KR20200033685A - Composition for Enhancing Immunity comprising Kalopanax Extract as effective component - Google Patents

Abstract

The present invention relates to a pharmaceutical composition and a food composition for enhancing congenital immunity, which contains a hot water extract of Kalopanax cortex as an active component. A composition for enhancing congenital immunity which contains the hot water extract of Kalopanax cortex as an active component exhibits a congenital immunity enhancing effect, thereby being able to be effectively used for preventing, alleviating, and treating various bacterial and viral infectious diseases.

Description

Composition for Enhancing Immunity comprising Kalopanax Extract as effective component

It relates to a pharmaceutical composition and a food composition for enhancing immunity, including the Haedongpi extract as an active ingredient.

Immunity refers to a mechanism for physiologically recognizing and excluding foreign and endogenous foreign substances in humans and animals, and maintaining homeostasis. Immunity can be broadly classified into two categories: innate immunity, which has been born since birth, and acquired immunity, which is acquired by adaptation to life.

Innate immunity, also called natural immunity, is characterized by nonspecific reaction to antigens and no specific memory action. Innate immune systems include skin and mucous tissues that block the invasion of antigens, acidic gastric acid, and complements present in the blood, and cells include macrophages and polymorphic nuclei responsible for phagocytosis. There are white blood cells (polymorphonuclear leukocyte) and K cells that can kill infected cells. In fact, most infections are defended by this congenital immunity. Acquired immunity is also called acquired immunity, it can remember the first invading antigen, react specifically when it invades again, can effectively remove the antigen, and serves to reinforce innate immunity.

In recent years, the field of innate immunity has attracted attention because activation of declared immunity can be a fundamental preventive method against various infectious disease pathogens. In addition, if the immunity is high, it is less susceptible to disease, recovery is quick even if it is diseased, and a healthy state can be maintained continuously. Therefore, it is very important to manage immunity to maintain health and prolong life. However, if you look at the lifestyle of modern people, stress, lack of exercise, irregular life rhythm, unbalanced eating habits, smoking, etc. can easily deteriorate immunity, and various pollution is widespread in the living environment, which also causes deterioration of immunity. many. Therefore, modern people should try to increase immunity. As such, studies on natural products to improve immunity (eg, Patent Document 1) have been conducted, but sufficient studies have not been conducted on thawed blood, and in particular, the effect of enhancing innate immunity using thawed blood extracts has not been studied.

Korean Patent Publication No. 10-2009-0126681

One aspect relates to a pharmaceutical composition for enhancing congenital immunity, comprising a hot water extract (Kalopanacis Cortex) as an active ingredient.

Another aspect relates to a food composition for enhancing congenital immunity, comprising a hot water extract of Haedongpi (Kalopanacis Cortex) as an active ingredient.

Another aspect relates to a feed composition for enhancing congenital immunity, comprising a hot water extract of Haedongpi (Kalopanacis Cortex) as an active ingredient.

Another aspect of the present invention relates to a method for enhancing congenital immunity of an animal, characterized in that a hot water extract of Kalopanacis Cortex is administered to an animal in need of enhancement of innate immunity.

Another aspect relates to a manufacturing method for obtaining a hot water extract of Kalopanacis Cortex.

One aspect of the present invention provides a pharmaceutical composition for enhancing congenital immunity, comprising a hot water extract (Kalopanacis Cortex) as an active ingredient.

Another aspect of the present invention provides a food composition for enhancing congenital immunity, comprising a hot water extract of Kalopanacis Cortex as an active ingredient.

Another aspect of the present invention provides a feed composition for enhancing congenital immunity, comprising a hot water extract of Kalopanacis Cortex as an active ingredient.

Another aspect of the present invention provides a method for enhancing congenital immunity in animals, characterized by administering a hot water extract (Kalopanacis Cortex) to an animal in need of enhancing innate immunity.

Another aspect provides a method for obtaining a hot water extract of Kalopanacis Cortex.

The composition according to one aspect confirmed the immune-enhancing action of the thawed blood hot water extract using macrophages, which are representative immune cells. The composition for enhancement of innate immunity, including the Haedongpi hot water extract as an active ingredient, can exhibit innate immunity-enhancing effects and be effectively used for prevention, improvement and treatment of various bacterial and viral infectious diseases. Therefore, it can be used as a drug for the prevention and treatment of diseases, including various bacterial and viral infectious diseases.

