JP2007254445A - New dipeptide, l-valyl-l-tryptophan and prolylendopeptidase inhibitor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new peptide having a prolylendopeptidase inhibitory activity obtained from a protein obtained by extracting the extracts of sea weeds and fine algae. <P>SOLUTION: This new sea weed and fine algae peptide obtained by extracting the dried powder of sea weeds (wakame sea weed, Cladosiphon okimuranus, Eisemia bicyclis, tangle, Sargassum fusiformis, Sargassum fulvellum, Surgassum horneri, sea laver, etc.,) and fine algae (chlorella and spirulina) with an aqueous weak alkaline solution, then performing ethanol fractionation to obtain the sea weed and fine algae protein, further splitting the same into peptide chains and isolating/purifying by a reverse phase HPLC to obtain the new sea weed and fine algae peptide which is an L-valyl-L-tryptophan having the prolylendopeptidase inhibitory activity, having an anti-amnesia effect in a living body and also having extremely low toxicity. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

The present invention relates to novel dipeptide and prolyl endopeptidase inhibitors.

A novel dipeptide, L-valyl-L-tryptophan, has advantages as a prolyl endopeptidase inhibitor and anti-amnesic agent.
Maruyama, S .; Et al .: Biosci. Biotech. Biochem. 60, 358-359 (1996). Kimura, K .; Et al .: Biosci. Biotech. Biochem. 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, peptide, and amino acid components of seaweed (Wakame, Okinawa Mozuku, Arame, Kombu, Hijiki, Honda Walla, Akamok, Nori, etc.) and microalgae (Chlorella, Spirulina) are relatively high in terms of nitrogen content. Although it is a component next to carbohydrates, there is only a research on non-patent chromoprotein [Non-Patent Document 3], and details regarding the protein, peptide, and amino acid components of seaweed and microalgae are completely unknown. Therefore, the prolyl endopeptidase inhibitory action on the protein, peptide and amino acid components of seaweed and microalgae and the anti-amnesic effect by oral administration are still unknown, and there is no report that development as a pharmaceutical has been advanced.

As a result of diligent research to solve the above-mentioned problems, the present inventor has found that seaweed (Wakame, Okinawa Mozuku, Alame, Kombu, Hijiki, Honda Walla, Akamok, Nori, etc.) and novel algae (Chlorella, Spirulina) The inventors have found that dipeptides have an anti-amnesic effect and have completed the present invention. That is, when a substance having a pharmacological action was searched from seaweed (Wakame, Okinawa mozuku, arame, kombu, hijiki, hondawala, akamoku, nori, etc.) and microalgae (chlorella, spirulina), this new dipeptide had a strong prolyl end. It was found to have a peptidase inhibitory action. And, as a result of diligent research to put this novel dipeptide into practical use as a medicine, this new dipeptide has an anti-amnesic effect and has usefulness as a prolyl endopeptidase inhibitor derived from natural products. I found it. The present invention is based on such knowledge. The seaweed and microalgae-derived peptide according to the present invention has a dipeptide structure represented by L-valyl-L-tryptophan, and is white powder at room temperature.

