KR102836700B1 - 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-enhancing composition containing a shiitake mushroom mycelia composite culture (HKSMM) as an effective ingredient and a method for producing a shiitake mushroom mycelia composite culture (HKSMM), and more specifically, to an anti-inflammatory or immune-enhancing composition containing a shiitake mushroom mycelia composite culture (HKSMM) as an effective ingredient which inhibits the expression of COX-2, iNOS, NF-κB, IL-1β, TNF-α, IL-4 and IL-6 and increases the expression of IL-2 and IFN-γ, and a method for producing a shiitake mushroom mycelia composite culture (HKSMM).

Description

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-enhancing composition containing a shiitake mushroom mycelia complex culture (hereinafter referred to as "HKSMM") as an effective ingredient, and a method for producing a shiitake mushroom mycelia complex culture (HKSMM), and more specifically, to an anti-inflammatory or immune-enhancing composition containing a shiitake mushroom mycelia complex culture (HKSMM) as an effective ingredient, which inhibits the expression of 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), IL-4 (interleukin-4), and IL-6 (interleukin-6) and increases the expression of IL-2 (interleukin-2) and IFN-γ (interferon-gamma). It relates to a method for producing a culture medium (HKSMM).

Inflammation is a normal, protective body defense mechanism that occurs locally in response to tissue damage caused by physical trauma, harmful chemicals, microbial infection, or irritating substances among metabolic products in the body. This inflammation is triggered by various chemical mediators produced by damaged tissue 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 removes pathogenic factors through the inflammatory response and regenerates damaged tissue to restore normal structure and function, but if not, it may progress to a disease state such as chronic inflammation.

Various biochemical phenomena are involved in the cause of inflammation in the body. Macrophages are cells with various functions, and they produce various cytokines and nitric oxide (NO) in response to chemical stimulation, playing an important role in the inflammatory response. In particular, inducible iNOS expressed in macrophages by cytokine stimulation such as lipopolysaccharide (LPS), interferon γ, and TNF-α produces a large amount of NO for a long time. This oxidative stress is known to promote the activity of NF-κB, a transcription factor of the inflammatory response that is suppressed by IκB. Activated NF-κB is known to translocate to the nucleus and promote the expression of genes that induce inflammatory responses, such as iNOS, COX-2, and various cytokines such as IL-1β and TNF-α, and inhibition of these factors 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-enhancing composition comprising a fermented coffee bean product fermented using Hongsukmyun, and Korean Patent No. 1604448 discloses an anti-inflammatory and immune-disease prevention and treatment pharmaceutical composition comprising a black rice bran extract as an effective ingredient. However, there has not yet been disclosed an anti-inflammatory or immune-enhancing composition comprising the shiitake mushroom mycelia complex culture of the present invention as an effective ingredient.

Accordingly, the inventors of the present invention completed the present invention by producing a shiitake mushroom mycelia composite culture (HKSMM) and confirming that the shiitake mushroom mycelia composite culture (HKSMM) exhibits anti-inflammatory or immune-enhancing effects by inhibiting the expression of COX-2, iNOS, NF-κB, IL-1β, TNF-α, IL-4 and IL-6 and increasing the expression of IL-2 and IFN-γ.

Republic of Korea Patent No. 10-1492174 (Announcement date: February 10, 2015) Republic of Korea Patent No. 10-1604448 (Announcement date: March 21, 2016)

Accordingly, the purpose of the present invention is to provide an anti-inflammatory or immune-enhancing health functional food composition containing, as an effective ingredient, a shiitake mushroom mycelia composite culture (HKSMM) in which a solid culture of shiitake mushroom mycelia and a liquid culture of shiitake mushroom mycelia are mixed.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating an inflammatory disease, which contains, as an effective ingredient, a shiitake mushroom mycelia composite culture (HKSMM) containing a solid culture of shiitake mushroom mycelia obtained by solid-cultivating shiitake mushroom mycelia and a liquid culture of shiitake mushroom mycelia obtained by liquid-cultivating shiitake mushroom mycelia.

