KR101116247B1 - Physiologically acitive materials from rice bran and process for preparation thereof - Google Patents

Abstract

The present invention relates to a physiologically active substance isolated from rice bran and a method for producing the same. The present invention relates to a method for producing a bioactive material, characterized in that the alkali extraction and treatment with starch hydrolase, a bioactive material produced by the method and a composition comprising the same.

The method for preparing a bioactive material according to the present invention has an effect of easily preparing a biologically active material having excellent immunological activity by inoculating an edible mushroom in a medium containing rice bran which has not been subjected to a separate pretreatment process. In addition, the physiologically active substance prepared according to the method of the present invention is very excellent in immunopotentiation activity such as macrophage activity, splenocyte growth promotion, intestinal immune activity and lymphocyte production promoting activity, and promotes immune activity in vivo to weaken immune It is effective in preventing and treating diseases caused, especially cancer.

Bioactive substance, rice bran, edible mushroom

Description

Physiologically acitive materials from rice bran and process for preparation etc.             

1 is a result of measuring the macrophage activity of the bioactive material prepared by using various strains and various production methods.

A: well without cells

B: negative control treated with saline solution

C: positive control treated with LPS

1 to 16: Experiment group 1 to Experiment group 16

Figure 2 is a result of measuring the spleen cell proliferation promoting activity of the bioactive material prepared by using various strains and various production methods.

A: well without cells

B: negative control treated with saline solution

C: positive control treated with Lipopolysacharide (LPS)

D: Positive control treated with ConA (Concanvalin A) glycoprotein

1 to 16: Experiment group 1 to Experiment group 16

Figure 3 is the result of measuring the intestinal immune activity of the bioactive material prepared using various strains and various production methods.

A: negative control treated with saline solution

B: positive control treated with LPS

1 to 16: Experiment group 1 to Experiment group 16

Figure 4 is the result of inoculating shiitake mushroom seedlings in culture medium containing several kinds of carbon sources and measuring the amount of extracellular biopolymer and glucosamine produced in the culture.

A: starch treatment group

B: dextrose treatment group

C: fructose treatment group

D: lactose treatment group

E: mannitol treatment group

F: maltose treated group

G: sorbitol treatment group

H: sucrose treatment group

I: xylose treatment group

5 is a result of measuring the amount of extracellular biopolymer and glucosamine generated in the culture after inoculating shiitake spawn spawn in a medium containing various nitrogen sources.

A: nitrate ammonium treatment group

B: Ammonium Chloride Treatment Group

C: Ammonium Phosphate Treatment Group

D: peptone treatment group

E: yeast extract treatment group

F: Soyton Treatment Group

G: polypeptone treatment group

6 is a result of inoculating shiitake spawn seedlings in culture medium containing potassium phosphate at various concentrations and measuring the amount of extracellular biopolymer and glucosamine produced in the culture.

7 is a result of measuring the amount of extracellular biopolymer and glucosamine produced in the culture after inoculating shiitake spawn seed in culture medium containing various kinds of enzymes.

A: physiological saline treatment group

B: LPS treatment group

C: ConA treatment group

D: no treatment group

E: biscozyme treatment group

F: rapidase treatment group

G: Cellulast Treatment Group

H: Ultraflow treatment group

I: amylase treated group

J: Econase treatment group

8 is a result of measuring the amount of biopolymer and glucosamine produced in the culture after inoculating shiitake spawn seed in the medium and incubated at various culture temperatures.

9 is the result of inoculating shiitake mushroom seedlings in the medium and incubating with different pHs to measure the amount of biopolymer and glucosamine produced in the culture.

10 is the result of inoculating shiitake spawn seed in the medium and measuring the amount of biopolymer and glucosamine produced in the culture according to the culture time.

A: negative control treated with saline solution

B: positive control treated with LPS

11A to 11D show the results of treating the splenocyte lymphocytes with the bioactive material of the present invention and measuring the amount of IL-6, GM-CSF, IFN-γ and IL-12, respectively.

A: cell free well

B: negative control treated with saline solution

C: positive control treated with LPS

D: positive control treated with ConA glycoprotein

E: bioactive substance treatment group of the present invention

12a to 12d are the results of treating the bioactive substances of the present invention in macrophages and measuring the production of IL-6, IL-12, TNF-α and IL-1B, respectively.

A: cell free well

B: negative control treated with saline solution

C: positive control treated with LPS

D: bioactive substance treatment group of the present invention

Figure 13a is a result of measuring the peritoneal macrophage activity of the mouse after intravenous or intraperitoneal administration of the bioactive substance of the present invention to the mouse.

A: cell free well

i.p .: intraperitoneal injection

i.v .: intravenous

Figure 13b is the result of measuring the peritoneal macrophage activity of the mouse after oral administration of the bioactive substance of the present invention to the mouse.

14 is a result of measuring the spleen lymphocyte activity of the mouse after intravenous or intraperitoneal administration of the bioactive substance of the present invention to the mouse.

A: cell free well

i.p .: intraperitoneal injection

i.v .: intravenous

Figure 15 shows the cancer metastasis inhibiting activity of mice transplanted with lung carcinoma following administration of the bioactive substance of the present invention.

Figure 16 shows the comparison of the macrophage activity of the physiologically active substance according to the present invention and the conventional immune enhancing substance.

A: cell free well

B: negative control treated with saline

C: positive control treated with LPS

D: bioactive substance treatment group of the present invention

E: Biobran ^？ treatment group

F: PSP ^？ Treatment group

G: vitamin C treatment group

The present invention relates to a bioactive material isolated from rice bran and a method for producing the same. In more detail, the present invention is characterized by inoculating an edible mushroom in a medium containing rice bran which has not been subjected to a separate pretreatment step, incubated in an optimized culture condition, and then alkali-extracting the culture and treating it with starch hydrolase. It relates to a method for producing a bioactive substance, a bioactive substance produced by the above method and a composition comprising the same.

Recently, many biopolymers having physiological activity produced from animal and plant raw materials and microorganisms have been separated and used industrially. The biopolymers include cellulose, pectin, agar, alginic acid, carrageenan, galactomannan, acacia gum, and chitin, chitosan, etc., which are produced from animal raw materials, and are produced from microbial culture. There are many different types.

It has been reported in the literature that β- (1,6) -glucan isolated from mushrooms such as Agaricus has anticancer activity by activating immune cells to stimulate the immune system (Mizuno T. et al., Agric. Biol. Chem ., 54, 2889-2896, 1990). In addition, β- (1,3) -glucan isolated from fungi has been reported to be involved in immune responses such as activating macrophages and inhibiting tumor cell metastasis (Sakurai T. et al., Immunophamacology , 30 , 157-166, 1995). Furthermore, mannan isolated from Candida albicans microorganisms acts as an immunosuppressive agent to suppress the inflammatory response (Vastsa GR et al., Dev. Comp. Immunol ., 23, 401-420, 1999). Glucuronoxylomannan isolated from Cryptococcus neoformans microorganisms is known to have an anti-inflammatory effect (Blackstock R., et al., Immunology , 92, 334-339, 1997). In addition, the anti-cancer activity of hemicellulose isolated from plants has recently been reported.

Such bioactive substances are widely used in food, biotechnology, medicine, cosmetics, and other industries.

