JP6731340B2 - Inhibitor of allergen activity and use thereof - Google Patents

Description

The present invention relates to an inhibitor of allergen activity that suppresses allergen activity.

An allergic reaction is generally an excessive immune reaction caused by a foreign substance as an allergen. In recent years, in particular, the number of patients causing allergic symptoms due to allergens such as mites and pollen has remarkably increased. Therefore, the search for effective substances that suppress allergic reactions is very important.

The allergic reaction is considered to occur, for example, by the following mechanism. First, proteins (allergens) derived from carcasses of mite, feces, pollen and the like adhere to the mucous membranes of human nose and throat and are recognized as foreign substances by lymphocytes. When the lymphocyte recognizes the allergen as a foreign substance, it produces an IgE antibody, and the produced IgE antibody is presented by binding to a receptor on the surface of mast cells. Thereby, when the same protein subsequently adheres to the mucous membrane, the protein acts as an allergen on the IgE antibody on the mast cells, and a chemical substance such as histamine is released from the mast cells. Then, these chemical substances cause allergic reactions, and cause allergic symptoms such as sneezing, runny nose/stuffy nose, itchy eyes, redness of eyes, and tear eyes.

In recent years, catechins (Patent Document 1), methylated catechins and the like have been reported as substances for suppressing the allergic reaction. The mechanism of action of these inhibitors is suppression of expression of FcεRI, which is an IgE receptor, and suppresses some reaction steps in a series of allergic reactions (Non-Patent Document 1).

However, as confirmed by the inventors of the present application, it was actually found that the above-mentioned catechin and methylated catechin cannot sufficiently suppress the occurrence of allergic reaction due to allergen.

JP-A-6-279273

Fujimura Y, Tachibana H, Yamada K, "A Tea Catechin Suppresses the Expression of the High-Affinity IgE Receptor FcεRI in Human Basophilic KU812 Cells", Journal of Agricultural and Food Chemistry, 4 May 2001, Vol. 49, No. 5 , P. 2527-2531

Therefore, an object of the present invention is to provide a new effective substance capable of maintaining safety and suppressing the activity of allergen.

To achieve the above object, the inhibitor of allergen activity of the present invention comprises a derivative of epigallocatechin gallate (EGCG) represented by the following chemical formula (1), or an isomer thereof or a salt thereof. And
In the chemical formula (1),
R ^{1 to} R ⁶ , R ¹² and R ¹⁴ are each a hydrogen atom, halogen, sodium, potassium or a linear or branched saturated or unsaturated acyl group, which may be the same or different, May be further substituted with one or more substituents, and at least one of R ^{1 to} R ⁶ , R ¹² and R ¹⁴ is the acyl group, and R ^{7 to} R ¹¹ , R ¹³ , R ¹⁵ And R ¹⁶ is a hydrogen atom, halogen, sodium or potassium, and may be the same or different.

The method for suppressing the allergen activity of the present invention is characterized by contacting the allergen with the above-mentioned inhibitor of the allergen activity of the present invention.

The method for suppressing an allergic reaction of the present invention is characterized by administering the above-mentioned inhibitor of the allergen activity of the present invention to a subject.

According to the present invention, an allergic reaction can be safely and effectively prevented. Therefore, the present invention can be said to be useful in the medical field, food hygiene field, and the like.

In Example 1 of this invention, it is a graph which shows the allergen activity inhibitory effect of an EGCG derivative with respect to mite allergen. 3 is a graph showing the allergen activity inhibitory effect of EGCG derivatives on spear allergens in Example 1 of the present invention. In Example 1 of this invention, it is a graph which shows the allergen activity inhibitory effect of methylated EGCG with respect to Spiria allergen.

<Inhibitor of allergen activity>
The inhibitor of the allergen activity of the present invention (hereinafter, also referred to as “activity inhibitor”) is, as described above, a derivative of epigallocatechin gallate represented by the following chemical formula (1), or an isomer thereof or their isomers. It is characterized by containing salt.
In the chemical formula (1),
R ^{1 to} R ⁶ , R ¹² and R ¹⁴ are each a hydrogen atom, halogen, sodium, potassium or a linear or branched saturated or unsaturated acyl group, which may be the same or different, May be further substituted with one or more substituents, and at least one of R ^{1 to} R ⁶ , R ¹² and R ¹⁴ is the acyl group, and R ^{7 to} R ¹¹ , R ¹³ , R ¹⁵ And R ¹⁶ is a hydrogen atom, halogen, sodium or potassium, and may be the same or different.

In addition, in the said Chemical formula (1), "A-D" is a description of each ring in epigallocatechin gallate. In the present invention, hereinafter, epigallocatechin gallate is referred to as “EGCG”, and a derivative of EGCG is referred to as “EGCG derivative”.

In the present invention, the “suppression of allergen activity” may be, for example, inactivation of the allergen activity, as well as suppression of the original activity of the allergen.

In the present invention, the EGCG derivative also includes, for example, isomers such as a salt of the compound represented by the chemical formula (1), tautomers, stereoisomers, optical isomers, geometric isomers, and a mixture of isomers. Be done. The salt is not particularly limited, and examples thereof include inorganic acid salts, organic acid salts, inorganic basic salts, organic basic salts, acidic or basic amino acid salts, and the like. The isomer can also be purified by a conventionally known separation method such as various chromatography. Further, in the present invention, the EGCG derivative also includes, for example, a compound produced by subjecting the compound represented by the chemical formula (1) to metabolism such as oxidation, reduction, hydrolysis, and conjugation.

