KR20240001411A - Composition for anti-inflammation or enhancement of immunity comprising mixed culture broth using shiitake mushroom mycelium as an effective ingredient, and manufacturing method of mixed culture broth using shiitake mushroom mycelium - Google Patents

Abstract

The present invention relates to an anti-inflammatory or immune-promoting composition containing shiitake mushroom mycelium complex culture (HKSMM) as an active ingredient and a method for producing shiitake mushroom mycelium complex culture (HKSMM), more specifically, COX-2, Shiitake mushroom mycelium complex culture (HKSMM), which inhibits the expression of iNOS, NF-κB, IL-1β, TNF-α, IL-4, and IL-6 and increases the expression of IL-2 and IFN-γ, is used as an active ingredient. It relates to a method for producing an anti-inflammatory or immune-promoting composition and shiitake mushroom mycelium complex culture (HKSMM) containing.

Description

Anti-inflammatory or immune-promoting composition containing shiitake mushroom mycelium complex culture (HKSMM) as an active ingredient and method for manufacturing shiitake mushroom mycelium complex culture (HKSMM) {Composition for anti-inflammation or enhancement of immunity comprising mixed culture broth using shiitake mushroom mycelium as an effective ingredient, and manufacturing method of mixed culture broth using shiitake mushroom mycelium}

The present invention relates to an anti-inflammatory or immune-promoting composition containing shiitake mushroom mycelium complex culture (hereinafter referred to as “HKSMM”) as an active ingredient and a method for producing shiitake mushroom mycelium complex culture (HKSMM), in more detail. COX-2 (cyclooxygenease-2), iNOS (inducible nitric oxide synthase), NF-κB (nuclear factor-kappa B), IL-1β (interleukin-1 beta), TNF-α (tumor necrosis factor-alpha), Shiitake mycelium complex culture that inhibits the expression of interleukin-4 (IL-4) and interleukin-6 (IL-6) and increases the expression of interleukin-2 (IL-2) and interferon-gamma (IFN-γ) It relates to an anti-inflammatory or immune-promoting composition containing (HKSMM) as an active ingredient and a method for producing shiitake mushroom mycelium complex culture (HKSMM).

Inflammation is the expression of a normal, protective defense mechanism in the body that appears locally against tissue damage caused by physical trauma, harmful chemicals, infection by microorganisms, or irritants in the body's metabolites. This inflammation is triggered by various chemical mediators produced from damaged tissues and migrating cells, and these chemical mediators are known to vary depending on the type of inflammatory process. In normal cases, the body neutralizes or eliminates pathogenic factors through an inflammatory response and regenerates damaged tissues to restore normal structure and function, but in other cases, it may progress to a disease state such as chronic inflammation.

Various biochemical phenomena are involved as causes of inflammation in living organisms. Macrophages are cells with various functions and play an important role in inflammatory reactions by producing various cytokines and NO (nitric oxide) in response to chemical stimulation. In particular, inducible iNOS, which is expressed in macrophages by cytokine stimulation such as lipopolysaccharide (LPS), interferon γ, and TNF-α, produces large amounts of NO for a long period of time. This oxidative stress is known to promote the activity of NF-κB, a transcription factor for the inflammatory response that is suppressed by IκB. Activated NF-κB is known to move to the nucleus and promote the expression of genes that induce inflammatory responses, such as iNOS, COX-2, and various types of cytokines such as IL-1β and TNF-α. Inhibiting these factors It is known to suppress inflammatory responses (Baeuerle et al., Annu. Rev. Immunol., 12:141-179, 1994).

Meanwhile, Korean Patent No. 1492174 discloses an anti-inflammatory and immune-boosting composition containing fermented green coffee beans fermented using red yeast, and Korean Patent No. 1604448 discloses a composition containing black rice bran extract as an active ingredient. Pharmaceutical compositions for preventing and treating anti-inflammatory and immune diseases are being disclosed. However, no anti-inflammatory or immune-promoting composition containing the shiitake mushroom mycelium complex culture of the present invention as an active ingredient has been disclosed yet.

Accordingly, the present inventors prepared a shiitake mushroom mycelium complex culture (HKSMM), and the shiitake mushroom mycelium complex culture (HKSMM) contained COX-2, iNOS, NF-κB, IL-1β, TNF-α, IL-4, and IL. The present invention was completed by confirming that it exhibits an anti-inflammatory or immune-enhancing effect by inhibiting the expression of -6 and increasing the expression of IL-2 and IFN-γ.

