KR20180071879A - Mushroom-garlic Extract for Hemocytopoiesis Function Enhancement and Radiation Protection - Google Patents

Abstract

The present invention relates to a composite composition having ingredients with reduction and removal of harmful active oxygen in a body and a protection effect of a living body by radiation and an immune activation enhancement effect in an extract obtained from mushroom-garlic as main ingredients, and a manufacturing method thereof and, more specifically, to a composite composition manufactured by mixing schizophyllan extracted from a culture medium of Schizophyllum commune and cycloalliin and allicin which are hot water extracts of garlic in optimal concentrations, a manufacturing method thereof, and a use thereof. The composite composition is made of natural materials selected from innoxuous plant materials to have less side effects and toxicity and better safety than conventional biochemical immunity-boosting medicines or body protection chemicals, suppresses oxidative body damage by chemicals and radiation, and enhances immune activation to be usefully used as radiation protection food, an anti-cancer therapeutic aid, or health supplement food.

Description

Mushroom-garlic Extract for Hemocytopoiesis Function Enhancement and Radiation Protection "

The present invention relates to a mushroom-garlic extract for enhancing hematopoietic function and radiation protection.

Radiation has been increasingly used in various fields, and research for the development of a radiation exposure prevention system that can overcome the risks of radiation damage caused by radiation workers has become an important social issue. Various ionizing radiation interacts with a substance in the process of permeating and absorbing the substance that constitutes the living body, causing unstable activity to the molecule or atom constituting the substance, resulting in abnormalities in cells or tissues due to changes in physical, And ultimately leads to biological barriers.

In physiological changes that occur in the living body during diagnosis and treatment using radiation, or in radiation workers, it is becoming a serious problem. In general, the essential condition of the radioprotective agent is that there is no action on normal tissues or negligibly small, and the protective effect on the tumor tissue is strong, so that the therapeutic or promoting effect should be clear.

In fact, the most important radiation protection agent is amifostin, which was developed in the US Army (Yuhas JM and Storer JB (1969): differentiae chemoprotection of normal and malignant tissue Jnatl cencer Inst 42: 331-335) The component is S-2 (3-aminoprophlamino) ethylphosphorothioic acid (hereinafter: WR-2721). The radiation protection effect of WR-2721 reported a DRR of 1.15 in the normal tissues of mice, with a 2.7% reduction factor (DRF) when using bone marrow as an index and a% transplantability as an index Since then, it has been noted that WR-2721, a thio-phosphate derivative of cysteamine, has low toxicity as a radiation-inhibiting agent and its clinical application is possible.

In fact, these radioprotective agents are usually in the form of free radical scavengers, or antioxidants, that remove unstable oxygen derivatives (ROS) generated in the body by irradiation, before they cause damage to the cells (WR-2721) has been reported to be the most effective anti-inflammatory agent in the animal studies. However, the adverse effects of the drug itself (nausea, vomiting, drowsiness, hypotension, hypocalcemia, , There have been many problems in practical generalization in terms of safety. However, it has been used clinically for low dose to reduce the incidence of dry mouth, esophagitis and pneumonia due to radiation in local radiation therapy of head and neck cancer patients or lung cancer patients. The use of post-exposure post-treatment type of prophylactic agents mainly used a mechanism such as bone marrow cell proliferation and immunity-enhancing action, but effective inhibitors have not been developed yet. Recently, to improve the defense effect and to reduce the side effects, There have been several studies using a combination of post-treatment and post-treatment. In the early days of development of radiation protection materials, the synthesis of thiol group-containing materials such as amifostin was focused. While this substance has the advantage of reducing the mortality rate, there are also some negative aspects such as drug toxicity. Therefore, the use of natural products that can exhibit antioxidant and immune enhancing effects without toxicity has attracted great interest (Drug Discovery Today. Volume 12, Numbers 19 / 20.October 2007). (KR 10-0519720, KR 10-0449655, KR 10-0440863) and Red Bit extract (Application No. 10-2014-0068468), which are known to have a traditional hematopoiesis. Among the plant-derived extracts, commercialized for use in the immunological enhancement and chemotherapy include schizophyllan, lentinan, grifolan, krestin (polysaccharide-peptide complex), mesima Mushroom mycelium), and β-Immunan (gingival mushroom) have been commercialized.

The present inventors isolated and purified a garlic mushroom extract composition having a more effective new immunity enhancement and radiation protection function and mixed them at various weight ratios to provide a higher safety than conventional biochemical immunity and radiation protection agents, And confirmed that it exhibits excellent radioprotective effect, thereby confirming that it is possible to comprehensively protect the main stem cells of the hematopoietic system and the regenerated tissue from radiation, etc. Thus, the present invention has been completed.

It is an object of the present invention to provide a non-toxic food extract which can be widely used for enhancing hematopoiesis and immune function, and which can be usefully used for preventing and overcoming radiation damage.

