KR20050051179A - Pharmaceutical composition comprising an extract of cordyceps militaris for enhancing immune system - Google Patents

Abstract

The present invention is a composition for immuno-stimulation comprising a Cordyceps militaris hot water extract, specifically, the militaris Cordyceps hot water extract of the present invention is T-cells, B-cells in mouse spleens receiving solid cancer And significantly increase the number of NK-cells, thereby prolonging life and antitumor effects through the enhancement of cellular and humoral mediated immune responses, and activating the host's immune function by increasing IL-2 secretion by the spleen. It can be usefully used as a pharmaceutical composition and health functional food for immune promotion.

Description

Pharmaceutical composition comprising an extract of Cordyceps militaris for enhancing immune system}

The present invention relates to a composition for immuno-stimulation comprising a Cordyceps militaris hot water extract.

The treatment of cancer using the regulation of the immune system, the body's defense system, can be classified as follows. First, the antigen-specific method using the memory function, which is a characteristic of the human immune system, is activated by the antigen. T cell (cell mediated immunity) or antibody (humoral immunity) to attack the exact target cell (external target) or the external microorganisms, etc. (vaccine) to form a defense system, and the second non-antigen and anti-cancer and anti- Cytokines such as interferon with proven viral effects, IL-2, known as T-cell growth factor, GM-CSF, which promotes the production of white blood cells, and TNF-α, which directly kills cancer cells There is a cytokine. These cytokines use cytokines, whose primary function is immunological effects, as soluble peptide regulatory factors that induce intercellular information. Function to regulate the immune system. Bispecific response modifiers (BRMs) are substances that exert a variety of physiological effects by stimulating the immune system to alter the host's biological response (Kath R, Herlyn M., Current Opinion in Immunol, 1 , pp863-866). , 1989: Herlyn M, Menrad A, Koprowski H., J. Natl. Cancer Inst., 82 , pp 1883-1889, 1990).

There are many types of cells involved in anticancer defense mechanisms, but there are two major mechanisms involved in direct cancer killing. First, cytotoxic T lymphocytes (CTLs) are thymus-dependent and antigen-specific. It is characterized by a very high immune memory and being controlled by a major histocampatibility complex antigen (MHC antigen). CTLs are very potent on cancer-specific immune responses, particularly virus-induced cancers, but do not play a central role in the first-line defense or anti-cancer resistance of cancer cells because they require a sensitization process. The second part of the anti-cancer defense mechanism is the function of natural killer cells and cells that are directly involved in the tumor monitoring system of the body, showing rapid assassination ability without antigen sensitization and anti-cancer activity against non-immune tumors. Therefore, there is a wide range of anticancer ability. It is also not restricted by MHC. Despite these commonalities, natural killer cells are not one cell and vary in phenotype and shape. Natural killer (NK) cells have the form of large granular lymphocytes (LCL) and are characterized by no rearrangement of T cell receptor genes. Therefore, NK cells, unlike T cells, do not recognize cancer cells through T cell receptors. NK cells are initially produced in the bone marrow but are important for the performance of mediated cytotoxicity. But it is also found in the spleen, lung, liver, abdominal cavity, small intestine and bronchial tissue. NK cells are distributed in many animals and are found in not only humans but also various invertebrates. The most important function of NK cells is their ability to resist tumor growth and metastasis. Recently, the main cells of the lymphokine activated killing (LAK) phenomenon are known as NK cells. In addition, NK cells are involved in the defense against infection, growth regulation of hematopoietic or lymphoid cells, and the production of various cytokines. That is, interferon, interleukin-1, interleukin-2, tumor necrosis material, lymphotoxic substance, B cell growth factor, etc. are produced by NK.

Some of the T lymphocytes of peripheral blood show spontaneous MHC unlimited cytotoxicity, and thus, NK and its characteristics are known to be very similar. These lymphocytes also express the same antigens as NK cells, making them difficult to distinguish. NK cells have much stronger toxicity than these lymphocytes, and T cell growth factor (interleukin-2) Induces the toxic capacity of lymphocytes. Initially, it was called LAK cells because they thought it was a unique action of NK and other lymphocytes. However, NK cells and MHC unlimited T lymphocytes are also involved in LAK function.

