KR100955075B1 - Composition for prevention and treatment of osteoporosis comprising silk peptide as an active ingredient - Google Patents

Abstract

The present invention relates to a composition containing a silk peptide, in particular the composition according to the present invention is effective in inhibiting the differentiation of osteoclasts, and provides a composition for the prevention and treatment of osteoporosis having an effect on the prevention and treatment of osteoporosis.

Silk fibroin, osteoclasts, osteoporosis, osteoblasts

Description

Composition for prevention and treatment of osteoporosis comprising silk peptide as an active ingredient}

The present invention relates to a composition for preventing and treating osteoporosis, which contains silk peptide as an active ingredient, which is highly effective for inhibiting osteoclast differentiation, which is associated with osteoporosis.

Human bones are mainly composed of osteoclasts that break bone and osteoblasts that make up bone. Among them, osteoporosis occurs when osteoclasts that break bone are stronger than osteoblasts.

Osteoporosis is a disease that anyone can get as a person ages. Especially, postmenopausal women are known to be more than twice as likely, and most of all, the density of the bones that form the human skeleton. It is known that is lowered. Human bone has been reported to decrease the bone density (bone density) of about 1% per year around 30 to 35 years. In particular, postmenopausal women lose 2-3 times the calcium in the body, 99% of the body's calcium is stored in the bones, and calcium is insufficient to maintain the structural phenomenon of getting out of the bones whenever needed. Therefore, in order to make up for the calcium deficiency with age, or taking calcium preparations, or in severe cases after menopause, estrogen, a female hormone, is prescribed by injection therapy to supplement calcium deficiency. However, these therapies have side effects such as very low absorption rate of calcium, bloating, constipation, and injection therapy of female hormone estrogen has been reported to have gynecological side effects.

Therefore, these minerals of calcium must be chelated by binding to peptides, which are protein degradants, so that they can be absorbed in the body, and functional calcium that can be absorbed in the body is all calcium-peptide-type calmodulin It is known to be able to be transported, stored and used in the body only by its structure.

Therefore, in order to supplement these problems, alternative therapies using active ingredients of natural products are required in reality, and the proper size of silk peptide forms the calcium-peptide to help the absorption of calcium in the body and further suppress the differentiation of osteoclasts. Alternatives can be made to natural products that can help.

With regard to osteoporosis patents, many osteoporosis related patents, such as Korean Patent Application No. 10-2003-7009671 and Korean Patent Application No. 10-1998-0710194, published on July 22, 2003, have been reported. The present invention provides a method for producing an osteoclastogenesis inhibitory factor and a method for genetically engineering a protein that binds to an osteoclastogenesis inhibitory factor genetically. However, even if these proteins are excluded from their efficacy, they may not be universalized due to the use of other techniques such as genetic engineering and may be subject to other usage restrictions.

Therefore, there is a continuous need to find natural proteins that are more comfortable and can directly inhibit osteoclast formation.

The present invention is effective to inhibit the differentiation of osteoclasts, and an object of the present invention is to provide a composition for preventing and treating osteoporosis having an effect on the prevention and treatment of osteoporosis.

The present invention to achieve the above object provides a composition for preventing and treating osteoporosis containing silk peptide as an active ingredient.

The composition for preventing and treating osteoporosis according to the present invention has an activity for inhibiting the differentiation of osteoclasts.

In the composition of the present invention, the silk peptide is preferably an acid of the silk or a hydrolyzate of the enzyme.

Silk peptides in the composition of the present invention comprises a degradation product of cocoon containing sericin.

In the composition of the present invention, the amino acid composition of the silk peptide is preferably 80% by weight or more of the total content of glycine, alanine and serine.

The amino acid composition of the silk peptide in the composition of the present invention is preferably 40 to 45% by weight glycine, 20 to 30% by weight alanine, and 10 to 20% by weight serine.

In the composition of the present invention, the silk peptide preferably has a weight average molecular weight of 100 to 3,500.

The present invention also provides an osteoclast differentiation inhibitor containing a silk peptide as an active ingredient.

The composition for preventing and treating osteoporosis according to the present invention includes various food compositions, such as health supplements, which have a function of preventing and treating osteoporosis as well as prophylactic and therapeutic agents for medical use.

The composition according to the present invention is effective in inhibiting the differentiation of osteoclasts, and is effective in the prevention and treatment of osteoporosis.

