KR101648330B1 - Composition containing collagen hydrolyzate having enhanced proliferation of osteoblast - Google Patents

Abstract

The present invention relates to a composition comprising a collagen degradation product capable of proliferating osteoblasts, and more particularly, to a composition for osteoblast proliferation comprising a functional peptide isolated and identified from a collagen degradation product as an effective ingredient. According to the present invention, it is possible to isolate and identify a functional peptide that promotes osteoblast proliferation from collagen or collagen degradation products. By confirming osteoblast proliferative activity of a synthetic peptide having the functional peptide sequence, To make it possible to apply the

Description

[0001] The present invention relates to a composition containing collagen hydrolyzate having the ability to proliferate osteoblast,

The present invention relates to a composition comprising a collagen degradation product capable of proliferating osteoblasts, and more particularly, to a composition for osteoblast proliferation comprising a functional peptide isolated and identified from a collagen degradation product as an effective ingredient.

In recent years, osteoporosis and various bone diseases have become a social problem in the aging population due to the fractures of the elderly and the busy modern life. The number of osteoporosis patients in Korea is estimated to be about 10 million, and it is expected to reach 16 million by 2010. The rate of osteoporosis is more than doubled as 42.6% of women in Korea show osteoporosis or osteopenia and progress to 50, 60, 70.

As a result of the diagnosis of osteoporosis in 660 women aged 30 ~ 70 in Korea, the number of osteopenia (BMD -1.0 ~ -2.5%) was 23.3 (35.3%) and osteoporosis (BMD -2.5) was 7.3% . In addition, osteoporosis was reported in 5.1% of fifties, 12.4% of fifties, and 26% of fifties. Osteoporosis is occurring in young people in the wrong eating habit, such as osteoporosis due to aging and osteoporosis as well as lack of calcium intake due to osteoporosis and female hormone deficiency over 65 years of age. In addition, the fracture rate of elementary and junior high school students has more than doubled over the past 20 years, and it is important to raise and manage the maximum bone mass through food during the growing period to prevent osteoporosis.

Nutrition plays a very important role in maintaining structural resilience of the bone against external physical load [Bonjour JP, J. Am Coll Nutr. 2005, 24 (6), 526S-536S]. Along with the intake of adequate vitamin D along with calcium intake, protein is an important nutrient for bone health and plays a role in the prevention of osteoporosis. In particular, animal proteins have been reported to promote chronic metabolic acidosis, ie, calciuric exacerbation of bone health and mineral destruction, but a decrease in protein intake may negatively affect bone health It turned out. Thus, a selective deficiency of protein can be attributed to the rapid reduction of bone mass, microstructure and bone strength, and the characteristics of osteoporosis. In elderly subjects, hip bone fracture was frequently observed in decreasing protein intake. In addition, calcium protein administration in these patients reduced bone loss after fracture, increased muscle strength, decreased complications, and reduced hospital admission. In laboratory or clinical trials, relatively high protein intake, either animal or vegetable protein, increases bone mineral content after ingestion, reduces bone fracture rates induced by osteoporosis, and protein ingestion promotes IGF-1 Which plays a positive role in skeletal development and bone formation. In conclusion, proteins, along with calcium and vitamin D, are essential for the prevention of osteoporosis.

Several calcium-fortified proteins or peptide materials have been developed to promote bone strength, promote growth, and prevent osteoporosis. Until now, casein phosphopeptide (CPP), which is derived from milk, is a typical example, and it is known that it improves the solubility of calcium upon ingestion to help calcium absorption.

CPP is produced by decomposition of casein in the digestive tract. The CPP chelates calcium to prevent precipitation of calcium phosphate form, thereby increasing the amount of calcium in the form of ion to increase calcium availability, thereby helping calcification of bone Have animal test results.

Among the egg proteins present in egg yolk, the researchers found that using fosotidine to enhance calcium availability promotes calcium absorption and promotes bone metabolism.

