KR20150144061A - A composition comprising an extract of Dendropanax morbifera for immunomodulating and immunopotentiating activity - Google Patents

Abstract

More particularly, the present invention relates to a composition comprising an extract of Hokutogi (Japanese White Lucid) extract as an active ingredient. In detail, the present invention relates to a composition comprising T cells and B It was confirmed that both cells significantly decreased the proliferative capacity (Experimental Example 1); The production of IL-2, IL-4, IL-10 and IFN-γ was increased in all experiments (experiment 2); In the experiment, it was confirmed that the LP-BM5 MuLV infection reduced oxidative stress caused by reactive oxygen species (Experiment 3), and the samples of the present invention showed strong immunity enhancement It was confirmed that the composition is useful as a pharmaceutical composition or health functional food for the prevention and treatment of immune diseases.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an immunomodulating or immunopotentiating agent for dendropanax morbifera,

The present invention provides a composition for prevention and treatment of an immune disease comprising an extract of Aspergillus oryzae as an active ingredient.

[Literature 1] Immunology. 49, 1992; Dig dis Sci. 52, pp 1890-1896, 2007; J Life Sci. 19, pp 479-485, 2009; Biol Pharm Bull. 27, pp 617-620, 2004

[Document 2] J. Allergy clin immunol. 9, pp 616, 1993; Immunology. 47, pp 75, 1982

[Literature 3] J Korean Soc Food Sci Nutr. 35 pp 1329-1335, 2006;

[4] J Korean Soc Food Sci Nutr 38, pp 423-429 2009;

[Literature 5] Korean J Plant Res 24, pp 105-112, 2011;

[Literature 6] J Korean Soc Food Sci Nutr 40, pp 379-384, 2011

[7] Kim HR, et al .. Chemical characteristics of the leaves and seeds of Korean Dendropanax ( Dendropanax morvifera Lev .). J. Korean Soc. Agric. Chem. Biotechnol. 43: 63-66 (2000),

Park, S., et al. Cellular antioxidant activity and whitening effects of dendropanax morbifera leaf extracts, Journal of Microbiology and Biotechnology, 41 (4): 407-415, 2013

[Literature 9] Ho JN et al., Inhibition of premature death by isothiocyanates through immune restorations in LP-BM5 leukemia retrovirus-infected C57BL / 6 mice. Biosci Biotechnol Biochem 75: 1234-1239, 2011

[10] Mosier DE, Animal models for retrovirus-induced immunodeficiency disease. Immunol Invest 15: 233-261, 1986

[Literature 11] Powrie F et al., Cytokine regulation of T-cell function: potential for therapeutic intervention. Immunol Today 14: 270-274, 1993

[12] Odeleye OE et al., Changes in hepatic lipid composition after infection by LP-BM5 murine leukemia virus causing murine AIDS. Life Sci 51: 129-134, 1992

[Literature 13] Erdelmeier I. et al., Reactions of N-methyl-2-phenylindole with malondialdehyde and 4-hydroxyalkenals. Mechanistic aspects of the colorimetric assay of lipid peroxidation. Chemical Research in Toxicology 11, 11841194, 1998

The human body has the characteristic of trying to maintain homeostasis, and it also works in the immune system. Immunization means a series of bio-defense reactions that occur when a substance other than its own components breaks the homeostasis of the body or threatens itself. It protects the body from harmful substances entering through the skin, digestive tract, and respiratory system. This is a defense system that removes external stimulants, or acts to reduce serious inflammation in the wound. All immune responses, including the inflammatory response activated by influx of pathogens in the body, return to normal after removal of the pathogen, but when the immune response is difficult, immune responses are significantly higher or lower than those of normal individuals do. Immune deficiency or deficiency of the immune function is called the immune system in this state, the immune response is not activated properly, the body does not respond properly to foreign substances, causing the infection. In contrast, immune hypersensitivity is a condition in which some immune responses overreact, resulting in an imbalance of the immune system resulting in an allergic reaction (Immunology, 49, 1992; Dig dis Sci., 52, pp 1890-1896, 2007; J Life Sci. 19, pp 479-485, 2009; Biol Pharm Bull. 27, pp 617-620, 2004).

