Immunopotentiating material comprising black yeast β-glucan and ginsenoside rg3 and preparation method thereof

Abstract

The immunopotentiating material includes black yeast β-glucan and ginsenoside Rg3, and the content of black yeast β-glucan is 3.5-4.8 g / L and the content of gisenoside Rg3 is 1.3-4.21 mg%. The immunopotentiating materia is prepared by carrying out the synthesis of black yeast β-glucan through the cultivation of black yeast (Aureobasidium pullulans) in the culture medium containing an extract of red ginseng simultaneously with the biotransformation of ginsenosides in the extract of red ginseng by using black yeast (Aureobasidium pullulans). The immunopotentiating materia can be used as an oral administration for anticancer, antitumor and immunopotentiating activities.

Description

[0001] Immunopotentiating material comprising black yeast p-glucan and ginsenoside Rg3 and preparation method thereof

[0002] Field of the Invention

[0003] The present invention relates to an immunopotentiating material which has anticancer, antitumor and immunopotentiating effect, and a preparation method thereof.

[0004] Background and Purpose of the Invention β -glucan is one of the leading materials for inhibiting malignant tumor like cancer and immunopotentiating effect. j3 -glucan serves to activate macrophages, NK cells, T cells, killer T cells and the like that attack infected cells and cancer cells in body, and exhibits effects of eliminating bacteria and foreign matter that have entered the body and suppressing the onset of disease by increasing immunity and resistance. Also, it has been reported that as a result of exhibiting these effects, β-glucan has functions such as suppressing allergies, suppressing malignant tumors such as cancer, reducing blood sugar levels, promoting urination, adjusting blood pressure, and reducing blood cholesterol levels and neutral lipid levels.

[0005] / 3 -1 ,3-1, 6-glucan attracts attention due to its high immunopotentiating effects among β-glucan, which is polysaccharide synthesizing and secreting in mushrooms and yeasts.

[0006] In specific condition of culture, black yeast synthesizes and secretes β-glucan out of the cell. The β-glucan has immunoregulatory effect due to its structural and immunological property. Black yeast β-glucan (APpG) is specific polysaccharide derived from only black yeast, in which the main chain consists of (3-1,3 bonds and 50-80% of the branched chain consists of P-1 , 6 bonds and its molecular weight is small (~100KDa).

[0007] In general, black yeast is cultured in a liquid culture medium which consists of sugar, glucose, rice bran and ascorbic acid for producing P-1, 3-1 , 6-glucan. In the prior methods for producing β-1 , 3-1 , 6-glucan, sugar and glucose are used as a carbon source for the growth of black yeast. Ginsenoside of red ginseng and aglycone are also among the materials which has an anticancer activity and a special efficacy in preventing cancer.

[0008] Ginsenoside of red gingeng is produced in the process of hydrolysis by heat treatment. The main ginsenoside contents of red ginseng are approximately Rg1 3.3mg / g, Re 2.0mg / g, Rb1 5.8mg / g, Rc 1.7mg / g, Rb2 2.3mg / g, and Rd 0.4mg / g.

[0009] The main active materials in preventing cancer are CK, Rg3, Rh2 which delay the development of cancer and vasculogenesis by effectively inhibiting the vascular endothelial cell growth factor depending neoangiogenesisand weakening a flow of epithelial cell.

[0010] Recently, increasing the content of the said ginsenoside which has high activity for anticancer comes to the fore as a pressing problem. In order to solve the problem, HPLC and enzyme treatment are used but the efficiency is low and much labor is required. Recently, biotransformation is mostly used.

[0011] Biotransformation is the biological method used to increase or decrease the content of some active ingredient of medical materials such as medical plants. Several methods for biotransformation are used but the method using microorganism is mostly used. In recent years, biotransformation method using several microorganisms (lactobacillus, ganoderma etc.) is used to increase the content of ginsenoside contained in ginseng.

[0012] In the prior art, the materials such as β-glucan and saponin of red ginseng having anticancer, antitumor and immunopotentiating effect and the preparation method therefor are disclosed but the effective method for preparing complex immunopotentiator having more excellent immunopotentiating effect has not been disclosed.

[0013] The purpose of the present invention is to overcome the drawbacks of the prior art and to provide a complex immunopotentiator including β-glucan and saponin of red ginseng which is effective for anticancer, antitumor and immunopotentiation and its effective preparation method.

[0014] Description of the Invention in a first aspect, there is provided an immunopotentiating material comprising black yeast β-glucan and ginsenoside Rg3 of red ginseng, as a complex immunopotentiator, which has excellent anticancer, antitumor and immunopotentiating effect.