On the other hand, rice bran is a by-product produced in the process of refining brown rice with white rice and refers to a powder consisting of rice bran and rice snow. On the other hand, almost all of the nutrients of rice is contained in rice bran. That is, rice bran contains abundant lipids, vitamin B group, high quality protein, and also contains abundant fiber and phosphorus. However, despite the excellent nutritional value as described above, rice bran is used only as rice bran oil, feed and fertilizer, or is treated as agricultural waste, and thus research on a wider industrial use method of rice bran is required.

Recently, it has been reported that there is a physiologically active substance having physiological activities such as immunopotentiation, antidiabetic and anticancer activity in the rice bran. However, the physiologically active substance of the rice bran is difficult to be used in living organisms because most of them are present in an insoluble form strongly bound to the plant cell wall. Therefore, there is a need for a process of separating the physiologically active substance from the plant cell wall and converting it into a water-soluble state so that it can be used in vivo.

In Korean Patent No. 344755, plant tissue raw material is extracted by hot water, filtered to obtain an aqueous solution, and the glucoamylase is treated to decompose starch to obtain a water-soluble polysaccharide, and to add and react the complex enzyme isolated from fungus and basidiomycetes. There has been disclosed a method for producing an immune enhancing substance.

In addition, Korean Patent Publication No. 2004-67175 discloses a hydrothermal extract after culturing basidiomycete using a plant tissue that has undergone a pretreatment process of treating amylase, a protease and a cellulase as a medium in an extract obtained by hydrothermal extraction of plant tissue. And a method for producing a complex polysaccharide having an immunoactive function by ethanol precipitation has been disclosed.

However, the above-mentioned prior arts allow plant tissue materials to be effectively subjected to the action of enzyme complexes separated from fungi and basidiomycetes, or effectively utilize other types of carbon sources except hemicellulose, which is an active ingredient contained in plant tissue materials. In order to proliferate, there is a disadvantage of separately pretreating plant tissue raw materials.

Meanwhile, until now Bioactive substances have been prepared using as a substrate a mycelium-derived polysaccharide of rice bran or water fungus obtained by pretreatment of plant tissue raw materials as described above. However, rice bran contains a large amount of other bioactive substances such as polyphenol components in addition to the water-soluble polysaccharide. Therefore, there is a need to manufacture a bioactive substance containing all of these.

Therefore, the present inventors inoculated an edible mushroom in a medium containing rice bran without undergoing a separate pretreatment process while studying a method of preparing a biologically active substance using rice bran and incubating the culture in an optimized culture condition, followed by alkali extraction. The present invention was completed by preparing a physiologically active substance having excellent immunity enhancing activity and anticancer activity by treatment with starch hydrolase.

Accordingly, it is an object of the present invention to provide a method for producing a bioactive material isolated from rice bran fermentation products.

In addition, another object of the present invention is to provide a bioactive material produced by the above method.

In addition, another object of the present invention to provide an immune enhancing composition comprising the physiologically active substance.

In addition, another object of the present invention to provide a composition for the prevention or treatment of cancer comprising the bioactive substance.

In order to achieve the above object, the present invention provides a method for producing a bioactive material separated from the rice bran fermentation.

The present invention also provides a bioactive material prepared by the above method.

The present invention also provides a composition for enhancing immunity comprising the physiologically active substance.

The present invention also provides a composition for preventing or treating cancer comprising the bioactive substance.

Hereinafter, the present invention will be described in detail.

In the present invention, "physiologically active substance" refers to a functional biopolymer having immuno-enhancing and anticancer activity prepared using a culture product such as polysaccharides generated during the cultivation process of rice bran and edible mushroom.

In one embodiment of the present invention, to establish a suitable production method for producing a bioactive material from rice bran fermentation, a bioactive material was prepared using various methods as follows. ① liquid culture → alkali extraction → amylase treatment method; ② solid culture → alkali extraction → amylase treatment method; ③ liquid culture → alcohol precipitation method; And ④ solid culture → hydrothermal extraction → alcohol precipitation method.

In order to prepare a physiologically active substance by various methods as described above and to establish the most preferable method, immunostimulatory activity of each sample was measured. As a result, it was found that the physiologically active substance prepared by using the liquid culture → alkali extraction → amylase treatment method and the solid culture → alkali extraction → amylase treatment method was the best in immune enhancing activity (see Example 1).

Therefore, the preparation method of the bioactive substance isolated from the fermented rice bran according to the present invention is (a) 0.5 to 10% (w / v) rice bran, 0.5 to 5% (w / v) sucrose and KH ₂ PO ₄ Inoculating an edible mushroom in a medium containing 0.1 to 1% (w / v) and incubating for 3 to 10 days under conditions of 10 to 50 ° C. and a pH of 5 to 7;

(b) alkali extracting the culture of step (a); And

(C) characterized in that it comprises the step of reacting by adding starch hydrolase to the alkali extract of step (b).

In step (a), the rice bran refers to a part including the rind, the endothelium, the whistle layer, and the endosperm constituting the outside of the brown rice, and can be easily obtained in the rice milling process. The rice bran is preferably dried and powdered, and is not subjected to a pretreatment process such as extraction or enzyme treatment separately.

In the step (a), the medium may be a liquid medium or a solid medium, preferably a liquid medium. In addition, the medium may further comprise a facchinase. Preferably, the pectin degrading enzyme is added to the medium in an amount of 0.2 to 1% (w / v). The facchinase is, for example, a commercially available rapidase (vision biocam). In addition, commercially available factolytic enzymes include, but are not limited to, pectinex, cytokinase, and the like.

Most preferably, the medium of the present invention is rice bran 5% (w / v), sucrose 2% (w / v), KH ₂ PO ₄ 0.2% (w / v) and 0.5% (w / v) rapidase. ) May be included.

Such a medium composition is established by the inventors from the experimental results for selecting the most suitable carbon source and nitrogen source for the preparation of the bioactive material and to determine the potassium phosphate content.

That is, in one embodiment of the present invention, the preparation of rice bran fermentation product using a medium containing various carbon sources and nitrogen sources, and after separating the physiologically active substance from the indicator of the extracellular biopolymer and the cell mass, which is an indicator of the physiologically active substance As a result of measuring the amount of glucosamine to be produced (see Examples <2-1> and <2-2>), starch, dextrose, lactose, sucrose as suitable carbon sources for the preparation of bioactive substances And xylose were selected. In particular, it was confirmed that it is most preferable to use sucrose of the carbon source (see Fig. 4). In addition, it was confirmed that it is not necessary to add a separate nitrogen source to the medium for the production of bioactive substances (see FIG. 5).

In addition, to determine the optimal concentration of potassium phosphate in one embodiment of the present invention to prepare a rice bran fermentation product using a medium to which potassium phosphate of various concentrations are added and the bioactive material is separated from the indicator of the bioactive material As a result of measuring the production amount of glucosamine as an indicator of extracellular biopolymers and cell masses (see Example <2-3>), a preferable addition amount of KH ₂ PO ₄ is 0.2 to 0.5% (w / v), more preferably. Was confirmed to be 0.2% (w / v) (see FIG. 6).

Furthermore, the present inventors estimate that the bioactive substance contained in rice bran is combined with starch, protein, and the like, and the rice bran fermentation product is prepared using a medium containing various enzymes in order to easily separate the active substance from starch and protein. After preparing and separating the bioactive material from it was measured macrophage activity (see Example <2-3>). As a result, it was shown that the macrophage activity was increased by 20% compared to the control group when the pectase, Rapidase, was added to the medium (see FIG. 7).