If the immune system is not properly regulated and becomes unbalanced, imbalance of cytokine in the body, unbalance of T / B cell proliferation, depletion of antioxidant nutrients may cause impairment of immune regulation and increase inflammatory substance secretion, And it can not cope with influx of pathogens, so that inflammation can be intensified. Thus, the immune response of the human body can be maintained in balance by regulating it normally, so immunological control is important for disease prevention and treatment (J. Allergy Clin Immunol. 9, pp 616, 1993; 1982).

The mouse AIDS model infected with LP-BM5 leukemia retrovirus (LP-BM5 MuLV) is known to induce an immune response similar to the human AIDS model and is commonly used for immunological evaluation in animal models. When AIDS is infected, the balance between Th1 type cytokines and Th2 type cytokines is destroyed, and normal immune response does not occur, and the immune system is destroyed and the immunity against the disease is lowered. In addition, there is a report that excessive oxygen production causes oxidative stress, damages DNA, cell membranes and proteins, causing cancer, as well as adult diseases and aging, resulting in premature death (J Korean Soc Food Sci Nutr. J Korean Soc Food Sci Nutr 40, pp 379-384, 2011; J Plant Res 24, pp 105-112, 2011; .

It is a dicotyledonous plant native to the southern coastal area of Korea and Jeju Island. It is an evergreen tree of the Araliaceae ( Dendropanax morbifera ) is a species that does not tolerate fallen leaves in the winter. If you bruise the bark, yellow resinous liquid comes out. The physiological activities such as anticancer activity and antioxidant activity of Hwangchulchu extract have been reported to be limited and they have been used in foods such as food additives and health functional supplements (Kim HR, et al .. Chemical characteristics of the leaves and the seeds of Korean Dendropanax ( Dendropanax morvifera Lev .). J. Korean Soc. Agric. Chem. Biotechnol. 43: 63-66 (2000) Park SN, et al. Cellular antioxidant activity and whitening effects of dendropanax morbifera leaf extracts, 41 (4): 407-415, 2013).

There is no disclosure or teaching at present of any of the above documents on the effect of immune control or immunostimulation through Horticultural extracts, especially fractions.

Accordingly, the present inventor has found an excellent immune modulating or immunostimulating activity of the fractions derived from Fusarium oxysporum sp. While investigating the activity against natural products.

In order to accomplish the above object, the present invention provides a pharmaceutical composition for immunomodulation or immunity enhancement comprising an extract of Aspergillus oryzae as an active ingredient.

Further, the present invention provides a health functional food for immunomodulation or immunity enhancement containing an extract of Aspergillus oryzae as an active ingredient.

As described above, the extract is a crude extract, a polar solvent-soluble extract, or a non-polar solvent-soluble extract.

The crude extract as defined herein may be dissolved in a solvent selected from water containing purified water, a lower alcohol having 1 to 4 carbon atoms such as methanol, ethanol, and butanol, or a mixed solvent thereof, preferably a water and ethanol mixed solvent, (V / v) ethanol, more preferably 10 to 50% (v / v) ethanol.

The non-polar solvent soluble extract fractions defined herein comprise extract fractions which are soluble in non-polar solvents which have been purified from the crude extracts of the present invention and purified only in extracts soluble in hexane, methylene chloride, chloroform or ethyl acetate, preferably ethyl acetate solvent.

The polar solvent-soluble extract as defined herein is prepared by removing the non-polar solvent-soluble fractions from the crude extract and washing with water, methanol, butanol or a mixed solvent thereof, preferably water or butanol, more preferably, And one extracted fraction.

The perennial tree as defined herein includes whole parts of branches, leaves, roots, etc., preferably branches or root parts.

Hereinafter, the present invention will be described in more detail.

The extracts of the present invention can be obtained by the following production methods.

For example, the present invention will be described in detail below.

The Horticultural Extract of the present invention can be prepared as follows. Water and ethanol mixed solvent, more preferably 10% ethanol, and more preferably 10% ethanol, water, methanol, ethanol, butanol or the like, or a mixed solvent thereof, And then subjected to ultrasonic extraction, hot water extraction, room temperature extraction or reflux extraction for 30 minutes to 48 hours, preferably 1 hour to 12 hours at a temperature of 30 to 150 ° C, preferably 40 to 100 ° C, , Preferably the hydrothermal extraction method is repeated about 1 to 20 times, preferably 2 to 10 times, and the extract is filtered, concentrated under reduced pressure, and dried to obtain the crude extract of the present invention.

