JPH0859697A - Peptide, prolylendopeptidase inhibitor containing the same, functional food and feed for animal - Google Patents

Abstract

PURPOSE: To obtain a new peptide containing a specific amino aicd sequence obtained from bovine brain, etc., having prolylendopeptidase inhibiting activity, useful as a reagent for studies and metabolism improver, etc., a physiologically functional food and an animal feed and used for treating and preventing dementia. CONSTITUTION: This new peptide can be obtained from bovine brain, etc., and contains a specific amino acid sequence and has amino acid sequences expressed by formulas I to VIII and has prolylendopeptitase inhibiting activity, and is useful as a reagent for studies and metabolism improver, etc., and useful as a prolylendopeptidase inhibitor for treating and preventing diseases such as dementia in which prolylendopeptidase plays a role and useful also as a component for physiologically functional foods and feeds for animals. The peptide is obtained by grounding bovine brain, etc., in frozen state, charging the ground brain, etc., in a frozen state, charging the ground brain into hot water, homogenizing the ground brain, centrifuging the homogenized material and purifying the resultant supernatant by ultrafilter membrane, gel permeation, ion exchange chromatography and high-performance liquid chromatography using a reserve phase column, etc.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel peptide, a prolyl endopeptidase inhibitor containing the same as a component, a functional food and an animal feed. More specifically, the present invention relates to a peptide which is present in the mammalian brain or other living body and has prolyl endopeptidase inhibitory activity, and a prolyl endopeptidase inhibitor containing the same, a functional food and an animal feed.

[0002]

2. Description of the Related Art In many cases, bioactive peptides in vivo contain proline. Proline having an imino group in the molecule forms an acid imide bond with other amino acids during peptide formation. Peptide chains generally bend at the site of proline, and when a large number of prolines are linked, they take a unique helix structure.Therefore, peptides around proline and proline-rich proteins (proline-rich proteins) should be treated with normal proteas. -It is less likely to be decomposed by ze. Therefore, proline is contained in many physiologically active peptides, and proline-rich protein is present in large amounts between plant cell walls and animal cells and plays an important role in maintaining the structure of living tissues. There is. Particularly in mammals, collagen involved in tissue structure maintenance and cell adhesion has been known for a long time, but recently, synapsin (Science, Vol. 259, which regulates the release of neurotransmitters in the nervous system). p78
0, 1993), the acidic domain of the human transcription factor CTF1 (J.
Biol. Chem., Vol. 268, p20866, 1993), NDPP-1 (Bi
ochim. Biophys. Acta, Vol.1132, p240, 1992), etc.
The discovery of proline-rich proteins and proline-rich peptides with important functions is being made one after another.

On the other hand, prolyl endopeptidase (EC 3.
4.21.26, hereinafter referred to as PEP) is a peptidase that specifically recognizes and cleaves the peptide bond on the carboxyl group side of the proline residue in the peptide chain, and is high in the hippocampal region of the brain, which is the place of memory fixation. Besides seeing enzyme activity,
It is known to be widely distributed in animal organs (Science,
Vol.173, p827, 1971, Molecular & Cellular Biochem
istry, Vol.30, p111,1980, Journal of Japan Society for Agricultural Chemistry, Vol.5
8, p1147, 1984). Also, a similar enzyme is Flavobacteriu
It has also been discovered in bacteria belonging to the genus m (J. Biol. Chem., Vol.
255, p4786, 1980). This enzyme is a neurotransmitter such as substance P, TRH, neurotensin, and brain vasopressin (Science, Vol.221, p1310, p1310,
1983, Nature, Vol.308, p276, 1984) and is inactivated, and it is suggested that this enzyme plays an important role in the regulation of brain function. In fact, Z-Gly-Pro-CH ₂ Cl, Z-Pro-prolinal, Z-Val
It was confirmed in animal studies that synthetic PEP inhibitors such as -prolinal and Boc-Pro-prolinal have anti-dementia action (Journal of the Japanese Society of Agricultural Chemistry, Vol.58, p1147, 1984, Chemistry and Biology, Vol.2).
5, p554, 1987), and then PEP inhibitors derived from microorganisms and food ingredients have also been reported (Abstracts of the Annual Meeting of the Pharmaceutical Society of Japan p.35, Agric. Biol. Chem., Vol.55, p825). , 1
991, J. Antibiotics, Vol.44, p956, 1991, JP-A-5-3
31072 publication). On the other hand, as an animal-derived endogenous PEP inhibitor, a substance having a molecular weight of 6500 derived from pig pancreas and a substance having a molecular weight of 7000 derived from rat brain have been reported (J. Pharm.
Dyn., Vol.5, p734, 1982, Chem. Pharm. Bull, Vol.3
3, p2445, 1985).

[0004]

As described above, the synthetic P
Although the treatment of dementia diseases using EP inhibitors has been studied, new useful dementia therapeutic agents, and thus PEP inhibitors, are constantly being sought. Further, not only as pharmaceuticals, functional foods that prevent symptoms such as dementia through daily intake have been demanded recently. As a PEP inhibitor used for such a purpose, an endogenous inhibitor produced by a mammal is most desirable from the viewpoint of reducing side effects, but most of the PEP inhibitors reported so far are It was chemically synthesized or extracted from plants. As the endogenous inhibitory substance, the above-mentioned substance having a molecular weight of 6500 is known, but its structure is still unclear and its molecular weight is large. Therefore, it is considered that there is a problem in absorbability into the body and stability. In addition, as mentioned above, proteins and peptides containing a large amount of proline from mammals have been discovered one after another, but their functions have not been sufficiently elucidated, and the functions of such substances in vivo, particularly the function for the brain. There is a need for a peptide useful as a research reagent for the purpose of the above-mentioned research, and a drug for the purpose of improving diseases associated with the function of proline-rich proteins and proline-rich peptides. The present invention solves such a problem,
The present invention provides an endogenous PEP inhibitory peptide having a PEP inhibitory action, which can be used not only as a research reagent but also as a drug, a functional food, etc., and a PEP inhibitor containing the peptide, a functional food, and It is also intended to provide animal feed.

