KR20110080984A - Immunocyte proliferation inhibitors comprising mung bean extract - Google Patents

Abstract

PURPOSE: An inhibitor for immunocyte proliferation, containing mung bean extract is provided to suppress IL-2 and IFN-gamma secretion and to prevent and treat immune diseases. CONSTITUTION: An inhibitor for immunocyte proliferation contains mung bean extract as an active ingredient. The mung bean extract is isolated by a solvent selected from the group consisting of water, alcohol of C1-4 carbon atoms, ethyl acetate, chloroform, ether, hexane, and dichloromethane. The immune diseases are autoimmune diseases or graft rejection of organ or tissue. The autoimmune disease includes rheumatoid arthrisis, systemic lupus erythematosus, diabetes, atopic dermatitis, psoriasis, smallpox, asthma, aphthous stomatitis, chronic fibrous thyroiditis, ulcerative colitis, polymyositis, autoimmune encephalomyelitis, and fibrofascitis.

Description

Immunocyte proliferation inhibitors comprising mung bean extract}

The present invention relates to an immune cell proliferation inhibitor containing mung bean extract, and more particularly to a composition for treating and preventing immune diseases comprising mung bean extract as an active ingredient.

In order for the immune response to the antigen to occur, T lymphocytes receive appropriate activation signals by antigen-presenting cells [Smith-Garvin et al., 2009], and sufficient effector through continuous cell proliferation. T lymphocytes should be produced. When an antigen presenting cell first recognizes a substance derived from outside, it expresses a major histocompatibility complex (MHC) and co-simulatory signal molecules containing an antigen. These two main signals stimulate the native T lymphocytes to proceed to the active phase. First, the antigen / MHC and TCR / CD3 complexes of T lymphocytes combine to deliver the primary stimulation signal [Dembic et al. ., 1986; Saito and Germain, 1987]. Signaling through the TCR / CD3 complex was performed by ITAM [Reth. 1989] through the phosphorylation of a variety of activation signals are transmitted downward (Imboden and Stobo, 1985). CD28, a representative co-simulatory signal molecule, is present in T lymphocytes and binds to CD80 and CD86 of antigen presenting cells to transmit stimulatory signals to T lymphocytes [Acuto and Michel, 2003]. Bound CD28 / CD80, CD86 induce activation of PI3K in T lymphocytes [Pages et al., 1994] and finally induce the activity of signals such as Akt, NF-kB [Narayan et al., 2006; Qiao et al., 2008]. When the necessary stimulus signals are delivered to T lymphocytes, T lymphocytes secrete various functions of cytokines (IL-2, IL-4, IFN-gamma, etc.) together with rapid cell proliferation, which are activated to induce immune responses. Appleman et al., 2000.

Based on this mechanism of immune response, immunosuppressive methods that interfere with each signaling system have been studied. Method of suppressing antigen transmission by antigen presenting cells, signaling process of T lymphocytes (Calcineurin-Ca ²⁺ signal) or intermediate signaling material, inhibition of cytokine function which is important for T lymphocyte activity, cells of T lymphocytes Methods for suppressing immunity have been used in various ways, such as inhibition of proliferation through cleavage.

However, currently used immunosuppressive agents have high cost and long-term side effects. At the present time, as organ transplantation and autoimmune diseases, which are used for immunosuppressive agents, are increasing, it is necessary to develop low-cost immunosuppressive agents that can overcome the existing problems.

Therefore, the present inventors have made diligent research efforts to overcome the problems of the prior art, and when the mung bean extract is treated to cells that activated immune cells, it inhibits IL-2 secretion and IFN-γ secretion ability of T lymphocytes. It was confirmed that the present invention can be completed.

Therefore, the main object of the present invention is to provide an extract of mung bean having the effect of inhibiting the IL-2 secretion and IFN-γ secretion ability of T lymphocytes involved in the immune response.

Another object of the present invention to provide a composition for treating and preventing immune diseases using the mung bean extract.

According to one aspect of the invention, the present invention provides a composition for the treatment and prevention of immune diseases comprising mung bean extract as an active ingredient.