The polar solvent or the non-polar solvent soluble extract of the present invention may further contain about 0.0005 to 50 times, preferably 0.05 to 5 times the volume (v / w%) of the crude extract, preferably 10 to 90% ), Followed by a conventional fractionation using hexane, methylene chloride, chloroform, or ethyl acetate, preferably an ethyl acetate solvent, followed by extraction with hexane, methylene chloride, chloroform or ethyl acetate, preferably ethyl acetate A nonpolar solvent-soluble extractable fraction which is soluble in a nonpolar solvent; And the non-polar solvent-soluble extract fractions are removed to obtain polar solvent-soluble extract fractions soluble in a polar solvent such as butanol and water.

The present inventors confirmed that the T cells and the B cells significantly decreased the proliferative capacity of the T cells and B cells in the spleen cells of the experimental animals, Experimental Example 1); The production of IL-2, IL-4, IL-10 and IFN-γ was increased in all experiments (experiment 2); In the experiment, it was confirmed that the LP-BM5 MuLV infection reduced oxidative stress caused by reactive oxygen species (Experiment 3), and the samples of the present invention showed strong immunity enhancement It was confirmed that the composition is useful as a pharmaceutical composition or health functional food for the prevention and treatment of immune diseases.

Accordingly, the present invention provides a pharmaceutical composition for immunomodulating or immuno-enhancing and a health functional food containing the extract of Wuchu nucifera obtained by the above-mentioned method as an active ingredient.

Also, it is a medicinal herb that has been used for a long time as an edible or herbal medicine, and thus the extract of the present invention has no toxicity and side effects.

The pharmaceutical composition of the present invention contains 0.1 to 50% by weight of the above extract relative to the total weight of the composition.

The pharmaceutical compositions comprising the extract of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the production of pharmaceutical compositions.

Examples of carriers, excipients and diluents that can be included in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The composition containing the extract of the present invention may be formulated in the form of powders, granules, tablets, capsules, oral preparations such as suspensions, emulsions, syrups and aerosols, external preparations, suppositories and sterilized injection solutions, Can be used.

More specifically, when formulating the composition, it can be prepared using a diluent or an excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, and the like. Solid formulations for oral administration include tablets, pills, powders, granules and capsules, which may contain at least one excipient such as starch, calcium carbonate, sucrose, ), Lactose, gelatin and the like.  

In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral use include suspensions, solutions, emulsions and syrups. In addition to water and liquid paraffin which are commonly used simple diluents, various excipients such as wetting agents, sweetening agents, fragrances and preservatives may be included. have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol and vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As a suppository base, witepsol, macrogol, tween 61, cacao paper, laurin, and glycerol gelatin can be used.

The preferred dosage of the extract of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the administration route and the period of time, but can be appropriately selected by those skilled in the art. However, for the desired effect, the extract of the present invention may be administered in an amount of 0.0001 to 100 mg / kg, preferably 0.001 to 100 mg / kg, once or several times per day. In the composition, the extract of the present invention may be formulated in an amount of 0.0001 to 50% by weight based on the total weight of the total composition.

The pharmaceutical composition of the present invention can be administered to mammals such as rats, mice, livestock, humans, and the like in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine and intracerebroventricular injections.

Further, the present invention provides a health functional food for immunomodulation or immunity enhancement containing an extract of Aspergillus oryzae as an active ingredient.

The extract of the present invention can be used variously for medicines, foods and beverages for the prevention and treatment of a target disease. Examples of foods to which the extract of the present invention can be added include various foods, beverages, gums, tea, vitamin complexes, and health supplement foods, and they can be used in the form of powders, granules, tablets, have.

The extract of the present invention can be added to food or beverage for the purpose of prevention and treatment of a target disease. At this time, the above-mentioned extract in food or beverage generally can be added to 0.01 to 15% by weight of the total food weight of the health food composition of the present invention, and 0.02 to 30 g, preferably 0.3 To 10 g.

The health beverage composition of the present invention may contain various flavors or natural carbohydrates such as ordinary beverages as an additional ingredient, as long as it contains the extract as an essential ingredient at the indicated ratio, and there is no particular limitation to the liquid ingredient. Examples of the above-mentioned natural carbohydrates include monosaccharides such as disaccharides such as glucose and fructose, polysaccharides such as maltose, sucrose and the like, such as dextrin, cyclodextrin and the like , Xylitol, sorbitol and erythritol. Natural flavors (tau martin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 of the composition of the present invention.

