JP2007126430A - New seaweed glycoprotein-derived peptide and prolylendopeptidase inhibitor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new peptide having a prolylendopeptidase inhibitory activity by extracting seaweed glycoprotein from seaweed extract and producing the same from the seaweed glycoprotein. <P>SOLUTION: This seaweed-derived glycoprotein is obtained by extracting the dried powdery material of the seaweeds (Undaria pinnatifida, Cladosiphon okamuranus, Eisenia bicyclis, Laminaria japonica, Hizikia fusiformis, Sarugassum fulvellum, Surgassum horneri, Porphyra yezoersis, etc.) with an aqueous weakly alkaline solution and then performing an ethanol fractionation. Further, the new seaweed glycoprotein-derived peptide obtained after cutting it to sugar chain and peptide chain and then isolating/purifying with a reverse phase HPLC is provided by having a peptide structure of an L-formed amino acid sequence of Ile-Lys-Pro-Gly-Val-Val-Val-Ala-Ala-Ala-Ile-Val-Gly-Gly-Tyr, and having a prolylendopeptidase inhibitory activity, also an amnesia resistant activity in a living body and an extremely low toxicity. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

The present invention relates to novel seaweed glycoprotein-derived peptides and prolyl endopeptidase inhibitors.

Seaweed glycoprotein-derived peptides have advantages as prolyl endopeptidase inhibitors and anti-amnesic agents.
Maruyama, S .; Et al .: Biosci. Biotech. Biochem. 60, 358-359 (1996). kimura, K .; Et al .: Biosci. Biotech. Blochem. 61, 1754-1756 (1997). Amano, H.M. Etc .: Nissui Magazine, 58, 291-299 (1992). Yoshimoto, T .; Et al: Biochem. Biophys. Acta, 569, 184-189 (1979).

Senile dementia is broadly classified into two types: Alzheimer's disease, and cerebrovascular dementia is classified into cerebrovascular dementia caused by cerebral infarction, intracerebral hemorrhage, subarachnoid hemorrhage and the like. It has been reported that the activity of an enzyme called prolyl endopeptidase is increased in the brain of patients with amnesia and dementia. If this enzyme is weakened, it can be expected to prevent or treat forgetfulness or dementia [ Non-patent document 1] [Non-patent document 2]. On the other hand, the protein of seaweed (Wakame, Okinawa Mozuku, Arame, Kombu, Hijiki, Honda Walla, Akamoku, Nori, etc.) has a high nitrogen content, and although it is quantitatively a component next to carbohydrates, There are only studies on chromoproteins [Non-Patent Document 3], and details on seaweed protein and seaweed glycoprotein are completely unknown. Therefore, the prolyl endopeptidase relating to the seaweed protein-derived and seaweed glycoprotein-derived peptides and the anti-amnesic effect by oral administration are still unclear, and there is no report that development as a pharmaceutical has been advanced.

As a result of diligent research to solve the above problems, the present inventor has found that the present invention has been obtained from glycoproteins of seaweed (Wakame, Okinawa Mozuku, Alame, Kombu, Hijiki, Honda Walla, Akamok, Nori, etc.). The present inventors have found that such a peptide has an anti-amnesic effect and have completed the present invention. That is, a substance having a pharmacological action is searched from a glycoprotein fraction of seaweed (Wakame, Okinawa mozuku, arame, kombu, hijiki, hondawala, akamoku, seaweed, etc.), and this novel seaweed glycoprotein-derived peptide is a strong prolyl endopeptidase. It was found to have an inhibitory effect. And, as a result of intensive research for practical application of this novel seaweed glycoprotein-derived peptide as a pharmaceutical, as a result, this novel seaweed glycoprotein-derived peptide has an anti-amnesic effect and prolyl endothelium derived from natural products. It was found useful as a peptidase inhibitor. The present invention is based on such knowledge. The seaweed glycoprotein-derived peptide according to the present invention has the following formula:
Ile-Lys-Pro-Gly-Val-Val-Val-Ala-Ala-Ala-Ile-Val-Gly-Gly-Tyr
Is a novel peptide represented by the amino acid sequence of L-form, and the property at room temperature is a white powder.