A novel dipeptide according to the present invention is a method of chemically synthesizing, or a method of separating and purifying from seaweed (Wakame, Okinawa Mozuku, Alame, Kombu, Hijiki, Honda Walla, Akamok, Nori, etc.) and microalgae (Chlorella, Spirulina) Can give. In the case of chemically synthesizing the novel dipeptide according to the present invention, it can be carried out 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 bonded to the body by peptide bonds from the carboxyl terminal side of the peptide. The synthetic dipeptide 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 dipeptide according to the present invention can be separated and purified from seaweed (Wakame, Okinawa mozuku, arame, kombu, hijiki, hondawala, akamoku, nori, etc.) and microalgae (chlorella, spirulina). In that case, for example, it can be performed as follows. The collected seaweed (Wakame, Okinawa Mozuku, Alame, Kombu, Hijiki, Honda Walla, Akamok, Nori, etc.) and microalgae (Chlorella, Spirulina) are thoroughly washed with water, sun-dried, and then using a high-speed grinder Finely pulverized to obtain dry powder of seaweed and microalgae. To this dry powder, water, a salt aqueous solution or a weak alkaline aqueous solution is added and a soluble component is extracted while wet grinding, and proteins derived from seaweed and microalgae are obtained from this extract by a protein separation method. Examples of protein separation methods used in the present invention include protein precipitation methods using ethanol, polyethylene glycol and other organic solvents, ammonium sulfate and trichloroacetic acid, ion exchanger adsorption methods, isoelectric precipitation methods, membrane separation methods, etc. Can be used alone or in combination. Furthermore, in order to increase the extraction rate of seaweed and microalgae-derived proteins, the viscous polysaccharides may be removed in advance by degradation or washing with an enzyme such as alginate lyase. The seaweed and microalgae-derived proteins separated and purified in this way should be cleaved into peptide chains by conventional methods such as bromocyan degradation and protease digestion, and then isolated and purified into peptide fragments by HPLC using a reversed-phase column. By this, seaweed and microalga-derived dipeptide can be obtained.

As the seaweed and microalgae used in the method for producing the novel seaweed and microalgae-derived dipeptide according to the present invention, any seaweed and microalgae may be used as long as the object of the present invention can be achieved. Okinawa mozuku, arame, kombu, hijiki, hondawala, akamoku, laver as red algae, chlorella as green algae, and spirulina as cyanobacteria. This novel seaweed and microalgae-derived dipeptide obtained as described above does not cause antibody production and does not cause anaphylactic shock when repeatedly administered intravenously. Furthermore, this novel seaweed and microalgae-derived dipeptide consisted arrangement of only L- amino acids after administration, because it is gradually degraded by proteases in vivo, toxicity is very low, the safety is very high (LD ₅₀ > 5000 mg / kg; rat oral administration). This novel seaweed and microalgae-derived dipeptide can be prepared into injections, tablets, capsules, granules, powders, and the like using additives such as commonly used excipients. Examples of the administration method include injection into a mammal (eg, human, dog, rat, etc.) or oral administration. The dosage is, for example, an amount of 0.01-100 mg of this novel seaweed and microalgae-derived dipeptide per animal 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. A sterile composition for injection is prepared by a conventional method using the novel seaweed and microalgae-derived dipeptide according to the present invention, such as water for injection, physiological saline and sugar alcohol injection solution such as xylitol and mannitol, propylene glycol, polyethylene glycol and the like. It can be dissolved or suspended in glycol to give 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 dipeptide derived from seaweed and microalgae is in the form of a lyophilized product or a dry powder, and can be used by dissolving in a normal solubilizing agent such as water or physiological saline at the time of use.

The novel seaweed and microalgae-derived dipeptide according to the present invention has an excellent prolyl endopeptidase 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 seaweed and microalgae-derived peptide according to the present invention has a dipeptide structure represented by L-valyl-L-tryptophan, and is white powder at room temperature. 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 suction filtered using diatomaceous earth and filter paper (Toyo Filter Paper No. 2) to obtain 700 mL of seaweed extract. 6 L of ethanol was added to this solution, and allowed to stand in a low temperature room (−5 ° C.) for 18 hours to precipitate the protein fraction, and centrifuged again (15000 rpm, 20 minutes) to obtain a wakame protein precipitate. This precipitate was subjected to reverse phase HPLC after cleaving wakame protein into peptide chains 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. A wakame peptide could 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 a peptide fragment having prolyl endopeptidase inhibitory activity. 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 wakame peptide according to the present invention has a dipeptide structure represented by L-valyl-L-tryptophan and is white powder at room temperature.