T lymphocytes or NK cells are functionally inactive in various cancer tissues of the human body. Recently, tumor infiltrating lymphocytes (TILs) are also known to have high cytotoxicity when incubated with IL-2, and clinical trials are already underway for anti-cancer immunotherapy using TILs.

Cordyceps sinensis belongs to the Cordyceps family of the fungus Cordyceps in the Aspergillus fungus and is widely distributed all over the world. Cordyceps sinensis, which has been used for medicinal purposes in China, is mainly dominated by Cordyceps sinensis, which is formed by larvae of bat moths. It lives in the highlands of 3,000 meters above sea level in Tibet. It is a medicinal plant with a fruiting body that develops only during the summer season and is not collected much (Samson RA., Studies in Mycology, 6 , pp31-36, 1974).

So far, 78 species have been collected and classified as Cordyceps sinensis in Korea. Representative caterpillars include Chrysalis Cordyceps Sinensis, Big Cicada Cordyceps, Caterpillar Black Cordyceps, Scarab Cordyceps, Snow Flower Cordyceps, Stink Bugs Cordyceps, Beetle Cordyceps, and Baekganggyun. Many types of cordyceps, including pupa, are expected to be used as artificial fruiting bodies. Bacillus and P. locusts are insect parasites that control pests that damage crops, but are highly likely to be developed as microbial insecticides. , Stink bug Cordyceps sinensis and Beetle insects are the most easily found in Korea, but are difficult to grow due to the slow growth of mycelia (Jung Bo-seop and Shin Mingyo, Hyang-Yae Daejeon, Yeonglimsa, p45-46, 1998).

Militaris michungris of the present invention ( Cordyceps militaris ) is based on the chrysalis of the tree buried in the basement to form a fruiting body of single or ancestors occur in the head of the host. Club-shaped fruiting bodies are orange, 2-5 cm long, 0.3-0.5 cm thick.

However, none of the above literature suggests or teaches the effect of the Militaris cordyceps sinensis extract of the present invention on immune function.

Therefore, the present inventors significantly increase the number of T-cells, B-cells and NK-cells in the mouse spleen of the militaris cordyceps sinensis hot water extract of the present invention, and also the IL-2 secretion ability by the spleen. The present invention has been completed by confirming the increase.

SUMMARY OF THE INVENTION An object of the present invention is to provide a pharmaceutical composition for immunostimulation, including Militaris cordyceps fungal water extract, which increases IL-2 secretion by T-cells, B-cells, NK-cells, and spleen to activate immune function of the host. To provide.

In order to achieve the above object, the present invention provides a pharmaceutical composition for immuno-stimulation comprising the extract of Millyceris cordyceps ( Cordyceps militaris ).

The millitaris cordyceps sec. Includes silkworm pupa or silkworm larvae millitaris cordyceps sec.

In addition, the millitaris Cordyceps Sinensis extract includes water and C ₁ to C ₄ lower alcohols such as methanol, ethanol, butanol and the like, and extracts available for a mixed solvent thereof, and are characterized by being extracted by hot water extraction.

Hereinafter, the present invention will be described in detail.

Millitaris Cordyceps Sinensis Extract of the present invention can be obtained as follows.

Millitaris cordyceps of the present invention commercially purchased silkworm pupa millitaris cordyceps vinegar after dispensing frozen silkworm pupae into a round plastic container, inoculated with liquid seedlings, cultivated silkworm larvae Militaris cordyceps worms Cultivated and grown by inoculating the liquid seedlings, and then pulverized cultivated Cordyceps sinensis, powdered, and then the volume of water, methanol, ethanol of about 2 to 20 times, preferably about 5 to 10 times the weight of Cordyceps sinensis , A polar solvent of C ₁ to C ₄ lower alcohols such as butanol or the like, or a mixed solvent having a mixing ratio of about 1: 0.1 to 1:10, preferably about 5 to 12 hours at 70 to 100 ° C. with water, Preferably, using an extraction method such as hot water extraction, cold needle extraction, reflux cooling extraction or ultrasonic extraction for 5 to 7 hours, preferably hot water extraction to vacuum A number of times by the supernatant may then, by following the procedure of 2 to 5 times to collect the supernatant to give a millimeter less other Cordyceps hot water extract was concentrated under reduced pressure.

