KR20110106760A - Composition for enhancement of immune function and improvement of hematopoiesis which comprises antler fermented with cordyceps militaris kctc 11455bp as an active ingredient, and a preparation method thereof - Google Patents

Abstract

The present invention relates to a composition for enhancing immune function and improving hematopoietic function, comprising antler fermented with Cordyceps militaris KCTC 11455BP as an active ingredient. In addition, the present invention relates to a pharmaceutical composition and a food composition for enhancing immune function and improving hematopoietic function comprising the composition. The composition of the present invention improves hematopoietic function and enhances immune function, and is effective for hematopoietic dysfunction and hematopoietic-related diseases when taking it, and is useful for constitution improvement and health promotion through immune function enhancement.

Description

Composition for enhancement of immune function and improvement of hematopoiesis which comprises antler fermented with Cordyceps militaris KCTC 11455 BP as an active ingredient, and a preparation method

The present invention relates to a composition for improving immune function and improving hematopoietic function, and a method for producing the same, comprising a deer antler fermented with a novel strain of genus Cordyceps having an active antler activity as an active ingredient.

Antler, Cervus cornu parvum ) is a plum blossom ( Cervus) nippon Temminck) or Maroc ( Cervus) elaphus Linnaeus) and the young horns of hairs of the same relatives and dense and unosteolyzed horns are the main products of the Yang Lok industry, which has been used for a long time in oriental countries such as China and Japan. Antlers are known to consume 80% of the world's production in Korea (Choi et. al ., 2005), most of the deer antlers (including rusts) currently used in Korea depend on imports. In 2000, 159 tons of antler were imported for medical purposes, and $ 16 million was spent in domestic production. It is just a kilogram. Korea is the world's largest importer of antler, mostly consumed in Korea.

Deer antler, along with ginseng, has long been used as the most representative tonic herb, mainly in Korea, China and Japan.In modern times, antler has been found to have lower blood pressure, hematopoietic function, hypercholesterolemia, and antistress effect. Kim and Park, 1982; Yong, 1976; Han 1970; Kang and Kim, 1989). Weaknesses of herbal medicines with tonic effects such as ginseng and antler are not only effective on specific organs, but they are not as strong as they are. et al ., 1994).

Recently, active studies on the bioactive components of deer antler have been conducted.The deer antler extract has been reported to have various effects such as immune function enhancement, antioxidant, anti-fatigue, antithrombosis, anti-aging, lowering blood pressure, and anti-inflammatory action. Cho et al., 2005).

However, deer antler has side effects such as diarrhea, and can be used in combination with herbal medicine with astringent action in herbal, in which case the expected effect may be lost or reduced. In addition, antler, which is a useful resource of oriental medicine, is dipped in alcohol for use in Chinese medicine, and then used in small pieces. There is a problem in that many active ingredients of antler are lost in this process.

On the other hand, no case of fermenting deer antler using a fungal strain of Cordyceps militaris. Therefore, the present inventors conducted this study to isolate the microorganisms of Cordyceps in order to increase the effective ingredient efficiency of fermented deer antler and cause a new pharmacological action, and as a result, Cordyceps militaris, a newly isolated strain from the fruiting body of the mushroom When fermentation of antler with KCTC 11455BP, it was found that the yield of fermented extract is much higher than that of the conventional Cordyceps militaris strain and the content of active ingredient can be greatly increased.

In addition, as a result of continuing the study on the antler fermentation fermented with the Cordyceps militaris KCTC 11455BP, the antler fermentation has high anti-complement activity, increase the cytokine (cytokine) production of macrophages , Lymphocytes were proliferated. The Cordyceps militaris KCTC 11455BP antler fermentation not only promotes red blood cell and white blood cell proliferation but also increases the proliferation of hematopoietic stem cells, thereby improving hematopoietic function. It was confirmed.

The immune system is a life phenomenon that maintains the body's homeostasis by removing various abnormalities caused naturally or by external factors (antigens) in vivo, and depending on the specificity of the antigen, innate immunity, nonspecific. immunity) and adaptive immunity (specific immunity). Innate immunity acts nonspecifically on antigens and involves immune cells such as alternative pathways of activation of the complement system, macrophage, and natural killer cells, as well as physical barriers such as skin and mucous membranes. do. In contrast, acquired immunity is an immune system that acts specifically on antigens that pass through the innate immune system, which is classified into humoral and cellular immunity. It is known that the classical pathway of activation of the complement system is involved in the blood reaction by the antibody. The other hand cellular immunity has _{cytotoxic T cell (T C),} helper T cell (T H), suppressor T cell (T S) action of other T cell after the release lymphokine such as is involved in T _C is bound to the surface antigen Activate or attract macrophage. In addition, T _H promotes the activation of B cells, and T _S is known to act to suppress the immune response (Kuby, 2006; Playfair, 1992). This immune system is tightly activated and tightly controlled by various types of cytokine (Benjamini, 2000). Macrophage is a mononuclear cell in which lysosomes develop and is an immune cell that is active in both phagocytosis and is involved in both innate and acquired immunity. Macrophage acts as an antigen-presenting (APC) that processes acquired phages to present and recognize some of the degraded antigens on their surface, inducing acquired immune cells to recognize and activate them (Cebra). and Shroff, 1994; James and Zeitz, 1994), as well as injury to autologous cells such as tumors (Kuby, 2006).

The complement system is a humoral immune system in which about 20 kinds of blood circulating proteins are involved, and is nonspecifically activated against antigens to form a MAC (membrane attack complex) that attacks the surface of bacteria and viruses to induce osmotic hemolysis. By killing target cells (Bienenstock and Befus, 1980; Hames and Glover, 1983). In particular, they are evaluated as biological defense mechanisms that act most rapidly on antigens among various immune systems in the human body (Moon et. al ., 1990), the activation of the complement system depends on the pathways leading to the activation of C ₃ factors (forming C ₃ a and C ₃ b), which are central to the role of the classical, alternative and lectin pathways. (lectin pathway). The activation of C ₃ factor by the classical pathway is activated in turn starting with C ₁ factor by the formation of immune complex of antigen and specific antibodies (IgM, IgG), so that C ₃ factor C ₃ a and C ₃ b Activation by the secondary path is the path through which C ₃ is activated by D factor, starting with the binding of C ₃ b and B factor without formation of immune complex. In addition, the lectin pathway is a pathway in which C ₃ is activated by binding mannan-binding lectin in the blood to polysaccharides including mannose on the surface of bacteria or viruses (Janaeway, 2000; Kim 1986). Classical route has Ca ⁺⁺ and Mg ⁺⁺ ions, the subpath known that are involved in Mg ⁺⁺ ions, each optionally (Janaeway, 1968). On the other hand, the complement system is known to act on infection defense, inflammatory defense, allergic reaction, etc., and research results show that the substances activating the complement system have an anti-cancer effect by acting on the activation of macrophage. Have been reported (Ito, 1986; Okuda et. al ., 1972).

Macrophage is a cell that regulates immune phenomena by secreting various cytokines in the process of phagocytosis and removal of bacteria or foreign substances, and plays a pivotal role in immune action against antigens.It is related to antigen presentation and nonspecific immunity of lymphocytes. The cells show direct injury activity. In addition, substances that react to TLR (tool-like receptors) (LPS or natural products) activate macrophage to stimulate secondary immune responses such as proliferation of T and B cells, macrophage activity for phagocytosis, and defense against microbial infection. It is known to produce cytokines such as IL-1, IL-6, IL-10, IL-12 and TNF-α.

IL-1, IL-6 and TNF-α are representative cytokines induced by macrophage, which play a pivotal role in the inflammatory response following bacterial infection and increase their amount in inflammatory lesions. IL-6 has been reported to be involved in the differentiation of T cells and B cells by cooperatively working with IL-1 and to have anticancer effects. TNF-α has cytotoxic and antiviral effects on certain cancer cells. It is known to play an important role in various biological reactions occurring in chronic inflammatory diseases. On the other hand, IL-12 is a cytokine that induces the activation of NK cells and Th1 type immune response is known to act to increase the reactivity to cellular foreign substances such as cancer cells. In addition, IL-10 cross-regulates the production of TNF, IL-1, chemokine and IL-12 by macrophage, thereby contributing to macrophages in T lymphocyte activation (T lymphocyte mediated specific immune inflammatory response). ) Is known to inhibit.

Recently, the possibility of effective use of complement system activator for disease prevention and treatment has been suggested, the interest in the development of immunotherapy for the purpose of improving and treating the immunocompromised state.