In the composition of the present invention, the mung bean extract is preferably extracted with one or more solvents selected from the group consisting of water, alcohol having 1 to 4 carbon atoms, ethyl acetate, chloroform, ether, hexane and dichloromethane.

The mung bean extract of the present invention is characterized by inhibiting IL-2 secretion and IFN-γ secretion ability of T lymphocytes. In a specific embodiment of the present invention, when quantitatively measured IL-2 and IFN-γ expressed and secreted from immune cells, the secretion amount of IL-2 and IFN-γ is reduced by treatment of the mung bean extract of the present invention. Confirmed.

In the composition of the present invention, the immune disease may be any disease caused by activation of T cells, and specifically, an autoimmune disease or an organ or tissue transplant rejection response.

The "autoimmune disease" of the present invention is a disease in which immune cells respond to host tissues and self-response to endogenous magnetic peptides, and are caused by abnormalities in systems regulating autoimmune responses. Collectively, a disease that occurs due to the activation of T cells in response to an antigen. T cells or B cells are randomly differentiated to have various specificities. In this process, cells that can be activated by specifically binding to self antigens can be generated, which is called self-recognition. Immune tolerance exists in the immune system of the human body to prevent self-awareness, and autoimmune diseases occur when immune tolerant in the body fails and the self-recognized immune cells are activated. Autoimmune diseases are caused by genetic factors (improper MHC expression), internal / external antigens, cytokine dysregulation, disruption of immunosuppressive functions, and organ defects.

The "organ or tissue transplant rejection response" of the present invention is a disease caused by a cell mediated immune response. The human body has a complex defense mechanism against foreign substances such as bacteria or viruses invading the body. These mechanisms that make up the immune system cannot distinguish disease-causing microorganisms from life-saving transplanted cells, and both of them are regarded as foreign substances and are challenged by the immune system to cause transplant rejection. T cell mediated responses are initiated when the recipient's lymphocytes meet the donor's MHC. In other words, immunization begins after host T cells meet branched cells in the transplanted organ or donor branched cells enter the recipient's lymph nodes. Activated CD4 T cells secrete cytokines during delayed hypersensitivity and vascular permeability. Increase the level and cause local infiltration of mononuclear cells such as lymphocytes and macrophages, and microvascular damage, tissue ischemia and graft destruction occur by the infiltrated macrophages. However, grafts may be safe if T lymphocytes cause inflammation and damage to the graft over several days or months and such cell mediated responses are suppressed.

In the composition of the present invention, the autoimmune disease is rheumatoid arthritis, systemic scleroderma, systemic lupus erythematosus, diabetes mellitus, atopic dermatitis, alopecia areata, psoriasis, asthma, asthma, aphthous stomatitis, chronic thyroiditis, ulcerative colitis, multiple myositis , Multiple sclerosis, autoimmune hemolytic anemia, autoimmune encephalomyelitis, fibromyalitis, temporal arteritis, and the like.

According to another aspect of the present invention, the present invention provides a method for inhibiting proliferation of immune cells, comprising treating mung bean extracts with immune cells in vitro, comprising the following steps.

a) separating RMA cells from the subject;

b) cell culturing the RMA cells;

c) treating the cultured cells with PMA and ionomycin;

d) treating the cultured cells mung bean extract.

PMA, ionomycin material of step c) is a material that stimulates T cells to activate the T cells act as the activation mechanism of the T cells by the actual antigen to create an environment in which an immune response occurs.

The composition for treating and preventing immune diseases of the present invention may be prepared by a method known in the pharmaceutical field for use as a medicament, and may be mixed with pharmaceutically acceptable carriers, excipients, diluents, etc., in powders, granules, tablets, and capsules. It may be prepared and used in formulations such as medicaments or injections. They can also be administered parenterally (eg, intravenously, subcutaneously, intraperitoneally or topically) or if administered.