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 suction filtered using diatomaceous earth and filter paper (Toyo Filter Paper No. 2) to obtain 630 mL of Okinawa mozuku extract. 6 L of ethanol was added to this solution, and allowed to stand in a low temperature room (−5 ° C.) for 18 hours to precipitate the protein fraction, and centrifuged again (15000 rpm, 20 minutes) to obtain a Okinawa mozuku protein precipitate. The Okinawa mozuku protein was cleaved into peptide chains by bromocyan decomposition or protease digestion according to a conventional method, followed by reverse phase HPLC. As the column, Develosil ODS-5 (4.5 mm ID × 25 cmL) manufactured by Nomura Chemical Co., Ltd. was used, and as the mobile phase, a concentration gradient method of 0.05% TFA to 25% acetonitrile / 0.05% TFA was performed, and a flow rate of 1 By conducting HPLC at a detection wavelength of 220 nm at 0.0 mL / min and isolating and purifying a peptide fragment having prolyl endopeptidase inhibitory activity, an Okinawa mozuku peptide could be obtained. 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 ABI. As a result, the Okinawa mozuku peptide according to the present invention has a dipeptide structure represented by L-valyl-L-tryptophan, and is white powder at room temperature.

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 arame extract. 6 L of ethanol was added to this solution, and allowed to stand in a low temperature room (−5 ° C.) for 18 hours to precipitate the protein fraction, and centrifuged again (15000 rpm, 20 minutes) to obtain an arame protein precipitate. This precipitate was subjected to reverse phase HPLC after cleaving the arame protein into peptide chains 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 as the mobile phase, a concentration gradient method of 0.05% TFA to 25% acetonitrile / 0.05% TFA was performed, and a flow rate of 1 By performing HPLC at a detection wavelength of 220 nm at 0.0 mL / min and isolating and purifying a peptide fragment having prolyl endopeptidase inhibitory activity, an arame peptide could be obtained. 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 ABI. As a result, the alame peptide according to the present invention has a dipeptide structure represented by L-valyl-L-tryptophan, and is white powder at room temperature.

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 kombu extract. 6 L of ethanol was added to this solution, and allowed to stand for 18 hours in a low-temperature room (−5 ° C.) to precipitate the protein fraction, and centrifuged again (15000 rpm, 20 minutes) to obtain a comb protein precipitate. The precipitate was cleaved into a peptide chain by a promocyan degradation method or a protease digestion method according to a conventional method, and then reverse phase HPLC was performed. As the column, Develosil ODS-5 (4.5 mm ID × 25 cmL) manufactured by Nomura Chemical Co., Ltd. was used, and as the mobile phase, a concentration gradient method of 0.05% TFA to 25% acetonitrile / 0.05% TFA was performed, and a flow rate of 1 A comb peptide could be obtained by performing HPLC at a detection wavelength of 220 nm at 0.0 mL / min and isolating and purifying a peptide fragment having prolyl endopeptidase inhibitory activity. 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 ABI. As a result, the comb peptide according to the present invention has a dipeptide structure represented by L-valyl-L-tryptophan, and is white powder at room temperature.

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 filtered with suction using diatomaceous earth and filter paper (Toyo Filter Paper No. 2) to obtain 680 mL of a hinoki extract. 6 L of ethanol was added to this solution, and allowed to stand in a low temperature room (−5 ° C.) for 18 hours to precipitate the protein fraction, and centrifuged again (15000 rpm, 20 minutes) to obtain a precipitate of hinoki protein. This precipitate was subjected to reverse phase HPLC after cleaving hiji protein into peptide chains 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 as the mobile phase, a concentration gradient method of 0.05% TFA to 25% acetonitrile / 0.05% TFA was performed, and a flow rate of 1 By performing HPLC at a detection wavelength of 220 nm at 0.0 mL / min and isolating and purifying a peptide fragment having prolyl endopeptidase inhibitory activity, a HIKI peptide could be obtained. 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 ABI. As a result, the hijiki peptide according to the present invention has a dipeptide structure represented by L-valyl-L-tryptophan and is white powder at room temperature.

