Dendritic cell activators

Abstract

Provided is a dendritic cell activator for generating or promoting an effective acquired immune response against cancer or infectious diseases, said dendritic cell activator containing, as an active ingredient, a naturally derived component that has been empirically confirmed to be safe after being eaten over the years. The present invention provides a dendritic cell activator containing, as an active ingredient, 5-(3, 5-dihydroxyphenyl)-gamma-valerolactone represented by formula (I), and a pharmaceutical, tonic, or food and drink containing, as an active ingredient, a compound represented by formula (I). The three-dimensional configuration of the wavy line can be any one of R configuration and S configuration. ] # imgabs0 #

Description

Technical Field

[0001] The present invention relates to a dendritic cell activator containing 5-(3,5-dihydroxyphenyl)-γ-valerolactone (hereinafter referred to as EGC-M5) represented by formula (I) as an active ingredient. The present invention also relates to pharmaceuticals, supplements, or foods and beverages using the dendritic cell activator for the purpose of enhancing immune function and preventing / or treating diseases including cancer and viral diseases. Background Art

[0002] The immunoregulatory functions of innate immunity and acquired immunity work synergistically in the body to produce a relatively important role in maintaining daily health. Therefore, the activation of immune function is considered to be particularly important for the treatment, prevention and health promotion of diseases. Various immune-activating substances have been discovered before regarding substances that promote immune activation in the body. However, with regard to side effects, many side effects are known, and some are frequent or serious, thus posing a challenge when conducting treatment. Therefore, when healthy people take them for the purpose of disease prevention, naturally derived immune-activating substances with a low risk of side effects and that can be taken daily are preferred, and the use of food ingredients for immune activation is being explored.

[0003] Previously, catechins, a type of plant polyphenol, have been reported to have a wide range of health benefits. Furthermore, the functional properties of various compounds that are metabolites of catechins by intestinal bacteria have been evaluated and reported.

[0004] The immune system, as an organism's defense mechanism, recognizes invading pathogens such as bacteria and viruses, or tumor cells that develop within the organism, as antigens and eliminates them. The innate immune system is innate, while the acquired immune system develops in response to foreign antigens.

[0005] If an antigen (pathogen or tumor) invades or appears in the body, the macrophages, natural killer cells, and dendritic cells responsible for the innate immune system will first work to eliminate the antigen. Next, the dendritic cells will present the captured antigen information to T cells or B cells, thereby inducing the production of killer T cells (CD8 + T cells), produce antibodies, and activate the acquired immune system.

[0006] Dendritic cells (DCs) play an important role as a bridge between the innate and acquired immune systems. DCs are derived from bone marrow hematopoietic stem cells, which differentiate into plasmacytoid dendritic cells (pDCs) through DC precursor cells (Bone marrow-derived dendritic cells: BMDCs). Figure 6Arrow B), or differentiation towards conventional dendritic cells (cDC) ( Figure 6 The arrow A) is generated.

[0007] Plasmacytoid DCs (pDCs) have a weak antigen presentation ability, but they have the function of secreting large amounts of type I interferon-α and β (IFN-α, β) in response to viral infection. In addition, by inducing naive CD4+ T cells (hereinafter referred to as naive CD4 + T cells) are towards the differentiation of IL-10 producing T cells, regulatory T cells (Treg), play the role of suppressing excessive immune response, prevent the generation of autoimmune diseases, inflammatory diseases, allergic reactions etc. Therefore, it is reported that: as a viral infection prevention or therapeutic agent based on inducing pDC proliferation, the safety of the lactic acid bacteria or culture (patent document 1) belonging to Lactobacillus helveticus (Lactobacillus helveticus), the lactobacillus kefiri (Lactobacillus kefiri) or its processed products (patent document 2) that improve the ability of DC to produce immunoregulatory cytokines is high, and is useful. However, although the enhanced acquired immune function by pDC can suppress excessive immune response (inflammation) by inducing regulatory T cells (Treg) or promoting the production of immunoregulatory cytokine IL-10 by pDC, it prevents secondary tissue damage, but constitutes the reason why the cell killing activity for antigens is reduced. Furthermore, pDCs accumulate in the bone marrow of multiple myeloma patients, inducing immunosuppression and tumor growth (Non-Patent Document 1). By promoting the proliferation and activation of Treg cells, they exacerbate immunosuppression and are reported to be associated with poor prognosis in some cancer types (Non-Patent Document 2). Therefore, strengthening the adaptive immune system solely through pDCs may have the disadvantage of reducing tumor clearance efficiency or even promoting tumor growth.

[0008] On the other hand, after activation, canonical DC (cDC) is distributed in lymphoid tissues (spleen, lymph nodes, bone marrow, etc.) and non-lymphoid tissues (lung, skin). cDC1 has the ability to induce CD8 through the cross-presentation pathway mediated by MHC-I receptors. + T cells ( Figure 7 The role of CD8T cells in differentiating into cytotoxic T cells (CTLs) Figure 7 Arrow a), promote differentiation into Th1 cells by secreting IL-12 ( Figure 7 cDC2 has the function of activating CD4 through antigen presentation mediated by MHC-II receptors. + T cells (helper T cells; Figure 7 It is recorded as CD4T. Figure 7 Arrow c) promotes differentiation into Th1 cells, Th2 cells, etc. according to the cytokine environment during antigen presentation. + When T cells are presented with antigens, their differentiation into Th1 cells is promoted. In addition, Th1 cells secrete IFN-γ, which enhances the cytotoxic activity of cytotoxic T cells (CTLs). Figure 7 Therefore, based on the promotion of differentiation from hematopoietic stem cells to canonical DCs (cDCs) accompanied by cDC activation ( Figure 6 The strengthening of the acquired immune system (arrow A) helps to effectively clear tumor cells and antigens such as parasitic microorganisms (bacteria, fungi, protozoa) and viruses that invade host cells from the outside, that is, to obtain antigen-specific cell killing activity, which can be expected to reduce the risk of disease and enhance immune response function.

[0009] Prior art literature

[0010] Patent Literature

[0011] Patent Document 1: Japanese Patent Application Laid-Open No. 2017-031109

[0012] Patent Document 2: Japanese Patent Application Laid-Open No. 2017-007983

[0013] Non-patent literature

[0014] Non-patent literature 1: Cancer Cell, 2009, Vol. 16, pp. 309-323.

[0015] Non-patent document 2: Cancer Res., 2012, Vol. 72, pp. 5240-5249 Summary of the Invention

[0016] The present invention aims to provide a dendritic cell activator for initiating or promoting an effective immune response against cancer or infection, or a pharmaceutical, supplement, or food or beverage for activating dendritic cells, containing as an active ingredient a naturally derived ingredient whose safety has been empirically confirmed through long-term consumption.

[0017] Focusing on strengthening the adaptive immune system through cDC activation and differentiation, the present inventors conducted intensive research on cDC differentiation and activation using naturally derived ingredients with extensive proven safety. They discovered that intestinal bacterial degradation products of catechins (Formula (I)) exhibited excellent activation of both cDC1 and cDC2. Furthermore, they discovered that cDC activation by this ingredient was not accompanied by suppression of cellular immune responses (Th2 cell induction) during antigen presentation to T cells. Consequently, the ingredient effectively enhanced the antigen elimination capacity (cytotoxic activity) of the adaptive immune system, leading to the completion of the present invention.

[0018] That is, the present invention includes the following contents.

[0019] [1] A dendritic cell activator, characterized in that:

[0020] The active ingredient is 5-(3,5-dihydroxyphenyl)-γ-valerolactone represented by formula (I).

[0021] [Chemical Formula 1]

[0022]

[0023] (In formula (I), the stereo configuration of the wavy line can be either R configuration or S configuration).