Edible mushroom in the step (a) is preferably a mushroom that can be used as food has proven stability. Specifically, the edible mushroom may be selected from the group consisting of Cordyceps militaris , Shiitake ( Lentinus edodes ), and leaf mushroom ( Grifola frondosa ). More preferably, it may be shiitake mushroom. In particular, Cordyceps sinensis is divided into club-shaped and coral-type according to the shape of the fruiting body, the shape of the fruiting body varies depending on the type and other conditions of insects that are hosts, the composition is also different.

The present inventors incubated the strains used as edible mushrooms and six strains used in fermented foods in a medium including rice bran to select strains for the production of physiologically active substances in one embodiment of the present invention, the rice bran fermentation product. Biologically active substances were separated from them and their biological activities were examined (see Example 1). As a result, it was confirmed that the use of Cordyceps sinensis, leaf fungus and shiitake mushrooms could produce bioactive substances with macrophage activity, splenocyte growth promoting activity and intestinal immune activity, especially when using shiitake mushrooms. It was found that the material exhibiting the highest physiological activity could be prepared (see FIGS. 1 to 3).

Cultivation in the step (a) is preferably carried out for 4 to 7 days at 20 ~ 40 ℃, pH 5.5 ~ 6.5 conditions. More preferably, it is incubated for 5 days under conditions of 20 degreeC and pH 5.5.

In one embodiment of the present invention inoculated shiitake mushrooms in the medium containing the rice bran and incubation by varying the culture temperature, culture pH and incubation time of the glucosamine to be the label of the in vitro biopolymers and biomass as a label of physiological activity By examining the production amount, it was possible to establish preferred culture conditions for the preparation of the bioactive substance (see Example 3).

In the step (b), the alkali extraction is performed by adding an alkaline solution to the culture of step (a) to adjust the pH to 9-12, stirring by extraction at 30-50 ° C. for 10-60 minutes, and then neutralizing by adding an acid. To perform. Examples of the alkaline solution may be selected from the group consisting of sodium carbonate, sodium bicarbonate, sodium hydroxide, potassium hydroxide, ammonium hydroxide and ammonia.

As the acid to be added for neutralization in the above, but not limited thereto, hydrochloric acid or citric acid may be used. The acid neutralization may be performed by adjusting the pH to 6-8 by adding hydrochloric acid or citric acid.

In step (c), starch hydrolase is added to the alkaline extract to remove starch components remaining in the alkali extract of step (d). Preferably, the starch hydrolase is added to the alkali extract at 0.5 to 5% (w / v), reacted at 30 to 60 ° C. for 1 to 5 hours, and then heat treated at 100 ° C. for 5 to 30 minutes to inactivate the enzyme. And cooled to room temperature. As the starch hydrolase, all kinds of enzymes capable of hydrolyzing starch may be used. Examples of the starch hydrolase include, but are not limited to, biscozyme or amylase.

The main component of the bioactive substance of the present invention prepared by the above method is a polysaccharide mixture. More specifically, the chemical composition of the bioactive material of the present invention is composed of 58.67% (w / w) neutral sugar, 32.17% (w / w) acidic sugar and 9.16% (w / w) protein. When analyzing the constituent sugar of the bioactive substance of the present invention using gas chromatography (GC), arabinose 21.77% (w / w), xylose 8.99% (w / w), glucose 22.01% (w / w) ), 22.86% (w / w) mannose and 18.61% (w / w) galactose, and other trace amounts of rhamnose and fucose (Example 6 and See Table 3).

The physiologically active substance of the present invention has immunopotentiating activity such as macrophage promoting activity, splenocyte proliferation activity and enteroimmunity promoting activity in vivo and in vitro (Example 1 and Example). 6). In addition, the physiologically active substance of the present invention has the activity of promoting the production of cytokines that promote the activation, growth and differentiation of immune cells in lymphocytes and macrophages of splenocytes (see Example 5 and Figure 14).

Furthermore, the physiologically active substance of the present invention has an activity of inhibiting tumor by promoting immune activation in vivo (see Example 7 and FIG. 15).

In addition, in one embodiment of the present invention was examined by comparing the macrophage activity of the physiologically active substance of the present invention and the conventional immune enhancing substance (see Example 8). As a result, it was confirmed that the physiologically active substance of the present invention has a better effect of promoting macrophage activity than the conventional immune enhancing substance (see Fig. 16).

Therefore, the bioactive material according to the present invention can be provided as an immune enhancing composition for enhancing immunity in vivo.

In addition, the physiologically active substance according to the present invention may be provided as a composition for the prevention or treatment of diseases caused by immune weakening by enhancing immunity in vivo.

Examples of diseases caused by the weakened immune system are not limited thereto. Autoimmune diseases such as rheumatoid arthritis, influenza, cancer, diabetes, high blood pressure and chronic diseases, and the like.

In particular, the bioactive material according to the present invention can be used as an active ingredient of a composition for preventing or treating cancer. Preferably, the bioactive material according to the present invention can be used for the prevention or treatment of lung cancer.

The immune enhancing composition or the composition for preventing or treating cancer may be in the form of a pharmaceutical composition or a food composition.

In the case of pharmaceutical compositions, the bioactive substances prepared according to the method of the present invention may be included alone or may further include one or more pharmaceutically acceptable carriers, excipients or diluents. As used herein, 'pharmaceutically acceptable' refers to a composition that is physiologically acceptable and does not normally cause an allergic or similar reaction when administered to a human.

The pharmaceutical composition of the present invention may be formulated into a preparation for oral or parenteral administration depending on the route of administration. The parenteral administration method is not limited thereto, but may be administered subcutaneously, intravenously, intramuscularly or intraperitoneally. Preferably the pharmaceutical composition of the present invention may be administered intraperitoneally or intravenously.

In the case of preparations for oral administration, the compositions of the present invention may be formulated using methods known in the art as powders, granules, tablets, pills, dragees, capsules, solutions, gels, syrups, slurries and suspensions, and the like. Can be. For example, oral formulations can obtain tablets or dragees by combining the active ingredients with solid excipients and then grinding them, adding suitable auxiliaries and processing them into granule mixtures. Examples of suitable excipients include sugars including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol and maltitol and starch, cellulose, including starch, corn starch, wheat starch, rice starch and potato starch, etc. Fillers such as cellulose, gelatin, polyvinylpyrrolidone, and the like, including methyl cellulose, sodium carboxymethylcellulose, hydroxypropylmethyl-cellulose, and the like. In addition, crosslinked polyvinylpyrrolidone, agar, alginic acid or sodium alginate and the like may optionally be added as a disintegrant. Furthermore, the pharmaceutical composition may further include an anticoagulant, a lubricant, a humectant, a perfume, an emulsifier, a preservative, and the like.

For parenteral administration, it may be formulated in the form of injections, creams, lotions, external ointments, oils, moisturizers, gels, aerosols and nasal inhalants in the art. These formulations are described in Remington's Pharmaceutical Science, 15th Edition, 1975. Mack Publishing Company, Easton, Pennsylvania 18042, Chapter 87: Blaug, Seymour, a prescription generally known in all pharmaceutical chemistries.

Suitable dosages of the pharmaceutical compositions according to the invention are about 10 mg / kg body weight / day to 1000 mg / kg body weight / day, more preferably about 15 mg / kg body weight / day to 500 mg / kg body weight / day. However, the dosage of the composition according to the present invention may be appropriately selected depending on various factors such as the route of administration, the age, sex, weight and severity of the patient.