[0015] The content of black yeast β-glucan is 3.5~4.8g / L and the content of ginsenoside Rg3 is 1.3~4.21mg% in the immunopotentiating material. The immunopotentiating material may further comprise the other ginsenoside of red ginseng such as Rd and Rgi.

[0016] In a second aspect, there is provided a preparation method of the immunopotentiating material comprising black yeast P-glucan and ginsenoside Rg3.

[0017] The present invention provides the preparation method that can effectively produce complex immunopotentiating material containing black yeast [3-glucan and ginsenoside of red ginseng which are effective for anticancer, antitumor and immunopotentiation by choosing a extract of red ginseng as a medium component for producing the black yeast [3-glucan.

[0018] In the present invention, black yeast β-glucan is produced by culturing black yeast (Aureobasidium pullulans) in the culture medium containing an extract of red ginseng and at the same time some ginsenosides of the extract of red ginseng are biotransformed into Rg3 by using black yeast (Aureobasidium pullulans), as a result, the complex immunopotentiating material containing black yeast [3-glucan and ginsenoside Rg3 is produced.

[0019] In the present invention, the culture medium for producing black yeast [3-glucan is constituted to increase the content of Rg3 which is especially effective for anticancer, antitumor and immunopotentiating among ginsenosides.

[0020] In the present invention, sugar which is traditional medium component for the production of β-1 , 3-1 , 6-glucan is not used or used less to create a environment favorable for producing more β-glucosidase needed for biotransformation of ginsenosides of red ginseng into ginsenoside Rg3.

[0021] In addition, the extract of red ginseng is used instead of rice bran as a nitrogen source to provide a environment of nitrogen deficiency more favorable for increasing production of black yeast β-glucan. Beside, the said culture medium makes it possible to overcome drawback difficult to separate rice bran from the culture after culturing in the traditional culture method. The culture medium of the invention for the production of biack yeast β-glucan comprises the extract of red ginseng and ascorbic acid, wherein the amount of ascorbic acid is 0.1 -0.2% of the mass of the extract of red ginseng.

[0022] The culture medium of the invention for the production of black yeast j3-glucan may further comprise sugar within the range of 0.5-1 % of the mass of the culture medium.

[0023] The extract of red ginseng is made by adding water as an extract solution 10-40 times of the mass of red ginseng and extracting at a temperature of 85-95°C for 2.5-1 Oh.

[0024] Seed culture is performed before the culture for the production of black yeast β-glucan.

[0025] In the seed culture, black yeast (Aureobasidium pullulans) is inoculated into PDA plate medium and is cultured at a temperature of 25°C for 4 days , then the colony taken by platinum loop is inoculated into PDA liquid medium and is shake-cultured at a stirring rotation rate of 125 rpm at a temperature of 25°C for 48h. This is used as a seed for the next culture.

[0026] In the next culture for the production of black yeast β-glucan, the seed culture solution is inoculated into the culture medium containing the extract of red ginseng, in an amount of 0.5% by weight of the culture medium, and an aering culture is performed at a temperature of 22-28 °C and a pH of 5.0 to 6.0 for 72~96h.

[0027] The immunopotentiating material of the present invention can be used as oral administration for anticancer, antitumor and immunopotentiation.

[0028] Example

[0029] The following examples are preferred embodiments of the immunopotentiating material of the invention and the preparation method thereof, and should not be understood as limiting the present invention.

[0030] In the seed cuture for preparing the seed culture solution of black yeast, black yeast (Aureobasidium pullulans) is inoculated into PDA plate medium and is cultured at a temperature of 25°C for 4 days, then the colony taken by platinum loop is inoculated into PDA liquid medium and is shake-cultured at a stirring rotation rate of 125 rpm at a temperature of 25°C for 48h.

[0031] Table 1. Examples of the present invention 1) Black yeast β-glucan content and ginsenoside content of the immunopotentiating material of the invention.

[0032] — Black yeast β-glucan content of the culture.

[0033] Black yeast β-glucan content of the culture is measured by phenol-sulphuric acid. The results are shown in Table 2. Table 2. Black yeast β-glucan content of the culture

[0034] As shown in Table 2, the content of black yeast β-glucan produced is 3.5-4.8g / L when black yeast is cultivated in the culture medium containing the extract of red ginseng. — Ginsenoside content of the culture

[0035] Ginsenoside content of the culture is measured by HPLC. The results are shown in Table 3.

[0036] Table 3. Ginsenoside content of the culture (mg%)

[0037]

[0038] As shown in Table 3, ginsenosides Rb1 , Rb2, Rc of the extract of red ginseng were transformed into an intermediate Rd and some of the intermediate Rd were finally transformed into Rg3 in the process of the cultivation and ginsenoside Rg3 content of the culture was increased by 2.3 times than before the cultivation in example 1 , by 3 times in example 2 and by 2.2 times in example 3.