In addition to the above, the extract of the present invention can also be used as a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and aging agents (cheese, chocolate, etc.), pectic acid and its salts, Salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages and the like. In addition, the extract or the compound of the present invention may contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The proportion of such additives is not so critical, but is generally selected in the range of 0 to about 20 parts by weight per 100 ml of the composition of the present invention.

As described above, the extract of the present invention showed that the T cells and the B cells significantly decreased the proliferative capacity through the measurement of the proliferative activity of the T cells and B cells in the spleen cells of the experimental animals, (Experimental Example 1); The production of IL-2, IL-4, IL-10 and IFN-γ was increased in all experiments (experiment 2); In the experiment, it was confirmed that the LP-BM5 MuLV infection reduced oxidative stress caused by reactive oxygen species (Experiment 3), and the samples of the present invention showed strong immunity enhancement It was confirmed that the composition is useful as a pharmaceutical composition or health functional food for the prevention and treatment of immune diseases.

FIG. 1 is a view showing a manufacturing process for separating the extract of Hwangchujang;
FIG. 2 is a graph showing the effect of T cells and B cells on the proliferative activity of spleen cells of Hwigulak extract or fraction;
FIG. 3 is a graph showing the effect on the production of IL-2 and IFN-y cytokines in the extracts or fractions of Wortmannia sp.
FIG. 4 is a graph showing the effect of IL-4, IL-10, and TNF-cytokine production on the level of IL-4, IL-10, and TNF-cytokine production;
FIG. 5 is a graph showing the effect of liver oil extract or fraction on oxidative damage of liver in an immunodeficient animal model. FIG.

Hereinafter, the present invention will be described in detail with reference to the following examples and experimental examples.

However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the contents of the present invention are not limited by the following Examples, Reference Examples and Experimental Examples.

Example   1. Yellowwood Crude extract  Produce

1-1. ethanol Crude extract  Produce

(2 kg) was cut into a size of 0.5 cm and pulverized. Then, it was placed in an extraction vessel (Ilshin Lab Co., Ltd, FD8512), 20 L of ethanol and 20 L of water were added, The extracts were heated (extraction temperature 45 ~ 55 ℃) and filtered with 3M paper to obtain an extract liquid. In order to prevent excessive sugar components from being extracted from the functional components, the extraction was carried out once for 6 hours. Subsequently, the obtained extract solvent was concentrated under reduced pressure using a vacuum evaporator (Sunil EYELA, Rotatory evaporator, N-1N) to obtain 96 g of ethanol extract (Hereinafter referred to as "DME").

1-2. water Crude extract  Produce

(2 kg) was cut into 0.5 cm size and pulverized. Then, 20 g of distilled water was added to the extraction vessel (Ilshin Lab Co., Ltd, FD8512), and the mixture was heated in a distillation circulating apparatus 95 ~ 100 ℃) and filtered with 3M paper to obtain an extract liquid phase. Extraction was carried out once for 6 hours to prevent excess sugar components from being extracted from the functional ingredients. The obtained extract solvent was then concentrated under reduced pressure using a vacuum evaporator (Sunil EYELA, Rotary evaporator, N-1N) to obtain 138 g of water extract (Hereinafter referred to as "DME").

Example   2. Yellowish wood polar solvent and Nonpolar solvent  Preparation of soluble extract (see Figure 1)

2-1. From the extract of Hwangchu - Hexane Fraction  Produce

A total of 1.6 L of distilled water was suspended in ethanol extract (80 g) obtained from Example 1 and suspended in a fractionating funnel. 1.6 L of n-hexane was added to extract the fraction. About one hour later, the n-hexane fraction was extracted from the supernatant to obtain n-hexane fraction. The fractionation procedure was repeated 3 times, after which the supernatant was concentrated under reduced pressure to give 6.8 g of n-hexane fraction. (Hereinafter referred to as "DMHE").

2-2. From ethyl acetate extract Fraction  Produce

The distilled water fraction obtained in the step of obtaining the n-hexane fraction was suspended in 1.5 L of distilled water, suspended in a fractionating funnel, and extracted with 1.5 L of ethyl acetate. After about 1 hour, the ethyl acetate fraction was extracted and the supernatant was taken to obtain the ethyl acetate fraction. The fractionation procedure was repeated three times, after which the supernatant was concentrated under reduced pressure to give 17.3 g of ethyl acetate fraction (hereinafter referred to as "DMEA").