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

In order to achieve the above-described purpose, the present invention provides an anti-inflammatory or immune-enhancing health functional food composition containing, as an effective ingredient, a shiitake mushroom mycelia composite culture (HKSMM) in which a solid culture of shiitake mushroom mycelia obtained by solid-cultivating shiitake mushroom mycelia and a liquid culture of shiitake mushroom mycelia obtained by liquid-cultivating shiitake mushroom mycelia are mixed.

In addition, the present invention provides a pharmaceutical composition for preventing or treating an inflammatory disease containing the above-mentioned shiitake mushroom mycelia complex culture (HKSMM) as an effective ingredient.

In addition, the present invention provides a method for producing the above-mentioned shiitake mushroom mycelia composite culture (HKSMM).

The shiitake mushroom mycelia 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 increases the expression of IL-2 and IFN-γ, and therefore can be usefully used in anti-inflammatory or immune-enhancing health functional foods and pharmaceuticals.

Figure 1 shows the contents of water (0% ethanol) and water-soluble ethanol extracts (30%, 50%, 70%, 95% ethanol) of the shiitake mushroom mycelia composite culture (HKSMM) sample of the present invention.
Figure 2 shows the inhibitory effect of water (0% ethanol) and water-soluble ethanol extracts (30%, 50%, 70%, 95% ethanol) of HKSMM sample on NO production in LPS-activated RAW264.7 cells of the present invention.
Figure 3 is a graph showing the measurement of cytotoxicity when HKSMM50 and AHCC are treated for LPS-activated RAW264.7 cells 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 represents a positive control.
Figure 4 is a graph showing the results of inhibiting NF-κB production when HKSMM50 is treated with LPS-activated RAW264.7 cells 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 represents a positive control.
Figure 5 is a graph showing the results of inhibiting NO production when HKSMM50 is treated on LPS-activated RAW264.7 cells of the present invention. AHCC represents a positive control.
Figure 6 is a graph showing the results of inhibition of iNOS (left) and COX-2 (right) protein expression when HKSMM50 is treated on LPS-activated RAW264.7 cells of the present invention. AHCC represents a positive control.
Figure 7 is a graph showing the results of reducing the production of IL-1β, TNF-a, IL-6, and IL-4 when HKSMM50 is treated on LPS-activated RAW264.7 cells of the present invention. AHCC represents a positive control.
Figure 8 is a graph showing the results of improving SOD and CAT activities when HKSMM50 is treated on LPS-activated RAW264.7 cells of the present invention. AHCC represents a positive control.
Figure 9 is a graph showing the results of cytotoxicity (cells viability (%)) when HKSMM50 and AHCC are treated for 24 hours on Jurkat cells of the present invention. AHCC represents a 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 and 6 hours. AHCC represents a positive control.
Figure 11 is a graph showing the results of cell proliferation (%) when HKSMM50 was treated for 3 and 6 hours on Jurkat cells activated with ConA (concanavalin A) of the present invention. AHCC represents a positive control.
Figure 12 is a graph showing the decrease in cytosolic NAFT (nuclear factor of activated T-cell) protein when HKSMM50 was treated for 3 and 6 hours in Jurkat cells activated with ConA of the present invention. AHCC represents a positive control.
Figure 13 is a graph showing the increase in nuclear NAFT protein when HKSMM50 was treated for 3 and 6 hours in Jurkat cells activated with ConA of the present invention. AHCC represents a positive control.
Figure 14 is a graph showing the results of increased IL-2 production when HKSMM50 was treated for 3 and 6 hours in Jurkat cells activated with ConA of the present invention. AHCC represents a positive control.
Figure 15 is a graph showing the results of increased production of IFN-γ when HKSMM50 was treated for 3 and 6 hours in Jurkat cells activated with ConA of the present invention. AHCC represents a positive control.
Figure 16 is a graph showing the results of inhibiting plasma iNOS and COX-2 activities of 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 means 500 mg/100g of HKSMM, T2 means 1,000 mg/100g of HKSMM, and T3 means 2,000 mg/100g of HKSMM.
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 represents negative control, PC represents positive control, AHCC represents negative control, T1 represents 500 mg/100g of HKSMM, T2 represents 1,000 mg/100g of HKSMM, and T3 represents 2,000 mg/100g of HKSMM.

