JP6860877B2 - Osteoblast differentiation promoter and bone formation promoter - Google Patents

Description

The present invention promotes osteoblast differentiation containing a tripeptide represented by X-Hyp-Gly (where X is an amino acid residue other than Gly, Hyp and Glu) or a medically acceptable salt thereof. Regarding agents and bone formation promoters.

Collagen is synthesized intracellularly as a polypeptide chain having a repeating structure of-(Gly-XY)-(in the formula, X and Y each indicate an amino acid residue which may be the same or different), and Y Most of the Pro located in is hydroxylated to hydroxyproline (Hyp). After that, three polypeptide chains form a triple helix structure and are secreted extracellularly, and then become collagen fibers that are deposited in various connective tissues such as skin, tendons, and bones, and are major extracellular matrix proteins. Functions as. When collagenase derived from Clostridium or Glymontia is allowed to act on collagen or gelatin (heat-denatured collagen) extracted from these connective tissues, a tripeptide represented by Gly-XY with an N-terminal of Gly is produced. These microbial collagenases are endoproteases that cleave between Y and Gly, and the N-terminus of the tripeptide is Gly. On the other hand, there is also a collagen / gelatin-derived tripeptide having a Gly C-terminal (Patent Document 1). Addition of ginger rhizome-derived enzyme to collagen or gelatin that cleaves the peptide bond between the amino acid residue adjacent to the C-terminal side of Pro or Hyp and the next amino acid residue is indicated by X-Hyp-Gly. It is said that it can produce peptides.

Examples of the tripeptide represented by X-Hyp-Gly include Ala-Hyp-Gly, Leu-Hyp-Gly, and Ph-Hyp-Gly. It has been reported that when a collagen hydrolyzate containing these tripeptides is orally ingested, the blood X-Hyp-Gly concentration is significantly increased as compared with the case where a control collagen hydrolyzate is orally ingested (non-patent). Document 1). In recent years, it has been reported that collagen-derived oligopeptides such as Pro-Hyp and X-Hyp-Gly type tripeptides are detected in blood at extremely high concentrations by oral ingestion of collagen hydrolysates. Attention is focused on the effects of collagen. The physiological actions of the X-Hyp-Gly type tripeptide include the collagen synthesis promoting effect of Ala-Hyp-Gly and Pro-Hyp-Gly (Patent Document 2), Glu-Hyp-Gly, Ser-Hyp-Gly, and Ala-. Elastin production promoting effect of Hyper-Gly, Leu-Hyp-Gly, Pro-Hyp-Gly (Patent Document 3), Gly-Hyp-Gly, Leu-Hyp-Gly, Ala-Hyp-Gly dipeptidyl peptidase IV inhibitory activity (Patent Document 4), Pro-Hyp-Gly, Glu-Hyp-Gly, Ala-Hyp-Gly, Ser-Hyp-Gly angiotensin-converting enzyme inhibitory effect (Non-Patent Document 2) and the like have been reported.

There are also reports of improvement of bone metabolism by ingestion of collagen hydrolyzate (Patent Document 5) and bone differentiation promoting effect of collagen hydrolyzate (Non-Patent Document 3 and Non-Patent Document 4). Further, it has been reported that Pro-Hyp has an effect of promoting bone differentiation instead of the collagen hydrolyzed composition (Non-Patent Document 5).

Further, there is also a proliferative chondrocyte or osteoblast proliferation promoter using Glu-Hyp-Gly (Patent Document 6). Glu-Hyp-Gly prepared by the peptide solid phase synthesis method was added to normal mouse osteoblast MC3T3-E1 at 10 nmol / ml, and alkaline phosphatase activity (hereinafter referred to as ALP activity) was measured. It is said that a high osteoblast differentiation promoting effect of 1.8 times was shown, and an osteoblast proliferation promoting rate of 1.7 times higher than that of the control was shown.

