JP7419416B2 - Allergen activity inhibitors and their uses - Google Patents

Description

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

An allergic reaction is generally an excessive immune reaction caused by a foreign substance becoming an allergen. In recent years, the number of patients suffering from allergic symptoms caused by allergens such as dust mites and pollen has increased significantly. For this reason, the search for effective substances that suppress allergic reactions is of great importance.

Allergic reactions are thought to occur, for example, by the following mechanism. First, proteins (allergens) derived from mite carcasses, feces, pollen, etc. adhere to the mucous membranes of the human nose and throat, and are recognized as foreign substances by lymphocytes. When the lymphocytes recognize the allergen as a foreign substance, they produce IgE antibodies, and the produced IgE antibodies bind to receptors on the surface of mast cells and are presented. As a result, when the same protein subsequently adheres to the mucous membrane, the protein acts as an allergen on the IgE antibody on the mast cell, and chemicals such as histamine are released from the mast cell. These chemical substances then induce allergic reactions, causing allergic symptoms such as sneezing, runny/stuffy nose, itchy eyes, bloodshot eyes, and watery eyes.

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

However, the inventors of the present application have confirmed that, in reality, the above-mentioned catechins and methylated catechins cannot sufficiently suppress the occurrence of allergic reactions caused by allergens.

Japanese Patent Application Publication No. 6-279273

Fujimura Y, Tachibana H, Yamada K, “A Tea Catechin Suppresses the Expression of the High-Affinity IgE Receptor FceRI in Huma n Basophilic KU812 Cells”, Journal of Agricultural and Food Chemistry, April 2001, Volume 49, No. 5 , p. 2527-2531

Therefore, the present invention aims to provide a new effective substance that can maintain safety and suppress allergen activity.

前記化学式（１）中、
Ｒ^１～Ｒ^６、Ｒ^１２およびＲ^１４は、それぞれ水素原子、ハロゲン、ナトリウム、カリウムまたは直鎖もしくは分枝状の飽和もしくは不飽和アシル基であり、同一でも異なっていてもよく、前記アシル基は、さらに１または複数の置換基で置換されていてもよく、前記Ｒ^１～Ｒ^６、Ｒ^１２およびＲ^１４の少なくとも１つが前記アシル基であり、Ｒ^７～Ｒ^１１、Ｒ^１３、Ｒ^１５およびＲ^１６は、水素原子、ハロゲン、ナトリウムまたはカリウムであり、同一でも異なっていてもよい。 In order to achieve the above object, the allergen activity suppressor of the present invention is characterized in that it contains a derivative of epigallocatechin gallate (EGCG) represented by the following chemical formula (1), an isomer thereof, or a salt thereof. shall be.

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, and may be the same or different, and each of the above acyl groups may be further substituted with one or more substituents, 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 are hydrogen atoms, halogens, sodium or potassium, and may be the same or different.

The method for suppressing allergen activity of the present invention is characterized by bringing the allergen activity suppressor of the present invention into contact with an allergen.

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

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

1 is a graph showing the allergen activity suppressing effect of EGCG derivatives on mite allergens in Example 1 of the present invention. 1 is a graph showing the allergen activity suppressing effect of EGCG derivatives on cedar allergen in Example 1 of the present invention. 1 is a graph showing the allergen activity suppressing effect of methylated EGCG on cedar allergen in Example 1 of the present invention.

前記化学式（１）中、
Ｒ^１～Ｒ^６、Ｒ^１２およびＲ^１４は、それぞれ水素原子、ハロゲン、ナトリウム、カリウムまたは直鎖もしくは分枝状の飽和もしくは不飽和アシル基であり、同一でも異なっていてもよく、前記アシル基は、さらに１または複数の置換基で置換されていてもよく、前記Ｒ^１～Ｒ^６、Ｒ^１２およびＲ^１４の少なくとも１つが前記アシル基であり、Ｒ^７～Ｒ^１１、Ｒ^１３、Ｒ^１５およびＲ^１６は、水素原子、ハロゲン、ナトリウムまたはカリウムであり、同一でも異なっていてもよい。 <Allergen activity inhibitor>
As mentioned above, the allergen activity inhibitor of the present invention (hereinafter also referred to as "activity inhibitor") is a derivative of epigallocatechin gallate represented by the following chemical formula (1), or an isomer thereof, or an isomer thereof. 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, and may be the same or different, and each of the above acyl groups may be further substituted with one or more substituents, 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 are hydrogen atoms, halogens, sodium or potassium, and may be the same or different.