The present invention can be modified in various ways and has various embodiments, and thus 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 purpose of the present invention, the present invention provides an anti-inflammatory or immune-enhancing health functional food composition containing a shiitake mushroom mycelia complex culture (HKSMM) as an effective ingredient.

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

The above-mentioned shiitake mushroom mycelia composite culture (HKSMM) contains a shiitake mushroom mycelia solid culture in which shiitake mushroom mycelia are solid-cultured and a shiitake mushroom mycelia liquid culture in which shiitake mushroom mycelia are liquid-cultured.

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

In addition, the ethanol extract is defined as including any one of an extract obtained by extraction treatment, a diluted or concentrated extract, a dried product obtained by drying the extract, or a controlled or purified product.

In addition, the above-mentioned shiitake mushroom mycelia complex culture (HKSMM) may inhibit the expression of COX-2, iNOS, NF-κB, IL-1β, TNF-α, IL-4 and IL-6, and increase the expression of IL-2 and IFN-γ, but is not limited thereto.

The above composition may be manufactured in any one formulation selected from powder, granules, pills, tablets, capsules, candy, syrup, and beverage, but is not limited thereto.

When the health functional food composition of the present invention is used 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 a conventional method. The effective ingredient may be used appropriately according to its intended use.

There is no special limitation on the type of the above health functional food. Examples of foods to which the above health functional food composition can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea drinks, alcoholic beverages, and vitamin complexes, and include all health foods in the conventional sense.

In addition, the health functional food composition of the present invention can be manufactured as a food, particularly a functional food. The functional food of the present invention can include ingredients that are usually added. For example, it includes proteins, carbohydrates, fats, nutrients, and seasonings. For example, when manufactured as a drink, in addition to the effective ingredient, a natural carbohydrate or a flavoring agent can be included as an additional ingredient. The natural carbohydrate is preferably a monosaccharide (e.g., glucose, fructose, etc.), a disaccharide (e.g., maltose, sucrose, etc.), an oligosaccharide, a polysaccharide (e.g., dextrin, cyclodextrin, etc.), or a sugar alcohol (e.g., xylitol, sorbitol, erythritol, etc.). The flavoring agent can use a natural flavoring agent (e.g., thaumatin, stevia extract, etc.) and a synthetic flavoring agent (e.g., saccharin, aspartame, etc.).

In addition to the above health functional food composition, it may further contain various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc.

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

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

The pharmaceutical composition of the present invention may further comprise a suitable carrier, excipient or diluent commonly used in the manufacture 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 an appropriate combination.

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, aerosols, etc., external preparations, suppositories, and sterile injection solutions, respectively, according to conventional methods. Carriers, excipients, and diluents that can be included in the pharmaceutical composition including the above-mentioned shiitake mushroom mycelia composite culture (HKSMM) include various compounds or mixtures including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. When formulated, it is prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrating agents, and surfactants that are commonly used. Solid preparations for oral administration include tablets, pills, powders, granules, and capsules, and these solid preparations are prepared by mixing the above-mentioned shiitake mushroom mycelia composite culture (HKSMM) with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups, and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives may be included. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solutions and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases may include witepsol, macrogol, Tween 61, cacao butter, laurin butter, and glycerogelatin.

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 a solid culture of shiitake mushroom mycelia (HKSMM) comprises the steps of producing a solid culture of shiitake mushroom mycelia by solid-cultivating shiitake mushroom mycelia on barley medium, and mixing the shiitake mushroom mycelia with defatted soybean powder, K ₂ HPO ₄ and MgSO _4. The method may include a step of producing a liquid culture of shiitake mushroom mycelia in a liquid medium mixed with a solid culture medium and a step of mixing the solid culture and the liquid culture of shiitake mushroom mycelia.