The present invention provides a pharmaceutical composition for immuno-stimulation containing the militaris Cordyceps sinensis hot water extract obtained as the active ingredient.

In addition, Militaris Cordyceps sinensis has been used for food, the extract of the present invention extracted from it is also a food without problems such as toxicity and side effects.

In order to examine the immune-promoting effect of the Militaris cordyceps sinensis hot water extract obtained by the above method, the militaris Cordyceps sinensis hot water extract of the present invention was intraperitoneally administered to a mouse transplanted with T-cells and B-cells in the mouse spleen. As a result of measuring the number of NK-cells, it was confirmed that significantly increased, and IL-2 secretion ability by the spleen also increased significantly.

The pharmaceutical composition for immunostimulation of the present invention comprises 0.1 to 50% by weight of the extract based on the total weight of the composition.

Pharmaceutical compositions comprising the extract of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

Pharmaceutical dosage forms of the extracts of the present invention may be used alone or in combination with other pharmaceutically active compounds, as well as in any suitable collection.

Pharmaceutical compositions comprising extracts according to the invention, in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterile injectable solutions, respectively, according to conventional methods. Can be formulated and used. Carriers, excipients and diluents that may be included in the composition comprising the extract include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and the solid preparations may include at least one excipient such as starch, calcium carbonate, sucrose in the extract. ) Or lactose, gelatin and the like are mixed. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

Preferred dosages of the extracts of the present invention vary depending on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. However, for the desired effect, the extract of the present invention is preferably administered at 0.0001 to 100 mg / kg, preferably at 0.001 to 100 mg / kg. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect.

The extract of the present invention can be administered to mammals such as mice, mice, livestock, humans, etc. by various routes. All modes of administration can be expected, for example by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

The present invention provides a health functional food comprising the hot water extract and a food acceptable food supplement additive exhibiting an immune enhancing effect. Examples of the food to which the Militaris Cordyceps Sinensis Hot Water Extract can be added include various foods, beverages, gums, teas, vitamin complexes, and health functional foods.

It may also be added to foods or beverages for the purpose of immunostimulating effects. At this time, the amount of the hot water extract in the food or beverage may be added to 0.01 to 15% by weight of the total food weight, the health beverage composition is added in a ratio of 0.02 to 5 g, preferably 0.3 to 1g based on 100 ml Can be.

The health functional beverage composition of the present invention is not particularly limited to other ingredients except for containing the hot water extract as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. . Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of natural carbohydrates is generally about 1-20 g, preferably about 5-12 g per 100 ml of the composition of the present invention.

In addition to the above, the hot water extract of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic and natural flavors, coloring and neutralizing agents (such as cheese, chocolate), pectic acid and salts thereof, alginic acid and Salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like. In addition, the hot water extracts of the present invention may contain pulp for the production of natural fruit juices and fruit juice drinks and vegetable drinks. These components can be used independently or in combination. The proportion of such additives is not so critical but is generally selected from the range of 0 to about 20 parts by weight per 100 parts by weight of the hot water extract of the present invention.

Below, The invention is illustrated in detail by the following examples and experimental examples.

However, the following Examples and Experimental Examples are only illustrative of the present invention, and the content of the present invention is not limited by the following Examples and Experimental Examples.

