KR20180090197A - Composition comprising the extract of buckwheat for immune activity - Google Patents

Abstract

The present invention relates to the use of buckwheat extract as an immunizing agent, and more specifically to a food composition for enhancing immune function and a pharmaceutical composition for enhancing immunity, which comprises buckwheat extract as an active ingredient. The buckwheat extract according to the present invention can increase the production of nitric oxide and increase the production of cytokines such as TNF-a or IL-6 to increase macrophage activity, thereby inducing immunity enhancing effect, , Increase the weight of the spleen and thymus, and increase the leukocyte count, and thus can be utilized as a pharmaceutical composition for diseases requiring immunity enhancement. In addition, the buckwheat extract of the present invention does not cause cytotoxicity and is stable in the body, so that it can be utilized as a composition for food for immunity enhancement.

Description

The present invention relates to a composition for enhancing immunity comprising buckwheat extract as an active ingredient,

The present invention describes the use of buckwheat extract as an immunity enhancer, and relates to a food composition and a pharmaceutical composition for enhancing immunity comprising buckwheat extract as an active ingredient.

The immune system can be divided into natural resistance, nonspecific immune system and specific immune system. Natural resistance (first line of defense) is an anatomical physiological element that blocks all invaders, including microorganisms, regardless of their type. Nonspecific immunity (second line of defense) is a phagocyte that breaks through natural resistance and removes intruders. A specific immune system is a highly developed immune system with the ability to distinguish between memory and non-immune systems. The immune system is composed of lymphocytes, and the specific immune system (the third line of defense).

In addition, the leukocyte constitutes a secondary or tertiary defense line, which breaks the primary line and takes charge of the foreign matter that enters the body. In the case of bacterial, viral infection or inflammatory reaction, the control of macrophage and lymphocyte activity determines the therapeutic effect It plays a pivotal role in Macrophage is a multifunctional cell that plays an important role in the immune system by producing various cytokines and NO in the context of oxidative stress. In particular, iNOS expressed by stimuli such as lipopolysaccharide (LPS), cytokine, and TNF-α in macrophages is known to produce large amounts of NO over a long period of time to promote NFκB activity. The production of reactive oxygen species (ROS) and nitric oxide (NO) such as superoxide anion (O2-), hydrogen peroxide (H2O2) by activated macrophages is not nonspecific Which is an important cytotoxic and cytostatic mechanism.

In addition, macrophages are microbicidal cells that exhibit antigen-presenting, tumoricidal, or microbial cells and are central to cell-mediated or humoral immunity. NO, produced by activated macrophages, acts as a nonspecific host defense mechanism, exhibiting bacterial and cancer cell proliferation inhibitory activity. In addition, macrophages have many lysosomes, which contain acid hydrolytic enzymes and peroxidases. It is also strongly attached to the glass surface and plastic surface and actively digests microorganisms and tumor cells. The cell has a cytokine receptor such as IFN-y. They produce cytokines such as complement components, interferons, interleukin-1 and tumor necrosis factors, and can be enhanced by a variety of cytokines produced from T-cells.

Therefore, many studies are currently being carried out to enhance health through immunity enhancement in the world. In recent years, problems of side effects have arisen on synthetic compounds, and studies have been focused on the development of materials capable of effectively obtaining immunity from natural products .

On the other hand, buckwheat is a perennial plant belonging to the Mallipulaceae, and its origin is grain grown in the Amur River in northern part of East Asia from the Manchu Baakalhu region. In Korea, it is used as a wild plant to cultivate when drought is inevitable, and it grows well on poor land, and has been given more meaning than a plant because it has been said to prevent ogre from the past.

Buckwheat has traditionally been used for medical treatments such as measles, ulcer gastroenteritis, bruises, and malignant boils, and has been reported to physiologically function to detoxify toxins and remove waste products. In addition, buckwheat contains a lot of routines with blood pressure-enhancing effect and has been clinically used because of its effectiveness in arteriosclerosis, pulmonary hemorrhage, bronchial asthma, hemorrhoids and cold treatment.

However, there is no report on the effect of buckwheat on immunity enhancement.

Korean Patent No. 10-0740716

Thus, the present inventors have completed the present invention by confirming that the buckwheat extract has an immunostimulating effect.

It is therefore an object of the present invention to provide a food composition for enhancing immune function comprising buckwheat extract as an active ingredient.

Another object of the present invention is to provide a pharmaceutical composition for enhancing immunity comprising buckwheat extract as an active ingredient.