Anemia is caused by a lack of circulating erythrocytes or a decrease in the amount of hemoglobin in erythrocytes, resulting in a decrease in oxygen transport capacity. RBC, hemoglobin, or hemotocrit in circulating blood decreases below normal. (Bruner et al., 1996). Depending on the cause, it is classified into hemorrhagic anemia, hypochromic anemia, aplastic anemia, and hemolytic anemia (Medical College of Seoul University. 1993). Anemia animal models can be induced by dietary factors such as low iron diet, vitamin B12 and folate deficiency, but it takes a long time to induce anemia using cyclophosphamide (CYP) or phenylhydrazine (PHZ). Induced (Chakrabarti et al., 1995; Valley, 2002). PHZ is a strong oxidant. PHZ metabolites such as ROS, phenyldiazene, phenylhydrazyl radical, and benzenediazonium ions produced by automatic oxidation cause oxidative damage to proteins and phospholipid membranes of red blood cells, and oxidize hemoglobin to cause severe hemolytic anemia. et al., 1975; Arduini et al., 1989; Hashmi and Saleemuddin, 1996). In general, anemia is used for the treatment of transfusions or synthetic recombinant human erythropoietin (rHuEPO), and drug administration can be expected to increase blood cell counts and improve symptoms, but the effects are temporary, and therefore, fundamental disease improvement cannot be expected (Feusner). and Hastings, 2002).

An object of the present invention is to provide a composition for enhancing immune function and improving hematopoietic function.

An object of the present invention is to provide a food composition for enhancing immune function and improving hematopoietic function.

An object of the present invention is to provide a pharmaceutical composition for enhancing immune function and improving hematopoietic function.

It is also an object of the present invention to provide a method for preparing a composition, food composition and pharmaceutical composition for enhancing immune function and improving hematopoietic function.

In order to achieve the above object, the present invention provides a composition for improving immune function and improving hematopoietic function, and a method for producing the same, containing the antler fermented with Cordyceps militaris KCTC 11455BP as an active ingredient. The present invention also provides a food composition for enhancing immune function and improving hematopoietic function, comprising the composition, and providing a pharmaceutical composition for improving immune function and improving hematopoietic function, and preparing methods thereof.

The composition containing the antler fermented with Codycept millitaris KCTC 11455BP of the present invention as an active ingredient improves the hematopoietic function and enhances the immune function of the doser, and is effective in hematopoietic dysfunction and hematopoietic related diseases when taking the same. It is effective in improving constitution and health by enhancing immune function.

1 is a change in the content of S-GAG and Salic acid according to the fermentation time of Cordyceps militaris KCTC 11455BP antler fermentation .
Figure 2 is a change in the total sugar and uronic acid content of the Cordyceps militaris (Cordyceps militaris) KCTC 11455BP antler fermentation with the fermentation time.
Figure 3 shows the change of anti-complement activity with the incubation time of Cordyceps militaris KCTC 11455BP.
Figure 4 shows the effect of calcium and magnesium on the anti-complement activity of Cordyceps militaris KCTC 11455BP antler fermentation.
(A) GVB ++ (Ca ++ and Mg ++)
(B) Mg ++-EGTA--GVB--(Mg ++ only)
(C) EDTA--GVB-(divalent metal ion free)
Anti-complement activity was expressed as a 50% inhibition of total complement hemolysis by the Mayer method, and polysaccharide-K (PSK), a known immunostimulatory polysaccharide derived from Coriolus versicolor, was used as a positive control. Each value is expressed as mean ± SD measured three times.
FIG. 5 shows cross-immunoelectrophoretic patterns of C3 factor degradation by Codycept millitaris KCTC 11455BP antler fermentation in the presence of Ca ions. Normal human serum was incubated at 37 ° C. for 30 minutes in samples with GVB ++ (A), MG ++-EGTA--GVB-(B) and EDTA--GVB-(C). Serum was subjected to immunoelectrophoresis using antibodies. Anode is on the left.
Figure 6 shows the lymphocyte proliferation activity of antler-free fermented extract.
Figure 7 shows the lymphocyte proliferation activity of Cordyceps militaris KCTC 11455BP antler fermentation.
Figure 8 shows the IL-6 production amount when the antler non-fermented extract is administered.
Figure 9 shows IL-6 production upon administration of Cordyceps militaris KCTC 11455BP antler fermentation.
Figure 10 shows the IL-12 production upon administration of the antler-free fermented extract.
Figure 11 shows IL-12 production upon administration of Cordyceps militaris KCTC 11455BP antler fermentation.
Figure 12 shows the production of TNF-alpha when the antler extract is not fermented.
FIG. 13 shows TNF-alpha production upon administration of Cordyceps militaris KCTC 11455BP antler fermentation. FIG.
14 shows the effect of antler fermentation on hemoglobin and hematocrit in blood in hemolytic anemia induced by PHZ.
15 shows the effect of antler fermentation on erythropoietin content in PHZ-induced hemolytic anemia rats.

The present invention provides a composition for enhancing immune function, containing antler, fermented with Cordyceps militaris KCTC 11455BP as an active ingredient.

In another aspect, the present invention provides a method for producing a composition for enhancing immune function, comprising the step of fermenting the antler with Cordyceps militaris KCTC 11455BP.

The present invention provides a composition for improving hematopoietic function containing antler fermented with Cordyceps militaris KCTC 11455BP as an active ingredient.

In addition, the present invention provides a method for producing a composition for improving hematopoietic function, comprising the step of fermenting the antler with Cordyceps militaris KCTC 11455BP.

Hereinafter, the present invention will be described.

In the case of fermenting the antler with Cordyceps militaris KCTC 11455BP of the present invention, the antler may be antler or rust, and the origin of antler, such as domestic, Japan, Russia, China, USA, Canada Either way is fine. When fermented with Cordyceps militaris KCTC 11455BP, antler may be fermented in the form of powder or extract.

The composition for enhancing immune function and / or improving hematopoietic function containing antler fermented with Cordyceps militaris KCTC 11455BP of the present invention as an active ingredient may be a food composition or a pharmaceutical composition.

The food and / or pharmaceutical composition for enhancing immune function is effective for autoimmune diseases such as cancer, multiple sclerosis, rheumatoid arthritis, Crohn's disease, autoimmune diseases such as diabetes, or allergies such as asthma, allergic rhinitis, conjunctivitis and atopic dermatitis. . However, even without such a specific disease, the food and / or pharmaceutical composition for enhancing immune function of the present invention may enhance the overall immunity of the body, thereby lowering the possibility of infection and development of various bacterial and viral diseases.

Pharmaceutical composition for enhancing immune function of the present invention is Zadaxin (Thymosinalpha-1), EP-HBS

(HBV Therapeutic Vaccine), HBV Core Antigen Vaccine, LPS, Con. It can be administered with an immunostimulant such as A or an immunomodulator such as Intron A (Interferon alpha-2b) or Pegasys (Peginterferon alfa-2a). In addition, the pharmaceutical composition for enhancing immune function of the present invention further comprises the immunostimulating agent or immunomodulator together with the composition for enhancing immune function containing the antler fermented with Cordyceps militaris KCTC 11455BP as an active ingredient. You may.

The food and / or pharmaceutical composition for improving hematopoietic function of the present invention may improve or reduce hematopoietic function to treat or alleviate anemia, erythrocytosis, or hematopoiesis, and to enhance physical fitness and maintain health by promoting overall body hematopoietic activity. It can help.

The pharmaceutical composition for improving hematopoietic function of the present invention is hemopoietic stimulant such as EPO (erythropoietin), darbepoetin alfa, Aranesp, filgrastim, lenograstim, dextran 40, recombinant human erythropoietin, methoxypolyethyleneglycol-epoetin beta, magnesium chloride, oprelvekin, sargramostim, hydroxyethyl starch, etc. It can be administered with hematopoietic agents. In addition, the pharmaceutical composition for improving hematopoietic function of the present invention may further include the hematopoietic stimulating agent together with the composition for improving hematopoietic function containing the antler fermented with Codycept millitaris KCTC 11455BP as an active ingredient.

The pharmaceutical composition for enhancing immune function and / or improving hematopoietic function containing the antler fermented with Cordyceps militaris KCTC 11455BP of the present invention as an active ingredient can be administered orally or parenterally, It can be used in the form. Preferred pharmaceutical preparations include oral preparations such as tablets, hard or soft capsules, solutions, suspensions, and the like, which are excipients, binders, in the case of conventional pharmaceutically acceptable carriers such as oral preparations, Disintegrating agents, lubricants, solubilizing agents, suspending agents, preservatives or extenders can be used.