[0005]

The present inventors have found that PEP (Mol. Ce) which specifically hydrolyzes a peptide containing proline.
ll. Biochem., Vol.30, p111, 1980) and aminopeptidase P-like enzymes that hydrolyze oligoproline sequences are mainly present in the brain, and PEP and aminopeptidase P substrates or inhibitors are present in the brain. We thought that there was an unknown peptide that could be a substance. Therefore, when a peptide serving as a substrate or an inhibitor of PEP was searched for from a solution obtained by extracting bovine brain with hot water, the peptide was purified, its structure was analyzed, and further studies were conducted. The inventors have found a peptide and completed the present invention.

That is, the peptide of the present invention is a peptide consisting of L-form amino acids and represented by any of the following amino acid sequences and salts thereof. Met-Pro-Pro-Pro-Leu-Pro-Ala-Arg-Val-Asp-Phe-Ser
-Leu-Ala-Gly-Ala-Leu-Asn Gly-Val-Gln-Val-Glu-Thr-Ile-Ser-Pro-Gly-Asp-Gly
-Arg-Thr-Phe-Pro-Lys Gly-Val-Gln-Val-Glu-Thr-Ile-Ser-Pro-Gly-Asp-Gly
-Arg Glu-Pro-Pro-Pro-Pro-Glu-Pro-Pro-Pro-Ile Met-Pro-Pro-Pro-Leu-Pro-Thr-Arg-Val-Asp-Phe-Ser
-Leu-Ala-Gly-Ala-Leu-Asn Met-Thr-Pro-Pro-Leu-Pro-Ala-Arg-Val-Asp-Phe-Ser
-Leu-Ala-Gly-Ala-Leu-Asn Met-Pro-Pro-Pro-Leu-Pro-Ala Met-Pro-Pro-Pro-Leu-Pro Further, the PEP inhibitor of the present invention is It comprises at least one of a peptide or a salt thereof as an active ingredient. Furthermore, the functional food and the animal feed of the present invention comprise at least one of the above-mentioned peptide or a salt thereof.

The salt of the peptide of the present invention includes an acid addition salt and a base addition salt. As the acid addition salt, a salt with a pharmaceutically acceptable acid (inorganic acid and organic acid), for example, hydrochloride, Hydrobromide, sulfate, nitrate, acetate, benzoate, maleate, fumarate, succinate, tartrate, citrate, oxalate, methanesulfonate, toluenesulfonate , Aspartate, glutamate and the like. The base addition salt is a salt with a pharmaceutically acceptable base (inorganic base and organic base), for example, a salt with an inorganic base such as sodium salt, potassium salt, ammonium salt, calcium salt, magnesium salt, aluminum salt and the like. , Salts with basic amino acids (eg, arginine, lysine, etc.) and the like.

The peptide of the present invention can be isolated from the brain of mammals such as cattle or other organs / tissues / body fluids, but usually, amino acids are introduced stepwise by an organic chemical synthesis method. It can be synthesized by a method. Alternatively, it can be produced by a genetic engineering method, a peptide synthesis method utilizing a reverse reaction of hydrolase, or enzymatic hydrolysis of natural protein.

The peptide of the present invention can be collected from a biological component containing the peptide according to a conventional method, and examples of such a method include a biological component containing the peptide such as bovine brain and hot water. It can be obtained by performing extraction and purifying the obtained extract by subjecting it to ultrafiltration membrane, gel filtration, ion exchange chromatography, high performance liquid chromatography (HPLC) using a reverse phase column, and the like.

Solid phase peptide synthesis or liquid phase peptide synthesis is known as a method for introducing amino acids stepwise by an organic chemical synthesis method. For example, Nobuo Izumiya et al., "Basics and Experiments of Peptide Synthesis", Maruzen It is described in detail in the issue. In liquid phase peptide synthesis, the carboxyl group of the amino acid to be located at the C-terminus is replaced with benzyl group (Bzl)
Protecting with a butyl group (t-Bu) etc., the amino group of the amino acid that should be located second from the C-terminus is t-butyloxycarbonyl
(Boc), protected with a benzyloxycarbonyl group (Z), etc., dissolved in a suitable solvent such as dimethylformamide (DMF), in the presence of dicyclohexylcarbodiimide (DCC) and 1-hydroxybenzotriazole (HOBT) 4 Incubate at ℃ for about 18 hours. Then, the amino-protecting group of the product is removed by a conventional method (using trifluoroacetic acid or the like), and the resulting dipeptide is reacted with the third amino acid (also having the amino group protected) in the same manner as above. Further, the same procedure is repeated to sequentially bond the required amino acids to obtain the target peptide with the protecting group attached. When the amino acid to be reacted has a side chain functional group, it needs to be protected prior to the peptide synthesis reaction. For example, the ω-carboxyl group of Glu is the benzyl ester (OBz
l) etc., the guanidyl group of arginine is
(Tos) to protect. After completion of the final reaction, these protective groups can be removed by catalytic reduction, hydrogen fluoride (HF) or the like to obtain the target peptide.