Republic of Korea Patent No. 10-1492174 (announcement date 2015. 02. 10) Republic of Korea Patent No. 10-1604448 (announcement date 2016. 03. 21)

Therefore, the purpose of the present invention is to use shiitake mushroom mycelium composite culture (HKSMM), which is a mixture of shiitake mushroom mycelium solid culture obtained by solid culturing shiitake mushroom mycelium and shiitake mycelium liquid culture obtained by liquid culturing shiitake mushroom mycelium, as an active ingredient. The aim is to provide a health functional food composition containing anti-inflammatory or immune-boosting properties.

Another object of the present invention is to contain shiitake mushroom mycelium complex culture (HKSMM), which contains shiitake mushroom mycelium solid culture obtained by solid culturing shiitake mushroom mycelium and shiitake mushroom mycelium liquid culture obtained by liquid culturing shiitake mushroom mycelium, as an active ingredient. The aim is to provide a pharmaceutical composition for preventing or treating inflammatory diseases.

Another object of the present invention is to provide a method for producing shiitake mushroom mycelium complex culture (HKSMM).

In order to achieve the above-described object, the present invention is a shiitake mushroom mycelium complex culture (HKSMM), which is a mixture of shiitake mushroom mycelium solid culture obtained by solid culturing shiitake mushroom mycelium and shiitake mushroom mycelium liquid culture obtained by liquid culturing shiitake mushroom mycelium. Provides an anti-inflammatory or immune-boosting health functional food composition containing as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases containing the shiitake mushroom mycelium complex culture (HKSMM) as an active ingredient.

Additionally, the present invention provides a method for producing the shiitake mushroom mycelium complex culture (HKSMM).

The shiitake mushroom mycelium complex culture (HKSMM) of the present invention inhibits the expression of COX-2, iNOS, NF-κB, IL-1β, TNF-α, IL-4, and IL-6, and inhibits IL-2 and IFN-γ. Since it increases the expression of , it can be usefully used in health functional foods and medicines for anti-inflammatory or immune-boosting purposes.

Figure 1 shows the content of water (0% ethanol) and water-soluble ethanol extract (30%, 50%, 70%, 95% ethanol) of the shiitake mushroom mycelium complex culture (HKSMM) sample of the present invention.
Figure 2 shows the NO production inhibitory effect of water (0% ethanol) and aqueous ethanol extract (30%, 50%, 70%, 95% ethanol) of HKSMM samples on RAW264.7 cells activated with LPS of the present invention. there is.
Figure 3 is a graph measuring cytotoxicity upon treatment with HKSMM50 and AHCC on RAW264.7 cells activated with LPS of the present invention. LPS-treated RAW264.7 cells were treated with various concentrations of HKSMM50 and cultured for 24 hours (5% CO2, 37°C). AHCC means positive control.
Figure 4 is a graph showing the results of inhibition of NF-κB production upon HKSMM50 treatment of RAW264.7 cells activated with LPS of the present invention. LPS-treated RAW264.7 cells were treated with various concentrations of HKSMM50 and cultured for 24 hours (5% CO2, 37°C). AHCC means positive control.
Figure 5 is a graph showing the results of suppressing NO production upon HKSMM50 treatment of RAW264.7 cells activated with LPS of the present invention. AHCC means positive control.
Figure 6 is a graph showing the results of inhibition of iNOS (left) and COX-2 (right) protein expression upon HKSMM50 treatment of RAW264.7 cells activated with LPS of the present invention. AHCC means positive control.
Figure 7 is a graph showing the results showing a decrease in the production of IL-1β, TNF-a, IL-6, and IL-4 upon HKSMM50 treatment of RAW264.7 cells activated with LPS of the present invention. AHCC means positive control.
Figure 8 is a graph showing the results of improving SOD and CAT activities upon HKSMM50 treatment of RAW264.7 cells activated with LPS of the present invention. AHCC means positive control.
Figure 9 is a graph showing the results of cytotoxicity (cells viability (%)) when treated with HKSMM50 and AHCC for 24 hours on Jurkat cells of the present invention. AHCC means positive control.
Figure 10 is a graph showing the results of cell proliferation (%) when Jurkat cells of the present invention were treated with HKSMM50 for 3 hours and 6 hours. AHCC means positive control.
Figure 11 is a graph showing the results of cell proliferation (%) when Jurkat cells activated with ConA (concanavalin A) of the present invention were treated with HKSMM50 for 3 hours and 6 hours. AHCC means positive control.
Figure 12 is a graph showing the decrease in cytosolic NAFT (nuclear factor of activated T-cell) protein when Jurkat cells activated with ConA of the present invention were treated with HKSMM50 for 3 and 6 hours. AHCC means positive control.
Figure 13 is a graph showing the increase in nuclear NAFT protein when Jurkat cells activated with ConA of the present invention were treated with HKSMM50 for 3 and 6 hours. AHCC means positive control.
Figure 14 is a graph showing the results of increased IL-2 production when Jurkat cells activated with ConA of the present invention were treated with HKSMM50 for 3 and 6 hours. AHCC means positive control.
Figure 15 is a graph showing the results of increased production of IFN-γ when Jurkat cells activated with ConA of the present invention were treated with HKSMM50 for 3 hours and 6 hours. AHCC means positive control.
Figure 16 is a graph showing the results of suppressing plasma iNOS and COX-2 activities in female Balb/c mice treated with HKSMM of the present invention. NC (negative control) means negative control, PC (positive control) means positive control, AHCC means negative control, T1 is HKSMM 500 mg/100g, T2 is HKSMM 1,000 mg/100g, and T3 is HKSMM 2,000 mg/100g. .
Figure 17 is a graph showing the results of reduction in plasma p-p65 and p-IκBα contents in female Balb/c mice treated with HKSMM of the present invention. NC means negative control, PC means positive control, AHCC means negative control, T1 means HKSMM 500 mg/100g, T2 means HKSMM 1,000 mg/100g, and T3 means HKSMM 2,000 mg/100g.