International Publication No. 2014/17474 Japanese Unexamined Patent Publication No. 2010-024200 Japanese Unexamined Patent Publication No. 2014-141450 International Publication No. 2012/102308 Japanese Unexamined Patent Publication No. 2013-124221 JP-A-2015-59087

Taga Y, et al., "Efficient absorption of X-hydroxyproline (Hyp)-Gly after oral administration of a novel gelatin hydrolysate prepared using ginger protease", J. Agric. Food Chem., (2016) 64 (14): 2962 -2970 Dynamics of human blood peptides and ACE inhibitory action after ingestion of chicken collagen hydrolyzate, Journal of Japanese Society of Food Science and Technology, 2009, Vol. 56, No. 6, p326-333 Guillerminet F, et al., "Hydrolyzed collagen improves bone metabolism and biomechanical parameters in ovariectomized mice: an in vitro and in vivo study", Bone, (2010) 46 (3): 827-834. Liu J, et al., "Bovine collagen peptides compounds promote the proliferation and differentiation of MC3T3-E1 pre-osteoblasts", PLoS One, (2014) 9 (6): e99920. Kimira Y, et al., "Collagen-derived dipeptide prolyl-hydroxyproline promotes differentiation of MC3T3-E1 osteoblastic cells", Biochem. Biophys. Res. Commun., (2014) 453 (3): 498-501.

In Non-Patent Document 5, Pro-Hyp promoted the differentiation of MC3T3-E1 cells, and in Patent Document 6, Glu-Hyp-Gly increased the ALP activity of MC3T3-E1 cells. It is desired to develop a peptide which has an excellent promoting action and can be industrially produced in a large amount.

In view of the above situation, the present invention provides an osteoblast differentiation-promoting agent containing a tripeptide represented by X-Hyp-Gly (X in the formula is an amino acid residue other than Pro, Gly, Hyp and Glu). The purpose is to do.

Another object of the present invention is to provide a bone formation-promoting agent containing a tripeptide represented by X-Hyp-Gly (X in the formula is an amino acid residue other than Gly, Hyp and Glu).

The present inventors have examined in detail the tripeptides represented by X-Hyp-Gly (X in the formula is an amino acid residue other than Pro, Gly, Hyp and Glu), and found that these promote osteoblast differentiation. We have found that it has an action and completed the present invention.

That is, the present invention is one or more peptides selected from the group consisting of Ala-Hyp-Gly, Ph-Hyp-Gly, Leu-Hyp-Gly, Ser-Hyp-Gly and Thr-Hyp-Gly , or medically thereof. It provides an agent for promoting the differentiation of osteoblasts, which contains an acceptable salt in Glycine.

The present invention also presents the osteoblast differentiation-promoting agent, wherein the tripeptide contains a peptide of either Ala-Hyp-Gly or Leu-Hyp-Gly, or a medically acceptable salt thereof. It is to provide.

Furthermore, the present invention provides a bone formation-promoting agent containing the osteoblast differentiation-promoting agent.

According to the present invention, bone differentiation containing one or more peptides selected from the group consisting of Ala-Hyp-Gly, Ph-Hyp-Gly, Leu-Hyp-Gly, Ser-Hyp-Gly and Thr-Hyp-Gly. Accelerators and the like are provided.

The present invention relates to one or more peptides selected from the group consisting of Ala-Hyp-Gly, Ph-Hyp-Gly, Leu-Hyp-Gly, Ser-Hyp-Gly and Thr-Hyp-Gly , or medically thereof. An osteoblast differentiation-promoting agent containing an acceptable salt. Hereinafter, the present invention will be described in detail.

(1) X-Hyp-Gly
The tripeptides used in the present invention are represented by X-Hyp-Gly, where X is an amino acid residue other than Pro, Gly, Hyp and Glu. Preferably, it is Ala-Hyp-Gly, Phe-Hyp-Gly, Leu-Hyp-Gly, Ser-Hyp-Gly, Thr-Hyp-Gly. In order to produce the tripeptide represented by the above formula, refer to Patent Document 4, and the peptide chain represented by-(Gly-XY)-of collagen or gelatin is derived from collagenase or yellow red mold as a primary enzyme. A protease may be allowed to act, and then a peptide that releases the amino acid from the N-terminal when an amino acid other than proline or hydroxyproline is present second from the N-terminal as a secondary enzyme may be allowed to act. In addition, a ginger rhizome-derived enzyme that cleaves the peptide bond between the amino acid residue adjacent to the C-terminal side of Pro or Hyp and the next amino acid residue is added, and the enzyme reaction alone makes it easier and more abundant. The peptide represented by X-Hyp-Gly can be produced. The peptides represented by X-Hyp-Gly can be prepared by purifying from these peptide compositions by liquid chromatography or other methods.