On the other hand, solid phase peptide synthesis can be carried out using a peptide synthesizer (for example, Model 430A manufactured by Applied Biosystems). In this method, phenylacetamidomethyl (PAM) resin to which the C-terminal amino acid of the target peptide is bound, that is, amino acid protected by Boc group on the N side of amino acid -OCH ₂ -PAM is sequentially bound by automatic control. It is possible to obtain a sample in which the protective group and PAM resin are bound to the peptide. Then, a scavenger such as anisole is added to this sample, and then HF is introduced and reacted at −2 ° C. for 1 hour to release the target peptide. The liberated peptide can be obtained as a powder by washing with anhydrous ether or the like, extracting with water containing acetic acid, freeze-drying, further purifying by high performance liquid chromatography, and drying under reduced pressure.

The peptide of the present invention can also be obtained by a genetic engineering method. For example, a DNA fragment encoding the amino acid sequence (1) to (4) above is synthesized, and this DNA fragment is incorporated into an appropriate expression vector by a conventional method. The desired peptide can be prepared by transforming an appropriate host with the expression vector, culturing the obtained transformant, and isolating and purifying from the culture. When the peptide of the present invention is expressed using bacteria and / or yeast as a host for the purpose of providing it as a functional food animal feed, the peptide of the present invention is used after being isolated and purified from the culture. Alternatively, killed bacteria and / or yeast or powder thereof may be used as it is.

The peptide of the present invention obtained by the above-mentioned method is, if necessary, an ultrafiltration membrane, gel filtration, ion exchange chromatography, high performance liquid chromatography (HP
It may be further purified by a conventional means such as LC). The salt of the peptide of the present invention can be prepared by adding an acid or base to the peptide according to a conventional method.

The peptide of the present invention can be used as a research reagent for researching biological functions, particularly brain functions. In addition, if its function is sufficiently clarified,
Especially, it can be used for treatment and prevention of brain diseases. Furthermore, since the peptide of the present invention has PEP inhibitory activity, it can be used as a medicine or functional food for treating / preventing diseases associated with PEP, particularly human dementia (amnesia). It can be used as a feed for functional animals for treating / preventing dementia in animals such as cows. Since the peptide of the present invention is originally a peptidic inhibitor existing in the brain of animals, it is highly safe and has a low molecular weight, so there are few problems such as absorbability. Therefore, the peptide of the present invention is
Research reagents, metabolic improvers (for example, dementia therapeutic agents, etc.),
It can be widely used as a functional food or animal feed.

The PEP inhibitor of the present invention comprises, as an active ingredient, at least one of the peptides (1) to (3) and salts thereof described above, and a general pharmaceutical preparation containing the peptide alone or usually together with at least one pharmaceutical adjuvant. And is administered parenterally (ie, intravenous injection, rectal administration, etc.) or orally. As such a pharmaceutical preparation, various forms can be selected according to the therapeutic purpose, and typical examples thereof include tablets, pills, powders, solutions, suspensions, emulsions, granules, suppositories, soft and hard capsules, Examples include injections (solutions, suspensions, etc.), patches, inhalants and the like. These formulations can be usually prepared by a conventional formulation means using carriers such as excipients, binders, humectants, disintegrants, surfactants, adsorbents, lubricants and the like. As the carrier used for formulation, for example, in the case of molding in the form of tablets, those conventionally known in this field can be widely used, for example, lactose, sucrose, sodium chloride, glucose, urea, starch, Excipients such as calcium carbonate, kaolin, crystalline cellulose, silicic acid, water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polyvinylpyrrolidone, etc. Agent, dry starch, sodium alginate, agar-free, laminaran powder, sodium hydrogen carbonate,
Calcium carbonate, polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, stearic acid monoglyceride, starch, disintegrants such as lactose, sucrose, stearin, cocoa butter, disintegration inhibitors such as hydrogenated oil, quaternary ammonium salt base, lauryl Surfactants such as sodium sulfate, humectants such as glycerin and starch, adsorbents such as starch, lactose, kaolin, bentonite and colloidal silicic acid, purified talc, stearate, boric acid powder, lubricants such as polyethylene glycol Examples thereof include agents. Further, the tablets may be tablets coated with a usual coating, if necessary, such as sugar-coated tablets, gelatin-coated tablets, enteric-coated tablets, film-coated tablets, double tablets, and multilayer tablets.

In the case of molding in the form of pills, those conventionally known in this field can be widely used as carriers, for example, excipients such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, kaolin and talc, Gum arabic powder,
Examples thereof include tragacanth powder, a binder such as gelatin and ethanol, and a disintegrant such as laminaran and agar. When prepared as an injection, the solution, emulsion and suspension are preferably sterilized and isotonic with blood. When the solution, emulsion and suspension are formed into a form, they are diluted. All the agents conventionally used in this field can be used, for example, water, ethyl alcohol,
Examples thereof include propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, and polyoxyethylene sorbitan fatty acid esters. In this case, sodium chloride, glucose or glycerin may be contained in the pharmaceutical preparation in an amount sufficient to prepare an isotonic solution, and an ordinary buffer (sodium hydrogen phosphate, citric acid, etc.) may be added. , A solubilizing agent, a soothing agent and the like, and if necessary, a coloring agent, a preservative and the like and other pharmaceuticals may be contained in the preparation.
The injection may be a freeze-dried preparation.