Since the present invention can be modified in various ways and have various embodiments, specific embodiments will be described in detail. Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings.

In order to achieve the object of the present invention, the present invention provides an anti-inflammatory or immune-promoting health functional food composition containing shiitake mushroom mycelium complex culture (HKSMM) as an active ingredient.

In the anti-inflammatory or immune-boosting health functional food composition of the present invention, the inflammation may include arthritis, rhinitis, hepatitis, keratitis, gastritis, enteritis, nephritis, bronchitis, pleurisy, peritonitis, spondylitis, pancreatitis, urethritis, cystitis, burn inflammation, and dermatitis. , may be any one selected from the group consisting of periodontitis, gingivitis, and neuroinflammation, but is not limited thereto.

The shiitake mushroom mycelium composite culture (HKSMM) contains a shiitake mushroom mycelium solid culture obtained by solid culturing shiitake mushroom mycelium and a shiitake mushroom mycelium liquid culture obtained by liquid culturing shiitake mushroom mycelium.

The shiitake mushroom mycelium complex culture (HKSMM) can be used as is or an extract extracted with water or ethanol. The ethanol extraction is preferably performed by extracting the shiitake mushroom mycelium complex culture with ethanol at a concentration of 0.1 to 99% by weight, and more preferably with ethanol at a concentration of 50% by weight.

In addition, the ethanol extract shall include any one of an extract obtained by extraction treatment, a diluted or concentrated liquid of the extract, a dried product obtained by drying the extract, or a crude product or purified product.

In addition, the shiitake mushroom mycelium complex culture (HKSMM) inhibits the expression of COX-2, iNOS, NF-κB, IL-1β, TNF-α, IL-4, and IL-6, and inhibits IL-2 and IFN- It may be, but is not limited to, increasing the expression of γ.

The composition may be manufactured in any one formulation selected from powder, granule, pill, tablet, capsule, candy, syrup, and beverage, but is not limited thereto.

When using the health functional food composition of the present invention as a food additive, the health functional food composition may be added as is or used together with other foods or food ingredients, and may be used appropriately according to conventional methods. The active ingredient may be used appropriately depending on its intended use.

There are no particular restrictions on the types of health functional foods. Examples of foods to which the health functional food composition can be added include meat, sausages, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, and tea. There are drinks, alcoholic beverages, and vitamin complexes, and it includes all health foods in the conventional sense.

Additionally, the health functional food composition of the present invention can be manufactured into food, especially functional food. The functional food of the present invention may contain commonly added ingredients. For example, it includes proteins, carbohydrates, fats, nutrients and seasonings. For example, when manufacturing a drink, natural carbohydrates or flavoring agents may be included as additional ingredients in addition to the active ingredient. The natural carbohydrates include monosaccharides (e.g., glucose, fructose, etc.), disaccharides (e.g., maltose, sucrose, etc.), oligosaccharides, polysaccharides (e.g., dextrins, cyclodextrins, etc.), or sugar alcohols (e.g., , xylitol, sorbitol, erythritol, etc.) are preferred. The flavoring agent may be a natural flavoring agent (e.g., thaumatin, stevia extract, etc.) or a synthetic flavoring agent (e.g., saccharin, aspartame, etc.).

In addition to the above health functional food composition, various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonic acid. It may further contain carbonating agents used in beverages.

Additionally, the present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases containing shiitake mushroom mycelium complex culture (HKSMM) as an active ingredient.

In the pharmaceutical composition for preventing or treating inflammatory diseases of the present invention, the inflammatory diseases include arthritis, rhinitis, hepatitis, keratitis, gastritis, enteritis, nephritis, bronchitis, pleurisy, peritonitis, spondylitis, pancreatitis, urethritis, cystitis, burn inflammation, and dermatitis. , may be any one selected from the group consisting of periodontitis, gingivitis, and neuroinflammation, but is not limited thereto.

