KR20050083369A - A proteoglycan, which is increased in immuno-activity of dendritic cells, from phellinus linteus and immunostimulator containing thereof - Google Patents

Abstract

The present invention relates to a protein polysaccharide (proteoglycan) that enhances the immune activity of dendritic cells from Phellinus linteus and an immunopotentiator containing the same as an active ingredient, more specifically, denaturation and dendritic cells of dendritic cells Protein polysaccharides of situational mushrooms to increase the expression of the surface factor of the cytokine, secretion of cytokine interleukin (IL) -12 of dendritic cells, proliferative capacity of T cells of dendritic cells and production of IL-2 from T cells, and It relates to the use as an immunopotentiator as an active ingredient.

Description

A proteoglycan, which is increased in immuno-activity of dendritic cells, from Phellinus linteus and Immunostimulator containing amounts, which increase the immune activity of dendritic cells.

The present invention relates to a protein polysaccharide that enhances the immune activity of dendritic cells from a situation mushroom and an immunopotentiator using the same. More specifically, the maturation of dendritic cells and the expression of surface factors of dendritic cells and cytokine IL- of dendritic cells It relates to proteinaceous polysaccharides and immunopotentiators of situational mushrooms to increase the secretion of 12, the proliferative capacity of T cells of dendritic cells and the production of IL-2 from T cells.

Dendritic cells are the most potent antigen-presenting cells and are the only cells that can induce a primary cellular immune response. They originate in the bone marrow and travel to all organs through the bloodstream in immature forms. Dendritic cells collect surrounding antigens from each tissue and go to lymphoid organs to present antigens to T lymphocytes. Immature dendritic cells are unable to activate T cells because they do not express CDs necessary for the transmission of accessory signals such as cluster determinant (CD) 40, CD54, and CD86. These undifferentiated dendritic cells are capable of phagocytosis, macropinocytosis, and macrophage mannose receptors, similar to the C-type lectin receptor, DEC-205 (CD ₂ 0). ₅ ) And receptors that mediate adsorptive endocytosis such as Fcγ and Fcε receptors are well expressed in cell membranes. Therefore, these differentiated dendritic cells can respond to nanomolar or picomolarity antigens, whereas other antigen-presenting cells respond to micromolar antigens. In the case of macrophages, the phagocytic antigen is degraded from the lysosome to the amino acid state and expresses a small amount of the major histocompatibility complexv-peptide conjugate on the cell surface. At a minimum, a sufficient amount of MHC-peptide conjugates can be expressed on the cell surface to remain stable for several days. As dendritic cells mature, an immune response is induced, which is regulated by several factors. In particular, bacteria and inflammatory products, polysaccharides that are cell wall components of bacteria, IL-1, granulocyte / macrophage-colony stimulating factor (TNF), and tumor necrosis factor (TNF)-promote all the dendritic cell maturation. Mature dendritic cells express high concentrations of NF-κB (nuclear transcription factor) -κB) transcription factors (Rel A / p65, Rel B, Rel C, p50, p52), which are responsible for various immune responses and inflammation It regulates the expression of the genes of the proteins involved in the reaction. Signaling through TNF-receptor families such as TNF-R (CD120a / b), CD40 and TRANCE / RANK (TNF-related activation induced-cytokine / receptor activator of NF-kB) activates NF-κB By stimulating dendritic cells to induce an immune response, pathogens or antigens are involved in the signaling pathways of TNF-R or TNF-R-associated factors (TRAFs). Therefore, when inducing and differentiating leukemia cells to dendritic cells, one dendritic cell is usually 100 to 3,000 by presenting antigens to T cells by expressing antigens related to their leukemia on the mass cell surface due to the characteristics of dendritic cells. Activates T cells in dogs. If each of these T cells is triggered by clonal expansion in the form of autocrine growth stimulation, it may induce anti-leukemic T-cell responses that are highly therapeutic. In fact, dendritic cells derived from GM-CSF, IL-4, and TNF-α from peripheral blood leukemia cells in patients with chronic myelogenous leukemia (CML) and acute myeloid leukemia (AML) may be anti-leukemic T cells. It has been reported to promote cell proliferation and enhance the cytotoxicity of these T cells.

Most current research focuses on dendritic cells activating T cells, but dendritic cells not only elicit an immune response to foreign antigens, but also immune tolerance of T cells similar to T cells tolerating their own antigens. ) May also result. Many tumor patients rarely elicit T cell responses specific to tumor antigens, probably because dendritic cells do not respond to tumor cells. Dendritic cells infiltrated with colorectal cancer or basal cell skin cancer are unable to express CD80 and CD86, thereby reducing the stimulatory activity of T cells. Therefore, maturation of dendritic cells and activation of cell surface factors of dendritic cells are absolutely necessary factors for immune promotion, including potent anti-tumor effects.