In the chemical formula (1), "A to D" represent each ring in epigallocatechin gallate. In the present invention, epigallocatechin gallate is hereinafter referred to as "EGCG", and a derivative of EGCG is referred to as "EGCG derivative".

In the present invention, "suppression of allergen activity" may include, for example, suppression of the original allergen activity, as well as inactivation of allergen activity.

In the present invention, the EGCG derivative includes, for example, isomers such as salts, tautomers, stereoisomers, optical isomers, geometric isomers, and isomer mixtures of the compound represented by the chemical formula (1). It will be done. The salt is not particularly limited and includes, for example, inorganic acid salts, organic acid salts, inorganic base salts, organic base salts, acidic or basic amino acid salts, and the like. The isomer can also be purified by conventionally known separation methods such as various chromatography methods. 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 includes carbonyl carbon and has 6 to 18 atoms, preferably 12 to 18 atoms, more preferably 12, 16, or 18, particularly preferably 12. The main chain length of the acyl group refers to the number of atoms in 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 mentioned above. But that's fine. The main chain length of the acyl group may be saturated or unsaturated, for example.

In R ¹ to R ⁶ , the number of atoms of the acyl group is not particularly limited. The number of atoms (for example, 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, 12 to 18, and specifically, For example, 8, 12, 16 or 18 are preferred. In addition, when the said acyl group is further substituted with the said substituent, it is preferable that the said carbon atom number is a number which does not include the carbon atom number of the said substituent, for example. Further, the unsaturated acyl group may be, for example, cis or trans.

The acyl group is not particularly limited, but for example, formyl group (C1), acetyl group (C2), propionyl group (C3), butyryl group (C4), isobutyryl group (C4), valeryl group (C5), isovaleryl group. 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), etc. "C" in parentheses in an acyl group indicates the number of carbon atoms including the carbonyl carbon.

Among the above acyl groups, for example, acyl groups shown in the following chemical formula are particularly preferred. Among the following acyl groups, the position of the unsaturated bond in the unsaturated acyl group 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, for example, it may be at any of the 2-5 positions and the 7-position. You can.

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

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 R 1 to R 6 may be the acyl group. The above may be the above-mentioned acyl group. When the acyl group exists at two or more locations, the acyl group at each location 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, for example, a hydrogen atom.

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

Further, in the chemical formula (1), it is preferable that at least one of R ¹ , R ² and R ³ of ring B is the acyl group, more preferably R ¹ , R ² and R ³ of ring B It is preferable that only one of them is an acyl group. In the chemical formula (1), it is preferable that at least one of R ⁴ , R ⁵ and R ⁶ in the D ring is the acyl group, and more preferably, at least one of R ⁴ , R ⁵ and R ⁶ in the D ring is the acyl group. Preferably, only one position is an acyl group.

In the chemical formula (1), the B ring rotates about the single bond between the B ring and the C ring, for example. Therefore, a derivative having an acyl group at R ¹ is substantially the same as a derivative having an acyl group at R ³ , for example. Further, in the chemical formula (1), the D ring rotates about the single bond between the D ring and the ester, for example. Therefore, a derivative having an acyl group at R ⁶ is substantially the same as a 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 hydrogen atoms, halogens, sodium (Na) or They are potassium (K), and may be the same or different. For example, as shown in the following chemical formula (2), a hydrogen atom is preferable. In the following chemical formula (2), for example, any of R ¹ to R ⁶ may be the above-mentioned acyl group. As a specific example, it is preferable that only one or two or more of R ¹ to R ⁶ be the above-mentioned acyl group, more preferably R ¹ , R ² , R ⁵ and R ⁶ Among them, it is preferable that only one or two or more locations are the above-mentioned acyl groups.

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. Specifically, for example, the hydrogen atom of the acyl group may be substituted with one or more substituents. , may be substituted with the above substituent. The substituents are not particularly limited, and include, for example, an alkyl group, an amino group, an alkylamino group, a dialkylamino group, and the like.

