TWI730244B - Method for using glucan and mushroom polysaccharide composition to promote the activity of natural killer cells or to promote the production of IgA or IgM antibodies - Google Patents

Abstract

A glucan and mushroom polysaccharide composition comprising 20-50% by weight of β-polydextrose concentrate, 20-50% by weight of Ganoderma lucidum mycelium extract, 1 to 50% by weight 5% Antrodia cinnamomea mycelium extract, 1 to 5% by weight Cordyceps mycelium extract, 1 to 5% by weight Phellinus igniarius fruiting body extract, 1 to 5% by weight Brazil Mushroom fruit body extract, 1 to 5% by weight of Maitake mushroom fruit body extract, and 1 to 5% by weight of Hericium erinaceus fruit body extract. A method of using dextran and mushroom polysaccharide composition to promote the activity of natural killer cells and a method of using dextran and mushroom polysaccharide composition to promote the production of IgA or IgM antibodies are also disclosed herein.

Description

Use dextran and mushroom polysaccharide composition to promote the vitality of natural killer cells Sexuality or methods to promote the production of IgA or IgM antibodies

The present invention relates to a polysaccharide composition; in particular, it refers to a glucan and mushroom polysaccharide composition that can enhance immune function and uses the glucan and mushroom polysaccharide composition to promote the activity or promotion of natural killer cells Method of IgA or IgM antibody production.

The polysaccharides present in mushrooms are known to have the effect of enhancing immune function. Taking Ganoderma lucidum as an example, it has been widely used as a raw material for nutrition and health foods for a long time. β-Glucan (β-Glucan) is a kind of polysaccharide, which is closely related to the immune function of polysaccharide.

In the past, polysaccharide health products included single-component products and compound products, and compound products have better immunomodulatory effects and are more favored by consumers. With the increasing demand of consumers for health foods in modern society, it is urgent to develop compound polysaccharide products with both flavor and health effects.

In view of this, the purpose of the present invention is to provide a glucan and mushroom polysaccharide composition, which can improve the immune function.

In order to achieve the above objective, the present invention provides a glucan and mushroom polysaccharide composition comprising 20-50% by weight of β-polydextrose concentrate and 20-50% by weight of Ganoderma lucidum Mycelium extract, 1 to 5% by weight Antrodia cinnamomea Mycelium extract, 1 to 5% by weight of Cordyceps mycelium extract, 1 to 5% by weight of Phellinus igniarius fruiting body extract, 1 to 5% by weight of Brazilian mushroom fruiting body extract , 1 to 5% by weight of Maitake mushroom fruit body extract and 1 to 5% by weight of Hericium erinaceus fruit body extract.

In order to achieve the above-mentioned object, the present invention also provides a method for producing glucan and mushroom polysaccharide composition, which comprises fermenting a cell fermentation product of a black yeast, a cell fermentation product of a Ganoderma lucidum, and an Antrodia cinnamomea The fermentation product of the bacteria, the fermentation product of the fungus of Trichosporum sinensis, the fruiting body powder of Phellinus igniarius, the fruiting body powder of Brazilian mushroom, the fruiting body powder of Maitake mushroom and the fruiting body powder of Hericium erinaceus, respectively The water is mixed in a weight ratio of 10:1 to 40:1 to form a mixture, and then the aforementioned mixture is stirred at 70 to 100°C for 2 to 6 hours, and then filtered to remove the solids. The filtrate is taken and heated and sterilized. Obtained a β-polydextrose concentrate, a Ganoderma lucidum mycelium extract, an Antrodia cinnamomea mycelium extract, a Cordyceps mycelium extract, a Phellinus igniarius fruiting body extract, a Brazilian mushroom fruiting body extract, Maitake mushroom fruit body extract and a Hericium erinaceus fruit body extract, and then all the aforementioned extracts are blended and concentrated to obtain the glucan and mushroom polysaccharide composition.