The most commonly used chemotherapy for the treatment of cancer patients is not only difficult to obtain satisfactory therapeutic effects such as complete eradication and metastasis prevention of cancer, but also serious side effects when the effective therapeutic dose is administered. Recently, in order to assist chemotherapy, immunochemotherapy has been attempted in animal models and cancer patients. Various biological response modifiers (BRMs), such as cytokines and bacterial products that can control the biological response of the host, have been used in immunochemotherapy of cancer patients. However, the genetically modified cytokines (IL-2, IL-12, and IFN (interferon) -γ), which are currently used for immunotherapy of cancer patients, are severely toxic and have a short half-life in vivo. In order to overcome the side effects due to hematopoietic cell death during the therapy and radiation therapy, the colony stimulating factor (colony stimulating factor) also has the disadvantage of proliferating only granulocytes and mononuclear cells. Therefore, in order to overcome these side effects, the development of new antitumor natural products and private medicines used in folk remedies are getting positive and successful results in foreign countries due to the effects on natural tumors and their economic effects. Recently, a systematic review of many natural products known as antitumor or various biological response modifiers has been actively conducted in Korea. It is attempting to develop new anticancer drugs using various polysaccharides such as lentinan, schizophyllan and low toxicity, and OK-432, and also induce anticancer cytokine production in vivo. Phospholipids such as edelfosine (ET18-OCH ₃ ) and substances that can increase immunity have been reported to have excellent anti-cancer effects, and studies of such phospholipids analogs have been actively conducted.

In the present invention, an object of the present invention is to provide a protein polysaccharide that enhances immune activity from Phellinus linteus . In other words, the situation-derived protein polysaccharides include denaturation of dendritic cells, expression of surface factors of dendritic cells, secretion of cytokine IL-12 of dendritic cells, proliferative capacity of T cells of dendritic cells and IL- from T cells. Confirmed to increase the production of 2 and confirmed the effect of enhancing the immune activity.

Another object of the present invention is to provide an immunopotentiator using the protein polysaccharide from Phellinus linteus as an active ingredient.

Therefore, the infrared spectrophotometric spectrum, ¹ H nuclear magnetic resonance spectrum, ¹³ C nuclear magnetic resonance spectrum of the substance which isolates and purifies the substance which enhances the immune activity of dendritic cells from the situation mushroom and enhances the immune activity of purely isolated dendritic cells. As a result of the consideration of polysaccharide, amino acid component analysis, etc., it was identified as a protein polysaccharide containing a large amount of amino acid having an acidic group. Therefore, to prepare a protein polysaccharide to enhance the immune activity of dendritic cells from Phellinus linteus and to develop an immune enhancer containing it as an active ingredient.

The present invention is to remove the precipitate obtained by treating the mushrooms with hot water, treated with ethanol and then centrifuged to dissolve the precipitate obtained by centrifugation, and then dialyzing only the supernatant and freeze-drying to separate the purified product by DEAE-cellulose chromatography and gel filtration chromatography. Protein polysaccharide to increase the immune activity of dendritic cells, characterized in that the extract comprises 41.6 mol% of mannose, 23.4 mol% of galactose, 23.8 mol% of glucose, 5.8 mol% of arabinose and 4.2 mol% of gyros. It is about.

The present invention also relates to an immunopotentiator using the protein polysaccharide as an active ingredient.

This invention will be described in more detail with reference to the following examples. However, the scope of the present invention is not limited to the Examples.

Example 1: Isolation and Purification of Protein Polysaccharides from Mushrooms

300 g of the fruiting mushroom fruit body was extracted 6 times in boiling water for 30 minutes. The extracted solution was allowed to stand in three volumes of 95% ethanol at 4 ° C. for 4 days and then centrifuged to recover the precipitate. The recovered precipitate was dissolved in distilled water, centrifuged at 10,000Xg for 20 minutes, and then the supernatant was obtained by dialysis for 4 days and lyophilized. The lyophilized purified product was dissolved in tertiary distilled water, and subjected to DEAE-cellulose anion exchange resin column (3x45 cm) chromatography. 2 M NaCl was used at a concentration gradient of 0.3 mL / min. Analysis by salicylic acid (dinitrosalicylic acid, DNS) method confirmed that the sugar contained in 0.3M-0.5M NaCl elution fraction (Fig. 1). The sugar fractions were collected and concentrated, followed by Sepharose CL-4B gel filtration column (1.5 × 10 ⁵ cm) chromatography (Sigma). At this time, distilled water was developed at a flow rate of 0.5 ml / min, corresponding to a molecular weight of about 150,000. The sugar-containing fractions were separated and purified from the eluted volume. In this case, in order to prepare a standard curve for molecular weight measurement, blue dextran (blue dextran, molecular weight 2,000,000), dextran D5251 (molecular weight 473,000), textran D1062 (molecular weight 41,272) (Sigma) was used (Fig. 2).

