Peptide-containing antiallergic composition

Abstract

An object of the present invention is to provide functional foods, pharmaceuticals, etc. that can effectively alleviate allergic symptoms in humans and are safe and reliable even when used orally. [Solution] Functional foods, medicines, etc. containing dipeptides containing glutamic acid as active ingredients, which can be obtained by variously processing crushed dried sardines, which are widely consumed in the diet, can be safely used by humans as oral medications, and can effectively alleviate allergic symptoms by inhibiting the degranulation of basophils, etc.

Description

[Technical Field]

[0001] The present invention relates to an oral agent or the like for alleviating allergic symptoms in humans and the like. [Background technology]

[0002] Currently, it is said that one in two Japanese people suffer from some kind of allergic disease, which is an increase from previous statistics. Typical examples of allergic diseases include atopic dermatitis, hay fever, asthma, and food allergies. For example, hay fever is a national disease, affecting one-third of Japanese people.

[0003] Hay fever is classified as a type I allergy, and like atopic dermatitis and food allergies, symptoms occur when the body's IgE antibodies bind to basophils and mast cells, which then react with allergens. When stimulated by an allergen, basophils and mast cells release granules that they had been holding inside the cell, and these granules contain substances that induce allergic symptoms, such as histamine and leukotriene, resulting in the eye and nasal symptoms typical of hay fever.

[0004] Antihistamines and other drugs are often used to alleviate these allergic symptoms, but they have many problems, such as side effects and a significant decrease in effectiveness with continued use. Therefore, many people would prefer to relieve their symptoms by consuming food ingredients that have the effect of alleviating allergic symptoms, rather than taking pharmaceutical ingredients that have little history in diet. For this reason, several research and development projects are underway to develop anti-allergic foods and anti-allergic drugs that use food-derived ingredients that are widely used in diets.

[0005] As such food-derived components, for example, a combination of β-lactoglobulin, a major protein in milk, and nobiletin, a citrus flavonoid (Patent Document 1), a product using tea leaf extract (Patent Document 2), and a product using potato shochu lees (Patent Document 3) have been proposed, but at present, these are still not sufficient.

[0006] Against this technological background, there has been a continuing demand in the industry for further development of anti-allergy foods and anti-allergy drugs that use food-derived ingredients that are widely consumed, particularly agents that use food-derived ingredients that can effectively inhibit the degranulation of basophils and mast cells. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Japanese Patent Application Laid-Open No. 2015-036369 [Patent Document 2] Japanese Patent Application Laid-Open No. 2014-114303 [Patent Document 3] Japanese Patent Application Laid-Open No. 2011-093815 Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention aims to provide an allergic symptom reliever (functional food, pharmaceutical, etc.) that can effectively relieve allergic symptoms in humans and is safe and reliable even when taken orally. [Means for solving the problem]

[0009] To achieve the above objective, the inventors conducted extensive research and discovered that crushed dried sardines, a commonly consumed food, were treated with protease to obtain a supernatant (enzyme-treated crushed dried sardines) or its dried product, which was then centrifuged. The resulting supernatant was then dialyzed through a dialysis membrane and sterilized by a filter (hereafter referred to as EDSE). This EDSE was then treated with acetonitrile at various concentrations, and the supernatant fraction was fractionated by reverse-phase chromatography under certain conditions to obtain a chromatogram. Fractions were collected and evaluated according to the detected peaks, and it was found that glutamic acid was highly abundant in all active fractions, leading to the present invention.

[0010] The following are examples of embodiments of the present invention. The present invention is not limited to these embodiments, and various modifications are possible within the scope of the technical concept of the present invention.

[0011] (1) An agent for alleviating allergic symptoms, characterized by containing a dipeptide containing glutamic acid as an active ingredient. (2) An agent for allergy symptom relief, the active ingredient of which is a dipeptide of glutamic acid-aspartic acid or aspartic acid-glutamic acid. (3) An agent for alleviating allergic symptoms, the active ingredient of which is a dipeptide of glutamic acid-serine or serine-glutamic acid. (4) An agent for alleviating allergic symptoms, the active ingredient of which is a dipeptide, glutamic acid-threonine or threonine-glutamic acid. (5) An agent for alleviating allergy symptoms, the active ingredient of which is a dipeptide of glutamic acid-glycine or glycine-glutamic acid. (6) An agent for alleviating allergy symptoms, the active ingredient of which is a dipeptide of glutamic acid-alanine or alanine-glutamic acid. (7) An agent for alleviating allergic symptoms, the active ingredient of which is a dipeptide of glutamic acid-lysine or lysine-glutamic acid. (8) An agent for relieving allergy symptoms, the active ingredient of which is the dipeptide histidine-glutamic acid. (9) An agent for alleviating allergy symptoms, the active ingredient of which is a dipeptide of glutamic acid-arginine or arginine-glutamic acid. (10) An agent for allergy symptom relief, the active ingredient of which is the dipeptide glutamic acid-glutamic acid. (11) An allergic symptom reliever or a method for producing the same, which contains as an active ingredient the non-adsorbed fraction obtained by centrifuging an enzyme-treated solution of crushed dried sardines, removing insoluble matter, obtaining a supernatant, removing molecules with a molecular weight of 500 or less, and treating the supernatant fraction with acetonitrile, or a method for producing the same. (12) The agent or the method for producing the same according to any one of (1) to (11), wherein the agent for alleviating allergic symptoms is a degranulation inhibitor. [Effects of the Invention]