Examples of the alkyl group include linear or branched alkyl groups having 1 to 6 carbon atoms, and preferably a methyl group. Further, 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 straight chain or branched alkyl groups having 1 to 6 carbon atoms, and dimethylamino group is preferred. These may be the same or different.

When the substituent is a group having a chain structure, specific examples include an alkyl group, an alkylamino group, a dialkylamino group, an alkoxy group, a carboxyalkyl group, an alkoxycarbonylalkyl group, an alkoxyalkyl group, and an alkenoxyalkyl group. In the case of groups, etc., there are no particular limitations, and they may be linear or branched. The same applies when a part of the substituents etc. contains a chain structure, for example, when a substituent in a substituted alkyl group or a substituted aryl group contains a chain structure. When an isomer exists in a substituent, etc., there is no particular restriction, and any isomer may be used. For example, when simply saying "propyl group", it can be either n-propyl group or isopropyl group, and when simply saying "butyl group", it can mean any of n-butyl group, isobutyl group, sec-butyl group, and tert-butyl group. 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. Furthermore, 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, and 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. Regarding groups containing an alkyl group in the structure or groups derived from an alkyl group (alkylamino group, dialkylamino group, alkoxy group, carboxyalkyl group, alkoxycarbonylalkyl group, alkoxyalkyl group, alkenoxyalkyl group, etc.) The same is true.

When the EGCG derivative according to the present invention has the acyl group at only one position among R ¹ , R ² , R ⁵ and R ⁶ in the formula (1) or the formula (2), for example, The acyl group is preferably, for example, the following acyl group. In this case, among R ¹ , R ² , R ⁵ and R ⁶ , those other than the acyl group include, for example, hydrogen, halogen, and alkali metals such as Na and K.

In the first form, when the acyl group is a linear saturated acyl group, the number of carbon atoms in the main chain is preferably in the range of 8 to 18, preferably in the range of 12 to 18. Examples include acyl groups having 12, 16 or 18 carbon atoms. The first form is suitable for allergens such as mites and pollen, for example.

As a second form, when the acyl group is an unsaturated acyl group, for example, the number of carbon atoms in the main chain is preferably in the range of 12 to 18, and the unsaturated bond has -C=C- at two positions. A specific example is an acyl group in which the main chain has 18 carbon atoms and -C=C- at two positions. The second form is suitable for allergens such as mites and pollen, for example.

As a third form, when the acyl group is a branched saturated acyl group, the number of carbon atoms in the main chain is preferably in the range of 6 to 20, and the number of carbon atoms in the branch chain is preferably in the range of 1 to 4, The number of branched chains is preferably 1 to 3, and a specific example is an acyl group in which the number of carbon atoms in the main chain is 6, the number of carbon atoms in the branched chain is 1, and the number of branched chains is 2. .

When 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), for example, The acyl group is preferably, for example, the following acyl group. In this case, among R ¹ , R ² , R ⁵ and R ⁶ , those other than the acyl group include, for example, hydrogen, halogen, and alkali metals such as Na and K.

As a fourth form, when the acyl group is a linear saturated acyl group, for example, the number of carbon atoms in the main chain of each of the two acyl groups is preferably in the range of 8 to 18. Examples include acyl groups having 8 carbon atoms in the chain. The fourth form is suitable for allergens such as mites and pollen, for example.

The number of EGCG derivatives in the present invention may be, for example, one type, or two or more types may be used in combination. For example, two or more types of EGCG derivatives having acyl groups 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 the EGCG derivatives in which R ¹ of the B ring is the above-mentioned acyl group, the EGCG derivatives in which R ² is the above-mentioned acyl group, and the EGCG derivatives in which R ³ is the above-mentioned acyl group, or It may be a mixture containing all three types, and among the EGCG derivatives in which R ⁴ of the D ring is the acyl group, the EGCG derivatives in which R ⁵ is the acyl group, and the EGCG derivatives in which R ⁶ is the acyl group, It may be a mixture containing any two or more types, or all three types. Further, it is a mixture of an EGCG derivative in which at least one of R ¹ to R ³ of the B ring is the above-mentioned acyl group, and an EGCG derivative in which at least one of R ⁴ to R ⁶ of the D ring is the above-mentioned acyl group. Good too.

