JP7584109B2 - Dipeptide and pharmaceutical composition containing same - Google Patents

Description

The present invention relates to a dipeptide and a pharmaceutical or food composition containing the same.

Fatigue generally has the main symptoms of tiredness and lethargy, but can also result in reduced athletic ability, sleep disorders, and decreased motivation. Short-term fatigue can be cured by resting, sleeping, and taking in nutrients, but chronic fatigue is difficult to cure due to long-term general fatigue, lethargy, and slight fever.

It is known that amino acid compositions (Patent Document 1), L-carnitine (Patent Document 2), ascorbic acid, etc. (Patent Document 3) are effective against fatigue. In addition, liver hydrolysates have been reported to activate AMPK and have anti-fatigue effects (Patent Document 4).

Japanese Patent Application Publication No. 9-124473 JP 2001-046021 A Japanese Patent Application Publication No. 6-327435 International Publication No. 2015/022927

Although liver hydrolysate is said to contain a large amount of amino acids and peptides, the actual active ingredients have not been identified.
Therefore, an object of the present invention is to provide a novel component having the effect of preventing and improving fatigue.

The inventors therefore conducted research to develop a new agent for preventing and improving fatigue, administered liver hydrolysate to animals to search for indigestible peptides that migrate into the bloodstream, and fractionated the liver hydrolysate using various columns to evaluate its efficacy. As a result, they discovered a dipeptide derived not from L-amino acids but from a specific D-amino acid, and found that this dipeptide has the effect of preventing and improving fatigue, thus completing the present invention.

That is, the present invention provides the following [1] to [9].

[1] (D) Ile-(D) Pro, (D) Leu-(D) Pro, (D) Pro-(D) Ile, (D) Pro-(D) Leu, (D) Val-(D )-Pro, (D)Pro-(D)Val, (D)Leu-(D)Hyp, (D)Ile-(D)Hyp, (D)Val-(D)Hyp, (D)Asp-( D) Ile, (D) Asp-(D) Val, (D) Asp-(D) Leu, (D) Asp-(D) Phe, (D) Il e-(L)Pro, (D)Leu-(L)Pro, (D)Pro-(L)Ile, (D)Pro-(L)Leu, (D)Val-(L)-Pro, (D ) Pro-(L) Val, (D) Leu-(L) Hyp, (D) Ile-(L) Hyp, (D) Val-(L) Hyp, (D) Asp-(L) Ile, (D ) A dipeptide selected from Asp-(L)Val, (D)Asp-(L)Leu, and (D)Asp-(L)Phe.
[2] (D) Asp-(D) Val, (D) Asp-(D) Ile, (D) Asp-(D) Leu, (D) Asp-(D) Phe, (D) Asp-(L 2. The dipeptide according to claim 1, wherein the dipeptide is selected from the group consisting of (D)Asp-(L)Ile, (D)Asp-(L)Leu, and (D)Asp-(L)Phe.
[3] A pharmaceutical composition comprising the dipeptide according to [1] or [2].
[4] The pharmaceutical composition according to [3], which is an agent for preventing and improving fatigue.
[5] A food composition containing the dipeptide according to [1] or [2].
[6] The food composition according to [5], which is a food composition for preventing and improving fatigue.
[7] Use of the dipeptide according to [1] or [2] for the manufacture of an agent for preventing and improving fatigue or a food for preventing and improving fatigue.
[8] The dipeptide according to [1] or [2] for preventing and improving fatigue.
[9] A method for preventing and improving fatigue, which comprises administering an effective amount of the dipeptide according to [1] or [2].

The dipeptide of the present invention is derived from D-amino acids and is therefore indigestible, persists in the blood for a long time, and has the effect of preventing and improving fatigue, making it useful as a pharmaceutical and food composition for preventing and improving fatigue.