The dosage of the pharmaceutical composition for enhancing immune function and / or improving hematopoietic function, which contains antler, fermented with Cordyceps militaris KCTC 11455BP of the present invention as an active ingredient, is determined by the condition, age, sex and It can be determined by a specialist depending on various factors such as complications, but can generally be administered at a dose of 0.1 mg to 10 g, preferably 10 mg to 1 g per kg of adult. In addition, it is intended to contain a daily dose of the pharmaceutical composition or a dose of 1/2, 1/3 or 1/4 thereof per unit dosage form, and may be administered 1 to 6 times a day. However, in the case of long-term intake for health and hygiene or health control, the amount may be below the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety.

The food composition for enhancing immune function and improving hematopoietic function containing antler fermented with Cordyceps militaris KCTC 11455BP of the present invention as an active ingredient may be a health functional food, a health supplement food, a functional food or a nutrient. May be, but is not limited to these. The food composition may be added as it is, or may be used in combination with other food or food compositions, including a composition containing antler fermented with Cordyceps militaris KCTC 11455BP, and may be suitably used according to a conventional method. The mixed amount of the active ingredient can be suitably determined depending on the purpose of use (prevention, health or therapeutic treatment). In general, the food composition of the present invention may be added in the amount of 0.1 to 70% by weight, preferably 0.1 to 60% by weight based on the raw materials in the manufacture of food or beverage. In the food composition, the effective dose of deer antler fermented with Codycept millitaris KCTC 11455BP may be used in accordance with the effective dose of the pharmaceutical composition, but for a long time for health and hygiene purposes or health control purposes In the case of intake may be below the above range, the active ingredient may be used in an amount above the above range because there is no problem in terms of safety.

 There is no particular limitation on the kind of food. The food composition comprising a composition for enhancing immune function and / or improving hematopoietic function containing the antler fermented with Cordyceps militaris KCTC 11455BP as an active ingredient is a tablet, a hard or soft capsule, a liquid, a suspension. Oral preparations, such as and the like, these preparations may be used in the form of acceptable conventional carriers, for example in the case of oral preparations, excipients, binders, disintegrants, lubricants, solubilizers, suspending agents, preservatives Or an extender or the like.

Examples of foods to which a composition for improving immune function and / or improving hematopoietic function containing antler fermented with Cordyceps militaris KCTC 11455BP as an active ingredient may be added to meat, sausage, bread, chocolate, Candies, snacks, confectionery, pizza, ramen, other dairy products including noodles, gum, ice cream, various soups, beverages, teas, drinks, alcoholic beverages and vitamin complexes, but are not limited to these types of foods. .

In another aspect, the present invention provides a method for producing the composition for improving immune function and / or hematopoietic function comprising the step of fermenting the antler with Cordyceps militaris KCTC 11455BP.

The method for preparing the composition for enhancing immune function may further comprise adding the immunostimulant. The step of adding the immunostimulating agent may be added before or after the step of fermenting the antler with Codycept millitaris KCTC 11455BP, and may be added with the antler fermentation.

In addition, the method for preparing a composition for improving hematopoietic function may further include adding the hematopoietic stimulant. The step of adding the hematopoietic stimulant may be added before or after the step of fermenting the antler with Codycept Millitaris KCTC 11455BP, and may be added with the antler fermentation.

Hereinafter, the present invention will be described in more detail by the following examples and experimental examples. However, the following examples and experimental examples are intended to illustrate the contents of the present invention, but the scope of the invention is not limited by the examples and the experimental examples.

Example 1 Isolation of Cordyceps militaris KCTC 11455BP

1. Strain assay with good growth from 5 strains of wild mushrooms

end. Isolation of Strains from Wild Mushrooms

Wild collected bacteria were fixed with a sterile tape on a Petri dish lid after sterilization of 2-3% sodium hypochloride in the fruiting body, and then separated from the spore spores of the mushrooms shown in Table 1 on the water agar (WA) medium. Potato dextrose agar (PDA) was cultured in a static culture to isolate the strain.

I. Cultivation of Strains

B-1. Cultivation in Deer Antler Powder Solid Medium

200 g of antler powder was added to suspend 200 mL of distilled water, and then agar 4 g was added thereto, followed by autoclaving at 121 ° C. for 15 minutes in a Petri dish to use as a solid medium. It was confirmed whether the growth of.

B-2. Cultivation in Solid Medium containing Deer Antler Extract

200 mL of distilled water was added to 10 g of antler, and the mixture was extracted under reflux at 100 ° C for 3 hours. The extract was recovered by centrifugation, and 150 mL of distilled water was added to the precipitate obtained by centrifugation, followed by secondary reflux extraction. The secondary reflux extract obtained by centrifugation and the primary extract were mixed and concentrated to obtain 80 mL of concentrate (7.5 brix). Add 120 mL of distilled water and 4 g of agar to 80 mL of concentrated extract, autoclave at 121 ° C. for 15 minutes, put in petri-dish, and use as a solid medium to spread the isolated strains and incubate at 25 ° C. for 5 days to grow the strains. It was confirmed.

All. Growth level measurement and strain selection

The growth of the strain according to the growth degree in the antler powder solid medium and antler extract solid medium is shown in Table 1 and Table 2, respectively.

Very strong strain growth: +++

Strain growth is strong: ++

Strain growth is usually: +

Weak strain growth:-+

Not growing at all:-

Strain Growth in Solid Medium Containing Deer Antler Extract

Strain Growth in Solid Medium Containing Deer Antler Powder

la. Identification of Strains

In Tables 1 and 2, strains were selected and isolated from Cordyceps militaris, which grew very strongly, and genomic DNA was extracted using a genomic DNA preparation kit (SGD62S120, Solgent Co. Ltd., Korea). Universal primers ITS 1 (5'-TCC GTA GGT GAA CCT GCG G-3 '), ITS 4 (5'-TCC TCC GCT TAT TGA TAT GC-3'), EF-Tag (SEF16 R250, Solgent Co. Ltd., Korea) was amplified by PCR (Applied Biosystem 9700, CA, USA). Purification using acetate and 70% ethanol was followed by sequencing kit (Big dye terminator cycle sequencing ready reaction kit) and ABI Prism 3730XL DNA analyzer (Applied Biosystems, CA, USA). The 18S rRNA sequences of the isolates were aligned with the sequences of the flexible strains obtained from the EMBL / DDBJ / PDB / GenBank sequence database and compared to their homology. The results are shown in Table 3. As a result, both the antler powder solid medium and the antler extract solid medium were The very active strain was Cordyceps militaris.

The Cordyceps militaris strain was deposited on January 16, 2009, with the accession number KCTC 11455BP to the Korea Institute of Bioscience and Biotechnology (KCTC).

Homology of 16S rDNA Sequences and Sequences of Cordyceps Militaris KCTC 11455BP

hemp. Mycological Characteristics of Isolated Strains

Cordyceps militaris (Cordyceps militaris) KCTC 11455BP strains according to the present invention is a fungal strain, unlike the conventional bacteriological properties of the known Cordyceps militaris (Cordyceps militaris) strain and nutrition for the carbon source The bacteriological characteristics were compared for the requirements.

There are only about three Cordyceps militaris strains that can be distributed at the Korea Institute of Bioscience and Biotechnology (KCTC), and among them, Cordyceps militaris strains derived from plant resources are KCTC 6472. (IFO 30377) Since it is only one strain that can be distributed, in the following experiment, Cordyceps militaris (Cordyceps militaris) KCTC 6472, which was sold at the BRC, was compared with a known strain.

In order to compare the bacteriological properties of the Cordyceps militaris KCTC 11455BP strain and the Cordyceps militaris KCTC 6472 strain according to the present invention, the nutritional requirements for the carbon source were measured.

Lily medium (Maltose 9.5 g / ℓ, aspargine 2.0 g / ℓ, KH ₂ PO ₄ 1.0 g / l, MgSO ₄ 7H ₂ O 0.5 g / l, Agar 20.0 g / l) were used as base medium, and the nutritional requirements of the two strains for the carbon source (10 g / l) were measured. After autoclaving the medium at 121 ° C. for 20 minutes, the strains were inoculated by removing the flora from a 5 mm cork borer. Inoculum was incubated for 15 days in a 25 ℃ incubator after measuring the colony diameter and mycelial density are shown in Table 4.

Nutritional Needs for Carbon Sources

aColony diameter (mm): measured after 15 days at 25 ° C

cMycelial density: +, thin: TT, moderate: +++, compact

As can be seen in Table 4, the mycelial growth of various carbon sources was similar to each other as a result of culturing the mycelium of Cordyceps millitaris KCTC 11455BP and Cordyceps millitaris KCTC 6472 at the same conditions. In Cordyceps mitaris KCTC 6472, the degree of growth of mycelium was higher than that of the cordyceps mitaris KCTC 11455BP according to the present invention, the density of mycelium growth was rather much higher than the cordyceps millitaris KCTC 11455BP according to the present invention.