FIG. 1 shows the toxicity test results of mushroom, garlic, and mixed extracts through MTT assay in Raw 264.7 according to an embodiment of the present invention.
FIG. 2 shows the results of measurement of NO production by each extract in Raw 264.7 according to an embodiment of the present invention.
FIG. 3 shows the results of B cell activity confirmation of PEC (peritoneal leukocyte) according to an embodiment of the present invention.
Figure 4 shows an experimental schedule according to one embodiment of the present invention.
Figure 5 shows an experimental schedule and an experimental set of immunoenhancing effects of SF-051 in a gamma-irradiation mouse model, according to one embodiment of the present invention.
FIG. 6 shows the results of checking the number of WBCs and the level of immunoglobulins by SF-051 in a γ-irradiation mouse model according to an embodiment of the present invention.
Figure 7 shows the results of confirming the activity and population change of immune cells in Thymus and PEC of the y-irradiation mouse model according to one embodiment of the present invention.
Figure 8 shows the results of confirming the activity and population change of immune cells in the Spleen of the y-irradiation mouse model according to one embodiment of the present invention.
FIG. 9 shows the results of confirming the activity and population change of immune cells in the LN of the γ-irradiation mouse model according to one embodiment of the present invention.
FIG. 10 shows the results of confirming the activity and population change of immune cells in the MLN of the γ-irradiation mouse model according to one embodiment of the present invention.
Figure 11 shows the results of cell death and cell cycle change of immune cells in Spleen of a y-irradiation mouse model according to one embodiment of the present invention.
Fig. 12 shows the results of confirming gene expression changes of various cytokines in Spleen of a y-irradiation mouse model according to an embodiment of the present invention.

The samples were lyophilized, pulverized and extracted with a solvent. The recoveries were changed from 80.5% to 88.8% at 10 min and 70 min, respectively. The extraction pressure increased to 15.2 MPa as the pressure increased. Optimum conditions for the separation of the zyphophyll were 10.1 MPa, 70 min at 30 ℃. Hot water extraction using an infrared extractor was also performed at 95-100 ° C. The extraction time was different for each mushroom. In another method, ultrasonic extraction was performed by adding ultrasonic wave output of 350W at 5 ℃ of distilled water and extraction at 90 ℃ for 30 minutes. The extract was subjected to methanol extraction. Protein was removed by precipitation with 20% w / v TFA. After removal of the protein, methanol was added at a ratio of 2: 1 (v / v) to obtain a precipitate, and a concentrated NaCl solution was further added to precipitate the precipitate more easily. The precipitate was washed with acetone or the like. Photographs of mushroom raw materials and treated extracts are shown below.

The content of β-glucan was determined by subtracting α-glucan from total glucan. First, 100 mg of pulverized sample, which was filtered through 100 mesh sieve, was added to 1.5 ml of 37% HCl in a tube, and then decomposed in a 30 ° C water bath for 45 minutes. Then, 10 mL of distilled water was added and vortexed and incubated at 100 ° C for 2 hours. Then, 10 mL of 2 N KOH was added while cooling at room temperature, and 100 mL of the solution was mixed with 200 mM sodium acetate buffer. To 0.1 mL of the supernatant, add 0.1 mL of exo-1,3-β-glucanase plus β-glucosidase dissolved in 200 mM sodium acetate buffer, 0.2 mL of acetate buffer as a reagent blank, 0.1 mL of D- 0.1 mL of acetate buffer and incubate at 40 ° C for 60 minutes. 3 mL of glucose oxidase / peroxidase mixture (GOPOD) was added, incubated at 40 ° C for 20 minutes, and absorbance was measured at 510 nm. α-Glucan was prepared by mixing 100 mg of the pulverized sample with 100 mesh sieve, adding 2 mL of 2 M KOH, and mixing for 20 minutes. Add 8 mL of 1.2 M sodium acetate buffer and mix. Add 0.2 mL of amyloglucosidase plus invertase, mix well and incubate in a water bath at 40 ° C for 30 minutes. To 0.1 mL of the supernatant, 0.1 mL of 200 mM sodium acetate buffer and 3 mL of GOPOD were added, incubated at 40 ° C for 20 minutes, and then measured at 510 nm absorbance.

The mushrooms were analyzed according to the AOAC method. Moisture was determined by weighing 0.5 g of each sample in a weighing dish and drying it at 105 ° C in a dry oven until the weight became constant. The crude protein content was calculated by multiplying the nitrogen content measured by Kjeldahl method by the nitrogen factor of 6.25. The crude fat content was determined by Soxhlet extraction method, and crude fiber Were obtained by the Henneberg Stohmann modification method. The contents of soluble nitrogen - free water were calculated by subtracting contents of moisture, crude ash, crude protein, crude fat and crude fiber in the total amount.