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

Example 1. Production of HKSMM mycelial complex culture

Shiitake mushroom mycelia composite culture (HKSMM) is manufactured by mixing (A) the solid culture of Shiitake mushroom mycelia on barley medium, followed by hot air drying and powderization, and (B) the liquid culture of Shiitake mushroom mycelia on a medium using defatted soybean flour as the main ingredient, followed by drying and powderization, in a weight ratio of 2:1. The specific manufacturing method is as follows.

1. Production of powder from solid culture of shiitake mushroom mycelia (A)

1) Preparation of barley medium: Wash barley 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 above medium (121℃, 2 hours).

3) Inoculation of shiitake mushroom spawn: Inoculate the medium with pre-cultured shiitake mushroom spawn.

4) Solid culture: Culture the shiitake mushroom fungi inoculated onto the medium in a solid culture medium (25°C) for more than 10 days.

5) Drying: Dry the solid culture of cultured shiitake mushroom mycelia using hot air (50°C).

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

2. Production of powdered liquid culture of shiitake mushroom mycelia (B)

1) Preparation of liquid medium: Mix defatted soybean powder (2.5 g/L), yolk sugar (25 g/L), K ₂ HPO ₄ (0.025 g/L), and MgSO ₄ (0.025 g/L) with water and add foam remover Foam Clean according to the instructions 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 yolk sugar, and K ₂ HPO ₄ 0.0025 parts by weight, MgSO ₄ It is prepared by mixing 0.0025 parts by weight of cellulose and 97.245 parts by weight of water.

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

3) Inoculation of shiitake mushroom spawn: Inoculate pre-cultured shiitake mushroom mycelia spawn into the liquid medium.

4) Liquid culture: After culturing for more than 10 days in a liquid culture medium (25°C), β-glucan is extracted (121°C, 1 hour), then defatted soybean powder is mixed at 10% (w/v) to the extract and then dried with hot air.

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

3. Preparation of a complex culture of shiitake mushroom mycelia

The above-mentioned solid culture powder of Shiitake mushroom mycelia (A) and liquid culture powder of Shiitake mushroom mycelia (B) are mixed at a weight ratio of 2:1 (w/w) to prepare a Shiitake mushroom mycelia composite culture (HKSMM).

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

Example 2. Composite culture of shiitake mushroom mycelia ( HKSMM ) according to processing in vitro Anti-inflammatory effects in

1. Preparation of samples for cell experiments

The HKSMM manufactured in Example 1 was freeze-dried by extracting the water (0% ethanol) extract and the extracts extracted with 30%, 50%, 70%, and 95% aqueous ethanol concentrations (Fig. 1). The extraction yields of these freeze-dried products with respect to HKSMM were 24.9% for the water extract, and 32.5%, 43.6%, 43.0%, and 23.1% for the 30%, 50%, and 70% ethanol extracts, respectively.

These lyophilized products were measured for the degree of inhibition of NO production in LPS-treated mouse macrophage cells, RAW264.7 cells, by the Griess method (Fig. 2). All of these lyophilized products inhibited NO production compared to LPS treatment, but the 50% ethanol extract (hereinafter referred to as 'HKSMM50') inhibited NO production the most. Therefore, HKSMM50 was used in the cell experiment.

2. Evaluation of cell viability of HKSMM50

We investigated at what concentration HKSMM50 exhibits cytotoxicity in LPS-treated RAW264.7 cells. LPS-treated RAW264.7 cells were treated with 5,000 ㎍/ml of the HKSMM50 and cultured for 24 hours, and cytotoxicity (cell viability) was measured using a CCK-8 kit (Fig. 3). Compared to LPS treatment, HKSMM50 did not affect cell growth up to a concentration of 5,000 ㎍/ml. AHCC, which was used as a positive control, also did not affect cell growth at concentrations up to 5,000 ㎍/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 ㎍/ml, and cultured for 24 hours. The content of NF-κB in the supernatant was measured using an ELISA kit (Fig. 4). AHCC 100 ㎍/ml was treated as a positive control.