In addition, since the composition for enhancing immunity containing the extract of hot water from thawed skin does not cause any toxicity or side effects, it can be safely used even for a long period of time for prevention purposes. Therefore, it can be used as a health functional food for the purpose of preventing and improving disease for improving and regulating immunity of a patient with reduced immunity or suppressed immunity, and can be used as various food materials such as dairy products and food and beverage.

In addition, since the composition containing the extract of hot water from the thawing skin can effectively and safely enhance the immunity of the animal by administering it to the animal necessary for enhancing innate immunity, it is widely used as a feed composition for immunity enhancement for the purpose of strengthening the anti-disease of the animal. Can be.

1 is a graph showing the effect on the cytotoxicity of the extract of Haedongpi hot water.
Figure 2 is a graph showing the effect on the cytotoxicity of 80% ethanol extract of haedongpi.
Figure 3 is a graph showing the effect on the production of nitrogen oxides of Haedongpi hot water extract.
4 is a graph showing the effect of 80% ethanol extract of hadongpi on nitric oxide production.
Figure 5 is a graph showing the effect on the expression level of the immune-related enzymes of Haedongpi hot water extract.
6 is a graph showing the effect of the hot water extract of Haedongpi on the production of cytokines.
7 is a graph showing the effect on the phagocytosis of Haedongpi hot water extract.

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

All technical terms used in the present invention, unless defined otherwise, are used in the sense as commonly understood by those skilled in the art in the relevant field of the present invention. In addition, although a preferred method or sample is described herein, similar or equivalent ones are included in the scope of the present invention. The contents of all publications described by reference herein are incorporated herein by reference in their entirety.

An aspect of the present invention provides a composition for enhancing innate immunity, comprising a hot water extract (Kalopanacis Cortex) as an active ingredient. The composition comprising the extract of haedongpi-may help to improve immunity and may enhance innate immunity.

The haedongpi (海桐 皮) (abbreviation name: Kalopanacis Cortex) (English name: Kalopanax Bark) refers to the stem bark of the Kalopanax pictus Nakai belonging to the Araliaceae family, and is also referred to as automatic blood (刺桐 皮). The decoction may be used by drying the skin, and may be an outpost, a leaf, a flower, a stem, a bud, or a combination thereof, and a dried outpost, a leaf, a flower, a stem, a bud, or the like. The thawed skin may be, for example, collected in early summer, scraped out thorns, and then dried. The thawed skin may be finely divided. In the examples described in the present specification, Haedong Phi, which was imported and manufactured by Hansol Pharmaceutical Co., Ltd. and sold in Herb Story, was purchased and used.

As used herein, the term "extract", regardless of the extraction method, extraction solvent, or form of the extract, can be obtained by a general extraction method in the art with a broad concept including substances obtained by extracting the components of natural products.

In one embodiment, the solvent for obtaining the defrosting extract may be extracted using a hydrophilic solvent (hydrophilic solvent). The hydrophilic solvent may be water, C ₁ to C ₁₀ alcohol, or a mixture thereof. In one embodiment, the extraction solvent of the defrosted blood extract may be water, a lower alcohol of C ₁ to C ₄ or a mixture thereof, preferably water as an extraction solvent.

In one embodiment, the solvent for obtaining the extract of thawed skin may use a hydrophilic solvent having a volume of 5 to 20 times, specifically 5 to 10 times the volume corresponding to 1 kg of thawed skin powder.

In one embodiment, the defrosted hot water extract can be obtained by adding hot water of 80 to 95 ° C, specifically 80 to 95 ° C, and more specifically 80 to 90 ° C. In one embodiment, the hot water is 80 ° C or more and less than 100 ° C, specifically 80 to 95 ° C, more specifically, 80 to 90 ° C includes distilled water, purified water or deionized water. The extract can be extracted to a point where about 1/2 of the initial volume is added after adding hot water. For example, the extraction time can be obtained by extracting for 1 to 10 hours, specifically 2 to 8 hours.