Aliquots of garlic samples were analyzed by liquid chromatography and ultraviolet spectrophotometry at 208 nm using 80% methanol solution adjusted to pH 3.0 with formic acid. At this time, the column temperature was 30 ° C, and the mobile phase was gradient solution of sodium dihydrogenphosphate and sodium heptanesulfonate (50:80) and acetonitrile solution. The average allyl content was found to be 15.84.

In addition, the effectiveness of the beneficial ingredients was investigated through the functional test of the useful ingredients as follows.

-. In vitro treatment of extracts using cytotoxicity test

-. In vivo treatment of combination extracts

-. Reactive oxygen species (ROS) and NO measurements

-. The cytotoxicity assay (51Cr release assay)

-. Gene expression analysis

-. Flow cytometry analysis

-. Observation of cell cycle and apoptosis changes

The following is a review of representative results of the various functional test results. In order to confirm the toxicity of mushroom, garlic, and mixed extracts and to establish the optimum treatment concentration, MTT assay was performed 48 hours after treatment of each extract with concentration in Raw264.7. As a result, mushroom and mixed extracts except garlic showed toxicity at 50 μg / ml, and the concentration of each extract was fixed to EC50: 12.5 μg / ml. (Fig. 1). In Raw264.7, extracts of Raw264.7 were treated at different concentration and ROS (Reactive oxygen species) and LPS (500ng), which stimulates the production of NO, were investigated in order to confirm the reduction effect of inflammatory markers by mushroom, garlic, / ml) to measure the amount of ROS and NO. It was confirmed that the amount of ROS increased by LPS was slightly decreased in the group treated with mushroom 1, mushroom 2, garlic, and mixed 2, and the amount of NO was significantly decreased in all the extracts compared with the control group 2).

The in-vivo experimental design was carried out by oral administration of mushroom 1, mushroom 2, garlic, mixed 1, and mixed extract 2 at 20 μg, 100 μg and 500 μg per mouse every two weeks for 3 weeks. As a result of measuring the weight change of experimental mice for 3 weeks, it was confirmed that the toxicity by oral administration of the extract was not observed in all groups (Fig. 4). After 3 weeks, the mice were sacrificed and the organs (lung, liver, kidney, stomach, heart, large intestine) were separated and the weight and length were measured.

Next, the activity of the immune cells in the major organs of the mouse model in which each extract was orally administered was examined. CD4 (CD4 T cell marker), CD8 (CD8 T cell maker), CD11b (macrophage marker), CD69 (activation marker), and the like were obtained by isolating spleen, LN and MLN, And analyzed by flow cytometry. As a result, the number of CD4 T cells was increased by the extract of mushroom 1, mushroom 2 and mixed 2 in spleen, and the number of CD8 T cells was increased by mushroom 2, garlic and mixed 2. . All extracts from LN showed increased numbers of CD4 T cells and macrophages. It was also confirmed that the number of CD4 T cells was increased by mushroom 1, mushroom 2 and mixed 1 extract in MLN, and that the number of CD8 T cells by mushroom 2 was increased. PEC was extracted from the mouse model of each extract, and analyzed by flow cytometry using fluorescence staining with B220 (B cell marker) and CD69 (activation marker) antibody. Fig. 3 shows the results thereof. The number of B cells was increased by all the extracts.

To investigate the efficacy of each extract in reducing immunity following irradiation, the following mouse models were prepared and tested. Changes in the number of white blood cells (WBC) in blood, IgG production in plasma, immune cell activity in spleen, immune cell activity in LN, immune cell activity in MLN, cell death in spleen And cell cycle changes in spleen. In addition, spleen was isolated at the sacrifice of mice administered with the candidate extract after oral administration, and the results of evaluation of expression of genes related to inflammation were confirmed.

● Identification of immunity enhancement by SF-051 in γ-irradiation mouse model

- γ-Irradiation mouse model was prepared and SF-051 was pretreated for 3 weeks before γ-irradiation in order to confirm immunostimulating effect by SF-051. After further treatment with SF-051 for 1 week, the mice were sacrificed and immunostimulating effect was confirmed (FIG. 5).

- In the γ-irradiation mouse model, the change in the body weight of the mice treated with SF-051 showed a slightly faster recovery rate after the γ-irradiation in the group treated with SF-051 than the γ-irradiated control group (FIG. 6A). The number of white blood cells (WBCs) in the blood after sacrifice of mice was then analyzed. As a result, the number of WBCs in the group irradiated with γ-irradiation was drastically decreased, and the decreased WBC was increased by SF-051 But did not differ from the control group (Fig. 6B). However, as a result of confirming the IgG level in blood, it was confirmed that the IgG level reduced by γ-irradiation was significantly increased by SF-051 (FIG. 6D). Further, as a result of checking the level of IgA in the mucosa, the level of IgA by SF-051 could not be confirmed (Fig. 6C). These results suggest that SF-051 does not affect mucosal immunity but may increase the activity of immune cells reduced by γ-irradiation.