The amount of the present peptide or a salt thereof to be contained in the pharmaceutical preparation of the present invention is not particularly limited and may be selected in a wide range, but usually 5 to 100%, particularly 10% in the whole composition.
70% is suitable. The administration method of the pharmaceutical preparation of the present invention is not particularly limited, and it may be administered according to various preparation forms, patient age, sex and other conditions, degree of disease and the like.
For example, tablets, pills, solutions, suspensions, emulsions, granules and capsules are orally administered. In the case of an injection, it may be administered intravenously alone or in a mixture with a normal replacement fluid such as glucose or amino acid, and if necessary, may be administered intramuscularly, intradermally, subcutaneously or intraperitoneally alone. The dose of the pharmaceutical preparation of the present invention is appropriately selected depending on the usage, the age of the patient, the sex and other conditions, the degree of disease, etc.
About 10 to 300 mg / kg body weight / day is suitable as an active ingredient, and the preparation can be administered once to several times a day in divided doses.

The functional food of the present invention comprises the above-mentioned peptide or a salt thereof, and as it is,
Alternatively, it is eaten as a functional food (or food material) for the purpose of treatment / prevention of dementia or the like by adding various nutrients or by adding it to foods and drinks. For example, after adding the appropriate auxiliaries mentioned above, using a conventional means, an edible form, such as granules, granules, tablets, capsules,
It may be formed into a paste or the like and provided for edible purposes, and various foods (for example, processed meat products such as ham and sausage, processed marine products such as kamaboko and chikuwa, dairy products such as butter and milk powder, bread and confectionery). ) Is used by adding it to water,
You may add and use it for drinks, such as fruit juice, milk, and a soft drink. The ingestion amount of the peptide of the present invention in the form of such a functional food is appropriately selected and determined according to age, weight, symptoms, degree of disease, form of food, etc., and is, for example, 10 per day.
Approximately 300 mg / kg body weight.

The animal feed of the present invention comprises the above-mentioned peptide or a salt thereof, and as it is,
Alternatively, various nutrients may be added or contained in foods and drinks to be fed as a functional feed for the purpose of treatment / prevention of dementia of animals such as dogs, cats and cattle. The feed can be prepared according to a conventional method according to the target animal, for example, in the case of dogs, a solid form,
It is prepared into pet food such as semi-paste and granules and jerky. Further, it may be in the form of an additive for drinking water. The intake amount of the peptide of the present invention in the form of animal feed is appropriately selected and determined depending on age, body weight, symptoms, degree of disease, form of feed and the like. The peptide of the present invention can be used not only as a feed for animals, but also as a drug for animals for the purpose of treatment / prevention of dementia and the like.

[0020]

INDUSTRIAL APPLICABILITY According to the present invention, a peptide having PEP inhibitory activity is provided and, based on the inhibitory activity, it can be used as a reagent for studying brain function, PEP inhibitor, functional food and the like. Further, the PEP inhibitor, functional food and animal feed of the present invention contain the above-mentioned peptide as an active ingredient, and according to the inhibitor, food and feed, P
It is possible to treat / prevent diseases associated with EP, such as dementia. In particular, the peptide of the present invention, which is an active ingredient, has the characteristics of being highly safe because it is an endogenous peptide, and excellent in absorbability because it is a low-molecular substance.

[0021]

EXAMPLES The present invention will be described below based on Examples, Test Examples and Preparation Examples, but the present invention is not limited to these Examples. Example 1 Separation / Purification of Peptide of the Invention from Bovine Brain 400 g of bovine brain crushed in a frozen state was put into hot water at 80 ° C., heated for 10 minutes, and then homogenized using a polytron. After removing the precipitate by centrifugation at 7,000 xg for 60 minutes and then at 27,000 xg for 30 minutes, the molecular weight cut-off was 10,0.
The product was passed through an ultrafiltration membrane PM-10 (Amicon Co., Ltd.) of 00 to collect low-molecular weight substances passing through. This sample was loaded on a Sephadex LH-20 column (Pharmacia, 26 x 900 mm), eluted with 20% methanol, and had a fraction having an inhibitory activity against microbial PEP (the measuring method is described in Test Example 1) (ie, 204-252 ml fractions) were collected. This fraction is then collected in DEAE Toyopearl 65
A 0M column (16 x 650 mm, manufactured by Tosoh Corporation) was loaded. Then, PEP inhibitory activity was observed in four of the flow-through fraction eluted with distilled water (eluted in 113 to 115 ml) and the fraction eluted with a linear concentration gradient of 0 to 1 M ammonium formate, and Five active fractions were collected.

The four active fractions eluted from the above column with a linear concentration gradient of ammonium formate were respectively subjected to high performance liquid chromatography (HPLC) using a μBondapak C18 column (3.9 x 300 mm, manufactured by Waters).
Fractionation was performed by linear gradient elution with 0-63% acetonitrile containing 0.1% trifluoroacetic acid. This active fraction was further analyzed by HPLC using an ion exchange column Shim-Pack PA-SP (manufactured by Shimadzu Corporation) with 0-0.5M sodium phosphate buffer (p
Fractionation by linear concentration gradient elution of H 6.5)
sphere C18 column (3.9 x 150mm, manufactured by Waters)
Using HPLC (0.7-63% with 0.1% trifluoroacetic acid)
Was eluted with acetonitrile) to obtain 3 kinds of active fractions (that is, fraction A eluted with acetonitrile concentration: about 26-34%, fraction B eluted with about 20% of acetonitrile, 17%
Fraction C) which elutes to the extent. Each of the obtained fractions was repurified by repeating HPLC twice with a μBondasphere C18 column. The three types of peptides that could be collected were dried under reduced pressure to give final preparations.