The novel peptide according to the present invention can be chemically synthesized or separated and purified from glycoproteins of seaweed (Wakame, Okinawa Mozuku, Alame, Kombu, Hijiki, Honda Walla, Akamoku, Nori, etc.). . In the case of chemically synthesizing the novel peptide according to the present invention, it can be performed by a usual synthesis method such as a liquid phase method or a solid phase method. It is preferable that the L-form amino acids corresponding to the amino acid residues are sequentially joined to the body by peptide bonds from the carboxyl terminal side of the peptide. The synthetic peptide thus obtained is cleaved from the porous solid support using trifluoromethanesulfonic acid, hydrogen fluoride, etc., and then the amino acid side chain protecting group is removed, and the reverse phase system is removed. And purified by a conventional method using high performance liquid chromatography (hereinafter abbreviated as HPLC).

As described above, the novel peptide according to the present invention can be separated and purified from seaweed (Wakame, Okinawa Mozuku, Alame, Kombu, Hijiki, Honda Walla, Akamok, Nori, etc.). It can be done as follows. Seaweed that has been collected (Wakame, Okinawa Mozuku, Alame, Kombu, Hijiki, Honda Walla, Akamok, Nori, etc.) is thoroughly washed with seawater, sun-dried, and then pulverized using a high-speed pulverizer to dry the dried seaweed powder. Get the body. To this dry powder, water, a salt aqueous solution or a weak alkaline aqueous solution is added, and soluble components are extracted while wet grinding, and a seaweed-derived glycoprotein is obtained from this extract by a glycoprotein separation method. Examples of the glycoprotein separation method used in the present invention include ethanol, polyethylene glycol and other organic solvents, glycoprotein precipitation method using ammonium sulfate and trichloroacetic acid, ion exchanger adsorption method, isoelectric point precipitation method, membrane separation method and the like. These can be used alone or in combination. Furthermore, in order to increase the extraction rate of seaweed glycoprotein, it is also possible to remove the viscous polysaccharide in advance by degradation or washing with an enzyme such as alginate lyase. The seaweed glycoprotein separated and purified in this way should be isolated and purified into peptide fragments by HPLC using a reversed-phase column after cleaving the sugar chain and peptide chain by bromocyan degradation, protease digestion or other conventional methods. Thus, a seaweed glycoprotein-derived peptide can be obtained.

As the seaweed used in the method for producing a novel peptide derived from a seaweed glycoprotein according to the present invention, any seaweed may be used as long as the object of the present invention can be achieved, preferably brown seaweed, seaweed, Okinawa mozuku, alame, It is good to use a paste as a kombu, hinoki, hondawala, akamoku, and red algae. The novel seaweed glycoprotein-derived peptide obtained as described above does not cause antibody production and does not cause anaphylactic shock when repeatedly administered intravenously. Moreover, since this novel seaweed glycoprotein-derived peptide has a sequence structure consisting of only L-amino acid and is gradually degraded by protease in vivo after administration, its toxicity is extremely low and safety is extremely high (LD ₅₀ > 5000 mg / kg; rat oral administration). This novel seaweed glycoprotein-derived peptide can be prepared into injections, tablets, capsules, granules, powders and the like using commonly used additives such as excipients. The administration method usually includes injection into a mammal having ACE (for example, human, dog, rat, etc.) or oral administration. The dose is, for example, 0.01-100 mg of this novel seaweed glycoprotein-derived peptide per animal body weight. The number of administration is usually about 1 to 4 times a day, but can be appropriately adjusted depending on the administration route.

The types of excipients, binders, and lubricants used in the various preparations are not particularly limited, and those used for ordinary injections, powders, granules, tablets, or capsules can be used. Examples of additives used in tablets, capsules, granules, and powders include the following. Examples of excipients include sugars such as crystalline cellulose, sugar alcohols such as mannitol, starches, anhydrous calcium phosphate and the like; binders such as starches and hydroxypropylmethylcellulose; and disintegrants such as carboxymethylcellulose and potassium salts thereof; Examples of the lubricant include stearic acid and its salts, talc, and waxes. In preparation of the preparation, flavoring agents such as menthol, citric acid and salts thereof, and fragrance can be used as necessary. Aseptic composition for injection is prepared by a conventional method using a novel seaweed glycoprotein-derived peptide according to the present invention, water for injection, physiological saline, sugar alcohol injection solution such as xylitol and mannitol, glycol such as propylene glycol and polyethylene glycol, etc. It can be dissolved or suspended in an injection. At this time, a buffer solution, a preservative, an antioxidant and the like can be added as necessary. The preparation containing the novel peptide derived from seaweed glycoprotein is in the form of a lyophilized product or a dry powder, and can be used by dissolving it in an ordinary solubilizing agent such as water or physiological saline at the time of use.