1 shows the anti-fatigue effect of the hydrophobic peptide fraction. 1 shows the anti-fatigue effect of a hydrophobic pyroglutamyl peptide fraction. 1 shows the anti-fatigue effect of the hydrophilic peptide fraction. 1 shows the anti-fatigue effect of a hydrophilic pyroglutamyl peptide fraction. 1 shows the anti-fatigue effect of Asp-Leu(Dα). 1 shows the anti-fatigue effect of Asp-Leu(Dβ). 1 shows the anti-fatigue effect of Asp-Val(Dα). 1 shows the anti-fatigue effect of Asp-Val(Dβ). 1 shows the anti-fatigue effect of Asp-Phe(Dα). 1 shows the anti-fatigue effect of Asp-Phe(Dβ).

The dipeptides of the present invention include (D)Ile-(D)Pro, (D)Leu-(D)Pro, (D)Pro-(D)Ile, (D)Pro-(D)Leu, (D)Val -(D)-Pro, (D)Pro-(D)Val, (D)Leu-(D)Hyp, (D)Ile-(D)Hyp, (D)Val-(D)Hyp, (D) Asp-(D) Ile, (D)Asp-(D)Val, (D)Asp-(D)Leu, (D)Asp-(D)Phe, (D)Ile-(L)Pro, (D)Leu-(L) Pro, (D)Pro-(L)Ile, (D)Pro-(L)Leu, (D)Val-(L)-Pro, (D)Pro-(L)Val, (D)Leu-(L )Hyp, (D)I (D)Asp-(L)Ile, (D)Asp-(L)Val, (D)Asp-(L)Leu, and (D Among these, (D)Asp-(D)Val and (D)Asp-(L)Phe are preferred from the viewpoints of transferability into the bloodstream upon oral administration, duration of action and anti-fatigue effect. ) Asp-(D)Ile, (D)Asp-(D)Leu, (D)Asp-(D)Phe, (D)Asp-(L)Val, (D)Asp-(L)Ile, (D Dipeptides selected from (D)Asp-(L)Leu and (D)Asp-(L)Phe are preferred, with (D)Asp-(D)Leu and (D)Asp-(L)Leu being more preferred.
Here, the designation (D) means that the amino acid is in the D-form, and the designation (L) means that the amino acid is in the L-form.
Of the α- and β-forms of the (D)- or (L)-dipeptides, the β-form is more preferred.

The dipeptide of the present invention can be produced by a conventional liquid phase peptide synthesis method or solid phase peptide synthesis method using the amino acid (D) as a raw material. For example, it can be produced by condensing an amino acid in which functional groups other than the α-amino group are protected with an amino acid in which functional groups other than the carboxy group are protected or an amino acid in which the carboxy group has been activated and functional groups other than the carboxy group are protected, and then removing the protecting groups.
Examples of the protecting group for the amino group of the amino acid include a benzyloxycarbonyl group, a tert-butoxycarbonyl group, a fluorenylmethoxycarbonyl group, etc. Examples of the protecting group for the carboxy group include a tert-butyl group, a benzyl group, etc. The condensation reaction can be carried out using a method using a condensing agent such as N,N'-dicyclohexylcarbodiimide, dicyclohexylurea, or the like, an active ester method using nitrophenol, N-hydroxysuccinimide, or the like, a mixed acid anhydride method, or the like.
After the condensation reaction is completed, the protecting group is removed, and in the case of the solid phase method, the bond between the C-terminus of the peptide and the resin is further cleaved. Furthermore, the peptide of the present invention is purified according to a conventional method.

The dipeptide of the present invention can be an acid addition salt or a base salt. Examples of acid addition salts include inorganic acid salts such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrobromic acid, and perchloric acid, and salts of organic acids such as citric acid, succinic acid, maleic acid, fumaric acid, malic acid, tartaric acid, p-toluenesulfonic acid, benzenesulfonic acid, methanesulfonic acid, and trifluoroacetic acid. Examples of base salts include alkali metal salts such as sodium, potassium, and lithium, and alkaline earth metal salts such as calcium and magnesium.

