of an oligosaccharide.

More specifically, in the first step, the mussel shell powder and the oligosaccharide are added to an acetic acid solution and mixed and stirred at 20 to 80 ° C for 1 to 10 times.

In addition, the method for preparing a water-soluble calcium complex may further include a third step of drying the mixture filtered at the second step at 60 to 90 캜.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

< Example  1> Mussel shell  And Fructooligosaccharide  Composite manufacturing

20 mL of glacial acetic acid and 180 mL of distilled water were mixed to prepare a 10% acetic acid solution. Then, 2 g of mussel shell powder and 2 g of fructooligosaccharide were added to 200 mL of the 10% acetic acid solution. Thereafter, the mixture was dissolved at 60 캜 for 10 minutes while stirring until the bubbles were exhausted. After 2 g each of mussel shell powder and fructooligosaccharide were added at intervals of 10 minutes, each powder was pushed into the foam so that the mussel shell powder placed on the foam was mixed well with the calcium acetate solution.

The above procedure was repeated 9 times to finally dissolve a total of 20 g of mussel shell powder and fructooligosaccharide, respectively. The resultant aqueous solution of the mussel shell powder / fructooligosaccharide conjugate solution was filtered and dried in an oven at 80 DEG C to obtain a dry powder of ionized mussel shell / fructooligosaccharide complex.

The mussel shell dried powder and the fructooligosaccharide powder were mixed at a ratio of 1: 1 by weight in the same manner as described above, without adding fructo-oligosaccharide, Dissolved mussel shell / fructooligosaccharide mixture.

< Example  2> water soluble Mussel shell  And Fructooligosaccharide  Solubility of complex agent

10 mL of distilled water (pH 7.0) was added to a 50 mL centrifuge tube, and 10 g of each of calcium carbonate, calcium hydroxide, mussel shell / fructooligosaccharide mixture and mussel shell / fructooligosaccharide combination powder were added, And left to dissolve.

Then, the residue was centrifuged at 12,000 × g for 15 minutes to obtain an insoluble residue. The residue was dried at high temperature to measure the mass. The solubility (w / v) in 100 mL of distilled water was determined from the measured values.

As a result of confirming the solubility in distilled water at room temperature, it was confirmed that calcium carbonate and calcium hydroxide were almost insoluble in water as shown in Table 2. In addition, the mussel shell / fructooligosaccharide mixture also showed a slightly lower solubility than the calcium carbonate, while the mussel shell / fructooligosaccharide powder had a solubility of 1.52%, which was much higher than that of calcium carbonate Solubility.

Solubility in water  ( % , w / v) Calcium carbonate powder 0.01 Calcium hydroxide powder 0.17 Mussel shell / Fructooligosaccharide mixture 0.28 Mussel shell / Fructooligosaccharide  Composite 1.52

< Example  3> Mussel shell  And Fructooligosaccharide  Compound ( FOS -Ca) Acute Toxicity

In order to confirm the acute toxicity of the mussel shell / fructooligosaccharide complex (FOS-Ca), the ionized mussel shell / fructooligosaccharide complex was added to the culture medium of the zebrafish embryo at 24 hours (24 hpf) mg / mL), and the embryo skin was removed to determine toxicity at 48 hpf. The heart rate was measured and the phenotype was confirmed.

The heart rate was measured 3 times repeatedly and statistically treated. Morphological changes were confirmed mainly by the presence of bleeding and trunk deformation.

As a result, when the zebrafish embryo was treated with the ionized mussel shell / fructooligosaccharide complex at a concentration of 0.01 to 0.1 / / mL as shown in Fig. 1, there was no significant difference in heart rate. In addition, typical morphological toxic effects such as bleeding or abnormal formation of hair, stomach, and tail have not been identified.

From the above results, it was confirmed that the mussel shell / fructooligosaccharide conjugate did not show toxicity based on heart rate, hemorrhage, and morphological changes.

< Example  4> Mussel shell  And Fructooligosaccharide  Compound ( FOS -Ca) Osteogenesis  Verify Active

When the yolk of the zebrafish embryo disappeared and the response to the food began to appear, a diet containing mussel shell / fructooligosaccharide conjugate (FOS-Ca) was administered once a day. At 21 days after the administration, the osteogenic differentiation was analyzed by staining with Alcian blue and Alizarin red.