[0024] [2] The dendritic cell activating agent according to [1], characterized in that

[0025] Used to promote differentiation into canonical dendritic cells (cDCs).

[0026] [3] The dendritic cell activating agent according to [2], characterized in that

[0027] The promotion of differentiation into canonical dendritic cells (cDCs) is accompanied by the activation of canonical dendritic cells (cDCs).

[0028] [4] The dendritic cell activating agent according to [2], characterized in that

[0029] Promote CD4 + T cells differentiate into Th1 cells and inhibit their differentiation into Th2 cells.

[0030] [5] The dendritic cell activating agent according to [2], characterized in that

[0031] Promote CD8 + T cells differentiate into cytotoxic T cells (CTLs), enhancing their cytotoxic activity.

[0032] [6] The dendritic cell activating agent according to [1] to [5], characterized in that

[0033] The dendritic cell activator is an enhancer of the acquired immune system.

[0034] [7] The dendritic cell activating agent according to [6], characterized in that

[0035] The acquired immune system is cellular immunity.

[0036] [8] The dendritic cell activating agent according to [6], characterized in that

[0037] The dendritic cell activator is an anticancer agent.

[0038] Effects of the Invention

[0039] Dendritic cell activators with 5-(3,5-dihydroxyphenyl)-γ-valerolactone as an active ingredient can enhance the acquired immune system by promoting differentiation from hematopoietic stem cells into canonical DCs (cDCs) accompanied by cDC activation. Furthermore, dendritic cell activators with 5-(3,5-dihydroxyphenyl)-γ-valerolactone as an active ingredient contribute to antigen elimination based on effective cellular immunity, i.e., antigen-specific cell-killing activity, against antigens such as tumor cells and parasitic microorganisms (bacteria, fungi, protozoa) and viruses that invade host cells from the outside, thereby reducing disease risk and enhancing immune response function. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is a graph showing the proportions of each dendritic cell subset in the bone marrow cells of mice to which EGC-M5 was intraperitoneally administered.

[0041] Figure 2 Graph showing the ratio of each dendritic cell subset when EGC-M5 was added to induce differentiation of mouse bone marrow cells into dendritic cells ( Figure 2 A) shows the expression of marker genes of each dendritic cell ( Figure 2 B) IL-12 gene expression and production ( Figure 2 C) chart.

[0042] Figure 3 Graph showing the expression of Th1 cell activation-related genes when EGC-M5 was added to mouse spleen cells ( Figure 3 A) Graph showing the production of IFN-γ, a cytokine secreted by Th1 cells ( Figure 3 B).

[0043] Figure 4The Th1 cell activation-related genes ( Figure 4 A), Th2 cell activation-related genes ( Figure 4 B) Graph of the expression of .

[0044] Figure 5 A is a graph showing the expression of CTL activation-related genes in spleen cells of colon cancer model mice to which EGC-M5 was intraperitoneally administered. Figure 5 B is a graph showing CD8 in each area of ​​tumor tissue + Graph showing the number of T cells (CTL).

[0045] Figure 6 Schematic diagram of the proposed pathway for the differentiation-promoting effect of EGC-M5 on bone marrow-derived precursor dendritic cells (BMDCs) toward cDCs.

[0046] Figure 7 Schematic diagram of the expected pathway of anti-tumor effects brought about by cDCs. DETAILED DESCRIPTION

[0047] The dendritic cell activator of the present invention contains 5-(3,5-dihydroxyphenyl)-γ-valerolactone represented by the following formula (I) as an active ingredient. The inclusion of 5-(3,5-dihydroxyphenyl)-γ-valerolactone as an active ingredient can promote the differentiation of hematopoietic stem cells into canonical dendritic cells (cDCs), accompanied by cDC activation.

[0048] [Chemical Formula 2]

[0049]

[0050] (In formula (I), the stereo configuration of the wavy line can be either R configuration or S configuration.)

[0051] use Figure 6 、 Figure 7 , the enhancement of the adaptive immune system by promoting differentiation into cDCs using the dendritic cell activator of the present invention will be described.

[0052] Figure 6 This is a schematic diagram of the proposed pathway for EGC-M5 to promote the differentiation of bone marrow-derived precursor dendritic cells (BMDC) into cDCs. Figure 6 Arrow A), or differentiate into plasmacytoid DC (pDC) ( Figure 6Arrow B). EGC-M5, the active ingredient of the dendritic cell activator of the present invention, acts on bone marrow-derived precursor dendritic cells (BMDCs) to increase the proportion of canonical DCs (cDCs) among the differentiated DCs, that is, to promote differentiation into cDCs.

[0053] Figure 7 This is a schematic diagram of the expected path of anti-tumor effects brought about by cDCs. Newly differentiated cDCs have not encountered foreign substances, and the expression levels of MHC-II class molecules and auxiliary signaling molecules as antigen presenting molecules are low. However, by introducing antigens, they become primordial T cells ( Figure 7 Mature cDCs (CD8 and CD4 T cells) present antigens to the cells, i.e., activated cells. EGC-M5, the active ingredient of the dendritic cell activator of the present invention, promotes antigen uptake and processing by cDCs, and antigen presentation via MHC class I and II, i.e., cDC activation. Activated cDCs present antigens to naive T cells (T cells that have never encountered an antigen) via MHC class I and II. Figure 7 cDCs are divided into two types: cDC1 that presents antigens with the help of MHC-I class and cDC2 that presents antigens with the help of MHC-II class. cDC1 presents the initial CD8 + T cells ( Figure 7 CD8 T) activation, promoting the differentiation towards cell-killing T cells (CTL) Figure 7 Arrow a), using IL-12 secretion to promote the initial CD4 + T cells ( Figure 7 CD4 T) differentiation towards Th1 cells ( Figure 7 Arrow b). cDC2 transfers the initial CD4 + T cells ( Figure 7 CD4 T) activation ( Figure 7 Arrow c) promotes differentiation towards Th1 cells in the presence of IL-12. Through IL-12 secretion by cDC1 and antigen presentation by cDC2 via MHC-II class, the initial CD4 + T cells ( Figure 7 Th1 cells differentiated from CD4 T cells secrete IFN-γ. IFN-γ further promotes the initial CD4 + T cells differentiate into Th1 cells and inhibit differentiation into Th2 cells that have the function of suppressing cellular immunity ( Figure 7 In addition, IFN-γ secreted by Th1 cells enhances the cytotoxic activity of cytotoxic T cells (CTLs) ( Figure 7d). Therefore, the dendritic cell activating agent of the present invention strengthens the adaptive immune system by promoting differentiation into cDCs.

[0054] In this specification, cell differentiation refers to the transformation into different cells through contact with other cells or stimulation by differentiation-inducing factors.

[0055] Hematopoietic stem cells formed in the bone marrow differentiate into either myeloid stem cells or lymphoid stem cells. Myeloid stem cells differentiate into erythrocytes, leukocytes, platelets, granulocytes (neutrophils, eosinophils, basophils), monocytes, macrophages, and dendritic cells, while lymphoid stem cells differentiate into lymphocytes (T cells, B cells, NK cells), which are a type of white blood cell.

[0056] In this specification, the differentiation of hematopoietic stem cells into canonical dendritic cells (cDCs) is induced ( Figure 6 The promotion of differentiation (arrow A) refers to increasing the proportion of canonical dendritic cells (cDCs) in dendritic cells differentiated from hematopoietic stem cells via bone marrow stem cells. The term "promotion of differentiation" as used herein has the same meaning as "differentiation promotion" and "differentiation induction."