The pharmaceutical composition of the present invention may further include appropriate carriers, excipients, or diluents commonly used in the preparation of pharmaceutical compositions.

The pharmaceutical dosage form of the composition according to the present invention can be used alone or in combination with other pharmaceutically active compounds, as well as in appropriate combinations.

The pharmaceutical composition according to the present invention can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, external preparations, suppositories, and sterile injection solutions, respectively, according to conventional methods. You can. Carriers, excipients, and diluents that may be included in the pharmaceutical composition containing the shiitake mycelium complex culture (HKSMM) include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, and acacia gum. , alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxy benzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. Various compounds or mixtures may be mentioned. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations contain at least one excipient, such as starch, calcium carbonate, It is prepared by mixing sucrose, lactose, and gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. there is. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, wethepsol, macrogol, Tween 61, cacao, laurin, glycerogelatin, etc. can be used.

The preferred dosage of the pharmaceutical composition of the present invention varies depending on the patient's condition and weight, degree of disease, drug form, administration route and period, but can be appropriately selected by a person skilled in the art.

The method for producing shiitake mushroom mycelium complex culture (HKSMM) includes the steps of producing a shiitake mycelium solid culture by culturing shiitake mushroom mycelium on a barley medium, and mixing shiitake mycelium with defatted soybean flour, K ₂ HPO ₄ and MgSO ₄ It may include preparing a shiitake mushroom mycelium liquid culture cultured in a mixed liquid medium and mixing the shiitake mushroom mycelium solid culture and liquid culture.

Hereinafter, the present invention will be described in more detail using examples.

Example 1. Preparation of shiitake mushroom mycelium complex culture (HKSMM)

Shiitake mushroom mycelium complex culture (HKSMM) is made by culturing shiitake mushroom mycelium on a barley medium as a solid, then drying it with hot air and powdering it (A), and shiitake mushroom mycelium on a medium containing defatted soybean meal as the main ingredient. It is prepared by mixing liquid culture, then drying and powdering (B) at a weight ratio of 2:1. The specific manufacturing method is as follows.

1. Manufacture of shiitake mycelium solid culture powder (A)

1) Preparation of barley medium: Wash the barley with water to remove foreign substances, soak in tap water for 24 hours, and leave in a strainer for 3-4 hours.

2) Sterilization of barley medium: Sterilize the medium (121°C, 2 hours).

3) Shiitake mushroom spawn inoculation: Inoculate pre-cultured shiitake mushroom spawn into the medium.

4) Solid culture: Shiitake mushrooms inoculated into the medium are cultured in a solid culture medium (25°C) for more than 10 days.

5) Drying: The cultured shiitake mushroom mycelium solid culture is dried with hot air (50°C).

6) Powder production: Powder (100 mesh) the dried shiitake mushroom mycelium solid culture.

2. Manufacture of shiitake mushroom mycelium liquid culture powder (B)

1) Preparation of liquid medium: Mix defatted soybean flour (2.5 g/L), yellow white sugar (25g/L), K ₂ HPO ₄ (0.025 g/L), and MgSO ₄ (0.025 g/L) with water and add foam remover. Apply Foam Cleaner according to the instructions for use to remove foam. At this time, based on 100 parts by weight of liquid medium, 0.25 parts by weight of defatted soybean powder, 2.5 parts by weight of yellow white sugar, K ₂ HPO ₄ 0.0025 parts by weight, MgSO ₄ It is prepared by mixing 0.0025 parts by weight and 97.245 parts by weight of water.

2) Sterilization of liquid medium: Sterilize the liquid medium (121℃, 2 hours).

3) Shiitake mushroom spawn inoculation: Inoculate pre-cultured shiitake mushroom mycelium spawn into the liquid medium.

4) Liquid culture: After culturing in a liquid incubator (25℃) for more than 10 days, β-glucan was extracted (121℃, 1 hour), and then defatted soybean powder was added at 10% (w/v) to the extract. After mixing, dry with hot air.

5) Powder production: Powder (100 mesh) the dried shiitake mushroom mycelium liquid culture.

3. Preparation of shiitake mycelium complex culture

Shiitake mushroom mycelium composite culture (HKSMM) is prepared by mixing the shiitake mushroom mycelium solid culture powder (A) and shiitake mycelium liquid culture powder (B) at a weight ratio of 2:1 (w/w).

The properties of the HKSMM prepared above are brown powder, β-glucan (mg/g): 110~200, lead (mg/kg): 1.0 or less, total arsenic (mg/kg): 1.0 or less, cadmium (mg/kg): 1.0 or less. kg): 0.5 or less, total mercury (mg/kg): 0.5 or less, total aflatoxin (sum of B1, B2, G1 and G2) (㎍/kg): 15.0 or less (B1 is 10.0 or less).