In the case of a food composition, one or more excipients and additives generally used in the field of preparing a food composition together with the bioactive material of the present invention may be easily prepared in various forms according to methods known in the art. In the above, the food composition of the present invention includes all forms such as functional food, nutritional supplement, health food and food additives.

For example, as a health food, the physiologically active substance of the present invention itself may be prepared in the form of tea, juice and drink for drinking, or granulated, encapsulated and powdered. In addition, it can be prepared in the form of a composition by mixing with the physiologically active substance of the present invention and known active ingredients known to have an immune enhancing or anticancer effect. In addition, in order to use the physiologically active substance of the present invention in the form of a food additive, it can be prepared in powder or concentrate form.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

&Lt; Example 1 >

Selection of strains and production methods for the production of bioactive substances

In order to select the strains and production conditions for the production of bioactive substances by using the strains used as edible mushrooms and six strains applied to fermented foods produced bioactive substances in different ways and then measuring their activity The most preferred strains and production methods were selected. As the physiological activity, immune activity such as macrophage activity, measurement of splenocyte proliferation of mice and intestinal immune activity were measured.

<1-1> Preparation of bioactive substances using various strains and production methods

A strain used in the experiment has a club-shaped Cordyceps stability used proved to food (Cordyceps militaris), coral type Cordyceps (Cordyceps militaris), Maitake mushrooms (Grifola frondosa), shiitake (Lentinus edodes), Aspergillus duck the ( Aspergillus oryzae ) and Aspergillus sojae were used. Production of the bioactive material using the strain was performed by four different methods as follows (Table 1).

Preparation of Rice Bran Fermentation with Different Strains and Production Methods Strain name Production method Experiment 1 Club-shaped Cordyceps sinensis ( Cordyceps militaris ) Liquid culture → alkali extraction → amylase treatment Experiment 2 Leaf Mushroom ( Grifola frondosa ) Liquid culture → alkali extraction → amylase treatment Experiment group 3 Coral Cordyceps Sinensis ( Cordyceps militaris ) Liquid culture → alkali extraction → amylase treatment Experimental Group 4 Shiitake mushrooms ( Lentinus edodes ) Liquid culture → alkali extraction → amylase treatment Experimental group 5 Club-shaped Cordyceps sinensis ( Cordyceps militaris ) Solid culture → alkali extraction → amylase treatment Experimental Group 6 Coral Cordyceps Sinensis ( Cordyceps militaris ) Solid culture → alkali extraction → amylase treatment Experimental group 7 Leaf Mushroom ( Grifola frondosa ) Liquid culture → Alcohol precipitation Experimental Group 8 Shiitake mushrooms ( Lentinus edodes ) Liquid culture → Alcohol precipitation Experimental Group 9 Club-shaped Cordyceps sinensis ( Cordyceps militaris ) Liquid culture → Alcohol precipitation Experimental group 10 Aspergillus Orize
( Aspergillus oryzae ) Liquid culture → Alcohol precipitation Experimental Group 11 Aspergillus sojae Liquid culture → Alcohol precipitation Experimental Group 12 Club-shaped Cordyceps sinensis ( Cordyceps militaris ) Solid culture → Hot water extraction → Alcohol precipitation Experimental group 13 Aspergillus Orize
( Aspergillus oryzae ) Solid culture → Hot water extraction → Alcohol precipitation Experimental group 14 Aspergillus sojae Solid culture → Hot water extraction → Alcohol precipitation Experimental group 15 Leaf Mushroom ( Grifola frondosa ) Solid culture → Hot water extraction → Alcohol precipitation Experimental Group 16 Shiitake mushrooms ( Lentinus edodes ) Solid culture → Hot water extraction → Alcohol precipitation

In the above liquid culture, the spawn of each strain was concentrated at a concentration of 5% (v / v) in sterile medium (pH 6.0) containing 5% (w / v) of rice bran and 0.2% (w / v) of KH ₂ PO ₄ . Inoculation was performed by incubating at 30 ° C. for 5 days. The seed was inoculated into each stock strain in a medium of PDA (potato dextrose agar) and incubated at 30 ° C. for 5 days, followed by 5% of rice bran (w / v), 0.25% of KH ₂ PO ₄ (w / v), and NaNO. ₃ was obtained by inoculating in a medium containing 0.15% (w / v), 0.1% (w / v) MgSO ₄ ~ 7H ₂ O and incubated at 30 ℃ for 5 days.

Solid culture was inoculated with 50 ml of the spawn in a solid medium containing 100 ml of distilled water in 80 g of rice bran and incubated at 25 ° C. for 20 days.

Alkali extraction was carried out by the following method. After distilled water 200ml was added to 50ml of each culture medium, the pH was adjusted to 10.0 with 20% sodium carbonate. This was extracted by stirring vigorously at 45 ° C for 30 minutes. The extract was then centrifuged at 3,000 rpm for 20 minutes to obtain a supernatant. Hydrochloric acid was added to the supernatant to adjust the pH to 7.0.

Alcohol precipitation was added to the culture medium of each strain 4 times ethanol and left at 4 ℃ for 12 hours. The precipitate was recovered by centrifugation at 12,000 g for 10 minutes and dried at 80 ° C. for 12 hours.

The amylase treatment was performed by adding 0.5 g of amylase to an alkali extract corresponding to 50 ml of the culture solution and reacting at 55 ° C. for 3 hours. The reaction was heated for 10 minutes to inactivate the enzyme and dialyzed in the dialysis membrane for 12 hours, and then centrifuged at 3,000 rpm to obtain a precipitate, which was dried.

Centrifugation of each extract prepared in the above manner to obtain only the supernatant (soluble fraction) It was lyophilized and dissolved in physiological saline at the same concentration and used as a sample for activity measurement.

<1-2> Macrophage activity measurement of each sample

Macrophage activity of each sample prepared in Example <1-1> was measured. The macrophage activity was measured by the following method using mouse peritoneal macrophages.

After injecting 2.5 ml of 5% thioglycollate medium into the abdominal cavity of ICR mice (6-8 weeks old, male, purchased from Daehan Biolink Co., Ltd.), the induced peritoneal macrophages were recovered within 72-96 hours, and then RPMI was obtained. Washed 2 to 3 times with 1640. The number of macrophages obtained above was adjusted to 1 × 10 ⁶ cells / ml, and 200 μl was dispensed into 96-well plates. This was incubated for 2 hours at 37 ° C. in a 5% CO ₂ incubator to form a macrophage monolayer, and then the concentration of the bioactive material of Example <1-1> was 10 μg / μl or 100 μg / ml in each well. Additional incubation was made for 24 hours. The negative control group was treated with saline and the positive control group was treated with LPS. It was then washed with RPMI 1640 and Triton X-100 was added to lyse the cell membrane. The free lysosomal phosphatase was reacted with the substrate ρ-NPP and the absorbance was measured at 405 nm using a microplate reader. At this time, the absorbance of the wells containing no cells was measured and used to correct the data of the experimental group.

As a result, all the experimental groups were found to have an effect of promoting the activity of macrophages compared to the negative control group using physiological saline. In addition, in the experimental group 1 to the experimental group 6, the effect of promoting the activity of macrophages was found to be similar or higher than the positive control treated with LPS. In particular, the experimental group 4 in which the shiitake mushroom was liquid cultured showed the highest effect of promoting macrophage activity (FIG. 1).

<1-3> measurement of splenocyte proliferation promoting activity of each sample

Splenocyte proliferation promoting activity of each sample prepared in Example <1-1> was investigated by measuring the activity of lymphocyte mitogen (mitogen, fissile material). The lymphocyte mitogen activity was measured by the following MTT assay method.