In the above results, it can be seen that Cordiscept millitaris KCTC 11455BP and the known strain Cordiscept millitaris KCTC 6472 according to the present invention have different utilizations for carbon sources when added to arabinose. You can see something else.

Example 2 Deer Antler Fermentation by Cordyceps Militaris KCTC 11455BP

The culture of Cordyceps militaris KCTC 11455BP strain isolated in Example 2 using 20 mL of PDB (potato dextrose broth) medium was performed as a preculture for 5 days at 25 ° C.

30 g of 50 mM phosphate buffer (pH 6.0) was added to 4 g of antler powder, which was autoclaved at 121 ° C. for 15 minutes, cooled to 25 ° C., and 1 mL of the whole culture solution of the Cordyceps militaris KCTC 11455BP strain was added. Incubated at 25 ℃ for 5 days. 60 mL of distilled water was added to the culture solution, followed by extraction at reflux for 3 hours at 100 ° C., followed by centrifugation to obtain a antler fermented extract.

Comparative Example 1 Deer Antler Fermentation by Cordyceps Militaris KCTC 6472

In the same manner as in Example 2, using a known strain Cordyceps militaris (Cordyceps militaris) KCTC 6472 strain to prepare a antler fermented extract to Comparative Example 1.

Experimental Example 1 Yield Comparison of Antler Extract

The yields of the antler extracts prepared in Example 2 and Comparative Example 1 were measured and shown in Table 5.

As can be seen in Table 5, the yield of the antler fermentation extract by Cordyceps militaris (Cordyceps militaris) KCTC 11455BP strain according to the present invention yield of the antler fermentation extract by the known Cordyceps militaris strain It can be seen that the yield of about 2 times better.

<Experiment 2> Comparison of active ingredient content of antler fermentation extract

S-GAG (sulgated-glycosaminglycan), an active ingredient of antler, is a substance that has increased interest as an indicator of osteoblast growth, osteoclast inhibition, osteoporosis prevention, and arthritis treatment, and Sialic acid determines the type of gangliosides. It is a major indicator component, Gangliosides is known to play a very important role in the neurotransmitter system of memory and learning ability enhancement cells, to compare the active ingredient content of the antler fermentation extract prepared in Example 2 and Comparative Example 1 The same experiment was conducted.

Analysis of the active ingredient for the antler fermentation extract was measured by the following method. Total sugar was measured by phenol-sulfuric acid method (Omran et al., 1989). Sulfated-glycosaminoglycan (S-GAG) was measured at 540 nm by dimethyl-methylene blue dye binding method (Farndale et al., 1982). The measurements are shown in Table 6, measured at 549 nm by Warren method (Warren, 1959) after hydrolysis with 0.1 N sulfuric acid at 80 ° C. for 1 hour.

Analysis of Active Ingredients for Deer Antler Extracts

As a result of analyzing the active ingredient content of the deer antler fermentation extract, as shown in Table 6, the content of the active ingredient Total sugar, S-GAG and the content of Sialic acid before and after fermentation of known codycept millitaris strain Although similar to the antler fermentation extract by, it was confirmed that significantly increased in the antler fermentation extract by the Cordyceps militaris KCTC 11455BP strain according to the present invention.

Therefore, the antler fermentation extract by the Cordyceps militaris KCTC 114455BP strain according to the present invention is very effective in promoting osteoblast growth, osteoclast inhibition, osteoporosis prevention effect, arthritis treatment agent, memory and learning ability enhancing neurotransmitter system It can be usefully used.

Experimental Example 3 Antioxidant Effect of Antler Ferment Extract

In order to determine the antioxidant activity effect of the antler fermentation extract prepared in Example 2 and Comparative Example 1 according to the present invention was carried out the following test.

Antioxidant activity was measured for scavenging activity and ABTS scavenging activity against DPPH radical. Scavenging activity against DPPH radicals was determined by the method of Blois (1958). 0.2 mL of the sample and 0.8 mL of 4 × 10 ^-4 M DPPH solution were added to the ethanol titration, mixed for 10 seconds, and left for 10 minutes.

ABTS scavenging activity was measured by modifying the method of Arnao et al. (Arnao et al. 2001). 1.0 mM AAPH (2,2'-azobis (2-amodino-propane) dihydrochloride) was dissolved in 2.5 mM ABTS (2,2'-azino-bis 3-ethylbenzenothiazolin-6-sulfonic acid) dissolved in 100 mM PBS buffer. After mixing, the reaction was carried out at 68 ° C. for 12 minutes. The concentration of the ABTS solution was adjusted to 0.650 ± 10.2 at 734 nm. The absorbance at 734 nm was measured while reacting 20 μl samples of various concentrations and 980 μl ABTS solution in a 37 ° C. water bath for 10 minutes. At this time, the concentration (IC ₅₀ ) of the extract to eliminate the radical 50% was determined.

The results of immediately scavenging activity and ABTS scavenging activity for the DPPH radical are shown in Table 7.

As can be seen in Table 7, the IC ₅₀ value indicating the degree of radical scavenging ability of the antler fermentation extract is higher than the antler fermentation extract obtained from the known antler fermentation extract according to the present invention in both ABTS radical scavenging ability and DPPH radical scavenging ability It was confirmed that the antioxidant activity effect of about 2-3 times was remarkably excellent.

Example 3 Changes in Components of Cordyceps Militaris KCTC 11455BP Antler Fermentation

As a result of measuring the changes in the composition of the antler fermentation by Codycept Millitaris KCTC 11455BP, the content of S-GAG increased as the incubation time increased (Fig. 1), while the sialic acid content increased as the incubation time increased. There was a tendency to decrease slightly and then increase again, especially up to 60-72 hours of incubation and then thereafter (Figure 1, Table 9). The total sugar and uronic acid contents increased with the incubation time, but the increase was observed after 36 hours of cultivation, and the increase was apparent in the total sugar content (Fig. 2). The pH of each culture time tended to increase as the culture time elapsed (Table 8). Therefore, 72 hours seems to be the most suitable when fermenting antler using Codycept Millitaris KCTC 11455BP.

Time (h) 0 12 24 36 48 60 72 84 pH 7.4 7.41 7.5 7.68 7.96 8.19 8.51 8.74

Cordyceps Militaris PH Change with Deer Antler Fermentation Time Using KCTC 11455BP

division 0 H 24H 36H 48H 60 H 72 H 84 H Uronic acid 106.1 ± 10.2 74.9 ± 3.6 90 ± 0.6 129.4 ± 0.6 146 ± 0.3 153.5 ± 3.3 165.7 ± 1.4 S-GAG 103.4 ± 10.6 264.2 ± 4.4 290.5 ± 3.6 276.3 ± 4.2 312.8 ± 20.6 358.3 ± 9.7 354 ± 4.1 Sialic acid 16.53 ± 0.15 15.87 ± 0.65 15.6 ± 0 12.77 ± 0.75 20.8 ± 0 20.1 ± 0.8 15 ± 0.9

Standard substance: Total sugar as glucose, Uronic acid as galacturonic acid, Sulfated-GAGs (glycosaminoglycan) as chondroitin sulfate, sialic acid as N-acetylneuraminic acid

Example 4 Immune Function Enhancing Activity of Cordyceps Militaris KCTC 11455BP

Experimental Example 1 Materials and Methods

Cordyceps Militaris KCTC  11455 BP  velvet Fermented  Produce

Deer antler used in this experiment was supplied from Kwangdong Pharm. The antler fermented product was obtained by injecting 40 g of antler powder into 300 mL of 50 mM phosphate buffer (pH 6.0) and inoculating Codycept Millitaris KCTC 11455BP, which was fermented at 25-30 ° C for 7 days. The antler powder which was not fermented at the same concentration was used as a sample. 300 mL of water was added to each sample, and the mixture was refluxed at 100 ° C. for 3 hours, and then the supernatant was recovered by centrifugation at 8,000 × g for 20 minutes. This was repeated twice, and the extracts were concentrated and lyophilized to obtain fermented antler samples and non-fermented antler samples, respectively.

Reagents and Laboratory Animals

RPMI medium 1640, Penicillin / Streptomycin, Fungizone, etc. used for lymphocyte proliferation and macrophage activity measurement experiment were purchased from Gibco BRL (Grand Island Co, NR, USA) and used for cell proliferation. Counting kit-8) was purchased from Dojindo, Japan. LPS (Lipopolysaccharide from E-coli 0127: B8) used as an immunological control was used by Sigma, and PSK (polysaccharide-K from Coriolus). versicolor ) is a commodity sold by Kwangdong Pharm ^. Soluble fraction was purified from and used. On the other hand, the amount of sensitized blood cells (IgM-hemolysis sensitized, EA cell) used in anti-complement activity was manufactured by Biotest, Japan, and the anti-human C ₃ used by cross-immunoelectrophoresis, was manufactured by Sigma. In addition, all reagents used in this experiment were commercial grade 1 or higher products.

