KR20110128542A - Composition for preventing or treating sjogren's syndrome, behcet's syndrome, ankylosing spondylitis or systemic lupus erythematosus comprising egcg as an active ingredient - Google Patents

Abstract

PURPOSE: A pharmaceutical composition containing EGCG(epigallocatechin gallate) is provided to suppress differentiation of cytotoxic Th17 cells and to prevent or treat Sjogren syndrome, Behcet syndrome, or lupus. CONSTITUTION: A composition for preventing or treating Sjogren syndrome, Behcet syndrome, or lupus contains EGCG(epigallocatechin gallate) compound or salt thereof as an active ingredient. The EGCG suppresses or reduces differentiation of undifferentiated T cells into Th17 cells. A method for activating regulatory T cells comprises a step of treating the EGCG compounds or salt thereof to the T cells(Treg) in vitro. A functional food for treating or preventing Sjogren syndrome, Behcet syndrome, or lupus contains the EGCG compound or salt thereof as an active ingredient. A composition for suppressing immunity contains the EGCG compounds or salt thereof as an active ingredient.

Description

Composition for preventing or treating Sjogren's syndrome, Behcet's syndrome, Ankylosing spondylitis or Systemic lupus erythematosus comprising EGCG as an active ingredient}

The present invention relates to a pharmaceutical composition containing EGCG (epigallocatechin gallate) as an active ingredient, and more particularly to a composition for preventing or treating Sjogren's syndrome, Behcet's syndrome, ankylosing spondylitis or lupus containing EGCG as an active ingredient. will be.

T cells are one of a group of cells that play a central role in the immune system as a biological defense system against various pathogens. T cells are produced in the thymus of the human body and undergo a series of differentiation processes to differentiate into T cells with unique characteristics.T cells, which have completed differentiation, are largely divided into type 1 auxiliary cells (Th1) and type 2 It is divided into helper cells (Th2). Among them, the main function of Th1 cells is involved in cell mediated immunity, Th2 cells are involved in humoral immunity, and in the immune system, these two cell populations are balanced with each other so that they are not activated with each other.

Therefore, most of the immune diseases can be attributed to the imbalance between these two immune cells, for example, abnormally increased activity of Th1 cells can cause autoimmune diseases, and abnormally increased activity of Th2 cells It is known that immune diseases occur due to hypersensitivity reactions.

Meanwhile, according to a recent study on the differentiation of Th1 cells, the existence of a new group of immunoregulatory T cells (Tregs), which can control Th1 cell activity, is known, and studies on the treatment of immune diseases using the same are emerging. In addition, since Treg cells inhibit the function of abnormally activated immune cells to control the inflammatory response, many experiments have been reported to treat immune diseases and inflammatory diseases by increasing the activity of Treg cells.

In addition to the Treg cells, another group made during the differentiation is Th17 cells, which are known to be formed through a process similar to the differentiation of Treg cells during the differentiation of undifferentiated T cells. That is, differentiation of Treg cells and Th17 cells is performed in the presence of TGF-β in common but does not require IL-6 in Treg cells, whereas IL-6 is present in combination with TGF-β in Th17 cells. Differentiate in situations In addition, differentiated Th17 cells are characterized by the secretion of IL-17.

Unlike Treg cells, Th17 cells have been found to be involved in the forefront of inflammatory reactions seen in immune diseases, maximizing the signal of inflammatory responses and accelerating disease progression. Therefore, in the case of autoimmune diseases which are not controlled by Treg cells among autoimmune diseases, development of therapeutic agents for autoimmune diseases that target the inhibition of Th17 cell activity has been highlighted.

Currently used immunosuppressive drugs are immunosuppressants that block signal transduction pathways in T cells. These immunosuppressive agents are toxic, infection, lymphoma, diabetes, tremor, headache, diarrhea, high blood pressure, and nausea. There is a problem that side effects such as renal failure occur.

In addition to the treatment of immune diseases by inhibiting the activation of T cells, a treatment for controlling the amount of cytokines secreted from immune cells and a therapeutic method using antibodies targeting cytokines secreted from immune cells have been developed. There is. However, this method has to take a long time to apply to the patients after the actual clinical trials, the method using the antibody has a problem that too much cost in the antibody manufacturing process.

Therefore, there is a need for the development of new immunological and inflammatory diseases treatment agents that have no side effects and are inexpensive and have excellent therapeutic effects.

Accordingly, an object of the present invention is to provide a composition for preventing or treating Sjogren's syndrome, Behcet's syndrome, ankylosing spondylitis or lupus, which contains an EGCG compound or a salt thereof as an active ingredient so as to effectively prevent or treat a disease caused by an abnormal immune response. It is.

Another object of the present invention is to provide a method for reducing or inhibiting the differentiation of undifferentiated T cells into Th17 cells in vitro, comprising treating EGCG with undifferentiated T cells.

Another object of the present invention is to provide a method of activating regulatory T cells comprising the step of treating EGCG with Regulatory T cells (Treg) in vitro.

Another object of the present invention is to provide a functional health food for the improvement or prevention of Sjogren's syndrome, Behcet's syndrome, ankylosing spondylitis or lupus symptoms containing an EGCG compound or a salt thereof as an active ingredient.