2-3. From the extract of Hwangchu - Butanol Fraction  Produce

The distilled water fraction obtained in the step of obtaining ethyl ethyl acetate fraction was concentrated under reduced pressure, and then a total of 1.2 L of distilled water was added to suspend the suspension. Then, 1.2 L of n-butanol saturated with water was added to extract the fraction. After about 1 hour, the n-butanol fraction was extracted from the supernatant obtained in the n-butanol fraction. The fractionation procedure was repeated three times, after which the supernatant was concentrated under reduced pressure to obtain 41.2 g of the n-butanol fraction (hereinafter referred to as "DMBU").

2-4. Water from the Houttuynia cordata extract Fraction  Produce

The distilled water fraction obtained in the process of obtaining the yellowing oil n-butanol fraction was concentrated under reduced pressure to obtain 14.7 g of a water fraction (hereinafter referred to as "DMWA").

Experimental Example   1. Experimental animal In splenocytes  T cells, B cells Proliferative ability  Reactivity measurement

Experiments were carried out as described below in order to confirm the effect of the above-mentioned examples on the immunological regulation of experimental animals (Ho JN et al., Inhibition of premature death by isothiocyanates through immune restorative in LP- BM5 leukemia retrovirus-infected C57BL / 6 mice. Biosci Biotechnol Biochem 75: 1234-1239, 2011).

A 4-week-old female C57BL / 6 mouse (20 g) supplied from Korea BioLink Experimental Livestock Breeding Center was adapted from rodent breeding room for one week, and normal state was observed during the adaptation period. The incubation environment was maintained at a temperature of 23 ± 3 ° C and a humidity of 50 ± 5%, with a light cycle of 12 hours. Body weight was measured for the animals that were judged to be healthy during the refinement period, and individuals close to the average body weight were selected and subjected to group separation using the random method.

LP-BM5 MuLV, a virus that breaks down the immune system in mice, was obtained from the culture medium of SC-1 cells to induce an immunocompromising animal model. The mice were injected twice with C57BL / 6 mouse, which is a pathogen-sensitive species, (Ho JN et al., Inhibition of premature death by isothiocyanates through immune restorations in LP-BM5 leukemia retrovirus-infected C57BL / 6 mice. Biosci Biotechnol Biochem 75: 1234-1239, 2011).

In the present study, 50 mg / kg of ethanol extract, 50 mg / kg of n-hexane fraction, and 50 mg / kg of EtOAc fraction were administered to normal control, immunodeficient, kg group, 50 mg / kg n-butanol fraction, and 50 mg / kg water extract fraction, respectively. All diets were made on the basis of AIN93G, and diet and negative water were supplied free. The diets containing the complexes were sacrificed after 12 weeks in total for 12 weeks. All test animals were fasted for 24 hours prior to autopsy.

In order to confirm the effect on splenocyte culture and T cell and B cell proliferation in animals, spleens isolated from animals were washed with PBS and cultured in a 0.45 μm cell strainer (BD Falcon 352340, BD Biosciences, San Jose, CA, USA ) Was used to make a single cell suspension. L-glutamine (Hyclone Laboratories, Logan, Utah, USA), 100 mg / L penicillin-stereptomycin (Hyclone Laboratories, Logan, Utah, USA) and 10% fetal bovine serum (Hyclone Laboratories, , Red blood cell lysing buffer (R7757, Sigma-Aldrich Co., St. Louis, Mo., USA) was used for the hemolysis of red blood cells, followed by washing with RPMI-1640 (Hyclone Laboratories, Logan, Utah, USA) The cells were suspended in 96-well plates at a density of 1 × 10 ⁶ cells / well (200 μL). To measure T cell proliferative capacity, 5 μg / mL of concanavalin A (ConA) (C0412, Sigma-Aldrich Co., St. Louis, Mo., USA) was treated with lipopolysaccharide (LPS) -Aldrich Co., St. Louis, Mo., USA) and cultured at 37 for 48 hours. After 48 hours, 20 μL of EZ-Cytox (DLS1212, Deilab INC, Korea) was dispensed and incubated for 4 hours at 37 ° C., and the absorbance was measured at 450 nm.