In R ^{1 to} R ⁶ , the main chain length of the acyl group is not particularly limited, but is, for example, 6 to 18 atoms including a carbonyl carbon, preferably 12 to 18, and more preferably 12, 16 or 18 and particularly preferably 12. The main chain length of the acyl group refers to the number of atoms of the longest chain in the acyl group, and includes, for example, nitrogen atoms, sulfur atoms, phosphorus atoms, oxygen atoms, boron atoms, etc. in addition to the carbon atoms. But it's okay. The main chain length of the acyl group may be, for example, saturated or unsaturated.

In R ^{1 to} R ⁶ , the number of atoms of the acyl group is not particularly limited. The number of atoms (for example, the number of carbon atoms) of the acyl group is, for example, 2 to 20, including carbonyl carbon, preferably 4 to 20, more preferably 8 to 18 and 12 to 18, and specifically, For example, 8, 12, 16 or 18 is preferable. When the acyl group is further substituted with the substituent, the number of carbon atoms is preferably a number that does not include the number of carbon atoms of the substituent. The unsaturated acyl group may be cis or trans, for example.

The acyl group is not particularly limited, and examples thereof include formyl group (C1), acetyl group (C2), propionyl group (C3), butyryl group (C4), isobutyryl group (C4), valeryl group (C5), isovaleryl. Group (C5), pivaloyl group (C5), hexanoyl group (C6), 2-ethylhexyl group (C8), octanoyl group (C8), geranoyl group (3,7-dimethylocta-2,6-dienoyl group) (C10) ), trans-8-methyl-6-nonenoyl group (C10), undecanoyl group (C11), lauroyl group (dodecanoyl group) (C12), tridecanoyl group (C13), 12-(dimethylamino)lauroyl group (12-( Dimethylamino)dodecanoyl group) (C14), farnesoyl group (3,7,11-trimethyldodeca-2,6,10-trienoyl group (C15), palmitoyl group (hexadecanoyl group) (C16), heptadecanoyl group (C17) ), stearoyl group (octadecanoyl group) (C18), linoleyl group (C18), linolenyl group (C18), nonadecanoyl group (C19), eicosanoyl group (icosanoyl group) (C20) and the like. The "C" within the brackets of an acyl group indicates the number of carbon atoms including the carbonyl carbon.

Among the above acyl groups, for example, an acyl group represented by the following chemical formula is particularly preferable. The position of the unsaturated bond in the unsaturated acyl group of the following acyl groups is not limited to these. As a specific example, the unsaturated bond (double bond) of the trans-8-methyl-nonenoyl group (C10) is not limited to the 6-position shown below, and is, for example, any of the 2-5-position and the 7-position. May be.

The type of the acyl group is not particularly limited and may be an unsaturated acyl group or a saturated acyl group as described above. The number of unsaturated bonds in the acyl group is not particularly limited, but is 1, 2, or 3, for example.

In the chemical formula (1), for example, only ^one of R ^{1 to} R ⁶ , R ¹² and R ¹⁴ or R ^{1 to} R ⁶ may be the acyl group, or any two of them. The above may be the acyl group. When two or more places are the above-mentioned acyl groups, the above-mentioned acyl groups in each part may be the same or different, for example. In the chemical formula (1), R other than the acyl group is not particularly limited and is preferably a hydrogen atom, for example.

In the chemical formula (1), the moiety of the acyl group of R ^{1 to} R ⁶ , R ¹² and R ¹⁴ or R ^{1 to} R ⁶ is not particularly limited. As a specific example, for example, at least one of R ¹ and R ² of the B ring and R ⁵ and R ⁶ of the D ring preferably has the acyl group, and in particular, R ¹ , R ² , R ⁵ and R It is preferable that any one of ⁶ has the acyl group. At this time, other R is not particularly limited and is preferably a hydrogen atom, for example.

Further, in Formula (1), it is preferable that at least one position of ^R 1, ^{R 2} and ^{R 3} of B ring is the acyl group, more preferably, ^R 1, ^{R 2} and ^{R 3} in the B ring Of these, it is preferable that only one position is an acyl group. In Formula (1), it is preferable that at least one position of ^R 4, ^{R 5} and ^{R 6} in the D ring is the acyl group, and more preferably, the D ring ^R 4, of ^{R 5} and ^{R 6} It is preferable that only one position is an acyl group.

In the chemical formula (1), the B ring rotates about a single bond between the B ring and the C ring, for example. Therefore, the derivative having an acyl group in R ¹ is, for example, substantially the same as the derivative having an acyl group in R ³ . Further, in the above chemical formula (1), the D ring rotates, for example, about a single bond between the D ring and the ester. Therefore, the derivative having an acyl group at R ⁶ is substantially the same as the derivative having an acyl group at R ⁴ , for example.

In the chemical formula (1), “R ^{7 to} R ¹¹ , R ¹³ , R ¹⁵ and R ¹⁶ ”, or “R ^{7 to} R ¹⁶ ”are, as described above, a hydrogen atom, halogen, sodium (Na) or It is potassium (K), which may be the same or different, and is preferably a hydrogen atom, for example, as shown in the following chemical formula (2). In the following chemical formula (2), for example, any of R ^{1 to} R ⁶ may be the acyl group. As a specific example, for example, only one or two or more of R ^{1 to} R ⁶ are preferably the acyl groups described above, and more preferably R ¹ , R ² , R ^5, and R ⁶ Of these, it is preferable that only one of them or two or more of them is the aforementioned acyl group.