The novel seaweed glycoprotein-derived peptide according to the present invention has an excellent prolyl enpeptidase inhibitory action and exhibits an anti-amnesic effect and an anti-dementia effect. Therefore, since the progression of dementia symptoms of mild to moderate Alzheimer type dementia and cerebrovascular dementia as a sequela of cerebral infarction can be delayed, it is useful as a preventive agent or symptom improving agent for amnesia.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention relates to a peptide having a peptide structure having the following amino acid sequence having utility as a pharmaceutical product, and a prolyl endopeptidase inhibitor containing the peptide as an active ingredient.
Ile-Lys-Pro-Gly-Val-Val-Val-Ala-Ala-Ala-Ile-Val-Gly-Gly-Tyr
(In the formula, each symbol representing an amino acid residue is based on a conventional display method in amino acid chemistry)
EXAMPLES Examples and test examples will be described below as examples, and the present invention will be described in more detail. However, the present invention is not limited to these examples.

Production Example 1
The dried wakame prepared by drying the wakame (Brown algae, Coleoptera, Cactiaceae, Wakame genus) frond was finely pulverized to 30 mesh with a high-speed pulverizer. To 40 g of this finely pulverized powder, 1 L of 0.1N sodium hydroxide solution was added and homogenized. After stirring at room temperature for 24 hours, the mixture was centrifuged (15000 rpm, 20 minutes) and further subjected to suction filtration using diatomaceous earth and filter paper (Toyo Filter Paper No. 2) to obtain 700 mL of a wakame glycoprotein-containing solution. 6 L of ethanol was added to this solution and left in a low temperature room (−5 ° C.) for 18 hours to precipitate the wakame glycoprotein fraction, and centrifuged again (15000 rpm, 20 minutes) to obtain a wakame glycoprotein precipitate. It was. This precipitate was subjected to reverse phase HPLC after cleaving the sugar chain and peptide chain of wakame glycoprotein by bromocyan decomposition method or protease digestion method according to a conventional method. As a column, Develosil ODS-5 (4.5 mID × 25 cmL) manufactured by Nomura Chemical Co., Ltd. was used, and as a mobile phase, 0.05% trifluoroacetic acid (hereinafter abbreviated as TFA) to 25% acetonitrile / 0.05%. A Wakame glycoprotein-derived peptide can be obtained by performing a concentration gradient method of TFA, performing HPLC at a flow rate of 1.0 mL / min, a detection wavelength of 220 nm, and isolating and purifying a peptide fragment having prolyl endopeptidase inhibitory activity. It was. The amino acid sequence of the thus obtained peptide fragment having prolyl endopeptidase inhibitory activity was determined using a protein sequencer type 477A manufactured by Applied Biosystems (ABI). As a result, the peptide according to the present invention has the following formula:
Ile-Lys-Pro-Gly-Val-Val-Val-Ala-Ala-Ala-Ile-Val-Gly-Gly-Tyr
It was confirmed that it is a novel peptide represented by the amino acid sequence of L-form represented by. The property at room temperature is a white powder.