Example 2. Shiitake mushroom mycelium complex culture ( HKSMM ) according to processing in vitro Anti-inflammatory effect in

1. Sample preparation for cell experiments

The water (0% ethanol) extract of HKSMM prepared in Example 1 and the extract extracted with aqueous ethanol at concentrations of 30%, 50%, 70%, and 95% were freeze-dried (Figure 1). In terms of extraction yield for HKSMM, the water extract was 24.9%, and the 30%, 50%, and 70% ethanol extracts were 32.5%, 43.6%, 43.0%, and 23.1%, respectively.

The degree of inhibition of NO production of these freeze-dried products in RAW264.7 cells, a mouse macrophage cell treated with LPS, was measured using the Griess method (Figure 2). All of these freeze-dried products suppressed the production of NO compared to LPS treatment, but the 50% ethanol extract (hereinafter referred to as 'HKSMM50') suppressed the production of NO the most. Therefore, HKSMM50 was used in cell experiments.

2. Evaluation of cell viability of HKSMM50

We investigated at what concentration HKSMM50 exhibits cytotoxicity in LPS-treated RAW264.7 cells. RAW264.7 cells treated with LPS were treated with 5,000 μg/ml of HKSMM50, cultured for 24 hours, and cell viability was measured using the CCK-8 kit (Figure 3). Compared to LPS treatment, there was no effect on cell growth up to a concentration of 5000 μg/ml of HKSMM50. AHCC, which was used as a positive control, did not affect cell growth at concentrations up to 5000 ㎍/ml. AHCC is a water-soluble substance mainly composed of β-glucan and α-glucan and is used as an immune enhancer or cancer treatment agent, so it was used as a positive control.

3. Anti-inflammatory effect of HKSMM50

1) Inhibition of NF-κB activity

First, the content of NF-κB, which acts upstream of the inflammatory response, was measured in RAW264.7 cells. LPS-treated RAW264.7 cells were treated with HKSMM50 at concentrations of 0, 20, 200, and 500 μg/ml, cultured for 24 hours, and the NF-κB content in the supernatant was measured using an ELISA kit (Figure 4 ). As a positive control, 100 μg/ml of AHCC was treated.

Compared to LPS treatment, HKSMM50 treatment significantly reduced NF-κB content (p<0.001). Treatment with 500 μg/ml of HKSMM50 reduced the content of NF-κB to the untreated level. This result means that HKSMM50 sample has a strong anti-inflammatory effect in RAW264.7 cells. AHCC, used as a positive control, also showed similar results to HKSMM50.

2) Effect of reducing NO content and PGE2 (prostaglandin E2) in HKSMM

The effect of HKSMM50 on NO and PGE2 content produced by RAW264.7 cells was investigated. LPS-treated RAW264.7 cells were treated with HKSMM50 at concentrations of 0, 20, 200, and 500 ㎍/ml, cultured for 24 hours, and the contents of NO and PGE2 in the supernatant were measured using an ELISA kit (Figure 5). As a positive control, 100 μg/ml of AHCC was treated. Compared to LPS treatment, HKSMM50 treatment significantly reduced the contents of NO and PGE2 in a concentration-dependent manner (p<0.001). In particular, the content of PGE2 was reduced to the untreated level by treatment with 500 ㎍/ml HKSMM50. AHCC 100 ㎍/ml used as a positive control was also similar to the same concentration of HKSMM50.

In Figure 6, to confirm the role of HKMSS50 in reducing the contents of NO and PGE2, RAW264.7 cells treated with LPS were treated with HKSMMf50 at concentrations of 0, 20, 200, and 500 μg/ml, incubated for 24 hours, and then added to the supernatant. Expression of iNOS and COX-2 proteins was examined by Western Blotting. As a positive control, AHCC was treated at concentrations of 0, 20, 200, and 500 μg/ml. Compared to LPS treatment, HKSMM50 treatment significantly reduced the expression of iNOS and COX-2 (p<0.001). The expression of iNOS was significantly reduced in a concentration-dependent manner by HKSMM50. COX-2 was also significantly reduced in a concentration-dependent manner, but there was some variation. When compared to the same treatment concentration of AHCC at the pretreatment concentration, HKSMM50 more strongly inhibited the expression of iNOS and COX-2.

These results indicate that HKSMM50 has an anti-inflammatory effect by reducing the contents of NO and PGE2 in RAW264.7 cells.