Another object of the present invention is to provide a composition for promoting the activity of natural killer cells comprising buckwheat extract as an active ingredient.

In order to achieve the above object, the present invention provides a food composition for enhancing immune function comprising buckwheat extract as an active ingredient.

In one embodiment of the present invention, the extract is a hot-water extract obtained by adding water to buckwheat; Or an extract comprising the polymer fraction obtained by lyophilizing the precipitate after adding the precipitate to the hot-water extract and precipitating the precipitate.

In one embodiment of the present invention, the buckwheat extract may increase macrophage activity, increase nitric oxide production, and increase the production of cytokines TNF-a or IL-6.

In one embodiment of the present invention, the buckwheat extract may have activity to increase the activity of NK cells, increase the weight of spleen and thymus, and increase leukocyte count.

The present invention also provides a pharmaceutical composition for enhancing immunity comprising buckwheat extract as an active ingredient.

In one embodiment of the present invention, the extract is a hot-water extract obtained by adding water to buckwheat; Or an extract comprising the polymer fraction obtained by lyophilizing the precipitate after adding the precipitate to the hot-water extract and precipitating the precipitate.

In one embodiment of the present invention, the buckwheat extract may increase macrophage activity, increase nitric oxide production, and increase the production of cytokines TNF-a or IL-6.

In one embodiment of the present invention, the buckwheat extract may have activity to increase the activity of NK cells, increase the weight of spleen and thymus, and increase leukocyte count.

The present invention also provides a composition for promoting natural killer cell activity comprising buckwheat extract as an active ingredient.

In one embodiment of the present invention, the extract is a hot-water extract obtained by adding water to buckwheat; Or an extract comprising the polymer fraction obtained by lyophilizing the precipitate after adding the precipitate to the hot-water extract and precipitating the precipitate.

The buckwheat extract according to the present invention can increase the production of nitric oxide and increase the production of cytokine TNF-a or IL-6 to promote macrophage activity, thereby inducing an immunity enhancing effect, It is possible to increase the weight of spleen and thymus and to increase the number of leukocyte. Thus, it can be utilized as a pharmaceutical composition ultimately. In addition, the buckwheat extract of the present invention does not cause cytotoxicity and is stable in the body, so that it can be utilized as a composition for food for immunity enhancement.

FIG. 1 shows the result of MTT analysis of the cytotoxicity of the buckwheat extract according to the present invention. In FIG. 1, 1a shows the results of extracting buckwheat maize, and 1b shows the results of hot water extraction of buckwheat.
FIG. 2 shows the result of analyzing the degree of nitric oxide production according to the treatment concentration of the buckwheat extract of the present invention. 2a shows the result of extracting the buckwheat maize, and 2b shows the result of the hot water extract of buckwheat.
FIG. 3 shows the result of analysis of the degree of production of TNF-a cytokine according to the treatment concentration of buckwheat extract, 3a shows the result of extracting the buckwheat extract, and 3b shows the result of the hot water extract of the buckwheat.
FIG. 4 shows the results of analysis of the degree of production of IL-6 cytokine by the treatment concentration of buckwheat extract, 4a shows the results of the alcohol extract of buckwheat, and 4b shows the results of the hot water extract of buckwheat.
FIG. 5 shows the results of measurement of changes in the spleen and thymus weight of the mouse group administered with buckwheat extract, the immunosuppressive drug administered mouse group, and the immunosuppressive adjuvant administered group.
FIG. 6 shows the results of analysis of total blood counts of the group administered with buckwheat extract, the group administered with immunosuppressant, and the group administered with immunosuppressive adjuvant.
FIG. 7 shows the results of analysis of NK cell activity and granzyme production according to the buckwheat extract treatment.

The present invention is characterized by providing a food composition for enhancing immune function and a pharmaceutical composition for enhancing immunity comprising buckwheat extract as an active ingredient.

In one embodiment of the present invention, the efficacy associated with immunological activity of buckwheat extract was analyzed, and the effect of macrophage activation was analyzed.

The activation effect of macrophages was confirmed by the production ability of NO and the cytokine production ability. The stimulating effect of NO production is an indicator of macrophage activation. NO generated in macrophages is eliminated by eliminating the invading pathogen and activating the basic host defense system . Especially, when Th1 and Th2 are divided into Th1 and Th2 cells during the differentiation stage of Th cells, they are known to help differentiate into Th1. It has also been reported that the cytotoxic activity of killing cancer cells, parasites or bacteria during the activation of leukocytes is shown by liberating NO. It is also known that NO is immunomodulating between T-cell and endothelial cells in bronchial inflammation and has strong platelet aggregation inhibitory action. In addition, the secretion of cytokines such as TNF-a and IL-6 is increased to induce immunity enhancing effects.

