Immunopotentiating substance comprising beta-glucan from black yeast and ginsenoside Rg3 and method for preparing the same

Abstract

This invention provides an immune-enhancing substance comprising black yeast β-glucan and ginsenoside Rg3, wherein the content of black yeast β-glucan is 3.5-4.8 g / L and the content of ginsenoside Rg3 is 1.3-4.21 mg%. This immune-enhancing substance is prepared as follows: black yeast is cultured in a culture medium containing red ginseng extract... Aureobasidium pullulans Synthesize β-glucan from black yeast, while using black yeast ( Aureobasidium pullulans Ginsenosides are bioconverted into Rg3 in red ginseng extract. This immune-enhancing substance can be administered orally for anti-cancer, anti-tumor, and immune-enhancing purposes.

Description

Technical Field

[0001] This invention relates to an immune-enhancing substance with anti-cancer, anti-tumor, and immune-enhancing effects, and its preparation method. Background Technology

[0002] Beta-glucan is one of the key substances for inhibiting malignant tumors such as cancer and for enhancing immunity. Beta-glucan activates macrophages, NK cells, T cells, and cytotoxic T cells that attack infected cells and cancer cells in the body, and exhibits functions such as clearing bacteria and foreign substances that have entered the body, enhancing immunity and resistance to inhibit disease. Furthermore, due to these effects, beta-glucan has been reported to have functions such as inhibiting allergies, inhibiting malignant tumors such as cancer, lowering blood sugar levels, promoting urination, regulating blood pressure, and lowering blood cholesterol and triglyceride levels.

[0003] Among β-glucans, β-1,3-1,6-glucan has attracted much attention due to its high immunomodulatory effect. It is a polysaccharide synthesized and secreted in mushrooms and yeast.

[0004] Under specific culture conditions, *Acer niger* synthesizes β-glucan and secretes it extracellularly. β-glucan possesses immunomodulatory effects due to its structural and immunological properties. *Acer niger* β-glucan (APβG) is a specific polysaccharide derived exclusively from *Acer niger*, in which the main chain is composed of β-1,3 bonds, and 50-80% of the branches are composed of β-1,6 bonds; its molecular weight is small (~100 kDa).

[0005] Generally, black yeast is cultured in a liquid medium consisting of sugar, glucose, rice bran, and ascorbic acid to produce β-1,3-1,6-glucan. In existing methods for producing β-1,3-1,6-glucan, sugar and glucose are used as the carbon source for black yeast growth.

[0006] Red ginseng contains ginsenosides and aglycones, which are among the substances that have anti-cancer activity and special anti-cancer effects.

[0007] The ginsenosides in red ginseng are produced during the hydrolysis process through heat treatment. The main ginsenoside contents of red ginseng are approximately: Rg1 3.3 mg / g, Re 2.0 mg / g, Rb1 5.8 mg / g, Rc 1.7 mg / g, Rb2 2.3 mg / g, and Rd 0.4 mg / g.

[0008] The main active substances for cancer prevention are CK, Rg3, and Rh2. They delay the development of cancer and angiogenesis by effectively inhibiting vascular endothelial growth factor-dependent angiogenesis and weakening epithelial cell migration.

[0009] Recently, increasing the content of ginsenosides, which possess high anti-cancer activity, has become an urgent issue. To address this problem, HPLC and enzymatic treatments have been used, but these are inefficient and require significant manpower. Currently, biotransformation is the most widely used method.

[0010] Biotransformation is a biological method used to increase or decrease the content of medicinal substances, such as certain active ingredients in medicinal plants. Several biotransformation methods have been used, but the most widely employed method utilizes microorganisms. In recent years, the biotransformation method using several microorganisms (Lactobacillus, Ganoderma lucidum, etc.) has been used to increase the content of ginsenosides in ginseng.

[0011] In the prior art, substances with anticancer, antitumor and immune-enhancing effects, such as β-glucan and red ginsenosides, and their preparation methods have been disclosed, but an effective method for preparing compound immune enhancers with superior immune-enhancing effects has not yet been disclosed.

[0012] The purpose of this invention is to overcome the shortcomings of the prior art and to provide a compound immune enhancer containing β-glucan and red ginsenosides, which is effective for anti-cancer, anti-tumor and immune enhancement, and to provide an effective preparation method for it. Summary of the Invention

[0013] In the first aspect, an immune-enhancing substance is provided, which contains black yeast β-glucan and ginsenoside Rg3 from red ginseng, as a compound immune enhancer, which has excellent anti-cancer, anti-tumor and immune-enhancing effects.