In R ^{1 to} R ⁶ of the chemical formula (1), the acyl group may be substituted with one or more substituents as described above, and specifically, for example, when the hydrogen atom of the acyl group is , May be substituted with the above-mentioned substituents. The substituent is not particularly limited, and examples thereof include an alkyl group, an amino group, an alkylamino group and a dialkylamino group.

Examples of the alkyl group include a linear or branched alkyl group having 1 to 6 carbon atoms, and a methyl group is preferable. The alkyl group in the alkylamino group includes, for example, a straight chain or branched alkyl group having 1 to 6 carbon atoms, and preferably a methylamino group. Examples of the alkyl group in the dialkylamino group include a linear or branched alkyl group having 1 to 6 carbon atoms, and preferably a dimethylamino group. These may be the same or different.

When the substituent or the like is a group having a chain structure, specifically, for example, an alkyl group, an alkylamino group, a dialkylamino group, an alkoxy group, a carboxyalkyl group, an alkoxycarbonylalkyl group, an alkoxyalkyl group, an alkenoxyalkyl group. In the case of a group or the like, it is not particularly limited and may be linear or branched. The same applies to the case where a part of the substituent or the like has a chain structure, for example, the case where the substituent in the substituted alkyl group or the substituted aryl group has a chain structure. When an isomer exists in the substituent or the like, it is not particularly limited and any isomer may be used. For example, when simply referring to "propyl group", it may be either n-propyl group or isopropyl group, and when simply saying "butyl group", any of n-butyl group, isobutyl group, sec-butyl group and tert-butyl group may be used. Well, when simply referred to as a "naphthyl group", it may be either a 1-naphthyl group or a 2-naphthyl group.

In the present invention, “halogen” refers to any halogen element. Examples of the halogen include fluorine, chlorine, bromine and iodine. Further, in the present invention, the “alkyl group” is not particularly limited. Examples of the alkyl group include methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group, tert-butyl group, pentyl group, hexyl group, heptyl group, octyl group. Group, nonyl group, decyl group, undecyl group, dodecyl group, tridecyl group, tetradecyl group, pentadecyl group, hexadecyl group, heptadecyl group, octadecyl group, nonadecyl group, icosyl group and the like. For groups containing alkyl groups in the structure or groups derived from alkyl groups (alkylamino groups, dialkylamino groups, alkoxy groups, carboxyalkyl groups, alkoxycarbonylalkyl groups, alkoxyalkyl groups, alkenoxyalkyl groups, etc.) The same is true.

In the case where the EGCG derivative in the present invention has the acyl group at any one position among R ¹ , R ² , R ⁵ and R ^{6 in} the formula (1) or the formula (2), The acyl group is preferably, for example, the following acyl group. In this case, examples of R ¹ , R ² , R ^5, and R ⁶ other than the acyl group include hydrogen, halogen, and alkali metals such as Na and K.

In the first embodiment, when the acyl group is a linear saturated acyl group, for example, the number of carbon atoms in the main chain is preferably in the range of 8 to 18 and 12 to 18, and specific examples include those of the main chain. Examples thereof include an acyl group having 12, 16 or 18 carbon atoms. In the first form, for example, the allergen is suitable for mites and pollen.

In the second embodiment, when the acyl group is an unsaturated acyl group, for example, the main chain preferably has 12 to 18 carbon atoms, and the unsaturated bond has -C=C- at two positions. As a specific example, an acyl group having a main chain of 18 carbon atoms and having -C=C- at two positions can be mentioned. In the second form, for example, the allergen is suitable for mites and pollen.

As a third aspect, when the acyl group is a branched saturated acyl group, for example, the main chain preferably has 6 to 20 carbon atoms, and the branched chain has preferably 1 to 4 carbon atoms. The number of branched chains is preferably 1 to 3, and specific examples thereof include an acyl group in which the main chain has 6 carbon atoms, the branched chain has 1 carbon atoms, and the branched chain has 2 carbon atoms. ..

In the case where the EGCG derivative in the present invention has the acyl group only at any two positions among R ¹ , R ² , R ⁵ and R ^{6 in} the formula (1) or the formula (2), The acyl group is preferably, for example, the following acyl group. In this case, examples of R ¹ , R ² , R ^5, and R ⁶ other than the acyl group include hydrogen, halogen, and alkali metals such as Na and K.

As a fourth mode, when the acyl group is a linear saturated acyl group, for example, each of the two acyl groups preferably has a main chain carbon number in the range of 8 to 18, and as a specific example, An acyl group having 8 carbon atoms in the chain may be mentioned. In the fourth form, for example, the allergen is suitable for mites and pollen.