As a result of the analysis of the production of nitric oxide and cytokine after treating the extract of buckwheat with macrophages in one embodiment of the present invention, it was found that the concentration of oxytocin and cytokine (TNF- a < / RTI > and IL-6).

Further, the present inventors confirmed that the extract of buckwheat can enhance the spleen and thymus weight through animal experiments, and confirmed that the buckwheat extract of the present invention has an effect of increasing the immunological organ index, Lt; RTI ID = 0.0 > activity. ≪ / RTI >

In addition, the buckwheat extract of the present invention activates natural killer cells (NK) cells. Natural killer cells (NK) are one of the innate immune cells and selectively cytotoxic to cancer cells or external invasive substances It is a cell. Unlike other immune cells, natural killer cells can instantaneously detect and eliminate external intrinsic substances such as cancer cells because they can distinguish foreign substances from normal cells through various immune receptors on the surface of natural killer cells. In addition, natural killer cells play a key role in regulating inflammation and immune responses through the production of cytokines such as IFN-γ and TNF-α, and natural killer cells play a role in dendritic cells, macrophages, It is also known to play a key role in regulating the immune response through direct interaction and indirect interaction through cytokines.

In this respect, according to one embodiment of the present invention, it has been confirmed that natural killer cell (NK) cells can be activated when the buckwheat extract of the present invention is treated, and furthermore, , The activity of NK cells was enhanced even when the extract was treated.

Therefore, since the buckwheat extract according to the present invention can activate natural killer cells, the present invention can provide a composition for promoting the activity of natural killer cells comprising buckwheat extract as an active ingredient.

As described above, natural killer cells (NK cells) are immune cells that play an important role in congenital immune responses by destroying cancer- or virus-infected cells (J. Immunol., 136: 3910- 3915, 1986, Melanoma Res., 13: 349-356, 2003; Lung Cancer, 35: 23-28, 2002). In addition, the immune system secretes cytokines through stimulation and recruits the immune system to perform an indirect immune response. These NK cell-destructive functions can be used to treat solid tumors using lymphocine activated killer cells (LAK) and tumor infiltration lymphocytes (TIL), or to treat donor lymphocytes Bordignon C, et al., Hematologia 84: 1110-1149, 1999) by performing an immunotherapy with an immunosuppressive agent (Tilden, AB et al., J. Immunol., 136: 3910-3915, 1986; New cell therapies have been attempted to prevent rejection of organ transplants. In addition, active defects of NK cells were observed in breast cancer (Konjevic G, et al., Breast Cancer Res. Treat., 66: 255-263, 2001), melanoma (Ryukey, et al., Melanoma Res. 356, 2003) and lung cancer (Villegas FR, et al., Lung Cancer, 35: 23-28, 2002), and NK cell therapy has been used to treat such diseases .

In addition, natural killer cells (NK cells) are present in about 10 to 15% of normal blood and have high killing ability when they react with nonself. In infecting cells with various viruses, bacterial infiltration, or abnormal cell formation, NK cells react nonspecifically and immediately to remove foreign matter. However, since the number of NK cells present in the body is not so large, it is necessary to develop a method capable of proliferating the number of effective cells necessary for showing a therapeutic effect, and it is necessary to develop a method for promoting the activation of NK cells Do.

In this respect, the buckwheat extract according to the present invention can effectively activate NK cells, and further, the NK cells treated with the buckwheat extract of the present invention can be used as a cell therapy agent.

Therefore, the buckwheat extract of the present invention can be used for the improvement and treatment of diseases related to NK cell dysfunction. Diseases associated with NK cell dysfunction include, but are not limited to, immunological diseases and cancers. Such cancers include blood cancer, liver cancer, lung cancer, kidney cancer, pediatric neurological cancer, and skin cancer.

In addition, it was confirmed that the extract of buckwheat of the present invention can enhance the leukocyte count in one embodiment of the present invention.

The leukocyte, as mentioned in the prior art, is responsible for the foreign body that enters the body and induces phagocytosis and induces immune defense function during bacterial, viral infection or inflammatory reaction.

From these results, the present inventors have found that the buckwheat extract of the present invention can effectively enhance the immunity enhancing action.