Furthermore, another object of the present invention is to provide an immunosuppressive composition containing an EGCG compound or a salt thereof as an active ingredient.

In order to achieve the above object, the present invention provides a composition for the prevention or treatment of Sjogren's syndrome, Behcet's syndrome, ankylosing spondylitis or Lupus disease, containing an EGCG (epigallocatechin gallate) compound or a salt thereof as an active ingredient.

In one embodiment of the present invention, the EGCG may be to reduce or inhibit the differentiation of undifferentiated T cells into Th17 cells.

In one embodiment of the present invention, the EGCG may be to promote or increase the activity or amplification of Regulatory T cells (Treg).

The present invention also provides a method of reducing or inhibiting the differentiation of undifferentiated T cells into Th17 cells in vitro, comprising treating undifferentiated T cells with an EGCG (epigallocatechin gallate) compound or salt thereof. .

In one embodiment of the present invention, the reduction or inhibition of differentiation of undifferentiated T cells into Th17 cells may be achieved through inhibition of production of IL-17 cytokines.

The present invention also provides a method for activating regulatory T cells comprising treating an EGCG (epigallocatechin gallate) compound or a salt thereof to Regulatory T cells (Treg) in vitro.

In one embodiment of the invention, the activated regulatory T cells may be to increase the expression of Foxp3.

The present invention also provides a functional health food for the improvement or prevention of Sjogren's syndrome, Behcet's syndrome, ankylosing spondylitis or Lupus disease, containing an EGCG (epigallocatechin gallate) compound or a salt thereof as an active ingredient.

Furthermore, the present invention provides an immunosuppressive composition containing an EGCG (epigallocatechin gallate) compound or a salt thereof as an active ingredient.

The EGCG compound according to the present invention has an excellent effect of inhibiting the differentiation of cytotoxic Th17 cells that produce and secrete inflammatory cytokines, immunomodulation having the characteristics of inhibiting the function of abnormally activated immune cells and controlling the inflammatory response. T cells (Tregs) are excellent in activating effects and can be used as pharmaceutical compositions and immunosuppressive agents that can prevent or treat Sjogren's syndrome, Behcet's syndrome, ankylosing spondylitis or lupus caused by abnormal regulation of immune responses. There is an effect that can be used as a composition for food that can improve and prevent the.

1 is a graph showing the results of measuring the secretion amount of IL-17 cytokines produced after treatment of splenocytes isolated from mice under Th17 differentiation conditions and EGCG at each concentration. to be.
Figure 2 in one embodiment of the present invention, the treatment of splenocytes isolated from the mouse with Th17 differentiation conditions and at the same time treated with EGCG at each concentration, the expression of IL-17 and Foxp3 expressed (mRNA) RT- As a result confirmed by PCR, 2a is the result of measuring the amount of IL-17 mRNA, 2b is the result of measuring the amount of Foxp3 mRNA.
Figure 3 shows the amplification of Th17 and Treg cells differentiated in the spleen tissue of each mouse in the EGCG-treated and untreated group of Lupus syndrome mice for CD4, CD25, Foxp3, IL-17 After immunostaining using the antibody, it shows a picture observed through a confocal microscope.
Figure 4 is stimulated splenocytes isolated from loops syndrome mice in the Treg differentiation conditions and at the same time the expression level of Foxp3 and Tregs that are expressed after EGCG treatment immunostained with antibodies to CD4, CD25, Foxp3 flow cells It shows the results observed through the analyzer (FACs, fluorescent-activated cell sorter).

The present invention provides a composition for the prevention or treatment of Sjogren's syndrome, Behcet's syndrome, ankylosing spondylitis or Lupus disease, which contains EGCG, that is, (-)-epigallocatechin gallate ((-)-epigallocatechin gallate) as an active ingredient. It has its features.

EGCG, the active ingredient of the composition according to the present invention, is a kind of polyphenols among the various active ingredients contained in the tea tree belonging to the fruit (茶 科), the main ingredient with the highest activity and is responsible for the effect of green tea. It is known as a substance. The EGCG has two triphenolic groups in its structure, which is known to play a powerful pharmacological action (Matsuo, N. et al., Allergy, 52 (1997) 58-64). The pharmacological action of EGCG is known to have mainly strong antioxidant action (Guo.G. et al., Biochim . Biophys , Acta , 1304 (1996) 210-222), antibacterial action and anti-mutant action.

In addition, Korean Patent Laid-Open Publication No. 2002-55735 discloses the use of EGCG as a preventive or therapeutic agent for neuronal cell damage to transient whole cerebral ischemia, and Korean Patent No. 449228 discloses EGCG prepared by reacting nicotinic acid with EGCG. Also disclosed is a cosmetic composition using a derivative of, and US Patent No. 633357 discloses an anticancer agent including epigallocatechin gallette and cocoa extract. It is known to be used. However, none of these prior documents mentions that EGCG can be used for the treatment of Sjogren's syndrome, Behcet's syndrome or Lupus disease.

Therefore, in the present invention, the EGCG compound can be used to prevent or treat Sjogren's syndrome, Behcet's syndrome, ankylosing spondylitis or lupus, and in particular, it inhibits the differentiation of Th17 cells in which EGCG is a pathogenic cell and promotes the activity of immunoregulatory T cells. It was first identified that the mechanism to induce a prophylactic or therapeutic effect of the disease.