[0057] As used herein, activation of canonical DCs (cDCs) refers to the transformation of newly differentiated, immature cDCs (those that have not encountered foreign substances and have low expression of auxiliary signaling molecules such as MHC class II) into mature cDCs. Upon maturation, cDCs express abundant MHC class II molecules that present antigenic peptides derived from viruses or tumors, exhibit increased expression of auxiliary signaling molecules, migrate to T cell areas in lymph nodes, and become capable of presenting antigens to naive T cells.

[0058] In this specification, cellular immunity is synonymous with T-cell immunity, which operates directly through the action of T cells and is distinct from humoral immunity, which is induced by antibodies. Cellular immunity refers to an immune response that directly attacks abnormal cells, such as microorganisms (bacteria, fungi, protozoa), virus-infected cells, and cancer cells, that reside within host cells.

[0059] The dendritic cell activator may be a dendritic cell activator containing 5-(3,5-dihydroxyphenyl)-γ-valerolactone as an active ingredient (hereinafter also referred to as "dendritic cell activator"), without particular limitation. The dendritic cell activator may be in any form, for example, a liquid form such as an aqueous solution, a turbid substance, or an emulsion; a semi-solid form such as a gel or paste; or a solid form such as a powder, granules, capsules, or tablets.

[0060] The dendritic cell activating agent may contain pharmaceutically acceptable additives such as excipients, binders, lubricants, disintegrants, preservatives, isotonicity agents, stabilizers, dispersants, antioxidants, colorants, flavoring agents, and buffers that are commonly used for formulation.

[0061] The dosage of the dendritic cell activator varies depending on age, body weight, symptoms, therapeutic effect, administration method, treatment duration, etc., but generally, it can be taken orally or parenterally once or multiple times a day, based on the content of the present compound as the active ingredient per adult, within the range of 0.1 mg to 1000 mg, preferably 1 mg to 500 mg.

[0062] Dendritic cell activators can be made into pharmaceuticals. As the pharmaceuticals, as long as the compound containing formula (I) is used as an active ingredient and is accompanied by the activation of standard DC and promotes the differentiation toward standard DC (cDC) (hereinafter also referred to as "pharmaceuticals"), there is no particular limitation. As the pharmaceuticals, the pharmaceuticals included in the Japanese Pharmacopoeia can be cited, and are pharmaceuticals for treating or preventing gastric cancer, colorectal cancer, lung cancer, prostate cancer, breast cancer, uterine cancer, esophageal cancer, liver cancer, malignant lymphoma and other cancers, atopic dermatitis, hay fever and other allergies, rheumatoid arthritis, psoriatic arthritis and other autoimmune diseases, leukemia and other blood diseases, hepatitis B, hepatitis C and other viral hepatitis caused by immune dysfunction, preferably pharmaceuticals for treating or preventing cancer, i.e. anticancer agents. In addition, because cellular immunity also works effectively for antigens such as host cell parasitic microorganisms (bacteria, fungi, protozoa), viruses and the like that invade from the outside, it is also preferably a therapeutic drug or preventive drug for them. For example, pharmaceuticals for treating or preventing diseases caused by all viruses such as influenza and herpes, intracellular parasitic bacteria (Tuberculosis Bacillus, Salmonella typhi, Treponema pallidum, Salmonella, Mycoplasma, Chlamydia, Legionella, etc.), intracellular parasitic fungi (Candida, Cryptococcus, Aspergillus, etc.), and parasitic protozoa (Toxoplasma gondii, Strongyloides stercoralis, etc.) are also preferred.

[0063] Examples of pharmaceutical preparation forms include tablets, granules, fine granules, pills, powders, capsules, lozenges, chewable tablets, liquids (drinks), and infusions. For external use, any form may be used as long as it is absorbed into the body from the skin surface, mucous membranes, and the like, and examples thereof include lotions, ointments, creams, gels, tapes, patches, aerosols, and aspirates.

[0064] Furthermore, pharmaceuticals may contain pharmaceutically acceptable additives such as excipients, binders, lubricants, disintegrants, preservatives, isotonicity agents, stabilizers, dispersants, antioxidants, colorants, flavoring agents, and buffers that are generally used for formulation.

[0065] The dosage of the pharmaceutical varies depending on age, body weight, symptoms, therapeutic effect, administration method, treatment duration, etc. Generally, the dosage of the pharmaceutical can be orally or parenterally administered once or multiple times a day, based on the content of the compound of formula (I) as the active ingredient per adult, within the range of 0.1 mg to 1000 mg, preferably 1 mg to 500 mg.

[0066] Dendritic cell activators can be used as quasi-drugs. These quasi-drugs are not particularly limited as long as they are designated by the Minister of Health, Labor and Welfare and are used to prevent discomfort such as nausea, bad breath, body odor, prevent prickly heat, ulcers, etc., prevent hair loss, or promote hair growth or hair removal. Examples of the dosage form of quasi-drugs include oral liquids, health drinks, vitamin-containing supplements, tablets, granules, liquids, lotions, ointments, creams, gels, tapes, patches, and aerosols.

[0067] As a supplement, there is no particular limitation as long as it is a supplement containing a compound of formula (I) as an active ingredient, which is accompanied by the activation of canonical DCs and promotes the differentiation toward canonical DCs (cDCs) (hereinafter referred to as "supplements"). The supplement can be in any form, for example, a liquid form such as an aqueous solution, a turbid substance, an emulsion; a semi-solid form such as a gel or a paste; or a solid form such as a powder, granules, capsules, or tablets.

[0068] In addition, supplements may include: amino acids such as leucine and valine; minerals such as zinc and calcium; vitamins such as vitamin A, vitamin B1, B2, B6, B12, vitamin C, vitamin D, vitamin E, beta-carotene, coenzyme Q10, etc.; and additives such as excipients, binders, lubricants, disintegrants, preservatives, isotonic agents, stabilizers, dispersants, antioxidants, colorants, flavoring agents, buffers, etc. commonly used in the preparation of supplements.

[0069] The amount of supplement intake varies depending on age, body weight, symptoms, therapeutic effect, administration method, treatment time, etc. Generally, it can be taken orally or parenterally once or multiple times a day, based on the content of the compound of formula (I) as the active ingredient per adult, within the range of 0.1 mg to 1000 mg, preferably 1 mg to 500 mg.

[0070] The food and drink of the present invention is any food and drink that contains a compound of formula (I) as an effective ingredient and is accompanied by the activation of standard DC and promotes the differentiation toward standard DC (cDC) (hereinafter also referred to as "this food and drink"), and is not particularly limited. The food and drink can be in any form, for example, in liquid form such as an aqueous solution, turbidity, or emulsion; in a semi-solid form such as a gel or paste; or in a solid form such as a powder, granules, capsules, or tablets.

[0071] Examples of food and beverages include: instant foods (instant noodles, cup noodles, soft canned food, canned food, microwaveable food, instant miso soup, canned soup, freeze-dried food, etc.); carbonated beverages, citrus (grapefruit, orange, lemon, etc.) juices, juice drinks, soft drinks containing juice, citrus pulp drinks, fruit drinks containing fruit pulp, vegetable drinks including vegetables such as tomatoes, green peppers, celery, melons, carrots, potatoes, and asparagus, soy milk / soy milk drinks, coffee drinks, tea drinks, powdered drinks, concentrated drinks, sports drinks, nutritional drinks, alcoholic beverages, tobacco, and other addictive beverages / addiction categories; wheat flour products such as macaroni / spaghetti, noodles, cake mix, fried chicken mix, bread mix, and dumpling wrappers; snacks such as caramel candies, chewing gum, chocolate, cookies, cakes / pies, snack biscuits, Japanese confectionery / rice confectionery / bean confectionery, and desserts; soy sauce, miso, sauces, Basic seasonings such as processed eggplant seasonings, flavorings, vinegars, and sweeteners; flavorings; compound seasonings / foods such as cooking powders, seasonings or spices for making curry, sauces, salad dressings, noodle soups, and spices; fats and oils such as butter, margarine, mayonnaise, and vegetable oils; milk / processed milk, milk beverages, yogurt, lactic acid bacteria beverages, cheese, ice cream, prepared milk powder, and cream; raw frozen foods , semi-cooked frozen foods, cooked frozen foods and other frozen foods; canned seafood, canned fruit / paste, fish ham / sausage, aquatic fish paste products, aquatic delicacies, dried aquatic products, simmered seafood and other aquatic product products; canned livestock / paste, canned livestock meat, canned fruit, jam / marmalade, pickles / boiled beans, dried agricultural products, oatmeal (cereal processed products) and other processed agricultural products; baby food, rice mixing, tea rice mixing and other commercially available foods, etc.