In another embodiment, the defrosted alcohol extract may be obtained by adding alcohol, for example, ethanol, to the defrosted blood, and reflux extraction and concentration under reduced pressure. The ethanol may be 50% (v / v) to 99% (v / v) aqueous solution, such as 80% (v / v) aqueous solution.

In one embodiment, the thawed skin extract may be crude extract, fraction, or a combination thereof. The crude extract is obtained by contacting the thawed skin with an extraction solvent, and contains a specific component, and is not separated. The fraction refers to a substance containing a specific component separated from the crude extract. The separation may be separation, chromatography or filtration using an organic solvent. The chromatography may be ion exchange chromatography, affinity chromatography, size exclusion chromatography, HPLC, or a combination thereof.

The extraction may be that the thawed skin is added to the extraction solvent and incubated. The thawed skin may be shredded or pulverized to have micronized particles. The thawed skin may be dried or washed. The incubation may be performed without stirring or stirring at room temperature to reflux temperature. The incubation temperature can be appropriately selected depending on the solvent selected. For example, it may be room temperature to reflux temperature, 30 ° C to reflux temperature, 40 ° C to reflux temperature, 50 ° C to reflux temperature, 60 ° C to reflux temperature, or 65 ° C to reflux temperature. The extraction time can be appropriately selected according to the extraction conditions. The extraction time may be 2 to 4 hours, for example, 2.5 to 3.5 hours. The extraction solvent may be 5 to 20 times, for example, 5 to 15 times, or 5 to 10 times the thawed skin. The extraction may be one or more times, for example, 1 to 5 times, 1 to 4 times, 1 to 3 times, 2 to 5 times, or 2 to 4 times. The obtained crude extract can be filtered, concentrated and dried. The drying may be dried under reduced pressure.

In one embodiment, the extract may be extracted under ultrasonic irradiation. The ultrasonic irradiation is room temperature, 10 to 25 ° C, 10 to 30 ° C, 10 to 40 ° C, 10 to 45 ° C, 10 to 50 ° C, 10 to 55 ° C, 10 to 60 ° C, 10 to 65 ° C, or 10 to 70 ° C It may be carried out at ℃. The irradiation time is 10 to 30 minutes. 10 minutes to 1 hour, 10 minutes to 2 hours, 10 minutes to 3 hours, 10 minutes to 4 hours, 10 minutes to 5 hours, 10 minutes to 6 hours, 10 minutes to 9 hours, 10 minutes to 12 hours, or 10 It may be performed for minutes to 24 hours. The ultrasonic irradiation may be performed in a sealed container.

In one embodiment, the extract may be further extracted using an organic solvent. The organic solvent may be ethyl acetate, hexane, dichloromethane, butanol, acetone, acetonitrile, or a combination thereof. The extraction may further include the step of chromatography the crude extract. The chromatography may be HPLC. The HPLC can be semi-preparative HPLC.

In one embodiment, the thawed skin hot water extract may be an extract obtained by adding 5 to 10 times the volume of distilled water to 1 kg of powdered thawed skin powder and heating at 80 to 90 ° C. until half the initial volume. The extract may then be obtained by filtering the powder using filter paper, followed by drying under reduced pressure and lyophilization.

As used herein, the term "enhanced innate immunity" is to enhance the natural immunity that reacts nonspecifically to the antigen, and in particular, it may be to improve the defense function by macrophages (macrophage) responsible for phagocytosis. In one embodiment, the composition has an effect of inhibiting infection and proliferation of various bacteria or viruses by increasing macrophage-mediated innate immunity, and thus may be a method for preventing, improving, and treating various infectious disease pathogens.

Nitrogen oxide (NO), iNOS, COX-2, cytokines (IL-1, IL-6, IL-12, and TNF-α, etc.), phagocytosis, complement system, NK Cells, etc. are presented (Health functional food functional evaluation guide, Ministry of Food and Drug Safety, Food and Drug Safety Evaluation Institute).