The EGCG derivative in the present invention may be, for example, one kind or a combination of two or more kinds. For example, two or more types of EGCG derivatives having an acyl group at different sites among R ^{1 to} R ⁶ may be used, or two or more types of EGCG derivatives having different acyl groups may be used. As a specific example, any two or more of an EGCG derivative in which R ¹ of the B ring is the acyl group, an EGCG derivative in which R ² is the acyl group, and an EGCG derivative in which R ³ is the acyl group, or It may be a mixture containing all three types, and among R4 of the D ring, an EGCG derivative in which R ⁴ is the acyl group, EGCG derivative in which R ⁵ is the acyl group, and EGCG derivative in which R ⁶ is the acyl group, It may be a mixture containing any two or more kinds, or all three kinds. A mixture of an EGCG derivative in which at least one of R ^{1 to} R ³ of the B ring is the acyl group, and an EGCG derivative in which at least one of R ^{4 to} R ⁶ of the D ring is the acyl group. Good.

The activity inhibitor of the present invention can be used for allergens as described above. The allergen causes, for example, an allergic reaction in humans and non-human animals, and is not particularly limited. Specific examples of the allergens include house dust, pollen, insect-derived proteins and plant-derived proteins. House dust includes, for example, animal skin (including dandruff); mites, worms, carcasses and feces thereof; fungi and bacteria. Examples of the animals include humans and non-human animals such as dogs and cats. Examples of the mites include leopard mites (dust mites) such as Dermatophagoides farinae, dust mites such as Dermatophagoides farinae, pest mites such as Plutella mites, and house dust mites such as dust mites. Examples of the pollen include pollen of plants such as cypresses such as cedar, asteraceae such as ragweed and mugwort, pine family such as pine, and grass family such as rice. Examples of the insect-derived protein include proteins such as cockroaches. Examples of the plant-derived protein include proteins such as soybean, egg, and milk.

The activity inhibitor of the present invention can be used, for example, for preventing the development of allergic reaction and treating the allergic reaction. Specifically, for example, the activity of the allergen can be suppressed by contacting the allergen with the activity inhibitor, and as a result, the onset of allergic reaction can be suppressed.

The activity-suppressing agent of the present invention only needs to contain the EGCG derivative, and its form is not limited at all. Examples of the form include liquids such as solutions and dispersions, solids, and powders. The dosage form is not particularly limited and can be appropriately set depending on the method of use, and examples thereof include liquid preparations, capsules, tablets, granules (fine granules), and powders.

Examples of the method of use include a method of administering the activity inhibitor to a subject that may cause an allergic reaction. The subject is, for example, an animal that may cause an allergic reaction due to an allergen. The administration method is not particularly limited, and examples thereof include parenteral administration and oral administration. Examples of the parenteral administration include transdermal administration, intraperitoneal administration, intravenous administration such as intravenous injection, intramuscular administration, subcutaneous administration such as subcutaneous injection, rectal administration, etc., and transdermal administration is preferable. .. The transdermal administration is not particularly limited and includes, for example, mucous membrane in addition to skin. The activity inhibitor of the present invention is, for example, according to these administration forms, for example, an ointment containing the EGCG derivative, a nasal drop, a nasal application drug, a nasal lavage drug, a mouthwash, a mouthwash, a sublingual drug, an internal drug, It can be administered as a disinfectant for hands and the like, or as an EGCG derivative or a solution or dispersion containing the same, using a nebulizer, an inhaler, an injection or the like. The EGCG derivative or a powder containing the same can be administered using, for example, a nebulizer, an inhaler, or the like.

Examples of the animal include human and non-human animals. Examples of the non-human animals include non-human mammals such as pigs, ferrets, rats, mice and cows, and birds such as ducks and chickens.

The activity inhibitor of the present invention, for example, before contacting the test substance and allergen such as the animal, it may be contacted with the test substance, after contacting the test substance and the allergen, the It may be brought into contact with the subject. Since it is possible to more effectively suppress the development of an allergic reaction in the subject by suppressing the allergen activity, like the former, before the subject and the allergen contact, the subject and the activity inhibitor. Contact is preferred.

Examples of the method of use include, in addition to the above, a method of treating a subject in which an allergen may be present with the activity inhibitor, and the subject is, for example, an environment in which an allergen may be present. The following are examples. In this case, the activity inhibitor of the present invention may be in the form of a detergent such as a hand-washing agent, a wiping agent, or a laundry agent containing the EGCG derivative. Thus, by the activity inhibitor of the present invention, for example, by treating the place where the allergen is likely to be present, such as hands and desks, the activity of the allergen present is suppressed and the onset of allergic reaction is prevented. Is also possible. The activity inhibitor of the present invention may be carried on, for example, clothes; bedding such as futons and pillows; furniture such as tatami mats, carpets, desks, chairs, beds, and masks. In addition, the activity inhibitor of the present invention may be used to treat a filter in, for example, an air conditioner or an air cleaner. In this case, for example, the activity inhibitor of the present invention may be supported on the filter, or the filter may be washed with the activity inhibitor of the present invention.

The subject may be, for example, the living body of the animal itself, cells and tissues collected from the living body, or cultures thereof. When the subject is, for example, the cells, tissues, or the like, the administration method of the activity inhibitor is not particularly limited, and examples thereof include addition to the medium.

In the activity suppressor of the present invention, the content of the EGCG derivative is not particularly limited and can be appropriately determined depending on, for example, the purpose of use and method of use. When the activity inhibitor of the present invention is a mouthwash, it is preferable that, for example, the EGCG derivative is contained in an amount of 10 to 100 μmol per dose. When the activity suppressor of the present invention is a nasal drop, it is preferable that it contains, for example, 10 to 100 μmol of the EGCG derivative per dose.