The peptides of the present invention may be solvates. Examples of solvates include solvates of water (in the case of hydrates), methanol, ethanol, isopropanol, etc.

The dipeptide of the present invention is indigestible, has high transferability into the blood after oral administration, has excellent durability, and has a fatigue prevention and improvement effect. Here, examples of fatigue prevention and improvement effects include motor function maintenance effect, motor function improvement effect, anti-fatigue effect, anti-stress effect, fatigue sensation reduction effect, chronic fatigue prevention effect, fatigue recovery effect, palpitations prevention and improvement effect, alertness prevention and improvement effect, shortness of breath prevention and improvement effect, and muscle pain prevention and improvement effect. Therefore, a composition containing the dipeptide of the present invention is useful as a pharmaceutical composition or a food composition.

The pharmaceutical composition of the present invention can be administered orally, transdermally, enterally, intravenously, etc., but oral administration is more preferred. Preparations for oral administration include liquids, tablets, powders, fine granules, granules, capsules, etc., with liquids and tablets being preferred, and liquids being more preferred.

To prepare these oral preparations, excipients such as lactose, mannitol, corn starch, and crystalline cellulose, binders such as cellulose derivatives, gum arabic, and gelatin, disintegrants such as calcium carboxymethylcellulose, lubricants such as talc and magnesium stearate, solubilizers such as nonionic surfactants, flavorings, sweeteners, stabilizers, pH adjusters, water, ethanol, propylene glycol, glycerin, and the like can be used. Coating agents such as hydroxymethylcellulose phthalate, hydroxypropylmethylcellulose acetate succinate, cellulose acetate phthalate, and methacrylate copolymers can also be used.

The pharmaceutical composition of the present invention may also contain other active ingredients, such as vitamin B1: thiamine, thiamine nitrate, thiamine hydrochloride, fursultiamine, bisbentiamine, benfotiamine, thiamine disulfide, dicethiamine, thiamine propyl disulfide, and derivatives thereof, vitamin _B2 : riboflavin and derivatives and salts thereof, vitamin _B3 : niacin, nicotinic acid, nicotinamide and derivatives and salts thereof, vitamin B5: panthenol, pantothenic acid and derivatives and salts thereof, vitamin B6: pyridoxine and derivatives and salts thereof, vitamin B7: niacin, nicotinic acid, nicotinamide and derivatives and salts thereof, vitamin B8: panthenol, pantothenic acid and derivatives and salts thereof, vitamin B9: pyridoxine, derivatives and salts thereof, vitamin B10: riboflavin and derivatives and salts thereof, vitamin B11: riboflavin and derivatives and salts thereof, vitamin B12: riboflavin and derivatives and salts thereof, vitamin _B13 : riboflavin and derivatives and salts thereof, vitamin B14: riboflavin and derivatives and salts thereof, vitamin _B15 : riboflavin and derivatives and salts thereof, vitamin _B16 : riboflavin and derivatives and salts thereof, vitamin B17: riboflavin and derivatives and salts thereof, vitamin B18: riboflavin and derivatives and salts thereof, vitamin B19: riboflavin and derivatives and salts thereof, vitamin B20: riboflavin and derivatives and salts thereof, vitamin B21: riboflavin and derivatives and salts thereof, vitamin B22: riboflavin and derivatives and salts thereof, vitamin B23: niacin, nicotinic acid, nicotinamide and derivatives and salts thereof, vitamin B24: riboflavin Category ₁₂ : cyanocobalamin and derivatives and their salts, other vitamins; vitamin A, vitamin C, vitamin E, vitamin K, vitamin P, diisopropylamine dichloroacetate, taurine, chondroitin sulfate, royal jelly, caffeine, turmeric, milk thistle, dandelion, burdock, garlic, chrysanthemum, yarrow, gardenia, sesame, Panax notoginseng, asparagus, onion, chicory, medicinal salvia, Korean thistle (artichoke), wolfberry, plants of the Fabaceae and Iridaceae families, quail, elba de passariño, sete sangria, Agametica japonicus, black tea, resveratrol, catechins, berberine, rosemary, bean extract, metformin, etc.