The structures of these inhibitors were analyzed by a protein sequencer 477A manufactured by Applied Biosystems. As a result, the structures of the peptides in the above-mentioned fractions A to C were as follows. Met-Pro-Pro-Pro-Leu-Pro-Ala-Arg-Val-Asp-Phe-Ser-Le
u-Ala-Gly-Ala-Leu-Asn (SEQ ID NO: 1) Gly-Val-Gln-Val-Glu-Thr-Ile-Ser-Pro-Gly-Asp-Gly-Ar
g-Thr-Phe-Pro-Lys (SEQ ID NO: 2) Gly-Val-Gln-Val-Glu-Thr-Ile-Ser-Pro-Gly-Asp-Gly-Ar
g (SEQ ID NO: 3)

Further, with respect to the flow-through fraction eluted from the DEAE-Toyopearl 650M column with distilled water, this fraction was further subjected to high performance liquid chromatography using a μBondapak C18 column (3.9 x 300 mm, manufactured by Waters). HP
LC) was fractionated by linear gradient elution of 0-63% acetonitrile containing 0.1% trifluoroacetic acid. This active fraction was further fractionated by HPLC using an ion exchange column Shim-Pack PA-SP (manufactured by Shimadzu Corporation) by elution with a linear concentration gradient of 0 to 0.5 M sodium phosphate buffer (pH 6.5), and μ
HPLC using a Bondasphere C18 column (3.9 x 150 mm, manufactured by Waters) was repeated twice (first elution with a concentration gradient of 0.7-63% acetonitrile containing 0.1% trifluoroacetic acid, second time 0.1% trifluoro). Contains acetic acid 1
Elution with a concentration gradient of 7.5-22% acetonitrile),
Purified. The collected peptide was dried under reduced pressure to give the final preparation. The structure of this peptide was analyzed by a protein sequencer 477A manufactured by Applied Biosystems. As a result, it was revealed that the peptide was the one shown below. Glu-Pro-Pro-Pro-Pro-Glu-Pro-Pro-Pro-Ile (SEQ ID NO: 4)

Example 2 SEQ ID NO: 1 (Met-Pro-Pro-Pro-Leu-Pro-Ala-Arg-Val-As
p-Phe-Ser-Leu-Ala-Gly- Ala-Leu-Asn)
Chemical synthesis of tide In a peptide synthesizer (430A type) manufactured by Applied Biosystems, 0.5 mmol of Boc-Asn-O-CH ₂ -PAM resin and 2 mmol of each Boc-Met (1 bottle), Boc-Pro ( 4), Boc
-Leu (3), Boc-Ala (3), Boc-Arg (Tos) (1),
Boc-Val (1 pc), Boc-Asp (OBzl) (1 pc), Boc-Phe (1
Book), Boc-Ser (Bzl) (1 pc), Boc-Gly (1 pc) cartridges, and Met-Pro-Pro-P by DCC anhydrous symmetry method
ro-Leu-Pro-Ala-Arg (Tos) -Val-Asp (OBzl) -Phe-Ser (Bzl)
The -Leu-Ala-Gly-Ala- Leu-Asn-O-CH 2 -PAM were synthesized. Boc is a t-butyloxycarbonyl group, Tos is a tosyl group, OBzl is a benzyloxy group, and Bzl is a benzyl group. All amino acids used here are in the L-form. Next, the above synthetic peptide resin was introduced into a hydrogen fluoride apparatus manufactured by Peptide Institute, and after adding 1.5 ml of anisole, 10 ml of hydrogen fluoride was introduced. After reacting at -2 ° C for 1 hour, hydrogen fluoride was removed under reduced pressure, the peptide was washed with anhydrous ether and chloroform alternately three times, and the peptide was dissolved in 60 ml of 2N acetic acid and freeze-dried. By this method, 200 mg of the desired title peptide was obtained as a white powder. The peptide was then purified by HPLC. The purification conditions by HPLC are shown below. Column: Merck LiChrospher RP-18 (e) (10 x 250
mm) Eluent: linear concentration gradient of 3.5 to 67% acetonitrile containing 0.1% trifluoroacetic acid Flow rate: 6 ml / min The synthesized peptide was hydrolyzed with 6N hydrochloric acid at 110 ° C for 24 hours, and was then analyzed by Hitachi 835 type amino acid analyzer. Further, the structure was confirmed by FAB-MS method using a JEOL HX-110 mass spectrometer by amino acid analysis. The retention time of the synthesized peptide by HPLC under the following conditions was 7.45 minutes, which was in agreement with that of the peptide purified from brain. HPLC conditions Column: μBondasphere C18 (3.9 x 150 mm) Eluent: 26-34% (30% containing 30% of 0.1% trifluoroacetic acid)
Min) Acetonitrile linear concentration gradient Flow rate: 1 ml / min

Example 3 SEQ ID NO: 4 (Glu-Pro-Pro-Pro-Pro-Glu-Pro-Pro-Pro-Il
Chemical synthesis of peptide shown in e) 0.5 mmol Boc-Ile-O-CH ₂ -PAM resin and 2 mmol Boc-Glu (OBzl) (2 mmol each) were applied to a peptide synthesizer (430A type) manufactured by Applied Biosystems. Books), Boc-Pro (7 books)
Glu (OBzl) -Pro-P using DCC anhydrous symmetry method
ro-Pro-Pro-Glu ( OBzl) -Pro-Pro-Pro-Ile-O-CH 2 -PAM was synthesized. Next, after introducing the above synthetic peptide resin into a hydrogen fluoride device manufactured by Peptide Institute and adding 3 ml of anisole,
30 ml of hydrogen fluoride were introduced. After reacting at -2 ° C for 1 hour, hydrogen fluoride was removed under reduced pressure to remove the peptide from anhydrous ether,
The peptide was dissolved in 60 ml of 2N acetic acid and lyophilized. By this method, 130 mg of the desired title peptide powder was obtained. The peptide was then purified by HPLC. The purification conditions by HPLC are shown below. Column: Merck LiChrospher RP-18 (e) (10 x 250
mm) Eluent: Gradient of 3.5 to 67% acetonitrile containing 0.1% trifluoroacetic acid Flow rate: 6 ml / min The synthesized peptide was hydrolyzed with 6N hydrochloric acid at 110 ° C for 24 hours and then analyzed by the Hitachi 835 amino acid analyzer for amino acid analysis. By conducting the analysis, the structure was further confirmed by the FAB-MS method using a JEOL HX-110 type mass spectrometer. Further, the synthesized peptide had a retention time of 4.04 minutes by HPLC using a reverse phase column shown below, which was almost the same as the retention time of 4.05 minutes of the peptide extracted from bovine brain. HPLC conditions Column: μBondasphere C18 (3.9 x 150 mm) Eluent: 17.5-22% (30% containing 0.1% trifluoroacetic acid)
Min) Acetonitrile linear concentration gradient Flow rate: 1 ml / min