[0014] The immune-enhancing substance contains 3.5-4.8 g / L of black yeast β-glucan and 1.3-4.21 mg% of ginsenoside Rg3. The immune-enhancing substance may also contain other ginsenosides from red ginseng, such as Rd and Rg1.

[0015] In a second aspect, a method for preparing an immune-enhancing substance comprising black yeast β-glucan and ginsenoside Rg3 is provided.

[0016] This invention provides a preparation method that, by selecting red ginseng extract as a culture medium component for manufacturing black yeast β-glucan, can effectively produce a complex immune-enhancing substance containing black yeast β-glucan and ginsenosides from red ginseng, which are effective for anti-cancer, anti-tumor, and immune-enhancing purposes.

[0017] In this invention, black yeast is cultured in a culture medium containing red ginseng extract. Aureobasidium pullulans To produce black yeast β-glucan, while using black yeast ( Aureobasidium pullulansSome ginsenosides in red ginseng extract were bioconverted into Rg3, resulting in the creation of a complex immune-enhancing substance containing black yeast β-glucan and ginsenoside Rg3.

[0018] In this invention, the culture medium used to produce black yeast β-glucan is configured to increase the content of Rg3, which is particularly effective in anti-cancer, anti-tumor and immune-enhancing effects among ginsenosides.

[0019] In this invention, sugars used in traditional culture medium components for the production of β-1,3-1,6-glucan are omitted or reduced to create an environment conducive to the production of more β-glucosidase, which is essential for the bioconversion of ginsenosides in red ginseng to ginsenoside Rg3.

[0020] Furthermore, red ginseng extract was used instead of rice bran as the nitrogen source to provide a nitrogen-deficient environment more conducive to increasing the yield of β-glucan in black yeast. Additionally, the culture medium overcomes the drawback of traditional culture methods where it is difficult to separate rice bran from the culture medium after cultivation.

[0021] The culture medium for producing black yeast β-glucan of the present invention comprises red ginseng extract and ascorbic acid, wherein the amount of ascorbic acid is 0.1-0.2% of the mass of red ginseng extract.

[0022] The culture medium for producing black yeast β-glucan of the present invention may also contain sugar in the range of 0.5-1% by weight of the culture medium.

[0023] Red ginseng extract is obtained by adding 10-40 times the weight of red ginseng to water as the extraction solution and extracting at 85-95℃ for 2.5-10 hours.

[0024] Seed culture is performed before it is used to produce black yeast β-glucan.

[0025] In seed culture, black yeast ( Aureobasidium pullulans The colonies were inoculated into PDA agar plates and cultured at 25°C for 4 days. Then, colonies picked with a platinum inoculation loop were inoculated into PDA liquid medium and cultured with shaking at 125 rpm at 25°C for 48 hours. This can be used as seed culture for the next generation.

[0026] In the next-generation culture for the production of black yeast β-glucan, the seed culture was inoculated into a medium containing red ginseng extract at a volume of 0.5% by weight of the medium and cultured under aeration at 22-28°C and pH 5.0-6.0 for 72-96 h.

[0027] The immune-enhancing substance of the present invention can be administered orally for anti-cancer, anti-tumor and immune enhancement purposes. Detailed Implementation

[0028] Example The following examples are preferred embodiments of the immune-enhancing substances and their preparation methods of the present invention, and should not be construed as limiting the present invention.

[0029] In the seed culture for preparing black yeast seed culture medium, black yeast ( Aureobasidium pullulans The colonies were inoculated into PDA agar plates and cultured at 25°C for 4 days. Then, colonies picked with a platinum inoculation loop were inoculated into PDA liquid medium and cultured at 25°C with shaking at 125 rpm for 48 h.

[0030] Table 1. Embodiments of the present invention

[0031] 1) The content of black yeast β-glucan and ginsenoside in the immune-enhancing substance of the present invention.

[0032] — Content of β-glucan in black yeast culture The content of β-glucan in black yeast cultures was determined by the phenol-sulfuric acid method.

[0033] The results are shown in Table 2.

[0034] Table 2. β-glucan content of black yeast cultures

[0035] As shown in Table 2, when black yeast is cultured in a medium containing red ginseng extract, the content of black yeast β-glucan produced is 3.5-4.8 g / L.

[0036] — Ginsenoside content in the culture The ginsenoside content of the culture was determined by HPLC. The results are shown in Table 3.

[0037] Table 3. Ginsenoside content in cultures

[0038] As shown in Table 3, the ginsenosides Rb1, Rb2, and Rc of the red ginseng extract were converted into intermediate Rd during the culture process, and some of the intermediate Rd was eventually converted into Rg3. The content of ginsenoside Rg3 in the culture in Example 1 increased by 2.3 times compared with that before culture, in Example 2 it increased by 3 times, and in Example 3 it increased by 2.2 times.