The dosage of the therapeutic agent according to the present invention may be appropriately selected according to the age, sex, weight, health condition, symptoms of the disease, administration time, administration method of the patient, preferably 0.01 to 100 mg per day per adult May be administered.

1 shows chromatogram results of mung bean extract.
2 shows cytokine expression transcriptional measurement results of T lymphocytes.
Figure 3 shows the result of measuring the secretion amount of IL-2 expressed and secreted from immune cells.
4 shows the results of measuring the secretion amount of IFN-γ expressed and secreted from immune cells.

Hereinafter, the present invention will be described in more detail with reference to Examples. Since these examples are only for illustrating the present invention, the scope of the present invention is not to be construed as being limited by these examples.

Example 1 Extraction Method and Extract Treatment Method of Mung Bean Extract

Mung beans used in the present invention were purchased from aquaculture products, and used for extraction after grinding the mung beans.

First, 1 L of n-hexane was added to 200 g of mung bean powder, and extracted with a reflux condenser for 3 hours to remove fat. After recovering the dried mung bean powder from which fat was removed, 1 L of 100% ethanol was added to perform secondary reflux extraction for 3 hours. After completing the extraction process, the sample was concentrated under reduced pressure at 40 ° C. or lower, and then lyophilized to powder. Then, the mung bean extract was separated using Diaion HP-20 (Mitsubishi chemicals, Japan) resin. After extracting reflux with ethanol (2 g) to the column filled with Diaion HP-20 resin (bed volume 400 ml) and adsorbed, 0, 20, 40, 60, 80, 100% ethanol was added to the step gradient mobile phase Elution was carried out at a rate of 8 ml / min, and 50 ml were collected using a fraction collector (EYELA DC-1500, Japan). The collected aliquots were analyzed for elution patterns at various wavelengths (210, 254, 280, 330, 450 nm), and finally chromatograms were obtained based on the absorption pattern at 254 nm. The chromatogram obtained is shown in FIG. 1.

As shown in Figure 1, mung bean extract shows the elution pattern of several fractions, in the present invention actually used a mung bean extract of F1 (fraction 1).

First, F1 mung bean extract was dissolved in PBS containing 10% DMSO (sigma), and then treated with cells by taking 1/100 of the total culture volume, and finally, a 37 ° C. incubator containing 5% CO ₂ with 0.1% DMSO. Incubated for 3 days. The cells were isolated from RMA (Lian et al., 1998), a mouse T lymphocyte cell line, and placed in a 96 well round tissue culture plate in 1 × 10 ⁵ / well cells in RPMI medium containing 10% calf serum. Incubated. In order to stimulate the extracted RMA cells in vitro, 50 ng / ml of PMA (sigma) and 500 ng / ml of ionomycin (sigma) were simultaneously treated. The two substances are signals that stimulate T cells to be activated. They act as an activation mechanism of T cells by actual antigens, creating an environment in which an immune response has occurred. You can see if it plays a role.

Example 2 Inhibition of Cytokine Expression Transcription of T Lymphocytes by Mung Bean Extract Treatment

Inverse electron chain polymerization (RT-PCR) was used to investigate the effects of mung bean extract on the cytokine expression transcription of T lymphocytes.

First, as shown in Example 1, after treatment with Mung bean extract on RMA cells, 200 μl of chloroform (Chloroform, sigma) was added per 1 ml of trizol (Trizol, invitrogen), and total RNA was isolated. The extracted mRNA (2 μg) was carried out by RT-PCR as a template to replicate the cDNA. Subsequently, primers were prepared according to the nucleotide sequences obtained from the gene database using the cDNA as a template, and each gene was obtained by using a chain polymerization (PCR). The prepared primers are shown in Table 1 below.

Table 1. Primers

Thereafter, each PCR product was loaded on 1.5% agarose gel (invitrogen) containing EtBr (sigma) to confirm cytokine expression transcription. At this time, the reverse transcriptase was used Superscript III (invitrogen), the polymerase was used Tag polymerase (gene craft). The cytokine expression transcription results are shown in FIG. 2.