Example 4 SEQ ID NO: 2 (Gly-Val-Gln-Val-Glu-Thr-Ile-Ser-Pro-Gl
y-Asp-Gly-Arg-Thr-Phe-Pro-Lys) and SEQ ID NO: 3 (Gly
-Val-Gln-Val-Glu-Thr-Ile-Ser-Pro-Gly-Asp-Gly-Arg)
Chemical Synthesis of Peptide Shown in Example 2 Similar to Example 2, it was synthesized by an anhydrous symmetry method by DCC using a peptide synthesizer (430A type) manufactured by Applied Biosystems, and the protecting group and the resin were cleaved with hydrogen fluoride. Thus, the desired title peptide was obtained.
Peptide purification and analysis conditions are the same as described above. Also,
The retention times by HPLC of the obtained peptides were consistent with those of peptides purified from brain.

Test Example 1 Measurement of PEP Inhibitory Activity The PEP inhibitory activity in Example 1 was measured by the following method 1), and P of the peptides obtained in Examples 2 to 4 was measured.
The EP inhibitory activity was measured by the following method 2). 1) Measurement of inhibitory activity against PEP derived from microorganisms PE derived from F. meningosepticum purchased from Seikagaku Corporation
P was dissolved in 0.1M phosphate buffer of pH 7.0 to prepare a 0.1 unit / ml enzyme solution. In addition, 2 mM Z-Gly-Pro-pNA (manufactured by Backchem, Z is a benzyloxycarbonyl group, pNA is paranitroanilide) was dissolved in the above phosphate buffer (containing 40% dioxane) to form a substrate solution. did. Sample solution containing the test peptide in a 1.5 ml plastic tube
Add 40 μl, and add 80 μl phosphate buffer and 40 μl substrate solution.
l was added and kept warm at 30 ° C for 10 minutes, then the PEP solution 4
0 μl was added and mixed well, and the reaction was carried out at 30 ° C. for 10 minutes. Then, the reaction was stopped by adding 200 μl of 1N-hydrochloric acid. After the reaction was stopped, paranitroaniline liberated by the enzymatic reaction was quantified by HPLC. The HPLC measurement conditions are as follows.

HPLC measurement conditions Column: Waters μBondasphere 5μ C8-300
A (3.9 x 150 mm) Elution: 53% acetonitrile containing 0.1% trifluoroacetic acid Detection: Absorption at 410 nm Such an experiment was performed multiple times, and the inhibition rate was calculated from the following formula. In the formula, A: peak area of para-nitroaniline without inhibitor B: peak area of para-nitroaniline with inhibitor added

2) Measurement of inhibitory activity against bovine brain-derived PEP
200 ml of 20 mM Tris-HCl buffer (pH 7.0) containing 10 mM EDTA and 10 mM 2-mercaptoethanol that had been sufficiently cooled (4 ° C) was added to 400 g of constant crushed bovine brain, and the container was homogenized using a polytron while cooling with ice. Later, 4 ℃, 30,000 xg
Centrifugation was performed for 20 minutes and the supernatant was collected. Then
Ammonium sulfate fractionation (50-80%) of the supernatant, followed by Q-Sepharose column chromatography, PBE94 column chromatography, and Superdex 200 column chromatography in order to purify brain PEP, and to prepare 0.1 unit / ml enzyme solution. did.
In addition, 2 mM Z-Gly-Pro-pNA was dissolved in the above Tris-HCl buffer solution (containing 40% dioxane) to obtain a substrate solution. Into a 1.5 ml capacity plastic tube, put 40 μl of the sample solution containing the test peptide, and add 80 μl of phosphate buffer, P above.
After 40 μl of EP solution was added and kept at 37 ° C. for 5 minutes, 40 μl of substrate solution was added and mixed well, and reacted at 37 ° C. for 10 minutes. Hereinafter, the measurement is performed under the same conditions as in the case of measuring the inhibitory activity against PEP derived from a microorganism, and the concentration of the peptide (IC ₅₀ ) when the inhibition rate is 50% and the dissociation constant (Ki) of the peptide-enzyme complex are measured. I asked. The results of measuring the inhibitory activity are shown in Table 1. As shown in Table 1, it was revealed that the peptides of the present invention represented by SEQ ID NOs: 1 to 4 have PEP inhibitory activity.