It can also be produced by a known peptide synthesis method such as "solid phase synthesis method" or "liquid phase synthesis method". The solid-phase synthesis method may be either the Fmoc method or the Boc method. Resin beads whose surface is modified with an amino group are used as a solid phase, and diisopropylcarbodiimide (DIC) is used as a condensing agent. The N-terminal of Gly, which corresponds to the C-terminal of the tripeptide, is protected by an Fmoc group and peptide-bonded to the amino group of the resin beads. The solid phase is washed with a solvent, and then the Fmoc group of Gly bonded to the solid phase is deprotected. After washing the resin beads with a solvent, Fmoc-Hyp-OH, DIC and HOBt are added to the reaction solution, respectively. After the reaction, the resin beads are washed with a solvent, the N-terminal Fmoc group is deprotected, and the resin beads are washed with a solvent. Then, Fmoc-X-OH, DIC and HOBt are added, respectively, to synthesize a peptide on a solid phase. After the synthesis, the N-terminal Fmoc group is removed, the dried resin beads obtained after drying under reduced pressure are treated with TFA, and the X-Hyp-Gly crude product is eluted from the resin beads into TFA. The obtained crude product is dissolved in purified water and purified by liquid chromatography or the like to isolate X-Hyp-Gly.

The tripeptide represented by X-Hyp-Gly may form a salt. The salt is preferably a medically acceptable salt. "Medically acceptable salt" refers to a salt form that is pharmacologically acceptable and is substantially non-toxic to the subject to whom it is administered. For example, there are non-toxic organic or inorganic acid salts. Examples of the inorganic acid include hydrochloride, sulfuric acid, and phosphoric acid. Organic acids include carboxylic acids, phosphonic acids and sulfonic acids, such as acetic acid, propionic acid, glycolic acid, lactic acid, hydroxybutyl acid, malic acid, maleic acid, malonic acid, salicylic acid, fumaric acid, amber acid and adipic acid. , Tartrate acid, citric acid, glutaric acid, 2- or 3-glycerophosphate, and other mineral acids known to those of skill in the art. Further, it may be a salt with an inorganic base or an organic base. Inorganic bases include alkali or alkaline earth metal (eg, sodium, potassium, lithium, calcium, or magnesium) hydroxides, ammonia, ammonium hydroxides, and the like. Further, examples of the organic base include alkylamine, hydroxyalkylamine, N-methylglucamine, benzylamine, piperidine, pyrrolidine and the like.

(2) Osteoblast Differentiation Promoting Agent The tripeptide represented by X-Hyp-Gly and a medically acceptable salt thereof can be used as an osteoblast differentiation promoting agent.
The formation of bone tissue is called ossification and is roughly divided into intramembranous ossification and endochondral ossification. Intramembranous ossification is observed in the skull and the like, and refers to a bone formation mode in which mesenchymal cells directly differentiate into osteoblasts to produce bone matrix. On the other hand, intrachondral ossification means that undifferentiated mesenchymal cells differentiate into cartilage cells to form cartilage primordia, cartilage cells differentiate into hypertrophic cartilage cells, and cells contained in the surrounding cartilage membrane are osteoblasts. A mode of bone formation that differentiates into cells and replaces the cartilage tissue calcified by these with bone tissue. It is common in that osteoblasts are involved in all bone formation processes. Bone formation is promoted when osteoblast differentiation is promoted.

The mouse calvaria-derived MC3T3-E1 cell is a precursor osteoblast line established from the skull of a fetal mouse. Alkaline phosphatase (ALP) is expressed when differentiation is induced into osteoblasts, so ALP activity is used as an index of osteoblast differentiation. Therefore, if a sample is added during MC3T3-E1 cell culture and the ALP activity at the time of sample addition to an untreated control is determined, the effect on osteoblast differentiation can be investigated. It is considered that the induction of osteoblast differentiation positively contributes to various bone formations constituting the living body and promotes bone formation. However, increased ALP activity and cell proliferation are not synonymous. In the general process of cell differentiation, differentiation occurs after proliferation has stopped, and the same applies to MC3T3-E1 cells. As shown in Examples described later, the X-Hyp-Gly promotes osteoblast differentiation and can therefore be used as an osteoblast differentiation promoter.