For staining, the zebrafish was anesthetized with 0.016% tricine and fixed with 3.7% neutral buffered formaldehyde. The zebrafish was washed 3 times for 5 minutes with 1 x PBS-T, and the zebrafish was transferred to 0.1% Alcian blue solution and stained for 4 hours to overnight.

After dyeing, dehydration was carried out in 70% ethanol and 30% PBST, 50% ethanol and 50% PBST, 30% ethanol and 70% PBST. Zebrafish was transferred to 0.05% trypsin, reacted for 1 hour, and washed 3 times with 1 × PBS-T for 5 minutes. The zebrafish was transferred to a 0.05% alizarin red solution, stained for at least 4 hours, and the degree of staining was confirmed with a microscope

As a result, as shown in FIG. 2, the degree of staining for cartilage and bone was increased in the group treated with the mussel shell / fructooligosaccharide (FOS-Ca) compared with the control group.

< Example  5> Water solubility according to oligosaccharide type Mussel shell  And the solubility of oligosaccharide conjugate

 In order to confirm the soluble mussel shell and the solubility of the oligosaccharide complex according to the type of oligosaccharide, the xylo-oligosaccharide was manufactured by Sam-In Chemical Co., the maltooligosaccharide by Inglide Korea, the isomaltooligosaccharide by the company, the galactooligosaccharide by biopam, The QuantiChrom ™ Calcium Assay Kit (CIDA-500) was purchased from Goma Biotech Co., Ltd., and the complex was produced in the same manner as in Example 1. The solubility of each oligosaccharide and water-soluble mussel shell complex Respectively.

First, 10 mL of distilled water (pH 7.0) was added to a 50 mL centrifuge tube, and then the mussel shell calcium (MsCa), the mussel shell / xylose oligosaccharide conjugate (XOS-Ca), the mussel shell / maltooligosaccharide complex (MOS- , Mussel shell / isomaltooligosaccharide complex (IMOS-Ca), mussel shell / galactooligosaccharide complex (GOS-Ca) and mussel shell / fructooligosaccharide complex (FOS-Ca) .

Thereafter, centrifugation was carried out at 12,000 × g for 15 minutes to obtain an insoluble residue. The residue was dried at high temperature to measure its mass, and the solubility (w / v) in 100 mL of distilled water was confirmed using the following equation.

Calcium solubility (%) = (Sample, g - Residue, g) / Sample, gx100

As a result of confirming the solubility in distilled water at room temperature, it was confirmed that calcium carbonate and calcium hydroxide were almost insoluble in water as shown in Fig. 3, while the solubility in water was found to be 0.01% and calcium hydroxide as 0.17%, while the mussels shell and oligosaccharide complex prepared through solubilization using oligosaccharide Solubility exceeded 70%. Among them, the solubilities of the mussel shell / galactooligosaccharide combination (GOS-Ca) and the mussel shell / fructooligosaccharide combination (FOS-Ca) were 79.57% and 82.87%, respectively, which were much higher than those of calcium carbonate and mussel shell calcium .

< Example  6> Mussel shell  And Galactooligosaccharide  Compound ( GOS -Ca) and Mussel shell  And Fructooligosaccharide  Compound ( FOS -Ca) to determine the level of blood calcium change

The mussel shell / galacto-oligosaccharide conjugate (GOS-Ca) and the mussel shell / fructooligosaccharide conjugate (FOS-Ca) were orally administered to BALB / c mice once daily for 5 days. Four hours after the last administration, the serum was sampled and used to compare the calcium concentration in the blood. The total serum calcium concentration was analyzed at 612 nm using the QuantiChrom ™ Calcium Assay Kit, which can measure calcium (Ca²) concentration using phenolsulphonephthalein dye.

As a result, as shown in FIG. 4, the control group administered with only vehicle showed 9.7 mg / dl of calcium in blood, while the mussel shell / galactooligosaccharide conjugate (GOS-Ca) and the mussel shell / fructooligosaccharide conjugate -Ca) showed a blood calcium concentration of 10.7 mg / dl and 11.6 mg / dl, respectively.