Example 1. Silkworm pupa and silkworm larvae Militaris cordyceps ( Cordyceps militaris ) Preparation of Hot Water Extract

The sample of Militaris Cordyceps was supplied from Mashville Co., Ltd. (Seongnam-si, Korea), and Silkworm pupa Militaris cordyceps ( Cordyceps militaris ) was used for dispensing frozen silkworm chrysalis in a round plastic container. The silkworm larva (Silkworm larva) was inoculated and inoculated with liquid spawn on the silkworm larva ( Cyceyceps militaris ). 8 L of distilled water was added to each 1 kg of cultivated Cordyceps sinensis and then heated at 95 ° C. for 6 hours. Repeat this process twice, and then filter twice with filter paper, and the filtrate was concentrated under reduced pressure and freeze-dried using a rotary vacuum concentrator to obtain silkworm pupa Militaris cordyceps (yield 170g) and silkworm caterpillar Militaris cordyceps ( Yield 170 g) to obtain a hot water extract.

Cordyceps sinensis samples in powder form were dissolved in a constant concentration of sterile distilled water, filtered through a 0.4 μm filter, and used in the following experiments.

Example 2 Analysis of Cordycepin Concentration in Silkworm Pupa and Silkworm Larva Militaris Cordyceps Sinensis

Cordycepin concentration analysis of cordyceps samples was carried out using the method of Cunningham et al. (Cunningham KG, Hutchinson SA. Et al., J. Chem. Soc., 51 , pp2299-2300, 1951). That is, the Cordyceps sinensis samples were boiled at 90 ° C. for 6 hours, filtered, and added with two volumes of acetone, and then left at 4 ° C. for 24 hours. The supernatant was collected by centrifugation at 5,000 × g for 10 minutes, the solvent was completely evaporated, then dissolved by adding distilled water and filtered using a 0.45 μm filter. Cordycepin content of the samples was analyzed by high performance liquid chromatography (HPLC) equipped with Altima C18 column (Shimatsu, Japan). It was measured at 260 nm using acetonitrile: water (3: 1) mixed solvent as an eluting solvent at a flow rate of 1.5 ml / min.

As a result, 0.2% and 0.4% of cordycepin were detected in silkworm pupa cordyceps and silkworm larvae. Cordycepin (3'-deoxyadenosine) used as a comparative standard was purchased from Sigma (St. Louis, MO, USA).

Experimental Example 1. Measurement of the weight of immune organs

1-1. Underarm Arm Cancer

A total of 64 male ICR mice (18-22 g body weight) were purchased from the Korea Experimental Animal Center and were adapted to laboratory environments for one week while breeding them as general solid feed (Samyang Yuji Feed Co., Ltd., Korea). The temperature of the feeding room was maintained at 22 ± 2.0 ℃ and the humidity was 55 ± 5.0%, and the contrast period was adjusted to 12 hours. The experimental animals adapted to the laboratory environment were divided into 8 groups and reared for 28 days. In the normal group (N), only saline was administered without cancer cells, and the control group (C). Physiological saline was administered after transplanting cancer cells. Silkworm chrysalis ms other less Cordyceps 50 group (CMP50) and silkworm pupa Milli other less Cordyceps after transplantation of the cancer cells when the 100 group (CMP100) other Each 50 ㎎ / ㎏ and 100 ㎎ / ㎏ silkworm pupa ms of less Cordyceps (Cordyceps militaris of silkworm pupa) extract was intraperitoneally administered. Silkworm larvae Militaris Cordyceps sinensis 50 groups (CML50) and silkworm larvae Militaris cordyceps sinensis 100 groups (CML100) after the transplantation of cancer cells 50 mg / kg and 100 mg / kg silkworm larvae Militaris Cordyceps sinensis extract ( Cordyceps, respectively) militaris of silkworm larva) was intraperitoneally administered. In the case of cordycepin group 1 (C1) and cordycepin group 2 (C2), cancer cells were transplanted, and then 1 mg / kg and 2 mg / kg of cordycepin were intraperitoneally administered, respectively.

To induce solid cancer in the armpits of mice, 0.2 ml (1.0 × 10 ⁶ cells / mouse) sarcoma-180 cell suspension, which is a sarcoma-180 cell sarcoma cell, was subcutaneously transplanted into the left armpit of the experimental animal. After 24 hours, 0.2 ml of saline, Militaris cordyceps extract or cordycepin were administered intraperitoneally for 10 consecutive days. All experimental animals were weighed two times, the start and end of the experiment (three weeks after cancer cell transplantation).