The binding capacity of calcium oligopeptide extracted from fish bone with calcium was increased by proteolytic enzymes, suggesting the possibility of its use as a calcium absorption enhancer [Jung et al., British Journal of Nutrition, Vol 95, p 124-128, 2006]. Studies have been reported to increase bone mineral density (BMD) and physical bone strength in soy milk [Omi et al., Journal of Nutritional Science and Vitaminology. Vol 40, p 201-211, 1994.] It is believed that this is due to the peptides contained in soybean milk. As a result of dividing into polymeric and low molecular weight peptide groups and dividing them into four groups into experimental animals causing osteoporosis, The mineral density (BMD) and bone strength were increased in soymilk, high molecular weight soybean peptide group and low molecular weight peptide group. In addition, calcium absorption in the small intestine was significantly increased. Based on these results, it was suggested that the peptides in soymilk increased the calcium uptake rate, which may have enhanced BMD and bone strength.

 Jung et al., British Journal of Nutrition, Vol. 95, pp. 124-128, 2006  Omi et al., ≪ / RTI > Journal of Nutritional Science and Vitaminology. Vol. 40, pp. 201-211, 1994.

Under these technical backgrounds, the present inventors have made intensive efforts to develop functional peptides having osteoblast proliferative ability and compositions for osteoblast proliferation comprising them.

Accordingly, an object of the present invention is to provide a composition for osteoblast proliferation comprising a functional peptide isolated and identified from a collagen degradation product as an active ingredient.

In order to solve the above technical problem,

According to one aspect of the present invention, there can be provided a composition comprising the peptide of SEQ ID Nos. 1 to 5 having osteoblast cell proliferation activity as an active ingredient.

In one embodiment, the composition may further comprise a hydrolyzate of collagen.

In one embodiment, the hydrolyzate of the collagen may comprise at least one of the peptides of SEQ ID NOS: 1-5.

In one embodiment, the composition comprises 100-500 μM of the peptide of SEQ ID NO: 1; 10 to 500 [mu] M of the peptide of SEQ ID NO: 2; 10 to 500 [mu] M of the peptide of SEQ ID NO: 3; 10 to 500 [mu] M of the peptide of SEQ ID NO: 4; And 100 to 500 [mu] M of the peptide of SEQ ID NO: 5.

According to the present invention, it is possible to isolate and identify a functional peptide that promotes osteoblast proliferation from collagen or collagen degradation products. By confirming osteoblast proliferative activity of a synthetic peptide having the functional peptide sequence, To make it possible to apply the

FIG. 1 shows a mass spectrometry result of a collagen degradation product containing a peptide according to an embodiment of the present invention.
FIGS. 2 to 5 show results of tandem spectrum identification of peptides isolated from collagen degradation products according to an embodiment of the present invention.
FIG. 6 shows the ability of MG-63 osteoblast treated with the candidate peptide identified according to an embodiment of the present invention to proliferate.
FIG. 7 shows MG-63 osteoclast proliferating ability according to the concentration of selected peptides according to an embodiment of the present invention.
8 is an S-shaped curve graph showing the ability of MG-63 osteoblasts to proliferate according to the concentration of the peptide shown in SEQ ID NO: 1.
9 is an S-shaped curve graph showing the ability of MG-63 osteoblasts to proliferate according to the concentration of the peptide shown in SEQ ID NO: 2.
10 is an S-shaped curve graph showing the ability of MG-63 osteoblasts to proliferate according to the concentration of the peptide shown in SEQ ID NO: 3.
11 is an S-shaped curve graph showing the ability of MG-63 osteoblasts to proliferate according to the concentration of the peptide shown in SEQ ID NO: 4.
12 is an S-shaped curve graph showing the ability of MG-63 osteoblasts to proliferate according to the concentration of the peptide shown in SEQ ID NO: 5.

Hereinafter, the present invention will be described in more detail.