From the above results, it was confirmed that both of GOS-Ca and FOS-Ca prepared under the reaction with oligosaccharide increase the calcium concentration in blood, and FOS-Ca has an excellent effect of increasing blood calcium concentration.

< Example  7> Mussels shell calcium  And Fructooligosaccharide Mixture (MsCa + FOS) and Mussel shell  And Fructooligosaccharide  Compound ( FOS -Ca) to determine the level of blood calcium change

Mortar shell calcium / fructooligosaccharide mixture (MsCa + FOS) and mussel shell / fructooligosaccharide conjugate (FOS-Ca) samples were administered to BALB / c mice once daily for 5 days, calculated as 12.5 mg of calcium per body weight . Serum was sampled 1, 2, and 4 hours after the last administration and the blood calcium levels were compared and analyzed. The total serum calcium concentration was analyzed at 612 nm using QuantiChrom ™ Calcium Assay Kit, a kit for measuring calcium (Ca²) concentration using phenolsulphonephthalein dye.

As a result, as shown in FIG. 5, there was 9.17 mg / dl of calcium in the control group administered with vehicle alone at 1 hour, and gradually increased to 8.4 mg / dl and 8.3 mg / (MsCa + FOS), the concentration of calcium in the blood increased to 9.2 mg / dl, 9.5 mg / dl and 10.0 mg / dl, respectively, over time. On the other hand, the amount of mussel shell / fructooligosaccharide complex (FOS-Ca) reached 10.2 mg / dl at 1 hour and 10.5 mg / dl and 11.0 mg / dl as time passed.

From the above results, it was confirmed that the FOS-Ca-derived calcium complex (FOS-Ca) solubilized based on fructooligosaccharide exhibits a remarkable bioavailability, and the superiority of the bioavailability compared to the mixture is merely due to the fact that fructo-oligosaccharide It is different from the previously reported that it is fed to bacteria to increase the uptake of calcium and may be suggested to be due to the improvement of solubility using various oligosaccharides.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Claims (14)

A mussel shell, and an oligosaccharide as an active ingredient. [4] The water-soluble calcium complex according to claim 1, wherein the nanocomposite is prepared by adding a mussel shell powder and an oligosaccharide to an acetic acid solution, and mixing and stirring. [3] The water-soluble calcium complex compound according to claim 2, wherein the mussel shell powder is a fine powder having an average diameter of 30 nm to 400 nm. [Claim 4] The method according to claim 1, wherein the oligosaccharide is any one selected from the group consisting of mannan oligosaccharide, fructooligosaccharide, soy oligosaccharide, galactooligosaccharide, isomaltooligosaccharide, xylooligosaccharide, agarooligosaccharide and maltooligosaccharide. Compound. The water-soluble calcium complex according to claim 1, wherein the water-soluble calcium complex comprises 10 to 90% by weight of a mussel shell and 10 to 90% by weight of an oligosaccharide. A pharmaceutical composition for preventing or treating a bone or joint disease comprising the water-soluble calcium complex compound of claim 1 as an active ingredient. [Claim 7] The pharmaceutical composition according to claim 6, wherein the bone or joint disease is selected from the group consisting of degenerative arthritis, rheumatoid arthritis, deformed arthritis, osteomalacia, osteoporosis, rickets and Paget's disease. [Claim 7] The pharmaceutical composition according to claim 6, wherein the water-soluble calcium complex agent exhibits bone formation or bone tissue regeneration. A health food for preventing or ameliorating a bone or joint disease comprising the water-soluble calcium complex compound of claim 1 as an active ingredient. Adding a mussel shell powder and an oligosaccharide to an acetic acid solution, mixing and stirring the mixture; And
And a second step of filtering the mixture. [Claim 11] The method of claim 10, wherein the acetic acid solution is 1 to 30% by weight. [Claim 11] The method according to claim 10, wherein the water-soluble calcium complex is mixed with 10 to 90% by weight of a mussel shell and 10 to 90% by weight of an oligosaccharide. [Claim 11] The method according to claim 10, wherein the first step comprises mixing the mussel shell powder and the oligosaccharide in an acetic acid solution and mixing and stirring at 20 to 80 DEG C for 1 to 10 times. [12] The method of claim 10, wherein the method further comprises a third step of drying the mixture filtered at the second step at 60 to 90 [deg.] C.

Images