Production Example 2
A dried Okinawa mozuku prepared by drying Okinawa mozuku (Brown algae, Nagamatsumo, Nagamatsumo, Okinawa mozuku) was finely pulverized to 30 mesh with a high-speed pulverizer. 1 L of 0.1N sodium hydroxide solution was added to 35 g of this finely pulverized powder and homogenized. After stirring at room temperature for 24 hours, the mixture was centrifuged (15000 rpm, 20 minutes), and further suction filtered using diatomaceous earth and filter paper (Toyo Filter Paper No. 2) to obtain 630 mL of a Okinawa mozuku glycoprotein-containing solution. To this solution was added 6 L of ethanol, and the mixture was allowed to stand for 18 hours in a low-temperature room (−5 ° C.) to precipitate the Okinawa mozuku glycoprotein fraction, and centrifuged again (15000 rpm, 20 min) to precipitate Okinawa mozuku glycoprotein. Got. This precipitate was subjected to reverse phase HPLC after cleaving the sugar chain and peptide chain of Okinawa mozuku glycoprotein by bromocyan decomposition method or protease digestion method according to a conventional method. As the column, Develosil ODS-5 (4.5 mm ID × 25 cmL) manufactured by Nomura Chemical Co., Ltd. was used, and from 0.05% trifluoroacetic acid (hereinafter abbreviated as TFA) to 25% acetonitrile / 0.05% as the mobile phase. By performing a TFA concentration gradient method, performing HPLC at a flow rate of 1.0 mL / min, a detection wavelength of 220 nm, and isolating and purifying a peptide fragment having prolyl endopeptidase inhibitory activity, a peptide derived from Okinawa mozuku glycoprotein can be obtained. did it. The amino acid sequence of the thus obtained peptide fragment having prolyl endopeptidase inhibitory activity was determined using a protein sequencer type 477A manufactured by Applied Biosystems (ABI). As a result, the peptide according to the present invention has the following formula:
Ile-Lys-Pro-Gly-Val-Val-Val-Ala-Ala-Ala-Ile-Val-Gly-Gly-Tyr
It was confirmed that it is a novel peptide represented by the amino acid sequence of L-form represented by. The property at room temperature is a white powder.

Production Example 3
The dried arame prepared by drying arame (Brown algae, Coleoptera, Compositae, Alameae) was finely pulverized to 30 mesh with a high-speed pulverizer. 1 L of 0.1N sodium hydroxide solution was added to 32 g of this finely pulverized powder and homogenized. After stirring at room temperature for 24 hours, the mixture was centrifuged (15000 rpm, 20 minutes) and further filtered with suction using diatomaceous earth and filter paper (Toyo Filter Paper No. 2) to obtain 610 mL of an alame glycoprotein-containing solution. 6 L of ethanol was added to this solution and left in a low temperature room (−5 ° C.) for 18 hours to precipitate the arame glycoprotein fraction, and centrifuged again (15000 rpm, 20 minutes) to obtain an arame glycoprotein precipitate. It was. This precipitate was subjected to reverse phase HPLC after cleaving the sugar chain and peptide chain of arame glycoprotein by bromocyan decomposition method or protease digestion method according to a conventional method. As the column, Develosil ODS-5 (4.5 mm ID × 25 cmL) manufactured by Nomura Chemical Co., Ltd. was used, and from 0.05% trifluoroacetic acid (hereinafter abbreviated as TFA) to 25% acetonitrile / 0.05% as the mobile phase. By performing a TFA concentration gradient method, performing HPLC at a flow rate of 1.0 mL / min, a detection wavelength of 220 nm, and isolating and purifying a peptide fragment having prolyl endopeptidase inhibitory activity, an arame glycoprotein-derived peptide can be obtained. It was. The amino acid sequence of the thus obtained peptide fragment having prolyl endopeptidase inhibitory activity was determined using a protein sequencer type 477A manufactured by Applied Biosystems (ABI). As a result, the peptide according to the present invention has the following formula:
Ile-Lys-Pro-Gly-Val-Val-Val-Ala-Ala-Ala-Ile-Val-Gly-Gly-Tyr
It was confirmed that it is a novel peptide represented by the amino acid sequence of L-form represented by. The property at room temperature is a white powder.