MTT analysis was performed by extracting the spleen from ICR mice (6-8 weeks old, male, purchased from Daehan Biolink Co., Ltd.), and transferring the spleen to a Petri dish containing RPMI-1640 medium (ice cold). Was placed on the patch and the cells were released from the spleen gang while pressing the rubber stopper of the syringe. This was filtered using a metal body (mesh # 200), washed three times with RPMI-1640 medium, and the cell concentration was adjusted to 5 × 10 ⁶ cells / ml. Dispense the cells into 96-well plates at 90 µl and add each sample of Example <1-1> at a concentration of 10 µg / ml or 100 µg / ml and incubate at 37 ° C. in a 5% CO ₂ incubator for 3 days. It was. Saline was added to the negative control, and LPS and B cell inducer extracted from E. coli were added to the positive control. ConA (Concanvalin-A) glycoprotein, a T cell inducer, was added. After that, MTT solution of 1 mg / mL concentration was added and reacted for 5 hours at 37 ° C. in a 5% CO ₂ incubator. After removing the supernatant, 100 μl of 0.04N HCl / isopropanol was added to dissolve for 5 minutes, and the same amount was added to water. Absorbance was measured at 570 nm. At this time, the absorbance of the wells containing no cells was measured and used to correct the data of the experimental group.

As a result, all the experimental groups showed higher lymphocyte mitogen activity than the physiological saline treated negative control group. In particular, the experimental group 1 and the experimental group 3 to the experimental group 6 showed higher mitogen-promoting activity than the positive control LPS treatment group, most of them showed a concentration-dependent trend (Fig. 2).

<1-4> Measurement of intestinal immune activity of each sample

Intestinal immune activity of each sample prepared in Example <1-1> was investigated by a known method for measuring the proliferation of bone marrow cells through stimulation of Peyer's patch cells (Hong). T et al., Phytomedicine , 5: 353-360, 1998). Intestinal immune activity measurements were performed using C3H / HeN mice (5-7 weeks old, female, purchased from Biolink, Inc.). Fire measures on the small intestinal wall of the mouse were carefully cut and transferred to a Petri dish containing HBSS. Then, a metal body (No. 100) was placed on the small incision and pressed with a syringe rubber stopper to destroy the tissue to release the cells from the fired intestine. The cell suspension was filtered through a metal body (No. 200), washed with RPMI 1640-FBS (RPM-1640 with 5% FBS), adjusted to a cell concentration of 2 × 10 ⁶ cells / ml, and placed into a 96 well plate. 180 μl were dispensed. Each sample of Example <1-1> was diluted to an appropriate concentration and added so that the final concentration was 100 μg / ml. Saline was added to the negative control and LPS was added to the positive control. After culturing for 5 days at 37 ℃, 5% CO ₂ incubator, the supernatant was recovered and reacted with the bone marrow cells to measure the proliferation of bone marrow cells. That is, bone marrow cells were recovered by injecting HBSS into the femoral bones of mice of the same species by a syringe. Then, adjust the concentration of the bone marrow cells to 2.5 × 10 ⁵ cells / ㎖ and dispense 100 μl into 96 well plate and add the fire-cell culture medium obtained above and 6 days in 37 ℃, 5% CO ₂ incubator Incubated. The proliferation of bone marrow cells was measured by adding a fluorescent reagent (Alamar Blue ^™ ) to the culture and measuring the fluorescence using a fluorescence meter (SPECTRAFluor Plus, TECAN Co. Ltd., Austria).

As a result, Experimental Group 1 to Experimental Group 6 showed higher intestinal immune activity than the positive control group. In particular, the experimental group 4 cultured shiitake mushroom showed the highest intestinal immune activity. In comparison, the experimental group 7 to the experimental group 16 showed similar or lower activity than the negative control treated with physiological saline (FIG. 3).

From the results of the experiment, it was found that the preferred method for producing the bioactive material was the method of extracting the alkali after liquid culture or solid culture and treating it with amylase again. In addition, it was found that the preferred strains for the production of physiologically active substances are Cordyceps sinensis, leaf mushrooms and shiitake mushrooms, in particular, it is most preferable to use shiitake mushrooms.

<Example 2>

Optimization of production media for the production of bioactive substances

<2-1> Determination of Carbon Source

Starch, dextrose, fructose, lactose, mannitol, maltose, sorbitol, sucrose as carbon sources in a basal medium containing 5% (w / v) rice bran and 0.2% (w / v) KH ₂ PO ₄ And xylose were each added at a concentration of 2% (w / v) and the pH was adjusted to 5.5. Shiitake spawn seed prepared in the same manner as in Example <1-1> was inoculated at a concentration of 5% (v / v) with respect to the whole medium and incubated at 30 ° C. for 5 days. As a control, a basic medium containing only 5% (w / v) rice bran and 0.2% (w / v) of KH ₂ PO ₄ was used.

After the incubation was completed, the amount of extracellular biopolymer (exo-biopolymer), which is an indicator of physiologically active substance, and glucosamine, which is an indicator of cell mass, was measured in each of the culture broths. The amount of the extracellular biopolymer was determined from the sum of total sugar content, acid sugar content and protein content in the culture medium. Total sugar content was analyzed using the phenol-sulfuric acid method (Chapin MF, Carbohydrate Analysis, a Practical Approach p. 2, IRL press, 1988), and acidic sugar content was determined using m-hydride as β-D-galacturonic acid as a standard. Measured by oxybiphenyl method (Blumenkrantz, N. & Asboe-Hansen, Anal. Biochem . 54: 484-489, 1973). In addition, the protein was analyzed using the Lowry method (Lowry OH, J Biol Chem , 193: 265-275, 1951).

As a result, the control group containing only rice bran and KH ₂ PO ₄ without the carbon source showed 7.2 mg / ml and 120 μg / ml of extracellular biopolymer and glucosamine, respectively. On the other hand, when the starch, dextrose, lactose, sucrose and xylose were added as the carbon source, the amount of extracellular biopolymers was higher than that of the control. In particular, when the sucrose was added as a carbon source, the amount of extracellular biopolymer was measured to be 16.3 mg / ml, which was the highest. In addition, the amount of glucosamine was also shown to be higher than the control (Fig. 4).

In order to prepare the bioactive substance of the present invention from the above experimental results, it is preferable to use starch, dextrose, lactose, sucrose and xylose as the carbon source of the production medium, and in particular, using sucrose It was found that it is preferable.

<2-2> Nitrogen Source Optimization

Ammonium nitrate, Ammonium chloride, Ammonium phosphate, Peptone, Yeast extract, Soytone (Ammonium nitrate) Various nitrogen sources, such as Soytone) and Polypeptone, were added at 0.5% (w / v), respectively. Shiitake spawn seed prepared in the same manner as in Example <1-1> was inoculated at a concentration of 5% (v / v) with respect to the whole medium and incubated at 30 ° C. for 5 days. At this time, the control group used a medium containing 2% (w / v) of sucrose in the basal medium without including a nitrogen source.

After the completion of the culture, the amount of extracellular biopolymer (exo-biopolymer) that is an indicator of physiologically active substance and glucosamine that is an indicator of cell mass is measured in the same manner as in Example <2-1>. Measured.