[0072] It should be noted that the characteristics of the above-mentioned dendritic cell activators are fully utilized, and examples include: (a) a dendritic cell activation method characterized by providing 5-(3,5-dihydroxyphenyl)-γ-valerolactone to a subject, (b) an immunoactivation method characterized by providing 5-(3,5-dihydroxyphenyl)-γ-valerolactone to a subject, (c) the use of 5-(3,5-dihydroxyphenyl)-γ-valerolactone as a dendritic cell activator, a medicine, a supplement or a food and drink, and (d) the use of 5-(3,5-dihydroxyphenyl)-γ-valerolactone in the preparation of a dendritic cell activator, a medicine, a supplement or a food and drink.

[0073] 5-(3,5-dihydroxyphenyl)-γ-valerolactone, which is an active ingredient in dendritic cell activators, pharmaceuticals, supplements, or foods and beverages, is a catechin metabolite, i.e., a compound produced from catechins by the action of microorganisms living in mammals such as humans, rats, mice, and pigs. Therefore, by using this compound, dendritic cell activators, pharmaceuticals, supplements, or foods and beverages with excellent safety can be prepared.

[0074] When the compound of formula (I) is contained in a dendritic cell activating agent, pharmaceutical, supplement, or food or beverage, the compound of formula (I) may be contained in a purified form, or a composition containing the compound of formula (I) may be contained in a crudely purified form.

[0075] The compound of formula (I) can be obtained using the well-known organic chemical synthesis method given in the following literature (SYNTHESIS, 9, 1512-1520, 2010), etc., for example, it can be prepared by using 3,5-(tert-butyldimethylsiloxy)bromobenzene (3,5-(tert-Butyldimethylsiloxy)bromobenzene) as a substrate and using seven reactions including Swern oxidation and Wittig reaction.

[0076] In addition, the compound of formula (I) can also be produced by a microbial transformation method using intestinal microorganisms. When producing the compound of formula (I) as a catechin metabolite by a microbial transformation method, the following method can be used: feces or cecal contents containing intestinal microorganisms of rats or humans are cultured to proliferate the intestinal microorganisms, the cultured microorganisms are suspended in a buffer solution, physiological saline, water, etc., and the catechins serving as a substrate are added to the suspension, followed by incubation.

[0077] Examples of catechins added as a matrix include (+)-epigallocatechin, (-)-epigallocatechin, and (-)-gallocatechin as non-gallate-type catechins; and (-)-gallocatechin gallate and (-)-epigallocatechin gallate as gallate-type catechins, with (-)-epigallocatechin being preferred.

[0078] To obtain the compound of formula (I), first, it is necessary to utilize microbial conversion to convert the catechins that will become the substrate into a compound represented by the following formula (II). Preferred examples of microorganisms having the ability to convert catechins into a compound represented by the following formula (II) include: Eggerthella lenta JCM9979 strain, Adlercreutzia equolifaciens MT4s-5 strain (trusted number FERM P-21738) and JCM14793 strain, Asaccharobacter celatus JCM14811 strain, and Slackia equolifaciens JCM16059 strain (see Biol. Pharm. Bull., 38, 325-330, 2015).

[0079] [Chemical Formula 3]

[0080]

[0081] (In formula (II), R1 and R2 each independently represent a hydroxyl group (OH) or a hydrogen atom (H), and the stereo configuration of the wavy line may be either R or S.)

[0082] It should be noted that, about obtaining the compound represented by formula (I), it is necessary to obtain the compound recorded in formula (II) R1 for H, R2 for OH compound. In this case, it is necessary to add hydrogen and / or formic acid to the culture medium. In addition, when hydrogen or formic acid are not added to the culture medium, it is also possible to make the coexistence of the microorganism with hydrogen and / or formic acid generating ability. As the example of such a microorganism, it is possible to enumerate: Escherichia coli (Escherichia coli), Butyricimonas bacteria, preferably Escherichia coli K12 strain (Escherichia coli K12), Butyricimonas virosa (Butyricimonas virosa) JCM15149, Butyricimonas synergistica (Butyricimonas synergistica) JCM15184, Butyricimonas paravirosa (Butyricimonas paravirosa) JCM18677 etc.

[0083] Preferred examples of microorganisms having the ability to convert a compound represented by formula (II) into a compound represented by formula (I) include: Flavonifractor bacteria (formerly known as Eubacterium bacteria and Clostridium bacteria), preferably Flavonifractor plautii (formerly known as Eubacterium plautii) ATCC 29863 strain, Flavonifractor plautii (formerly known as Eubacterium plautii) MT42 strain (trusted number FERM P-21765), and Flavonifractor plautii (formerly known as Clostridium orbiscindens) ATCC 49531 strain.

[0084] By adding catechins and / or a catechin-containing substance as a substrate to the culture suspension or culture medium in which the microorganism having the ability to convert catechins into a compound represented by formula (II) and the microorganism having the ability to convert the compound represented by formula (II) into a compound represented by formula (I) coexist, and incubating the suspension under anaerobic conditions, the above-mentioned substance containing the present compound can be easily obtained. Regarding the incubation treatment method, there is a method in which the substance obtained by culturing the above-mentioned microorganisms and collecting the bacteria is suspended in a buffer solution, physiological saline, water, etc. and a substrate is added, or a method in which the substrate is added during the cultivation of the above-mentioned microorganisms or after a certain period of time after the start of the cultivation. In addition, by adding a substrate to the culture medium in which the above-mentioned microorganisms are grown and incubating the suspension under anaerobic conditions, the compound of formula (I) can also be easily obtained.

[0085] In the case of cultivating the above-mentioned microorganism given, be inoculated in the nutrient source culture medium that this microorganism can grow, cultivate under anaerobic condition.Be used for obtaining the microorganism culture of cultivating thalline and the microorganism culture under the existence of matrix and can adopt the cultural method of common anaerobic microorganism.In addition, will cultivate thalline and be collected after incubating under the existence of above-mentioned matrix, also preferably carry out under anaerobic condition.As the culture medium used in cultivating, as long as the culture medium that above-mentioned microorganism can grow gets final product, is not particularly limited, for example, can utilize GAM broth (Nissui Pharmaceutical (strain) system) etc.

[0086] The culture conditions can be appropriately selected within the range in which the above-mentioned microorganisms can grow. Typically, the conditions are pH 6.0-7.5 and 35-40°C, preferably pH 6.5-7.3 and 37-39°C. The culture time is typically 24-120 hours, preferably 48-72 hours. The various culture conditions described above can be appropriately modified depending on the type and characteristics of the microorganisms used, external conditions, etc., and optimal conditions can be selected.

[0087] When producing catechin metabolites using microbial conversion methods, extracts containing multiple catechin metabolites are obtained during production. However, through repeated purification, high-purity catechin metabolites can be obtained. Similar to organic synthesis methods, various known purification methods can be selected and combined to achieve the desired purity. For example, solvent extraction using ethyl acetate, diethyl ether, or butanol, desorption using synthetic resin adsorbents, column chromatography using silica gel, or high-performance liquid chromatography can be used alone or in appropriate combinations to adjust the content of catechin metabolites in the extract.