Production Example 4
A dried comb that was prepared by drying a kombu (Brown algae, Compositae, Combraceae, Konbu spp.) Was finely pulverized to 30 mesh using a high-speed pulverizer. To 42 g of this finely pulverized powder, 1 L of 0.1N sodium hydroxide solution was added and homogenized. After stirring at room temperature for 24 hours, the mixture was centrifuged (15000 rpm, 20 minutes), and further subjected to suction filtration using diatomaceous earth and filter paper (Toyo Filter Paper No. 2) to obtain 720 mL of a kumbu glycoprotein-containing solution. 6 L of ethanol was added to this solution and left in a cold room (−5 ° C.) for 18 hours to precipitate the kumbu glycoprotein fraction, and centrifuged again (15000 rpm, 20 min) to obtain a kumbu glycoprotein precipitate. It was. This precipitate was subjected to reverse phase HPLC after cleaving the sugar chain and peptide chain of the comb glycoprotein by bromocyan decomposition method or protease digestion method according to a conventional method. As the column, Develosil ODS-5 (4.5 mm ID × 25 cmL) manufactured by Nomura Chemical Co., Ltd. was used, and from 0.05% trifluoroacetic acid (hereinafter abbreviated as TFA) to 25% acetonitrile / 0.05% as the mobile phase. By performing the TFA concentration gradient method, performing HPLC at a flow rate of 1.0 mL / min detection wavelength of 220 nm, and isolating and purifying peptide fragments having prolyl endopeptidase inhibitory activity, it is possible to obtain peptides derived from kombu glycoproteins. It was. The amino acid sequence of the thus obtained peptide fragment having prolyl endopeptidase inhibitory activity was determined using a protein sequencer type 477A manufactured by Applied Biosystems (ABI). As a result, the peptide according to the present invention has the following formula:
Ile-Lys-Pro-Gly-Val-Val-Val-Ala-Ala-Ala-Ile-Val-Gly-Gly-Tyr
It was confirmed that it is a novel peptide represented by the amino acid sequence of L-form represented by. The property at room temperature is a white powder.

Production Example 5
Dried hijiki prepared by drying hijiki (Brown algae, Hibamata, Hondaidae, Genus genus) was finely pulverized to 30 mesh with a high-speed pulverizer. 1 L of 0.1N sodium hydroxide solution was added to 39 g of this finely pulverized powder and homogenized. After stirring at room temperature for 24 hours, the mixture was centrifuged (15000 rpm, 20 minutes), and further subjected to suction filtration using diatomaceous earth and filter paper (Toyo Filter Paper No. 2) to obtain 680 mL of a hydride glycoprotein-containing solution. 6 L of ethanol was added to this solution and left in a low-temperature room (−5 ° C.) for 18 hours to precipitate a hinoki glycoprotein fraction, and centrifuged again (15000 rpm, 20 minutes) to obtain a precipitate of hinoki glycoprotein. It was. This precipitate was subjected to reverse phase HPLC after cleaving the sugar chain and peptide chain of the hiji glycoprotein by bromocyan decomposition or protease digestion according to a conventional method. As the column, Develosil ODS-5 (4.5 mm ID × 25 cmL) manufactured by Nomura Chemical Co., Ltd. was used, and from 0.05% trifluoroacetic acid (hereinafter abbreviated as TFA) to 25% acetonitrile / 0.05% as the mobile phase. By performing a TFA concentration gradient method, performing HPLC at a flow rate of 1.0 mL / min, a detection wavelength of 220 nm, and isolating and purifying peptide fragments having prolyl endopeptidase inhibitory activity, it is possible to obtain a peptide derived from a hiji glycoprotein. It was. The amino acid sequence of the thus obtained peptide fragment having prolyl endopeptidase inhibitory activity was determined using a protein sequencer type 477A manufactured by Applied Biosystems (ABI). As a result, the peptide according to the present invention has the following formula:
Ile-Lys-Pro-Gly-Val-Val-Val-Ala-Ala-Ala-Ile-Val-Gly-Gly-Tyr
It was confirmed that it is a novel peptide represented by the amino acid sequence of L-form represented by. The property at room temperature is a white powder.