Accordingly, the present invention can provide a food composition for enhancing immune function comprising buckwheat extract as an active ingredient.

In the present invention, the buckwheat extract may be prepared by drying the buckwheat according to a conventional extraction method. The buckwheat may be extracted by adding C1 to C4 lower alcohol or a mixed solvent thereof such as water, methanol or the like to the dried buckwheat, Extracting by cold extraction, hot water extraction, ultrasonic extraction and reflux cooling extraction. The extract can be obtained as a buckwheat extract of the present invention after filtration under reduced pressure through a filter, followed by lyophilization by concentration of the filtrate.

Preferably, the buckwheat extract according to the present invention may be a hot-water extract obtained by adding water to buckwheat. Alternatively, the buckwheat extract may be a polymer fraction obtained by lyophilizing the precipitate after adding the precipitate to the obtained hot- That is, a fraction containing a polysaccharide component.

More preferably, the buckwheat extract may be a hot-water extract of buckwheat obtained by adding 4 to 20 times volume of buckwheat water and reflux-extraction at a temperature of 60 to 100 ° C for 3 to 8 hours, or the hot- More preferably 80 to 90% of the crude polysaccharide fraction may be precipitated for 12 to 24 hours, more preferably 15 to 20 times as much as the buckwheat weight, Or 2 to 4 hours at room temperature, or the polysaccharide fractions derived from the buckwheat may be precipitated for 12 hours by addition of 80% alcohol to the hot-water extract. In the present invention, the polysaccharide fraction fraction derived from buckwheat was referred to as a " alcohol extract ".

In particular, the present inventors analyzed the immunopotentiating effect of the extract of buckwheat hot water and the extract of the buckwheat containing the polysaccharide component obtained by adding the alcohol to the hot water extract. As a result, both the hot water extract and the alcohol extract increased the activity of macrophages , Increased the production of nitric oxide, increased the production of cytokines TNF-a or IL-6, and also significantly enhanced the activity of NK cells.

Foods to which the extract of the present invention can be added include, for example, various foods, beverages, gums, tea, vitamin complexes, and health functional foods. It can also be added to foods or beverages for the purpose of enhancing the immunity. At this time, the amount of the extract in the food or beverage may generally be 0.01 to 50% by weight, preferably 0.01 to 15% by weight, of the total food weight of the health functional food composition of the present invention, , Preferably from 0.3 to 1 g, based on the total weight of the composition.

The health functional food of the present invention includes forms such as tablets, capsules, pills, and liquids. The health beverage composition of the present invention has no particular limitations on other components other than those containing the above extract as an essential ingredient in the indicated ratios and may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. 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 And sugar alcohols such as 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 ml 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 thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages and the like. In addition, the extract 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 parts by weight of the composition of the present invention.

In addition, the pharmaceutical composition containing the extract of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the production of pharmaceutical compositions.

The pharmaceutical composition containing the extract according to 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 Examples of the carrier, excipient and diluent which can be contained in the composition containing the extract 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. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, 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 talc are also used. Examples of the liquid preparation for oral administration include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . 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, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

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 is preferably administered at 0.0001 to 100 mg / kg per day, preferably 0.001 to 10 mg / kg per day. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.

The extract 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, muscular, subcutaneous injection.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are for further illustrating the present invention, and the scope of the present invention is not limited to these examples.

≪ Example 1 >

Production of buckwheat extract

<1-1> Production of hot water extract of buckwheat

Buckwheat was used in the form of Fagopyrum esculentum Moench in Hanwi herb. It was dried in a drying oven at 50 ℃ and pulverized to less than 0.5 mm using a pulverizer. Then, water corresponding to 20 times volume was added to 100 g of buckwheat, and the mixture was refluxed for 3 hours at a temperature of 80 캜 to obtain a hot water extract of buckwheat. The resulting extract was filtered, concentrated under reduced pressure using a rotary vacuum evaporator, and then lyophilized for 72 hours to obtain a final buckwheat water extract. The buckwheat water extract was subjected to BWE (Buckwheat water extract) .

<1-2> Preparation of alcohol extract of buckwheat containing polysaccharide component

The buckwheat was used in the form of Fagopyrum esculentum Moench in the Korean herb, and dried in a drying oven at 50 ° C and then pulverized to a size of 1.0 mm or less using a pulverizer. Then, water corresponding to 20 times volume was added to 1000 g of buckwheat and refluxed for 3 hours at a temperature of 80 ° C to obtain a buckwheat hot water extract. Then, the extract was filtered and concentrated under reduced pressure to 1/10 of the extract using a rotary vacuum evaporator. Then, 80% of the extract was added to the concentrated hot-water-extracted buckwheat hot-water extract for 24 hours to precipitate The precipitate was lyophilized to obtain a buckwheat extract of the buckwheat. The buckwheat extract obtained by the above method contains polysaccharide as a main component and is described as BPF (Buckwheat polysaccharide fraction) in the following.