The activity inhibitor of the present invention can be used against allergens as described above. The allergen is, for example, one that causes an allergic reaction in humans and non-human animals, and is not particularly limited. Specific examples of the allergen include house dust, pollen, insect-derived proteins, plant-derived proteins, and the like. House dust includes, for example, animal skin (including dander); mites, their insect bodies, their carcasses, and their feces; molds, bacteria, and the like. Examples of the animals include humans and non-human animals such as dogs and cats. The mites include, for example, Leopard dust mites (house dust mites), such as P. elegans, Pleurotus mites, such as the woolly mite, Thread mites, such as the common dust mite, Dust mites, such as the house dust mite, and the like. Examples of the pollen include pollen from plants such as Cypressaceae such as cedar, Asteraceae such as ragweed and Artemisia, Pinaceae such as pine, and Poaceae such as rice. Examples of the insect-derived proteins include proteins from cockroach and the like. Examples of the plant-derived proteins include proteins such as soybean, egg, and milk.

The activity inhibitor of the present invention can be used, for example, to prevent the onset of allergic reactions as well as to treat allergic reactions. Specifically, for example, by bringing an allergen into contact with the activity inhibitor, the activity of the allergen can be suppressed, and as a result, the onset of an allergic reaction can be suppressed.

The activity inhibitor of the present invention only needs to contain the above-mentioned EGCG derivative, and its form is not limited at all. Examples of the form include liquids such as solutions and dispersions, solids, and powders. Moreover, 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), powders, and the like.

Examples of the method of use include a method of administering the activity inhibitor to a subject who is likely to develop an allergic reaction. The subject is, for example, an animal that is likely to cause an allergic reaction due to an allergen. The administration method is not particularly limited, and includes 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 preferably transdermal administration. . The transdermal administration is not particularly limited, and includes, for example, not only the skin but also mucous membranes. The activity inhibitor of the present invention can be used, for example, depending on the form of administration thereof, such as ointments, nasal drops, nasal ointments, nasal washes, mouth washes, gargles, sublingual preparations, oral preparations, etc., containing the EGCG derivatives. It can be administered as a disinfectant for hands, etc., and it can also be administered as an EGCG derivative or a solution or dispersion containing it using a nebulizer, aspirator, injection, etc. Further, 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 humans 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 may be brought into contact with the subject, such as the animal, before the allergen comes into contact with the subject, or may be brought into contact with the allergen after the subject comes into contact with the allergen. It may also be brought into contact with the subject. Since the onset of an allergic reaction in the subject can be more effectively suppressed by suppressing allergen activity, in the former case, the activity inhibitor is administered to the subject before the subject comes into contact with the allergen. It is preferable to bring them into contact.

Other methods of use include, for example, a method in which a specimen in which an allergen may exist is treated with the activity inhibitor, and the specimen is placed in an environment in which an allergen may exist. Examples include those described below. In this case, the activity inhibitor of the present invention may be in the form of a cleaning agent, such as a hand wash, a wiping agent, or a laundry agent, containing the EGCG derivative. In this way, by treating areas where allergens are thought to be present, such as hands and desks, with the activity inhibitor of the present invention, the activity of existing allergens can be suppressed and the onset of allergic reactions can be prevented. is also possible. Furthermore, the activity inhibitor of the present invention may be supported on, for example, clothing; bedding such as futons and pillows; furniture such as tatami mats, carpets, desks, chairs, and beds; and masks. In addition, for example, filters in air conditioners or air cleaners may be treated with the activity inhibitor of the present invention. 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.

Further, the subject may be, for example, the living body of the animal itself, or cells and tissues collected from the living body, or cultures thereof. When the subject is, for example, the cell, tissue, etc., the method of administering the activity inhibitor is not particularly limited, and includes, for example, addition to a medium or the like.

In the activity inhibitor of the present invention, the content of the EGCG derivative is not particularly limited, and can be determined as appropriate depending on the purpose and method of use, for example. When the activity inhibitor of the present invention is a gargle, it is preferable that it contains, for example, 10 to 100 μmol of the EGCG derivative per dose. Furthermore, when the activity inhibitor of the present invention is a nasal spray, it is preferable that it contains, for example, 10 to 100 μmol of the EGCG derivative per dose.