(3) Bone Formation Promoting Agent The X-Hyp-Gly or a medically acceptable salt thereof can be used as a bone formation promoting agent. As described above, the tripeptide represented by X-Hyp-Gly and its medically acceptable salt have an osteoblast differentiation promoting action, and this differentiation promoting action can promote bone formation. ..

(4) Dosage form, etc. Osteoblast differentiation promoters and bone formation promoters can be administered orally or parenterally. In the case of oral administration, the tripeptide represented by X-Hyp-Gly or a medically acceptable salt thereof may be used as it is as a powder, a granule, a pill, a coating agent whose surface is processed with a sugar coating or the like, or a coating agent. It may be a capsule filled in a capsule, an oral solution or suspension dissolved in an appropriate solvent, an emulsion obtained by emulsifying with an emulsifier, or the like. In the case of parenteral administration, there are lotions, ointments, patches (pups), injections, suppositories, nasal drops and the like.

The osteoblast differentiation promoters and bone formation promoters include excipients, disintegrants, lubricants, preservatives, antioxidants, surfactants, pH regulators, moisturizers, etc., depending on the dosage form. Thickeners, inorganic fillers, binders, diluents, colorants, fragrances, UV absorbers and the like may be added. The X-Hyp-Gly is not limited to the one obtained by decomposing and purifying a collagen-containing food, and a protein hydrolyzate containing X-Hyp-Gly may be used without purification.

The dose of the above peptide when used as an osteoblast differentiation promoter or bone formation promoter varies depending on the condition, body weight, dosage form, administration route, etc. of the subject, but in the case of oral administration per day for adults, It is 1 to 5000 mg, preferably 2 to 2000 mg, and more preferably 5 to 500 mg. When directly administered to the affected area, it is 0.1 to 500 mg, preferably 0.2 to 200 mg, and more preferably 0.5 to 50 mg. The preparation may be administered in 1 to several divided doses per day, or may be administered once in 1 to several days.

The above-mentioned osteoblast differentiation-promoting agent and bone formation-promoting agent may be added to foods and drinks. Such foods and drinks include juices and other beverages containing vegetables, fruits, lactic acid bacteria, and semi-fluid foods such as jelly, yogurt, pudding, and ice cream, and are kneaded with other ingredients to prepare solid foods. You may.

Next, the present invention will be specifically described with reference to examples, but these examples do not limit the present invention in any way.

(Manufacturing example 1)
The exodermis of ginger rhizome was removed, shredded and stored frozen at −30 ° C., and 5 times the amount (weight / volume) of cold acetone was added to the frozen ginger and sufficiently crushed with a homogenizer. The crushed solution was filtered to separate the residue, which was washed and filtered again with 5 times the amount of cold acetone, and then air-dried at room temperature to obtain ginger enzyme powder.
A bovine skin-derived type I collagen solution was prepared by heating at 60 ° C. for 30 minutes, and hydrochloric acid was added to adjust the pH to 4.0, and dithiotreitol was added to make a 5% gelatin solution. Was prepared. To this, 1/10 amount of the ginger enzyme powder was added, and the mixture was reacted at 50 ° C. for 16 hours with shaking. After completion of the reaction, the ginger enzyme powder was removed by filtration, and the filtrate was further ultrafiltered (Vivaspin 20-10K, manufactured by GE Healthcare). The recovered filtrate was freeze-dried to obtain a peptide composition. The weight average molecular weight of this peptide composition was 590. Then, the analysis was performed under the following LC / MS measurement conditions, and the amount of major X-Hyp-Gly type tripeptide produced was quantified. Table 1 shows the amount of major tripeptide produced per 1 g of gelatin.

(result)
A large amount of various X-Hyp-Gly type tripeptides were produced from gelatin by using an enzyme derived from ginger rhizome. Among them, the amount of Ala-Hyp-Gly produced was 7.49 mg / g, and the amount of Leu-Hyp-Gly produced was 7.99 mg / g, which was particularly high compared to other X-Hyp-Gly type tripeptides. It has been shown.