The present invention also provides a method for using a glucan and mushroom polysaccharide composition to promote the activity of natural killer cells, comprising: providing the glucan and mushroom polysaccharide composition, which comprises 20-50% by weight % Beta -polydextrose concentrate, 20-50% by weight of Ganoderma lucidum mycelium extract, 1-5% by weight of Antrodia cinnamomea mycelium extract, 1-5% by weight of Cordyceps sinensis Silk body extract, 1 to 5% by weight of Phellinus igniarius fruiting body extract, 1 to 5% by weight of Brazilian mushroom fruiting body extract, 1 to 5% by weight of Maitake mushroom fruiting body extract, and 1 to 5% by weight of Hericium erinaceus fruit body extract; culture a target cell and a spleen cell in an incubator, where the number of the target cell: the number of the spleen cell = 1:100 or 1: The ratio of 200 is mixed in the glucan and mushroom polysaccharide composition; and the spleen cells are taken and analyzed to obtain the activity of the natural killer cells.

The present invention further provides a method for promoting the production of IgA or IgM antibodies by using a glucan and mushroom polysaccharide composition, comprising providing the glucan and mushroom polysaccharide composition, which comprises 20-50% by weight % Beta -polydextrose concentrate, 20-50% by weight of Ganoderma lucidum mycelium extract, 1-5% by weight of Antrodia cinnamomea mycelium extract, 1-5% by weight of Cordyceps sinensis Silk body extract, 1 to 5% by weight of Phellinus igniarius fruiting body extract, 1 to 5% by weight of Brazilian mushroom fruiting body extract, 1 to 5% by weight of Maitake mushroom fruiting body extract, and 1 to 5% by weight of a Hericium erinaceus fruit body extract; adding the glucan and mushroom polysaccharide composition to an anticoagulated whole blood; and determining the IgM or IgA contained in the anticoagulated whole blood The value of the antibody.

Figure 1 is the sensitization process of the experimental animals; Figure 2 is the weekly average weight change chart of the experimental animals; Figure 3 is the phagocytic cell activity chart of the experimental animals; Figure 4 is the natural killer cell activity chart of the experimental animals; and Figure 5 is the blood of the experimental animals The content of non-specific antibody IgA and IgM.

In order to explain the present invention more clearly, the preferred embodiments are described in detail in conjunction with the drawings as follows.

The glucan and mushroom polysaccharide composition of the present invention are extracted from a variety of mushrooms. The main formula includes a β-polydextrose concentrate, a Ganoderma lucidum mycelium extract, and an Antrodia cinnamomea mycelium extract , A Cordyceps mycelium extract, a Phellinus igniarius fruiting body extract, a Brazilian mushroom fruiting body extract, a Maitake mushroom fruiting body extract and a Hericium erinaceus fruiting body extract.

The preparation method of the glucan and mushroom polysaccharide composition of the present invention will be described below.

(1) The method of obtaining polysaccharides from the fermentation products of bacteria

1.1 Preparation of bacterial fermentation broth

The strains are respectively inoculated with carbon source (e.g. glucose, sucrose or mannitol) 0.5 to 5.0 weight percent, nitrogen source (e.g. yeast extract, yeast protein or soy protein) 0.1 to 1.5 weight percent, and other trace substances (e.g., trace amounts) Elements and inorganic substances), in a medium with a pH between 5.0 and 6.5, cultured with aeration and stirring at a temperature of 20 to 30°C for 2 to 7 days to obtain the fermentation product of the bacteria.

1.2 Obtain polysaccharide extract

After the bacterial fermentation product and water are mixed into a mixture in a weight ratio of 10:1 to 40:1, the mixture is stirred at 70 to 100°C for 2 to 6 hours, and the mixture is filtered by pressure to remove solids. The filtrate is heated and sterilized to obtain a polysaccharide extract.

(2) Ways to obtain polysaccharides from fruiting bodies

2.1 Preparation of fruit body mixture

After mixing the fruit body powder and water in a weight ratio of 10:1 to 40:1 to form a mixture, the mixture is stirred at 70 to 100°C for 2 to 6 hours.

2.2 Obtain polysaccharide extract

The aforesaid mixture is filtered separately to remove solids, and the filtrate is taken and heat-sterilized to obtain a polysaccharide extract.

(3) Determination method of polysaccharide specifications

3.1 Calculation of β-glucan content

After adding β-polydextrose concentrate to an appropriate volume of buffer, mix well, then add α-amylase, protease and amyloglucosidase three enzymes in sequence after treatment, Precipitation with 4 times the volume of alcohol, the β-glucan is separated from the solution by sedimentation, the precipitate is collected and washed with alcohol and dried. The dried precipitate is treated with strong acid and high temperature hydrolysis, and then measured after neutralization with acid and alkali Glucose, and calculate the β-glucan content.