As a result of the above experiment, normal mice (T cells: 7.8 ± 0.5 10 * 3 cpm, B cells: 6.3 ± 10 cps) were immunodominant (T cells: 2.4 ± 0.4 10 * 3 cpm, B cells: 0.7 10 * 3 cpm) significantly decreased T-cell and B-cell proliferation (P <0.05). In contrast, in the ethanol extract (T cell: 3.9 ± 0.4 10 * 3 cpm, B cell: 3.710 * 3 cpm) and ethyl acetate fraction (T cell: 5.610 * 3 cpm, B cell: 5.210 * 3 cpm) (P <0.05) compared with the control group. (T cells: 3.5 ± 0.3 10 * 3 cpm, B cells: 3.910 * 3 cpm) significantly increased T cell proliferative potency (P <0.05) in the n-butanol fraction administration group (FIG.

Therefore, it was confirmed that the ethanol extracts and ethyl acetate fractions of Hwangbukchil showed positive effects on immune regulation by inhibiting the proliferative capacity of T cells and B cells reduced by LP-BM5 MuLV infection.

Experimental Example   2. Measurement of cytokine production

In order to confirm the effect on the cytokine production level of the above-mentioned samples, an experiment was conducted as described below (Mosier DE, Animal models for retrovirus-induced immunodeficiency disease. Immunol Invest 15: 233-261, 1986).

In order to examine the effect of the test animals on the cytokine production level after the autopsy of Experimental Example 1, splenocytes were dispensed in a 96-well plate at a concentration of 1 × 10 ⁶ cells / well in an amount of 200 μL per well, IL-10 and IFN-γγ were stimulated by treatment with 5 μg / mL of LPS (L2880, Sigma-Aldrich Co., St. Louis, Aldrich Co., St. Louis, Mo., USA) to stimulate the production of TNF-a. IL-2, IL-4, IL-10 and TNF-a were incubated for 24 hours and IFN-γγ was cultured for 72 hours. The amount of cytokine in the supernatant was measured using a DuoSet sandwich ELISA mouse kit (R & D System, McKinley Place NE, USA). After incubation for 1 day in a 100-μL aliquot of the primary antibody specific for each cytokine in a 96-well plate for ELISA, the cells were washed three times with washing buffer (R & D System, McKinley Place NE, After washing the primary antibody with PBS (0.05% Tween 20 in PBS), the plate was washed with 1% BSA in PBS (R & D System, McKinley Place NE, IL-4, -10, TNF-a) or 0.1% BSA in TBST (IL-2, IFN-γγ) for 2 hours and washed with washing buffer. After incubation for 2 hours, 100 μL of the culture solution for the standard curve and the seeded spleen cells was incubated for 2 hours. After completion of the reaction, the cells were washed with washing buffer and diluted with secondary antibody in assay buffer. 100 μL aliquots are added to each well and treated for 2 hours. After completion of this procedure, wash the plate with washing buffer and incubate with 100 μL of substrate reagent (R & D System, McKinley Place NE, MN, USA) for color development. Measure absorbance at 570 nm, The amount of cytokine produced in the mice was calculated.

CD4 + Th cells include Th1 cells that produce Th1 type cytokines and Th2 cells that produce Th2 type cytokines. Th1 type cytokines mainly stimulate phagocytosis by increasing macrophage activity and Th2 type cytokines stimulate B cell activity and increase antibody production. In addition, Th1-type cytokines have been reported to be decreased and Th2-type cytokines are increased in LP-BM5 MuLV infection (Powrie F et al., Cytokine regulation of T-cell function: potential for therapeutic intervention. Immunol Today 14: 270-274, 1993).

As a result, IL-2 and IFN-γγ production of Th1 type cytokines were compared with those of normal group (IL-2: 148 ± 18 pg / mL, IFN-γγ: 186 ± 12 pg / mL) (P <0.05) in the mice (IL-2: 42 ± 10 pg / mL, IFN-γγ: 68 ± 11 pg / mL). On the other hand, the ethanol extract group (IL-2: 108 ± 11 pg / mL, IFN-γ: 98 ± 8 pg / mL) and the ethyl acetate fraction , IFN-γ: 143 ± 13 pg / mL) significantly increased the production of IL-2 and IFN-γ (P <0.05). IL-2 production was significantly increased only in the n-butanol fraction (IL-2: 64 ± 7 pg / mL, IFN-γ: 77 ± 9 pg / mL) (FIG.