[0039] 2) The immune activity and toxicity of the immune-enhancing substances of the present invention.

[0040] Animals and Experimental Design Pathogen-free purebred mice (8 weeks old) and mice (weighing 20-22 g) were obtained from the Experimental Animal Center of Pyongyang Medical University, North Korea.

[0041] During the experiment, the mice had access to food and water at all times. The temperature was maintained at 20±2°C and the humidity at 55%. This study has been approved by the Animal Experiment Ethics Committee of the Department of Basic Medical Sciences, Pyongyang Medical University.

[0042] (1) Immunological activity test Mice were randomly divided into 5 groups (n=10 per group). Groups 1, 2, and 3 (treatment groups) received the immune-enhancing substance of this invention at doses of 10, 20, and 40 mg / kg / day, respectively. The positive control group received 40 mg / kg / day of red ginseng aqueous extract (1:1 v / v). The normal control group received an equal volume of physiological saline. All treatments were administered orally for 14 days.

[0043] — Relative weight of spleen and thymus Twenty-four hours after the last administration, the animals were weighed and then euthanized by decapitation. The spleen and thymus were immediately removed and weighed. The spleen and thymus index was calculated using the following formula: Spleen and thymus index = Spleen or thymus weight / Body weight × 100.

[0044] — Lymphocyte proliferation assay Three mice were selected for each group for the lymphocyte proliferation assay. The spleens of the euthanized mice were aseptically removed in cold Hanks solution using scissors and forceps, gently homogenized, and then passed through a cell sieve to obtain a single-cell suspension. The red blood cells in the cell mixture were washed with hypotonic hemolysis and resuspended in RPMI 1640 medium supplemented with 10% bovine serum albumin, to a final density of 3 × 10⁻⁶ cells / mL. 6 mL - ¹. Then, 1 mL of spleen cell solution was seeded into a 24-well plate containing 75 µL of Con A, and Con A was replaced with 75 µL of deionized water as a control. The plate was incubated at 37°C in a 5% CO2 atmosphere for 3 days. After 68 h of incubation, 0.7 mL of solution was removed, and 0.7 mL of RPMI-1640 and 50 µL of MTT (5 mg / mL) were added to each well, followed by incubation for another 4 h. Then, 1 mL of acidic isopropanol was added to each well and mixed thoroughly. The contents of each well in the 24-well plate were added to three wells of a 96-well plate. The absorbance at 570 nm was measured using a microplate reader.

[0045] — CD 4+ and CD 8+ Detection of T lymphocytes Mouse spleen cells were homogenized, sieved, and then centrifuged to obtain a single-cell suspension. Individual cells were counted and adjusted to a concentration of 8 × 10⁻⁶ cells / mL. 6 mL -1 Then, 200 µL of cells were centrifuged at 1500 rpm for 8 min at 4°C. The cells were resuspended in 100 µL PBS. Next, 2.5 µL of anti-CD3-PerCP, 2.5 µL of anti-CD4-PE, and 1 µL of anti-CD8-FITC were added to stain the cells. The stained cells were incubated at room temperature in the dark for 30 min, washed with PBS, and then centrifuged at 1500 rpm for 5 min at 4°C. The cells were then resuspended in 500 µL PBS and analyzed using flow cytometry.

[0046] — Serum IL-2 and IFN-γ concentration test Mouse blood was collected in 1.5 mL centrifuge tubes at 37°C without anticoagulant for 2 h, followed by centrifugation at 3000 rpm for 15 min at 4°C. Serum was collected for cytokine concentration determination. IL-2 and IFN-γ concentrations were determined using an ELISA kit according to the manufacturer's instructions. Mouse serum samples were diluted 1:4 and incubated in 96-well microtiter plates.

[0047] (2) Subchronic toxicity test Eighty male and female mice were randomly divided into four groups (n=20 per group; 10 male mice and 10 female mice) for subchronic toxicity testing. Different doses (300, 600, or 1200 mg / kg) of the immune-enhancing substance of this invention were dissolved in distilled water and administered to the different groups of mice daily by gavage, while the control group received only distilled water. General activity, toxic effects, and mortality were monitored daily for 60 consecutive days.

[0048] Statistical analysis Results are expressed as mean and SEM. Data were analyzed using SPSS 16.0 using one-way ANOVA, and differences between means were assessed using Dunnett's multiple comparison test. A p-value <0.05 was considered statistically significant.