Experimental results showed that the extracellular biopolymer production was the highest at about 16 mg / ml for the control group without the nitrogen source. On the other hand, when the peptone, yeast extract and polypeptone were added, the amount of extracellular biopolymer was lower than that of the control, but the amount of glucosamine was high. In particular, when the yeast extract was added, the amount of glucosamine was 183 μg / ml, showing the highest cell mass, but the amount of extracellular biopolymer was 13.2 mg / ml, which was lower than that of the control group without the nitrogen source (Fig. 5). ).

Since it contains various nitrogen sources in addition to carbohydrates in rice bran itself, it is thought that another nitrogen source is not required for the preparation of bioactive substances. Therefore, since the added nitrogen source does not significantly affect the production of extracellular biopolymers, a separate nitrogen source is not added.

<2-3> Determination of Optimal Concentration of Potassium Phosphate

Potassium phosphate (KH ₂ PO ₄ ) added in the basal medium is known as a nutrient used in mushroom cultivation. In order to determine the optimal concentration of potassium phosphate, the concentration of KH ₂ PO ₄ in the base medium to which 2% sucrose was added was 0.05% (w / v), 0.1% (w / v) and 0.2% (w / v) and 0.5% (w / v), respectively. Shiitake spawn seedlings prepared in the same manner as in Example <1-1> were inoculated at a concentration of 5% with respect to the whole medium and incubated at 30 ° C. for 5 days. At this time, as a control, a medium in which only 5% (w / v) of rice bran and 2% (w / v) of sucrose was added without adding KH ₂ PO ₄ was used.

After completion of the culture, the amount of exo-biopolymer that is an indicator of a bioactive substance and glucosamine that is an indicator of cell mass in each culture medium was measured in the same manner as in Example <2-1>. Measured.

Experimental results showed that the amount of extracellular biopolymer was 16.2 mg / ml for the control group without adding KH ₂ PO ₄ . In comparison, KH ₂ PO ₄ When added at the concentrations of 0.2% (w / v) and 0.5% (w / v), it was measured at 16.8 mg / ml and 16.9 mg / ml, respectively, indicating that the amount of extracellular biopolymer was slightly higher than that of the control group. There was no significant difference from the control group. In addition, the amount of glucosamine also appeared to be no difference between the control group and the experimental group (Fig. 6).

Although the _{KH 2 PO 4 0.2% (w} / v) The addition did not indicate a significant difference in productivity of extracellular raw polymer than the additive-free control group, since a slight increase KH ₂ PO ₄ The optimum concentration was determined to be 0.2% (w / v).

<2-4> Selection of Optimal Enzyme

Bioactive substances contained in rice bran were estimated to exist in the form of combined with starch and protein. In order to facilitate the decomposition of substances such as proteins or starch, which are associated with bioactive substances, cellulase, pectinase, amylase (Cululase), pectinase (amylase) in medium containing KH ₂ PO ₄ and sucrose 0.5% (w / v) of various enzymes such as amylase) were added to each of them, and the seed mushroom spawn prepared in the same manner as in Example <1-1> was inoculated at a concentration of 5% with respect to the whole medium, and then 30 ° C. Incubated for 5 days. As an enzyme, a commercially available enzyme was used. In other words, carbohydrate hydrolysis enzyme Viscozyme, pectin degrading enzyme (Rapidase), cellulose degrading enzyme (Celluclast), beta glucan degrading enzyme Ultraflo (Ultraflo), starch hydrolase Phosphorus amylase and xylan (cellulose) hydrolase Econase were used.

As a control, a medium without an enzyme was used. After the culture was completed, the degree of each culture on the macrophage activity was measured in the same manner as in Example <1-2>.

As a result of the experiment, the addition of rapidase to the medium showed a 20% increase in macrophage activity compared to the case without the addition of enzymes (FIG. 7).

Therefore, a medium containing rice bran, KH ₂ PO ₄ , sucrose and rapidase was selected as an optimal medium for preparing a bioactive substance.

<Example 3>

Optimization of Culture Conditions for the Preparation of Bioactive Substances

<3-1> Optimization of incubation temperature

Example <1-to a production medium comprising 5% (w / v) rice bran, 2% (w / v) sucrose and 0.2% (w / v) KH ₂ PO ₄ selected through Example 2. Shiitake spawn prepared in the same manner as 1> was inoculated at a concentration of 5% (v / v) and incubated at 20 ° C., 30 ° C., 40 ° C. and 50 ° C. for 5 days, respectively. After the culture was completed, the amount of the ex vivo biopolymer and glucosamine in the culture was measured in the same manner as in Example <2-1>.

As a result of the experiment, the in vitro biopolymer content was the highest as 18.2mg / ml when incubated at 20 ℃, the amount of the in vitro biopolymer was decreased as the culture temperature was increased. On the other hand, the cell mass was found to be the highest when cultured at 30 ℃ the amount of biopolymers in vitro was 16.4mg / ㎖. The amount of in vitro biopolymer was almost the same as that of the in vitro biopolymer at 40 ° C, but the amount of glucosamine, which is an indicator of cell mass, was lower than that at 30 ° C. When incubated at 50 ° C., both the amount of ex vivo biopolymer and glucosamine were lower than 20 ° C. (FIG. 8).

From the results of the experiment, it was found that a suitable incubation temperature for the production of physiologically active substance is 20 ~ 40 ℃, the most preferred temperature is 20 ℃.

<3-2> Optimization of Culture pH

Example <1-to a production medium comprising 5% (w / v) rice bran, 2% (w / v) sucrose and 0.2% (w / v) KH ₂ PO ₄ selected through Example 2. Shiitake spawn seedlings prepared in the same manner as 1> were inoculated at a concentration of 5% (v / v), and the pH of the medium was adjusted to 4.5, 5.5, 6.5 and 7.5, respectively, and incubated at 20 ° C. for 5 days. After the incubation was completed, the amount of ex vivo biopolymer and glucosamine in the culture was measured in the same manner as in Example <2-1>.

As a result, the in vitro biopolymer content was found to be the highest when the pH of the culture medium was adjusted to 5.5, and then the pH of the culture medium was adjusted to 6.5. The glucosamine content was found to be the highest when the pH of the culture medium was adjusted to 6.5 (FIG. 9).

From the results of the experiment it was found that the preferred pH range for the production of physiologically active substance is 5.5 ~ 6.5, the optimum pH is 5.5.

<3-3> Optimization of incubation time

PH of the production medium containing 5% (w / v) of rice bran, 2% (w / v) of sucrose and 0.2% (w / v) of KH ₂ PO ₄ selected through Example 2 was adjusted to 5.5. After inoculating shiitake mushroom spawn prepared in the same manner as in Example <1-1> at a concentration of 5% (v / v) and incubating for 7 days, samples were taken at daily intervals and in culture according to the culture time. The in vitro biopolymer content and macrophage activity change were measured. The in vitro biopolymer content was measured in the same manner as in Example <2-1>, macrophage activity was measured in the same manner as in Example <1-2> to determine the preferred incubation time.

Experimental results showed that the in vitro biopolymer content was the highest at 5 days and 7 days incubation, there was no significant difference between the two incubation times. Macrophage activity was highest in culture for 5 days and then high in culture for 4 or 7 days (Figure 10).

From the results of the experiment it was found that the preferred incubation time is 4 days to 7 days, the optimum incubation time was 5 days.

<Example 4>

Analysis of physiologically active substances prepared according to the method of the present invention

The shiitake mushroom culture cultured in Example <3-3> for 5 days was alkali-extracted and treated with amylase in the same manner as in Example <1-1> to obtain a bioactive material. The chemical composition of the bioactive substance was analyzed by the following method.