All technical terms used in the present invention are meant to be understood by those skilled in the art to which the present invention pertains in general, unless otherwise defined. Also, preferred methods or samples are described in this specification, but similar or equivalent ones are also included in the scope of the present invention. The contents of all publications cited herein are hereby incorporated by reference in their entirety.

Justice

As used herein, the term "peptide" means a linear molecule formed by combining amino acids with each other by peptide bonds. The peptides of the present invention can be prepared according to synthetic methods known in the art, for example, the solid phase synthesis technique (Merrifield, J. Amer. Chem. Soc. 85: 2149-54 (1963 , Stewart et al., Solid Phase Peptide Synthesis, 2nd ed., Pierce Chem. Co .: Rockford, 111 (1984)).

As used herein, the term "osteoblast" refers to a cell capable of calcining bone tissue by synthesizing and secreting a bone matrix and depositing inorganic salts such as calcium or magnesium ions on the matrix, Neovascularization, bone growth or bone regeneration. The osteoblast cells are buried in the bone tissue formed by themselves when the osteogenesis proceeds, and become osteocytes. The osteoblast proliferation degree is directly proportional to the degree of osteogenesis, osteogenesis or bone regeneration, and the degree of osteoblast proliferation at the site where osteogenesis occurs And the degree of bone formation (time or bone density, etc.) can be improved by increasing the proliferation of osteoblasts at the site.

As used herein, the term "collagen" is a light protein which is mainly present in bones and skin of animals, and is also distributed in cartilage, organs, membranes, etc., and is a fibrous solid state protein, also called collagen. The collagen is a triple helical structure in which three polypeptide chains are twisted, and particularly, a protein having a high content of hydro-proline. The collagen is difficult to be degraded by proteases such as trypsin, and can be degraded by enzymes such as collagenase. As a method for separating the collagen from an animal tissue, a method of treating with trypsin / hyaluronidase is mainly used after extraction with an organic solvent and acid or alkali treatment.

According to one aspect of the present invention, there can be provided a composition comprising the peptide of SEQ ID Nos. 1 to 5 having osteoblast cell proliferation activity as an active ingredient.

(SEQ ID NO: 1) SGPPGPPGPAGKEG

(SEQ ID NO: 2) PSGIPGLQGPAGPIGPQ

(SEQ ID NO: 3) GNAGIPGAVGLP

(SEQ ID NO: 4) GIKGPIGPPGQPGPPGP

(SEQ ID NO: 5) GPAGPRGEVGLPG

It is to be appreciated by those skilled in the art that the sequence information expressing the peptide according to the present invention has been described in order to express the peptide structure of the present invention for convenience and modifications thereof belong to the present invention. For example, it is apparent to those skilled in the art that modifications of the peptide sequences shown in SEQ ID NOS: 1 to 5 are also within the scope of the present invention.

In one embodiment, the N-terminal or C-terminal of the peptide of the present invention is an acetyl group, a fluorenylmethoxycarbonyl group, a formyl group, a palmitoyl group, a myristyl group, a stearyl group, a polyethylene glycol (PEG) A protecting group selected from the group consisting of amino acids can be additionally combined.

In one embodiment, the N-terminal or C-terminal of the peptide of the present invention may be modified with a hydroxy group (-OH) or an amino group (-NH ₂ ), and more preferably a hydroxyl group. Modification of the amino acid can greatly improve the stability of the peptide of the present invention. Here, the term "stability" means in vivo stability and storage stability (e.g., room temperature storage stability). The protecting group may function to protect the peptide according to the present invention from attack of a protein cleaving enzyme in vivo.

In one embodiment, the peptide is derived from collagen. The peptide has higher osteoblast proliferative activity than native collagen protein and has a short sequence (20 or fewer amino acid sequences) by degradation of native protein or protein synthesis. Since the peptide alone possesses osteoblast proliferative activity, it can be useful for the development of functional foods related to bone growth and / or regeneration.