The activity inhibitor of the present invention may further contain additives, bases, etc., as appropriate, depending on the dosage form and method of use. Examples of the additives include excipients, binders, lubricants, disintegrants, colorants, flavoring agents, flavoring agents, emulsifiers, surfactants, solubilizing agents, suspending agents, and tonicity agents. , buffering agents, preservatives, antioxidants, stabilizers, absorption enhancers, etc. These addition ratios are not particularly limited, and 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 contain other agents having anti-allergenic activity, such as organic anti-allergen agents such as tannic acid, and inorganic anti-allergen agents such as zeolite and silver. etc. can be mentioned. Moreover, these may be included 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, conventionally known methods such as organic synthesis, chemical synthesis using enzymes, etc. can be employed. The chemical synthesis method using the enzyme is not particularly limited, but includes, for example, the method using lipase disclosed in WO2007/105280. That is, this is a method in which an enzymatic reaction is carried out with lipase using EGCG and an acyl group donor as substrates in an organic solvent to acylate EGCG. According to this method, for example, EGCG can be selectively acylated. Note that, although a method using lipase will be illustrated below as an example, the present invention is not limited to the method for producing an EGCG derivative.

As the lipase, for example, IUB No. 3.1.1.3. lipase can be used. Specific examples include lipases derived from the genus Aspergillus such as Aspergillus niger ; lipases derived from the genus Candida such as Candida rugosa , Candida cylindracea , Candida antarctica ; lipases derived from the genus Pseudomonas such as Pseudomonas fluorescens , Pseudomonas cepacia , Pseudomonas stutzeri ; lipases derived from the genus Alcaligenes ; Burkholderia cepacia lipase derived from the genus Burkholderia , such as lipase derived from the porcine pancreas, and the like. These can also be prepared by conventionally known methods, but for example, Lipase AS “AMANO”, Lipase AYS “AMANO”, Lipase PS “AMANO”, Lipase AK “AMANO” 20, Lipase AH “AMANO” (all products) Lipase MY, Lipase OF, Lipase PL, Lipase PLC, Lipase PLG, Lipase QLM, Lipase QLC, Lipase QLG, Lipase SL, Lipase TL (all product names: manufactured by Meito Sangyo Co., Ltd.), Lipase Commercial products such as PPL, L4777 Lipase acrylic resin from Candida Antarctica, and L3126 Lipase from porcine pancreas (all product names: manufactured by Sigma-Aldrich) can also be used. The physicochemical properties of each commercially available product are as described in the respective product instructions, and enzymes exhibiting similar physicochemical properties can also be used.

Furthermore, a lipase having any of the physicochemical properties and enzymatic properties of (1) to (8) as shown below may be used.
(1) Molecular weight 35,000, isoelectric point 4.10 (e.g., derived from Aspergillus niger )
(2) Molecular weight 64,000, isoelectric point 4.30, inactivated by treatment at 80°C for 10 minutes (for example, derived from Candida rugosa )
(3) Optimum pH 8, optimal temperature 60°C, particularly stable in the pH range of 4 to 10, particularly stable below 70°C (for example, derived 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 state below 37°C (for example, derived from Candida cylindracea , Candida rugosa )
(5) Molecular weight 30,000, isoelectric point 4.5, optimum pH 8-9.5, pH stability 7-10, optimum temperature 50°C, especially stable below 40°C (for example, derived from Alcaligenes )
(6) Molecular weight 31,000, isoelectric point 4.9, optimum pH 7-9, pH stability 6-10, optimum temperature 65-70°C, particularly stable below 50°C (for example, derived from the genus Alcaligenes )
(7) Molecular weight 31,000, isoelectric point 5.2, optimum pH 7-9, pH stability 6-10, optimum temperature 65-70°C, particularly stable below 60°C (for example, Pseudomonas cepacia- derived, Burkholderia (derived from cepacia )
(8) Molecular weight 27,000, isoelectric point 6.6, optimum pH 7-8, pH stability 6-9, optimum temperature 50°C, especially stable below 40°C (for example, derived from Pseudomonas stutzeri )

The organic solvent is not particularly limited, and for example, acetonitrile, acetone, dimethylformamide (DMF), dimethyl sulfoxide (DMSO), etc. 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. Examples of such an organic solvent include the aforementioned acetonitrile (logP value: -0.45 to 0.28). 0.19), acetone (logP value: -0.16 to 0.19), DMF (logP value: -1.01 to 0.28), and 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 a solvent, a person skilled in the art can select a solvent that satisfies the above parameters. Note that logP is 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, expressed as a common logarithm. It is a general parameter that indicates the hydrophobicity of a substance.