3.2 Detection of polysaccharide concentration

The β-polydextrose concentrate, Ganoderma lucidum mycelium extract, Antrodia cinnamomea mycelium extract, Cordyceps mycelium extract, Phellinus igniarius fruiting body extract, Brazilian mushroom fruiting body extract, Maitake mushroom fruiting body extract and The Hericium erinaceus fruiting body extract is diluted to appropriate concentration and injected into the dialysis membrane (specification: MW6000~8000), and dialysis is performed with flowing water for 48 hours (flow rate: 0.2L/min). The dialysis liquid system uses phenol-sulfuric acid Method (Phenol-sulfuric acid assay) to determine the concentration of polysaccharides. When sugars encounter strong acids, the hydroxyl groups on the structural formula combine with phenols to produce an orange liquid. Therefore, the colorimetric method can be used to determine the absorbance value to detect the concentration of its polysaccharides.

(4) Examples

In this example, black yeast ( Aureobasidium pullulans , BCRC number 930184), Ganoderma lucidum , Antrodia camphorate , Hirsutella sinensis , and Phellinus linteus , The fruiting bodies of Brazilian mushroom ( Agaricus blazei murill ), maitake mushroom ( Grifola frondosa ) and Hericium erinaceus ( Hericium erinaceus ) were respectively prepared in accordance with the aforementioned method and contained the following formula:

Among them, the black yeast glucan (single prescription group) is used as the control formula in this example, where the single prescription group is to concentrate β-polydextrose concentrate to specifications: polysaccharide>6g/L, glucan>5g /L. The black yeast glucan and mushroom polysaccharide composition (compound group) are used as the experimental group formula in this example. Among them, this compound group is the first combination of Antrodia cinnamomea mycelium extract, cordyceps mycelium extract, and Phellinus igniarius The fruiting body extract and the Brazilian mushroom fruiting body extract are mixed at a ratio of 1:1:1:1, and then concentrated to a polysaccharide >5g/L to obtain a mixture A, and then the Maitake mushroom fruiting body extract and monkey The head mushroom fruit body extract is mixed at a ratio of 1:1 and concentrated to a polysaccharide> 5g/L to obtain a mixture B. Finally, β-polydextrose concentrate and Ganoderma lucidum mycelium extract are added to the aforementioned mixture A And mixed solution B, and then mixed and concentrated to polysaccharide>6g/L, which is the black yeast glucan and mushroom polysaccharide composition of this example (compound group).

The glucan and mushroom polysaccharide composition of the present invention includes: 44% by weight of β-polydextrose concentrate, 44% by weight of Ganoderma lucidum mycelium extract, 2% by weight of Antrodia cinnamomea mycelium extract, 2% by weight of Cordyceps mycelium extract, 2% by weight of Phellinus igniarius fruiting body extract, 2% by weight of Brazilian mushroom fruiting body extract, weight percentage 2% Maitake mushroom fruit body extract and 2% Hericium erinaceus fruit body extract.

In order to evaluate the ability of the glucan and mushroom polysaccharide composition of the present invention to promote the proliferation of immune cells, the following animal experiments were used to evaluate specific and non-specific immunomodulatory effects.

(1) Design of test group and dosage of test substance

6-week-old BALB/c female mice were used for animal experiments. As shown in Table 1, the experimental groups were designed with water control group, black yeast glucan low-dose group (hereinafter referred to as the single low-dose group), and black yeast glucan low-dose group. Glycan high-dose group (hereinafter referred to as single high-dose group), black yeast glucan and compound mushroom polysaccharide low-dose group (hereinafter referred to as compound low-dose group), and black yeast glucan and compound mushroom polysaccharide high-dose Group (hereinafter referred to as the compound high-dose group), the number of test animals in each group is 8; the water control group is only given deionized water, and the single prescription and compound low-dose groups are all dosed to 1 times the recommended dose of the human body. Both the single and compound high-dose groups are 3 times the recommended dose for humans. The recommended dose for humans is 30 mL/day for a 60 kg person. The dose conversion factor 12.3 per kg body weight of the animal and the human body is used to convert the dose for each group of mice. The test animals were given the test substance and negative control solution (i.e. deionized water) by gastric tube orally every day for 9 consecutive weeks with a volume of 20 mL/kg.