Production Example 6
A dried Honda Walla prepared by drying Honda Walla (Brown Algae, Hibamata, Honda Wallaceae, Honda Walla) was finely pulverized to 30 mesh with a high-speed pulverizer. To 44 g of this finely pulverized powder, 1 L of 0.1N sodium hydroxide solution was added and homogenized. After stirring at room temperature for 24 hours, the mixture was centrifuged (15000 rpm, 20 minutes), and further subjected to suction filtration using diatomaceous earth and filter paper (Toyo Filter Paper No. 2) to obtain 740 mL of a solution of Honda walla glycoprotein. 6 L of ethanol was added to this solution and left for 18 hours in a low-temperature room (−5 ° C.) to precipitate the hondawala glycoprotein fraction, and centrifuged again (15000 rpm, 20 min) to obtain a hondawala glycoprotein precipitate. It was. This precipitate was subjected to reverse phase HPLC after cleaving the sugar chain and peptide chain of Honda Wallacoprotein by bromocyan decomposition method or protease digestion method according to a conventional method. As the column, Develosil ODS-5 (4.5 mm ID × 25 cmL) manufactured by Nomura Chemical Co., Ltd. was used, and from 0.05% trifluoroacetic acid (hereinafter abbreviated as TFA) to 25% acetonitrile / 0.05% as the mobile phase. By performing a TFA concentration gradient method, performing HPLC at a flow rate of 1.0 mL / min, a detection wavelength of 220 nm, and isolating and purifying a peptide fragment having prolyl endopeptidase inhibitory activity, a peptide derived from Honda walla glycoprotein can be obtained. It was. The amino acid sequence of the thus obtained peptide fragment having prolyl endopeptidase inhibitory activity was determined using a protein sequencer type 477A manufactured by Applied Biosystems (ABI). As a result, the peptide according to the present invention has the following formula:
Ile-Lys-Pro-Gly-Val-Val-Val-Ala-Ala-Ala-Ile-Val-Gly-Gly-Tyr
It was confirmed that it is a novel peptide represented by the amino acid sequence of L-form represented by. The property at room temperature is a white powder.

Production Example 7
A dried red scallop prepared by drying red scallops (Brown algae, Hibamata, Hondawala, Honda genus) was finely pulverized to 30 mesh with a high-speed pulverizer. 1 L of 0.1N sodium hydroxide solution was added to 38 g of this finely pulverized powder and homogenized. After stirring at room temperature for 24 hours, the mixture was centrifuged (15000 rpm, 20 minutes), and further subjected to suction filtration using diatomaceous earth and filter paper (Toyo Filter Paper No. 2), to obtain 710 mL of an akamoku glycoprotein-containing solution. 6 L of ethanol was added to this solution and left in a low temperature room (−5 ° C.) for 18 hours to precipitate the red saccharoglycoprotein fraction, and centrifuged again (15000 rpm, 20 minutes) to obtain a red saccharoglycoprotein precipitate. It was. This precipitate was subjected to reverse phase HPLC after cleaving the sugar chain and peptide chain of akamoku glycoprotein by bromocyan decomposition method or protease digestion method according to a conventional method. As the column, Develosil ODS-5 (4.5 mm ID × 25 cmL) manufactured by Nomura Chemical Co., Ltd. was used, and from 0.05% trifluoroacetic acid (hereinafter abbreviated as TFA) to 25% acetonitrile / 0.05% as the mobile phase. By performing TFA concentration gradient method, HPLC at a flow rate of 1.0 mL / min, detection wavelength of 220 nm, and isolating and purifying peptide fragments having prolyl endopeptidase inhibitory activity, peptides derived from akamoku glycoprotein can be obtained. It was. The amino acid sequence of the thus obtained peptide fragment having prolyl endopeptidase inhibitory activity was determined using a protein sequencer type 477A manufactured by Applied Biosystems (ABI). As a result, the peptide according to the present invention has the following formula:
Ile-Lys-Pro-Gly-Val-Val-Val-Ala-Ala-Ala-Ile-Val-Gly-Gly-Tyr
It was confirmed that it is a novel peptide represented by the amino acid sequence of L-form represented by. The property at room temperature is a white powder.