1-2. Immune organ weight measurement

To investigate the effect of militaris cordyceps hydrothermal extract and cordycepin intraperitoneal on the weight of immune-related organs in mice transplanted with solid cancer, experiments were performed by cervical dislocation on the 21st day after transplantation of tumor cells into the armpits. Animals were killed and the thymus, spleen and liver removed and weighed.

As a result, there was no significant difference in weight gain, liver and spleen weight between the normal group without transplanting cancer cells and the control group receiving saline after transplantation. Liver and spleen weights of Militaris cordyceps hydrothermal extract or cordycepin group were not statistically significant compared to the control group, but showed a tendency to increase. 63.4 ± 6.2 mg, which was lower than normal group (77.6 ± 4.1 mg) without cancer cells, and thymus weight of CMP50 and CM100 group was 87.6 ± 12.5 mg and 90.8 ± 6.2 mg, respectively. Compared with 38-43% (p <0.05). Thymus weight of CML50 and CML100 groups was also significantly increased (p <0.05) compared to the control group receiving physiological saline (p <0.05), and the thymus weight of the cordycepin group (78 to 83 mg) was 15 to 30% compared to the control group. There was a tendency to be higher but no statistical significance was observed (p> 0.05). The results are shown in Table 1 below.

group Dose (mg / kg / d) Weight gain (g / 3 wks) Liver weight (g) Spleen Weight (g) Thymus weight (mg) Jeongsan-gun Saline solution 10.7 ± 0.5 1.65 ± 0.04 0.21 ± 0.02 77.6 ± 4.1 ^ab Control Saline solution 12.1 ± 0.3 1.63 ± 0.06 0.24 ± 0.01 63.4 ± 6.2 ^b CMP 50100 7.9 ± 0.38.8 ± 0.5 1.67 ± 0.061.77 ± 0.12 0.26 ± 0.010.28 ± 0.02 87.6 ± 12.5 ^a 90.8 ± 6.2 ^a CML 50100 9.2 ± 0.58.5 ± 0.2 1.56 ± 0.081.57 ± 0.06 0.29 ± 0.040.29 ± 0.01 89.0 ± 6.4 ^a 91.2 ± 5.4 ^a Cordycepin 12 8.9 ± 0.29.4 ± 1.2 1.76 ± 0.121.79 ± 0.17 0.26 ± 0.010.24 ± 0.02 82.6 ± 3.0 ^ab 73.2 ± 4.6 ^ab

Experimental Example 2 Measurement of Immune Function

2-1. Preparation of Splenocytes

After administration of tumor cells to the left armpit of Experimental Example 1, spleens of mice bred for 21 days were extracted and washed twice with HBSS (Hank's Balance Salt Solution, GIBCO BRL, Gaithersburg, MD, USA) at 4 ° C. . Transfer it to a 30 mm cell culture dish, add HBSS, and remove spleen cells using a 40 μm nylon cell strainer (Becton Dickinson, Mountain View, Calif., USA) and repeat filtration to remove small tissue fragments. It was. A lysis solution (a solution of 50% diethylene glycol and 15% formaldehyde mixed with distilled water at a ratio of 1: 10 v / v) was added to the released splenocytes and left at room temperature for 10 minutes. The remaining red blood cells were hemolyzed. The precipitate obtained after centrifugation was washed with HBSS and suspended in RPMI 1640 culture containing 10% FBS.