(IL-4: 187 ± 18 pg / mL) compared to the normal control (IL-4: 64 ± 11 pg / mL, IL-10: 89 ± 9 pg / mL, TNF- IL-10 and TNF-α levels were significantly increased in the presence of IL-10, IL-10, IL-10 and TNF- (P < 0.05). These results demonstrate that the LP-BM5 MuLV infection caused Th1 / Th2 type cytokines imbalance when associated with a decrease in Th1 type cytokines. The cytokines increased by immunodeficiency were ethanol extract (IL-4: 141 ± 13 pg / mL, IL-10: 189 ± 22 pg / mL, TNF-α: 240 ± 15 pg / mL), ethyl acetate fraction (IL-4: 144 ± 12 pg / mL, IL-4: 108 ± 17 pg / mL, IL-10: 147 ± 21 pg / mL, TNF- IL-10: 183 ± 17 pg / mL, TNF-α: 269 ± 9 pg / mL).

This decrease in Th2 type cytokines is thought to inhibit Th1 type cytokines, and may help to maintain immune homeostasis by reducing Th1 / Th2 imbalance.

Experimental Example   3. The liver Oxidative  Damage measurement

In order to confirm the effect of the above-mentioned examples on the oxidative damage of the liver in the immunodeficient animal model, the following experiment was conducted by using the method described in the literature (Odeleye OE et al., Changes in hepatic lipid composition after infection LP-BM5 murine leukemia virus causing murine AIDS. Life Sci 51: 129-134, 1992).

In order to measure lipid peroxidation production by oxidative stress, Erdelmeier et al., Reactions of N-methyl-2-phenylindole with malondialdehyde and 4-hydroxyalkenals, Mechanistic aspects of lipid peroxidation. in Toxicology 11, 11841194, 1998), the oxidative damage was observed by measuring thiobarbituric acid reactive substance (TBARS). The liver was cut and homogenized using tris-HCl buffer (Sigma-Aldrich Co., St. Louis, Mo., USA) and centrifuged at 12,000 rpm for 20 minutes in a centrifuge (micro 17R, Hanil, Korea) And 300 μL of the supernatant was taken. The supernatant was treated with 2-thiobarbituric acid (TBA) (Sigma-Aldrich Co., St. Louis, Mo., USA) USA) at 95 ° C for 20 minutes. The absorbance of the TBA reaction product was measured at 532 nm and its value was expressed as 100% in the normal group.

It is known that oxidative stress caused by reactive oxygen species increases rapidly during infection with LP-BM5 MuLV, and it is known that hepatocyte inflammation is induced and liver disease such as fatty liver occurs (Odeleye OE et al., Changes in hepatic lipid composition after infection by LP-BM5 murine leukemia virus causing murine AIDS. Life Sci 51: 129-134, 1992). The oxidative damage of 100 ± 9.8% in the normal control group was significantly increased to 284 ± 24.7% in the immunodeficient mouse group (P <0.05). (P <0.05) (223 ± 17.9% in ethanol extract group, 164 ± 19.7 U / mL in ethyl acetate fraction and 208 ± 11.2% in n-butanol fraction).

The results of this study showed that ethanol extract, ethyl acetate fraction, and n - butanol fraction of H. japonica increased lymphocyte proliferation and cytokine imbalance and liver oxidative damage in immunodeficient animal models. Therefore, it can be expected to use as a domestic immunomodulator material.

The preparation examples of the pharmaceutical composition containing the extract of the present invention will be described, but the present invention is not intended to be limited thereto but is specifically explained.

Formulation example  One. Sanje  Produce

DME ------------------------------------------------ 300 mg

Lactose hydrate ----------------------------------------- 100 mg

Talk ------------------------------------------------ 10 mg

The above components are mixed and filled in airtight bags to prepare powders.

Formulation example  2. Preparation of tablets

DMW ----------------------------------------------- 300 mg

Corn starch ---------------------------------------- 100 mg

Lactose baggage ---------------------------------------- 100 mg

Magnesium stearate --------------------------------- 2 mg

After mixing the above components, tablets are prepared by tableting according to the usual preparation method of tablets.

Formulation example  3. Preparation of capsules

DMHE ---------------------------------------------- 300 mg

Microcrystalline cellulose ------------------------------------ 3 mg

Lactose Luggage --------------------------------------- 14.8 mg

Magnesium stearate ------------------------------- 0.2 mg

The above components are mixed according to a conventional capsule preparation method and filled in gelatin capsules to prepare capsules.

Formulation example  4. Preparation of injections

DMEA ---------------------------------------------- 300 mg

Di-mannitol ----------------------------------------- 180 mg

Sterile sterilized distilled water for injection ------------------------------- 2974 mg

Na ₂ HPO ₄ 12H ₂ O ---------------------------------------- 26 mg

(2 ml) per 1 ampoule in accordance with the usual injection method.