Compared with LPS treatment, HKSMM50 treatment significantly reduced NF-κB content (p<0.001). HKSMM50 500 ㎍/ml treatment reduced NF-κB content to the untreated level. This result suggests 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) NO content and PGE2 (prostaglandin E2) reduction effect of HKSMM

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

In Fig. 6, to confirm the role of HKMSS50 in reducing the contents of NO and PGE2, HKSMMf50 was treated at concentrations of 0, 20, 200, and 500 ㎍/ml in RAW264.7 cells treated with LPS, and cultured for 24 hours. The expression of iNOS and COX-2 proteins in the supernatant was examined by Western blotting. AHCC was treated at concentrations of 0, 20, 200, and 500 ㎍/ml as a positive control. 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 of HKSMM50. COX-2 was also significantly reduced in a concentration-dependent manner, but there was some variation. When HKSMM50 was compared with AHCC at the same treatment concentration at the pretreatment concentration, HKSMM50 inhibited the expression of iNOS and COX-2 more strongly.

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

4. Inhibition of pro-inflammatory cytokines production by HKSMM50

The effect of HKSMM50 on the contents 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 μg/ml and cultured for 24 h. The contents of IL-1β, TNF-α, IL-4, and IL-6 in the supernatant were measured using an ELISA kit (Fig. 7). AHCC 100 μg/ml was treated as a positive control.

Compared to LPS treatment, HKSMM50 treatment significantly decreased the contents of four cytokines in a concentration-dependent manner (p<0.05 - p<0.001). In particular, HKSMM50 500 ㎍/ml treatment decreased the contents of TNF-α and IL-6 to the level of the untreated group. AHCC 100 ㎍/ml, used as a positive control, was similar to the same concentration of HKSMM50. These results imply that the HKSMM50 sample has an anti-inflammatory effect by decreasing 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) activity

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

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

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

Thus, HKSMM50 exhibits anti-inflammatory effects by inhibiting the translocation of NF-κB in the cytoplasm to the nucleus, suppressing the expression of COX-2 (reducing PGE2 production), and suppressing the expression of iNOS (reducing NO production) in LPS-activated mouse macrophage cells, RAW264.7.

Example 3. According to treatment with HKSMM mycelia complex culture in vitro Immune-boosting effect in

1. Sample

The shiitake mushroom mycelia complex culture (HKSMM) manufactured in Example 1 was extracted with 50% concentration of aqueous alcohol, and the extract was freeze-dried and used as a sample (HKSMM50).

2. Measurement of Jurkat cell viability of HKSMM50

Jurkat cells were treated with HKSMM50 at concentrations of 0 to 100 μg/ml and cultured for 24 hours, after which cytotoxicity was measured using the MTT assay. Jurkat cells are a type of cancer cell line, a cell line obtained from a patient with T cell leukemia (Acute T cell leukemia), and a single (clone) cell population. By giving some stimuli such as ConA (concanavalin A) to Jurkat in an unactivated state, the characteristics of Th1 or Th2 can be expressed.

The above measurement results showed that there was no significant decrease even at a concentration of 100 μg/mL of HKSMM50 (Fig. 9). Jurkat cells were treated with AHCC, a positive control, at a concentration of 0 to 100 μg/mL, the same as HKSMM50, and cultured for 24 hours, after which cytotoxicity was measured using an MTT assay. As a result, there was no significant decrease even at a concentration of 100 μg/mL of AHCC (Fig. 9).

3. Measurement of cell proliferation of HKSMM50

To confirm the proliferation ability of HKSMM50 on Jurkat cells, 90 ㎕ of Jurkat was dispensed into each 96-well plate at a concentration of 2 × 10 ³ cells/well and stabilized for 24 hours. Then, 10 ㎕ of the medium prepared to have a final concentration of 25, 50, and 100 μg/㎖ of the HKSMM50 sample was added, cultured for 3 and 6 hours, and the cell proliferation rate was measured (the positive control was AHCC 50 μg/㎖). The cases where Jurkat cells were activated by treating with ConA and cases where ConA was not treated were compared.