In addition to being used as medicines, the compositions of the present invention can also be used as functional foods such as quasi-drugs, foods for specified health uses, sports drinks, beverages for rehabilitation, and pet foods.

The content of the dipeptide in the pharmaceutical composition or food composition of the present invention varies depending on the dosage form, but is usually preferably 0.001 to 10% by mass, more preferably 0.001 to 5% by mass. The daily dosage of the dipeptide in the pharmaceutical composition or food composition of the present invention is preferably 10 mg to 1000 mg, more preferably 20 mg to 800 mg, and even more preferably 50 mg to 800 mg.

The present invention will now be described in detail with reference to examples, but the present invention is not limited to these examples.

Example 1 (Fractionation of Liver Hydrolysate)
(1) Fractionation of Peptides and Pyroglutamyl Peptides A strong cation exchange resin (AG50) was packed in an Econo Column (2.5 x 20 cm), and the resin was equilibrated with 10 mM HCl. 1 g of liver hydrolysate (Sample A) was dissolved in 20 mL of 10 mM HCl. This solution was added onto the resin, and 20 mL of the non-flowing fraction was collected (non-flowing fraction 1). Then, 20 mL of 10 mM HCl was added onto the resin, and 20 mL of the non-flowing fraction was collected. This operation was repeated 19 times (non-flowing fractions 2 to 20). The absorbance (230 nm) of non-flowing fractions 1 to 20 was measured, and the elution of the peptides was confirmed.
Next, 20 mL of 50% ammonia solution was added onto the resin, and 20 mL of the adsorbed fraction was collected. This procedure was repeated 20 times (adsorbed fractions 1 to 20). The absorbance (230 nm) of the adsorbed fractions 1 to 20 was measured to confirm the elution of the peptide.
The flow-through fraction (pyroglutamyl peptide fraction) and the adsorbed fraction (peptide fraction) were concentrated under reduced pressure using an evaporator.

(2) Hydrophilic and hydrophobic fractionation A solid-phase extraction column (Sep-Pak) was equilibrated with 10 mM HCl. The pyroglutamyl peptide fraction was passed through the column, and the non-adsorbed fraction was eluted and collected (hydrophilic pyroglutamyl peptide fraction). Then, a 60% acetonitrile solution containing 10 mM HCl was passed through the column, and the adsorbed fraction was eluted and collected (hydrophobic pyroglutamyl peptide fraction).
The peptide fraction was also subjected to these procedures.
The four fractions obtained were freeze-dried.

Example 2
(1) Quantitative experiment of indigestible peptides in liver hydrolysate 1) 2.5 mg of liver hydrolysate was dissolved in 1 mL of 50 mM Tris-HCl.
2) Pancreatin (0.1 mg), leucine aminopeptidase (2.45 units), and carboxypeptidase (7.7 units) were added to 1) and subjected to an enzymatic reaction (37° C., 24 hours).
3) The enzyme was removed by ultrafiltration (10K).
4) The mixture from 3) was passed through a strong cation exchange resin (AG50) packed in a spin column, and the flow-through fraction was collected (pyroglutamyl peptide fraction).
5) 200 μL of 3) (peptide fraction) and 4) (pyroglutamyl peptide fraction) were fractionated and collected by size exclusion HPLC (SEC Fr. 35-44).
6) For the peptide fraction, SEC Fr. 35-44 was dried and converted to AccQ. For the pyroglutamyl peptide fraction, SEC Fr. 35-44 was used as it was.
7) The structures of the resistant peptides were determined by LC-MS/MS analysis.
Column: Inertsil ODS-3
Eluent: 0.1% formic acid and 80% acetonitrile containing 0.1% formic acid. Analytical method: For the peptide fraction, the AccQ fragment (m/z = 171.1) is specifically detected (precursor ion scan analysis), and then the structure is estimated by MS/MS analysis. A standard is synthesized and identified and quantified by MRM analysis.
For the pyroglutamyl peptide fraction, peaks are detected by total scan ion analysis, the structure is estimated by MS/MS analysis, and a standard is synthesized and identified and quantified by MRM.