Therefore, the composition comprising the functional peptide having osteoblast cell proliferating ability according to the present invention is in the form of a composition for inducing and / or improving osteogenesis, growth and / or regeneration by improving osteoblast proliferating ability at a site where osteogenesis occurs Can be provided. In addition, the composition may be provided as a food having an osteoblast cell proliferation ability, a food additive, an adjuvant, an outpatient drug, a medicament or a pharmaceutical additive.

In one embodiment, the composition may further comprise a hydrolyzate of collagen. When the composition comprising the functional peptide having the osteoblast cell proliferating ability according to the present invention is used as a food having an osteoblast cell proliferation ability, a food additive, an adjuvant, an outpatient drug, a medicinal drug or a pharmaceutical additive, osteoblast proliferative activity or osteoblast growth, Or other functional peptides for enhancing regeneration ability. In this case, the other functional peptide may be in the form of a composition containing the peptide itself or a peptide.

In one embodiment, a peptide of SEQ ID NO: 1, a peptide of SEQ ID NO: 2 of 10 to 500 M, a peptide of SEQ ID NO: 3 of 10 to 500 M, a peptide of 10 to 500 M A peptide represented by SEQ ID NO: 4, and a peptide represented by SEQ ID NO: 5 of 100 to 500 M.

Hereinafter, the present invention will be described in more detail by way of examples. However, these embodiments are not intended to limit the scope of the present invention.

Experimental Method

One . The step of hydrolysis of collagen

Ponchio collagen was hydrolyzed by direct treatment with the microbial protein hydrolytic enzymes Alcalase, Flavourzyme, Neutrase or Protamex. A solution containing 2 mg / ml or 5 mg / ml of collagen protein was prepared in 10 mM sodium phosphate buffer. Flavourzyme, Neutrase and Protamex were treated at pH 7 and Alcalase at pH 8. The ratio of hydrolytic enzyme and collagen was 2.5: 1. The reaction mixture was incubated at 50 < 0 > C. Enzymatic hydrolysis was terminated by heating in boiling water for 5 minutes and partial samples were immediately recovered at each incubation or treatment time (2, 4, 6 and 24 hours). The sample was filtered through a 0.45 μm filter and stored at -20 ° C.)

2. Separation of peptides from collagen degradation

Separation of the peptides from the collagen degradation products was carried out with Eksigent Express HT (micro HPLC) (Dublin, CA, USA) and AB Sciex, Triple-TOF 5600 (Framingham, MA 01701, USA) Peptides were selected from the largest to the lowest (see FIG. 1). Mass spectrometry chromatograms were extracted through the m / z value of the peptides. The largest amount of peptide was detected at 7.4 min and was found to have a mass of 480.2422 m / z and a charge of +2 valence.

3. Identification of isolated peptides

Identification of the peptides isolated from the collagen degradation products was first identified using a protein sequence database (Swiss-prot database) and Protein Pilot (AB Sciex), and then identified as tandem spectra. When the sequence of the selected peptide is theoretically fragmented, the N-term containing fragment is labeled as y-ion (y1, y2, y3 .... yn) and the C-term containing fragment (b1, b2, b3 ..... bn). The search score was calculated through matching with the actual tandem spectrum, and the peptide sequence was determined to be the highest value (see FIGS. 2 to 5).

The sequences of the candidate peptides (total 11) selected as a result of the above identification are shown in Table 1 below.

No. designation order Remarks P-1 COL_PEP_01 GLPGLLGNPGPLG P-2 COL_PEP_02 AGGIIRIGLG P-3 COL_PEP_03 AGPPGPTGPAGPPGFPG P-4 COL_PEP_04 PGPAGSRGAPGPQGP P-5 COL_PEP_05 GLPGSRGERGLPG P-6 COL_PEP_06 IDGRPGPIGPA P-7 COL_PEP_07 SGPPGPPGPAGKEG SEQ ID NO: 1 P-8 COL_PEP_08 PSGIPGLQGPAGPIGPQ SEQ ID NO: 2 P-9 COL_PEP_09 GNAGIPGAVGLP SEQ ID NO: 3 P-11 COL_PEP_11 GIKGPIGPPGQPGPPGP SEQ ID NO: 4 P-12 COL_PEP_12 GPAGPRGEVGLPG SEQ ID NO: 5

Example

1. Osteoblast proliferative activity of candidate peptide isolated and identified from collagen degradation product

P-7 (SEQ ID NO: 1), P-8 (SEQ ID NO: 1) and P-9 (SEQ ID NO: 1) 1), P-11 (SEQ ID NO: 1) and P-12 (SEQ ID NO: 1) peptides.