[0039] 2) Immunological Activities and toxicity of the immunopotentiating material of the invention.

[0040] Animal and Experimental Design

[0041] Pathogen-free purebred mice (8 weeks old) and mice (weighing 20-22 g) were obtained from the Laboratory Animal Centre of the Pyongyang University of Medical Sciences.

[0042] During the experiment, feed and water were available to rats at any time. The temperature was maintained at 20 ±2 °C and the humidity was 55%. The study was approved by the Ethics Committee for Animal Experimentation, Faculty of Basic Medicine, Pyongyang University of Medical Sciences.

[0043] (1) Immunological Activities test

[0044] The mice were randomly divided into 5 groups (10 mice per group). The Exp-1 , Exp-2 and Exp-3 treatment groups received the immunopotentiating material of the invention 10, 20, 40mg / kg / day respectively. Positive control group received 40 mg / kg / day water extract (1 :1 VAX) of Red ginseng. The normal group received equal volume of physiological saline solution. All treatments were administered intragastrically for 14 days.

[0045] — Relative Spleen and Thymus Weight

[0046] At 24 h after the last agent administration, the animals were weighed and then sacrificed via decapitation. Spleens and thymuses were immediately removed and weighed. The spleen and thymus indexes were calculated using the following equation: spleen and thymus indexes=spleen or thymus weight / body x 100.

[0047] — Lymphocyte Proliferation Assay

[0048] Three mice of each group were selected to perform the lymphocyte proliferation assay . The mouse spleens were aseptically removed from the sacrificed mice using scissors and forceps in cold Hanks, gently homogenized, and then passed through a cell strainer to obtain single-cell suspensions. The erythrocytes in the cell mixture were washed via hypo-osmotic hemolysis, and the cells were resuspended to a final density of 3 x 106mL'1in RPMI 1640 medium supplemented with 10% bovine serum albumin. Then, 1 mL of the spleen cell solution was seeded into a 24-well plate containing 75 pL of Con A, and Con A was replaced with 75 pL of deionized water as the control. The plates were cultured at 37 °C in 5% CO2 atmosphere for 3 days. After incubation for 68 h, 0.7 mL of solution was removed, and 0.7 mL of RPMI-1640 and 50 pL of MTT (5 mg / mL) were added to each well and incubated for another 4 h. Then, 1 mL of acid isopropyl alcohol was added into each well and evenly mixed. The content of each well of the 24-well plate was added into three wells of a 96-well plate. The absorbance at 570 nm was obtained on a microplate reader.

[0049] — Detection of CD4+and CD8+T Lymphocytes

[0050] The mouse spleens cells were homogenized, sieved, and then centrifuged to obtain single-cell suspensions. The single cells were counted and adjusted to 8 x 106mL-1. Then, 200 pL of cells were centrifuged at 1500 rpm for 8 min at 4 °C. The cells were resuspended in 100 pL of PBS. Afterward, 2.5 pL of anti-CD3~PerCP, 2.5 pL of anti-CD4-PE, and 1 pL of anti-CD8-FITC were applied to stain the cells. The stained cells were incubated at room temperature for 30 min in the dark, washed with PBS, and then centrifuged at 1500 rpm for 5 min at 4 °C. Then, the cells were resuspended in 500 pL of PBS and analyzed using a flow cytometer. — Serum IL-2 and IFN-y Concentration Assay

[0051] The blood of the mice was collected in a 1.5 mL centrifuge tube without an anticoagulant for 2 h at 37 °C and then centrifuged at 3000 rpm for 15 min at 4 °C. The serum was collected to measure cytokine concentrations. The concentrations of IL-2 and IFN-y were assayed using an ELISA kit in accordance with the manufacturer’s instructions. Mouse serum samples were diluted at 1 :4 and then incubated in 96-well microtiter plates.

[0052] (2) Sub chronic toxicity test

[0053] 80 mice of either sex were randomly divided into 4 groups (n=20; 10 males and 10 females per group) and employed in the test of sub chronic toxicity. Different doses of the immunopotentiating material of the invention (300, 600, or 1200 mg / kg) were dissolved in distilled water and administered daily by lavage to different groups of mice, while the control group received only distilled water. General activity, toxic manifestations and mortality were monitored daily for 60 days.

[0054] Statistical analysis

[0055] Results were expressed as the mean and SEM. Data were analysed by one-way analysis of variance (ANOVA) using SPSS 16.0 and the differences between the means assessed using Dunnet’s multiple range test. A P value of < 0.05 was taken as the level of statistical significance.

