JP2012116819A - Memory enhancing agent and method for improving memorizing power - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a memory enhancing agent containing a peptide exhibiting memory enhancing action and a method for improving memorizing power.SOLUTION: The memory enhancing agent includes a peptide expressed by Leu-Arg-Pro-Val-Ala. There is further provided a functional food containing the memory enhancing agent as an active component.

Description

  The present invention relates to memory enhancers and methods for enhancing memory.

  In recent years, with the progress of an aging society, dementia associated with aging has become a major social problem, and the development of substances that treat or prevent this is desired. As a learning accelerator expected to treat or prevent dementia, the present inventors have reported that the peptide represented by Arg-Ile-Tyr is effective (Patent Document 1). . However, since dementia has great individual differences in its pathology, manifestation mode, and degree, there is a further need for the development of active substances that can improve symptoms accordingly.

JP 2006-8572 A

  As a result of intensive studies, the present inventors have found that a peptide represented by Leu-Arg-Pro-Val-Ala has an excellent memory enhancing action, and completed the present invention. That is, an object of the present invention is to provide a memory enhancing agent containing a peptide exhibiting a memory enhancing action and a method for enhancing memory using the memory enhancing agent.

  The present invention relates to a memory enhancer comprising a peptide represented by Leu-Arg-Pro-Val-Ala, and further to a functional food or nutritional supplement containing the memory enhancer as an active ingredient Regarding food. In addition, the method for enhancing memory of the present invention is characterized by using a peptide represented by Leu-Arg-Pro-Val-Ala.

  The memory enhancer of the present invention is easy to prepare because it has few amino acid residues and does not contain special amino acids. Nevertheless, it is a memory enhancer that exhibits an excellent memory enhancing action and is effective for oral administration.

It is the graph which showed the result of the passive avoidance experiment by the intraperitoneal administration of the LRPVA peptide in Example 1, 2, and the comparative example 1, * shows a significant difference of p <0.05. It is the graph which showed the result of the passive avoidance experiment by the intraperitoneal administration of the LRP peptide in Comparative Examples 2-6. It is the graph which showed the result of the passive avoidance experiment by the oral administration of the LRPVA peptide in Example 3, 4 and Comparative Example 7, and * shows the significant difference of p <0.05.

  The peptide of the present invention is a peptide represented by Leu-Arg-Pro-Val-Ala. Here, Leu means leucine, Arg means arginine, Pro means proline, Val means valine, Ala means alanine, and these amino acids are all in the L-form. This peptide is hereinafter referred to as LRPVA peptide.

  The LRPVA peptide can be produced by a peptide synthesis method as described below. Peptide synthesis is performed by a liquid phase method or a solid phase method, and in each case, a raw material having a reactive carboxyl group corresponding to one of two kinds of fragments that are bisected at an arbitrary position of the peptide bond, and the other fragment A method using an active ester such as 2- (1H-Benzotriazole-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) or a carbodiimide The condensation is performed by a method.

  Functional groups that should not participate in the reaction in this condensation are protected by protecting groups. Examples of the amino-protecting group include benzyloxycarbonyl (Bz), t-butyloxycarbonyl (Boc), p-biphenylisopropyloxycarbonyl, 9-fluorenylmethyloxycarbonyl (Fmoc) and the like. Examples of the protecting group for the carboxyl group include groups capable of forming alkyl esters, benzyl esters, etc. In the solid phase method, the carboxyl group at the C-terminal of the peptide is a chlorotrityl resin, chloromethyl resin, oxymethyl resin, P -It is bound to a carrier such as an alkoxybenzyl alcohol resin. The condensation reaction is performed using an N-protected amino acid active ester or peptide active ester in the presence of a condensing agent such as carbodiimide.

  After completion of the condensation reaction, the protecting group is removed, but in the case of the solid phase method, the bond between the C-terminus of the peptide and the resin is further cleaved, and the product is purified according to the usual method. Examples of the purification method include reverse phase liquid chromatography, ion exchange chromatography, affinity chromatography and the like.

  The LRPVA peptide can also be produced by cleaving a protein or (poly) peptide containing the peptide sequence with a cleavage reagent such as peptidase.

  Analysis of the synthesized peptide is performed by a protein sequencer, LC-MS, or the like that reads an amino acid sequence from the C-terminal by Edman degradation.

  The administration route of the memory enhancing agent of the present invention may be any of oral administration, intravascular administration and rectal administration, but oral administration is preferred. The dose of the memory enhancer for humans cannot be generally stated depending on the administration method, patient's symptoms and age, etc., but is usually 0.01 to 1000 mg, preferably 0.1 to 100 mg at a time, 1 to 3 per day. It is preferable to set the time. Thus, a method for enhancing memory ability is provided by administering a memory enhancing agent containing an LRPVA peptide to a patient by the various administration means described above.

  Memory enhancers are usually administered in the form of a formulation. As the carrier and auxiliary agent used in the preparation, a substance that is commonly used in the preparation field and does not react with the LRPVA peptide is used.