Example 2 Analysis of Protein Polysaccharides Isolated from Strain Mushrooms

The protein polysaccharide isolated from the situation mushroom in Example 1 was investigated by using the DNS method and the Bradford (bradford) method to examine the content of sugar and protein, respectively. As a result, the sugar was about 72.5%, and the protein was confirmed to be 22.3% protein polysaccharide (Table 1). The isolated protein polysaccharide was acid hydrolyzed at 110 ° C. for 1 hour using 2M trifluoroacetic acid (TFA), and they were then subjected to HPLC (Waters, Sugar-Pak column, 7.8 × 300 mm), and 30% aceto. Nitrile was developed as a developing solvent at a flow rate of 1 ml / min and analyzed using an ELSD detector (evaporative light scattering detector, Waters). As a result, it was confirmed that the situation mushroom protein polysaccharide contains a large amount of mannose, galactose, and glucose (Table 2). Polysaccharides isolated from mushrooms were acid-hydrolyzed in 6N HCl for 1 hour and then analyzed for constituent amino acids using a Biochrom 20 Amino acid analyzer (BD). As a result, aspartic acid, threonine, serine, glutamic acid, glycine, and alanine were identified in large amounts (Table 3).

In order to confirm the structure of protein polysaccharides, the absorption wavelengths of their infrared spectrophotometric spectra (FT-IR) were compared with each other using beta glucan (Sigma) of Pleurotus ostreatus as a standard (FIG. 3a, 3b), as in beta glucan (β-glucan) was able to identify the typical sugar ring, and many flexural structures were confirmed. In addition, in order to see their sugar binding patterns, ¹ H nuclear magnetic resonance spectra, ¹³ C nuclear magnetic resonance spectra were measured (Figs. 4A, 4B), the protein polysaccharides isolated from the situation mushrooms sugar binding α- and β- Two types of coexistence could be confirmed, and much of the methylation was confirmed.

Party(%) protein(%) Protein Polysaccharides 75.2 22.3

Contents Mannose Galactose Glucose Arabinose Xylose Content (mol%) 41.6 23.4 23.8 5.8 4.2

Amino acid Content (mol%) Aspartic acid 9.0 Throneine 6.9 Serine 11.8 Glutamic acid 11.1 Glycine 12.0 Alanine 12.7 Cysteine 0.7 Valine 5.1 Methionine 1.0 Isoleucine 2.9 Leucine 5.6 Tyrosine 1.6 Phenylalanine 8.4 Histidine 1.2 Lysine 4.2 Arginine 2.8 Proline 3.0

Example 3 Effect on Cell Surface Factor Expression of Dendritic Cells

Dendritic cells derived from C57BL / 6 mice were isolated and dispensed at a concentration of 1 × 10 ⁶ cells / ml, followed by incubation with GM-CSF and IL-4 at 10 ng / ml. After 1 day and 3 days of culture, unattached cells were washed and removed to increase the purity of dendritic cells. Bone marrow cells were cultured for 7 days and treated with the situation mushroom extract protein polysaccharide of Example 1 at the last 24 hours, FITC (Fluorescein Isothiocyanate) -CD11c, which is a specific surface factor of dendritic cells, and PE, a surface factor strongly expressed after maturation. R-Phycoerythrin) -CD80, PE-CD86, PE-MHC I, PE-MHC II were stained and analyzed using a flow cytometer.

To investigate the effect on the maturation of dendritic cells derived from C57BL / 6 mice, the surface factor changes of dendritic cells were investigated after treatment of lipopolysaccharide (LPS) with S. mushroom protein polysaccharide and control for 24 hours. As can be seen in Figure 5a, as a result of processing the situation mushroom protein polysaccharides, it can be seen that a variety of cell surface factors are very strongly expressed from dendritic cells. The results indicate that the situation mushroom polysaccharides strongly promote the maturation of dendritic cells. In addition, as shown in Figure 5b after treatment with polymyxin B (polymyxin B, PB), an LPS inhibitor, the cell surface factor was strongly expressed even after the extract. On the other hand, it was confirmed that this effect is strongly suppressed when PB is treated simultaneously with LPS. This is not a result of LPS contamination that may occur during the extraction of natural products, but a result that proves the effect of pure extract. In view of the above results, it was confirmed that the protein polysaccharide of the present invention matures dendritic cells, the most important cells that connect innate immunity and acquired immunity, without LPS contamination, thereby increasing the expression of surface factors.