4. Inhibition of production of pro-inflammatory cytokines by HKSMM50

The effect of HKSMM50 on the content of several cytokines produced by RAW264.7 cells was investigated. LPS-treated RAW264.7 cells were treated with HKSMMf50 at concentrations of 0, 20, 200, and 500 ㎍/ml, cultured for 24 hours, and IL-1β, TNF-α, IL-4, and IL-6 contained in the supernatant. The content was measured using an ELISA kit (Figure 7). As a positive control, 100 μg/ml of AHCC was treated.

Compared to LPS treatment, HKSMM50 treatment significantly reduced the content of four cytokines in a concentration-dependent manner (p<0.05 - p<0.001). In particular, treatment with 500 μg/ml of HKSMM50 reduced the contents of TNF-α and IL-6 to the untreated level. AHCC 100 ㎍/ml used as a positive control was also similar to the same concentration of HKSMM50. This result means that the HKSMM50 sample has an anti-inflammatory effect by reducing the contents of IL-1β, TNF-α, IL-4, and IL-6 in RAW264.7 cells.

5. Effect of HKSMM50 on increasing antioxidant enzyme SOD and catalase (CAT) activities

In Figure 8 RAW264.7 cells treated with LPS were treated with HKSMM50 at concentrations of 0, 20, 200, and 500 μg/ml and cultured for 24 hours. The activities of SOD and CAT contained in the supernatant are shown. SOD enzyme activity was significantly decreased by LPS treatment compared to untreated (p<0.001). However, HKSMM50 treatment increased SOD activity to the untreated level. AHCC also obtained similar results.

CAT enzyme activity was also significantly decreased by LPS treatment compared to untreated (p<0.001). However, HKSMM50 treatment increased CAT activity in a concentration-dependent manner, increasing it to the untreated level. AHCC also obtained similar results.

These results indicate that HKSMM50 increases the activities of antioxidant enzymes SOD and CAT in RAW264.7 cells.

In this way, HKSMM50 inhibits the movement of NF-κB in the cytoplasm to the nucleus in RAW264.7, a mouse macrophage cell activated with LPS, inhibits the expression of COX-2 (reduces PGE2 production), and inhibits the expression of iNOS. It has an anti-inflammatory effect by reducing NO production.

Example 3. According to treatment with shiitake mushroom mycelium complex culture (HKSMM) in vitro Immune-boosting effect in

1. Sample

The shiitake mushroom mycelium complex culture (HKSMM) prepared in Example 1 was extracted with 50% concentration of water-soluble alcohol, freeze-dried, and used as a sample (HKSMM50).

2. Measurement of Jurkat cytotoxicity (cell viability) of HKSMM50

Jurkat cells were treated with HKSMM50 at a concentration of 0 to 100 μg/ml, cultured for 24 hours, and cytotoxicity was measured by MTT assay. Jurkat cells are a type of cancer cell line, obtained from a patient with T cell leukemia (Acute T cell leukemia), and are a single cell group. By applying several stimuli, such as ConA (concanavalin A), to a non-activated Jurkat, it can develop Th1 or Th2 characteristics.

As a result of the above measurement, there was no significant decrease even at a concentration of 100 μg/mL of HKSMM50 (FIG. 9). AHCC, a positive control for Jurkat cells, was treated in the same concentration as HKSMM50 at a concentration of 0 to 100 μg/mL, cultured for 24 hours, and cytotoxicity was measured by MTT assay. As a result, there was no significant decrease even at the concentration of AHCC 100 μg/mL (FIG. 9).

3. Measurement of cell proliferation of HKSMM50

To confirm the proliferative ability of HKSMM50 on Jurkat cells, 90 ㎕ of Jurkat was dispensed into each ⁹⁶ well plate at a concentration of 2 And 10 μl of medium prepared to 100 μg/ml was added, cultured for 3 hours and 6 hours, and cell proliferation rate was measured (positive control was AHCC 50 μg/ml). The cases in which Jurkat cells were activated by treating them with ConA were compared with those in which Jurkat cells were not treated with ConA.

1) When ConA is not processed (Figure 10)

As a result of treating Jurkat cells with HKSMM50 at different concentrations and examining cell proliferation according to treatment time, cell proliferation increased in all concentrations. It increased at 3 and 6 hours at all concentrations, and AHCC, a positive control, also increased similarly to HKSMM50.

2) When ConA is processed (Figure 11)

As a result of treating Jurkat cells with HKSMM50 at different concentrations and examining cell proliferation according to treatment time, there was an increase at all concentrations in the 3-hour treatment, but there was a significant increase in the 100 μg/ml treatment (p<0.01), and 3 in the 6-hour treatment. Similar results were obtained from time processing. AHCC 50 μg/ml treatment, which is a positive control, was similar to the same treatment effect as HKSMM50.