In addition, the experimental animal BALB / c mouse (5-6 weeks old) used in this experiment was purchased from Korea Animal Tech, and then subjected to experiments for a week. Breeding conditions were maintained at 23 ± 3 ℃, humidity 55-70%, water and feed in the form of free feeding.

Experimental Example 2 Changes in Immune Activity of Cordyceps Militaris 11455BP Antlers

Normal serum ( normal human serum , NHS Manufacturing

Blood from healthy adults is collected and left to coagulate for about 15 minutes at room temperature, and then the coagulated blood is cut and left to stand at room temperature for about 5 minutes. The blood was left to stand again at 4 ° C. for about 20 minutes, and then centrifuged (3,000 rpm, 20 minutes, 4 ° C.) to separate the serum, and then, 1 mL was dispensed into a micro centrifuge tube and stored at -70 ° C. for freezing. Used for.

Fermentation time and Concentration Anticomplement  Active measurement

Anti-complement activity was measured by a complement fixation test method based on the degree of erythrocyte hemolysis caused by the complement remaining after complement consumption by the sample using the Mayer method.

Samples dissolved in distilled water at different concentrations were mixed with GVB ⁺⁺ (containing gelatin veronal buffer, pH 7.4, 0.1% gelatin, 0.15 mM Ca ⁺⁺ , 0.5 mM Mg ⁺⁺ ) and serum of normal persons, respectively, at 37 ℃. First reaction was carried out for 30 minutes at. 350 μl of GVB ⁺⁺ was added to the reaction solution, and serial dilutions thereof were carried out up to 10 to 160 times, followed by 750 μl of GVB ⁺⁺ and positive sensitized blood cells (IgM-sensitizated sheep erythrocyte, EA cell, 1 × 10 ⁸ cells / 250 µl of the solution) was added thereto, followed by secondary reaction at 37 ° C. for 60 minutes, and 2.5 mL of PBS (phosphate buffered saline, pH 7.4) was added to stop the reaction. The reaction solution was centrifuged at 2,000 rpm for 10 minutes, and the obtained supernatant was measured for absorbance at 412 nm to measure residual hemolytic activity. Anti-complement activity was inhibited by 50% total complement hemolysis (TCH ₅₀ ,%) of the negative control group that reacted only with normal serum and GVB ⁺⁺ and distilled water (inhibition of 50% total complement hemolysis, ITCH ₅₀ ,%). ). The positive control group was compared by using PSK (polysaccharide-K), an immune enhancer derived from fingering mushrooms.

<Equation 1>

ITCH ₅₀ (%) = TCH ₅₀ of sample / TCH ₅₀ of control × 100

Anti-complement activity is Cordyceps Militaris From day 1 of KCTC 11455BP culture, the activity of the control group was about 2 times higher than that of the control group. In the experiment, the extraction amount of the active substance was the highest at the 3rd day of culture. In order to obtain KCTC 11455BP deer antler fermentation, it is preferable to culture for at least 3 days (Fig. 3).

Cordyceps Militaris In order to measure the immunological activation patterns of KCTC 11455BP deer antler fermentation by concentration, anti-complement activity by concentration was measured according to Mayer's method. Coriolus was the positive control. versicolor ) -derived immunoactive polysaccharide PSK (polysaccharide-K) was used, and distilled water without a sample was used as a negative control. Activation capacity was confirmed by setting the degree of activation in the negative control group to ITCH ₅₀ 0. As a result, Codycept Militaris KCTC 11455BP deer antler fermentation showed a relatively good activity of more than about 40% at a concentration of 1,000 μg / mL, which was lower than the positive control group added the same concentration (Fig. 4). In general, polysaccharides exhibiting anti-complementary activity of 40% or more at a concentration of 1,000 μg / mL are known to be commonly recognized for their pharmacological properties (Kabat and Meyer, 1964), and thus, the fermented extracts are considered to have excellent complement activation. In addition, as a result of experiments with different concentrations of fermentation at 1,000, 500, and 100 μg / mL, the anti-complement activity was found to be Cordyceps militaris. It was confirmed that the increase in proportion to the concentration of KCTC 11455BP antler fermentation (Fig. 4).

Cordyceps Militaris KCTC  11455 BP  velvet Fermented Complement  Review of Activation Path

1. Review of complementary system activation pathway using metal ions

In order to determine the active path of the complement system GVB ⁺⁺ buffer and Ca ⁺⁺ ions are selectively removed by Mg ⁺⁺ -EGTA-GVB ^- a buffer, Ca ⁺⁺ and Mg ⁺⁺ ions to remove all EDTA-GVB ^- A buffer was prepared, mixed with the sample and NHS, respectively, and reacted at 37 ° C. for 30 minutes. Each reaction solution was activated again for 60 minutes at 37 ℃, 2.5 mL of PBS was added again, centrifuged at 2,000 rpm for 10 minutes and the supernatant was measured for absorbance at 412 nm to measure the residual hemolytic activity to compare the activation of complement system. .

The activation pathway of the complement system is largely composed of the classical pathway and the alternative pathway (Wang et al ., 2003), both Ca ⁺⁺ and Mg ⁺⁺ are involved in the activity of the classical pathway (Tsukagosh et. al ., 1984) It is reported that only Mg ⁺⁺ is selectively involved in the secondary path (Marcus et. al ., 1971). Therefore, when comparing the activity of the reaction system in which a specific metal ion is removed and the basic reaction system, it is possible to predict the complement activation pathway of the sample. Cordyceps Militaris found to have excellent complement activity Anti-complement activity was measured in a reaction system in which a specific metal ion was removed from the deer antler extract fermented with KCTC 11455BP.

Was Ca ^++, as shown in Figure 5 is in the selective removal of a reaction system Ca ⁺⁺ and Mg ⁺⁺ that is approximately 20% activity compared to the primary reaction system in the presence both appeared in a dose-dependent, Ca ⁺⁺ and Mg ^It was confirmed that the activity was almost lost in the reaction system ⁺⁺ all removed. This means that the activation of the complement system of antler extract is via both the classical and the secondary paths.

2. Review of complement system activation pathway using 2D immunoelectrophoresis

According to Shimura et al., Two-dimensional immunoelectrophoresis was performed to confirm the complement system activation pathway. ^{GVB ++ buffer, Mg ++ -EGTA-} GVB - buffer the GVB-EDTA ^- by mixing an equal volume of a normal human serum or purified polysaccharide sample in each buffer (50 ㎕) was cooled after 30 minutes of reaction at 37 ℃. 5 μl each was loaded into a 1% agarose gel plate (5 × 5 cm) well prepared by dissolving the reaction solution in barbital buffer (pH 8.6), and primary electrophoresis (75 mA / plate) was performed at 4 ° C. for about 3 hours. Was carried out. Subsequently, secondary electrophoresis (25 mA / plate) was performed at 4 ° C. for about 15 hours on an agarose gel plate containing 1% anti-human C ₃ . The gel is to discoloration after about 10 minutes, stained with bromophenol blue deployed by checking the sedimentation line (line precipitation) was observed for activation of C _3.

Anti-complement activity measurement by the Mayer method is a measure of the consumption of complement by activation of the complement system of the sample component in the first reaction step, even if a specific inhibitory component other than the activation of complement in the sample, high anti-complement activity (Kim et. al ., 2002). In general, when the high and minor pathways of the complement system are activated, C ₃ , the most important component of the complement system activation, is decomposed into C ₃ a and C ₃ b, followed by subsequent activation of C ₅ and antigens of C ₆ to C ₉ . The binding system activates the complement system. Therefore, by confirming whether C ₃ is activated using two-dimensional immunoelectrophoresis, it is possible to confirm whether the anti-complement activity of the sample confirmed by the Mayer method is due to complement system activation or a complement inhibitory factor.

Cordyceps Militaris The antler extract fermented with KCTC 11455BP was reacted in the basic reaction system and the reaction system in which the specific metal ion was removed, and the degradation of C ₃ was observed. From the well reaction system in which both Ca ⁺⁺ and Mg ⁺⁺ are present, The second settler was higher than the first settler. From the well the first settling line is caused by C ₃ and the second settling line is caused by C ₃ a and C ₃ b, which is Codycept Militaris It means that the anti-complement activity of the antler extract fermented with KCTC 11455BP is not caused by the complement inhibitor. On the other hand, in the reaction system in which all metal ions were removed, only the first settler line appeared clearly and the second settler line did not appear. In addition, in the reaction system in which only Ca ⁺⁺ was selectively removed, the height of the second settler was relatively lower than that of the normal reaction system. This indicates that C ₃ activation occurred even when the classical pathway was inhibited. It is the result confirming that complement system activity of deer antler extract fermented with KCTC 11455BP appears through both the classical path and the secondary path.