In one embodiment, the composition comprising the defrosted hot water extract increases the ability to produce nitric oxide (NO) associated with an immune response. In the past, in relation to nitrogen oxides, NO has been considered as a harmful gas that is generated by combustion of fuel and causes air pollution. However, more than 20 years ago, it is known that NO occurs in the human body and has various physiological or pathological functions. Important functions of NO are known as immunoregulation, cytotoxicity, relaxation of blood vessels through relaxation of smooth muscle, inhibition of platelet aggregation, and neurotransmission.

In the human body, NO is produced by nitric oxide synthase (NOS), and specifically, L-arginine (amino acid) is produced by oxidation by NOS. There are three isotypes of NOS, specifically eNOS (endothelial NOS) and nNOS (neuronal NOS), and secretible iNOS (inducible NOS). nNOS and eNOS are generally expressed as constitutive NOS (cNOS) because they are always expressed, and iNOS is called inducible NOS (iNOS) because they are transcribed and expressed only when there is a specific stimulus.

Unlike cNOS (nNOS, eNOS), iNOS (inducible NOS) is not normally expressed, but is expressed by transcriptional regulation when there are stimuli such as cytokines. It is known that iNOS does not rely on calcium-calmodulin and produces large amounts of NO for a long time (hours to days) once expressed. iNOS was found in macrophages, and can be expressed in macrophages, hepatocytes, osteoclasts, dendritic cells, astrocytes, epithelial cells, etc., and is known to have anti-cancer and anti-microbial effects in the immune system. NO produced in iNOS is mainly involved in immune response regulation and cytotoxicity. NO is produced from macrophage activity, and this NO is a representative example of cytotoxicity in tumor cells, lymphocytes, microorganisms, and the like. As a ligand for inducing transcription of iNOS, IFNγ, TNFα, IL-1, lipopolysaccharide (LPS) (bacterial endotoxin), CpG motif, and the like are known.

Prostaglandin performs various physiological functions such as immune response, hormone excretion, vasodilation, and blood cell speed, moisture and sleep control, as well as maintaining neuronal function. Prostaglandin E ₂ (PGE ₂ ) is known to promote a Th2 type immune response, as well as a role as a mediator of a well-known inflammatory response. Cyclooxygenase-2 (COX-2) is one of the coenzymes of COX and is an important enzyme for prostaglandin synthesis such as PGE ₂ .

Macrophage (macrophage) is the main cell responsible for innate immunity. When an antigen invades, it feeds or secretes toxins, destroying, removing the antigen, and delivering the antigen to lymphocytes, causing an immune response. In particular, phagocytosis is one of the essential functions of macrophages and refers to the action of feeding external antigens. This action neutralizes the invading antigen with a non-specific immune response, while at the same time presenting a portion of the antigen to other immune cells to drive subsequent immune responses. Macrophages have many lysosomes, which contain acidic hydrolase and peroxidase, and actively phagocytose microorganisms and tumor cells. In addition, macrophages produce cytokines such as complement components, interferon, interleukin-1 and tumor necrosis factor, have cytokine receptors, and function can be enhanced by various cytokines produced from T-cells.

Cytokine (cytokine) is a term that refers to a protein that is secreted by immune cells. Cytokines can secrete from cells and then affect other or secreted cells themselves. In other words, it induces the proliferation of macrophages or promotes differentiation of secretory cells themselves. Representative cytokines that activate macrophages include IL-1β, IL-6, IL-12, and TNF-α.

For example, IL-1β activates T cells by allowing T cells to increase production of several cytokines, such as IL-2 or its receptors. In addition, IL-1β is known to proliferate B cells, mature B cells, and allow macrophages to secrete more IL-1, IL-6, IL-8, TNF, GM-CSF, and PGE2. . IL-6 affects adaptive immunity and is known to be involved in the proliferation of B cell lineages and secretion of antibodies. IL-12 acts on NK cells and is known to increase the amount of TH1 cells. TNF-α is known to increase the antiviral activity of macrophages.

The term "treatment" as used herein means to improve the pathological symptoms affecting the host. The term "improvement" means any action that at least reduces the extent of symptoms, such as symptoms, related to the condition being treated with the administration of said food composition, such as a dietary supplement. The term "prevention" refers to all actions that indicate immunity enhancing activity by administration (or application) of the composition and inhibit or delay the occurrence of disease caused by bacteria or viruses.