[0088] Hereinafter, the present invention will be described in further detail based on Production Examples and Examples of the compound of formula (I), but the present invention is not limited to these Examples.

[0089] Example

[0090] <Production of the compound of formula (I)>

[0091] (Production Example 1: Production of (R)-5-(3,5-dihydroxyphenyl)-γ-valerolactone in the coexistence of Eggerthella tarda JCM9979 strain, Flavonoids platysporum ATCC49531 strain, and Escherichia coli K12 strain)

[0092] Eggerthella tarda JCM9979 was inoculated into 30 mL of GAM broth (manufactured by Nissui Pharmaceutical Co., Ltd.) and anaerobically cultured at pH 7.2 and 37°C for 48 hours to prepare pre-culture solution 1. Separately, Escherichia coli K12 and Eubacterium prausnitzii ATCC49531 were anaerobically cultured in 10 mL of GAM broth at pH 7.2 and 37°C for 24 hours to prepare pre-culture solution 2. To 100 mL of GAM broth containing 290 mg of (-)-epigallocatechin, 30 mL of pre-culture solution 1 of JCM9979 and 10 mL of pre-culture solutions 2 of Escherichia coli K12 and ATCC49531 were added, and anaerobically cultured at pH 7.2 and 37°C for 72 hours. 1 mL of the resulting culture medium was collected and centrifuged at high speed (15,000 × g, 10 minutes, 4°C) to remove bacterial cells. The supernatant was analyzed using the following LC / MS analysis conditions to confirm the production of 5-(3,5-dihydroxyphenyl)-γ-valerolactone and 5-(3,5-dihydroxyphenyl)-4-hydroxyvaleric acid. The LC / MS analysis conditions are as follows.

[0093] (LC / MS conditions)

[0094] Column: Capcellpak C18 MG (2.0 id × 100.0 mm, 5 μm, (manufactured by Shiseido Co., Ltd.)

[0095] Flow rate: 0.2 mL / min

[0096] Column temperature: 40°C

[0097] Solvent

[0098] Solvent A: water: acetonitrile: acetic acid (100:2.5:0.1 volume ratio (v / v / v))

[0099] Solvent B: water: acetonitrile: methanol: acetic acid (35:2.5:65:0.1 volume ratio (v / v / v / v))

[0100] Gradient conditions: 0 min A: 100% B: 0%, 3 min A: 100% B: 0%, 25 min A: 0% B: 100%, 25.1 min A: 100% B: 0%, 33 min A: 100% B: 0%

[0101] Detector: PDA and mass analyzer

[0102] Interface: ESI

[0103] Polarity: Negative

[0104] The culture solution was centrifuged at high speed (10,000 × g, 20 minutes, 20°C) to remove the bacteria. 5M hydrochloric acid water was added to the obtained supernatant, adjusted to pH 2.0, and maintained at 80°C for about 2 hours to dehydrate and condense the 5-(3,5-dihydroxyphenyl)-4-hydroxypentanoic acid contained in the supernatant and convert it into 5-(3,5-dihydroxyphenyl)-γ-valerolactone. The reaction solution was extracted three times with 200 mL of ethyl acetate, and the ethyl acetate phases were combined and concentrated using an evaporator. The obtained concentrated solution was subjected to preparative HPLC. The preparative HPLC conditions are as follows.

[0105] (Preparative HPLC conditions)

[0106] Column: Capcellpak MG (20i.d. × 150mm, 5μm, (manufactured by Shiseido Co., Ltd.)

[0107] Flow rate 15mL / min

[0108] Solvent:

[0109] Solvent A: acetonitrile: methanol: water: acetic acid (5:5:90:0.3 volume ratio (v / v / v))

[0110] Solvent B: acetonitrile: methanol: water: acetic acid (5:65:30:0.5 volume ratio (v / v / v))

[0111] Gradient conditions: 0 min A: 80% B: 20%, 5 min A: 80% B: 20%, 20 min A: 10% B: 90%, 25 min A: 1% B: 90%, 26 min A: 80% B: 20%, 35 min A: 80% B: 20%

[0112] Detector: UV270nm

[0113] After separation and collection, the fractions containing the target metabolite were analyzed using the aforementioned LC / MS analysis conditions. The fractions were then concentrated to dryness using an evaporator. 5 mL of pure water was added to the dried product, and the product was concentrated again to dryness. This process was repeated three times to completely remove the acetic acid from the fractions. Finally, the dried product, dissolved in a small amount of pure water, was lyophilized to yield 45 mg of 5-(3,5-dihydroxyphenyl)-γ-valerolactone.

[0114] <Example 1: Effect of Intraperitoneal Administration of EGC-M5 on the Composition of Dendritic Cells Formed in the Bone Marrow>

[0115] (a) Feeding methods and drug administration methods

[0116] After pre-raising 6-week-old male C57BL / 6J mice, they were divided into two groups: the EGC-M5 administration group and the control group (10 mice in each group). For the EGC-M5 administration group, EGC-M5 was dissolved in PBS buffer containing 10% DMSO to obtain a test solution, and the test solution was intraperitoneally administered daily at a dose of EGC-M5 10 mg / kg bw. For the control group, PBS buffer containing 10% DMSO was intraperitoneally administered daily. After 12 days of administration, the mice were slaughtered using blood collection under isoflurane anesthesia and the thigh bones were removed.

[0117] (b) Preparation of bone marrow cells

[0118] The femur was placed in a 5 mL culture dish containing RPMI-1640 medium (Fuji Film Wako Pure Chemical Industries, Ltd.). Myelin was collected using a 2.5 mL syringe and a 26G needle. The tissue fragments were removed by filtration and centrifuged for 5 minutes. The cell count was then measured using a hemocytometer.

[0119] (c) Composition analysis of each dendritic cell

[0120] Bone marrow cells were adjusted to 1.0 × 10 7 cells / mL, mixed with an equal amount of 2% PFA (4% paraformaldehyde·phosphate buffer (Fuji Film Wako Pure Chemical Industries, Ltd.) diluted with water for injection), and allowed to stand at 4°C for 20 minutes. After centrifugation for 5 minutes, the supernatant was removed and resuspended with 5% FBS-TPBS (0.2% Tween-PBS (Fuji Film Wako Pure Chemical Industries, Ltd.)). 50 μL / well was dispensed into 96-well V-bottom plates and allowed to stand at room temperature for 30 minutes. Each antibody given in Table 1 was added at 50 μL / well, allowed to stand at room temperature for 1 hour, and the cells were fluorescently labeled. Afterwards, 50 μL of the cell suspension and 250 μL of PBS were mixed, and the proportion (%) of cDC1, cDC2, and pDC in dendritic cells was determined by flow cytometry. At this time, based on the intensity of the fluorescent labeling, cells that are CD3-negative and CD11c-positive are selected as "dendritic cells". Among the "dendritic cells", cells that are CD103-positive and strongly IA / IE (MHC-II)-positive are defined as "cDC1", cells that are CD11b-positive and strongly IA / IE (=MHC-II)-positive are defined as "cDC2", and cells that are B220-positive and weakly IA / IE (MHC-II)-positive are defined as "pDC".

[0121] Table 1 shows the antibodies (cDC1, cDC2, and pDC) used to measure the dendritic cells by flow cytometry.

[0122] Table 1

[0123]

[0124] (d) Statistical processing

[0125] The obtained measurement results were expressed as mean values ​​and standard errors (SE), and the significant difference between the control group and the EGC-M5-administered group was investigated using Student's t-test. The significance level was set at *P<0.05.