[0012] According to the present invention, it is possible to provide a safe and reliable oral agent for alleviating allergic symptoms in humans, etc., and oral administration of this agent, etc., can effectively alleviate allergic symptoms by inhibiting basophil degranulation and promoting cytokine production in macrophages, etc. As a result, the quality of life (QOL) of allergy patients, etc. can be further improved.

[0013] The present invention also has technical significance in that it has been possible to identify the active substance itself that exhibits the aforementioned effect, and it is now possible to use the active substance directly as an active ingredient, which has the advantages of further increasing the efficacy of the agent and facilitating purification, etc. [Brief explanation of the drawings]

[0014] [Figure 1] 1 is a graph showing the degranulation inhibitory activity of EDSE. In the figure, two asterisks indicate p<0.01 vs. control. [Figure 2] The data was obtained by treating EDSE with an 80% acetonitrile solution and subjecting the resulting supernatant fraction to reverse phase chromatography. [Figure 3] 3 is a graph showing the degranulation inhibitory activity of the fractions obtained by reversed-phase chromatography in Fig. 2. In the figure, one asterisk indicates p<0.05, and two asterisks indicate p<0.01 vs. control. [Figure 4] 1 is a graph showing the degranulation inhibitory activity of various amino acids. In the figure, one asterisk indicates p<0.05, and two asterisks indicate p<0.01 vs. control. [Figure 5] 1 is a graph showing the degranulation inhibitory activity of glutamic acid multimers. In the figure, two asterisks indicate p<0.01 vs. control. [Figure 6] 1 is a graph showing the degranulation inhibitory activity of various dipeptides. In the figure, one asterisk indicates p<0.05, and two asterisks indicate p<0.01 vs. control. [Figure 7] 1 is a graph showing the degranulation inhibitory activity of Glu-Asp and Asp-Glu. In the figure, two asterisks indicate p<0.01 vs. control. [Figure 8] 1 is a graph showing the degranulation inhibitory activity of Glu-Ser and Ser-Glu. In the figure, one asterisk indicates p<0.05, and two asterisks indicate p<0.01 vs. control. [Figure 9] 1 is a graph showing the degranulation inhibitory activity of Glu-Thr and Thr-Glu. In the figure, one asterisk indicates p<0.05, and two asterisks indicate p<0.01 vs. control. [Figure 10] 1 is a graph showing the degranulation inhibitory activity of Glu-Gly and Gly-Glu. In the figure, two asterisks indicate p<0.01 vs. control. [Figure 11] 1 is a graph showing the degranulation inhibitory activity of Glu-Ala and Ala-Glu. In the figure, two asterisks indicate p<0.01 vs. control. [Figure 12] 1 is a graph showing the degranulation inhibitory activity of Glu-Lys and Lys-Glu. In the figure, two asterisks indicate p<0.01 vs. control. [Figure 13] 1 is a graph showing the degranulation inhibitory activity of Glu-His and His-Glu. In the figure, two asterisks indicate p<0.01 vs. control. [Figure 14] 1 is a graph showing the degranulation inhibitory activity of Glu-Arg and Arg-Glu. In the figure, two asterisks indicate p<0.01 vs. control. DETAILED DESCRIPTION OF THE INVENTION

[0015] In the present invention, a dipeptide, particularly a dipeptide containing glutamic acid, is used as an active ingredient of an allergic symptom alleviating agent or the like.

[0016] The dipeptide can be obtained, for example, as follows. First, a supernatant or a dried product thereof is obtained by treating pulverized dried sardines with a protease. The pulverized dried sardines refers to dried sardines or other small fish that have been boiled and then dried, and then pulverized. In particular, it is preferable to use pulverized dried sardines that have been treated with a grinder, hammer mill, or the like to finely pulverize them.