[0056] Results

[0057] (1 ) Immunological Activities

[0058] — Effect on Spleen and Thymus Indexes in Mice

[0059] Table 4. Effects on spleen and thymus indexes in mice Each value represents the mean ± SEM of 10 per group. *p<0.05 as compared with control group. A p<0.05 as compared with positive control group

[0060] The effects on the spleen and thymus indexes in mice are shown in Table 4. Significant differences in the spleen and thymus indexes were detected between all the Exp groups and the control group (p< 0.05). These indexes in Exp-3 group were higher than those in Positive control group (p<0.05), whereas those in Exp-2 and Exp-3 groups were numerically higher than that in the Positive control group (p > 0.05).

[0061] — Effect on Spleen Lymphocyte Proliferation in Mice

[0062] The effects on the A570 values are shown in Table 5. The A570 values in all Exp groups were significantly higher than those in the control group (p <0.05), whereas those in the Exp-2 and Exp-3 groups were significantly higher than those in the Exp-1 and Positive control groups (p < 0.05). No significant difference was detected between the Exp-2 and Exp-3 groups (p > 0.05).

[0063] Table 5. Effect on the spleen lymphocyte proliferation in mice

[0064] Each value represents the mean ± SEM of 10 per group. *p<0.05 as compared with control group. A p<0.05 as compared with positive control group

[0065] — Effect on CD4* and CD8* T Lymphocytes

[0066] The effects on the CD4* and CD8* T lymphocytes are shown in Table 6.

[0067] The percentages of CD4* T cells in all Exp groups and positive group were significantly increased compared to control group (p < 0.05). In contrast, the percentages of CD8* T cells in all Exp groups and positive group were significantly decreased compared to control group(p < 0.05). The percentages of CD4* T cells and CD8+T ceils in ail Exp groups were significantly changed compared to positive group (p < 0.05).

[0068] Table 6. Effects on CD4+and CD8+T Lymphocytes Each value represents the mean ± SEM of 10 per group. *p<0.05 as compared with control group. A p<0.05 as compared with positive control group

[0069] — Effect on IL-2 and IFN-y Secretion in Serum in Mice

[0070] Table 7. Effects on IL-2 and IFN-y levels in mice Each value represents the mean ± SEM of 10 per group. *p<0.05 as compared with control group. A p<0.05 as compared with positive control group

[0071] The effects on the serum concentrations of IL-2 and IFN-y are shown in Table 7. The serum IL-2 concentrations in ali Exp groups were significantly higher than those in the positive and control groups (p < 0.05), but no significant difference was detected between positive and control groups (p < 0.05). The serum IFN-y concentration in all Exp groups and positive group was significantly higher than those in control groups (p < 0.05).

[0072] (2) Subchronic toxicity In the study of the subchronic toxicity, the treated mice as well as the control mice of both sexes appeared generally healthy throughout the experimental period. No mortality was recorded and no toxicity signs were detected in any of the treated mice. The subchronic toxicity indicated that the oral administration of the agent up to 1200 mg / kg dose caused neither visible sign of toxicity nor mortality to experimental animals. Determined LD50 of the agent is more than 1200 mg / kg.

Claims

Claims1. An immunopotentiating material, comprising biack yeast β-glucan and ginsenoside Rg3, wherein the content of black yeast |3- glucan is 3.5-4.8g / L and the content of ginsenoside Rg3 is 1.3~4.21mg%.

2. A preparation method of the immnopotentiating material of ciaim 1, characterized in that the synthesis of black yeast |3-glucan by the culture of Aureobasidium pullulans in the culture medium containing an extract of red ginseng and the biotransformation of ginsenosides in the extract of red ginseng into Rg3 by using Aureobasidium pullulans are carried out at the same time.

3. The preparation method of the immunopotentiating material according to claim 2, wherein the culture medium containing an extract of red ginseng comprises the extract of red ginseng and ascorbic acid, and the amount of ascorbic acid is 0.1 -0.2% of the mass of the extract of red ginseng.

4. The preparation method of the immunopotentiating material according to claim 3, wherein the extract of red ginseng is made by adding water as an extract solution 10-40 times of the mass of red ginseng and extracting at a temperature of 85-95 ’C for 2.5-1 Oh.

5. The preparation method of the immunopotentiating material according to any one of claim 2 to 4, in the culture for the production of black yeast P-glucan, the seed of black yeast is inoculated into the culture medium containing the extract of red ginseng, in an amount of 0.5% by weight of the culture medium, and an aering culture is performed at a temperature of 22-28’0 and a pH of 5.0 to 6.0 for 72-96h.

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