[0126] (e) Test results

[0127] like Figure 1 As shown, regarding dendritic cells formed in the mouse bone marrow, the proportion of standard dendritic cells (cDC1 and cDC2) increased significantly in the EGC-M5-treated group compared to the control group, while the proportion of plasmacytoid dendritic cells (pDC) did not change.

[0128] The present results indicate that administration of EGC-M5 has an effect of promoting the differentiation of hematopoietic stem cells into standard dendritic cells (cDC1 and cDC2) in the process of differentiation of hematopoietic stem cells into dendritic cells in the bone marrow of a living body.

[0129] <Example 2: Effects of EGC-M5 on Differentiation and Activation of Bone Marrow-Derived Progenitor Dendritic Cells (BMDCs)>

[0130] (a) Mice used and breeding methods

[0131] Six-week-old male C57BL / 6J mice were pre-raised and then slaughtered by blood sampling under isoflurane anesthesia to obtain the thigh bones.

[0132] (b) Preparation of bone marrow cells

[0133] Bone marrow cells were isolated from the femur using the same method as in Example 1 (b).

[0134] (c) Differentiation of bone marrow-derived precursor dendritic cells (BMDCs) in the presence of EGC-M5

[0135] Bone marrow cells containing precursor dendritic cells (BMDCs) were cultured in R10 (10% inactivated FBS, 20 ng / mL GM-CSF, 50 μM 2-mercaptoethanol-containing RPMI) medium containing EGC-M5 at a concentration of 2.0 × 10 5cells / mL and seeded into a 10mL culture dish for suspension cells. After 72 hours of incubation at 37°C and 5% CO2, 10mL of R10 medium containing 0, 1, or 10μM EGC-M5 was added. After 48 hours of incubation, 10mL of the culture supernatant was recovered and centrifuged, and the pellet was suspended in 10mL of R10 medium containing 0, 1, or 10μM EGC-M5 and returned to the original culture dish. After 48 hours of incubation, the precursor dendritic cells (BMDCs) were differentiated into immature dendritic cells using the same treatment as before. After culturing for an additional 48 hours, the cells were harvested and resuspended in maturation induction medium (10% inactivated FBS, 10 ng / mL GM-CSF, 50 μM 2-mercaptoethanol, 1 μg / mL LPS) containing EGC-M5 at 0, 1, or 10 μM concentrations. The cells were then seeded into 10 mL culture dishes for adherent cells to convert the immature dendritic cells into mature dendritic cells (activated cDCs). After culturing for 24 hours, the cells and culture supernatant were harvested.

[0136] (d) Composition analysis of each dendritic cell

[0137] The cells obtained in step (c) were recovered and the proportions (%) of cDC1, cDC2, pDC, and activated DC in dendritic cells were measured using flow cytometry in the same manner as in step (c) of Example 1. Activated DCs were defined as cells that were IA / IE (MHC-II) positive among dendritic cells. The antibodies used in the measurement of activated DCs are shown in Table 2.

[0138] Table 2 shows the antibodies (MHC-II + DC).

[0139] Table 2

[0140]

[0141] (e) Analysis of IL-12 production, cDC and pDC gene expression

[0142] The IL-12 concentration in the culture supernatant obtained in step (c) was measured using the ELISA method. Mouse IL-12p70 DuoSet ELISA (DY419-05, R&D Systems) and DuoSet Ancillary Reagent Kit 2, 5 Plate (DY008, R&D Systems) were used for the measurement. In addition, RNA was extracted from the cells obtained in step (c) and the expression of the RNA was detected using Prime Script. TMcDNA was synthesized using the RT Reagent Kit (Takara Bio). The synthesized cDNA was mixed with PCR primers for each gene detection and SsoAdvanced Universal SYBR Green Supermix (BIO-RAD). The expression level of each gene was measured using real-time PCR. Actb was used as an internal standard. The primer sequences for each gene are as follows.

[0143] (cDC1 marker)

[0144] Xcr1:Forward 5'-ACATGATACCCATGGGGAAGT-3'

[0145] Reverse 5'-GTGCACGAAGTGTTGCTTTG-3'

[0146] Cd103:Forward 5'-GCCGTGATCCAGACTGAGTTTGAT-3'

[0147] Reverse 5'-ATGGCTGAGGCGGTCTTAGTGACT-3'

[0148] (cDC2 marker)

[0149] Cd11b:Forward 5'-CCACTCATTGTGGGCAGCTC-3'

[0150] Reverse 5'-GGGCAGCTTCATTCATCATGTC-3'

[0151] (pDC marker)

[0152] Bst2:Forward 5'-ACATGGCGCCCTCTTTCTATCACT-3'

[0153] Reverse 5'-TGACGGCGAAGTAGATTGTCAGGA-3'

[0154] (Cytokine IL-12 gene)

[0155] Il-12a: Forward 5'-TCTGGTACATCTTCAAGTCCTCATAGA-3'

[0156] Reverse 5'-TACTAGAGAGACTTCTTCCACAACAAGAG-3'

[0157] (Endogenous regulatory genes)

[0158] Actb:Forward 5'-CATCCGTAAAGACCTCTATGCCAA-3'

[0159] Reverse 5'-ATGGAGCCACCGATCCACA-3'

[0160] (f) Statistical processing

[0161] The results are expressed as mean and standard error (SE), and significant differences were investigated using Dunnett's test. The significance level was set at *P<0.05 and **P<0.01.

[0162] (g) Test results

[0163] As a result of the analysis of the composition of each dendritic cell in (d), in the group where precursor dendritic cells (BMDC) were treated with 10 μM EGC-M5, the proportion of cDC1 and cDC2 in the dendritic cells increased significantly compared to the control group, but the proportion of pDC did not change. On the other hand, activated DC (MHC-II + The proportion of DC) increased significantly ( Figure 2 (A)).

[0164] Next, as a result of the analysis of gene expression levels of cDC1, cDC2, and pDC markers in (e), the group treated with 10 μM EGC-M5 showed a significant increase in gene expression of cDC1 markers (XCR1, CD103) and cDC2 marker (CD11b) compared to the control group. No significant change was observed in gene expression of the pDC marker (BST2). Figure 2 (B)).

[0165] Furthermore, as a result of the analysis of the cytokine IL-12 gene expression and production in (e), in the 1 μM EGC-M5-treated group, an increasing trend in IL-12a gene expression was observed, and in the 10 μM EGC-M5-treated group, an increasing trend in IL-12 production was observed ( Figure 2 IL-12 is a cytokine mainly secreted by activated cDC1. Therefore, it is believed that the increase in IL-12 gene expression and production depends on the increase in activated cDC1.

[0166] These results indicate that EGC-M5 directly acts on precursor dendritic cells (BMDCs), promoting their differentiation into standard dendritic cells (cDC1 and cDC2) and activating immature cDCs to mature cDCs.

[0167] Examples 1 and 2 demonstrate the potential of intraperitoneal administration of EGC-M5 and its addition to bone marrow-derived pro-dendritic cells to promote the differentiation and activation of cDC1 and cDC2 pro-dendritic cells. Therefore, subsequent experiments evaluated the effects of EGC-M5 on Th1 cells and cytotoxic T cells (CTLs) activated by the promotion of cDC1 and cDC2 differentiation.

[0168] <Example 3: Effect of EGC-M5 on Activation of Th1 Cells in Mouse Spleen Cells>

[0169] (a) Rearing methods

[0170] Six-week-old male C57BL / 6J mice were pre-raised and then slaughtered by blood collection under isoflurane anesthesia to remove the spleen.