2-2. Immune cell count

The percentage of T-cells, B-cells and NK-cells present in the spleen of mice was determined using indirect immunofluorescence (Skehan P, Storeng R. et al., J. Nat. Cancer Inst., 82 , pp 1107-1112, 1990). Specifically, 300 μl of mouse splenocytes prepared to the number of 6 × 10 ⁶ cells / ml were added to two tubes, centrifuged at 1,000 × g for 5 minutes, and then 0.1% sodium azide. Suspended by adding 500 μl of PBS (phosphate buffered saline, pH 7.2) contained therein, and centrifuged at 1,000 × g for 5 minutes to obtain a cell precipitate. Monoclonal antibody (PE-labled antimouse CD8 + FITC-labeled antimouse CD4 and PE-labeled antimouse CD19 + FITC-labeled antimouse NK1.1) in PBS solution containing 0.1% sodium azide and 2% FBS (Becton Dickinson, Mountain View , USA) were mixed to each concentration of 10 ㎍ / ㎖ 50 ㎕ added to the cell precipitate, and incubated for 30 minutes on ice. 500 μl of an ice-cold PBS solution containing 0.1% sodium azide was added, washed, and centrifuged. After removing the supernatant, add 300 µl of a fixed buffer (0.1% sodium azide + 1% paraformaldehyde in PBS solution) to the cell precipitate and mix well, followed by flow cytometry (FACScan, Becton Dickinson, Mountain View, USA). The number of immune cells was analyzed using.

As a result, in the spleen lymphocyte ratio of the normal group, CD4 + and CD8 + T-cells were present at 35.6 ± 1.0% and 17.7 ± 0.9%, respectively, indicating a ratio of about 2.0 CD4 + / CD8 + T-cells. The number of CD4 + and CD8 + T-cells in the control group was 20.2 ± 2.2% and 11.2 ± 0.8%, respectively, which was significantly lower than that of the normal group (p <0.05), and the CD4 + / CD8 + ratio was also lower than that of the normal group. However, no statistical significance was observed (p> 0.05). On the other hand, in the CMP50 and CMP100 groups, the number of CD4 + T-cells was 29.7 ± 2.2% and 31.5 ± 1.3%, respectively, which was significantly higher than the control group, and the CD8 + T-cell ratio was also 15.6 ± 0.6% and 15.7 ± 1.6%. Significantly higher than the control group (p <0.05). The CD4 + T-cell counts (26.5 ± 0.6% and 27.5 ± 0.7%) and CD8 + T-cell counts (14.3 ± 0.8% and 15.9 ± 1.3%) in the CML50 and CML100 groups were also significantly higher than those in the control group (p <0.05). On the other hand, in the cordycepin-administered group, the number of CD4 + and CD8 + T-cells in the spleen did not show a significant difference from the control group. The ratio of CD4 + / CD8 + T-cell counts was not significantly different between all experimental and control groups because both the CD4 + and CD8 + T-cell counts were significantly increased in the experimental group compared to the control group. The number of CD19 cells corresponding to B-cells tended to decrease in the control group (34.6 ± 2.1%) with cancer cells compared to the normal group (40.8 ± 1.3%). In the CMP50, CMP100 and CML100 groups, There was a tendency to increase compared to the control (p <0.05). Spleen NK-cell counts were 4.0 ± 0.6% and 3.7 ± 0.5% in the CMP50 and CMP100 groups, 96-110% higher than the control group (1.9 ± 0.2%) (p <0.05). Spleen NK cell counts in the CML50 group (3.1 ± 0.3%) and CML100 group (3.6 ± 0.2%) were significantly increased by 63 to 89% compared to the control group (p <0.05). 31-36% increased significantly (p <0.05). As a result of the above immune cell count, silkworm pupae or silkworm larvae Militaris cordyceps sinensis hot water extract was found to be more effective in increasing the immune cell count of the spleen than cordycepin single substance. 2 is shown.