As shown in Figure 2, it can be observed that the amount of transcription of IL-2 and IFN-γ is reduced in the RMA cell line, a mouse T lymphocyte cell line cultured in a medium containing mung bean extract, even when treated with PMA and ionomycin. Significant differences are also observed in comparison with (GAPDH). The GAPDH is a gene whose expression level does not change well depending on the conditions of the cell, and was used as an intracellular reference in the cytokine expression level investigation of T lymphocytes of the present invention.

Example  3. ELISA  Used IL -2 suppression of secretion capacity

To determine the secretion capacity of IL-2 by immune cells treated with mung bean extract, an indirect sandwich ELISA method was used. The culture of the cells was carried out under the same conditions as in Example 1, and analyzed three days after the culture with the culture (supernatant).

First, a capture antibody (anti-mouse IL-2, BD Bioscience) was coated on an ELISA plate, and the culture solution was added and reacted at 4 ° C. for 12 hours. To identify bound cytokines, IL-2 expressed and secreted from immune cells was quantitatively measured using biotinylated anti-mouse IL-2 (BD Bioscience) and SA-HRP (Jackson ImmunoResearch Inc.). The measurement results are shown in FIG. 3.

As shown in FIG. 3, it can be observed that the amount of IL-2 secretion in the RMA cell line, which is a mouse T lymphocyte cell line, cultured in a medium containing mung bean extract, also decreased when treated with PMA and ionomycin.

Example  4. ELISA  Used INF -γ secretion ability

In order to determine the secretion capacity of IFN-γ of immune cells by mung bean extract treatment, an indirect sandwich ELISA method was used. The culture of the cells was carried out under the same conditions as in Example 1, and analyzed three days after the culture with the culture (supernatant).

First, a capture antibody (anti-mouse IFN-γ, BD Bioscience) was coated on an ELISA plate, and the culture solution was added and reacted at 4 ° C. for 12 hours. In order to identify bound cytokines, IFN-γ expressed and secreted from immune cells was measured quantitatively using biotinylated anti-mouse IFN-γ (BD Bioscience) and SA-HRP (Jackson ImmunoResearch Inc.). The measurement results are shown in FIG. 4.

As shown in FIG. 4, it can be observed that the secretion amount of IFN-γ in the RMA cell line, which is a mouse T lymphocyte cell line, cultured in a medium containing mung bean extract, also decreased when treated with PMA and ionomycin.

As described above, according to the present invention, it is possible to inhibit IL-1 secretion and IFN-γ secretion ability of T lymphocytes using mung bean extract. Therefore, the mung bean extract can be used to inhibit the proliferation of immune cells, and is expected to be used as a composition for treating and preventing immune diseases caused by autoimmune diseases or immune diseases such as transplantation rejection of organs or tissues.

Claims (6)

Immune disease treatment and prevention composition comprising mung bean extract as an active ingredient.
The method of claim 1, wherein the mung bean extract is treated with water, alcohol having 1 to 4 carbon atoms, ethyl acetate, chloroform, ether, hexane and dichloromethane, characterized in that extracted with one or more solvents selected from the group consisting of Composition.
According to claim 1, wherein the mungbean extract is a composition for the treatment and prevention of immune diseases, characterized in that the suppression of IL-2 secretion and IFN-γ secretion ability of T lymphocytes.
According to claim 1, wherein the immune disease autoimmune disease or organ disease or tissue transplant rejection response, characterized in that the composition for the treatment and prevention of immune diseases.
The method of claim 4, wherein the autoimmune diseases include rheumatoid arthritis, systemic scleroderma, systemic lupus erythematosus, diabetes mellitus, atopic dermatitis, alopecia areata, psoriasis, asthma, asthma, aphthous stomatitis, chronic thyroiditis, ulcerative colitis, multiple myositis, Multiple sclerosis, autoimmune hemolytic anemia, autoimmune encephalomyelitis, fibromyalitis, temporal arteritis comprising a composition for treating and preventing immune diseases.
A method for inhibiting immune cell proliferation comprising treating mung bean extract to immune cells in vitro.

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