[0171] (b) Analysis of T cell activation-related gene expression in splenocytes in the presence of EGC-M5

[0172] The spleen was placed in a 5 mL culture dish filled with RPMI-1640 medium and the tissue was ground using a glass slide to disperse the cells. The tissue fragments were removed by filtration and centrifuged for 5 minutes. The supernatant was removed and the filtration was performed based on BDPharm Lyse TM Hemolysis was performed using Lysing Buffer (Becton, Dickinson and Company). Lysing Buffer was diluted 10-fold with water for injection (Otsuka Pharmaceutical). The cells were washed twice with RPMI-1640 medium and the cell count was measured using a hemocytometer. 10% FBS-RPMI1640 medium was used to prepare 1.0×10 7 cells / mL and seeded in a 24-well plate. EGC-M5 was then added at final concentrations of 0, 1, and 10 μM. After incubation at 37°C and 5% CO2 for 72 hours, RNA was extracted using the RNeasy Mini kit (QIAGEN) and analyzed using Prime Script. TM cDNA was synthesized using the RTReagent Kit (Takara Bio). The synthesized cDNA was mixed with various primers and SsoAdvanced Universal SYBR Green Supermix (BIO-RAD), and the expression of genes involved in Th1 cell activation was evaluated by real-time PCR. Actb was used as an internal standard.

[0173] Il-12a is mainly a gene for cytokines produced by activated cDC1. IL-12 has the function of acting on the initial CD4 + T cells differentiate into Th1 cells. T-bet is a cytokine such as IL-12 that interacts with the initial CD4 + T-bet is a transcription factor expressed by activating the signal transduction pathway when it binds to the receptor on T cells. It enhances the expression of IFN-γ gene and promotes the proliferation of naive CD4 + Differentiation of T cells into Th1 cells. TNF-α and IFN-γ are cytokines secreted by Th1 cells and are known to induce CTL activation.

[0174] The primer sequences for each gene are as follows.

[0175] (Cytokine IL-12 gene)

[0176] Il-12a: Forward 5'-TCTGGTACATCTTCAAGTCCTCATAGA-3'

[0177] Reverse 5'-TACTAGAGAGACTTCTTCCACAACAAGAG-3'

[0178] (Th1 cell differentiation-related genes)

[0179] T-bet: Forward 5'-AACCAGTATCCTGTTCCAGC-3'

[0180] Reverse 5'-TGTCGCCACTGGAAGGATAG-3'

[0181] (Cytokine TNF-α gene)

[0182] Tnf-α: Forward 5'-GAGGCACTCCCCCAAAAGAT-3'

[0183] Reverse 5'-CGATCACCCCGAAGTTCAGT-3'

[0184] (Cytokine IFN-γ gene)

[0185] Ifn-γ:Forward 5'-GCTTCCTGAGGCTGGATTC-3'

[0186] Reverse 5'-GGATGCATTCATGAGTATTGG-3'

[0187] (Endogenous regulatory genes)

[0188] Actb:Forward 5'-CATCCGTAAAGACCTCTATGCCAA-3'

[0189] Reverse 5'-ATGGAGCCACCGATCCACA-3'

[0190] (c) Evaluation of IFN-γ production

[0191] Splenocytes were isolated from the spleen in the same manner as in (b) and the culture medium was adjusted to 1.0×10 7 cells / mL and seeded in a 96-well plate. EGC-M5 was then added at final concentrations of 0, 1, and 10 μM. After 72 hours of incubation at 37°C and 5% CO2, the culture supernatant was recovered and IFN-γ production was measured by ELISA. The assay used the Mouse IFN-gamma DuoSet ELISA (DY485-05, R&D Systems) and the separately available DuoSet Ancillary Reagent Kit 2, 5 Plates (DY008, R&D Systems).

[0192] (d) Statistical processing

[0193] The results are expressed as mean and standard error (SE), and significant differences were investigated using Dunnett's test. The significance level was set at *P<0.05 and **P<0.01.

[0194] (e) Test results

[0195] like Figure 3 As shown, the expression levels of IL-12a, T-bet, TNF-α, and IFN-γ genes in splenocytes in the 10 μM EGC-M5 treatment group were significantly higher than those in the control group ( Figure 3 (A)) and IFN-γ production ( Figure 3 (B)) increased significantly. It can be seen that EGC-M5 promotes the growth of naive CD4 + T cells differentiate into Th1 cells. Furthermore, it was shown that EGC-M5 enhances the production of cytokines secreted by Th1 cells by increasing the expression of TNF-α and IFN-γ genes. Furthermore, it was shown that EGC-M5 enhances the cytotoxic activity of cytotoxic T cells (CTLs) by increasing IFN-γ production.

[0196] The results show that EGC-M5 has the following effects: + The differentiation of T cells into Th1 cells enhances the production of cytokines secreted by Th1 cells, thereby increasing the cytotoxic activity of cytotoxic T cells (CTLs) acted upon by the produced IFN-γ, thereby strengthening cellular immunity.

[0197] <Example 4: Effect of Intraperitoneal Administration of EGC-M5 on Activation of Th1 Cells in the Spleen in Colon Cancer Model Mice>

[0198] (a) Feeding methods and drug administration methods

[0199] After 6-week-old male C57BL / 6J mice were pre-fed, 1.0×10 6 Subcutaneous transplantation was performed using a cells / mouse method. Four days after transplantation, the animals were divided into two groups: the EGC-M5 administration group and the control group (10 animals in each group). For the EGC-M5 administration group, 200 μL of PBS buffer containing 10% DMSO and EGC-M5 prepared at a dosage of 10 mg / kg bw of EGC-M5 was intraperitoneally administered daily. For the control group, PBS buffer containing 10% DMSO was intraperitoneally administered daily. 16 days after transplantation, the animals were slaughtered by blood sampling under isoflurane anesthesia, and the spleen and tumor tissue were taken.

[0200] (b) Analysis of cytokine gene expression levels related to T cell differentiation

[0201] The spleen was placed in a 5 mL culture dish containing 5 mL of RPMI1640 culture medium. Next, the spleen was ground using a frosted glass slide and filtered with a 70 μm cell strainer (FALCON). After centrifugation at 2,500 rpm for 5 minutes, the supernatant was removed, and the suspension was suspended with 1 mL of Lysing Buffer (manufactured by BD) and allowed to stand on ice for 1 minute. PBS2 mL was added to stop the reaction, and after centrifugation at 2,500 rpm for 5 minutes, the supernatant was removed, and the suspension was suspended with 10 mL of RPMI1640 culture medium. Centrifugation was performed again at 2,500 rpm for 5 minutes, the supernatant was removed, and the suspension was suspended with RPMI1640 culture medium to determine the number of cells. In addition, the RNA of the cells was extracted using RNeasy Mini kit (QIAGEN) and the RNA was extracted using Prime Script TMcDNA was synthesized using the RT Reagent Kit (Takara Bio). The synthesized cDNA was mixed with various primers and Sso Advanced Universal SYBR Green Supermix (BIO-RAD) and the expression of genes involved in Th1 and Th2 cell activation was evaluated by real-time PCR. Actb was used as an internal standard. The primer sequences are as follows.