On the other hand, the immune system controls specific immune responses to autoantigens under normal conditions, and also suppresses immune responses against external antigens. For example, the pregnant woman's response to the fetus and the chronic infection state Reaction. These phenomena are known to be induced by clonal deletion, clone anergy and active control by immunoregulatory T cells (Treg) as a mechanism by which antigen specific immunotolerance can be induced. Investigating some patients who accidentally acquired immunotolerance against transplanted antigens or experimentally induced animal models showed that all three of these mechanisms are involved in immunological tolerance. Is attracting attention as an important cell involved in controlling almost all immune responses of living body such as autoimmune, tumoral immunity, infectious immune response as well as transplantation immune response.

The immunoregulatory T cells, ie immunoregulatory T lymphocytes (Tregs), which have recently been found to be present, can be largely divided into natural and adaptive Treg cells, and CD4 + CD25 + T cells, which are natural Tregs, are referred to as thymus It is given immunosuppressive function when it is newly created in, and is present in 5 ~ 10% of peripheral CD4 + T lymphocytes of normal individuals. The mechanism of immunosuppression of this cell is not yet known, but it has recently been discovered that the expression control factor of the gene, Foxp3, plays an important role in the differentiation and activity of the cell. In addition, peripheral natural T cells can be differentiated into cells that exhibit immunosuppressive effects upon stimulation of autologous or external antigens under certain circumstances, which are called adaptive or inducible Tregs and secrete IL-10. These include Tr1, Th3 and CD8 Ts that secrete TGF-β.

In addition, as described in the prior art, T cells also differentiate into Th17 cells through differentiation in addition to Treg cells. Th17 cells are made in the presence of TGF-β in common with Treg cells, but in the case of Treg cells, IL While it does not require -6, Th17 cells are characterized by differentiating in the presence of IL-6 with TGF-β and secreting IL-17.

In addition, Th17 cells are characterized by having cytotoxicity that maximizes the signal of the inflammatory response to accelerate disease progression. Therefore, inhibition of differentiation or activity into Th17 cells is one of the ways to treat immune diseases.

In this regard, the present inventors confirmed that the EGCG compound of the present invention can treat immune or inflammatory diseases by regulating T cells, specifically, differentiating T cells into Treg cells and Th17 cells. According to one embodiment of the present invention, the splenocytes isolated from the mouse are treated with EGCG compounds at different concentrations and at the same time as the treatment conditions for stimulating undifferentiated T cells present in the spleen can differentiate into Th17 cells. Then, the amount of IL-17 cytokine expressed in the cell was measured. In general, the inflammatory cytokine IL-17 is produced from Th17 cells and can measure the differentiation of undifferentiated T cells into Th17 cells through the amount of IL-17 produced.

As a result, the control group without EGCG was differentiated into Th17 cells by Th17 cell differentiation conditions, and the production of IL-17 cytokines was significantly increased compared to the group without any treatment. In the group, the production of IL-17 cytokines was found to be markedly reduced compared to the group not treated with EGCG. In addition, the amount of IL-17 cytokine produced was shown to decrease in proportion to the concentration of treated EGCG (see FIG. 1).

Therefore, the present inventors have found that the EGCG compound reduces or inhibits the differentiation of undifferentiated T cells into Th17 cells, which are pathogenic cells producing IL-17, an inflammatory cytokine.

In addition, according to another embodiment of the present invention, in order to investigate the effect of the EGCG compound on the activity or proliferation of immunoregulatory T cells (Treg) spleen cells isolated from the mouse to stimulate the conditions that can be differentiated into Th17 cells The EGCG compound was treated with each concentration and cultured at the same time, and then the degree of amplification or activity of differentiated Treg cells was determined by measuring the amount of expressed Foxp3, which is an immunoregulatory T derived from the thymus. As a transcriptional factor mainly present in Regulatory T cells and present in cells with CD4 + CD25 + labeled antigens, its function is low reactivity to antigens upon antigen recognition against T cells expressing Foxp3. Among the CD4 + CD25- T cells that do not express Foxp3 differentiated from the thymus at the same time, they may be used for T cells that can potentially cause autoimmunity. Therefore, it has a role as a suppressor T cell that suppresses IL-2 production and cell division. In addition, Foxp3 is not only IL-2, but also IL-4, which is influenced by the transcription factor NFAT on CD25-T cells through regulatory T cells expressing Foxp3 and cell-cell contact therewith. It has been shown to function to inhibit transcriptional regulation such as IFN-gamma. Therefore, in the case of T cells expressing Foxp3 having such a function, it is applied to the treatment of immune diseases through the action of inhibiting or regulating the immune response, and furthermore, the CD4 T cells expressing Foxp3 present in humans. To apply self-antigen specific T cell clones to cell therapy by increasing their number through high concentration of IL-2 cytokine and combination treatment with anti-CD3 and anti-CD28 antibodies There have been attempts. Therefore, confirming the expression of Foxp3 is an indicator to measure the activity or amplification of Treg cells.

As a result, in the EGCG-treated group, the mRNA level of Foxp3 expressed in Treg cells was significantly increased compared with the EGCG-treated group, and the increase was proportional to the concentration of EGCG treated. (See Figure 2).

