JP2835504B2 - Novel peptides and activated oxygen inhibitors - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a peptide having a peptide structure of the following amino acid sequence which is useful as a medicament, and an activated oxygen inhibitor containing such a peptide as an active ingredient. Gln-Gln-Pro-Ile-Gln-Ala (wherein each symbol representing an amino acid residue is represented by a convention used in amino acid chemistry.)

[0002]

2. Description of the Related Art Diseases involving activated oxygen are widely present, such as inflammation such as burns and arthritis, reperfusion injury, side effects of anticancer drugs, radiation injury, peptic ulcer, bacterial shock, cachexia, and autoimmune diseases. I do. Diseases caused by a large amount of activated oxygen generated by activation of neutrophils and macrophages are all covered. Generally, oxygen includes oxygen essential for animals (triplet oxygen molecule: ³ O ₂ ) and radicals (activated oxygen) generated under specific conditions or when the body is upset.
And exists. Radicals can be directly or indirectly (in the form of peroxidation) in cell membranes, intracellular granule membranes, or DNA.
And alters and damages various cell components. The radicals are produced in the body, the type superoxide anion ^(- _O 2 ·), singlet oxygen ⁽¹ _O 2 ·),
There are hydroxyl radicals (.OH) and the like. Among superoxide anion ^(- O 2 _·) causes peroxidation acts on unsaturated fatty acids in cell membranes, the oxidizing power to lipid is said to be higher several thousand times the essential oxygen to animals. Superoxide dismutase as activated oxygen inhibitor (SOD, enzyme number EC 1.15.1.1)
In McCord, J. et al., 1969. M.
& Fridovich, I .; : J. Biol. Che
m. , 244,6049 (1969)] by an enzyme that acts is found, superoxide anion oxygen molecules occurs is one-electron reduction ^(- _{O 2} ·) the disproportionation _{^{2 - O 2 · + 2H +}} → Catalyzes H ₂ O ₂ + O ₂ . When the human body is normal, SOD works to suppress the generation of superoxide anions. This SOD
The activity decreases with aging, that is, the activity decreases from the middle age to the old age, and the increase or decrease of the SOD activity depends on the aging of the living body,
It is also called a barometer of canceration. Such SO
When the D activity decreases, it becomes difficult to suppress the generation of radicals and S
It is necessary to take up an activated oxygen inhibitor which supplements OD or scavenges and removes radicals. On the other hand, the activated oxygen inhibitory action of polypeptides ranging from amino acids to proteins as water-soluble antioxidants inhibits the contact between oxygen molecules and unsaturated fatty acids by encapsulating fats and oils by peptides, and the lipid peroxy radical (LOO) It is thought to suppress the generation of ()), and acts on radicals (LOO) generated during the oxidation of fats and oils (L), such as the antioxidant action of BHA (butylhydroxyanisole) and BHT (dibutylhydroxytoluene) Thus, it is distinguished from the radical scavenging action that stops the chain reaction of oxidation. _{LOO · + AH 2 → LOOH +} AH · 2AH · → 2AH 2 + A or LOO · + AH · → LOOH + A (AH2; anti-oxidant) on the basis of such a background, anti-cancer, today there is no silver bullet for anti-aging, from the environment Removes or inactivates DNA damage factors, mutation factors, carcinogens, aging factors, etc.
Studies and studies on activated oxygen inhibitors exhibiting an activated oxygen free radical scavenging action and an antioxidant action have been advanced.

[0003]

In the above-mentioned prior art, SOD as an activated oxygen inhibitor is difficult to produce and the availability of raw materials is limited. Vitamin E, vitamin C, catechins, etc. However, there is a drawback that the activated oxygen inhibitory action is not sufficient in the experiment using, and an activated oxygen inhibitor having a stronger action is demanded. In addition, most of activated oxygen inhibitors exhibiting an active oxygen free radical scavenging action and an antioxidant action are mostly produced by chemical synthesis, and even natural substances using materials from plants and animals. Even if they are derived, many of them are made by using chemical substances that harm the human body during the manufacturing process or by reacting some of the products with the chemical substances. Very little is known about the amino acid sequence and antioxidant activity of polypeptides ranging from amino acids to proteins as water-soluble antioxidants, and Yamaguchi et al. [New Food Industry, Vol. 31, pp. 18-22 (1989)] Has a stronger antioxidant activity than amino acids and proteins, and recently Takushoku et al. [Journal of the Japanese Society of Agricultural Chemistry, Vol. It only reports oxidized peptides. There is no report that these activated oxygen agents having an active oxygen free radical scavenging action and an antioxidant action are still being developed as pharmaceuticals.