1) When ConA was not treated (Fig. 10)

When HKSMM50 was treated at various concentrations in Jurkat cells and cell proliferation was investigated according to treatment time, cell proliferation increased at 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 was treated (Fig. 11)

When HKSMM50 was treated at various concentrations in Jurkat cells and cell proliferation according to treatment time was investigated, it increased in the 3-hour treatment at all concentrations, and it significantly increased in the 100 μg/ml treatment (p<0.01). Similar results were obtained in the 6-hour treatment as in the 3-hour treatment. The positive control, AHCC 50 μg/ml treatment, showed a treatment effect similar to that of 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, inducing the expression of IL-2 and IFN-γ mRNA. Therefore, the production of IL-2 and IFN-γ is utilized as an indicator of immune enhancement. Therefore, in order to measure the cytosolic 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, then treated with ConA and cultured for 2 hours. The cells were harvested and the content of NFAT in the cytosol and nucleus was analyzed by Western blotting.

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

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

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

Treatment of ConA-activated Jurkat cells with HKSMM50 sample dephosphorylated cytosolic NFAT and translocated it to the nucleus (Fig. 12, Fig. 13). Therefore, to confirm that HKSMM50 sample increases the production of immunostimulatory cytokines in Jurkat cells, ConA-activated Jurkat cells were treated with HKSSM50 sample (0, 25, 50, and 100 μg/ml), and the IL-2 (Fig. 14) and IFN-γ (Fig. 15) contents were measured after 3 and 6 hours of incubation. The positive control AHCC was treated at 100 μg/ml.

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

In Fig. 15, the IFN-γ content was higher in the ConA and HKSMM50 sample treatments than in the control group at 3 and 6 hours of culture, and the HKSMM50 sample treatments of 50 μg/ml and 100 μg/ml significantly increased the IFN-γ content compared to the ConA treatment (p<0.001). The IFN-γ increase by AHCC (100 μg/ml) treatment was not different from the 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 flavonoid, and HKSMM50 exhibits an immune-enhancing effect that promotes the production of IL-2 and IFN-γ by moving NAFT from the cytosol to the nucleus. Therefore, HKSMM containing 14% or more of β-glucan has an immune-enhancing effect and can be used as an immune-enhancing composition.

Example 4. Treatment with HKSMM (HKSMM) mycelial complex culture in vivo In Anti-inflammatory effect

1. Sample: HK shiitake mushroom mycelia (HKSMM)

2. Experimental animals and treatment

Female Balb/c mice were treated with 10 normal chow-fed negative control (NC) groups, 10 AHCC-fed positive control (PC) groups, and HKSMM-fed groups T1 (500 mg/100 g), T2 (1,000 mg/100 g), and T3 (2,000 mg/100 g). The mice were given ad libitum food and water that had passed through a microfilter and an ultraviolet sterilizer. The serum of the experimental animals was separated by centrifugation after collecting blood at 4 and 6 weeks and leaving it on ice for 20 min. The animal housing was maintained in a constant temperature and humidity environment with a 12-h lighting cycle. The animal experiments were approved by the IACUC, Gyeongnam Environmental Toxicology Headquarters, Korea Institute of Toxicology (Approval No. 2108-0004).

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

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

iNOS and COX-2 produce the inflammatory cytokines NO and PGE2, respectively. 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. The results showed that it was significantly lower in the 4- and 6-week treatment groups than in the NC group. In addition, HKSMM treatment lowered the content of p-IKB in the 4- and 6-week treatment groups than in the NC treatment. Flavonoids have anti-inflammatory effects by reducing the expression of p-65 and IκBα. Therefore, it is suggested that β-glucan and total flavonoids of HKSMM increase the immune and anti-inflammatory functions by inhibiting the expression of NF-κB in Balb/c mice.