Therefore, through the above results, the present inventors found that the EGCG compound acts to inhibit or reduce the differentiation of undifferentiated T cells into Th17 cells and to activate or amplify regulatory T cells (Treg). Could know.

Therefore, EGCG of the present invention can prevent or treat diseases related to immunity or inflammation through the action of promoting or increasing the activity or amplification of Regulatory T cells (Tregs) and / or inhibiting the differentiation into Th17 cells, which are pathogenic cells. There are features that can be.

In the present invention, the immune or inflammation-related diseases that can be prevented or treated by the pharmacological action of EGCG may preferably be Sjogren's syndrome, Behcet's syndrome, ankylosing spondylitis or lupus disease.

The Sjogren's syndrome is a chronic autoimmune disease in which lymphocytes penetrate into the exocrine glands that secrete liquids out of the human body, resulting in reduced saliva and tear secretion, which are characteristic of dry mouth and dry eye, resulting in abnormalities in the immune system protecting the human body from the outside. The attack on one's body appears. The cause of Sjogren's syndrome is genetic cause, abnormal immune response to infection, autonomic nervous system disorder, hormonal abnormality. . Currently, the method of treating Sjogren's syndrome is in the treatment of exocrine gland symptoms, that is, by using substitutes such as artificial tears, saliva, vaginal lubricants, etc. to reduce the discomfort of the patient and cyclosporine in addition to artificial tears. Eye drops are used to improve dry eye symptoms, and in severe cases, procedures such as tear point ablation or external eyelid sutures may be performed. Another treatment is the treatment of symptoms outside the exocrine glands, which use nonsteroidal anti-inflammatory drugs or antimalarial drugs used for joint pain and muscle pain.

In addition, Behcet's syndrome is a chronic inflammatory disease that can involve various organs such as skin, blood vessels, gastrointestinal tract, central nervous system, heart and lung, in addition to oral ulcers, genital ulcers, and ocular symptoms. It is vasculitis. The exact cause of Behcet's syndrome is not yet known, but it has been known for a long time that the immune response is activated by the addition of environmental factors in patients with genetic predisposition, resulting in various symptoms. Therefore, by suppressing excessive immune response and at the same time reducing the inflammatory response can prevent or treat the symptoms of Behcet's syndrome.

In addition, the lupus, called systemic lupus erythematosus, is a chronic autoimmune disease that occurs mainly at young age, including women of childbearing age, and the immune system that protects the body from the outside causes abnormalities and rather attacks the human body. This causes inflammatory reactions throughout the body, including the skin, joints, kidneys, lungs, and nerves. Lupus goes through a chronic course, and symptoms may worsen and relieve over time. These lupus are known to be caused by a combination of several genes, hormones and environmental factors, but the specific relationship is still unclear. Lupus, on the other hand, does not yet have a cure, so it suppresses excessive immune responses and only reduces or prevents symptoms of the disease.

 In addition, in the case of ankylosing spondylitis, the rigidity means a change in the joints after a long period of inflammation to slow the movement of the joints, spondylitis is an illness in which the spine is inflamed. Therefore, ankylosing spondylitis can be said to be an 'inflammation and slow motion of the spine'. Ankylosing spondylitis is the most common disease in the group called 'serum-negative spondyloarthropathy', in which the rheumatoid factor is negative. Onset. Osteoarthritis is characterized by inflammation of the ligaments and tendons that attach to the bone, such as the heel and forebone. In addition to joints, other organs such as the eyes, gastrointestinal tract, lungs, heart, kidneys and prostate gland can be affected. Currently, exercise therapy, surgery and immunosuppressive agents are used for the treatment of ankylosing spondylitis.

As described above, Sjogren's syndrome, Behcet's syndrome, ankylosing spondylitis and lupus disease are diseases caused by abnormalities of the immune system, particularly excessive immune response or inflammatory response. The disease can be prevented or treated.

Therefore, EGCG according to the present invention is capable of activating or amplifying Regulatory T cells (Tregs) that can normally regulate excessive immune responses, and at the same time, have an excellent activity of inhibiting Th17 cell differentiation, which secretes inflammatory cytokines. This can prevent or treat Sjogren's syndrome, Behcet's syndrome, ankylosing spondylitis or lupus.

In the present invention, the activity of the regulatory T cells means that all the mechanisms of the regulatory T cells (Treg), ie, Treg cells including both natural and adaptive Treg cells in vivo. It is meant to be promoted or promoted, which means that an immunomodulatory action such as an immunosuppressive reaction is promoted or promoted so that an immune response in a living body is maintained in a normal state.

In addition, the term "expansion" refers to the differentiation and proliferation of undifferentiated T cells into regulatory T cells, 'differentiation' refers to a phenomenon in which structures or functions are specialized while the cells divide and grow, In other words, the shape or function of a cell or tissue of an organism is changed in order to perform a given task, and 'proliferation' refers to the division of cells and the expansion of homogeneous ones. It means to increase.

Therefore, the present invention can provide a composition for preventing or treating Sjogren's syndrome, Behcet's syndrome, ankylosing spondylitis or Lupus disease containing EGCG as an active ingredient.

In addition, the EGCG according to the present invention may be epigallocatechin gallate represented by Formula 1 below (Epigallocatechin gallate, EGCG, gallate-3-epigallocatechin).