[0049] result (1) Immune activity — Effects on spleen and thymus index in mice Table 4. Effects on spleen and thymus index in mice

[0050] Each value represents the mean ± SEM of 10 samples in each group. △ indicates that compared with the control group, p<0.05. △ indicates that compared with the positive control group, p<0.05.

[0051] Table 4 shows the effects on spleen and thymus indices in mice. Significant differences were found in spleen and thymus indices between all experimental groups and the control group (p<0.05). These indices in experimental group 3 were higher than those in the positive control group (p<0.05), while these indices in experimental groups 2 and 3 were numerically higher than those in the positive control group (p>0.05).

[0052] — Effects on the proliferation of mouse splenic lymphocytes Table 5 shows the effect on A570 values. The A570 values ​​of all experimental groups were significantly higher than those of the control group (p<0.05), while the A570 values ​​of experimental groups 2 and 3 were significantly higher than those of experimental group 1 and the positive control group (p<0.05). No significant difference was detected between experimental groups 2 and 3 (p>0.05).

[0053] Table 5. Effects on the proliferation of mouse spleen lymphocytes

[0054] Each value represents the mean ± SEM of 10 samples in each group. △ indicates that compared with the control group, p<0.05. △ indicates that compared with the positive control group, p<0.05.

[0055] — For CD 4+ and CD 8+ Effects of T lymphocytes Table 6 shows the CD 4+ and CD 8+ The influence of T lymphocytes.

[0056] Compared with the control group, CD was significantly lower in all experimental and positive groups. 4+ The percentage of T cells was significantly increased in all experimental and positive groups (p<0.05). In contrast, compared with the control group, CD4+ was significantly lower in all experimental and positive groups. 8+ The percentage of T cells was significantly reduced in all experimental groups (p<0.05). Compared with the positive group, CD4+ was significantly lower in all experimental groups. 4+ T cells and CD4 8+ The percentage of T cells changed significantly (p<0.05).

[0057] Table 6. Effects on CD4+ and CD8+ T lymphocytes

[0058] Each value represents the mean ± SEM of 10 samples in each group. △ indicates that compared with the control group, p<0.05. △ indicates that compared with the positive control group, p<0.05.

[0059] — Effects on serum IL-2 and IFN-γ secretion in mice Table 7. Effects on IL-2 and IFN-γ levels in mice

[0060] Each value represents the mean ± SEM of 10 mice in each group. △ indicates that compared with the control group, p<0.05. △ indicates that compared with the positive control group, p<0.05.

[0061] Table 7 shows the effects on serum IL-2 and IFN-γ concentrations. Serum IL-2 concentrations were significantly higher in all experimental groups than in the positive and control groups (p<0.05), but no significant difference was detected between the positive and control groups (p<0.05). Serum IFN-γ concentrations were significantly higher in all experimental and positive groups than in the control group (p<0.05).

[0062] (2) Subchronic toxicity In the subchronic toxicity study, the treated mice and both male and female control mice remained generally healthy throughout the experiment. No deaths or toxic symptoms were recorded in the treated mice. The subchronic toxicity study demonstrated that oral doses of up to 1200 mg / kg of this formulation did not induce any obvious toxic symptoms or cause death in the experimental animals. The LD50 of this formulation was determined to be greater than 1200 mg / kg.

Claims

1. An immune-enhancing substance comprising black yeast β-glucan and ginsenoside Rg3, wherein, The content of β-glucan in black yeast is 3.5-4.8 g / L, and the content of ginsenoside Rg3 is 1.3-4.21 mg.

2. A method for preparing an immune-enhancing substance, wherein the immune-enhancing substance is the immune-enhancing substance according to claim 1, wherein, In a culture medium containing red ginseng extract, black yeast ( Aureobasidium pullulans ) Synthesize black yeast β-glucan, and simultaneously use black yeast to bioconvert ginsenosides into Rg3 in red ginseng extract.

3. The method for preparing the immune-enhancing substance according to claim 2, wherein, The culture medium containing red ginseng extract comprises red ginseng extract and ascorbic acid, wherein the amount of ascorbic acid is 0.1-0.2% of the mass of red ginseng extract.

4. The method for preparing the immune-enhancing substance according to claim 3, wherein, The red ginseng extract is prepared by adding 10-40 times the weight of red ginseng to water as the extraction solution and extracting at a temperature of 85-95℃ for 2.5-10 hours.

5. A method for preparing the immune-enhancing substance according to any one of claims 2 to 4, wherein, In the culture used to produce black yeast β-glucan, black yeast seeds are inoculated into the culture medium containing the red ginseng extract at a weight of 0.5% of the culture medium and cultured under aeration at a temperature of 22-28°C and a pH of 5.0 to 6.0 for 72-96 h.

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