&Lt; Example 2 >

Cytotoxicity analysis of buckwheat extract

To determine whether the buckwheat extract of the present invention obtained in Example 1 was toxic to cells, cytotoxicity was examined in vitro. For this, Raw 264.7 cells, a murine macrophage cell line, were cultured in KCLB. Cells were placed in a flask for cell culture, and 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin U / ml) was added and incubated in an incubator maintained at 37 ° C, 5% CO _2, 95% humidity condition.

The cytotoxicity was assessed by MTT assay, in which the dehydrogenase in mitochondria of cells with intact metabolic pathways was identified as yellow soluble tetrazoliumsalt [3- (4,5-dimethylthiazol-2-yl) -2-5-diphenyltetrazolium bromide] ) Is reduced to a water-insoluble dark purple MTT formazan crystal. It is a method of evaluating cytotoxicity by measuring absorbance at an appropriate wavelength (mainly 500-600 nm). LPS (serotype: E. coli 055: B5, Sigma Co., St. Louis, Mo., USA) was used as an indicator of immune response and the extent of the reaction was measured by increasing or decreasing cell proliferation. RAW 264.7 cells were divided into 96 wells at a concentration of 1 × 10 ⁵ cells / well and cultured for 24 hours. Then, the buckwheat extract of the present invention was added to the culture medium at a concentration of 0.5, 1, 5 and 10 μg / After incubation for 20 hours, MTT at 0.5 mg / mL was added and incubated for 4 hours at 37 ° C. DMSO (dimethyl sulfoxide) was added to measure the absorbance at 540 nm using a microplate reader. Was calculated by the following equation.

Cell viability (%) = sample absorbance / control absorbance × 100

As shown in FIG. 1, the cell survival rates in the groups treated with 0.5, 1, 5, and 10 μg / mL of alcohol extract (BPF) were 112.5 ± 6.0%, 98.4 ± 0.5% 3.4%, 109.5 ± 5.6%, and 98.9 ± 5.2%, respectively, indicating that the extract of the present invention had no significant effect on cytotoxicity (see FIG. 1A).

The cell survival rates were 103.1 ± 2.5%, 98.6 ± 5.6%, 94.3 ± 6.3% and 89.7 ± 6.7%, respectively, in the group treated with water extract (BWE) at 0.5, 1, 5 and 10 μg / % Or more, indicating that the extract of the present invention did not significantly affect cytotoxicity (see Fig. 1B).

&Lt; Example 3 >

Analysis of nitrogen oxides (NO) and cytokine production ability of buckwheat extract

<3-1> Nitric oxide production assay

After collecting the cell supernatant from the cell test group used in the above example, the amount of NO (nitric oxide) in the substance secreted by the immune cell activity was confirmed with the cell supernatant. The production of NO produced by cells exposed to inflammatory mediators is known to contribute to biofilm and tissue damage induced by free radicals and is known to increase as a result of various inflammatory diseases. The activity of NO was determined by the Griess reaction of nitrate (NO ₃ ^- ) and nitrite (NO ₂ ^- ) production by metabolism. Specifically, RAW 264.7 cells were divided into 96 wells at a concentration of 1 × 10 ⁵ cells / cultured for 24 hours in a while, and then, the extract of the present invention, 0.5, 1, 5, treated and LPS was treated with 1 μg / mL concentration to 10 μg / mL concentration in the following 37 ° C, under the conditions of 5% CO ₂ And cultured for 20 hours. Then, the cell supernatant and the cell supernatant were added to the same amount of griess reagent and reacted for 15 minutes. Absorbance was measured at 540 nm using a microplate reader. The NO content was determined by preparing a standard curve with sodium nitrite (NaNO ₂ ).