Production Example 8
The dried laver prepared by drying the laver (Rhodophyceae, Ganodernaceae, Ganodermaceae, Amanori) was finely pulverized to 30 mesh with a high-speed pulverizer. 1 L of 0.1N sodium hydroxide solution was added to 32 g of this finely pulverized powder and homogenized. After stirring at room temperature for 24 hours, the mixture was centrifuged (15000 rpm, 20 minutes), and suction filtered using diatomaceous earth and filter paper (Toyo Filter Paper No. 2) to obtain 620 mL of a nori glycoprotein-containing solution. 6 L of ethanol was added to this solution and left in a low temperature room (−5 ° C.) for 18 hours to precipitate the nori glycoprotein fraction, and centrifuged again (15000 rpm, 20 minutes) to obtain a nori glycoprotein precipitate. It was. This precipitate was subjected to reverse phase HPLC after cleaving the sugar chain and peptide chain of Nori glycoprotein by bromocyan decomposition method or protease digestion method according to a conventional method. As the column, Develosil ODS-5 (4.5 mm ID × 25 cmL) manufactured by Nomura Chemical Co., Ltd. was used, and from 0.05% trifluoroacetic acid (hereinafter abbreviated as TFA) to 25% acetonitrile / 0.05% as the mobile phase. Noriglycoprotein-derived peptides can be obtained by performing a TFA concentration gradient method, performing HPLC at a flow rate of 1.0 mL / min, a detection wavelength of 220 nm, and isolating and purifying peptide fragments having prolyl endopeptidase inhibitory activity. It was. The amino acid sequence of the thus obtained peptide fragment having prolyl endopeptidase inhibitory activity was determined using a protein sequencer type 477A manufactured by Applied Biosystems (ABI). As a result, the peptide according to the present invention has the following formula:
Ile-Lys-Pro-Gly-Val-Val-Val-Ala-Ala-Ala-Ile-Val-Gly-Gly-Tyr
It was confirmed that it is a novel peptide represented by the amino acid sequence of L-form represented by. The property at room temperature is a white powder.

In addition, when a novel seaweed glycoprotein-derived peptide according to the present invention is formulated as a prolyl endopeptidase inhibitor, for example, into a tablet, it may be treated according to a conventional method, for example, as follows: (1) Seaweed sugar 8g of protein-derived peptide, (2) 82g of lactose, (3) 28g of corn starch, (4) 1.5g of magnesium stearate as raw materials, first mix (1), (2) and 10g of corn starch, from 7g of corn starch The resulting paste is granulated with the paste, 11 g of corn starch and (4) are added to the granule, and the resulting mixture is compressed with a compression tablet machine to produce 1000 tablets.

Production Example 9
This example is an example of production by a synthesis method.
Ile-Lys-Pro-Gly-Val-Val-Val-Ala-Ala-Ala-Ile-Val-Gly-Gly-Tyr Synthesis Method Solid Phase Method Using Peptide Automatic Synthesizer Model 430A manufactured by Applied Biosystems The peptide was synthesized by As the solid support, a resin obtained by chloromethylating a styrene divinylbenzene copolymer (polystyrene resin) was used. First, according to the amino acid sequence of the peptide, as usual, the C-terminal tyrosine was reacted with chloromethyl resin to obtain a peptide binding resin. As the amino acid at this time, a t-Boc amino acid protected with a t-butoxycarbonyl (hereinafter abbreviated as t-Boc) group was used. Next, this peptide-bonded resin was suspended in a mixed solution composed of ethanedithiol and thioanisole, stirred at room temperature for 10 minutes, added with trifluoroacetic acid under ice cooling, and further stirred for 10 minutes. Trifluoromethanesulfonic acid was added dropwise to the mixture, and the mixture was stirred at room temperature for 30 minutes. Then, anhydrous ether was added to precipitate the product, and the precipitate was washed several times with anhydrous ether. And dried. The crude synthetic peptide thus obtained was dissolved in distilled water and then purified by HPLC using a reverse phase system column C ₁₈ (5 μm). (A) 0.1% TFA-containing distilled water and (B) 0.1% TFA-containing acetonitrile solution were used as the mobile phase, and (A) liquid was flowed at a flow rate of 1 by a concentration gradient method of 81% → 55% in 59 minutes. Chromatography was performed at 5 mL / min. The elution fraction detected with an ultraviolet wavelength of 216 nm and exhibiting the maximum absorption was collected and lyophilized to obtain the target synthetic peptide (IKPGVVVAAAIVGGY).

As a result of analyzing this synthetic peptide by amino acid analysis, it was confirmed that the amino acid sequence was a peptide having the amino acid sequence structure shown above. Furthermore, when the prolyl endopeptidase inhibitory activity of this synthetic peptide was measured, the IC ₅₀ value was 5.34 μM.
The novel seaweed glycoprotein-derived peptide according to the present invention obtained by such synthesis was confirmed to have a pharmacological effect by the following test.