<Formula 1>

The EGCG compounds according to the invention can also be used in the form of salts, preferably pharmaceutically acceptable salts. The salt is preferably an acid addition salt formed by a pharmaceutically acceptable free acid, and an organic acid and an inorganic acid may be used as the free acid. The organic acid is not limited thereto, citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, fumaric acid, formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, metasulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, Glutamic acid and aspartic acid. In addition, the inorganic acid includes, but is not limited to, hydrochloric acid, bromic acid, sulfuric acid and phosphoric acid.

The EGCG compounds according to the present invention can be used that are isolated from nature or prepared by chemical synthesis known in the art.

Preferably, those obtained from nature can be used using a method of extracting and separating conventional substances, and more preferably those separated from green tea can be used. However, the EGCG compounds which can be used in the present invention are not limited to those obtained from the above method. In one embodiment of the present invention was used to buy what is sold in the market.

As a suitable solvent that can be used to extract the EGCG compound from nature, water or an organic solvent may be used. Preferably, purified water, methanol, ethanol, propanol, isopropanol, Butanol, acetone, ether, benzene, chloroform, ethyl acetate, methylene chloride, hexane and cyclohexane Various solvents of can be used alone or in combination.

In addition, the separation and purification of the EGCG compound of the present invention from the extract obtained by the above method is a method known in the art, for example, column chromatography filled with various synthetic resins, such as silica gel (silica gel) or activated alumina (alumina) ( column chromatography) and high performance liquid chromatography (HPLC) may be used alone or in combination. However, the extraction and separation and purification method of the active ingredient is not necessarily limited to the above method.

On the other hand, Sjogren's syndrome, Behcet's syndrome, ankylosing spondylitis or lupus disease, in which the EGCG compound according to the present invention can induce a therapeutic effect, is a disease belonging to an immune disease or an inflammatory disease, wherein the "immune disease" is a component of the mammalian immune system. A disease that causes, mediates or otherwise contributes to the pathology of a mammal. In addition, stimulation or interruption of the immune response refers to a disease having a compensatory effect on the progression of the disease, and in the present invention, particularly refers to diseases caused by an overactive immune response.

In addition, one of the most important traits of all normal individuals is that they do not deleteriously react with the antigenic substances that make up self, while non-self antigens can recognize and react to eliminate them. Have the ability to The non-response of the body to autoantigens is called immunologic unresponsiveness or tolerance.

However, when there is a problem in inducing or maintaining self-tolerance, an immune response occurs to autoantigens, and this causes an attack on one's own tissue. The disease caused by this process is called an "autoimmune disease". do.

In addition, "inflammatory disease" refers to tumor necrosis factor-α (TNF-α) and IL-1 (excessively stimulated by the immune system such as macrophages due to excessive stimulation of the human immune system due to harmful stimuli such as inflammation-induced factors or irradiation). interleukin-1), IL-6, prostaglandin (prostagladin), luecotriene or nitric oxide (nitric oxide, NO) refers to a disease caused by proinflammatory substances (inflammatory cytokines).

Therefore, the composition according to the present invention can be used as a pharmaceutical composition capable of preventing or treating Sjogren's syndrome, Behcet's syndrome, ankylosing spondylitis or lupus disease.

The term 'treatment', unless stated otherwise, means to reverse, alleviate, inhibit, or prevent the progression or alleviation of the disease or condition to which the term applies, or one or more symptoms of the disease or condition. As used herein, the term 'treatment' refers to the act of treating when 'treating' is defined as above. Thus, in mammals, the "treatment" or "therapy" of Sjogren's syndrome, Behcet's syndrome, ankylosing spondylitis or lupus may comprise one or more of the following:

(1) inhibits the growth of Sjogren's syndrome, Behcet's syndrome, ankylosing spondylitis or lupus, ie, inhibits its development,

(2) prevent the spread of Sjogren's syndrome, Behcet's syndrome, ankylosing spondylitis or lupus,

(3) relieves Sjogren's syndrome, Behcet's syndrome, ankylosing spondylitis or lupus,

(4) prevent recurrence of Sjogren's syndrome, Behcet's syndrome, ankylosing spondylitis or lupus, and

(5) Palliating the symptoms of Sjogren's syndrome, Behcet's syndrome, ankylosing spondylitis or lupus.

The composition for preventing or treating Sjögren's syndrome, Behcet's syndrome, ankylosing spondylitis or lupus according to the present invention comprises a pharmaceutically effective amount of an EGCG compound or a salt thereof alone or in combination with one or more pharmaceutically acceptable carriers, excipients or diluents. It may include. The pharmaceutically effective amount herein refers to an amount sufficient to prevent, ameliorate and treat the symptoms of Sjogren's syndrome, Behcet's syndrome, ankylosing spondylitis or lupus.

The pharmaceutically effective amount of the EGCG compound or salt thereof according to the present invention is 0.5 to 100 mg / day / kg body weight, preferably 0.5 to 5 mg / day / kg body weight. However, the pharmaceutically effective amount may be appropriately changed depending on the degree of Sjogren's syndrome, Behcet's syndrome, ankylosing spondylitis or lupus symptoms, the patient's age, weight, health condition, sex, route of administration and duration of treatment.