The activity suppressor of the present invention may further contain additives, bases and the like, depending on the dosage form and method of use, for example. Examples of the additives include excipients, binders, lubricants, disintegrants, coloring agents, flavoring agents, flavoring agents, emulsifiers, surfactants, solubilizing agents, suspending agents, isotonic agents. , Buffers, preservatives, antioxidants, stabilizers, absorption promoters and the like. The addition ratio of these is not particularly limited, and they can be added within a range that does not impair the effects of the EGCG derivative.

The activity inhibitor of the present invention may further include other agents having antiallergen activity, and specific examples include, for example, organic antiallergen agents such as tannic acid, zeolite, and inorganic antiallergen agents such as silver. Etc. Further, these may be contained in the activity inhibitor of the present invention or may be used in combination with the activity inhibitor of the present invention.

The method for producing the EGCG derivative in the present invention is not particularly limited. As the method, a conventionally known method such as an organic synthesis method or a chemical synthesis method utilizing an enzyme can be employed. The chemical synthesis method using the enzyme is not particularly limited, and examples thereof include a method using lipase disclosed in WO2007/105280. That is, it is a method of acylating EGCG by performing an enzymatic reaction with lipase using EGCG and an acyl group donor as substrates in an organic solvent. According to this method, for example, EGCG can be selectively acylated. In the following, a method using lipase will be described as an example, but the present invention is not limited to the method for producing an EGCG derivative.

Examples of the lipase include IUB No. 3.1.1.3. Lipase can be used. Specific examples, Aspergillus sp lipase derived from such Aspergillus niger; Candida rugosa, Candida cylindracea , Candida sp lipase derived from such Candida antarctica; Pseudomonas fluorescens, Pseudomonas cepacia , Pseudomonas sp lipase derived from such Pseudomonas stutzeri; Alcaligenes sp lipase; Burkholderia cepacia Burkholderia sp lipase and the like; lipase derived from porcine pancreas and the like. These can be prepared by a conventionally known method, for example, Lipase AS “AMANO”, Lipase AYS “AMANO”, Lipase PS “AMANO”, Lipase AK “AMANO” 20, Lipase AH “AMANO” (all products Name: Amano Enzyme Inc.), Lipase MY, Lipase OF, Lipase PL, Lipase PLC, Lipase PLG, Lipase QLM, Lipase QLC, Lipase QLG, Lipase SL, Lipase TL (all brand names: Meito Sangyo Co., Ltd.), Lipase Commercial products such as PPL, L4777 Lipase acrylic resin from Candida Antarctica, and L3126 Lipase from porcine pancreas (all trade names: manufactured by Sigma-Aldrich) can also be used. The physicochemical properties of each commercial product are as described in the respective product manuals, and an enzyme having the same physicochemical properties can be used as well.

Further, it may be a lipase having any of the physicochemical and enzymatic properties of (1) to (8) shown below.
(1) Molecular weight 35,000, isoelectric point 4.10 (for example, from Aspergillus niger )
(2) Inactivated by treatment with a molecular weight of 64,000, an isoelectric point of 4.30, and 80°C for 10 minutes (for example, from Candida rugosa )
(3) Optimal pH 8, optimum temperature 60°C, particularly stable in the range of pH 4 to 10, particularly stable below 70°C (for example, from Pseudomonas fluorescens )
(4) Molecular weight 60,000, optimum pH 6-7, pH stability 3-8, optimum temperature 40-50°C, particularly stable in solution at 37°C or lower (for example, derived from Candida cylindracea, derived from Candida rugosa )
(5) Molecular weight of 30,000, isoelectric point of 4.5, optimum pH of 8 to 9.5, pH stability of 7 to 10, optimum temperature of 50°C, particularly stable at 40°C or lower (for example, derived from Alcaligenes genus).
(6) Molecular weight 31,000, isoelectric point 4.9, optimum pH 7-9, pH stability 6-10, optimum temperature 65-70°C, particularly stable at 50°C or lower (for example, derived from Alcaligenes genus).
(7) Molecular weight 31,000, isoelectric point 5.2, optimum pH 7-9, pH stability 6-10, optimum temperature 65-70°C, particularly stable at 60°C or lower (for example, derived from Pseudomonas cepacia , Burkholderia (from cepacia )
(8) Molecular weight 27,000, isoelectric point 6.6, optimum pH 7-8, pH stability 6-9, optimum temperature 50°C, particularly stable at 40°C or lower (for example, derived from Pseudomonas stutzeri )

The organic solvent is not particularly limited and, for example, acetonitrile, acetone, dimethylformamide (DMF), dimethylsulfoxide (DMSO) and the like can be used. Further, for example, an organic solvent having a parameter indicating hydrophobicity (logP value) in the range of −0.35 to 0.28 may be used. As such an organic solvent, the above-mentioned acetonitrile (logP value: −0.45 to −0.45) may be used. 0.19), acetone (logP value: -0.16 to 0.19), DMF (logP value: -1.01 to 0.28), DMSO (logP value: -1.35 to 0.28) can give. In addition to these, conventionally known solvents that satisfy the above parameters can be used. Since the logP is a value specific to the solvent, those skilled in the art can select a solvent that satisfies the above parameters. Note that logP is a logarithm of the concentration ratio of the target substance in the octanol layer and the water layer when equilibrium is reached when the target substance is added to a mixed solution of octanol and water. Is generally used as a parameter indicating the hydrophobicity of a substance.