As described above, the composition for preventing and treating osteoporosis according to the present invention can prevent and treat osteoporosis by inhibiting osteoclast formation when applied to the human body using silk peptide as an active ingredient.

Silk peptides used in the present invention may include any conventional protein degradation products that have been used as edible proteins. For example, the silk peptide may be used from the cocoon, and a silkworm containing a sericin or a non-fibrillated dog may be used.

Preferably, those derived from silk fibroin are used. Acid, base or enzymatic hydrolyzate of silk fibroin, acid, base or enzymatic hydrolyzate of silk sericin, and the like, are not particularly limited. Addition amount of these active ingredients is different depending on the component, for example, depending on the component may be appropriately added within the range of 0.001 to 100.0% by weight, but is not necessarily limited thereto.

The amino acid composition of the silk fibroin hydrolyzate is 40 to 45% by weight of glycine, preferably 40 to 45% by weight, 20 to 30% by weight of alanine, preferably 25 to 27% by weight and 10 to 20% by weight of serine, preferably Preferably, 11 to 18% by weight is added. In addition, the sum of these three amino acid compositions is preferably 80% by weight or more. Moreover, these weight average molecular weights are about 100-3,500, Preferably it is the range of 200-1,500 in the meaning which is not restrict | limited.

Silk itself contains serine, tyrosine, and the like, which is known to have skin regeneration effect on its own (see Korean Patent No. 0366390, 0315168, etc.). From the skin regeneration effect of the silk, in addition to the effect of promoting the penetration of the active ingredient in vivo can be expected to further improve the effect of promoting the formation of osteoblasts. In addition, the molecular weight silk peptide of the appropriate size, so when taken, can also maximize the absorption of the body.

Silk peptides according to the invention can be obtained through the following process. After preparing and washing the raw fibers that cannot be fibrillated as raw materials, an acid or alkali hydrolysis method or an enzyme hydrolysis method may be used, and an enzyme hydrolysis method is preferably used. Peptides obtained as a result of hydrolysis are neutralized to approximately the neutral region, and the neutralized solution is filtered and concentrated under reduced pressure. In a preferred embodiment according to the present invention it can be used to powder the obtained concentrate as an aqueous solution or a method of high pressure reduction injection or freeze drying.

In addition, a pre-solution containing the silk peptide composition according to the present invention may be prepared, and may be added to a composition for treating or preventing osteoporosis in the form of the pre-solution.

The composition containing the silk peptide according to the present invention may be mixed with general osteoporosis symptomatic and prophylactic agents containing any active ingredient that may be used to inhibit the formation of osteoclasts, and the formulation may include liquid, It may be in the form of a drink, candy, syrup, tablet, chewing gum, granule-type powder, and the like, and these products may be prepared according to a commercial method. These active ingredients include ingredients that can be added to the effects of osteoporosis, in particular osteoclast formation inhibition closely related to osteoporosis and further to various agents for promoting and treating osteoblast formation.

Hereinafter, the content of the present invention will be described in more detail with reference to Examples. However, these examples are only presented to understand the content of the present invention, and the scope of the present invention should not be construed as being limited to these embodiments.

Example 1 Preparation of Compositions Effective for Inhibiting Osteoclasts

Preparation of silk peptide compositions effective for osteoclast inhibition begins with the process of dissolving silk. 100 g of the incapacitated dogs which cannot be fibrillated were prepared. 500 ml of pure distilled water was added to this keratin dog, and it boiled at 100 degreeC for 40 minutes. When this operation was repeated twice and washed three times, pure silk fibroin fibers from which sericin was dissolved were recovered. When converted to the ratio of the initial weight to the initial weight, it indicates that 23% by weight was lost, it can be confirmed that the pure silk fibroin. 100 g of this pure fibroin fiber was added to 240 ml of 0.1 N aqueous hydrochloric acid solution, and then subjected to 200 ° C. for 1 hour using an ultrahigh pressure reactor to completely hydrolyze. The hydrolyzed silk aqueous solution was neutralized with a 0.5 M NaOH solution to pH 7.0. In order to remove the salt (NaCl) generated afterwards, the salt was completely removed using an electric desalination apparatus. At this time, the aqueous solution was light brown, and glycine, alanine, and serine were contained in a large amount. Sodium chloride, which may occur during the neutralization process, was found to have a slightly sweet taste. This was filtered using a precision nonwoven fabric and concentrated and powdered using a concentrated reduced pressure or lyophilizer [Sample-1].