[0202] (Cytokine Gene)

[0203] Il-12a: Forward 5'-TCTGGTACATCTTCAAGTCCTCATAGA-3'

[0204] Reverse 5'-TACTAGAGAGACTTCTTCCACAACAAGAG-3'

[0205] Il-12b:Forward 5'-AACTTGAGGGAGAAGTAGGAATGG-3'

[0206] Reverse 5'-GGAAGCACGGCAGCAGAATA-3'

[0207] Il-2:Forward 5'-CTTCAAGTCCCACTTCAAGCT-3'

[0208] Reverse 5'-CCATCTCCTCAGAAAGTCCACC-3'

[0209] Ifn-γ:Forward 5'-GCTTCCTGAGGCTGGATTC-3'

[0210] Reverse 5'-GGATGCATTCATGAGTATTGG-3'

[0211] Il-4:Forward 5'-TCCTCACAGCAACGAAGAAC-3'

[0212] Reverse 5'-CAAGCATGGAGTTTTCCCATG-3'

[0213] Il-5:Forward 5'-TCAGCTGTGTCTGGGCCACT-3'

[0214] Reverse 5'-TTATGAGTAGGGACAGGAAGCCTCA-3'

[0215] Il-6:Forward 5'-GGCCTTCCCTACTTCACAAG-3'

[0216] Reverse 5'-ATTTCCACGATTTCCCAGAG-3'

[0217] Il-10:Forward 5'-GACCAGATGGACAACATACTGATAA-3'

[0218] Reverse 5'-GACCAGCTGGACAACATACTGCTAA-3

[0219] (Endogenous regulatory genes)

[0220] Actb:Forward 5'-CATCCGTAAAGACCTCTATGCCAA-3'

[0221] Reverse 5'-ATGGAGCCACCGATCCACA-3'

[0222] (c) Statistical processing

[0223] The results are expressed as mean and standard error (SE), and significant differences were investigated using Student's t-test. The significance level was set at *P<0.05 and **P<0.01.

[0224] (d) Test results

[0225] like Figure 4 As shown, regarding the spleen of colon cancer model mice, in the EGC-M5 administration group, the initial CD4 + The expression levels of IL-12a and IL-12b genes, which are the genes that differentiate T cells into Th1 cells, increased. In addition, the expression levels of IL-2 and IFN-γ genes, which are the cytokines secreted by Th1 cells, also increased ( Figure 4 On the other hand, the expression levels of cytokines Il-4, Il-6, and Il-10 genes produced by Th2 cells, which have an opposite effect to Th1 cells, were reduced in the EGC-M5-administered group ( Figure 4 (B)).

[0226] The results showed that the administration of EGC-M5 promoted the proliferation of naive CD4 + T cells differentiate into Th1 cells and inhibit differentiation into Th2 cells, which have opposite effects to Th1 cells. Figure 7 e).

[0227] <Example 5: Effects of Intraperitoneal Administration of EGC-M5 on Activation of Cytotoxic T Cells (CTLs) and Tumor Infiltration in Colon Cancer Model Mice>

[0228] (a) Evaluation of the effect of EGC-M5 on CTL activation

[0229] The spleen obtained in Example 4 was placed in a 5 mL culture dish containing 5 mL of RPMI1640 culture medium. Next, the spleen was ground using a frosted slide and filtered using a 70 μm cell strainer (FALCON). After centrifugation at 2,500 rpm for 5 minutes, the supernatant was removed, and the cell suspension was suspended with 1 mL of Lysing Buffer (manufactured by BD) and allowed to stand on ice for 1 minute. PBS 2 mL was added to stop the reaction, and after centrifugation at 2,500 rpm for 5 minutes, the supernatant was removed, and the cell suspension was suspended with 10 mL of RPMI1640 culture medium. Centrifugation was performed again at 2,500 rpm for 5 minutes, the supernatant was removed, and the cell suspension was suspended with RPMI1640 culture medium to determine the cell number. In addition, RNA of the cells was extracted using RNeasy Mini kit (QIAGEN) and the RNA was extracted using Prime Script TM cDNA was synthesized using the RT Reagent Kit (Takara Bio). The synthesized cDNA was mixed with various primers and SsoAdvanced Universal SYBR Green Supermix (BIO-RAD) and the expression of genes involved in CTL activation was evaluated by real-time PCR. Actb was used as an internal standard. The primer sequences are as follows.

[0230] Perforin: Forward 5'-GAGAAGACCTATCAGGACCA-3'

[0231] Reverse 5'-AGCCTGTGGTAAGCATG-3'

[0232] Grzb:Forward 5'-CCTCCTGCTACTGCTGAC-3'

[0233] Reverse 5'-GTCAGCACAAAGTCCTCTC-3'

[0234] Actb:Forward 5'-CATCCGTAAAGACCTCTATGCCAA-3'

[0235] Reverse 5'-ATGGAGCCACCGATCCACA-3'

[0236] (b) CD8 in tumor tissue + Evaluation of T cell infiltration

[0237] The tumor tissue obtained in Example 4 was immersed in 4% paraformaldehyde for 1 week. After that, paraffin blocks and tissue sections were prepared by coordinating pathology. After deparaffinization using Lemosol (Fuji Film Wako Pure Chemical Industries, Ltd.), heat treatment was performed using ImmunoSaver (Nissin EM) to activate the antigen. 5% FBS-Sodium azide (0.01% sodium azide-PBS (Fuji Film Wako Pure Chemical Industries, Ltd.)) was used to treat at room temperature for 1 hour, and after blocking, anti-CD8alpha antibody (abcam, ×200) was used to treat at 4°C overnight. After that, Alexa Flour488 F(ab')2fragment of goatanti-IgG (thermofisher, ×500), Hoechst 33342, trihydrochloride, trihydrate (H3570, thermofisher, ×10000) were used to treat at 4°C for 1 hour to perform staining. After the tissue was sealed with VECTASHIELD (VECTORLABPRATORIES) and a cover glass, it was observed using a multifunctional fluorescence labeling microscope BZ-X700 (KEYENCE), and the number of green fluorescent cells (CD8 + T cells).

[0238] (c) Statistical processing

[0239] The results are expressed as mean and standard error (SE), and significant differences were investigated using Student's t-test. The significance level was set at **P<0.01.

[0240] (e) Test results

[0241] like Figure 5 As shown in (A), intraperitoneal administration of EGC-M5 to colon cancer model mice increased the expression of perforin and Grzb genes, which are cytotoxic substances produced by cytotoxic T cells (CTLs), in spleen cells. In addition, administration of EGC-M5 confirmed that CD8 + Significant accumulation of T cells (CTL), CD8 + The number of T cells increased significantly after EGC-M5 administration ( Figure 5(B)). This result suggests that EGC-M5 may activate CTLs and promote cell infiltration into tumor tissues, thereby increasing the attack power of CTLs against cancer cells (enhancing cellular immunity).

[0242] Industrial applicability

[0243] The present dendritic cell activating agent, the present pharmaceutical product, the present supplement, or the present food and drink promotes differentiation into canonical dendritic cells that initiate or promote adaptive immune responses and activates canonical dendritic cells, and is therefore useful in the field of prevention or treatment of cancer or infectious diseases.

Claims

1. A dendritic cell activator, characterized in that Contains 5-(3,5-dihydroxyphenyl)-γ-valerolactone represented by formula (I) as an active ingredient, [Chemical Formula 1] In formula (I), the stereo configuration of the wavy line may be either R configuration or S configuration.

2. The dendritic cell activator according to claim 1, characterized in that Used to promote differentiation into canonical dendritic cells (cDCs).

3. The dendritic cell activator according to claim 2, characterized in that The promotion of differentiation into canonical dendritic cells (cDCs) is accompanied by the activation of canonical dendritic cells (cDCs).

4. The dendritic cell activator according to claim 2, characterized in that Promote CD4 + T cells differentiate into Th1 cells and inhibit their differentiation into Th2 cells.

5. The dendritic cell activator according to claim 2, characterized in that Promote CD8 + T cells differentiate into cytotoxic T cells (CTLs), enhancing their cytotoxic activity.

6. The dendritic cell activator according to claims 1 to 5, characterized in that The dendritic cell activator is an enhancer of the acquired immune system.

7. The dendritic cell activator according to claim 6, characterized in that The acquired immune system is cellular immunity.

8. The dendritic cell activator according to claim 6, characterized in that The dendritic cell activator is an anticancer agent.

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