  Specifically, for example, lactose, glucose, mannitol, dextrin, cyclodextrin, starch, sucrose, magnesium aluminate metasilicate, synthetic aluminum silicate, sodium carboxymethylcellulose, hydroxypropyl starch, carboxymethylcellulose calcium, ion exchange resin, methylcellulose , Gelatin, gum arabic, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, polyvinyl alcohol, light anhydrous silicic acid, magnesium stearate, talc, tragacanth, bentonite, bee gum, titanium oxide, sorbitan fatty acid ester, sodium lauryl sulfate, glycerin, Fatty acid glycerin ester, refined lanolin, glycero gelatin, polysorbate, macro Lumpur, vegetable oils, waxes, liquid paraffin, white petrolatum, fluorocarbons, nonionic surfactants, such as propylene glycol.

  Examples of the dosage form include tablets, capsules, granules, powders, syrups, suspensions, suppositories, ointments, creams, gels, patches, inhalants, injections, and the like. These preparations are prepared according to a conventional method.

  Specifically, in order to produce a solid preparation for oral administration, an active ingredient and an excipient component such as lactose, starch, crystalline cellulose, calcium lactate, silicic acid anhydride, etc. are mixed to form a powder, Further, if necessary, a binder such as sucrose, hydroxypropylcellulose, polyvinylpyrrolidone, a disintegrant such as carboxymethylcellulose and carboxymethylcellulose calcium is added and wet or dry granulated to form granules. In order to produce a tablet, these powders and granules may be tableted as they are or after adding a lubricant such as magnesium stearate or talc. These granules or tablets should be coated with an enteric solvent base such as hydroxypropylmethylcellulose phthalate, methacrylic acid-methyl methacrylate polymer, etc., and coated with an enteric solvent preparation, or with ethylcellulose, carnauba wax, hydrogenated oil, etc. You can also. In order to produce a capsule, a hard capsule is filled with a powder or granule, or an active ingredient is left as it is or dissolved in glycerin, polyethylene glycol, sesame oil, olive oil, etc., and then coated with a gelatin film to form a soft capsule. can do.

  In order to produce liquid preparations for oral administration, an active ingredient and a taste-masking agent such as sucrose, sorbitol, and glycerin can be dissolved in water to add a transparent syrup, and then add essential oil, ethanol, etc. to make an elixir. Further, gum arabic, tragacanth, polysorbate 80, sodium carboxymethyl cellulose and the like may be added to form an emulsion or suspension. Further, it may be dissolved or suspended in another appropriate medium instead of water. If necessary, colorants, preservatives and the like may be added to these liquid preparations.

  In order to produce an injection for intravascular administration, the active ingredient is adjusted with a pH adjusting agent such as hydrochloric acid, sodium hydroxide, lactose, lactic acid, sodium, sodium monohydrogen phosphate, sodium dihydrogen phosphate, chloride, if necessary. Dissolved in distilled water for injection together with isotonic agents such as sodium and glucose, buffering agents, preservatives, etc., filtered aseptically and filled into ampoules, or further frozen with mannitol, dextrin, cyclodextrin, gelatin, etc. It may be dried and used as an ergonomic injection. In addition, an emulsion for injection can be prepared by adding reticin, polysorbate 80, polyoxyethylene hydrogenated castor oil and the like to the active ingredient and emulsifying in water.

  To produce a rectal dosage form, the active ingredient is moistened with a suppository base such as cacao butter, fatty acid tri, di and monoglycerides, polyethylene glycol, etc., dissolved and poured into a mold and cooled, or the active ingredient is made of polyethylene. What is necessary is just to coat | cover with a gelatin film | membrane after melt | dissolving in glycol, soybean oil, etc.

  Furthermore, the memory enhancing agent of the present invention can be administered in the form of a functional food or a dietary supplement (so-called supplement). The memory enhancer of the present invention can be blended in foods and seasonings that are generally eaten and consumed, and such foods and drinks are produced by ordinary methods and are particularly limited. It is not a thing.

  These preparations, functional foods, and dietary supplements preferably contain the LRPVA peptide in an amount of 0.01% by weight or more, and further 0.5 to 70% by weight. These preparations and the like may also contain other ingredients with therapeutic value.

  EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples. However, the present invention is not limited to these examples. The test group was expressed as P <0.05 when it was significant at a significance level of 5% with respect to the control group.

Synthesis Example 1 [Synthesis of LRPVA peptide]
0.6 g of commercially available Fmoc-Ala-Wang resin (substitution rate: 0.5 meq / g) was dispensed into a reaction vessel of a PS3 type peptide synthesizer (manufactured by Protein Technologies), and the LRPVA peptide was synthesized as follows. It was.