(2) Sensitized mice

As shown in Figure 1, after 3 weeks of administration of the test substance, ovalbumin (OVA) was used as the immunogen to sensitize the test mice, and an injection volume of 100μL was given by subcutaneous injection at a dosage of 3μg OVA and Freund's complete Adjuvant (complete Freund's adjuvant) mixture, 2 weeks later, 100μL of 12μg OVA and incomplete Freund's adjuvant mixture were injected subcutaneously again for the second sensitization, and 100μL subcutaneously injected again 2 weeks later The mixed solution of 12μg OVA and incomplete Freund's adjuvant is the third sensitization. Blood was collected from the eye socket before and after sensitization for total antibody and OVA specific antibody Analysis. After the experiment, the mice were sacrificed, and blood and spleen cells were collected for immune function evaluation and analysis, including the determination of immune cell proliferation, immune cell hormone secretion, immune cell types, and serum antibody concentration, etc., to evaluate the glucan and mushroom of the present invention The specific and non-specific immunomodulatory effects of polysaccharide composition.

(3) Observation of experimental animals

After 9 weeks of feeding, there were no significant differences in body weight, absolute and relative weight of the spleen, and the number of spleen cells between the groups, indicating that the two test substances had no adverse effects on the growth of mice, as shown in Table 2, Table 3 and Figure 2. .

(4) Non-specific immunomodulatory effect

4.1 Phagocyte activity test

Phagotest kit was used to detect the phagocytic activity of phagocytes in the blood. On the 4th week, take 100μl of anticoagulated whole blood from the collected blood and mix it with 20μl of opsonized FITC-labelled E.coli , and place it in a 37℃ water bath as the experimental group or on ice as the negative control group for 10 minutes, then Place on ice to stop phagocytosis, add 100μl of ice-cold quenching solution, and mix well. Centrifuge and clean. Add 200μl of DNA staining solution, place on ice, and react for 10 minutes in the dark, and then analyze by flow cytometer within 60 minutes. Phagocytosis activity (%) = phagocytosis of the experimental group (%)-phagocytosis of the negative control group (%).

As shown in Table 4 and Figure 3, the activity of phagocytic cells in the blood of the single-prescription low-dose group was significantly higher than that of the water control group (p<0.05). Although the average value of the single-prescription high-dose group was also higher than that of the water control group, it was not statistically significant. Significant difference. There was no significant difference between the low-dose compound group and the high-dose group and the water control group.

4.2 Natural killer cell activity test

Use LIVE/DEAD cell-mediated cytotoxicity kit to detect the ability of natural killer cytotoxicity to kill target cells YAC-1. First label the YAC-1 cells with the dye DIOC ₁₈ , incubate in a 37°C, 5% CO ₂ incubator for 4 hours, and then mix the target cells: spleen cells at a ratio of 1:100 or 200 as the experimental group. ℃, 5% CO ₂ incubator for 4 hours. The negative control group replaced spleen cells with RPMI 1640+10% FBS. Analyze by flow cytometer. Natural killer cell activity (%) = (cell death rate in the experimental group)-(cell death rate in the negative control group).

As shown in Table 5 and Figure 4, the compound low-dose group and high-dose group can significantly promote the activity of natural killer cells (p<0.05).

4.3 Immune cell proliferation test

Add 100μl/well of medium or 2.5μg/ml Concanavalin A (Con A), or 10μg/ml lipopolysaccharide (LPS), or 200μg/ml OVA to the 96-well culture plate, and repeat each treatment three times. Then add 100μl/well of 4×10 ⁶ cells/ml spleen cell suspension, place it in a 37°C, 5% CO ₂ incubator, and incubate for 48 or 72 hours. Then add 20μl of MTT reagent (5mg/ml) to each hole, and continue to incubate for 4 to 6 hours. Centrifuge at 250×g for 10 minutes. Aspirate and discard 200μl/well supernatant. Add 200μl/well of dimethyl sulfoxid (DMSO), shake for 5 minutes, and measure the absorbance of _{A at 570 nm with an ELISA reader.} Stimulation index (SI)=(A ₅₇₀ nm treatment-A ₅₇₀ nm medium control)/(A ₅₇₀ nm cell only-A ₅₇₀ nm medium control).

As shown in Table 6, it was found from the experimental results that there was no difference between the groups regardless of whether it was treated with Con A or LPS.