[0004]

Means for Solving the Problems The present inventors searched for a substance having a pharmacological action from a decomposition solution of a protease of wheat gluten and found that the novel hexapeptide has a strong activated oxygen inhibitory action. . And, they intensively studied to put this peptide into practical use as a medicine. As a result, the present inventors have found that this peptide has an active oxygen free radical scavenging action and an antioxidant action, and is useful as an activated oxygen inhibitor derived from natural products. The present invention is based on such findings. Hereinafter, the present invention will be described in detail. The novel peptide according to the present invention is a novel hexapeptide represented by an L-form amino acid sequence represented by the following formula: Gln-Gln-Pro-Ile-Gln-Ala, and is a white powder at room temperature.

[0005] The above-mentioned hexapeptide may be chemically synthesized or separated and purified from a decomposed solution of wheat gluten protease. When chemically synthesizing the novel peptide according to the present invention, a conventional peptide synthesis method such as a liquid phase method or a solid phase method is used to convert a C-terminal (carboxyl terminal side) of the peptide to a polymeric solid support. It is preferable that the L-form amino acids corresponding to the amino acid residues are sequentially bonded by peptide bonds. Then, the synthetic peptide thus obtained is cleaved from the polymeric solid support using trifluoromethanesulfonic acid, hydrogen fluoride, or the like, and then the protecting group of the amino acid side chain is removed. Can be purified by a usual method using a column described above.

[0006] As described above, the novel hexapeptide according to the present invention can be separated and purified from the degradation solution of the protease of wheat gluten. In that case, for example, the following procedure is performed. Can be. The wheat gluten portion containing the novel peptide is removed and hydrolyzed. The hydrolysis is performed according to a conventional method. For example, when hydrolyzing with a protease such as pepsin, wheat gluten is further hydrolyzed if necessary, and then adjusted to the optimal value of the enzyme, and the enzyme is added and incubated. Then, if necessary, after neutralization, the enzyme is inactivated to obtain a hydrolyzed solution. The hydrolyzed solution is filtered using a filter paper and / or celite to remove insoluble components, and the obtained filtrate is filtered using a semipermeable membrane such as cellophane or the like with a suitable solvent (for example, Tris-HCl buffer, After sufficient dialysis in a neutral buffer such as a phosphate buffer, the solution containing the components in the filtrate and having passed through the semipermeable membrane is subjected to a strongly acidic cation exchange resin (for example, Dow Chemical Co., Ltd.). Dowex 50W etc.)
And a fraction containing a component having activated oxygen inhibitory activity is obtained from the adsorbed fraction, and the obtained activated oxygen inhibitory activity fraction is subjected to cation exchange gel filtration (for example, SP-Sphadex C-Pharmacia). 25), and the obtained activated oxygen inhibitory active fraction is further subjected to reverse phase HP
Fractionate by LC.

[0007] This novel hexapeptide does not induce antibody production and does not cause anaphylactic shock when repeatedly administered intravenously. Also it consists array structure of this peptide L- amino acids only, after administration, because it is gradually degraded by proteases in vivo toxicity is extremely low safety is very high (LD _50> 5000mg /
kg: oral administration to rats). Hexapeptide according to the present invention, injections using additives such as commonly used excipients,
It can be prepared into tablets, capsules, granules, powders and the like. The method of administration usually includes injection into mammals (eg, humans, dogs, rats, etc.) deficient in SOD or oral administration. The dose may be, for example, 0,0 kg of the hexapeptide per 1 kg of the animal body weight.
It is an amount of 01 to 10 mg. The frequency of administration is usually 1
It is about 4 to 4 times, but can be appropriately prepared depending on the administration route. The types of excipients, binders, disintegrants, lubricants, etc. used in the above various formulations are not particularly limited, and those used in ordinary injections, powders, granules, tablets or capsules are used. can do.