First, 2M trifluoroacetic acid was added to the physiologically active substance and hydrolyzed at 121 ° C. for 1.5 hours to convert to neutral sugar and aldonic acid. Then, neutral and acidic sugar contents were analyzed, respectively. Neutral sugar content was measured by phenol-sulfuric acid (Chapin MF, Carbohydrate Analysis , a Practical Approach p.2, IRL press, 1988), and acidic sugar content was β-D-galacturonic acid as standard. It was measured by m-hydroxybiphenyl method (Blumenkrantz, N. & Asboe-Hansen, Anal. Biochem. 54: 484-489, 1973). NaBH _{4 was} then added to the sample to reduce to altitol and aldonic aicd, respectively. The resulting alditol was converted to an alditol acetic acid derivative using acetic anhydride and analyzed for constituent sugars by GLC. The analysis conditions of gas-liquid chromatography (GLC) are shown in Table 2, and the sugar components in the bioactive substances were analyzed by comparing the retention time of the standard and the retention time of the sample. The mole% of the constituent sugars was calculated from the area ratio of the peaks and the molecular weight of the alditol acetic acid derivative of each constituent sugar. In addition, the protein content of the bioactive substance was quantified by Lowry method using bovine albumin as a standard (Lowry OH et al., J. Biol. Chem ., 193: 265-275, 1951). .

GLC analysis conditions Analytical device GC M600D (Young-Lin Co. Ltd., Korea) Detector Flame ionization detector (FID) (Young-Lin Co. Ltd., Korea) column SP-2380 Capillary Columns (Supelco USA) Column size 0.25mm ㅧ 30m ㅧ 0.2㎛ Film Thickness Oven temperature 60 ° C (1 minute) → 220 ° C (12 minutes) → 250 ° C (15 minutes)
30 ℃ / min 8 ℃ / min Injection temperature 250 ℃ Detector temperature 250 ℃ Carrier gas N2 (1.5 ml / min) recorder D520B Integrator (Young-Lin Co. Ltd., Korea)

As a result, the neutral sugar content of the bioactive substance according to the present invention was quantified as 58.67%, the acid sugar content was 32.17%, and the protein content was 9.16%.

In addition, as a result of analyzing the constituent sugar of the bioactive substance of the present invention, arabinose, xylose, glucose, mannose and galactose were detected, and trace amounts of rhamnose and fucose were detected. Among them, mannose and glucose content were found to be the most contained 22.86% and 22.01%, respectively. Next, arabinose and galactose were quantified at 21.77% and 18.61%, respectively.

Analysis result of physiologically active substance according to the present invention Chemical composition % (W / w) Neutral 58.67 Acid sugar 32.17 protein 9.16 Per composition Rhamnose 4.08 Fucose 1.67 Arabinoz 21.77 Xylose 8.99 Glucose 22.01 Manno Ozu 22.86 Galactose 18.61

Example 5

Cytokine Production Promoting Activity of Biologically Active Compounds Prepared According to the Method of the Present Invention

<5-1> Cytokine Production Promoting Activity in Splenocyte Lymphocytes

The physiologically active substance of Example 4 was treated and cultured to spleen cell lymphocytes prepared in the same manner as in Example <1-3> at a concentration of 10 μg / ml or 100 μg / ml, and the culture was obtained by centrifugation. The amount of cytokine produced by using the supernatant as a sample was measured. As cytokines, production of IL-6, IL-12, GM-CSF and IFN-γ was measured. At this time, the negative control was treated with saline to the lymphocytes, the positive control was treated with LPS to the lymphocytes.

Cytokine production was measured using a sandwich ELISA (enzyme-linked immunosorbent assay) method. First, one day before measuring the level of cytokine production, anti-IL-6, IL-12, GM-CSF, IFN-γ monoclonal antibodies (anti-mouse IL-2, IL-6, IL-12, GM-CSF, IFN-γ monoclonal antibody (R & D systems Inc. USA) was mixed with coating buffer (0.1M carbonate, pH 9.5), aliquoted into 100-well 96-well ELISA plates (Nunc ^TM immunoplate) and allowed to react overnight at 4 ° C. Attached to well surface. The next day, the plate was washed three times with PBST (PBS containing 0.05% Tween 20), followed by dispensing 200 μl of assay diluent (PBS with 10% FBS) and leaving it at room temperature for 1 hour. The surface of the wells that did not attach were blocked. Then wash each well three times with PBST and dispense 50 μl of standard recombinant IL-6, IL-12, GM-CSF, IFN-γ (R & D systems Inc. USA) or sample and add 50 assay diluents. Aliquots were incubated for 90 minutes at room temperature. When the incubation was completed, the reaction supernatant was removed, 50 μl of the assay diluent was dispensed, and the well surface was blocked for 30 minutes and washed five times with PBST. Conjugates of anti-cytokine monoclonal antibody and avidin-horseradish peroxidase conjugated with biotin `to the treated plate were diluted in an assay diluent and allowed to react for 1 hour. After removing the supernatant present in each well, washed 7 times with PBST and incubated for 30 minutes by the addition of TMB substrate (3, 3 ', 5, 5' tetramethylbenzidine and hydrogen peroxide, Pharmingen) 50μl The reaction was stopped by the addition of 2N H ₂ SO ₄ , and the absorbance was measured at 405 nm using a microplate reader (Molecular Devices, USA).

As a result, the bioactive substance according to the present invention was shown to promote the production of IL-6, IL-12, GM-CSF and IFN-γ compared to the negative control. In more detail, the experimental results, IL-6, GM-CSF and IFN-γ was produced in much larger amounts than the negative control (Figs. 11b to 11d). In particular, it was shown that IL-12 is produced in a greater amount compared to the positive control treated with LPS (Fig. 11E).

<5-2> Cytokine Production Promoting Activity in Macrophages

The supernatant obtained by treating the physiologically active substance of Example 4 to a concentration of 10 μg / ml or 100 μg / ml in the macrophages of Example <1-2> and culturing the culture was then produced as a sample. The amount of cytokines taken was measured. That is, the production amount of IL-6, IL-12, TNF-α and IL-1B was measured in the same manner as in Example <5-1>.

Experimental results showed that the production of all types of cytokines was increased when the bioactive substance was treated at a concentration of 100 μg / ml compared to the negative control. However, the treatment of the physiological activity at low concentrations of 10 μg / ml showed little effect on the production of IL-6 and TNF-α (FIGS. 12A and 12C). IL-12 showed a similar degree to the positive control treated with LPS (FIG. 12B), and IL-1B produced the same amount as the bioactive substance of the present invention, although its production was lower than that of the positive control. It could be confirmed that the increase (Fig. 12d).

<Example 6>

In vivo immunopotentiation activity of physiologically active substances prepared according to the method of the present invention

<6-1> Macrophage Activity

In order to measure the in vivo immuno-enhancing activity of the physiologically active substance according to the present invention, the physiologically active substance prepared in Example 4 was prepared in C3H / HeN mice (6-8 weeks old, female, purchased from Biolink Co., Ltd.). Once daily for 5 days through intravenous or abdominal cavity at concentrations of 0, 2.5, 5.0 and 25 mg / kg Administered. In addition, the mice were orally administered with the bioactive substances of Example 4 at concentrations of 25, 50 and 250 mg / kg for 5 days. On day 6 of sample administration, mice were killed to separate macrophages from the abdominal cavity. Macrophages activity was examined in the same manner as in Example <1-2> using the isolated macrophages. Physiological saline was administered to the control group.