[0031]

[Table 1]

Example 5 PEP Activity of Similar Peptide of Peptide of SEQ ID NO: 1 As shown in Table 1, the peptide of SEQ ID NO: 1 has a strong PEP inhibitory effect. Peptides are glial fibrillary acidic
3 of protein (glial fibrillary acidic peptide, GFAP)
It was revealed that they are peptide residues corresponding to Nos. 8 (Met) to 55 (Asn). GFA depending on animal species
Since the sequences of P Nos. 38 to 55 are slightly different, in order to investigate the effect of this difference on the PEP inhibitory activity,
A peptide having the amino acid sequence of the 38th to 55th of human GFAP (SEQ ID NO: 5, alanine at the 7th position of SEQ ID NO: 1 is replaced with threonine) and 38th to 5th of mouse GFAP
A peptide having the amino acid sequence of No. 5 (SEQ ID NO: 6, the second proline in SEQ ID NO: 1 was replaced with threonine) was synthesized, and the PEP inhibitory activity thereof was measured.

SEQ ID NO: 5 (Met-Pro-Pro-Pro-Leu-Pro-T
hr-Arg-Val-Asp-Phe-Ser-Leu-Ala-Gly-Ala-Leu-Asn) and SEQ ID NO: 6 (Met-Thr-Pro-Pro-Leu-Pro-Ala-Arg-Val-A)
Chemical synthesis of peptide represented by sp-Phe-Ser-Leu-Ala-Gly-Ala-Leu-Asn) and measurement of PEP inhibitory activity In the same manner as in Example 2, a peptide synthesizer manufactured by Applied Biosystems (430A type) Was synthesized by the anhydrous symmetry method using DCC, and the protecting group and the resin were cleaved with hydrogen fluoride to obtain the desired title peptide.
Peptide purification and analysis conditions are the same as described above. Regarding the peptide obtained above, according to Test Example 1-2),
The inhibitory activity against bovine brain-derived PEP was measured. The results are shown in Table 2.

[0034]

[Table 2]

As shown in Table 2, SEQ ID NOs: 5 and 6
Peptide of P is almost equivalent to the peptide of SEQ ID NO: 1
EP inhibitory activity was observed and 3 of GFAP was observed regardless of animal species.
The peptide having the amino acid sequence of 8th to 55th is PEP
It became clear that it has an inhibitory effect.

Example 6 Preincubation of PEP with the peptide of SEQ ID NO: 1
As shown in Table 1, the peptide of SEQ ID NO: 1 is PEP
It has been shown to have inhibitory activity, but this peptide is a substance having a PEP inhibitory action in the true sense, or is actually a substrate of PEP and only apparently exhibits an inhibitory action. To confirm that
The preincubation time of EP with the peptide of SEQ ID NO: 1 was extended to 60 minutes and its inhibitory activity was tested over time. Table 3 shows the results. In addition, in order to confirm the presence or absence of decomposition of the peptide at this time, the reaction solution after the preincubation for 60 minutes was subjected to reverse phase chromatography, each peak was collected, and each was subjected to structural analysis by a protein sequencer. It was

[0037]

[Table 3]

As shown in Table 3, no change was observed in the PEP inhibitory activity of the peptide of SEQ ID NO: 1 even after preincubation for 60 minutes. From this, it became clear that the peptide of SEQ ID NO: 1 is a peptide having a PEP inhibitory action. On the other hand, as a result of subjecting the reaction solution after incubation to reverse phase chromatography, about 10 to 20% of the peptide was degraded, and the peptide consisting of 6 residues on the N-terminal side of the peptide of SEQ ID NO: 1 (SEQ ID NO: It was found that 7) was generated. This indicates that the peptide of SEQ ID NO: 1 is a substrate for PEP. Thus, it was revealed that the peptide of SEQ ID NO: 1 is a substrate of PEP and is degraded by PEP. However, as described above, the peptide of SEQ ID NO: 1 inhibits PEP even when preincubated with PEP for 60 minutes. Since there was no change in activity,
The present inventors speculated that the peptide of SEQ ID NO: 7 may also have a PEP inhibitory action. Based on this assumption,
The peptide of SEQ ID NO: 7 was synthesized and its PEP inhibitory activity was measured. For comparison, a peptide consisting of 7 residues on the N-terminal side of the peptide of SEQ ID NO: 1 (SEQ ID NO: 8) and a peptide consisting of 5 residues on the N-terminal side (Met-Pro-
Pro-Pro-Leu (hereinafter referred to as comparative peptide) was also synthesized, and the PEP inhibitory activity thereof was measured.

Example 7 SEQ ID NO: 7 (Met-Pro-Pro-Pro-Leu-Pro), SEQ ID NO: 8 (Me
t-Pro-Pro-Pro-Leu-Pro-Ala) peptide and ratio
Chemical synthesis of comparative peptide and measurement of PEP inhibitory activity As in Example 2, the peptide was synthesized by the anhydrous symmetry method by DCC using a peptide synthesizer (430A type) manufactured by Applied Biosystems, and protected with hydrogen fluoride to give a protecting group. The desired title peptide was obtained by cleaving the resin.
Peptide purification and analysis conditions are the same as described above. Also,
The retention time of the obtained peptide of SEQ ID NO: 7 by HPLC was in agreement with those of the peptide purified from brain. For the peptides obtained above, the inhibitory activity against bovine brain-derived PEP was measured according to Test Example 1-2).
The results are shown in Table 4.