Experimental Example 3 Lymphocyte Proliferation Activity of Cordyceps Militaris KCTC 11455BP Antler Fermentation

BALB / c (♀, 6 weeks old) mice were killed by oral dislocation and spleens were removed under aseptic conditions. Lymphocytes were pulverized (100 mesh) and filtered (200 mesh) on PBS using a stainless steel mesh. Obtained. 5 mL of 0.2% NaCl was added for 15-30 seconds to destroy the mixed red blood cells, washed 2-3 times with RPMI 1640, and then adjusted to 1 × 10 ⁶ cells / mL using a Hemacytometer. Lymphocytes were stained with 0.2% trypan blue (Sigma Chemical. Co. Ltd., USA) to count only living cells. 180 μl of isolated spleen lymphocytes and 20 μl of PBS lysed sample were dispensed (1.44 × 10 ⁵ cells / 0.2 mL / well) into a flat-bottomed 96-well microplate, followed by 72 hours in a 37 ° C., 5% CO ₂ incubator. Incubated. Lymphocyte proliferation stimulation activity was measured by dispensing 10 μl of CCK-8 (cell counting kit-8, Dojindo Co. Ltd., Japan) per well, and reacting at 37 ℃ for 5 hours in a 5% CO2 incubator for 450 nm. Absorbance was measured at to determine the extent of proliferation.

Lymphocytes obtained from spleen of BALB / c mouse were fermented antler extract and cordyceps militaris. KCTC 11455BP antler fermented product was added to measure the proliferation results are shown in Figure 6 and 7, respectively. No growth was observed in the non-fermented deer antler extract when 100% of the lymphocyte proliferation (positive control group) was cultured with media alone, and the same pattern was observed when boosted with LPS and conA. It was confirmed that the simple antler extract had no lymphocyte proliferation activity. Cordyceps Militaris, on the other hand KCTC 11455BP deer antler fermentation increased lymphocyte proliferation in a concentration-dependent manner, especially at 500 μg showed more than 30% proliferative activity compared to the no addition control. This lymphocyte proliferative activity was more pronounced when added with LPS and conA, which resulted in Codycept millitaris It shows that KCTC 11455BP antler fermentation has mitogen activity to directly proliferate mature immune cells. Therefore, when exposed to antigen from the outside, Codycept Militaris By administering KCTC 11455BP antler fermentation, it is possible to effectively induce defense against antigen by increasing the number of effector cells that induce an immune response against the antigen.

Experimental Example 4 Effect of Cordyceps Militaris 11455BP Antler Fermentation on Cytokine Cytokine Production

Preparation of Macrophage Culture

RPMI 1640 was added to the antler fermentation sample solution prepared at 1 mg / mL concentration using PBS, and serially diluted in multiples to a concentration of 0.12 μg / mL at 500 μg / mL and 100 μl each on a flat-bottomed 96-well microplate. Busy. Inject 2 mL of 5% thioglycollate medium into the abdominal cavity of BALB / c (♀, 6 weeks) mouse and add 100 μl of induced macrophage (2.25 × 10 ⁵ cells / mL of RPMI 1640) for 72 hours. Incubation was performed for 24 hours in a 37 ° C., 5% CO ₂ incubator. After incubation, 150 μl of the cell culture solution was recovered by centrifugation at 1,500 rpm for 5 minutes and the content of cytokine induced in the supernatant was measured.

Sandwich ELISA On by cytokine  Measure

The content of cytokine produced by macrophages was analyzed using a sandwich enzyme-linked immunosorbent assay (ELISA).

Antibodies of each cytokine were diluted in a coating buffer, coated on a flat-bottomed 96-well microplate, and then left at 4 ° C for 12 hours. The coated microplate was washed three times using washing buffer (PBS with 0.05% tween 20, PBST), and 200 μl of assay diluent (PBS with 10% FBS or 2% skin milk) was added and left for 1 hour. The non-stick well surface was blocked. After the blocking was completed, each well was washed three times again using a washing buffer, and 50 μl of each of the standard dilutions (recombinant mouse cytokine) and immune cell culture medium diluted in each well was dispensed. This was allowed to stand at room temperature for 2 hours, washed with washing buffer and treated with 100 μl of detection antibody (in assay diluent). After treatment with 100 μl of Enzyme reagent (avidin-horseradish peroxidase conjugate) for 30 minutes at room temperature, add 100 μl of substrate solution [tetramethylbenzidine (TMB) and hydrogen peroxide] and react for 30 minutes in the dark. 1 MH ₃ PO ₄ or 2 N NH ₂ SO ₄ )] was measured at 450 nm.

result

Non Fermented Antler Extract and Cordyceps Militaris KCTC 11455BP Antler Ferment Direct stimulation of cytokine production in macrophages by In vitro measurements, the non-fermented deer antler extract did not stimulate the production of IL-6, IL-10, IL--12 and TNF--α at all (Figs. 8, 10 and 12), whereas Codycept Millie Taris KCTC 11455BP antler fermentation promoted the production of IL-6, IL-10, IL-12 and TNF-α, and the production of these cytokines was determined by Cordyceps millitaris. It was confirmed that it is proportional to the concentration of KCTC 11455BP antler fermentation product.

Cordyceps Militaris for IL-6 KCTC 11455BP deer antler fermentation began to increase from the concentration of 0.49 μg / mL and showed a maximum yield at a low concentration of 7.81 μg / mL (Fig. 9, Table 10).

group
normal
sample concentration (pg / mL)
LPS 0.12 0.49 1.95 7.81 31.25 125 500 Non Fermented Deer Antler Extract 0 0 0 0 0 0 0 32.3 ± 1.2 2100 ± 110.4 Cordyceps Militaris KCTC 11455BP Antler Ferment 0 0 150.3
± 10.2 1650.8
± 0.5 2110.3
± 10.3 2000.8
± 50.4 1981.3
± 30.5 1950.7 ±
20.3 2100
± 110.4

Comparison of Induction Capacity of IL-6 from Unfermented Antler Extract and Cordyceps Militaris KCTC 11455BP Antler Extract

Cordyceps Militaris for IL-10 It was confirmed that the antler extract fermented with KCTC 11455BP has a production capacity at high concentration. In addition, in the case of IL--12, the non-fermented deer antler extract showed little production induction ability (Fig. 10), while the Codycept millitaris KCTC 11455BP deer antler fermentation showed a rapid increase in IL--12 production at concentrations above about 2 μg / mL, and at 500 μg / mL, 2.2 times the yield of LPS as a control was observed. (FIG. 11, Table 11). Therefore, Codycept Militaris KCTC 11455BP deer antler ferment seems to have activity in cell mediated immunity such as activation of NK cells and activation of cytotoxic T lymphocytes (CTL) through induction of Th1 type immune responses.

Group Normal Sample concentration (pg / mL) LPS 0.12 0.49 1.95 7.81 31.25 125 500 Non Fermented Deer Antler Extract 20.5
± 2.3 20.2
± 1.9 19.6
± 2.3 17.6
± 1.2 20.8
± 2.1 21.3
± 0.9 21.9
± 1.4 20.9
± 1.0 310.0
± 3.8 Cordyceps Militaris KCTC 11455BP Antler Ferment 20.5
± 2.3 21.4
± 0.5 20.4
± 0.3 50.9
± 2.4 136.9
± 20.3 192.3
± 10.4 339.7
± 5.7 662.4
± 2.5 310.0
± 3.8

Comparison of Induction Capacity of IL-12 Production from Unfermented Antler Extract and Cordyceps Militaris KCTC 11455BP Antler Extract

TNF--α was also observed in production in the non-fermented deer antler extract (Fig. 12), but Codycept millitaris In the case of KCTC 11455BP antler fermentation, the production-inducing capacity increased in a concentration-dependent manner at about 2 μg / mL or more (FIG. 13, Table 12).