Meanwhile, the above detailed description should not be construed as restrictive in all respects but should be considered as illustrative. The scope of the present invention should be determined by a 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)

Contains a shiitake mushroom mycelia complex culture, which is a mixture of a solid culture of shiitake mushroom mycelia and a liquid culture of shiitake mushroom mycelia, as an effective ingredient.
The above-mentioned shiitake mushroom mycelia solid culture is a shiitake mushroom mycelia solid culture obtained by solid-cultivating shiitake mushroom mycelia in a barley medium, and the above-mentioned shiitake mushroom mycelia composite culture is a shiitake mushroom mycelia liquid culture obtained by liquid-cultivating shiitake mushroom mycelia in a liquid medium mixed with defatted _soybean powder, _K2HPO4 , and _MgSO4 .
An anti-inflammatory or immune-enhancing health functional food composition characterized in that the above shiitake mushroom mycelia liquid culture is a shiitake mushroom mycelia liquid culture produced by culturing in a liquid culture vessel, extracting β-glucan, and then mixing it with defatted soybean flour.
In the first paragraph,
An anti-inflammatory or immune-enhancing health functional food composition characterized in that the above shiitake mushroom mycelia complex culture is an extract obtained by extracting the above shiitake mushroom mycelia complex culture with water or aqueous ethanol.
In the first paragraph,
The above shiitake mushroom mycelia complex culture is an extract obtained by extracting the above shiitake mushroom mycelia complex culture with water or 50% concentration ethanol,
An anti-inflammatory or immune-enhancing health functional food composition characterized in that the above-mentioned shiitake mushroom mycelia complex culture inhibits the expression of COX-2, iNOS, NF-κB, IL-1β, TNF-α, IL-4 and IL-6, and increases the expression of IL-2 and IFN-γ.
An anti-inflammatory or immune-enhancing health functional food composition, characterized in that in claim 1, the composition is manufactured in any one formulation selected from powder, granules, pills, tablets, capsules, candy, syrup, and beverage.
Contains a shiitake mushroom mycelia complex culture, which is a mixture of a solid culture of shiitake mushroom mycelia and a liquid culture of shiitake mushroom mycelia, as an effective ingredient.
The above-mentioned shiitake mushroom mycelia solid culture is a shiitake mushroom mycelia solid culture obtained by solid-cultivating shiitake mushroom mycelia in a barley medium, and the above-mentioned shiitake mushroom mycelia composite culture is a shiitake mushroom mycelia liquid culture obtained by liquid-cultivating shiitake mushroom mycelia in a liquid medium mixed with defatted _soybean powder, _K2HPO4 , and _MgSO4 .
A pharmaceutical composition for preventing or treating an inflammatory disease, characterized in that the above shiitake mushroom mycelia liquid culture is a shiitake mushroom mycelia liquid culture prepared by culturing in a liquid culture vessel, extracting β-glucan, and then mixing it with defatted soybean flour.
In paragraph 5,
A pharmaceutical composition for preventing or treating an inflammatory disease, characterized in that the above shiitake mushroom mycelia complex culture is an extract obtained by extracting the above shiitake mushroom mycelia complex culture with water or ethanol.
In paragraph 5,
The above shiitake mushroom mycelia complex culture is an extract obtained by extracting the above shiitake mushroom mycelia complex culture with water or 50% concentration ethanol,
The above-mentioned shiitake mushroom mycelia complex culture is characterized by inhibiting the expression of COX-2, iNOS, NF-κB, IL-1β, TNF-α, IL-4 and IL-6, and increasing the expression of IL-2 and IFN-γ, a pharmaceutical composition for preventing or treating an inflammatory disease.
A step for producing a solid culture of shiitake mushroom mycelia by cultivating shiitake mushroom mycelia on barley medium;
A step for producing a liquid culture of shiitake mushroom mycelia by cultivating the shiitake mushroom mycelia in a liquid medium mixed with defatted soybean powder, K ₂ HPO ₄ and MgSO ₄ ; and
A step of mixing the solid culture and liquid culture of the above shiitake mushroom mycelia;
A method for producing a shiitake mushroom mycelia complex culture, characterized in that the step of producing the above shiitake mushroom mycelia liquid culture includes a step of producing a shiitake mushroom mycelia liquid culture by mixing in defatted soybean flour after extracting β-glucan after culturing in a liquid culture vessel.