[0008] Examples of additives used in tablets, capsules, granules and powders include the following. As excipients, sugars such as crystalline cellulose, sugar alcohols such as mannitol, starch, anhydrous calcium phosphate, etc .; as binders, starches, hydroxypropylmethyl cellulose, etc .; as disintegrants, carboxymethyl cellulose and its potassium salts Lubricating agents include stearic acid and its salts, talc and waxes. In preparing the preparations, odorants such as menthol, citric acid and salts thereof, and fragrances can be used as necessary. A sterile composition for injection is prepared by dissolving the novel hexapeptide according to the present invention in water for injection, physiological saline and a sugar alcohol injection such as xylitol or mannitol, or a glycol such as propylene glycol or polyethylene glycol by an ordinary method. It can be suspended to prepare an injection. At this time, buffers, preservatives,
An antioxidant and the like can be added as needed. The preparation containing the novel hexapeptide of the present invention may be in the form of a lyophilized product or a dry powder, and may be used by dissolving it with a usual dissolving agent, for example, water or physiological saline when used.

[0009] Activated oxygen is generated in phagocytes such as macrophages, and has a role of decomposing foreign substances that have been engulfed by phagocytes. However, when activated oxygen is excessively produced, it is secreted out of cells. Disturb other organizations. The novel hexapeptide according to the present invention has an excellent activated oxygen inhibitory action,
Activated oxygen Free radical scavenging and antioxidant effects, decomposes excess active oxygen that causes tissue damage and protects tissues.For example, it is effective as an anti-inflammatory agent for arthritis and rheumatism. In addition,
It is also useful for Behcet's disease, myocardial infarction and the like.

[0010]

The present invention will be described in more detail with reference to the following examples, which are production examples and test examples. Production Example 1 Deionized water (1.65 l) was added to 330 g of wheat gluten and homogenized. 9.9 g of pepsin was added to the obtained wheat gluten homogenate, and the pH was adjusted to 2.0 to 37.
Incubated for 20 hours at ° C. The pepsin hydrolyzate of wheat gluten homogenate prepared in this manner was used as Di
Ultrafiltration was performed using an aflow membrane (YM-10 type membrane manufactured by Amicon, fractionated molecular weight 10,000). The obtained filtrate is D
Chromatography was performed using a column packed with Owex 50W × 4 (H ⁺ ). Washed with deionized water, elution the eluate was concentrated performed in _2N-NH 4 OH. This concentrated solution was subjected to chromatography using a Scphadex G-25 column to fractionate a low-molecular peptide fraction (fraction number 2).
4-41) were separated. The column chromatograph is shown in FIG. The low molecular peptide fraction was concentrated to obtain a wheat gluten peptide solution. Further, this peptide solution was added to SP-
Chromatography was performed on a Scphadex C-25 (H ⁺ ) column to obtain SP-1 as each peptide fraction.
Fractions (fraction numbers 17-37), SP-2 fractions (fraction numbers 38-59) and SP-3 fractions (fraction numbers 60-80)
Was isolated. The column chromatograph is shown in FIG. Each of these peptide fractions is freeze-dried to obtain peptide powder (hereinafter referred to as wheat gluten peptide).
18.6 g of the SP-1 fraction, 19.9 g of the SP-2 fraction and 23.3 g of the SP-3 fraction were obtained. Among the fractionated wheat gluten peptides, the peptide powder of the SP-3 fraction having high activating oxygen inhibitory activity was dissolved in deionized water (5 mg / 25 μιη) and then subjected to HPLC. The conditions were as follows: Dcvclosil ODS manufactured by Nomura Chemical Co., Ltd.
-5 (φ4.6 mm ID × 25 cmL) and 0.05% trifluoroacetic acid (hereinafter abbreviated as TFA) to 25% acetonitrile / 0.05% T as a mobile phase.
By the concentration gradient method in FA, the flow rate was 1.0 ml / min,
Chromatography was performed at a detection wavelength of 220 nm to obtain a peptide fragment having a strong activated oxygen inhibitory action at an elution time of 48.5 minutes. The result is as shown in FIG. The amino acid sequence of the peptide having an activated oxygen inhibitory effect obtained in this manner is obtained by using a protein sequencer 47 manufactured by Applied Biosystems (ABI).
Determined using type 7A. As a result, it was confirmed that the peptide was a hexapeptide represented by an L-form amino acid sequence represented by the following formula: Gln-Gln-Pro-Ile-Gln-Ala. When the wheat gluten peptide according to the present invention is formulated as an activated oxygen inhibitor, for example, in tablets, it may be treated according to a conventional method, for example, as follows: (1) 13 g of peptide, (2) lactose 87
g, (3) 29 g of corn starch, (4) 1 g of magnesium stearate, and (1), (2) and 17 g of corn starch were first mixed and granulated together with a paste made from 7 g of corn starch.
g of corn starch and (4) are added, and the resulting mixture is tableted with a compression tablet machine to produce 1,000 tablets.