As a result, NO production was increased in RAW 264.7 cells treated with 0.5, 1, 5, and 10 μg / mL of buckwheat hot water extract and alcohol extract, respectively. The concentrations of the extracts of the buckwheat extracts were 12.92 ± 1.1 μM, 14.46 ± 0.8 μM, 16.42 ± 2.6 μM and 16.23 ± 1.5 μM, respectively, at the levels of 0.5, 1, 5 and 10 μg / (Fig. 2a). &Lt; tb &gt; &lt; TABLE &gt; In addition, the NO production was increased depending on the treatment concentration in the case of the hot-water extract, the NO content was 11.89 ± 2.2 μM in the LPS-activated cells, and the level of BWE 5 μg / And at a concentration of 10 μg / mL, NO production was higher than that of the control group. When BWE was treated at 0.5, 1, 5, and 10 μg / mL, it increased to 10.58 ± 2.5 μM, 11.18 ± 3.4 μM, 11.96 ± 3.3 μM and 13.58 ± 1.0 μM, respectively (see FIG.

Therefore, the extract of buckwheat of the present invention can enhance the activity of macrophages, which play an important role of the immune system, through the increase of production of NO.

<3-2> Analysis of production ability of cytokine (TNF-α, IL-6)

Tumor necrosis factor (TNF) -a is one of inflammatory mediators, a proinflammatory cytokine, which is considered to be a potent immune mediator in many infectious diseases or autoimmune diseases, which is produced by macrophages, activated T cells and mast cells, , Lymphocytes, and mononuclear cells. Interleukin-6 (IL-6) is involved in the regulation of hematopoietic function and immune response. It acts synergistically with other cytokines to induce a synergistic effect. It stimulates B cells, &Lt; / RTI &gt; To determine whether the extract of buckwheat of the present invention has an effect of increasing TNF-α and IL-6 production in addition to NO production, commercial competitive enzyme immunoassay kit (R & D system, Minneapolis, USA) And the amount of cytokines such as TNF-α and IL-6.

To the 96 wells pre-incubated with anti-mouse TNF-α, 50 μl of culture medium or TNF-α conju- gate was added to each of the cells used in the experiment and reacted at room temperature for 2 hours. After washing 4 times with washing buffer, 100 μl of substrate solution was added and reacted under the condition of blocking light for 30 min. Then, 100 μl of stop solution was treated and absorbance was measured at 450 nm using a microplate reader. IL-6 was also applied to the experiment in the same manner as described above.

As shown in FIG. 3, TNF-α production was 4864.1 ± 91.6 pg / mL when treated with LPS alone, and TNF-α was significantly higher than that of the normal group. The extract of buckwheat was 0.5 , TNF-α production was increased depending on the treatment concentration of extracts. The concentrations of TNF-α, TNF-α and TNF-α in the extracts were 2049.41 ± 156.0, 3160.1 ± 531.8, 4676.7 ± 167.5, and 4881.8 ± 64.0 pg / In the case of buckwheat extract 5 and 10 μg / mL, the level of TNF-α was similar to that of the negative control treated with LPS alone, indicating that the buckwheat extract had a high effect on TNF-α expression ).

As a result of measuring IL-6 production, LPS alone showed 7190.6 ± 96.3 pg / mL of TNF-α, which was significantly higher than normal group. The extract of buckwheat extract was 0.5, 1, 5 and 10 μg / mL was 526.9 ± 67.2, 2021.2 ± 129.0, 7026.9 ± 409.7, and 8734.6 ± 133.5 pg / mL, respectively. At the concentrations of 0.5 and 1 μg / mL, IL-6 was produced at a lower level than the negative control 5 μg / mL, and it was found to be about 1544.0 μg / mL at the concentration of 10 μg / mL. Therefore, it was confirmed that the buckwheat extract of the present invention activates macrophages and promotes the production of IL-6 (see Fig. 4A).

In addition, the production of TNF-α in the hot-water extract of buckwheat produced 4864.1 ± 91.6 pg / mL of LPS alone, which was significantly higher than that of the normal group, and the BWE was 0.5, 1, 5, The production of TNF-α was similar to that of the LPS-treated group when the concentration was 10 μg / mL, which was 3133.6 ± 44.9, 3480.0 ± 374.6, 4366.4 ± 296.2 and 4696.3 ± 226.4 pg / 1 &lt; / RTI &gt; cells, thereby enhancing immune responses (see FIG. 3B). In addition, IL-6 production was found to be 7190.6 ± 96.3 pg / mL when treated with LPS alone. IL-6 production was significantly higher than that of the normal group, especially when LPS was treated at a concentration of 10 μg / IL-6 production was higher than that of one group. When treated with BWE at 0.5, 1, 5, and 10 μg / mL, 3064.6 ± 124.7, 5771.3 ± 126.7, 6948.3 ± 84.5, and 7480.2 ± 183.4 pg / mL, respectively, were found in the macrophages activated by BWE (See FIG. 4B). In addition, the present inventors have found that IL-6 production is enhanced by enhancing the immune activity.