(Method for measuring proliferative capacity of osteoblast)

P-9, P-11, and P-12, respectively, are selected from the group consisting of Blank, P-1, P-2, P-3, P-4, P- After the treatment, the proliferative activity of MG-63 osteoblast was measured to be 100.0 ± 5.6%, 115.2 ± 43.0%, 110.9 ± 12.9%, 114.6 ± 7.7%, 113.4 ± 3.4%, 111.7 ± 5.3%, 125.9 ± 5.6% , 160.6 ± 30.0%, 173.4 ± 27.5%, 162.1 ± 4.4%, 157.9 ± 6.5% and 166.9 ± 17.1%, respectively. P-7 (SEQ ID NO: 1), P-8 (SEQ ID NO: 1), P-9 (SEQ ID NO: 1), P-11 (SEQ ID NO: 1) and P- It was confirmed that one sample group had higher osteoblast proliferative capacity than the other treatment groups.

2. Osteoblast proliferative activity was determined by the concentration of the selective peptide

P-8 (SEQ ID NO: 1), P-9 (SEQ ID NO: 1), P-11 (SEQ ID NO: 1) and P- 12 (SEQ ID NO: 1) peptides, the candidate peptides were independently treated with MG-63 osteoblasts at concentrations of 500 μM, 100 μM, 10 μM and 1 μM, (See Fig. 7). The measurement method is as described above.

The osteoblast proliferative activity of the P-7 peptide (SEQ ID NO: 1) was 157.8 ± 12.6%, 160.6 ± 30.0%, 116.3 ± 10.5% and 111.3 ± 12.1% at the concentrations of 500 μM, 100 μM, 10 μM and 1 μM, respectively Respectively. 8 shows the osteoblast proliferative activity according to the concentration of P-7 peptide (SEQ ID NO: 1) in an S-shaped curve graph. From the above results, it was confirmed that the maximum value of osteoblast proliferative activity by the P-7 peptide (SEQ ID NO: 1) appears near the concentration of 100 μM, and below 10 μM, there is no significant difference from the blank control.

The osteoblastic proliferative activity of P-8 peptide (SEQ ID NO: 2) was 140.7 ± 50.7%, 173.4 ± 27.5%, 145.2 ± 15.7%, and 121.2 ± 10.2% at the concentrations of 500 μM, 100 μM, 10 μM and 1 μM, respectively Effect. 9 shows the osteoblast proliferative activity according to the concentration of the P-8 peptide (SEQ ID NO: 2) in an S-shaped curve graph. From the above results, it was confirmed that the maximum value of the osteoblast proliferative activity by the P-8 peptide (SEQ ID NO: 2) appears near the concentration of 100 μM and that the concentration of 10-500 μM effectively has a higher ability to proliferate than the blank control.

The osteoblastic proliferative activity of the P-9 peptide (SEQ ID NO: 3) was 178.4 ± 22.5%, 162.1 ± 4.4%, 149.9 ± 8.4% and 124.5 ± 5.0% at the concentrations of 500 μM, 100 μM, 10 μM and 1 μM, respectively Respectively. 10, the osteoblast proliferating ability according to the concentration of the P-9 peptide (SEQ ID NO: 3) is shown by an S-shaped curve graph. From the above results, it was confirmed that the maximum value of the osteoblast proliferative activity by the P-9 peptide (SEQ ID NO: 3) appears at a concentration of 500 μM and that the concentration of 10-500 μM effectively has a higher proliferative activity than the blank control.