As used herein, the term "included as an active ingredient" means to include an amount sufficient to achieve a congenital immunity-enhancing efficacy or activity using an extract of Haedongpi. The present invention is a composition comprising an extract extracted from a natural plant material, haedongpi, or a compound separated therefrom, so that there is no adverse effect on the human body even when administered in excess, the person skilled in the art has an upper or lower quantitative limit of the thawed blood extract contained in the composition or a compound isolated therefrom. Can be carried out by selecting within an appropriate range.

The composition may include the defrosted hot water extract in an amount effective for treatment, prevention or improvement purposes. The effective amount can be appropriately selected depending on the cell or individual selected by those skilled in the art. The effective amount is 0.1 mg to 1,000 mg per composition, for example, 0.1 mg to 500 mg, 0.1 mg to 100 mg, 0.1 mg to 50 mg, 0.1 mg to 25 mg, 1 mg to 1,000 mg, 1 mg to 500 mg, 1 mg to 100 mg, 1 mg to 50 mg, 1 mg to 25 mg, 5 mg to 1,000 mg, 5 mg to 500 mg, 5 mg to 100 mg, 5 mg to 50 mg, 5 mg to 25 mg, 10 mg To 1,000 mg, 10 mg to 500 mg, 10 mg to 100 mg, 10 mg to 50 mg, or 10 mg to 25 mg.

The composition may include the thawed skin hot water extract in an effective concentration for treatment, prevention or improvement purposes. The effective concentration can be appropriately selected depending on the cell or individual selected by those skilled in the art. The effective concentration includes 1 to 80% by weight, for example, 5 to 70% by weight, 10 to 50%, 20 to 40% by weight, or 5 to 20% by weight, based on the total weight of the composition, including thawed blood hot water extract It may be.

The composition may be a pharmaceutical composition, food composition or feed composition. The composition may further include a pharmaceutically, food, or animal-acceptable excipient or carrier. The composition may be added as an active ingredient to pharmaceuticals, health functional food or feed.

There are no particular restrictions on the type of food. Examples of foods to which the above substances can be added include various dairy products such as milk, yoplait, beverages, teas, drinks, alcoholic beverages and vitamin complexes, and snacks such as bread, chocolate, candy, confectionery, etc. All foods in the sense of phosphorus are included.

The composition may be used alone or in combination with other pharmaceutical, food or feed ingredients, and can be suitably used according to conventional methods. The mixing amount of the active ingredient can be appropriately determined according to the purpose of use (prevention, health or therapeutic treatment). In general, when preparing food or feed, the composition of the present invention is 15 parts by weight or less, 10 parts by weight or less, 1 to 15 parts by weight, 1 to 10 parts by weight, 1 to 5 parts by weight, based on the total weight, Or it may be added in an amount of 0.1 to 15 parts by weight. However, in the case of long-term intake for health and hygiene purposes or for health control purposes, the amount may be below the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range.

The pharmaceutical, food or feed composition may also include nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, sweeteners, preservatives, glycerin, It may contain additives such as alcohol, carbonic acid used in carbonated beverages, or a combination thereof. These additives may be appropriately selected from 0.01 to 0.1 parts by weight per 100 parts by weight of the composition.

The composition can be formulated in oral or parenteral dosage forms. Oral dosage forms can be granules, powders, liquids, tablets, capsules, dry syrups, or combinations thereof. The parenteral dosage form can be an injection or an external preparation for skin. The external skin preparation may be a cream, gel, ointment, skin emulsifier, skin suspension, transdermal delivery patch, drug containing bandage, lotion, or combinations thereof.

In one embodiment, administration of the composition can be administered by methods known in the art. Administration may be administered directly to an individual by any means, for example, by intravenous, intramuscular, oral, transdermal, mucosal, intranasal, intratracheal or subcutaneous administration. You can. The administration can be administered systemically or locally.

The composition may include a pharmaceutically acceptable diluent or carrier. The carrier may be an excipient, a disintegrant, a binder, a lubricant, or a combination thereof. The excipient may be microcrystalline cellulose, lactose, low-substituted hydroxycellulose, or a combination thereof. The disintegrant may be sodium starch glycolate, calcium monohydrogen phosphate anhydride, or a combination thereof. The binder may be polyvinylpyrrolidone, low-substituted hydroxypropyl cellulose, hydroxypropyl cellulose, or a combination thereof. The lubricant may be magnesium stearate, silicon dioxide, talc, or a combination thereof.