In addition, the pharmaceutically acceptable refers to a composition that is physiologically acceptable and does not cause an allergic reaction such as gastrointestinal disorders, dizziness or the like when administered to humans. Examples of such carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, Polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In addition, fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers and preservatives may be further included.

In addition, the pharmaceutical compositions of the present invention may be formulated using methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. The formulations may be in the form of powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatine capsules, sterile injectable solutions, sterile powders.

In addition, the composition for preventing or treating Sjogren's syndrome, Behcet's syndrome, ankylosing spondylitis or lupus in accordance with the present invention can be administered via various routes including oral, transdermal, subcutaneous, intravenous or intramuscular, the dosage of the active ingredient being administered It can be appropriately selected according to various factors such as the route, age, sex, weight of the patient and the severity of the patient, and the composition for preventing or treating Sjogren's syndrome, Behcet's syndrome, ankylosing spondylitis or lupus according to the present invention is Sjogren's syndrome, Behcet It may be administered in combination with known compounds having the effect of preventing, ameliorating or treating the symptoms of syndrome, ankylosing spondylitis or lupus.

Accordingly, the present invention can provide a medicament for preventing or treating Sjogren's syndrome, Behcet's syndrome, ankylosing spondylitis or lupus, comprising a pharmaceutical composition containing an EGCG compound or a salt thereof as an active ingredient, and the present invention further provides an EGCG compound or The composition for immunosuppression containing the salt as an active ingredient can be provided.

In addition, the present invention can provide a method for reducing or inhibiting the differentiation of undifferentiated T cells into Th17 cells in vitro, comprising treating an EGCG compound to undifferentiated T cells, wherein the undifferentiated T cells Reduction or inhibition of differentiation into Th17 cells can be achieved through inhibition of production of IL-17 cytokines.

In addition, the present invention may provide a method for activating regulatory T cells comprising the step of treating an EGCG compound in a regulatory T cell (Treg) in vitro, and activating according to the present invention. Said regulatory T cells have increased expression of Foxp3.

The method of reducing or inhibiting the differentiation of undifferentiated T cells into Th17 cells in vitro and the method of activating regulatory T cells in the in vitro according to the present invention is a method for treating a cell with an EGCG compound. The EGCG compound may be directly treated in the culture medium, or the composition containing the EGCG compound according to the present invention as an active ingredient may be treated in the culture medium. In this case, the concentration of the EGCG compound that can be treated in the culture medium may be treated so that the final concentration is 1uM ~ 100uM, in one embodiment of the present invention cells so that the concentration of the EGCG compound is the final 1uM, 10uM and 50uM Was added to the culture medium.

Furthermore, the composition for preventing or treating Sjogren's syndrome, Behcet's syndrome, ankylosing spondylitis or lupus according to the present invention is excellent in the activity or amplification of immune regulatory T cells (Treg), and differentiates Th17 cells, which are pathogenic cells producing inflammatory cytokines. In addition to the excellent effect of inhibiting, there is no toxicity and side effects for the drug can be used safely even when taking long-term, especially the EGCG compound which is an active ingredient contained in the composition of the present invention is mostly in natural plants, such as green tea It exists, and when using the EGCG compound obtained from nature, there is a characteristic more stable to the body.

Therefore, the present invention can provide a food composition which can improve or prevent the symptoms of Sjogren's syndrome, Behcet's syndrome, ankylosing spondylitis or lupus containing an EGCG compound or a salt thereof as an active ingredient, the composition for food according to the present invention Silver can be easily utilized as foods that are effective in improving or preventing Sjogren's syndrome, Behcet's syndrome, ankylosing spondylitis or lupus symptoms, such as main ingredients, side ingredients, food additives, functional foods or beverages.

As used herein, the term “food” refers to a natural product or processed product containing one or more nutrients, and preferably means a state in which it can be directly eaten through a certain processing step, It includes all foods, food additives, functional foods and drinks.

Foods to which the food composition according to the present invention may be added include, for example, various foods, beverages, gums, teas, vitamin complexes, functional foods, and the like. In addition, in the present invention, food includes special nutritional products (e.g., formulated milk, young, infant food, etc.), processed meat products, fish products, tofu, jelly, noodles (e.g. ramen, noodles, etc.), bread, health supplements, seasonings. Foods (e.g. soy sauce, miso, red pepper paste, mixed soy sauce), sauces, confectionery (e.g. snacks), candy, chocolates, gums, ice creams, dairy products (e.g. fermented milk, cheese, etc.), other processed foods, kimchi, Pickled foods (various kimchi, pickles, etc.), beverages (e.g., fruit drinks, vegetable drinks, soy milk, fermented beverages, etc.), natural seasonings (e.g. ramen soup, etc.) are not limited thereto. The food, beverage or food additive may be prepared by a conventional production method.

In addition, the term "functional food" refers to the control of biological defense rhythms and disease prevention of food groups or food compositions that have added value to the food by using physical, biochemical, or biotechnological techniques to act and express the function of the food for a specific purpose. It means a food that is designed and processed to fully express the body's regulatory function regarding recovery and the like, and specifically, it may be a health functional food. The functional food may include food acceptable food additives, and may further include appropriate carriers, excipients and diluents commonly used in the manufacture of functional foods.