Formulation example  5. Liquid  Produce

DMBU ---------------------------------------------- 300 mg

Ising Party -------------------------------------------- 10 g

D-mannitol -------------------------------------------- 5 g

Purified water ----------------------------------------------

Each component was added and dissolved in purified water according to the usual liquid preparation method, and the lemon flavor was added in an appropriate amount. Then, the above components were mixed, and purified water was added thereto. The whole was added with purified water to adjust the total volume to 100 ml, And sterilized to prepare a liquid preparation.

Formulation example  6. Manufacture of health food

DMWA --------------------------------------------- 1000 mg

Vitamin mixture ---------------------------------------

Vitamin A Acetate -------------------------------- 70 ug

Vitamin E ------------------------------------------ 1.0 mg

Vitamin B ₁ ---------------------------------------- 0.13 mg

Vitamin B ₂ ------------------------------------------ 0.15 mg

Vitamin B ₆ ------------------------------------------- 0.5 mg

Vitamin B ₁₂ ------------------------------------------ 0.2 mg

Vitamin C ------------------------------------------- 10 mg

Biotin --------------------------------------------- 10 ug

Nicotinic acid amide 1.7 mg

Folic acid ----------------------------------------------- 50 ug

Calcium pantothenate ------------------------------------- 0.5 mg

Inorganic mixture ---------------------------------------

Ferrous sulfate ---------------------------------------- 1.75 mg

Zinc oxide ----------------------------------------- 0.82 mg

Magnesium carbonate ------------------------------------- 25.3 mg

Potassium phosphate monohydrate 15 mg

Secondary 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 mixture is comparatively mixed with a composition suitable for health food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional method for producing healthy foods , Granules can be prepared and used in the manufacture of health food compositions according to conventional methods.

Formulation example  7. Manufacture of health drinks

DMEA ---------------------------------------------- 300 mg

Vitamin C -------------------------------------------- 15 g

Vitamin E (powder) ------------------------------------- 100 g

Lactic Acid Iron ------------------------------------------- 19.75 g

Zinc oxide ------------------------------------------- 3.5 g

Nicotinic acid amide 3.5 g

Vitamin A ------------------------------------------- 0.2 g

Vitamin B ₁ ----------------------------------------- 0.25 g

Vitamin B ₂ ------------------------------------------ 0.3 g

Water ------------------------------------------------- - Quantity

The above components were mixed according to a conventional health drink manufacturing method, and the mixture was stirred and heated at 85 ° C for about 1 hour. The solution was filtered to obtain a sterilized 2-liter container, which was sealed and refrigerated It is used in the production of the health beverage composition of the present invention.

Although the compositional ratio is relatively mixed with a component suitable for a favorite drink, it is also possible to arbitrarily modify the compounding ratio according to the regional or national preference such as the demand class, the demanding country, and the use purpose.

Claims (8)

A medicinal composition for immunomodulation or immunity enhancement, comprising an extract of Aspergillus oryzae as an active ingredient. The method of claim 1, wherein
Wherein the extract is a crude extract, a polar solvent-soluble extract or a non-polar solvent-soluble extract. The method according to claim 2, wherein
Wherein the crude extract is an extract which is soluble in a solvent selected from water containing purified water, a lower alcohol having 1 to 4 carbon atoms such as methanol, ethanol, butanol or a mixed solvent thereof. The method according to claim 2, wherein
Wherein the non-polar solvent-soluble extract comprises extracts obtained by extracting the crude extract of claim 3 in a non-polar solvent obtained by extracting only the extract soluble in hexane, methylene chloride, chloroform, or ethyl acetate solvent. The method according to claim 2, wherein
Wherein the polar solvent-soluble extract comprises an extract obtained by removing the non-polar solvent-soluble fractions from the crude extract of claim 3, and extracting the remaining water with a solvent selected from methanol, butanol or a mixed solvent thereof. Wherein said woodweed comprises whole parts such as branches, leaves, roots, and the like. Health food containing immunoglobulin extract as an active ingredient for immune control or immune enhancement. The method of claim 7, wherein
The health functional food is a health functional food in the form of a food, a powder, a granule, a tablet, a capsule, a syrup, a beverage, a gum, a tea, a vitamin complex, or a health functional food.

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