In order to confirm the effect of SF-051 on the differentiation of immune cells in the γ-irradiation mouse model, cells of Thymus and PEC (peritoneal excudate cells), which are important for T cell and B cell differentiation, The population of immune cells was confirmed using an analyzer. As a result, it was confirmed that in the γ-irradiation mouse model, SF-051 increased the differentiation of CD8 + T cells and that the differentiation of CD4 + T cells was restored to a normal level than the γ-irradiation control (FIG. 7A). In PEC, it was not confirmed that the population of B cells reduced by γ-irradiation was restored by SF-051 (FIG. 7B). These results indicate that the number of immune cells reduced by γ-irradiation is likely to be recovered by Thymus by SF-051.

In order to confirm the effect of SF-051 on the activity of immune cells in the γ-irradiation mouse model, cells of Spleen, Lymph node (LN), Mesenteric lymph node (MLN) And the activity and population of immune cells were confirmed. As a result, the population of CD4 + T cells, CD8 + T cells, and dendritic cells in Spleen was increased by SF-051 compared to γ-irradiated control, and the activity of NK cells was increased. However, there was no difference in macrophages (Fig. 8). As a result of confirming the activity and population of immune cells in LN, the number of NK cells was increased by SF-051, but there was no significant difference in activity. And no difference in the remaining immune cells (FIG. 9). In MLN, the activity of CD4 + T cells and NK cells was increased, but CD8 + T cells, macrophages and dendritic cells were not affected (FIG. 10). These results indicate that SF-051 mainly affects the number and activity of NK cells and also affects T cells. However, SF-051 does not affect other macrophages and dendritic cells.

In the γ-irradiation mouse model, the population of T cells and NK cells was increased by SF-051. Cells were isolated from the spleen of γ-irradiation mouse model, and cell death and cell cycle changes were confirmed by flow cytometry. As a result, the cell death of the immune cells was increased by γ-irradiation, and the increased cell death was observed to be reduced by SF-051 (FIG. 11A). In addition, the cell cycle analysis revealed that the S-phase was increased in the group treated with SF-051 (Fig. 11B) compared with the group treated with only γ-irradiation. Cells were separated from the spleen of the γ-irradiation mouse model and the expression of various cytokine genes was analyzed by Real-time PCR (FIG. 12). IL-2, IL-4, IL-6, and TGF-β to determine the effects of SF-051 on immune cell activity and population changes in inflammatory cytokines in γ- And the expression of inflammatory cytokines such as TNF-α was confirmed by RT-PCR. As a result, it was confirmed that the expression of inflammatory cytokines was reduced in the SF-051-treated mouse group as compared with the control group. IFN-γ, a cytokine secreted by NK cells and associated with antiviral, anti-cancer, and immunomodulation, was confirmed to be increased by SF-051 due to γ-irradiation. IL-17, which promotes the proliferation of T cells, is increased in the SF-051 group and IL-10, which suppresses the immune response, than the γ-irradiation control group. These results are consistent with increased population and activation of NK cells in flow cytometry. These results indicate that the cell death and cell cycle of immune cells are regulated by SF-051, and the expression of inflammatory cytokines is regulated, thereby reducing the inflammatory response.

Claims (7)

A composition for promoting hematopoietic function and radiation protection comprising mushroom extract and garlic extract.
The method according to claim 1,
Wherein the mushroom extract is extracted from at least one mushroom selected from the group consisting of Ganoderma lucidum, Shiitake mushroom, Cordyceps mushroom, and White mushroom.
The method according to claim 1,
Wherein the method for extracting mushroom extract is at least one extraction method selected from a pressurized water extraction method, a hot water extraction method, and an ultrasonic extraction method.
The method of claim 3,
A composition for promoting hematopoietic function and radiation protection, which comprises separating a mushroom from a mushroom through an extraction method of the mushroom extract.
The method according to claim 4,
Wherein the pressurized water extracting method comprises separating the mizorium from the mushroom under the condition of a temperature of 25 to 35 DEG C, a pressure of 8 to 12 MPa, and an extraction time of 60 to 70 minutes.
The method according to any one of claims 1 to 5,
Wherein the garlic extract comprises 10 to 20 mg / g of an alginate.
A method of preparing a composition for promoting hematopoietic function and radiation protection comprising mushroom extract and garlic extract,
Characterized in that the mushroom extract is extracted by a pressurized water extraction method and is separated from the mushroom under conditions of a temperature of 25 to 35 DEG C, a pressure of 8 to 12 MPa, an extraction time of 60 to 70 minutes, A method for preparing a radioprotective composition.

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