In addition, in the present invention, the "beverage" refers to a generic term for drinking to quench thirst or to enjoy a taste and includes a functional drink. The beverage is not particularly limited in addition to the composition for improving or preventing the Sjogren's syndrome, Behcet's syndrome, ankylosing spondylitis or lupus symptoms as an essential ingredient in the indicated proportions and various flavors or Natural carbohydrates and the like may be included as additional components.

Furthermore, in addition to the above, food containing a composition for food for the improvement or prevention of Sjogren's syndrome, Behcet's syndrome, ankylosing spondylitis or lupus symptoms of the present invention may contain various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural Flavoring agents such as flavoring agents, colorants and fillers (such as cheese and chocolate), pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonated drinks The carbonation agent used may be contained, and the above components may be used independently or in combination.

In the food containing the composition for food of the present invention, the amount of the composition according to the present invention may comprise from 0.001% to 90% by weight of the total food weight, preferably from 0.1% to 40% by weight In the case of a beverage, it may be included in a ratio of 0.001g to 2g, preferably 0.01g to 0.1g based on 100ml, in the case of long-term intake for health and hygiene purposes or health control purposes It may be less than the above range, and the active ingredient is not limited to the above range because it may be used in an amount above the above range because there is no problem in terms of safety.

Hereinafter, the present invention will be described in more detail with reference to Examples. It will be apparent to those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not limited to these embodiments.

< Example  1>

Th17 In a cell EGCG On by IL -17 Production Inhibitory Activity Assay

The present inventors investigated whether the EGCG compound is active in inhibiting the production of the inflammatory cytokine IL-17 in Th17 cells, which are pathogenic cells. For this, first, spleen cells were obtained from the mouse, that is, the spleen extracted from the mouse was finely chopped spleen tissue using a glass slide, and then red blood cells in the spleen were removed with a erythrocyte hemolysis solution. Splenocytes were obtained by adding PBS buffer solution and washing by centrifugation. Thereafter, 2 ug / mL of anti-CD3 antibody, 1 ug / mL of anti-CD28 antibody, 10 ug / mL of anti-IFNr antibody, and anti-IL-4 were conditions for stimulating Th17 cells. After 10 ug / mL of the antibody and 2 ng / mL of TGF beta were simultaneously treated, the cells were cultured for 3 days, and the cells were cultured by treating the cells with concentrations of 0 uM, 1 uM, 10 uM, and 50 uM, respectively. Then, after obtaining a culture of the cells incubated for 3 days, the amount of IL-17 cytokine present in the culture was measured by ELISA method known in the art.

As a result, as shown in FIG. 1, IL-17, an inflammatory cytokine produced from Th17 cells, which are pathogenic cells, was found to be inhibited by the EGCG compound of the present invention. It has been shown that production is reduced.

< Example  2>

PCR  Through the way Treg  In a cell EGCG Impact analysis

Controlled T lymphocytes, or Treg (regulatory T lymphocytes) cells, are known to play a key role in the maintenance of immune tolerance (Nat. Rev. Immunol. 2009, 7: 305) and are produced primarily in the thymus and are transcription factors Expression of Foxp3 is required and Foxp3 is known to play a role in inhibiting the production of inflammatory cytokines. In order to investigate whether the EGCG compound according to the present invention has an action of activating the activity of Treg cells which are immunoregulatory cells, the present inventors stimulated spleen cells of mice with Th17 cell stimulation conditions of Example 1, The expression levels of IL-17 and Foxp3 expressed from the cells, ie, mRNA, were measured by reverse transcriptase polymerase reaction. That is, EGCG was treated with 0 ng / ml, 1 ng / ml, 10 ng / ml and 50 ng / ml for each cell under Th17 differentiation conditions, and then RNA was purified from the cells according to methods known in the art. After each extraction, polymerase chain reaction was carried out using primers capable of amplifying Foxp3 and IL-17. The reaction compound for RT-PCR was 25 μl in total, and the composition of the solution was 2.5 μl of 10 × reaction. Each primer pair having a buffer, 0.5 mM dNTP and the sequence shown in Table 1 was used, and amplification was performed at 94 ° C. 30 seconds in the denaturation step and 60 ° C. 30 seconds at the annealing step using a Dual-bay Thermal cycler system instrument. , The reaction was repeated 25 times for 30 seconds at 72 ℃.

 Primer sequence primer Primer sequence SEQ ID NO: IL-17 sense 5’-CCACTCTGTAGCACCCCAATG-3 ’ One IL-17 antisense 5’-CCTGGAGATGTAGCCCTGGTC-3 ’ 2 Foxp3 sense 5’-ATGCCTCCTCTTCTTCCTTGA-3 ’ 3 Foxp3 antisense 5’-TGAGAAGGGCAGGGCACAAT-3 ’ 4

As a result, as shown in FIG. 2, the expression of IL-17 was found to gradually decrease in concentration-dependent manner by the treatment of EGCG, while the expression of Foxp3 was gradually increased in concentration-dependent manner by the treatment of EGCG.