4. NFAT (nuclear factor of activated T-cells) expression of HKSMM50

When Jurkat cells are activated by ConA, they produce immunoregulatory cytokines such as IL-2 and IFN-γ. When ConA binds to the T-cell receptor (TCR) of Jurkat cells, NFAT is activated and induces the expression of IL-2 and IFN-γ mRNA. Therefore, the production of IL-2 and IFN-γ is used as an indicator of immune enhancement. Therefore, to measure the cytosol and nuclear distribution of NFAT in Jurkat cells, Jurkat cells were treated with HKSMM50 (0, 25, 50, and 100 μg/ml) and AHCC (100 μg/ml) for 1 hour after treatment with ConA. The cells were treated and cultured for another 2 hours, the cells were recovered, and the NFAT content contained in the cytosol and nucleus was analyzed by Western blotting.

In Figure 12, the cytosolic NFAT/β-actin ratio was 1.7 in the control group, but decreased to 1.3 with ConA treatment. This means that NFAT moved from the cytosol to the nucleus with ConA treatment. This content ratio of the HKSMM50 sample treated with 25 μg/ml was similar to that of the ConA treatment, but the content ratio of the HKSMM50 sample treated with 50 μg/ml and 100 μg/ml was significantly reduced (p<0.001). The effect of AHCC (100 μg/ml) treatment was similar to that of the HKSMM50 sample at the same concentration. Therefore, this result means that NFAT moved from cytosol to the nucleus through HKSMM50 sample processing.

In Figure 13, the nuclear NFAT/β-actin ratio was 1.1 in the control group, but significantly increased to 1.7 with ConA treatment, meaning that the NFAT increased by ConA treatment was moved from the cytosol to the nucleus. This ratio significantly increased in a concentration-dependent manner for HKSMM50 sample treatment (p<0.05). However, these ratios were lower than those in the ConA treatment (p<0.05), but higher than those in the control group (p<0.05). The effect of AHCC (100 μg/ml) treatment was similar to that of HKSMM50 (100 μg/ml). This result means that HKSMM50 treatment moved NFAT protein from the cytosol to the nucleus.

5. Increased production of IL-2 and IFN-γ in HKSMM50

Treatment of Jurkat cells activated with ConA with HKSMM50 dephosphorylated NFAT in the cytosol and caused it to move to the nucleus (Figures 12 and 13). Therefore, to confirm that HKSMM50 samples increase the production of immunostimulatory cytokines in Jurkat cells, Jurkat cells activated with ConA were treated with HKSSM50 samples (0, 25, 50, and 100 μg/ml) for 3 hours and 6 days. IL-2 (FIG. 14) and IFN-γ (FIG. 15) contents were measured after culturing for a time. Positive control AHCC was treated at 100 μg/ml.

As shown in Figure 14, the IL-2 content was increased in ConA and HKSMM50 samples treated for 3 and 6 hours compared to the control treatment (p<0.05). At both culture times, the IL-2 content in HKSMM50 samples treated with a concentration of 50 μg/ml or more was significantly increased compared to ConA treatment (p<0.05~0.001). The effect of AHCC (100 μg/ml) treatment on increasing IL-2 content was not significantly different from that of ConA treatment.

In Figure 15, the increase in IFN-γ content by treatment with ConA and HKSMM50 samples at 3 and 6 hours of culture was higher than that in the control, and treatment with 50 μg/ml and 100 μg/ml HKSMM50 samples increased the level of IFN-γ compared to treatment with ConA. -γ content was significantly increased (p<0.001). The increase in IFN-γ by AHCC (100 μg/ml) treatment was not different from treatment with the same HKSMM50 concentration.

According to the present invention, HKSMM50, a 50% concentration water-soluble ethanol extract of HKSMM, contains 161.0 mg/g of β-glucan and 2.1 mg/g of total flavonoids, and HKSMM50 moves NAFT from the cytosol to the nucleus. In that it exhibits an immune-boosting effect that enhances the production of IL-2 and IFN-γ, HKSMM containing more than 14% of β-glucan has an immune-boosting effect and can be used as an immune-boosting composition.

Example 4. According to treatment with shiitake mushroom mycelium complex culture (HKSMM) in vivo in anti-inflammatory effect

1. Sample: HK shiitake mushroom mycelium (HKSMM)

2. Experimental animals and treatment

To female Balb/c mice, 10 negative controls (NC) in the regular feed group, 10 positive controls (PC) in the AHCC feed group, HKSMM feed groups T1 (500 mg/100 g), T2 (1,000 mg/100 g), and Treated with T3 (2,000 mg/100 g). Feed prepared according to the concentration and water that had passed through a fine filter and an ultraviolet water sterilizing device were freely fed. Serum from experimental animals was collected at the 4th and 6th weeks, left on ice for 20 min, and then separated by centrifugation. The animal rearing room was reared in a constant temperature and humidity environment with lighting on a 12-hour cycle. Animal testing was conducted with approval from the IACUC of the Gyeongnam Environmental Toxicology Center of the Korea Institute of Toxicology (KIT) (approval number: 2108-0004).