As a result, the intravenous administration of the bioactive material of the present invention showed the highest macrophage activity in the group administered at a concentration of 25 mg / kg, the macrophage activity showed a tendency to increase with increasing the concentration of the administration. On the other hand, when the physiologically active substance was intraperitoneally administered, the macrophage activity was found to be much higher than when administered intravenously, and the macrophage activity was increased according to the administration concentration (FIG. 13A).

On the other hand, oral administration of the physiologically active substance of the present invention showed that the macrophage activity was significantly increased at the concentrations of 50 and 250 mg / kg compared to the control group, and there was no significant difference between the two treatment concentrations (FIG. 13B).

<6-2> Spleen Lymphocyte Activity

In the same manner as in Example <6-1>, mice were injected intravenously or intraperitoneally with a bioactive substance. At this time, the control group was injected with saline solution. Lymphocytes were isolated from the spleen after the mice were killed. The isolated lymphocytes were examined for splenic lymphocyte activity by performing MTT assay in the same manner as in Example <1-3>.

As a result, the intravenous injection of physiologically active substance showed the maximum activity in the 5mg / kg administration group and was significantly different from the control group. In the case of the 25 mg / kg administration group showed similar activity to the 5 mg / kg administration group.

Intraperitoneal injection of physiologically active substance showed higher activity in the 2.5 mg / kg administration group compared to the control group, and lymphocyte activity increased with the increase of the administration concentration, indicating the maximum activity in the 25 mg / kg administration group (FIG. 14).

<Example 7>

Cancer metastasis inhibitory activity of physiologically active substances prepared according to the method of the present invention

As shown in the above experimental results, it was confirmed that other biologically active substances in the present invention are excellent in various immune activities in vitro and in vivo, and thus, the biologically active substances may exhibit an activity of inhibiting tumors by promoting immune activation in vivo. There is this.

In this regard, the cancer metastasis inhibiting activity of the physiologically active substance of Example 4 was investigated. The control group, the ^Biobran? Arabinoxylan ^(Republic of Korea Patent No. 344755), immune polysaccharides derived from mushroom fingering ^{PSP? (PSP} Extracts Inc. Canada) was used. Cancer metastasis inhibitory activity was investigated using an animal tumor metastasis model using lung carcinoma. As experimental animals, Balb / c mice (3 weeks old, males) were purchased from Biolink Co., Ltd. and used for 1 week in a laboratory environment. The mice were inoculated with a colon carcinoma cell line colon26-M3.1 (Seoul Cell Line Bank) through the tail vein to transplant the tumor. On the other hand, two days before transplanting the tumor cells into the mouse and one day after the tumor cells were implanted intravenously in the amount of 6.25mg / kg, 25mg / kg and 50mg / kg of the bioactive material of Example 4 14 days Later, mice were killed to remove lungs, which are target organs of tumors, and fixed in Bowin's solution. The colonies of tumors formed in lungs were counted and cancer metastasis inhibitory activity was calculated using the following equation.

Cancer metastasis inhibitory activity = (tumor population number of control group-tumor population number of experimental group) / control group tumor population x 100

As a result of the experiment, all groups to which the physiologically active substance of the present invention showed cancer metastasis inhibiting activity were more than 75% compared to the control group to which physiological saline was administered. In particular, even at a low concentration of 6.25 mg / kg administration group showed a high cancer metastasis inhibitory activity of more than 80% (Fig. 15).

<Example 8>

Comparison of macrophage activity between physiologically active substance according to the present invention and conventional immune enhancing substance

Biobran ^? (Korean Patent No. 344755), arabinoxsilane produced from rice bran from Daiwa, Japan, prepared according to the method of the present invention, PSP ^? (PSP Extracts Inc.) Canada's macrophage activity was measured and compared in the same manner as in Example <1-2>.

As a result, the macrophage activity of the physiologically active substance according to the present invention was found to be the highest and almost similar to that of the LPS-treated group as a positive control group. In contrast, Biobran ^？ and PSP ^？ showed high macrophage activity compared to the negative control but showed very weak activity compared to the positive control (FIG. 16).

Therefore, it was confirmed that the immune enhancing activity of the physiologically active substance prepared according to the method of the present invention was the best.

 As described above, the method for producing a physiologically active substance according to the present invention as described above through the inoculation of edible mushrooms in a culture medium containing a rice bran which has not been subjected to a separate pretreatment process to facilitate a physiologically active substance having excellent immunity. It is effective to make. In addition, the physiologically active substance prepared according to the method of the present invention is very excellent in immunopotentiation activity such as macrophage activity, splenocyte growth promotion, intestinal immune activity and lymphocyte production promoting activity, and promotes immune activity in vivo to weaken immune It is effective in preventing and treating diseases caused, especially cancer.

Claims (10)

(a) Cordyceps militaris in a medium containing 0.5 to 10% (w / v) rice bran, 0.5 to 5% (w / v) sucrose and 0.1 to 1% (w / v) KH ₂ PO _4. ), Inoculated with an edible mushroom selected from the group consisting of shiitake mushrooms ( Lentinus edodes ) and leaf mushrooms ( Grifola frondosa ) and incubated for 3 to 10 days under conditions of 10-50 ° C. and pH 5-7; (b) alkali extracting the culture of step (a); And (C) a method of producing a bioactive material isolated from the rice bran fermentation product comprising the step of reacting by adding starch hydrolase to the alkali extract of step (b). The method of claim 1, wherein the medium of step (a) further comprises 0.2-1% (w / v) pectinase. delete According to claim 1, Alkali extraction in step (b) is adjusted to pH 9 ~ 12 by adding an alkaline solution to the culture of step (a) and extracted by stirring for 10 to 60 minutes at 30-50 ℃ Method characterized by neutralizing. The method of claim 4, wherein the alkaline solution is selected from the group consisting of sodium carbonate, sodium bicarbonate, sodium hydroxide, potassium hydroxide, ammonium hydroxide and ammonia. The method of claim 4, wherein the acid neutralization is characterized in that the pH is adjusted to 6-8 by adding hydrochloric acid or citric acid. The method according to claim 1, wherein the starch hydrolase in step (c) is biscozyme or amylase. delete (a) Cordyceps militaris in a medium containing 0.5 to 10% (w / v) rice bran, 0.5 to 5% (w / v) sucrose and 0.1 to 1% (w / v) KH ₂ PO _4. ), Inoculated with an edible mushroom selected from the group consisting of shiitake mushrooms ( Lentinus edodes ) and leaf mushrooms ( Grifola frondosa ) and incubated for 3 to 10 days under conditions of 10-50 ° C. and pH 5-7; (b) alkali extracting the culture of step (a); And (c) adding a starch hydrolase to the alkali extract of step (b) and reacting. (a) Cordyceps militaris in a medium containing 0.5 to 10% (w / v) rice bran, 0.5 to 5% (w / v) sucrose and 0.1 to 1% (w / v) KH ₂ PO _4. ), Inoculated with an edible mushroom selected from the group consisting of shiitake mushrooms ( Lentinus edodes ) and leaf mushrooms ( Grifola frondosa ) and incubated for 3 to 10 days under conditions of 10-50 ° C. and pH 5-7; (b) alkali extracting the culture of step (a); And (c) adding a starch hydrolase to the alkaline extract of step (b) and reacting the same; preparing a composition for preventing or treating cancer metastasis.

Images