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 suction filtered using diatomaceous earth and filter paper (Toyo Filter Paper No. 2) to obtain 740 mL of Honda Walla Extract. 6 L of ethanol was added to this solution, and allowed to stand in a low temperature room (−5 ° C.) for 18 hours to precipitate the protein fraction, and centrifuged again (15000 rpm, 20 minutes) to obtain a precipitate of Honda walla protein. This precipitate was subjected to reverse phase HPLC after cleaving Honda walla protein into peptide chains 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 as the mobile phase, a concentration gradient method of 0.05% TFA to 25% acetonitrile / 0.05% TFA was performed, and a flow rate of 1 By performing HPLC at a detection wavelength of 220 nm at 0.0 mL / min and isolating and purifying a peptide fragment having prolyl endopeptidase inhibitory activity, a Honda walla peptide could be obtained. 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 ABI. As a result, the Honda wallapeptide according to the present invention has a dipeptide structure represented by L-valyl-L-tryptophan, and is white powder at room temperature.

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 suction filtered using diatomaceous earth and filter paper (Toyo Filter Paper No. 2) to obtain 710 mL of red mock extract. 6 L of ethanol was added to this solution, and allowed to stand in a low temperature room (−5 ° C.) for 18 hours to precipitate the protein fraction, and then centrifuged again (15000 rpm, 20 minutes) to obtain a precipitate of akamoku protein. This precipitate was subjected to reverse phase HPLC after cleaving the akamoku protein into peptide chains 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 as the mobile phase, a concentration gradient method of 0.05% TFA to 25% acetonitrile / 0.05% TFA was performed, and a flow rate of 1 By performing HPLC at a detection wavelength of 220 nm at 0.0 mL / min and isolating and purifying a peptide fragment having a prolyl endopeptidase inhibitory activity, an akamoku peptide could be obtained. 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 ABI. As a result, the akamoku peptide according to the present invention has a dipeptide structure represented by L-valyl-L-tryptophan, and is white powder at room temperature.

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 paste extract. 6 L of ethanol was added to this solution, and allowed to stand in a low temperature room (−5 ° C.) for 18 hours to precipitate the protein fraction, and again centrifuged (15000 rpm, 20 minutes) to obtain a protein paste. After this precipitate was cleaved into a peptide chain by a bromocyan decomposition method or protease digestion method according to a conventional method, reverse phase HPLC was performed. As the column, Develosil ODS-5 (4.5 mm ID × 25 cmL) manufactured by Nomura Chemical Co., Ltd. was used, and as the mobile phase, a concentration gradient method of 0.05% TFA to 25% acetonitrile / 0.05% TFA was performed, and a flow rate of 1 By performing HPLC at a detection wavelength of 220 nm at 0.0 mL / min and isolating and purifying a peptide fragment having prolyl endopeptidase inhibitory activity, a noripeptide could be obtained. 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 ABI. As a result, the noripeptide according to the present invention has a dipeptide structure represented by L-valyl-L-tryptophan, and is white powder at room temperature.

Production Example 9
Chlorella (Chlorophyceae, Chlorococcidae, Oocystisaceae, Chlorella sp.) Dried finely pulverized powder (68 g) was homogenized by adding 1 L of 0.1N sodium hydroxide solution. 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 940 mL of chlorella extract. 6 L of ethanol was added to this solution, and allowed to stand in a low temperature chamber (−5 ° C.) for 18 hours to precipitate the protein fraction, and centrifuged again (15000 rpm, 20 minutes) to obtain a chlorella protein precipitate. This precipitate was subjected to reverse phase HPLC after cleaving the chlorella protein into peptide chains 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 as the mobile phase, a concentration gradient method of 0.05% TFA to 25% acetonitrile / 0.05% TFA was performed, and a flow rate of 1 Chlorella peptide could be obtained by performing HPLC at a detection wavelength of 219 nm and isolating and purifying a peptide fragment having prolyl endopeptidase inhibitory activity. 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 ABI. As a result, the chlorella peptide according to the present invention has a dipeptide structure represented by L-valyl-L-tryptophan, and is white powder at room temperature.