Production Example 2 This example is based on Gln-Gln-Pro-Ile-Gln-A
This is a production example of a la by a synthesis method. The hexapeptide was synthesized by a solid phase method using a peptide synthesizer model 430A manufactured by Applied Biosystems (ABI).
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 hexapeptide, a C-terminal alanine was reacted with a chloromethyl resin in the usual manner to obtain a peptide-bound resin. The amino acid at this time is t-butoxycarbonyl (hereinafter, t-butoxycarbonyl).
Abbreviated as -Boc. ) -Protected t-Boc amino acid was used. Next, this peptide-bonded resin was suspended in a mixture of ethanediol and thioanisole, stirred at room temperature for 10 minutes, and then added with trifluoroacetic acid under ice-cooling.
Stir for another 10 minutes. Trifluoromethanesulfonic acid was added dropwise to the mixture, and the mixture was stirred at room temperature for 30 minutes. The product was precipitated by adding anhydrous ether and separated.The precipitate was washed several times with anhydrous ether, and then dried under reduced pressure. And dried. The unpurified synthetic peptide thus obtained was dissolved in distilled water, and then reversed-phase column C ₁₈ (5 μ).
Purified by HPLC using (A) as mobile phase
Using distilled water containing 0.1% TFA and (B) acetonitrile solution containing 0.1% TFA, solution (A) was 9% in 20 minutes.
The flow rate was 1.4 ml / mi by the concentration gradient method of 3% → 71%.
n. UV wavelength 214n
The elution fraction showing the maximum absorption was detected and collected and freeze-dried to obtain the desired synthetic hexapeptide.

Analysis of the synthetic hexapeptide by mass spectrum confirmed that it was a hexapeptide having the amino acid sequence structure of the following formula: Gln-Gln-Pro-Ile-Gln-Ala. The result of this mass spectrum is as shown in FIG. The hexapeptide of the present invention obtained by synthesis is in vitro (in a test tube) shown below.
The test confirmed the activated oxygen free radical scavenging effect and the antioxidant effect, thereby confirming the activated oxygen inhibitory effect.

[0013] Test Example 1 (Measurement of activated oxygen free radical scavenging activity) Cypridina over luciferin derivative (CLA) singlet oxygen ⁽¹ _O 2 ·), superoxide anion ^(- O
₂ ) is an effective chemiluminescent reagent that specifically detects
The inventors [Agric. Biol. Chem. , 55,
157-160 (1991)] Superoxide dismutase and (SOD) and CLA by quenching experiments using the quencher by a method ^- reaction rate of the O ₂ · is calculated.
CLA (C ₁₃ H ₁₁ ON ₃ , manufactured by Tokyo Chemical Industry Co., Ltd., final concentration 1.39 × 10 ^{−7 to} 4.64 × 10 ⁻⁸ ) solution 10 μm
1, albumin (50 mg / ml, manufactured by Sigma Chemical Co.) 50
0 μl, xanthine oxidase (1.45 unit /
ml, manufactured by Sigma Chemical Co., Ltd.) in a cylindrical quartz cell (inner diameter: 14 mm, height: 60 mm), placed in a sample chamber of a luminometer (Aloka BLR-102B, manufactured by Hamamatsu Photonics), and transferred to a 3 mM hypoxanthine solution 2
00 μl was injected and chemiluminescence was measured from the bottom of the cell by single photon counting. In the absence and, if the quencher is present ^- the ratio of O 2 _· emission intensity _(I 0 _/ I)
Is I ₀ / I = 1 + [k ₃ / (k ₁ + k ₂ [CLA])]
× [Q]. Here [Q] is activated oxygen inhibitor, _{k 1} is ^- O ₂ · quenching rate constant, _{k 2} is the reaction rate constant of -O ₂ · and [CLA], _{k 3} is a -O ₂ · [Q]
Shows the reaction rate constant. Table 1 shows the activated oxygen inhibitory activity (quenching rate) of the peptide fraction derived from wheat gluten according to the present invention, which exhibits an activated oxygen free radical scavenging effect.