Therefore, the inventors of the present invention have found that the buckwheat extract of the present invention can effectively activate macrophages, which play an important role in the immune system as a part of the host defense mechanism and recognize the foreign substance intrusion first, Activation of these macrophages was found to result in increased production of nitric oxide and increased production of cytokines (TNF-α and IP-6).

Therefore, the hot-water extract of the buckwheat of the present invention and the alcohol extract containing the polysaccharide ingredient can inhibit the growth of cancer cells and various harmful microorganisms due to the promotion of the macrophage activation. Through the activation of the macrophages, nitric oxide and cytokine (TNF- α and IP-6) to increase the immune response in the early phase of the immune response in the body without stimulation from the external antigens.

<Example 4>

Measurement of spleen and thymus weight change in immunosuppressed animal model by buckwheat extract treatment

The immunological enhancement effect of the buckwheat extract was confirmed in animal models. The animals used for the experiment were obtained from 6-week-old female Balb / c mice raised in an aseptic environment and purchased from Sarron Bio Co., , And was used for experiments for about 1 week before the experiment. The mice were maintained at a temperature of 22 ± 2 ° C and humidity of 40-60%, and the light and dark cycle was adjusted at intervals of 12 hours. The normal mouse group raised under the above conditions was classified into the following five groups.

Animal model group Remarks Normal group (Nor) Normal mouse test group CTX group The mice treated with the immunosuppressant cyclophosphamide CFX group A group of mice treated with CFX (cold-fx) BPF 50 mg / kg group The group of mice treated with 50 mg / kg of buckwheat extract of the present invention (oral administration for 4 weeks) BPF 100 mg / kg group The group of mice treated with 100 mg / kg of buckwheat extract of the present invention (oral administration for 4 weeks)

That is, the normal group and the immunosuppression model group (CTX group; model control group) were treated with distilled water, the positive control group was treated with CFX 50 mg / kg, and the BPF treatment group was treated with the buckwheat extract of the present invention 50, 100 mg / kg for 4 weeks. For the immunosuppressed animal model experiment, physiological saline was administered intraperitoneally for 7 days once a day for one week before the end of the experiment. The positive control (CFX group) and the BPF group, including the immunosuppression model group (model control group) mg / kg was administered intraperitoneally to the mice after immunosuppression. Twenty-four hours after the last administration, mice were harvested and the spleen thymus of each animal was harvested to measure the proliferative capacity of the splenocytes, cytokine, and leukocytes in the peripheral blood. Then, the spleen and thymus were aseptically removed, and the weight was measured. Then, to eliminate the variation according to the difference in the weight of the experimental animals, the spleen and the thymus were weighed to standardize them, And thymus indices were calculated.

Spleen or thymus index = spleen or thymus weight (mg) / mouse weight (g)

The weight of the spleen was decreased by 18.5% in the CTX group and 36.9, 32.5 and 41.4% in the CFX, 50-BPF, and 100-BPF groups, respectively, as compared with the CTX group. In addition, the weight of the thymus was decreased by 51.3% in the CTX group and 46.8, 47.4 and 57.6% in the CFX, 50-BPF, and 100-BPF groups, respectively, as compared with the CTX group.

The spleen not only filters the blood but also acts as a secondary lymphoid tissue. It is an important organ that removes the blood antigen and activates B cells and T cells. Therefore, changes in the number of splenocytes can be considered to indicate changes in lymphocytes. In addition, since the increase in the weight of the spleen and thymus is recognized as an important index for the increase in the nonspecific immune response and the specific immune response, the buckwheat extract according to the present invention increases the weight of the spleen and thymus , It was found that buckwheat extract can play a role as an immune enhancer in the change of activity of the major organs of the immune system.

&Lt; Example 5 >

Analysis of Total Leukocyte Count by Buckwheat Extract Treatment

Bacterial or traumatic signals activate pro-inflammatory cytokines and chemokines in various cells and plasma, leading to increased adhesion of leukocytes and endothelial cells. Therefore, in order to analyze changes in leukocyte counts in an animal model according to the treatment with buckwheat extract, the inventors of the present invention conducted animal mouse experiments in the above-mentioned Examples, and they were harvested under ether anesthesia and collected from the portal vein. White blood cells were analyzed by Green Cross Medical Foundation.