Production Example 10
1 L of 0.1N sodium hydroxide solution was added to 57 g of dried pulverized powder of Spirulina (Cyanophyceae, Nemoptera, Auremidae, Spirulina) 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 920 mL of Spirulina extract. 6 L of ethanol was added to this solution, and allowed to stand for 18 hours in a low temperature chamber (−5 ° C.) to precipitate the protein fraction, and centrifuged again (15000 rpm, 20 minutes) to obtain a spirulina protein precipitate. This precipitate was subjected to reverse phase HPLC after cleaving the spirulina protein into a peptide chain 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 as the mobile phase, a concentration gradient method from 0.05% TFA to 25% acetonitrile / 0.05% TFA was performed, and the flow rate was 0. Spirulina peptide could be obtained by performing HPLC at a detection wavelength of 221 nm at .9 mL / min and isolating and purifying a peptide fragment having prolyl endopeptidase inhibitory activity. 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 ABI. As a result, the spirulinapeptide according to the present invention has a dipeptide structure represented by L-valyl-L-tryptophan, and is white powder at room temperature.

In addition, when the novel seaweed and microalgae-derived dipeptide according to the present invention is formulated as a prolyl endopeptidase inhibitor, for example, into a tablet, it may be treated, for example, as follows: {circle around (1)} seaweed And 7 g of dipeptide derived from microalgae, (2) 71 g of lactose, (3) 30 g of corn starch, and (4) 1.2 g of magnesium stearate. First, mix (1), (2) and 11 g of corn starch. It granulates with the paste made from corn starch, 12g corn starch and (4) are added to this granule, and the obtained mixture is compressed with a compression tablet machine, and 1000 tablets are manufactured.

Production Example 9
This example is an example of production by a synthesis method.
Synthesis method of L-valyl-L-tryptophan The peptide was synthesized by a solid phase method using an automatic peptide synthesizer type 430A manufactured by Applied Biosystems. 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 tryptophan was reacted with chloromethyl resin to obtain a peptide-bonded 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 is suspended in a mixed solution composed of ethanedithiol and thioanisole, stirred at room temperature for 10 minutes, added with trifluoroacetic acid (hereinafter abbreviated as TFA) under ice cooling, and further stirred for 10 minutes. did. 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 flow rate was 0% by a concentration gradient method of 76% → 24% in 88 minutes. Chromatography was performed at 9 mL / min. The elution fraction detected at the ultraviolet wavelength of 220 nm and exhibiting the maximum absorption was collected and lyophilized to obtain the target synthetic peptide (L-valyl-L-tryptophan).

As a result of analyzing this synthetic peptide by amino acid analysis, it was confirmed that the amino acid sequence was a dipeptide 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 1.21 μM.
The novel seaweed and microalgae-derived dipeptides according to the present invention obtained by such synthesis were confirmed to have pharmacological effects by the tests shown below.

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及び微細藻類由来ジペプチドは、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、この新規な海藻及び微細藻類由来ジ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、この新規な海藻及び微細藻類由来ジペプチドは、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 (L-valyl-L-tryptophan) according to the present invention is a passive avoidance learning device for rats, that is, a foot shock stress system (MFS-01, manufactured by Muromachi Kikai Co., Ltd.). Type). 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: L-valyl-L-tryptophan 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 residence time (seconds) until the rat descends to the floor, how much the synthetic peptide derived from seaweed glycoprotein (L-valyl-L-tryptophan) administered before learning prevents this memory loss. saw.

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

Claims (2)

      A novel dipeptide having a peptide structure represented by L-valyl-L-tryptophan.       A prolyl endopeptidase inhibitor comprising a novel dipeptide having a peptide structure represented by L-valyl-L-tryptophan as an active ingredient.