[0040]

[Table 4]

As shown in Table 4, the peptide of SEQ ID NO: 7 showed a PEP inhibitory activity almost equivalent to that of the peptide of SEQ ID NO: 1. In addition, PE that is weak against the peptide of SEQ ID NO: 8
P inhibitory activity was observed. On the other hand, the comparative peptide, which is a peptide lacking the C-terminal Pro of SEQ ID NO: 7, is PE
No P inhibitory activity was observed. From this, the peptide of SEQ ID NO: 7 was considered to be the PEP inhibitory active site of SEQ ID NO: 1. Therefore, the peptide of SEQ ID NO: 7 was preincubated with PEP, and its inhibitory activity was tested over time. The results are shown in Table 5. In addition, in order to confirm the presence or absence of decomposition of the peptide at this time, the reaction solution after the preincubation for 60 minutes was subjected to reverse phase chromatography, each peak was collected, and each was subjected to structural analysis by a protein sequencer. It was

[0042]

[Table 5]

As shown in Table 5, the peptide of SEQ ID NO: 7 showed no reduction in PEP inhibitory activity even after preincubation with PEP for 60 minutes. As a result of subjecting the reaction solution after incubation to reverse phase chromatography, substantially no degradation of the peptide of SEQ ID NO: 7 was observed. From the above, it was revealed that the peptide of SEQ ID NO: 7 is not a substrate but a peptide having a true PEP inhibitory action. The peptide of SEQ ID NO: 1 is a substrate for PEP, but the peptide of SEQ ID NO: generated by the PEP inhibitory action and the degradation caused by the steric hindrance action of the residue from the 7th to the C-terminal side. It was considered that the PEP inhibitory action was expressed by the peptide of 7.

Preparation Example 1 Peptide of SEQ ID NO: 1 5 mg Magnesium stearate 5 mg Corn starch 20 mg Lactose 174.5 mg According to a conventional method, the mixture having the above composition was tableted to give tablets.

Preparation Example 2 Peptide of SEQ ID NO: 4 5 mg Magnesium stearate 5 mg Corn starch 20 mg Lactose 174.5 mg According to a conventional method, the mixture having the above composition was tableted to give tablets.

Preparation Example 3 Peptide of SEQ ID NO: 7 5 mg Magnesium stearate 5 mg Corn starch 20 mg Lactose 174.5 mg According to a conventional method, the mixture having the above composition was tableted to give tablets.

Preparation Example 4 After mixing the peptide of SEQ ID NO: 1 with the meat for wiener sausage at a ratio of 10 g per 50 kg of the meat,
Fill the sausage casing according to the usual method, smoke,
It was sterilized, and after cooling, it was packaged to obtain a wiener sausage.

Preparation Example 5 A peptide of SEQ ID NO: 1 was mixed with a jerky type meat for pet food at a ratio of 5 g per 50 kg of the meat, and then molded using an extrusion molding apparatus according to a conventional method. ,
It was smoked, heat-sterilized and dried to obtain a pet food jerky having a water activity of 0.84.

[0049]

[Sequence Listing] SEQ ID NO: 1 Sequence length: 18 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Met Pro Pro Pro Leu Pro Ala Arg Val Asp Phe Ser Leu Ala Gly 1 5 10 15 Ala Leu Asn 18

SEQ ID NO: 2 Sequence length: 17 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe 1 5 10 15 Pro Lys 17

SEQ ID NO: 3 Sequence length: 13 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg 1 5 10 13

SEQ ID NO: 4 Sequence length: 10 Sequence type: Amino acid Topology: Linear Sequence type: Peptide

SEQ ID NO: 5 Sequence length: 18 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Met Pro Pro Pro Leu Pro Thr Arg Val Asp Phe Ser Leu Ala Gly 1 5 10 15 Ala Leu Asn 18

SEQ ID NO: 6 Sequence length: 18 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Met Thr Pro Pro Leu Pro Ala Arg Val Asp Phe Ser Leu Ala Gly 1 5 10 15 Ala Leu Asn 18

SEQ ID NO: 7 Sequence length: 6 Sequence type: Amino acid Topology: Linear Sequence type: Peptide

SEQ ID NO: 8 Sequence length: 7 Sequence type: Amino acid Topology: Linear Sequence type: Peptide

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical indication location C07K 7/08 C12N 9/99 (72) Inventor Hideoki Tanaka 1-3 cg Higashi, Tsukuba City, Ibaraki Prefecture Institute of Biotechnology, Industrial Technology Institute of Technology (72) Inventor, Oka Omori, 3-3 Midorigahara, Tsukuba, Ibaraki Prefecture, Central Research Laboratory, Nippon Ham Co., Ltd.

Claims (4)

[Claims] 1. A peptide composed of L-amino acids and represented by the following amino acid sequence and salts thereof. Met-Pro-Pro-Pro-Leu-Pro-Ala-Arg-Val-Asp-Phe-Ser
-Leu-Ala-Gly-Ala-Leu-Asn Gly-Val-Gln-Val-Glu-Thr-Ile-Ser-Pro-Gly-Asp-Gly
-Arg-Thr-Phe-Pro-Lys Gly-Val-Gln-Val-Glu-Thr-Ile-Ser-Pro-Gly-Asp-Gly
-Arg Glu-Pro-Pro-Pro-Pro-Glu-Pro-Pro-Pro-Ile Met-Pro-Pro-Pro-Leu-Pro-Thr-Arg-Val-Asp-Phe-Ser
-Leu-Ala-Gly-Ala-Leu-Asn Met-Thr-Pro-Pro-Leu-Pro-Ala-Arg-Val-Asp-Phe-Ser
-Leu-Ala-Gly-Ala-Leu-Asn Met-Pro-Pro-Pro-Leu-Pro-Ala Met-Pro-Pro-Pro-Leu-Pro 2. A prolyl endopeptidase inhibitor containing at least one of the peptide according to claim 1 or a salt thereof as an active ingredient. 3. A functional food containing at least one of the peptide according to claim 1 or a salt thereof. 4. An animal feed containing at least one kind of the peptide according to claim 1 or a salt thereof.