(2) Experiment on transfer of peptides into blood when liver hydrolysate was administered to rats 1) A single dose of an aqueous solution of liver hydrolysate was administered to Wistar rats (6 weeks old, male) (10 g/60 kg).
2) 30 and 60 minutes after administration, blood was collected from the abdominal vena cava under isoflurane anesthesia to obtain plasma. The digestive tract (duodenum to ileum) was then removed and the luminal contents were eluted with 10 mL of physiological saline. Three volumes of ethanol were added to the plasma and digestive tract contents (stored at -20°C until analysis).
3) Plasma and digestive tract contents were dried over ethanol and subjected to AccQ conversion.
4) The indigestible peptides identified in (1) were identified and quantified by MRM analysis.
The dipeptide concentrations in the blood are shown in Table 1.

As a result, it was found that (D)Asp-(D)Val, (D)Asp-(D)Phe, (D)Asp-(D)Ile, (D)Asp-(D)Leu, (D)Asp-(L)Val, (D)Asp-(L)Ile, (D)Asp-(L)Leu, and (D)Asp-(L)Phe have good transferability into the blood when orally administered and persist in the blood for a long period of time.

Example 3 (Synthesis of dipeptide)
1) Add the following reagents to a recovery flask in the following order and react with stirring (4°C overnight):
(i) H-Leu-OtBu・HCl
(ii) DMF
(iii) TEA
(iv) Boc-Asp(OtBu)-OH (Lα form)
(v) HOBt
(vi) EDL·HCl
For other isomers, the following protected amino acids are used:
Boc-D-Asp(OtBu)-OH (Dα body)
Boc-Asp-OtBu (Lβ body)
Boc-D-Asp-OtBu (Dβ body)
2) Remove the DMF using an evaporator.
3) Dissolve in ethyl acetate and transfer to a separatory funnel.
4) Add 5% sodium bicarbonate and stir, then remove the aqueous layer. (x2)
5) Add 10% citric acid and stir, then remove the aqueous layer. (x2)
6) Add saturated saline and stir, then remove the aqueous layer.
7) The ethyl acetate layer is collected and dehydrated by adding sodium hydrogen sulfate.
8) The ethyl acetate layer is collected by filtration and concentrated using an evaporator.
9) Add petroleum ether and dry the resulting precipitate. (If no precipitate forms, proceed to 10))
10) Add 4M HCl/dioxane to the dried material and react with stirring (4°C, 24-48 hours).
11) Remove the 4M HCl/dioxane using an evaporator.
12) Diethyl ether is added, the resulting precipitate is disrupted and washed with ultrasound, and the ethereal supernatant is removed with decane. (x3)
13) Add diethyl ether and leave to stand (4°C, overnight).
14) Diethyl ether is added, the resulting precipitate is disrupted and washed with ultrasound, and the ethereal supernatant is removed with decane. (x3)
15) Dry the precipitate.