4.4 Cytohormone analysis test

Add culture medium, Con A (final concentration: 2.5 μg/ml) or OVA (final concentration: 100 μg/ml) to the 24-well culture plate, and add spleen cells to make the total number of ^{cells per well 5×10 6} cells/well. The cell culture supernatant was collected 24 and 48 hours later. Afterwards, the sandwich enzyme-linked immunosorbent assay (sandwich-ELISA) was used to test the interferon- γ (Interferon γ, IFN- γ ) and interleukin-5 (Interleukin-5, IL-5) content.

As shown in Table 7, there was no significant difference among the other groups except that the Con A-stimulated IL-5 of the compound low-dose group was significantly lower than that of the water control group (p<0.05).

4.5 Test of antibody content in blood

The anticoagulated whole blood stored at 4°C was used to measure the total antibodies of IgG, IgM, and IgA contained in it by sandwich-ELISA. As shown in Table 8 and Figure 5, the blood antibody test results at week 8 showed that the compound high-dose group can significantly promote the production of IgA and IgM antibodies (p<0.05).

(5) Specific immunomodulatory effect

The concentration of OVA-specific IgG antibody contained in anticoagulated whole blood stored at 4°C was measured by sandwich-ELISA. The 96-well plate was coated with 10μg/ml OVA (dissolved in 0.05M Sodium carbonate buffer at pH 9.6). liquid).

Referring again to Table 8, in terms of the production of specific antibody anti-OVA IgG, there was no difference between the single low-dose group and high-dose group, compound low-dose group and high-dose group and the water control group.

Also refer to Table 6 and Table 7, in the two indicators of OVA-specific lymphocyte proliferation and OVA-specific cytokine, single low-dose group and high-dose group, compound low-dose group and high-dose group and water control group no difference.

According to the above, black yeast glucan, black yeast glucan and compound mushroom polysaccharide all have a regulatory effect on non-specific immune response.

The above are only the preferred and feasible embodiments of the present invention. Any equivalent changes made by applying the specification of the present invention and the scope of the patent application should be included in the patent scope of the present invention.

Claims (4)

A method for using glucan and mushroom polysaccharide composition to promote the activity of natural killer cells, comprising: providing the glucan and mushroom polysaccharide composition, which contains one of 20 to 50% β by weight percentage -Polydextrose concentrate, 20-50% by weight of Ganoderma lucidum mycelium extract, 1 to 5% by weight of Antrodia cinnamomea mycelium extract, 1 to 5% by weight of Cordyceps sinensis mycelium extract , 1 to 5% by weight of Phellinus igniarius fruiting body extract, 1 to 5% by weight of Brazilian mushroom fruiting body extract, 1 to 5% by weight of Maitake mushroom fruiting body extract, and 1 to 5% by weight A 5% Hericium erinaceus fruit body extract; culture a target cell and a spleen cell in an incubator, where the number of the target cell: the number of the spleen cell is mixed at a ratio of 1:100 or 1:200 In the glucan and mushroom polysaccharide composition; and taking the spleen cells for analysis to obtain the activity of the natural killer cells. The method for using glucan and mushroom polysaccharide composition to promote the activity of natural killer cells as described in claim 1, wherein the β-polydextrose concentrate contains the β-polydextrose concentrate of black yeast. A method for using glucan and mushroom polysaccharide composition to promote the production of IgA or IgM antibodies, comprising: providing the glucan and mushroom polysaccharide composition, which comprises one of 20 to 50% by weight β -polydextrose concentrate, 20-50% by weight of Ganoderma lucidum mycelium extract, 1 to 5% by weight of Antrodia cinnamomea mycelium extract, 1 to 5% by weight of Cordyceps sinensis mycelium extract Liquid, 1 to 5% by weight of Phellinus igniarius fruiting body extract, 1 to 5% by weight of Brazilian mushroom fruiting body extract, 1 to 5% by weight of Maitake mushroom fruiting body extract, and 1 by weight To 5% of the Hericium erinaceus fruiting body extract; add the glucan and mushroom polysaccharide composition to the anticoagulated whole blood; and determine the value of the IgM or IgA antibody contained in the anticoagulated whole blood . The method for promoting the production of IgA or IgM antibodies using a glucan and mushroom polysaccharide composition as described in claim 3, wherein the β-polydextrose concentrate comprises the β-polydextrose concentrate of black yeast.

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