Example 4 Effect on Cytokine Expression in Dendritic Cells

In order to measure the amount of IL-12 and IL-10 produced from dendritic cells, the cells were stained using Cytofix / cytoperm kit (PharMingen), and analyzed using a flow cytometer. In particular, IL-12 is known to be an important data for measuring the functional maturation of dendritic cells. For these experiments, dendritic cells derived from C57BL / 6 mice were first isolated and dispensed at a concentration of 1 × 10 ⁶ cells / ml, followed by incubation with GM-CSF and IL-4 at 10 ng / ml. After 1 day and 3 days of culture, unattached cells were washed and removed to increase the purity of dendritic cells. Bone marrow cells are incubated for 7 days, and the cytokine (IL-12, IL-10) secreted by dendritic cells is treated with Cytofix / Cytoperm kit in the last 24 hours. Analyzed.

As shown in FIG. 6, the situation mushroom polysaccharides increased IL-12 production to a level comparable to that of the positive control LPS, and had no effect on the expression level of IL-10. This could indirectly explain that the situation mushroom polysaccharides promote IL-12 of dendritic cells and induce functional maturation of these cells.

Example 5 Investigation of T Cell Proliferation of Dendritic Cells

To investigate how the expression of cell surface factors increased by the situational polysaccharides on the maturation of allogeneic T cells, the control group, the LPS-treated group, and the polysaccharide-treated group were compared. . Dendritic cells (1 × 104 cells) derived from bone marrow cells of C57BL / 6 mice were treated with 50 μg / ml mitomycin to block maturation and allow expression of surface factors only, followed by T of genetically different BALB / c mice. T cell proliferation was measured using 3 [H] incorporation. At the same time, the amount of IL-2 produced by these T cells was also measured using an enzyme linked immunosolvent assay (ELISA).

After treatment with the extract as shown in Figures 7a, 7b mature dendritic cells were found to strongly induce the proliferation of T cells (Fig. 7a). In addition, the production of IL-2 from T cells was significantly increased (FIG. 7B). Therefore, it was found that dendritic cells treated with these polysaccharides strongly induce T cell activation and enhance antitumor effects.

As described above, the present invention was confirmed that the protein polysaccharide isolated from Phellinus linteus is a substance that enhances the immune activity of dendritic cells. Therefore, it is possible to formulate an immunopotentiator using the protein polysaccharide as an active ingredient, and it is also possible to provide a type of immunopotentiator different from conventional immunopotentiators.

Figure 1 shows the DEAE-cellulose (diethylaminoethyl-cellulose) anion exchange resin chromatography results of the situation mushroom extract. ●: sugar concentration of fraction by DNS (2-hydroxy-3,5-dinitrosalicylic acid) method, ▲: NaCl concentration gradient.

Figure 2 shows the Sepharose CL-4B chromatography of the situation mushroom sugar fraction of the present invention.

Figure 3a shows the infrared spectrophotometric spectrum (FT-IR) of beta glucan derived from Pleurotus eryngii.

Figure 3b shows a situation mushroom-derived protein polysaccharide infrared spectrophotometric spectrum (FT-IR) of the present invention.

Figure 4a shows the ¹ H nuclear magnetic resonance spectrum of the situation mushroom-derived protein polysaccharide of the present invention.

Figure 4b shows a ¹³ C nuclear magnetic resonance spectra of the situation mushroom-derived protein polysaccharide of the present invention.

5a is a graph illustrating the change of surface factor expression of dendritic cells by treatment of situation mushroom protein polysaccharide ( Phellinus linteus , PL) of the present invention and LPS (lipopolysaccharide) as a control group to determine the effect of maturation of dendritic cells.

Figure 5b is a graph of the surface factor expression changes of dendritic cells treated with polymyxin B (polymyxin B, PB) after treatment of the situation mushroom protein polysaccharide (PL) and LPS of the present invention.

Figure 6 shows the effect of the situation mushroom protein polysaccharide of the present invention on cytokine production of dendritic cells.

Figure 7a shows the effect of the situation mushroom protein polysaccharide of the present invention on the T cell proliferation capacity of dendritic cells.

Figure 7b shows the effect of the situation mushroom protein polysaccharide of the present invention on increased production of IL-2 from T cells.

Claims (2)

The resultant mushrooms were extracted by hot water extraction, ethanol treatment, centrifugation to dissolve precipitates, centrifugation, dialysis and lyophilization of supernatant, and purification by DEAE-cellulose chromatography and gel filtration chromatography. Immune activity of dendritic cells, characterized in that 75.2%, protein content 22.3%, constituents of constituent sugar is 41.6 mol% mannose, 23.4 mol% galactose, 23.8 mol% glucose, 5.8 mol% arabinose and 4.2 mol% gyros. Situation of mushroom-derived protein polysaccharide. An immunopotentiator using the protein polysaccharide of claim 1 as an active ingredient.