group Dose (mg / kg / d) CD4 + T-cells (%) CD8 + T-cells (%) CD4 + / CD8 + ratio (%) CD19B-cells (%) NK cells (%) Normal Saline solution 35.6 ± 1.0 ^a 17.7 ± 0.9 ^a 2.0 ± 0.1 40.8 ± 1.3 ^ab 2.9 ± 0.2 ^bc Control Saline solution 20.2 ± 2.2 ^c 11.2 ± 0.8 ^d 1.8 ± 0.2 34.6 ± 2.1 ^bc 1.9 ± 0.2 ^c CMP 50100 29.7 ± 2.2 ^ab 31.5 ± 1.3 ^ab 15.6 ± 0.6 ^ab 15.7 ± 1.6 ^ab 1.9 ± 0.22.0 ± 0.1 39.3 ± 3.2 ^ab 44.0 ± 3.0 ^a 4.0 ± 0.6 ^a 3.7 ± 0.5 ^a CML 50100 26.5 ± 0.6 ^ab 27.5 ± 0.7 ^ab 14.3 ± 0.8 ^c 15.9 ± 1.3 ^ab 1.9 ± 0.11.9 ± 0.1 35.4 ± 0.9 ^bc 40.8 ± 1.3 ^ab 3.1 ± 0.3 ^bc 3.6 ± 0.2 ^ab Cordycepin 12 24.5 ± 0.7 ^bc 23.9 ± 0.9 ^bc 12.3 ± 0.4 ^cd 12.4 ± 1.3 ^cd 2.0 ± 0.11.9 ± 0.2 32.4 ± 2.5 ^c 36.8 ± 2.4 ^bc 2.5 ± 0.1 ^bc 2.6 ± 0.9 ^bc

1-3. Interleukin-2 (lL-2) Concentration Measurement

Concanavalin-A (concanavalin-A, Sigma, USA) was added to the mouse splenocytes prepared at a concentration of 5 × 10 ⁶ cells / ml to a concentration of 100 μg / ml, and then in a 37 ° C., 5% CO ₂ incubator. Incubated for 48 hours. Cultures were harvested and the concentration of IL-2 was measured using the Interleukin-2 (IL-2) kit (BD Bioscience, USA). 50 μl of the ELISA dilution solution provided in the kit was dispensed into a 96-well plate, and then 50 μl each of the IL-2 standard solution provided in the culture sample or the kit was added and left at room temperature for 2 hours. The reaction solution in the 96-well plate was removed using an aspirator, washed five times with PBS, and then mixed with a pre-mixed reaction solution (anti-mouse IL-2 monoclonal antibody + avidin-horse radish peroxidase conjugated enzyme). ) 100 μl was added and allowed to stand at room temperature for 1 hour. The reaction solution in the well was removed using an aspirator and washed five times. 100 µl of 3,3 ', 5,5'-tetramethylbenzidine (TMB) used as a substrate was added to a 96-well plate, followed by room temperature. Incubate for 30 minutes. 50 씩 of each solution was added to each well, and the absorbance was measured at 450 nm using an ELISA reader (Perkin Elmer, Wallac 1420, Turkin, Finland).

As a result, 481 ± 10.9 pg / ml was found in the control group transplanted with solid cancer, which was significantly decreased compared to the normal group (869 ± 11.8 pg / ml). IL-2 secretion by splenocytes was decreased in the CMP50 and CMP100 groups. 41.8% and 51.4% were significantly increased (p <0.05) compared with the control group receiving physiological saline, and IL-2 secretion by splenocytes of CML50 and CML100 groups was also significantly increased by 33.3% and 33.4%. (p <0.05). In the C1 and C2 groups that received cordycepin, IL-2 secretion was significantly increased compared to the control group (p <0.05), but the increase was more than that of the silkworm pupae or silkworm larvae Militaris cordyceps. Less could be confirmed (see FIG. 1).

Experimental Example 3. Statistical Processing

Statistical analysis of all data was carried out using the SAS (Statistical Analysis system) PC package (package), the analysis value is presented as the average ± SEM. The effects of two different Cordyceps sinensis hot water extracts or cordycepin at different concentrations were evaluated at p <0.05 level by one way ANOVA, and if statistical significance was observed, the difference in the mean value of each group was Verified by Duncan's multiple range test. On the other hand, the significance of the difference in cell viability observed in Sacoma-180 cell lines and control cells treated with silkworm pupae cordyceps, silkworm larvae fungus, or cordycepin at various concentrations was shown by Student's t. -test) at the p <0.05 level.

Although the example of the pharmaceutical composition containing Cordyceps sinensis hydrothermal extract of the present invention will be described, the present invention is not intended to limit the present invention, but is intended to be described in detail.

Formulation Example 1 Preparation of Powder

Cordyceps Sinensis Extract Dry Powder 300 mg

Lactose 100 mg

Talc 10 mg

The above ingredients are mixed and filled in an airtight cloth to prepare a powder.