In the present invention, the acyl group (R-CO-) donor include carboxylic acid vinyl ester (R-CO-O-CH = CH 2) and the like. Examples of the acyl group include the linear or branched saturated or unsaturated acyl groups described above.

When DMF is used in the enzyme reaction solution, the addition ratio of EGCG is not particularly limited, but is, for example, 0.2 to 100 mmol/L, preferably 0.5 to 50 mmol/L, more preferably 0.5. ~20 mmol/L. The addition ratio of the acyl group donor is not particularly limited and can be appropriately determined depending on the addition ratio of EGCG in the reaction solution, for example. As a specific example, the addition ratio (molar ratio) of EGCG and the acyl group donor is, for example, 1:1 to 1:10, preferably 1:1 to 1:5, and more preferably 1:1 to 1:1. : 3. Further, the addition ratio of lipase in the reaction solution can be appropriately determined depending on, for example, the addition ratio of EGCG or an acyl group donor, the specific activity of lipase, and the like, but is not particularly limited, and, for example, to EGCG 1 mmol/L, for example, , 500 to 50,000 U/L, preferably 500 to 5,000 U/L, and more preferably 1,000 to 2,500 U/L.

The conditions of the enzyme reaction are not particularly limited, but the reaction temperature is, for example, in the range of 45 to 75°C. The reaction time can be appropriately determined depending on the amount of the substrate or the enzyme and is not particularly limited, but is, for example, 30 minutes to 24 hours (1440 minutes), preferably 1 hour (60 minutes) to 3 hours (180 minutes). ), more preferably 1.5 hours (90 minutes) to 3 hours (180 minutes).

A basic catalyst may be further added to the reaction solution. Examples of the basic catalyst include tertiary amines such as triethylamine, pyridine, and the like. The addition ratio of the basic catalyst in the reaction solution is not particularly limited, but is, for example, 5 to 720 mmol/L, preferably 12 to 240 mmol/L, more preferably 12 to 48 mmol/L.

The position where the acyl group is introduced in EGCG can be selected, for example, depending on the type of lipase used. The number of acyl groups introduced into EGCG can be determined, for example, by the type of organic solvent used and the reaction time. For example, as the hydrophobicity of the organic solvent is relatively high (the hydrophilicity is relatively low), the number of introduced acyl groups can be relatively reduced, and as the hydrophilicity of the organic solvent is relatively high ( The lower the hydrophobicity), the greater the number of acyl groups that can be introduced. The number of acyl groups introduced can also be adjusted by mixing two or more kinds of organic solvents. As a specific example, for example, it is preferable to use acetonitrile or the like when introducing one acyl group, and for example, acetone or acetonitrile is used when introducing one or two acyl groups. For example, when introducing 3 to 5 acyl groups, DMSO, DMF or the like is preferably used.

Further, even when the same organic solvent is used, the number of acyl groups to be introduced can be adjusted by combining with the control of temperature time and reaction temperature. Examples thereof will be shown below, but the present invention is not limited thereto. When DMF is used, two acyl groups are selected for EGCG by, for example, setting the reaction temperature in the range of about 57° C. to about 70° C. and increasing the reaction temperature (for example, about 3 to 5 hours). To obtain the derivative introduced in a selective manner, while lowering the reaction temperature (for example, 57° C. to about 5° C. lower) and shortening the reaction time (for example, about 1 to 3 hours). By this, one acyl group can be selectively introduced. Further, by using a mixed solvent in which acetone and DMF are mixed in the same amount (mass), one acyl group can be selectively introduced into EGCG.

Further, the number of acyl groups to be introduced can be increased by adding the above-mentioned basic catalyst to the reaction solution. In this case, which site in EGCG the acyl group is further introduced depends on, for example, the regioselectivity of lipase.

The yield of the EGCG derivative by the enzyme reaction can be relatively improved by setting the reaction temperature relatively high, for example. The reaction temperature is usually 45 to 75°C as described above, but from the viewpoint of improving the yield, it is preferably 57 to 75°C, more preferably 57 to 70°C. Particularly, when the reaction temperature is 57 to 70° C., the yield of the EGCG acylated derivative can be about 35 to 45%. In addition, the said yield means the ratio (conversion efficiency) of the EGCG acylated derivative (for example, all monoacylated derivatives) when EGCG used for reaction is made into 100%.

In the present invention, as the EGCG derivative, for example, as described above, any one kind may be used, or a mixture of two or more kinds may be used. When one kind of EGCG derivative is isolated from the mixture, it can be prepared by a conventionally known method using, for example, chromatography.

<Method of suppressing allergen activity>
The method for suppressing the allergen activity of the present invention is characterized by contacting the allergen with the activity suppressor of the present invention as described above. The suppressing method of the present invention is characterized by using the above-mentioned activity suppressing agent of the present invention, and other configurations, conditions and the like are not limited at all. The activity inhibitor of the present invention and the method of using the same are the same as described above, for example, and all of them can be incorporated.

In the present invention, the method of contacting the allergen with the activity inhibitor is not particularly limited. As described above, for example, it may be administered to a subject, and the allergen possessed by the animal may be contacted with the activity inhibitor, or the subject in which the allergen may be present is treated with the activity inhibitor. By doing so, the allergen may be brought into contact with the activity inhibitor. In the former, the subject is, for example, an animal (human or non-human animal) as described above, and in the latter, the subject is, for example, an environment in which an allergen may be present. As examples, the above-mentioned examples of clothes, bedding and the like can be incorporated.