[0017] The pulverized dried sardines are then treated with a protease. The protease used is preferably a food-grade protease derived from a microorganism, with an optimal pH in the acidic range (e.g., pH 2 to 6), and is particularly preferably an acidic protease derived from the filamentous fungus Aspergillus. An example of a commercially available protease is Denapsin 2P (derived from Aspergillus niger, product of Nagase ChemteX Corporation).

[0018] Protease treatment is carried out, for example, as follows: First, 1 to 10 times (preferably 3 to 5 times) the amount of water is added to the sardine powder, and the pH is adjusted to 2 to 6 as necessary. Then, an enzyme is added at 0.05 to 3.0% (preferably 0.1 to 1.0%), and the reaction is carried out at the optimum temperature for the enzyme, 20 to 55°C (preferably 30 to 50°C), for 0.5 to 30 hours (preferably 1 to 20 hours). The enzymatic reaction is preferably carried out with stirring, but this is not limiting as long as the reaction proceeds smoothly.

[0019] After protease treatment, neutralization is performed if necessary, and the enzyme is inactivated by holding at a temperature of 70°C or higher (preferably 90-100°C) for 2-60 minutes (preferably 5-30 minutes). The dried sardine enzyme-treated solution obtained in this manner is then subjected to a treatment to remove insoluble solids. This dried sardine pulverized enzyme-treated solution is centrifuged, for example, at 70 krpm for 20 minutes to remove unwanted materials. The resulting supernatant is dialyzed, for example, using a dialysis membrane with a molecular weight cutoff of 100-500, and then filter-sterilized to obtain a sample (the EDSE described above).

[0020] Next, further impurities are removed from the EDSE. The EDSE is treated with, for example, 80% acetonitrile to obtain a supernatant. This EDSE 80% acetonitrile supernatant fraction is then fractionated by reverse-phase chromatography. After the 80% acetonitrile supernatant fraction is loaded onto a column, 0% acetonitrile (ultrapure water) is passed through the column, and the acetonitrile concentration is then increased stepwise to 20, 40, and 60%. A chromatogram is then obtained, and fractions are collected according to the detected peaks. This allows the desired dipeptide to be obtained. Furthermore, as long as the desired dipeptide is capable of exhibiting the effects of the present invention, the method of production or acquisition does not matter. For example, it may be obtained by chemical synthesis or the like.

[0021] The dipeptide obtained as described above may be subjected to concentration treatment, sterilization, disinfection treatment, powdering (drying), etc. as needed. Powdering can be carried out by standard methods such as spray drying, drum drying, and freeze drying. This allows preparations in powder form to be obtained.

[0022] The present invention may be in the form of not only powders as described above, but also granules, tablets, capsules, powders, sticks, liquids, gels, pastes, syrups, etc., and is not particularly limited as long as it is an orally administrable form. Furthermore, the present invention may also be formulated by appropriately combining one or more of the known adjuvants commonly used in the pharmaceutical technology field, such as excipients, binders, disintegrants, lubricants, flavorings, solubilizers, suspending agents, and coating agents, within a range that does not impair the effects of the active ingredient. Taking into consideration the form, composition, etc., the active ingredient of the present invention may be produced by appropriately combining the adjuvants and the like throughout the process from the raw material stage to the finished product.

[0023] The method of use and dosage of the allergic symptom reliever of the present invention may be determined appropriately depending on the purpose of use (prophylactic, therapeutic, or health care use), the age of the subject, the dosage form, etc., and it is suitable to orally administer 0.1 to 50 mg / kg / day, preferably 1.0 to 30 mg / kg / day of the dipeptide, etc. to humans for, for example, 30 days or more. However, since there is no problem with the safety of the active ingredient, there is no problem at all even if it is used in an amount greater than the above range.

[0024] The present invention relates to orally administering to humans an allergic symptom reliever containing a dipeptide containing glutamic acid as an active ingredient to relieve allergic symptoms, and includes not only pharmaceuticals (medicinal agents) but also health supplements, health functional foods, supplements, etc., which have specific functions and forms and are taken for the purpose of maintaining health, etc., and have a specified dose (effective amount) and method of use of the active ingredient, and can be taken in that amount per unit package. Food compositions (including quasi-drugs) that are clearly distinguished from those used solely as foods are also included.

[0025] In this way, by using an allergy symptom alleviating agent containing a dipeptide or the like as an active ingredient and orally administering it to an allergy patient, it becomes possible to alleviate allergy symptoms safely and effectively.