[Table 1] The activated oxygen inhibitory activity value (reaction rate constant k ₃ ) of the novel hexapeptide according to the present invention, which indicates an activated oxygen free radical scavenging action, is 3.5 × 10 ⁻⁶ M ⁻¹ sec ⁻¹ . The activated oxygen inhibition activity value (reaction rate constant k3) showing the activated oxygen free radical scavenging action of the standard SOD is 3.47 × 10 ⁻⁸ M ⁻¹ sec ⁻¹ .

Test Example 2 (Measurement of antioxidant action) As a measure of antioxidant action, the reaction solution was linoleic acid 51.1.
mg, ethanol 4.052 ml, 0.05 M phosphate buffer (pH 7.0) 4.0 ml, deionized water 1.948
1 to 3 m of peptide having antioxidant action
g was added to adjust the total amount to 10 ml. This solution was sealed in a test tube with a screw, left in a thermostat at 50 ° C., and the peroxide value of linoleic acid was measured every 24 hours by the iron-rodan method. That is, 0.1 ml of the reaction solution. 9.7 ml of 75% ethanol solution, 0.1 m of 30% rhodamon ammonium solution
3.5% hydrochloric acid solution containing 0.02 M ferric chloride
After adding 1 ml and reacting for 3 minutes, absorbance 500 n
m was measured. At that time, the absorbance at 500 nm was 0.35.
The number of days up to was defined as the number of days of induction (days). FIG. 5 shows the activated oxygen inhibitory activity (induction days) showing the antioxidant activity of the wheat gluten-derived peptide fraction according to the present invention.
The activated oxygen inhibitory activity value (day of induction) showing the antioxidant effect of the novel hexapeptide according to the present invention is 1 day for 1 mg of hexapeptide relative to 6.5 days for 2 mg of tocopherol.
4 days. As a result of the above test, the novel hexapeptide according to the present invention has a activating oxygen free radical scavenging action and an antioxidant action, and thus shows a significant activating oxygen inhibitory action in an in vitro (in vitro) test. confirmed. Therefore, the hexapeptide according to the present invention is useful as a therapeutic or preventive drug for patients with ischemic heart disease, rheumatoid arthritis and severe burns who are the targets of activated oxygen inhibitors. Incidentally, the hexapeptide according to the present invention,
In a peptide structurally represented by its amino acid sequence, it can also be employed in the structure.

[Brief description of the drawings]

FIG. 1 is a diagram showing the results of separation and purification of an activated oxygen-inhibiting peptide by Sephadex G-25 column chromatography in Production Example 1 of a wheat gluten pepsin hydrolyzate according to the present invention. In the figure, as markers, insulin having a molecular weight of 6,000, insulin B chain having a molecular weight of 3,500, insulin A chain having a molecular weight of 2,550,
Bacitracin with a molecular weight of 1,450 and glycine with a molecular weight of 75 were used.

FIG. 2 shows SP-Sephadex C-25 (H ⁺ ) in Wheat gluten peptide according to the present invention in Production Example 1.
It is a figure which shows the result of separation purification of the activated oxygen inhibition peptide by column chromatography.

FIG. 3 Production Example 1 of wheat gluten peptide according to the present invention
FIG. 4 is a view showing the results of separation and purification of a fragment of an activated oxygen-inhibiting hexapeptide by reverse-phase HPLC in FIG.

FIG. 4 is a view showing a mass spectrum of the synthetic hexapeptide obtained in Production Example 2 of the hexapeptide according to the present invention.

FIG. 5

[Explanation of symbols]

It is a figure which shows the induction | guidance | derivation days (day) of the SP fraction (1, 2, 3 mg) in the production example 1 of the wheat gluten peptide which concerns on this invention, and shows an active oxygen inhibitory effect while showing an antioxidant effect.

Claims (2)

(57) [Claims] 1. A novel hexapeptide having a peptide structure based on the amino acid sequence of L-form represented by the following formula: Gln-Gln-Pro-Ile-Gln-Ala. 2. Activation comprising a novel hexapeptide having a peptide structure based on the amino acid sequence of L-form represented by the following formula: Gln-Gln-Pro-Ile-Gln-Ala as an active ingredient. Oxygen inhibitors.