Therefore, through the above results, the present inventors confirmed that the EGCG compound according to the present invention suppresses cell differentiation into Th17, which is a pathogenic cell, whereas the expression of Foxp3 is increased, thereby regulating EGCG as an immunoregulatory Treg. It has been found that the action of activating cells can prevent or treat inflammatory diseases such as Sjogren's syndrome, lupus syndrome, ankylosing spondylitis and Behcet's syndrome.

< Example  3>

For animal models EGCG Impact analysis

Furthermore, the present inventors more clearly confirmed the effect of EGCG confirmed in Example 2 with respect to an animal model, that is, after dividing the mouse group causing lupus syndrome into two groups, one group of EGCG was identified. Intraperitoneal injections were made 10 times at a concentration of 10 uM, and the other group was one that was not injected with EGCG as a control. After administration, spleen tissues were isolated from each mouse group in the same manner as in Example 1, and then compared with Th17 cell group and Treg cell group for immunoassay with CD4 and IL-17 antibodies to confirm Th17 cell differentiation. In order to confirm Treg cells, CD4, CD25 and Foxp3 antibodies were immunostained and observed by confocal microscopy.

As a result, as shown in Figure 3, when treated with EGCG in the Lupus syndrome mouse group, it was confirmed that the Treg cells were activated in the spleen, that is, the red stained CD4 and blue stained CD25 is expressed as purple In the nucleus, green stained Foxp3 was expressed more than the control group. On the other hand, when the degree of Th17 cell differentiation was measured, green IL-17 was expressed more than the group treated with the EGCG-treated control group.

Therefore, the present inventors have found that the EGCG of the present invention has the effect of activating Treg cells in the tissues of Lupus syndrome while reducing the differentiation of Th17 cells, which are pathogenic cells. It was found that lupus syndrome can be prevented or treated.

< Example  4>

EGCG On by Treg  Increase effect of cells

More specifically, in order to confirm the effect of increasing the Treg cells by the EGCG of the present invention, the present inventors obtained splenocytes in the same manner as in Example 1 from the mice of Lupus syndrome, and then Treg cells were differentiated by treatment with 2 ug / ml of CD3 antibody and 5 ng / ml of TGF beta. At this time, EGCG was added at a concentration of 10 ng / ml, followed by incubation for 72 hours. As a control, those without addition of EGCG were used. In order to evaluate whether there is a difference in the Treg cell population by EGCG after the culture, immunostaining with antibodies to CD4, CD25, Foxp3 by a method known in the art, and then a fluorescence-activated cell sorter (FACs) Treg cell populations were analyzed.

As a result, as shown in Figure 4, when treated with 10ng / ml EGCG, Foxp3 expression was 21.57, while the untreated control group was 2.69. These results indicate that the treatment of EGCG can increase Treg cells by about 10 times or more compared to the untreated group.

So far I looked at the center of the preferred embodiment for the present invention. Those skilled in the art will appreciate that the present invention can be implemented in a modified form without departing from the essential features of the present invention. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the scope will be construed as being included in the present invention.

<110> Industry Academic Cooperation Foundation of Catholic University <120> Composition for preventing or treating Sjogren's syndrome,          Behcet's syndrome, Ankylosing spondylitis or systemic lupus          erythematosus comprising EGCG as an active ingredient <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> IL-17 sense primer <400> 1 ccactctgta gcaccccaat g 21 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> IL-17 antisense primer <400> 2 cctggagatg tagccctggt c 21 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Foxp3 sense primer <400> 3 atgcctcctc ttcttccttg a 21 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> Foxp3 antisense primer <400> 4 tgagaagggc agggcacaat 20

Claims (9)

A composition for preventing or treating Sjogren's syndrome, Behcet's syndrome, ankylosing spondylitis or lupus containing an EGCG (epigallocatechin gallate) compound or a salt thereof as an active ingredient. The method of claim 1,
The EGCG is a composition for preventing or treating Sjogren's syndrome, Behcet's syndrome, ankylosing spondylitis or lupus, characterized in that to reduce or inhibit the differentiation of undifferentiated T cells into Th17 cells. The method of claim 1,
The EGCG is a composition for preventing or treating Sjogren's syndrome, Behcet's syndrome, ankylosing spondylitis or lupus, characterized in that it promotes or increases the activity or amplification of Regulatory T cells (Treg). A method of reducing or inhibiting differentiation of undifferentiated T cells into Th17 cells in vitro, comprising treating undifferentiated T cells with an EGCG (epigallocatechin gallate) compound or salt thereof. The method of claim 4, wherein
Reduction or inhibition of differentiation of undifferentiated T cells into Th17 cells is achieved through the inhibition of production of IL-17 cytokines, which reduces or inhibits the differentiation of undifferentiated T cells into Th17 cells in vitro. Way. A method of activating regulatory T cells comprising treating an EGCG (epigallocatechin gallate) compound or salt thereof in a regulatory T cell (Treg) in vitro. The method of claim 6,
Activated regulatory T cells are a method of activating regulatory T cells, characterized in that the expression of Foxp3 is increased. Functional health food for the improvement or prevention of Sjogren's syndrome, Behcet's syndrome, ankylosing spondylitis or Lupus disease, containing an EGCG (epigallocatechin gallate) compound or a salt thereof as an active ingredient. An immunosuppressive composition comprising an EGCG (epigallocatechin gallate) compound or a salt thereof as an active ingredient.

Images