In the present invention, examples of the acyl group (R-CO-) donor include carboxylic acid vinyl ester (R-CO-O-CH=CH ₂ ). In addition, examples of the acyl group include linear or branched saturated or unsaturated acyl groups as 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, for example, depending on the addition ratio of EGCG in the reaction solution. As a specific example, the addition ratio (molar ratio) of EGCG and acyl group donor is, for example, 1:1 to 1:10, preferably 1:1 to 1:5, more preferably 1:1 to 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 and acyl group donor, the specific activity of lipase, etc., and is not particularly limited. , 500 to 50,000 U/L, preferably 500 to 5,000 U/L, more preferably 1,000 to 2,500 U/L.

The conditions for the enzymatic 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, for example, depending on the amount of substrate and 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 further be 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 at which the acyl group is introduced in EGCG can be selected depending on, for example, the type of lipase used. Furthermore, the number of acyl groups introduced into EGCG can be determined depending on, for example, the type of organic solvent used and the reaction time. For example, the higher the hydrophobicity of the organic solvent is (the lower the hydrophilicity is), the more the number of introduced acyl groups can be relatively reduced; As the hydrophobicity is relatively lower), the number of acyl groups introduced can be relatively increased. Furthermore, the number of acyl groups introduced can also be controlled by using a mixture of two or more types of organic solvents. As a specific example, for example, when introducing one acyl group, it is preferable to use acetonitrile, etc. For example, when introducing 1 to 2 acyl groups, it is preferable to use acetone, acetonitrile, etc. For example, when introducing 3 to 5 acyl groups, it is preferable to use DMSO, DMF, etc.

Furthermore, even when using the same organic solvent, the number of acyl groups to be introduced can be adjusted by controlling the temperature time and reaction temperature. Examples are shown below, but are not limited thereto. When using DMF, two acyl groups can be 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 (e.g., about 3 to 5 hours). can preferentially obtain derivatives that have been introduced in a similar manner, while lowering the reaction temperature (e.g., about 5° C. below 57° C.) and shortening the reaction time (e.g., about 1 to 3 hours). One acyl group can be selectively introduced. Alternatively, one acyl group can be selectively introduced into EGCG by using a mixed solvent containing the same amount (mass) of acetone and DMF.

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 the lipase.

The yield of the EGCG derivative by the enzymatic reaction can be relatively improved, for example, by setting the reaction temperature relatively high. As mentioned above, the reaction temperature is usually 45 to 75°C, 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%. Note that the yield refers to the ratio (conversion efficiency) of EGCG acylated derivatives (for example, all monoacylated derivatives) when EGCG used in the reaction is taken as 100%.

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

<Method for suppressing allergen activity>
As described above, the method for inhibiting allergen activity of the present invention is characterized by bringing the activity inhibitor of the present invention into contact with an allergen. The suppression method of the present invention is characterized by using the activity inhibitor of the present invention, and other configurations, conditions, etc. are not limited at all. The activity inhibitor of the present invention and its method of use are, for example, the same as those described above, 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 mentioned above, for example, the activity inhibitor may be administered to a subject to bring the allergen possessed by the animal into contact with the activity inhibitor, or a subject in which the allergen may be present may be treated with the activity inhibitor. By doing so, the allergen and the activity inhibitor may be brought into contact with each other. 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 where an allergen may be present, and specific examples As such, the above-mentioned examples of clothing, bedding, etc. can be used.

<How to suppress allergic reactions>
As described above, 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. The present invention is characterized by using the activity inhibitor of the present invention, and other configurations, conditions, etc. are not limited at all. The activity inhibitor of the present invention and its method of use are, for example, the same as those described above, and all of them can be incorporated. Furthermore, the method for suppressing allergic reactions of the present invention can also refer to the description in the method for suppressing allergen activity of the present invention, for example.