<How to suppress allergic reaction>
The method for suppressing an allergic reaction of the present invention is characterized by administering the activity inhibitor of the present invention to a subject as described above. The present invention is characterized by using the activity inhibitor of the present invention, and other constitutions, conditions and the like are not limited at all. The activity inhibitor of the present invention and the method of using the same are the same as described above, for example, and all of them can be incorporated. In addition, the description of the method for suppressing an allergen activity of the present invention can be applied to the method for suppressing an allergic reaction of the present invention.

The method for suppressing an allergic reaction of the present invention can treat a disease derived from an allergic reaction in the subject by administering the activity inhibitor of the present invention. Therefore, the method for suppressing an allergic reaction of the present invention can also be referred to as, for example, a method for treating an allergic disease. In the present invention, “treatment” includes prevention of the disease, improvement of the disease, and improvement of prognosis of the disease, and may be any.

The allergic disease is not particularly limited, and examples thereof include hay fever, allergic rhinitis, urticaria, animal allergy, food allergy, insect allergy to insects such as cockroaches, and house dust allergy.

The timing of administration of the activity inhibitor to the subject is not particularly limited, and may be before contact with the allergen or after contact with the allergen, for example.

<Use of inhibitors for allergic activity>
The present invention is the use of the activity inhibitor of the present invention for suppressing an allergic reaction, and the use of the activity inhibitor of the present invention for treating an allergic disease. The present invention also relates to the use of the activity inhibitor of the present invention for producing a drug for allergic reaction, and the use of the activity inhibitor of the present invention for producing a drug for allergic diseases.

Next, examples of the present invention will be described. However, the present invention is not limited to the following examples.

As compounds, an acylated EGCG derivative corresponding to the example, EGCG corresponding to the comparative example, and methylated catechin were prepared.

(1) Acylated EGCG Derivative In the chemical formula (2) below, an EGCG derivative in which any one or two of R ^{1 to} R ⁶ are the following acyl groups was used (all manufactured by Protectia). In addition, R ^{1 to} R ⁶ other than the acyl group are hydrogen atoms.

EGCG-C12, EGCG-C16, EGCG-C18, EGCG-C18DE and EGCG-EH are an acylated derivative of R ¹ , an acylated derivative of R ² , an acylated derivative of R ^{5 and} an acylated derivative of R ⁶ , respectively. It is a mixture of four types. EGCG-C8×2 is an acylated derivative of R ¹ and R ⁵ , an acylated derivative of R ¹ and R ⁶ , an acylated derivative of R ² and R ⁵ , an acylated derivative of R ² and R ^6. Of R ¹ and R ² , an acylated derivative of R ¹ and R ³ , an acylated derivative of R ⁴ and R ^5, and an acylated derivative of R ⁴ and R ⁶ . It is a mixture.

(2) EGCG
In the chemical formula (2), a natural EGCG (trade name: Sanphenon EGCG, manufactured by Taiyo Kagaku Co., Ltd.) in which all of R ^{1 to} R ⁶ are hydrogen atoms (non-acyl groups) was used.

(3) Methylated catechin In the above formula (2), monomethylated catechin (manufactured by Protectia Co.) in which only R ⁵ is a methyl group (non-acyl group), and dimethylated in which R ² and R ⁵ are methyl groups Catechin (manufactured by Protectia) was used. In addition, R ^{1 to} R ⁶ other than the methyl group are hydrogen atoms.

[Example 1]
The ability of the acylated EGCG derivative to suppress the activity against various allergens was confirmed.
(1) Suppression test of mite allergen activity The Derbecco phosphate physiological buffer solution (D-PBS(-), sum of Kona deer mite extract (trade name: Mite Extract Df, manufactured by Cosmo Bio Co., Ltd.) containing no calcium ion and magnesium ion was used. A mite allergen solution was prepared by diluting the solution with a final concentration of 100 μg/mL with Kojun Pure Chemical Co., Ltd.). A DMSO solution containing 10 mmol/L of the acylated EGCG derivative was diluted with a phosphate physiological buffer solution (PBS) to a final concentration of 111 μmol/L to prepare an example sample.

50 μL of the mite allergen solution and 450 μL of the sample were mixed in a tube, and this was incubated at room temperature for 2 hours. Then, the mite allergen activity remaining in the mixed solution was measured using a mite allergen measurement ELISA kit (trade name: mite allergen (Derf1) measurement kit, manufactured by Nichi Nichi Pharmaceutical Co., Ltd.). The measurement by the kit was performed according to the instruction manual.

As a control, PBS or a 1% DMSO solution (hereinafter referred to as 1% DMSO) was used as a control sample instead of the above-mentioned example sample, and the mite allergen activity was measured in the same manner. In the comparative example, natural EGCG was used as the comparative example sample instead of the above-mentioned example sample, and a sample was prepared in the same manner to measure mite allergen activity.

Then, the relative value (%) of the mite allergen activity in each of the Examples and Comparative Examples was calculated with the control mite allergen activity as 100%, and this was defined as the mite allergen activity residual rate (%). In this example, two measurements were individually performed for each sample, and the average value was used as the result.