Formulation Example 2 Preparation of Tablet

Cordyceps Sinensis Extract Dry Powder 50 mg

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, tablets are prepared by tableting according to a conventional method for preparing tablets.

Formulation Example 3 Preparation of Capsule

Cordyceps Sinensis Extract Dry Powder 50 mg

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare capsules.

Formulation Example 4 Preparation of Injection

Cordyceps Sinensis Extract Dry Powder 50 mg

Appropriate sterile distilled water for injection

pH adjuster

According to the conventional method for preparing an injection, the amount of the above ingredient is prepared per ampoule (2 ml).

Formulation Example 5 Preparation of Liquid

Cordyceps Sinensis Extract Dry Powder 100 mg

10 g of isomerized sugar

5 g of mannitol

Purified water

After dissolving each component in purified water according to the usual method of preparing a liquid solution, adding lemon flavor appropriately, mixing the above components, adding purified water, adjusting the whole to 100 ml by adding purified water, and then filling into a brown bottle. The solution is prepared by sterilization.

Formulation Example 6 Preparation of Healthy Food

Cordyceps Sinensis Extract Dry Powder 1000 mg

Vitamin mixture proper amount

     70 μg of Vitamin A Acetate

Vitamin E 1.0 mg

Vitamin B ₁ 0.13 mg

Vitamin B ₂ 0.15 mg

Vitamin B ₆ 0.5 mg

0.2 μg of vitamin B ₁₂

Vitamin C 10 mg

10 μg biotin

Nicotinic Acid 1.7 mg

Folate 50 ㎍

Calcium Pantothenate 0.5mg

Mineral mixture

Ferrous Sulfate 1.75 mg

Zinc Oxide 0.82 mg

Magnesium carbonate 25.3 mg

Potassium monophosphate 15 mg

Dibasic calcium phosphate 55 mg

Potassium Citrate 90 mg

Calcium Carbonate 100 mg

Magnesium chloride 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixtures is mixed with a component suitable for a health food in a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional health food manufacturing method. The granules may be prepared and used for preparing a health food composition according to a conventional method.

Formulation Example 7 Preparation of Healthy Drink

Cordyceps Sinensis Extract Dry Powder 1000 mg

Citric acid 1000 mg

100 g oligosaccharides

Plum concentrate 2 g

1 g of taurine

Add 900 ml of purified water

After mixing the above components according to a conventional healthy beverage manufacturing method, and then stirred and heated at 85 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed and sterilized and then refrigerated and stored in the present invention For the preparation of healthy beverage compositions.

Although the composition ratio is mixed with a component suitable for a favorite beverage in a preferred embodiment, the composition ratio may be arbitrarily modified according to regional and ethnic preferences such as demand hierarchy, demand country, and usage.

As described above, the militaris Cordyceps sinensis extract of the present invention increases IL-2 secretion by T-cells, B-cells, NK-cells, and the spleen to activate the immune function of the host. It can be usefully used as a pharmaceutical composition and health functional food for immune promotion.

1 is a result of measuring the effect of the administration of militaris Cordyceps Sinensis extract on IL-2 production by splenocytes of mice transplanted with solid cancer.

Claims (6)

Pharmaceutical composition for immunostimulation comprising extracts of Millythas Cordyceps militaris . The pharmaceutical composition according to claim 1, wherein the Militaris cordyceps is silkworm pupa or silkworm larvae Militaris cordyceps. [Claim 2] The pharmaceutical composition according to claim 1, wherein the extract of Millitaris Cordyceps Sinensis is water and an extract available for C ₁ to C ₄ lower alcohols such as methanol, ethanol, butanol and the like and a mixed solvent thereof. [Claim 2] The pharmaceutical composition according to claim 1, wherein the extract Militaris Cordyceps Sinensis is extracted by hot water extraction. Health functional food comprising militaris Cordyceps sinensis hot water extract and food acceptable food supplement additives exhibiting an immune enhancing effect. The health functional food of Claim 5 which is a health drink.