Test Example 1 (Prolyl Endopeptidase Inhibitory Activity Measurement Method)
Enzyme: Prolyl endopeptidase (manufactured by Seikagaku Corporation, enzyme number EC 3.4.21.26) 0.1 U / mL, synthetic substrate Z-Gly-L-Pro-pNA (manufactured by Seikagaku Corporation) 2 mM , And Yoshimoto et al. [Non-patent Document 4]. That is, 2000 μL of 0.1 M phosphate buffer (pH 7.0), 100 μL of a test solution, and 100 μL of an enzyme solution (containing 40% dioxane) were sequentially added, and preincubated at 30 ° C. for 5 minutes. Further, 100 μL of the substrate solution was added and incubated at 30 ° C. for 10 minutes. Thereafter, the reaction was stopped by adding 500 μL of 1N HCl, and the absorbance at 410 nm was measured. The inhibition rate was calculated from the following equation, where Es is the absorbance in the test solution, Ec is the value when the buffer solution is added instead of the test solution, and Eb is the value when the reaction stop solution is added and reacted in advance. .
Inhibition rate (%) = (Ec−Es) / (Ec−Eb) × 100
The inhibitory activity IC ₅₀ value of the prolyl endopeptidase inhibitor was expressed as the concentration (M) of the test solution necessary to inhibit the enzyme activity of prolyl endopeptidase by 50% (inhibition rate).

以上の試験の結果、本発明に係る新規な海藻糖タンパク由来ペプチドは、ｉｎ ｖｉｔｒｏ（試験管内）においてプロリルエンドペプチダーゼ阻害活性を有し、ｉｎ ｖｉｖｏ（生体内）においても有意な抗健忘症効果を示すことが確認された。従って、この新規な海藻糖タンパク由来ペプチドは痴呆症の治療又は予防薬として有用である。尚、この新規な海藻糖タンパク由来ペプチドは、構造的にそのアミノ酸配列を部分構造とするペプチドにおいて、構造中に採用することもできる。Test Example 2 (Anti-amnesic effect test using rats)
The anti-amnesic effect test of the synthetic peptide derived from seaweed glycoprotein (IKPGVVVAAAIVGGY) according to the present invention is performed using a rat passive avoidance learning device, that is, a foot shock stress system (MFS-01, MFS-01). It was. The experimental animals were purchased from Kyushu Co., Ltd., 4 male male rats (average body weight 160 g), 3 rats each in 3 groups, and then a passive avoidance learning test was conducted. Rats orally administered with saline as an intact control (saline group), rats orally administered with 0.2 mg / kg donepezil hydrochloride (manufactured by Eisai Co., Ltd.) as a control, and seaweed glycoprotein-derived Experiments were conducted using rats (synthetic peptide group: IKPGVVVAAAIVGGY group) to which 200 mg / kg of synthetic peptide was orally administered. Each of these three groups of rats is placed on a table, and a current is passed from the moment of getting down to the floor, returning to the table to avoid an electric shock, and passive avoidance learning until it no longer gets down to the floor. Next, when 20 mg / kg of scopolamine that causes dementia is administered to these three groups of rats, the previous electric shock is forgotten and the patient comes down to the floor. By measuring the stay time (seconds) until the rat descended to the floor, it was examined how much the synthetic peptide derived from seaweed glycoprotein (IKPGVVVAAAIVGGY) administered before learning prevented this memory loss.

As a result of the above tests, the novel seaweed glycoprotein-derived peptide according to the present invention has a prolyl endopeptidase inhibitory activity in vitro (in vitro) and a significant anti-amnesic effect also in vivo (in vivo). It was confirmed that Therefore, this novel seaweed glycoprotein-derived peptide is useful as a therapeutic or prophylactic agent for dementia. In addition, this novel seaweed glycoprotein-derived peptide can be employed in the structure in a peptide having a partial structure of its amino acid sequence.

Claims (2)

    A novel seaweed sugar having a peptide structure based on the amino acid sequence of the L-form represented by the following formula: Ile-Lys-Pro-Gly-Val-Val-Val-Ala-Ala-Ala-Ile-Val-Gly-Gly-Tyr Protein-derived peptide.     A novel seaweed sugar having a peptide structure based on the amino acid sequence of the L-form represented by the following formula: Ile-Lys-Pro-Gly-Val-Val-Val-Ala-Ala-Ala-Ile-Val-Gly-Gly-Tyr A prolyl endopeptidase inhibitor comprising a protein-derived peptide as an active ingredient.