The results are shown in FIG. FIG. 1 is a graph showing the residual rate (%) of allergen activity of mite allergen. In FIG. 1, the Y-axis is the residual rate of allergen activity (%), C12 is EGCG-C12, C16 is EGCG-C16, C18 is EGCG-C18, C18DE is EGCG-C18DE, EH is EGCG. -EH, C8x2 shows the result of EGCG-C8x2. In FIG. 1, the lower the residual rate of mite allergen activity, the better the effect of suppressing mite allergen activity by the sample. As shown in FIG. 1, natural EGCG showed almost no activity suppressing effect on mite allergen, whereas EGCG derivatives (C12, C16, C18, C18DE, EH, C8×2) were It showed a significant activity-suppressing effect on mite allergens. Among them, C12 and C18DE showed a very excellent activity suppressing effect on mite allergen, and in particular, C12 suppressed mite allergen activity up to about 10%, and it was found that it is extremely excellent.

(2) Sugi allergen activity suppression test Cedar pollen antigen (trade name: purified cedar pollen antigen Cryj1, manufactured by Funakoshi Co., Ltd.) was diluted with the D-PBS(-) to a final concentration of 500 ng/mL, and a cedar allergen solution was obtained. Was prepared. Further, the example sample of the acylated EGCG derivative prepared in (1) above was used.

50 μL of the pearl millet allergen solution and 450 μL of the sample were mixed in a tube, and this was incubated at room temperature for 2 hours. Then, the cedar allergen activity remaining in the mixed solution was measured using a cedar pollen antigen measurement ELISA kit (trade name: cedar pollen antigen (Cryj1) measurement kit, manufactured by Nichinichi Pharmaceutical Co., Ltd.). The measurement by the kit was performed according to the instruction manual. The control and the comparative example were the same as those in (1) above.

The results are shown in FIG. FIG. 2 is a graph showing the residual rate (%) of allergen activity of Spiria allergen. In FIG. 2, the Y-axis is the residual rate of allergen activity (%), C12 is EGCG-C12, C16 is EGCG-C16, C18 is EGCG-C18, C18DE is EGCG-C18DE, and C8×2 is , EGCG-C8×2. In FIG. 1, the lower the residual rate of allergen allergen activity, the better the effect of suppressing the activity of allergic allergens by the sample. As shown in FIG. 2, natural EGCG showed almost no activity-suppressing effect on spear allergens, whereas EGCG derivatives (C12, C16, C18, C18DE, C8×2) were effective against spear allergens, respectively. Showed a significant activity suppressing effect.

In addition, as a comparative example, the ability to suppress sugi allergen activity was confirmed by using the methylated catechin, which has been reported to have an antiallergic effect, instead of the acylated EGCG derivative. In this comparative example sample, the methylated catechin (monomethylated catechin or dimethylated catechin) was used in place of the acylated EGCG derivative, and the final concentration was 50 μmol/L or 100 μmol/L. It was prepared in the same manner as the sample. Then, using the sample of Comparative Example, the sperger allergen activity was measured in the same manner as described above, and the residual rate of allergen activity was obtained. The residual rate of the allergen activity was determined as a relative value (%) with the allergen activity of 1% DMSO as 100%.

The results are shown in FIG. FIG. 3 is a graph showing the residual rate (%) of allergen activity. In FIG. 3, the Y-axis is the residual rate of allergen activity (%), mM EGCG is monomethylated catechin, dM EGCG is dimethylated catechin, and μM (μmol/L) is the methylated catechin in the sample. Indicates the concentration. In FIG. 3, the lower the residual rate of the spiny allergen activity, the better the effect of suppressing the activity of the spiny allergen. As shown in FIG. 3, the methylated catechins had an allergen activity residual rate of more than 100% at any concentration, and did not show an activity suppressing effect on the allergen.

According to the present invention, an allergic reaction can be safely and effectively prevented. Therefore, the present invention can be said to be useful in the medical field, food hygiene field, and the like.

Claims (7)

An inhibitor of allergen activity of an allergen, which comprises an epigallocatechin gallate derivative represented by the following chemical formula (1), or an isomer thereof or a salt thereof:
In the chemical formula (1),
R ^{1 to} R ⁶ are
Each is a hydrogen atom, halogen, sodium, potassium or lauroyl group , and may be the same or different ,
Before SL any one of ^{R 1,} R 2, ^{R 5} and R ⁶ is a the lauroyl group,
R ^{7 to} R ¹⁶ are a hydrogen atom, halogen, sodium or potassium, and may be the same or different. In ^{R 6} from R ^1, any one of R ^1, R 2, ^{R 5} and ^{R 6,} is said lauroyl group, others, a hydrogen atom, an inhibitor of allergen activity according to claim 1. R ⁷ to R ¹⁶ is a hydrogen atom, an inhibitor of allergen activity according to claim 1 or 2. The allergen activity inhibitor according to any one of claims 1 to 3 , wherein the allergen is a red bean allergen. The inhibitor of allergen activity according to any one of claims 1 to 4 , wherein the inhibitor of allergen activity is an inhibitor of mite allergen or spear allergen. A method for suppressing allergen activity, which comprises contacting the allergen with the inhibitor for allergen activity according to any one of claims 1 to 5 . A method for suppressing an allergic reaction, which comprises administering the allergen activity inhibitor according to any one of claims 1 to 5 to a non-human animal.