3. Inhibitory effect of iNOS and COX-2 expression in Balb/c mice of HKSMM (Figure 16)

iNOS was significantly lower in the PC group than in the NC group at week 4, but tended to be higher in the experimental groups T1, T2, and T3. However, at week 6, the experimental groups T1, T2, and T3 showed a concentration-dependent significant difference and decreased iNOS expression compared to the NC group. In the case of COX-2, the results in the T1 group at week 4 were significantly lower than those in the NC group, but there was no significant difference in other experimental groups. However, at week 6, the T3 group showed significantly lower COX-2 expression.

iNOS and COX-2 produce inflammatory cytokines NO and PGE2, respectively. This suggests that HKSMM has an anti-inflammatory effect by suppressing the production of NO and PEG2 by suppressing the expression of iNOS and COX-2 in Balb/c mice.

4. Effect of HKSMM on regulation of NF-κB signaling pathway (Figure 17)

After feeding HKSMM to Balb/c mice for 4 and 6 weeks, serum was collected and p-p65 was measured. As a result, p-p65 was significantly lower in the 4 and 6 week treatments compared to the NC group. Additionally, HKSMM treatment lowered the p-IKB content at 4 and 6 weeks compared to NC treatment. Flavonoids have an anti-inflammatory effect by reducing the expression of p-65 and IκBα. Therefore, this suggests that β-glucan and total flavonoids in HKSMM increase immune and anti-inflammatory functions by suppressing the expression of NF-κB in Balb/c mice.

Meanwhile, the above detailed description should not be construed as restrictive in all respects and should be considered illustrative. The scope of the present invention should be determined by reasonable interpretation of the appended claims, and all changes within the equivalent scope of the present invention are included in the scope of the present invention.

Claims (8)

An anti-inflammatory or immune-boosting health function containing shiitake mushroom mycelium complex culture as an active ingredient, which is a mixture of shiitake mushroom mycelium solid culture, which is a solid culture of shiitake mushroom mycelium, and a shiitake mycelium liquid culture, which is a liquid culture of shiitake mushroom mycelium. Food composition.
According to paragraph 1,
The shiitake mushroom mycelium complex culture is an anti-inflammatory or immune-boosting health functional food composition, characterized in that the shiitake mushroom mycelium complex culture is extracted with water or aqueous ethanol.
According to paragraph 1,
The shiitake mushroom mycelium complex culture is an extract obtained by extracting the shiitake mushroom mycelium complex culture with water or 50% concentration ethanol,
The shiitake mushroom mycelium complex culture inhibits the expression of COX-2, iNOS, NF-κB, IL-1β, TNF-α, IL-4 and IL-6, and the expression of IL-2 and IFN-γ. A health functional food composition for anti-inflammatory or immune-boosting purposes, characterized in that it increases immunity.
The anti-inflammatory or immune-boosting health functional food composition according to claim 1, wherein the composition is manufactured in a formulation selected from powder, granule, pill, tablet, capsule, candy, syrup, and beverage.
A pharmaceutical for the prevention or treatment of inflammatory diseases containing as an active ingredient a shiitake mycelium complex culture, which is a mixture of shiitake mushroom mycelium solid culture, which is a solid culture of shiitake mushroom mycelium, and a shiitake mycelium liquid culture, which is a liquid culture of shiitake mushroom mycelium. Composition.
According to clause 5,
The shiitake mushroom mycelium complex culture is a pharmaceutical composition for preventing or treating inflammatory diseases, characterized in that the shiitake mushroom mycelium complex culture is extracted with water or ethanol.
According to clause 5,
The shiitake mushroom mycelium complex culture is an extract obtained by extracting the shiitake mushroom mycelium complex culture with water or 50% concentration ethanol,
The shiitake mushroom mycelium complex culture inhibits the expression of COX-2, iNOS, NF-κB, IL-1β, TNF-α, IL-4 and IL-6, and the expression of IL-2 and IFN-γ. Pharmaceutical composition for preventing or treating inflammatory diseases characterized by increasing
Preparing a shiitake mushroom mycelium solid culture by culturing shiitake mushroom mycelium on a barley medium;
Shiitake mushroom mycelium was mixed with defatted soybean meal, K ₂ HPO ₄ and MgSO ₄ Preparing a shiitake mushroom mycelium liquid culture cultured in a liquid medium mixed with; and
A method for producing a shiitake mushroom mycelium complex culture comprising mixing the shiitake mushroom mycelium solid culture and liquid culture.