The osteoblast proliferative activity of the P-11 peptide (SEQ ID NO: 4) was 183.6 ± 12.7%, 157.9 ± 6.5%, 177.2 ± 4.2% and 111.3 ± 16.5% at the concentrations of 500 μM, 100 μM, 10 μM and 1 μM, respectively Respectively. 11 shows the osteoblast proliferative ability according to the concentration of P-11 peptide (SEQ ID NO: 4) in an S-shaped curve graph. From the above results, it was confirmed that the maximum value of osteoblast proliferating ability by P-11 peptide (SEQ ID NO: 4) appears at a concentration of 500 μM and that the concentration of 10-500 μM effectively has a higher proliferative activity than the blank control.

The osteoblastic proliferative activity of P-12 peptide (SEQ ID NO: 5) was 175.5 ± 15.4%, 166.9 ± 17.1%, 119.7 ± 9.7% and 113.0 ± 2.3% at the concentrations of 500 μM, 100 μM, 10 μM and 1 μM, respectively Respectively. 12 shows the osteoblast proliferative activity according to the concentration of the P-12 peptide (SEQ ID NO: 5) in an S-shaped curve graph. From the above results, it was confirmed that the maximum value of osteoblast proliferative activity by the P-12 peptide (SEQ ID NO: 5) appears at a concentration of 500 μM and that the concentration of 10-500 μM has a significantly higher ability to proliferate than the blank control.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, It will be obvious that it is not. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

                         SEQUENCE LISTING <110> RURAL DEVELOPMENT ADMINISTRATION   <120> Composition containing collagen hydrolyzate having enhanced        proliferation of osteoblast <130> NPF-25000 <160> 5 <170> PatentIn version 3.2 <210> 1 <211> 14 <212> PRT <213> Artificial <220> <223> Synthetic peptides having enhanced proliferation of osteoblast <400> 1 Ser Gly Pro Pro Gly Pro Pro Gly Pro Ala Gly Lys Glu Gly 1 5 10 <210> 2 <211> 17 <212> PRT <213> Artificial <220> <223> Synthetic peptides having enhanced proliferation of osteoblast <400> 2 Pro Ser Gly Ile Pro Gly Leu Gln Gly Pro Ala Gly Pro Ile Gly Pro 1 5 10 15 Gln      <210> 3 <211> 12 <212> PRT <213> Artificial <220> <223> Synthetic peptides having enhanced proliferation of osteoblast <400> 3 Gly Asn Ala Gly Ile Pro Gly Ala Val Gly Leu Pro 1 5 10 <210> 4 <211> 17 <212> PRT <213> Artificial <220> <223> Synthetic peptides having enhanced proliferation of osteoblast <400> 4 Gly Ile Lys Gly Pro Ile Gly Pro Pro Gly Gln Pro Gly Pro Pro Gly 1 5 10 15 Pro      <210> 5 <211> 13 <212> PRT <213> Artificial <220> <223> Synthetic peptides having enhanced proliferation of osteoblast <400> 5 Gly Pro Ala Gly Pro Arg Gly Glu Val Gly Leu Pro Gly 1 5 10

Claims (5)

100 to 500 [mu] M of the peptide of SEQ ID NO: 1;
10 to 500 [mu] M of the peptide of SEQ ID NO: 2;
10 to 500 [mu] M of the peptide of SEQ ID NO: 3;
10 to 500 [mu] M of the peptide of SEQ ID NO: 4; And
100 to 500 [mu] M of the peptide of SEQ ID NO: 5; As an active ingredient,
Wherein each of the peptides has osteoblastic cell proliferation ability.
The method according to claim 1,
Wherein the peptide is produced by hydrolysis of collagen.
The method according to claim 1,
Wherein the composition further comprises a further hydrolyzate of collagen to enhance osteoblast proliferative activity, osteogenesis, bone growth and bone regeneration ability.
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