100 g of pure fibroin fiber, which is the same sample, was added to 7000 ml of distilled water, and 5% by weight of protease (protease) was added, followed by nitrogen gas control at 50 ° C. for 1 hour, and then at pH 7.0 at 50 to 55 ° C. After maintaining the high-pressure vacuum state, the enzyme was hydrolyzed by treatment at 50 ° C. for 120 hours using an ultrahigh pressure reactor. Thereafter, the residue was removed with a precision nonwoven fabric or the like to obtain an aqueous solution of enzyme hydrolysis. This solution was likewise concentrated and powdered using a concentrated reduced pressure or lyophilizer [Sample-2].

In order to obtain pure silk fibroin with high molecular weight, silk fibroin was obtained from the silkworm central silk gland. Silkworm (B.mori) 5 commands a dissecting sukjam of 6-7 days and then carefully walked two cases the central silsaem wicker tray of ice water prepared in advance 0 ℃, it is possible to obtain a large amount of ultrapure liquid fibroin were gathered in the central silsaem . This liquid pure fibroin solution was diluted as it was and powder was obtained using a lyophilizer [Sample-3].

Using each of these samples, the composition amino acid analysis was carried out using a dedicated automatic amino acid analyzer for all amino acid analysis.

Table 1 Amino Acid Composition (wt%) of Silk Peptides for Osteoporosis Prevention and Treatment

As shown in Table 1, in particular, in the case of Sample 1 and Sample-2, the content of the three amino acid compositions were all about 42 to 44% by weight of glycine, about 26% by weight of alanine, and about 11 to 18% by weight of serine. It is over 80% by weight.

In addition, the weight average molecular weight (Mw) was measured using a gel permeation chromatography (GPC) apparatus on the basis of the three samples obtained.

As shown in FIG. 1, the GPC molecular weight of Sample-1 is Mw 240, Sample-2 is Mw 1,050, and Sample-3 is Mw 108,100. Especially in the case of sample-2, it shows that the appropriate molecular weight is around 1,000.

Example 2 Osteoclast Differentiation Inhibitory Effect

On the basis of each of the three samples prepared in Example 1 it was examined whether the differentiation of osteoclasts. The cells were incubated with raw 264.7 mouse macrophage cells (osteoblast progenitor cells) at 37 ° C. and 5% CO ₂ , and then treated with RANKL, a derivative, at a concentration of 30 ng / ml to induce differentiation into osteoclasts. Normal cells will obviously differentiate into osteoclasts and, if silk peptides are treated, if differentiation into osteoclasts is clearly inhibited, they will be able to prevent differentiation into osteoclasts.

Figure 2 shows a cell photograph when the silk peptides of different molecular weight and amino acid composition prepared above were treated to osteoclasts.

As shown in FIG. 2, the weight average molecular weight Mw 240 of the sample-1, particularly the sample-2, showed a definite effect on the inhibition of osteoclast differentiation at a weight average molecular weight Mw of 1,050.

Therefore, it was possible to identify specific molecular weight bands and silk peptides that are effective in inhibiting osteoclast differentiation according to the present invention. Therefore, according to the present invention, it is possible to prepare a composition effective for the treatment and prevention of osteoporosis, as well as to industrialize the mixture with other osteoporosis agents and the like in the future.

As described above, although described with reference to a preferred embodiment of the present invention, those skilled in the art will be variously modified and modified within the scope of the present invention without departing from the spirit and scope of the invention described in the claims below. It will be appreciated that it can be changed.

1 is a result of measuring the GPC molecular weight of the silk peptide according to the present invention.

Figure 2 shows a picture of the osteoclasts treated with several different sized silk peptides prepared according to the present invention.

Claims (9)

It contains an amino acid composition of 40 to 45% by weight glycine, 20 to 30% by weight alanine, and 10 to 20% by weight serine, and contains an acid or enzymatic hydrolyzate of a silk peptide having a weight average molecular weight of 200 to 1500 as an active ingredient. Osteoporosis prevention and treatment composition having the differentiation inhibitory activity of the osteoclasts. delete delete delete delete delete delete delete delete

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