Group Normal Sample concentration (pg / mL) LPS 0.12 0.49 1.95 7.81 31.25 125 500 Non Fermented Deer Antler Extract 0
± 12.3 0 0 0 0 0 0 0 1210.2
± 20.4 Cordyceps Militaris KCTC 11455BP Antler Ferment 0
± 12.3 0 0 0 290.3
± 12.6 618.2
± 3.7 821.8
± 4.2 980.6
± 4.5 1210.2
± 20.4

Comparison of TNF-alpha Production Induction Capacity of Unfermented Antler Extract and Cordyceps Militaris KCTC 11455BP Antler Extract

Example 5 Hematopoietic Function Improvement Activity of Cordyceps Militaris KCTC 11455BP Antler Fermentation

Experimental Example 1 Materials and Methods

Experimental animal

Four-week-old female Sprague-Dawley rats were purchased from Narabaitech Co., Ltd. (Daejeon, South Korea) and supplied with sufficient standard feed and water. : 00 ～ 20: 00) Contrast was automatically adjusted to adapt to the laboratory environment.

Antler extract and Cordyceps Militaris KCTC  11455 BP  velvet Fermented  Produce

The antler fermented product was obtained by injecting 40 g of antler powder into 300 mL of 50 mM phosphate buffer (pH 6.0), inoculated with Codycept Millitaris KCTC 11455BP, and fermenting at 25-30 ° C. for 5 days. Using the same concentration of the fermented antler powder solution as a sample, 300 mL of water was added thereto, and the mixture was refluxed at 100 ° C. for 3 hours, and the supernatant was recovered by centrifugation at 8,000 rpm for 20 minutes. After extraction, 150 mL of water was added to the residue, followed by extraction for 3 hours. This process was repeated two times, and the extracted extract was concentrated to obtain 100 mL (7.5 brix) of concentrate, which was used as a fermented antler extract and non-fermented antler extract, respectively.

Anemia Induction and Drug Administration

The experimental group was normal control (Control), PHZ treatment anemia control (PHZ-control), PHZ treatment antler extract administration group (PHZ-AE), PHZ treatment antler fermentation product (Cordyceps mitaris KCTC 11455BP antler fermentation product, PHZ-FAB) The administration group, that is, the antler extract and the antler fermented product after the PHZ treatment were divided into 4 groups, each of which was administered 200 mg / kg. In the PHZ treatment group, 10 mg / kg of PHZ dissolved in physiological saline was injected into the tail vein of rats for 4 days. The antler extract and cordyceps millitaris KCTC 11455BP antler fermentation were orally administered for one week after the end of the PHZ administration period. Administered.

Statistical processing

For statistical analysis, ANOVA was performed using the SPSS statistical package program (statistical package social science, version 14.0). The significance between the treatment groups was Duncan's multiple range test at p <0.05.

Experimental Example 2 Effect of Cordyceps Militaris KCTC 11455BP Antler Fermentation on Dietary Efficiency

The diet and water supply of all experimental animals were determined by free intake. Dietary intake was measured daily during the experimental period and body weight was measured every two days. The food efficiency ratio (FER) was calculated by dividing the weight gain during the total experiment period from the start date to the end date by the dietary intake.

Table 13 shows the final body weight, dietary intake, and dietary efficiency after the end of the experiment. Body weight gain was slightly decreased in PHZ-treated anemia-inducing group (PHZ-control), but there was no significant difference. The antler extract group showed a similar increase as the control group, and the weight gain was high in the antler extract and cordyceps militaris KCTC 11455BP antler fermentation group, but there was no significant difference. In the results of this study, weight loss and dietary efficiency of the PHZ group were similar to those of Han et al. (2003), which showed weight loss and low dietary efficiency in the experiments that caused anemia with the chemical drug cyclophosphamide. It was determined that the reaction of free radicals produced by the automatic oxidation of PHZ, a strong oxidant, caused the oxidative damage of tissues due to oxidative damage of tissues (Nicolas et al., 2002).

Experimental group Control PHZ-control PHZ-AE PHZ-FAC Dietary Intake
(g / day) 10.87 ± 2.23 10.21 ± 1.08 10.52 ± 1.13 10.17 ± 1.16 Weight gain
(g / day) 5.21 ± 0.57 5.01 ± 0.75 5.48 ± 0.67 5.52 ± 0.83 Dietary efficiency 0.48 ± 0.03 0.49 ± 0.07 0.52 ± 0.06 0.54 ± 0.07

Control group: untreated group, PHZ-control: group treated with PHZ-HCl (10 mg / kg, iv) for 4 days, PHZ-AE: antler extract, PHZ-FAC: cordyceps millitaris KCTC 11455BP antler fermentation 1 week after PHZ administration (200 mg / kg, po) (p <0.05, significantly different values are given in different letters, and mean ± SD (n = 6)), dietary efficiency = weight gain Dietary Intake

Experimental Example 2 Effect of Cordyceps Militaris KCTC 11455BP Antler Fermentation on Blood

Blood was collected from vacutainne coated with anticoagulant EDTA. Whole blood includes red blood cell (RBC), hemoglobin (Hb), hematocrit (Hct), mean corpuscular volume (MCV), mean corpuscular hemoglobin (mean corpuscular hemoglobin) concentration, MCHC) and the like were measured using an automatic blood cell counter (ADVIA 120, Bayer, Tarrytown, NY, USA).

Hemoglobin Content and Hematocrit Levels

14 is Hemoglobin and hematocrit (hematocrit) were measured in whole blood treated with anticoagulant. The hemoglobin concentration was 12.47 ± 2.12 g / dL in the hemolytic anemia induced by hematopoietic anemia after 4 days of administration of PHZ, whereas the control group which did not induce hemolytic anemia was 13.21 ± 2.32 g / dL. Antlers induced anemia with PHZ showed 14.97 ± 1.05 g / dL, and the PHZ-FAC group, which received the Codycept millitaris KCTC 11455BP antler fermentation group, was 15.81 ± 0.83 g / dL, compared to the hemoglobin group. Levels increased significantly (p <0.05).

Hematocrit levels, expressed as a percentage of the volume of red blood cells in whole blood, were significantly lower in the PHZ-treated group (37.3 ± 1.31%) than in the control group (40.2 ± 2.41%) (p <0.05). On the other hand, the group treated with antler extract and cordyceps millitaris KCTC 11455BP deer antler fermentation significantly increased hematocrit level than the group treated with PHZ, especially in the group treated with cordyceps millitaris KCTC 11455BP antler fermentation. The increase effect was noticeable (Table 14).

Therefore, oxidative red blood cell membrane protein and lipid peroxidation of plasma membranes were prevented by cordyceps militaris KCTC 11455BP deer antler fermentation PHZ to prevent erythrocyte damage and to achieve normal hematopoietic effect.

Group hemoglobin
(g / dL) Hematocrit
(%) Control 13.21 ± 2.32 ^ab 40.2 ± 2.41 ^b PHZ-control 12.47 ± 2.12 ^a 37.3 ± 1.31 ^a PHZ-AE 14.97 ± 1.05 ^ab 42.4 ± 1.24 ^bc PHZ-FAC 15.81 ± 0.83 ^b 43.6 ± 1.06 ^c

Control group: untreated group, PHZ-control: group treated with PHZ-HCl (10 mg / kg, iv) for 4 days, PHZ-AE: antler extract, PHZ-FAC: cordyceps millitaris KCTC 11455BP antler fermentation Group (200 mg / kg, po) administered 1 week after PHZ dosing (p <0.05, significantly different values are written in different letters, and the mean ± SD (n = 6))

Hematological analysis

Table 15 shows the measurement results of hematological markers of anticoagulant-treated whole blood. In the study, the number of erythrocytes in the PHZ-administered group decreased compared to the control group (p <0.05). The decrease in hemoglobin and hematocrit levels due to erythrocyte reduction is shown in Table 14. In the PHZ-FAC group treated with cordyceps militaris KCTC 11455BP antler fermentation, the number of red blood cells was significantly higher than that of the PHZ group (p <0.05). There was no difference. There was also no significant difference between the antler extract and the antler extract.

Leukocyte count was not significantly different between groups. Deer Antler extract and Cordyceps militaris KCTC 11455BP deer antler fermentation showed less increase in leukocyte levels than in the PHZ group, and was maintained at the same level as the normal control group.

Reticulocytes are generally known to increase in number with chronic hemoglobin reduction due to hemolytic anemia or hemolytic anemia (Xiea et al., 2006). The number of reticulocytes in PHZ-control induced anemia with PHZ was 10.25 ± 1.51%, which was higher than 6.51 ± 1.10% of normal control (p <0.05). When the antler extract and cordyceps millitaris KCTC 11455BP antler fermentation were administered, the reticulocyte count decreased compared to the PHZ-administered group. It showed an effect. Other mean hemoglobin (MCH), mean blood cell volume (MCV), and mean red blood cell hemoglobin concentration (MCHC) showed no difference between groups.