The method for suppressing allergic reactions of the present invention can treat diseases resulting from allergic reactions in the subject, for example, by administering the activity inhibitor of the present invention. Therefore, the method for suppressing allergic reactions of the present invention can also be called, for example, a method for treating allergic diseases. In the present invention, "treatment" includes, for example, prevention of the disease, improvement of the disease, and improvement of the prognosis of the disease, and any of these may be used.

The allergic disease is not particularly limited, and includes, for example, hay fever, allergic rhinitis, urticaria, animal allergy, food allergy, insect allergy to insects such as cockroaches, house dust allergy, and the like.

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

<Use of inhibitors of allergic activity>
The present invention is the use of the activity inhibitor of the present invention to suppress allergic reactions, and the use of the activity inhibitor of the present invention to treat allergic diseases. The present invention also provides the use of the activity inhibitor of the present invention for the production of a medicament for allergic reactions, and the use of the activity inhibitor of the present invention for the production of a medicament 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, acylated EGCG derivatives corresponding to Examples, EGCG and methylated catechin corresponding to Comparative Examples were prepared.

(1) Acylated EGCG derivative In the following chemical formula (2), EGCG derivatives in which one or both of R ¹ to R ⁶ are the following acyl groups were used (all manufactured by Protectia). Note that R ¹ to R ⁶ other than the acyl group were hydrogen atoms.

EGCG-C12, EGCG-C16, EGCG-C18, EGCG-C18DE and EGCG-EH are R ¹ acylated derivatives, R ² acylated derivatives, R ⁵ acylated derivatives and R ⁶ acylated derivatives, respectively. It is a mixture of four types. Furthermore, 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, and an acylated derivative of R ¹ and R ⁶ . acylated derivatives, acylated derivatives of R ¹ and R ² , acylated derivatives of R ¹ and R ³ , acylated derivatives of R ⁴ and R ⁵ , and acylated derivatives of R ⁴ and R ⁶ . It is a mixture.

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

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

[Example 1]
The ability of acylated EGCG derivatives to suppress activity against various allergens was confirmed.
(1) Suppression test for mite allergen activity The Leopard mite extract (trade name: Mite Extract Df, manufactured by Cosmo Bio) was added to Dulbecco's phosphate physiological buffer (D-PBS(-), Japanese A mite allergen solution was prepared by diluting the solution with Hikari Pure Chemical Industries, Ltd. to a final concentration of 100 μg/mL. Further, a DMSO solution containing 10 mmol/L of the acylated EGCG derivative was diluted with phosphate physiological buffer (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. Thereafter, the mite allergen activity remaining in the mixture was measured using an ELISA kit for measuring mite allergen (trade name: mite allergen (Derf1) measurement kit, manufactured by Nichinichi Pharmaceutical Co., Ltd.). Measurements using the kit were performed according to the instructions for use.

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

Then, assuming that the mite allergen activity of the control was 100%, the relative values (%) of the mite allergen activities of the Examples and Comparative Examples were calculated, and this was defined as the residual rate of mite allergen activity (%). In this example, each sample was individually measured twice, and the average value was used as the result.

These 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 allergen activity residual rate (%), C12 is EGCG-C12, C16 is EGCG-C16, C18 is EGCG-C18, C18DE is EGCG-C18DE, and EH is EGCG -EH, C8×2 indicates the results of EGCG-C8×2. 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 Figure 1, natural EGCG showed almost no activity-suppressing effect against mite allergens, whereas EGCG derivatives (C12, C16, C18, C18DE, EH, C8×2) each showed It showed a significant inhibitory effect on the activity of mite allergen. Among them, C12 and C18DE showed an extremely excellent activity-inhibiting effect on mite allergen, and in particular, C12 suppressed mite allergen activity to about 10%, which was found to be extremely excellent.

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

50 μL of the cedar allergen solution and 450 μL of the sample were mixed in a tube, and this was incubated at room temperature for 2 hours. Thereafter, the cedar allergen activity remaining in the mixture was measured using an ELISA kit for cedar pollen antigen measurement (trade name: cedar pollen antigen (Cryj1) measurement kit, manufactured by Nichinichi Pharmaceutical Co., Ltd.). Measurements using the kit were performed according to the instructions for use. In addition, the control and comparative example were the same as in (1) above.