  First, the above resin is placed in a reaction vessel, the Fmoc group of the resin is removed in 20 ml of dimethylformamide containing 20% by volume piperidine, 1 mmol of Fmoc-Val and 1 mmol of HBTU as an activator are added in 10 ml of 0 ml. Fmoc-Val was coupled to the resin by adding a solution dissolved in dimethylformamide containing 4M N-methylmorpholine to the reaction vessel and stirring at room temperature for 20 minutes. After washing the obtained resin with dimethylformamide, the Fmoc group was removed again in 20 ml of dimethylformamide containing 20% by volume piperidine, and then the C-terminal was removed in the same manner as in the above-mentioned method of coupling Fmoc-Val. Fmoc-Pro, Fmoc-Arg (Pmc), and Fmoc-Leu were sequentially coupled to obtain a Leu-Arg (Pmc) -Pro-Val-Ala resin. The resin in 10 ml of deprotection solution (82% by volume trifluoroacetic acid, 5% by volume thioanisole, 3% by volume ethanedithiol, 2% by volume ethylmethyl sulfide, 3% by volume phenol, 5% by volume water). Stir at room temperature for 4 hours to release the peptide from the resin.

40 ml of cold ether was added thereto to precipitate the peptide, and further washed with cold ether three times to obtain a crude peptide. This was developed and purified by a high-performance liquid chromatography using an ODS column (Cosmosil 5C ₁₈ -ARI, 4.6 × 150 mm) with a linear concentration gradient of acetonitrile containing 0.1% by weight of trifluoroacetic acid, and Leu-Arg− A peptide having the sequence Pro-Val-Ala was obtained. This product was analyzed by a protein sequencer (“type 492” manufactured by Applied Biosystems) and confirmed to have the above sequence.

Synthesis Example 2 [Synthesis of LRP peptide]
Fmoc-Arg (Pmc) and Fmoc-Leu were coupled sequentially from the C-terminal in the same manner as in Synthesis Example 1 except that Fmoc-Pro-Wang resin (substitution rate 0.5 meq / g) was used. After obtaining Leu-Arg (Pmc) -Pro resin, the peptide was released from the resin. The crude peptide obtained by the same method as in Synthesis Example 1 was purified to obtain a peptide having the Leu-Arg-Pro sequence (hereinafter referred to as LRP peptide). In addition, it analyzed by the protein sequencer like the synthesis example 1, and confirmed that it was said arrangement | sequence.

Examples 1-2 and Comparative Examples 1-6
Using the peptides obtained above, a test for enhancing learning memory was performed by the following method.

(Learning memory enhancement effect)
The effect on learning memory ability was examined by passive avoidance experiments using a step-through device. When a ddy mouse (male, weight 28 ± 1 g) is placed in the light room of a device divided into two light and dark rooms, the mouse enters the dark room because it prefers dark places. When entering the dark room, an electric shock (0.25 mA, 5 seconds) is given from the floor to educate that the dark room is dangerous (training trial). Immediately after the training trial, the LRPVA peptide or LRP peptide was intraperitoneally administered at the dosage shown in FIGS. 1 and 2, and the test trial was conducted 24 hours later. That is, the learning memory ability of the mouse in the presence or absence of the peptide was compared by re-inserting the mouse into the same apparatus and measuring the time (second) that the mouse stayed in the bright room. The results are shown in FIG. 1 and FIG.

Examples 3 to 4 and Comparative Example 7
Except that the test was conducted by oral administration of the dose shown in FIG. 3, the test for enhancing the learning memory was conducted in the same manner as in Example 1. The results are shown in FIG.

  As shown in FIG. 1 and FIG. 3, it can be seen that administration of the LRPVA peptide prolonged the stay in the light room and enhanced the learning and memory ability of the mice. In the examples and comparative examples, n = 12, and 600 seconds was taken as the cutoff value in this experiment. When the U test was performed by the Mann and Whitney method, the group (Example 2) in which the LRPVA peptide was intraperitoneally administered 150 μg / kg was 5% more dangerous than the control group (Comparative Example 1). A significant difference was observed (P <0.05). In addition, the same tendency was observed with 50 μg / kg administration (Example 1) although there was no significant difference. The group that was orally administered 15 mg / kg (Example 4) also showed a significant difference (P <0.05) with a risk rate of 5% compared to the control group (Comparative Example 7), 1.5 mg / kg. A similar tendency was observed even when administered with kg (Example 3), although there was no significant difference.

  On the other hand, as shown in FIG. 2, the LRP peptide did not have a learning memory enhancing action (Comparative Examples 2 to 6). The reason why the LRPVA peptide has a learning memory enhancing action is not known at present, and further research is necessary for its elucidation.

  The memory enhancer of the present invention is excellent in learning and memory enhancing action, and is useful not only for treatment and prevention of dementia but also for various uses that require improvement of memory ability, excluding medical use and medical use. For example, it is useful for health foods represented by foods for specific health use.

Claims (4)

A memory enhancer comprising a peptide represented by Leu-Arg-Pro-Val-Ala. A functional food containing the memory enhancer according to claim 1 as an active ingredient. A dietary supplement containing the memory enhancer according to claim 1 as an active ingredient. A method for enhancing memory using a peptide represented by Leu-Arg-Pro-Val-Ala.

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