(1) LC / MS measurement conditions High-performance liquid chromatograph: 1200Series (Agilent Technologies),
Mass spectrometer: 3200QTRAP (AB Sciex),
Analytical column: Ascentis Express F5 5 μm, 4.6 mm i.d. × 250 mm (SUPELCO),
Column temperature: 40 ° C,
Mobile phase: Solution A; 10 mM ammonium acetate, Solution B; 100% acetonitrile,
Gradient condition:
0-7.5 minutes: Solution A 100%,
7.5 to 20 minutes: Solution A 100 to 25%; Solution B 0 to 75%,
20 to 25 minutes: Solution A 25%; Solution B 75%,
25 to 30 minutes: Solution A 100%,
Flow velocity: 0.4 mL / min,
(2) Mass spectrometry conditions:
Ionization: ESI, positive,
Analysis mode: Multiple Reaction Monitoring (MRM) mode,
Ion spray voltage: 3kV,
Ion source temperature: 700 ° C

(Example 1)
Using mouse calvaria-derived osteoblast line MC3T3-E1 cells, the osteoblast differentiation promoting effect of each X-Hyp-Gly type tripeptide shown in Table 1 was evaluated. As the tripeptides shown in Table 1, those chemically synthesized by a custom synthesis service of Anygen Co., Ltd. were used.
MC3T3-E1 cells were prepared in 24-well cell culture plates under the condition of ^{5 × 10 4} cells / well in alpha MEM containing 10% fetal bovine serum. The next day, after confirming that the cells were confluent, the medium was differentiated medium (10% dialed fetal bovine serum (manufactured by Thermo Scientific), 100 μg / mL ascorbic acid, alpha MEM containing 10 mM glycerol 2-sodium phosphate. ), And the tripeptide shown in Table 1 was added to a final concentration of 200 nmol / mL. As a control, the same amount of distilled water was added instead of the tripeptide. The differentiation medium was changed every two days, and the tripeptides shown in Table 1 were added to a final concentration of 200 nmol / mL. After culturing for 6 days, the cells were washed with phosphate buffered saline (PBS) and fixed with 3.7% formaldehyde.

After washing with PBS, Tris buffered saline containing Tween 20, and TNM solution containing Tween 20 (0.1M Tris-HCl, pH 9.5, 0.1M NaCl, 0.05M MgCl ₂ ), NBT / BCIP (18.75 mg) / ML p-nitroblue tetrazolium chloride, 9.4 mg / mL 5-bromo-4-chloro-3-indrill phosphate p-toluidine salt (solution of 67% methyl phospoxide (v / v)) in TNM solution It was diluted 50 times with, and 300 μL was added to each well for ALP staining. The area showing ALP positive in the well was quantified by Image J.

It was repeated twice independently at N = 3, and the mean value and standard error of a total of 6 samples were shown by relative evaluation with 1 as the control. In the t-test, it was set that there was a statistically significant difference with respect to the control at p <0.05. The results are shown in Table 2.

(Comparative Example 1)
The ALP activity was calculated in the same manner as in Example 2 except that Pro-Hyp (manufactured by Bachem) was used instead of the tripeptide. The results are shown in Table 2.

(result)
Addition of each X-Hyp-Gly type tripeptide and Pro-Hyp to MC3T3-E1 cells increased ALP activity, respectively. Furthermore, the increase in ALP activity was 1.4-fold in Pro-Hyp compared to the control, whereas all the tripeptides showed a further increase in ALP activity. Since MC3T3-E1 cells express ALP when they are induced to differentiate into osteoblasts, it is considered that each tripeptide has a high effect of inducing differentiation of osteoblasts. Among them, Ala-Hyp-Gly and Leu-Hyp-Gly were shown to have a particularly high osteoblast differentiation-inducing effect.

Claims (3)

One or more peptides selected from the group consisting of A la-Hyp-Gly, Ph-Hyp-Gly, Leu-Hyp-Gly, Ser-Hyp-Gly and Thr-Hyp-Gly, or medically acceptable thereof. containing salt, differentiation promoting agent of osteoblasts. Before Kipe peptide is, Ala-Hyp-Gly or peptide is either Leu-Hyp-Gly or containing the medically acceptable salts, differentiation promoter of osteoblasts according to claim 1 .. An osteogenesis-promoting agent comprising the osteoblast differentiation-promoting agent according to claim 1 or 2.