Control PHZ PHZ-AE PHZ-FAC Red blood cells (106 mm ³ ) 6.85 ± 0.55 ^c 5.71 ± 0.17 ^a 6.09 ± 0.11 ^ab 6.44 ± 0.36 ^bc White blood cell (103 mm ³ ) 4.61 ± 1.10 5.53 ± 1.69 5.18 ± 1.37 4.62 ± 1.89 Reticulocytes (%) 6.51 ± 1.10 ^a 10.25 ± 1.51 ^b 8.90 ± 1.20 ^ab 7.85 ± 1.98 ^ab MCH (pg) 18.88 ± 0.58 18.60 ± 0.42 18.97 ± 0.45 18.42 ± 0.67 MCV (fL) 65.65 ± 2.10 64.05 ± 1.66 66.67 ± 0.89 65.26 ± 2.13 MCHC (g / dL) 27.31 ± 0.44 26.73 ± 1.08 26.77 ± 0.87 26.56 ± 0.75

Control: untreated group, PHZ-control: treated with phenylhydrazine-HCl (10 mg / kg, iv) for 4 days, PHZ-AE: antler extract, PHZ-FAC: cordyceps millitaris KCTC 11455BP antler fermentation Group (200 mg / kg, po) administered 1 week after PHZ dosing (p <0.05, significantly different values are written in different letters, and the mean ± SD (n = 6))

Erythropoietin ( EPO ) content

Serum EPO concentration was measured by RIA method (Radioimmunoassay) using EPO-TracTM 1251 RIA kit (DiaSorin, Stillwater, USA) (COBRA 5010 II, Quantum, Packard, Meriden, USA), δ-aminolevulinic acid of liver tissue Dehydrates (ALAD) Activity Russell and Coleman's method (Russell and Coleman, 1963) was modified to use 10% liver tissue solution. 150 μl of liver tissue was added to a tube containing 50 μl of 0.3 M glutathione and 400 μl of 0.1 M phosphate buffer (pH 6.8) for 30 minutes at 37 ° C. After the reaction, 150 μl of 0.1 M δ-aminolevulinic acid as a substrate solution was added thereto, followed by reaction for 30 minutes, and then the reaction was terminated by adding 10% 1.5 mL TCA (containing 0.1 M HgCl ₂ ). After centrifugation at 4,000 rpm for 10 minutes, 1.5 mL of 5% TCA and 1.5 mL Ehrlich reagent were added to 0.5 mL supernatant, and allowed to stand for 10 minutes. The absorbance was measured at 555 nm.

In anemia, erythrocytes and hemoglobin are reduced, resulting in a decrease in arterial blood oxygen pressure, which in turn increases erythropoietin in the kidneys, promoting red blood cell production in the bone marrow (Mc Mullin et al., 1989). The effect of antler extract on serum EPO content is shown in FIG. 15. The serum EPO content of the PHZ group, which induced hemolytic anemia with PHZ administration, was 42.93 ± 3.94 mU / mL, which was higher than the normal control group (37.21 ± 2.72 mU / mL) (p <0.05). Increasing erythropoietin, a hematopoietic hematopoietic factor, can increase the number of erythrocytes in the bone marrow and increase reticulocyte counts.

As a result of examining the effects of the antler extract and cordyceps millitaris KCTC 11455BP antler fermentation on hemolytic anemia, the serum EPO content of the cordyceps millitaris KCTC 11455BP deer antler fermentation group (PHZ-FAC) was 32.48 ± 2.48 mU antler extract administration group (PHZ-AE) was 36.89 ± 1.89 mU / mL. Therefore, Cordyceps Militaris KCTC 11455BP deer antler fermentation was found to be excellent in the effect of reducing EPO in hemolytic anemia (Table 16).

Group Control PHZ-control PHZ-AE PHZ-FAC EPO (mU / mL) 37.21 ± 2.72b 42.93 ± 3.94c 36.89 ± 0.89b 32.48 ± 2.48ab

Control: untreated group, PHZ-control: treated with PHZ-HCl (10 mg / kg, iv) for 4 days, PHZ-AE: antler extract, PHZ-FAB: cordyceps millitaris KCTC 11455BP antler fermentation Group (200 mg / kg, po) administered 1 week after PHZ dosing (p <0.05, significantly different values are written in different letters, and the mean ± SD (n = 6))

δ- Aminolevulinic acid dehydrates ( ALAD ) activation

δ-Aminolevulinic acid dehydrates are enzymes that catalyze the synthesis of porphobilinogen from δ-aminolevulinic acid, the initial process of hemoglobin synthesis. Anemia due to erythrocyte hemolysis inhibits the activity of δ-aminolevulinic acid dehydrates (ALAD), which is involved in hemoglobin synthesis. ).

The effects of antler extract and cordyceps millitaris KCTC 11455BP antler fermentation on PHZ-induced hemolytic anemia showed that the absorbance values of ALAD activity of PHZ-administered anemia-induced group was 0.410 ± 0.030 lower than that of the normal control group. ALAD enzyme activity increased upon administration. In addition, the administration of cordyceps millitaris KCTC 11455BP antler fermentation was higher in the increase of ALAD enzyme activity than the antler extract, and the antler fermentation showed similar ALAD enzyme activity as the normal control (Table 17).

Group Control PHZ-control PHZ-AE PHZ-FAC OD value / mg protein 0.410 ± 0.030 ^c 0.332 ± 0.010 ^a 0.377 ± 0.008 ^b 0.381 ± 0.009 ^bc

Control: untreated group, PHZ-control: treated with PHZ-HCl (10 mg / kg, iv) for 4 days, PHZ-AE: antler extract, PHZ-FAB: cordyceps millitaris KCTC 11455BP antler fermentation Group (200 mg / kg, po) administered 1 week after PHZ dosing (p <0.05, significantly different values are written in different letters, and the mean ± SD (n = 6))

conclusion

According to the above results, the PHZ-induced hemolytic anemia-induced PHZ group had a significant decrease in hemoglobin level, hematocrit level, and red blood cell count compared to the control group, and reticulocyte count was significantly increased. On the other hand, hemoglobin, hematocrit, and erythrocyte counts were increased in the antler extract and cordyceps millitaris KCTC 11455BP antler fermentation group, especially in the antler fermentation group.

The serum EPO content increased by the administration of PHZ was found to be significantly recovered in the cordyceps millitaris KCTC 11455BP antler fermentation administration group, δ-ALAD activity was also excellent in the antler fermentation. Therefore, it was confirmed that Cordyceps Militaris KCTC 11455BP deer antler fermentation has an excellent effect on hematopoiesis.

Example 6 Toxicity Test

As a result of the acute toxicity test of the Codycept Millitaris KCTC 11455BP antler fermentation of Experimental Example 1 of Example 5, 50% lethal dose during oral administration and intraperitoneal injection was no more than 2 g / kg body weight.

The composition containing the antler fermented with Codycept millitaris KCTC 11455BP of the present invention as an active ingredient improves the hematopoietic function and enhances the immune function of the doser, and is effective in hematopoietic dysfunction and hematopoietic related diseases when taking the same. It is expected to help improve constitution and health by enhancing immune function.

Korea Research Institute of Bioscience and Biotechnology KCTC11455 20090116

Claims (12)

Cordyceps militaris (Cordyceps militaris) A composition for enhancing immune function containing the antler fermented with KCTC 11455BP as an active ingredient.
The method of claim 1,
The composition is a composition for enhancing immune function, characterized in that the food composition or pharmaceutical composition.
The method of claim 2,
The food is a health functional food, health supplements, functional food, nutritional composition for enhancing immune function, characterized in that the nutrient.
The method of claim 2,
The pharmaceutical composition is an immune function enhancing composition, characterized in that it further comprises an immunostimulant.
The method according to any one of claims 1 to 4,
A composition for enhancing immune function, which is in the form of a tablet, a hard or soft capsule, a liquid or a suspension.
Method of producing a composition for enhancing immune function, comprising the step of fermenting antler with Cordyceps militaris KCTC 11455BP.
Cordyceps militaris (Cordyceps militaris) A composition for improving hematopoietic function containing the antler fermented with KCTC 11455BP as an active ingredient.
The method of claim 7, wherein
The composition is a composition for improving hematopoietic function, characterized in that the food composition or pharmaceutical composition.
The method of claim 8,
The food is a health functional food, health supplements, functional food, nutritional composition for improving hematopoietic function, characterized in that the nutrient.
The method of claim 8,
The pharmaceutical composition is a composition for improving hematopoietic function, characterized in that it further comprises a hematopoietic stimulant.
The method according to any one of claims 7 to 10,
A composition for improving hematopoietic function, which is in the form of a tablet, a hard or soft capsule, a liquid or a suspension.
Method of producing a composition for improving hematopoietic function comprising the step of fermenting the antler with Cordyceps militaris KCTC 11455BP.

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