On the other hand, it is known that the administration of a large amount of CTX causes a decrease in leukocyte and neutrophil count due to immunosuppression. In this experiment, the immunosuppressive mouse model was prepared with CTX 100 mg / kg, and CFX and BPF were orally administered. The leukocyte count in the blood was analyzed by the Green Cross Medical Foundation and analyzed. As shown in FIG. 6, The number of leukocytes was decreased by 37.6% compared to the normal group, and the CFX group increased by 23.6% compared to the CTX group, and the 50-BPF and 100-BPF increased by 27.3% and 42.1%, respectively. Therefore, it can be seen from the results that the extract of the present invention can effectively increase the number of leukocytes degraded by CTX, and that the ability to recover the immune activity is excellent.

&Lt; Example 6 >

Analysis of NK cell activity by buckwheat extract treatment

The cytotoxicity of NK (natural killer) cells was analyzed by measuring lactate dehydrogenase (LDH) liberated from NK cell-destroyed tumor cells YAC-1 (NK-sensitive cell line). To determine the changes in NK cell activity, the spleen of mice fed a 100 mg / kg buckwheat extract for 2 weeks was harvested and used. 1 × 10 ³ cells / well of each well were dispensed into 96-well plates and used as effector cells. YAC-1 cells were used as target cells and the ratio of effector cells to target cells was adjusted to 12.5: 1, 25: 1, 50: 1 and cultured for 6 hours. After incubation, the cells were centrifuged at 900 g for 10 min. After mixing with the supernatant and lactate dehydrogenase (LDH) solution (Pierce Biotechnology), the absorbance at 680 nm was measured using a microplate reader and the degree of YAC- %). The concentration of granzyme B, which is a substance released by NK cells at the same time as the NK cell activity, was measured.

NK cell cytotoxic activity (%) = [(effector cell spontaneous release target cell spontaneous release) / (target cell maximum release target spontaneous release)] × 100

As shown in FIG. 7, in the group treated with the buckwheat extract of the present invention, the activity of NK cells was significantly increased compared to that of the normal group (P <0.01). When the cell ratio was 25: 1, the activity was about 6 times higher than that of the normal group and about 4 times higher when the cell ratio was 50: 1, which was statistically significant (p <0.01). In addition, the concentration of granzyme B, a substance that NK cells excrete during immune function, was measured at the cell concentration of 12.5: 1, 25: 1, and 50: 1 compared with the normal group, 42.2, 36.4, 32.4 % Increase.

From the above experimental results, it was found that the NK cell activity of the buckwheat extract of the present invention was the highest at a cell concentration of 12.5: 1, and the extract of the present invention effectively promoted NK cell activity and immune activation, It is possible to maintain and improve health by activating the body defense mechanism.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Claims (10)

A food composition for enhancing immune function comprising buckwheat extract as an active ingredient. The method according to claim 1,
Wherein the extract is a hot-water extract obtained by adding water to buckwheat; Or an extract comprising the fraction of the polymer obtained by lyophilizing the precipitate after precipitating the alcohol by adding the alcohol to the hot-water extract. The method according to claim 1,
Wherein the buckwheat extract increases the activity of macrophages, increases the production of nitric oxide, and increases the production of cytokines TNF-a or IL-6. The method according to claim 1,
Wherein the buckwheat extract has an activity of increasing the activity of NK cells, increasing the weight of spleen and thymus, and enhancing leukocyte count. A pharmaceutical composition for enhancing immunity comprising buckwheat extract as an active ingredient. 6. The method of claim 5,
Wherein the extract is a hot-water extract obtained by adding water to buckwheat; Or an extract comprising the fraction of the polymer obtained by lyophilizing the precipitate after the precipitate is added to the hot-water extract and the precipitate is added thereto. 6. The method of claim 5,
Wherein the buckwheat extract increases the activity of macrophages, increases nitric oxide production, and increases the production of cytokines TNF-a or IL-6. 6. The method of claim 5,
Wherein the buckwheat extract has activity to increase the activity of natural killer cells (NK cells), increase the weight of the spleen and thymus, and increase the leukocyte count. A composition for promoting natural killer cell activity comprising buckwheat extract as an active ingredient. 10. The method of claim 9,
Wherein the extract is a hot-water extract obtained by adding water to buckwheat; Or a fraction containing a polymer obtained by lyophilizing a precipitate by adding a precipitate to the hot-water extract and adding the precipitate to the extract.

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