These results are shown in FIG. 2. FIG. 2 is a graph showing the residual rate (%) of allergen activity of cedar allergen. In FIG. 2, the Y axis is the allergen activity residual rate (%), C12 is EGCG-C12, C16 is EGCG-C16, C18 is EGCG-C18, C18DE is EGCG-C18DE, C8×2 is , shows the results of EGCG-C8×2. In FIG. 1, the lower the residual rate of cedar allergen activity, the better the activity suppression effect of the sample on cedar allergen. As shown in Figure 2, natural EGCG showed almost no activity-suppressing effect on cedar allergen, whereas EGCG derivatives (C12, C16, C18, C18DE, C8×2) each showed an effect on cedar allergen. showed a significant activity-suppressing effect.

Furthermore, as a comparative example, instead of the acylated EGCG derivative, the methylated catechin, which has been reported to have an antiallergic effect, was used to confirm its ability to suppress activity against cedar allergen. This comparative example sample is the same as the example described above, except that the methylated catechin (monomethylated catechin or dimethylated catechin) was used instead of the acylated EGCG derivative, and the final concentration was 50 μmol/L or 100 μmol/L. Prepared in the same manner as the sample. Then, using the comparative sample, the cedar allergen activity was measured in the same manner as described above, and the allergen activity residual rate was determined. In addition, the allergen activity residual rate was determined as a relative value (%) with the allergen activity of 1% DMSO as 100%.

These results are shown in FIG. 3. FIG. 3 is a graph showing the residual rate (%) of cedar allergen activity. In FIG. 3, the Y axis is the residual rate of allergen activity (%), where mM EGCG is monomethylated catechin, dM EGCG is dimethylated catechin, and μM (μmol/L) is the amount of methylated catechin in the sample. Indicates concentration. In FIG. 3, the lower the residual rate of cedar allergen activity, the better the activity suppression effect on cedar allergen. As shown in FIG. 3, the residual rate of allergen activity of methylated catechin exceeded 100% at all concentrations, and it did not exhibit an activity-suppressing effect on allergens.

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

Claims (9)

An allergen activity inhibitor for use in suppressing the allergenic activity of cedar allergen,
It is characterized by containing an epigallocatechin gallate derivative represented by the following chemical formula (1), an isomer thereof, or a salt thereof,
An agent for suppressing the allergenic activity of an allergen, wherein the isomer is a tautomer, stereoisomer, optical isomer, or geometric isomer:

In the chemical formula (1),
R ¹ to R ⁶ are
Each is a hydrogen atom, sodium, potassium, or a linear or branched saturated or unsaturated acyl group, which may be the same or different;
The acyl group may be further substituted with one or more substituents,
At least one of R ¹ to R ⁶ is the acyl group,
R ¹² and R ¹⁴ are a hydrogen atom, sodium or potassium, and may be the same or different,
R ⁷ to R ¹¹ , R ¹³ , and R ¹⁵ to R ¹⁶ are hydrogen atoms or halogens, and may be the same or different. The allergenic activity suppressor according to claim 1, wherein the acyl group is at least one of a linear saturated acyl group and a linear unsaturated acyl group. The allergen activity suppressor according to claim 2, wherein in R ¹ to R ⁶ , the acyl group has 8 to 18 carbon atoms. The allergen activity suppressor according to claim 2, wherein in R ¹ to R ⁶ , the acyl group has 12 to 18 carbon atoms. The allergen activity suppressor according to any one of claims 1 to 3, wherein the acyl group is at least one selected from the group consisting of an octanoyl group, a palmitoyl group, a stearoyl group, and a linolenyl group. The allergenic activity suppressor according to any one of claims 1 to 4, wherein at least one of R ¹ , R ² , R ⁵ and R ⁶ is the acyl group. The allergen activity suppressor according to claim 6, wherein the acyl group is at least one selected from the group consisting of palmitoyl group, stearoyl group, and linolenyl group. The allergenic activity suppressor according to any one of claims 1 to 4, wherein at least two of the R ¹ , R ² , R ⁵ and R ⁶ are the acyl groups. The allergen activity suppressor according to claim 8, wherein the acyl group is an octanoyl group.