Example 4
[Test Method]
(1) Animals used For the experiment, ddY male mice (Japan SLC) weighing 28-32 g (26 g at time of delivery) were used, and were kept in a constant environment of 22±2°C room temperature, 55±5% humidity, and a 12-hour light/dark cycle (light period: 7:00-19:00, dark period: 19:00-7:00) until the experiment. The animals were kept in plastic cages (30 cm long x 20 cm wide x 13 cm high), and were allowed to consume solid food and tap water ad libitum except during the experiment.
(2) Drugs used and preparation method Hydrophobic peptide fraction, hydrophobic pyroglutamyl peptide fraction, hydrophilic peptide fraction, and hydrophilic pyroglutamyl peptide fraction were dissolved in physiological saline and administered intraperitoneally (i.p.) at 0.1 mL per 10 g body weight. The administration schedule was as follows: before forced walking, physiological saline or hydrophobic peptide fraction, hydrophobic pyroglutamyl peptide fraction, hydrophilic peptide fraction, and hydrophilic pyroglutamyl peptide fraction were administered i.p., and after 3 hours of forced walking, the animals were acclimated to the observation cage for 15 minutes, and then physiological saline or hydrophobic peptide fraction, hydrophobic pyroglutamyl peptide fraction, hydrophilic peptide fraction, and hydrophilic pyroglutamyl peptide fraction were administered i.p.
(3) Forced walking load: The mice were placed in an electric rotating cage measuring 37 cm in diameter and 35.5 cm in depth, and forced to walk at a speed of one rotation per 25 seconds for 3 hours.
(4) Measurement of spontaneous motor activity Spontaneous motor activity was evaluated by placing each mouse individually in a plastic cage (24 cm length × 17 cm width × 12 cm height) and allowing it to adapt to the environment for 15 minutes. After that, the amount of planar motor activity over a 90-minute period was automatically quantified using a Supermex.

〔result〕
The results are shown in Figures 1 to 4. As can be seen from Figures 1 to 4, the hydrophobic peptide fraction and the hydrophobic pyroglutamyl peptide fraction showed superior anti-fatigue effects compared to the hydrophilic peptide fraction.

Example 5
(1) Animals used For the experiment, male ddY mice (Japan SLC) weighing 28-32 g (26 g at time of delivery) were used, and were kept in a constant environment of 22 ± 2°C room temperature, 55 ± 5% humidity, and a 12-hour light/dark cycle (light period: 7:00-19:00, dark period: 19:00-7:00) until the experiment. The mice were kept in plastic cages (30 cm long x 20 cm wide x 13 cm high), and were allowed to consume solid food and tap water ad libitum except during the experiment.
(2) Drugs and preparations: The dipeptides were dissolved in saline and administered intraperitoneally (i.p.) at 0.1 mL per 10 g body weight. The administration schedule was as follows: saline or dipeptide was administered i.p. before forced walking, and after 3 hours of forced walking, the animals were acclimated to the observation cage for 15 minutes, and then saline or dipeptide was administered i.p.
(3) Forced walking load: The mice were placed in an electric rotating cage measuring 37 cm in diameter and 35.5 cm in depth, and forced to walk at a speed of one rotation per 25 seconds for 3 hours.
(4) Measurement of spontaneous motor activity Spontaneous motor activity was evaluated by placing each mouse individually in a plastic cage (24 cm length × 17 cm width × 12 cm height) and allowing it to adapt to the environment for 15 minutes. After that, the amount of planar motor activity over a 90-minute period was automatically quantified using a Supermex.
(5) Statistical Processing The experimental results were shown as the mean and standard error (S.E.M.). The significant difference test was performed using Fisher's PLSD method after analysis of variance. A P value of 5% or less was determined to be significant. Stat view-J 5.0 for Windows (registered trademark) was used for this test.

〔result〕
The results are shown in Figures 5 to 10. As is clear from Figures 5 to 10, Asp-Leu (Dα form), Asp-Leu (Dβ form), Asp-Val (Dα form), Asp-Val (Dβ form), and (D)Asp-Phe (Dα, Dβ) showed excellent anti-fatigue effects.

Claims (2)

A fatigue prevention and improvement agent comprising a dipeptide selected from (D)Asp-(D)Leu, (D)Asp-(D)Val, (D)Asp-(D)Phe , (D)Asp-(L)Leu, (D)Asp-(L)Val, and (D)Asp- ( L)Phe . A food composition for preventing and improving fatigue, comprising a dipeptide selected from (D)Asp-(D)Leu, (D)Asp-(D)Val, (D) Asp-(D)Phe, (D)Asp-(L)Leu, (D)Asp-(L)Val, and (D)Asp- ( L)Phe .