[0026] Hereinafter, examples of the present invention will be described, but the present invention is not limited to these examples, and various modifications are possible within the technical concept of the present invention. [Example]

[0027] (Production of enzyme-treated crushed dried sardines) A liquid (enzyme-treated liquid of pulverized dried sardines) was produced by treating pulverized dried sardines with protease using the following method, which is the premise of the product of the present invention.

[0028] To the dried sardine powder, 3.7 times the weight of water was added, and the pH was adjusted (pH 3.3-4.3). Then, 0.3% protease (Denapsin 2P: Nagase ChemTech Corporation) was added and treated at the enzyme's optimum temperature (40-50°C) for 15-20 hours. The mixture was then neutralized (pH adjusted to 5.2-5.6) and heated (heated at 97-99°C for 30 minutes) to inactivate the enzyme, yielding an enzyme-treated dried sardine powder solution.

[0029] (Production of sample (EDSE)) Next, EDSE, which is the sample used in the present invention and tests, was produced by the following method.

[0030] The enzyme-treated solution of crushed dried sardines obtained above was centrifuged at 70 krpm for 20 minutes to remove insoluble matter. The resulting supernatant was dialyzed for desalting. The dialysis conditions were 24 hours at 4°C using a dialysis membrane with a molecular weight cutoff of 100 to 500 Da (molecular weight cutoff: 100 to 500 Da). The solution was then filter-sterilized to obtain the sample. This is EDSE.

[0031] (Separation of dipeptides and amino acids) EDSE was then treated to give the desired dipeptide and amino acid as follows:

[0032] EDSE was fractionated by reversed-phase chromatography of the 80% acetonitrile supernatant fraction. Specifically, the 80% acetonitrile EDSE supernatant fraction was fractionated by reversed-phase chromatography under the following conditions. Column: YMC-Pack ODS-AQ column (6.0 mm ID x 150 mm, S 5 μm, 12 nm) Mobile phase: 0, 20, 40, 60% acetonitrile / ultrapure water (Stepwise increase in acetonitrile concentration) Flow rate: 1.0mL / min Column temperature: 40℃ Sample injection volume: 100 μL

[0033] After loading the 80% acetonitrile supernatant onto the column, 0% acetonitrile (ultrapure water) was passed through the column, and then the acetonitrile concentration was increased stepwise to 20, 40, and 60%. The resulting chromatogram is shown in Figure 2. The desired dipeptides and amino acids were obtained by collecting fractions according to the detected peaks. [Example]

[0034] (EDSE degranulation inhibitory activity) In this application, the experimental method for examining degranulation inhibitory activity is as follows. Specifically, the inhibitory activity against antigen-induced degranulation (histamine release) in rat basophilic cell line RBL-2H3 cells was examined. 5% FBS-DMEM medium was used. Because quantifying histamine is very complicated, in this experiment degranulation was evaluated using the release of β-hexosaminidase, which is present in granules together with histamine, as an indicator. The WST-8 method was used to measure cell viability.

[0035] EDSE was added to the culture medium of RBL-2H3 cells to examine its inhibitory effect on antigen-induced degranulation. The results showed that EDSE inhibited degranulation without cytotoxicity (Fig. 1). [Example]

[0036] (Analysis of active fractions obtained by reversed-phase chromatography fractionation) Example 1 Eight fractions F1 to F8 were collected according to the peaks detected in the chromatographic chart obtained in FIG. The degranulation inhibitory activity of the collected fractions was evaluated, and significant degranulation inhibitory activity was found in F1, F2 (acetonitrile 0%), and F6 (acetonitrile 20%) (Fig. 3). [Example]

[0037] (Analysis of Amino Acid Composition of Active Fractions) The amino acid composition of the supernatant fraction of the EDSE treated with 80% acetonitrile and the active fractions F1, F2, and F6 obtained by fractionation by reversed-phase chromatography was analyzed. The results are shown in Table 1.

[0038] [Table 1]

[0039] As shown in Table 1, amino acids such as aspartic acid (Asp), glutamic acid (Glu), alanine (Ala), leucine (Leu), histidine (His), and taurine (Tau) were present in relatively high amounts in the active fractions. Glutamic acid was particularly abundant in all active fractions, and alanine was also present in relatively high amounts. [Example]

[0040] (Degranulation inhibitory activity of amino acid monomers) The degranulation inhibitory activity of the six amino acids detected in high amounts in the EDSE-active fraction, as shown in Table 1, was examined. The results are shown in Figure 4.

[0041] That is, although aspartic acid, glutamic acid, alanine, leucine, histidine, and taurine were examined, as shown in FIG. 4, none of the amino acids exhibited degranulation inhibitory activity in the monomer form. [Example]

[0042] (Degranulation inhibitory activity of glutamic acid polymers) Based on the results shown in Table 1, glutamic acid was present in relatively high amounts in all active fractions, so we evaluated the degranulation inhibitory activity of dimers and polymers (molecular weight 12,000 or more). The results are shown in Figure 5.

[0043] That is, although the glutamic acid monomer did not exhibit degranulation inhibitory activity (Fig. 4), the dimer exhibited concentration-dependent degranulation inhibitory activity (Fig. 5), but the polymer did not exhibit activity.

[0044] This revealed that glutamic acid has no activity as a monomeric amino acid, but is active when it becomes a homodimeric dipeptide, and that the activity is lost when it becomes a further polymer. [Example]

[0045] (Degranulation inhibitory activity of dipeptides) Next, various dipeptides were evaluated for their activity. First, as shown in Figure 6, no activity was observed in six dipeptides: Gly-Tyr, Gly-Gly, Leu-Leu, Val-Tyr, Val-Trp, and Ile-Tyr.

[0046] Furthermore, the degranulation inhibitory activity and cytotoxicity of the following 16 dipeptides, which were prepared by combining amino acids with a high content of glutamic acid, were evaluated. (A) Glu-Asp (glutamic acid-aspartic acid), Asp-Glu (aspartic acid-glutamic acid), (B) Glu-Ser (glutamic acid-serine), Ser-Glu (serine-glutamic acid), (C) Glu-Thr (glutamic acid-threonine), Thr-Glu (threonine-glutamic acid), (D) Glu-Gly (glutamic acid-glycine), Gly-Glu (glycine-glutamic acid), (E) Glu-Ala (glutamic acid-alanine), Ala-Glu (alanine-glutamic acid), (F) Glu-Lys (glutamic acid-lysine), Lys-Glu (lysine-glutamic acid), (G) Glu-His (glutamic acid-histidine), His-Glu (histidine-glutamic acid), (H) Glu-Arg (glutamic acid-arginine), Arg-Glu (arginine-glutamic acid)

[0047] The degranulation inhibitory activity and cytotoxicity of each dipeptide are shown in Figures 7 to 14. As a result, although there were some differences in the strength of activity, it was confirmed that dipeptides other than Glu-His inhibited degranulation without cytotoxicity, and particularly strong activity was observed in Asp-Glu and Glu-Asp.

[0048] Furthermore, when the activity of dipeptides consisting of the same amino acid combinations was compared, it was suggested that those with glutamic acid at the C-terminus exhibited stronger degranulation inhibitory activity, particularly Ala-Glu, Lys-Glu, His-Glu, and Arg-Glu.

[0049] Based on the above, the supernatant fraction of the enzyme-treated sardine crushed product (EDSE) was treated with 80% acetonitrile, and the supernatant was collected and purified by reversed-phase chromatography. Activity was confirmed in several fractions. Based on the amino acid composition analysis of the active fractions obtained by fractionating EDSE by reversed-phase chromatography, the degranulation inhibitory activity of amino acids and dipeptides was evaluated. Many dipeptides containing glutamic acid were found to inhibit degranulation. Furthermore, it was suggested that dipeptides with glutamic acid at the C-terminus exhibited stronger activity when using dipeptides with the same amino acid combination.

[0050] The above studies have shown that a specific dipeptide can inhibit basophil degranulation and alleviate allergic symptoms in humans, etc. Furthermore, continuous daily intake of a prescribed amount of this dipeptide is safe and does not cause any side effects.

[0051] The present invention can be summarized as follows.

[0052] The present invention relates to a peptide-containing antiallergic composition, and aims to provide functional foods, pharmaceuticals, etc. that can effectively alleviate allergic symptoms in humans and are safe and reliable even when used orally.

[0053] Furthermore, functional foods and pharmaceuticals containing dipeptides containing glutamic acid as active ingredients, which can be obtained by various processes of crushed dried sardines, which are widely consumed in the diet, can be safely used by humans as oral medications, and can effectively alleviate allergic symptoms by inhibiting the degranulation of basophils.

Claims

1. An allergy symptom reliever whose active ingredient is the dipeptide alanine-glutamic acid.

2. An allergy symptom reliever whose active ingredient is the dipeptide lysine-glutamic acid.

3. An allergy symptom reliever whose active ingredient is the dipeptide histidine-glutamic acid.

4. An allergy symptom reliever whose active ingredient is the dipeptide arginine-glutamic acid.

5. An allergy symptom reliever whose active ingredient is the dipeptide glutamic acid-glutamic acid.

6. The agent according to any one of claims 1 to 5, wherein the agent for alleviating allergic symptoms is a degranulation inhibitor.

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