Another aspect provides a method of enhancing the innate immunity of an individual, including the step of administering the composition to the individual, and preventing, ameliorating, or treating diseases caused by reduced immunity. In one embodiment, a method of enhancing innate immunity is provided, comprising administering a hot water extract (Kalopanacis Cortex) to a mammal in need of enhanced innate immunity. In another embodiment, a method for enhancing congenital immunity of an animal is provided by administering a hot water extract of Kalopanacis Cortex to an animal in need of enhancing innate immunity. The animals include livestock such as mammals other than humans, such as cattle, horses, pigs, dogs, sheep, goats, or cats.

Another aspect provides a method for obtaining a composition comprising a hot water extract from Haedongpi (Kalopanacis Cortex). In one embodiment, the method of preparing a composition comprising the defrosted hot water extract,

A step of obtaining a solution of thawed blood by adding distilled water to the powder (Kalopanacis Cortex);

Heating the solution at a temperature of 80 to 120 ° C;

A first extraction step of concentrating the heated solution under reduced pressure to obtain a first extract; And

It may include a second extraction step of obtaining a second extract by freeze-drying the first extract.

The step of obtaining the thawed blood solution may be obtained by adding 5 to 20 times the volume of distilled water having a volume of 1 kg of thawed blood powder.

The heating step of the solution may be performed by heating the thawed blood solution at a temperature of 80 to 120 ° C, for example, 80 to 90 ° C.

The step of obtaining the first extract may be performed by concentrating the heated solution under reduced pressure at a temperature of 40 ° C. or less and a pressure condition of 0 to 100 mbar.

The step of obtaining the second extract may be performed by freeze-drying the first extract at a temperature of -120 ° C or less for 24 hours or less, for example, at a temperature of -120 ° C for 18 to 24 hours.

The manufacturing method is various types using a solid stationary phase, such as standard methods currently used in the field, such as column / flash, vacuum and high performance liquid chromatography (CC, VLC, HPLC). The method may further include using solid-liquid chromatography or counter-current chromatography (CCC).

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1: Preparation of Haedongpi hot water extract

Haedongpi (Kalopanacis Cortex) (made in China) was imported and manufactured by Hansol Pharmaceutical Co., Ltd., and then pulverized and powdered 1 kg after purchasing the dried products sold by Herb Story. To 1 kg of the powdered thawed skin powder, distilled water of 5 to 10 times the corresponding volume was added as a solvent and heated at a temperature of 80 to 90 ° C. until half of the initial volume. Subsequently, the powder was filtered using filter paper, and then dried under reduced pressure and then freeze-dried to obtain an extract.

Comparative Example 1: Preparation of 80% ethanol extract of Haedongpi

It was prepared in the same manner as in Example 1, except that 80% ethanol was used as a solvent to obtain 80% ethanol extract of thawed skin.

In the following experimental example, an immune response experiment was performed using Raw 264.7 cells, which are macrophages of mice. Cells were cultured using DMEM medium containing 10% FBS, and 96 well or 6 well plates were used for the experiment. Statistical processing was analyzed using Student t-test, and statistical significance was recognized when the p value was less than 0.05. In evaluating the efficacy of increasing immunity excluding cytotoxicity, the first control group was compared (#) with the 1g / mL lipopolysaccharide (LPS) treated group as a positive control group and the DMSO treated group without the negative control group treated with the extract, followed by the negative control group and the extract treatment. Groups were compared (*).

Experimental Example 1. Cytotoxicity test

Cytotoxicity experiments were performed using the MTT method using the thawed skin extract prepared in Example 1 and Comparative Example 1. 2 x 10 ⁵ cells were dispensed into each well with 100 uL medium and attached to a 96 well plate for 20 to 24 hours. Thereafter, the medium was exchanged for serum-free (starvation process). After 2 hours, treated with hot water extract of thawed blood or 80% ethanol extract of thawed blood to the final concentration, and after 20 to 22 hours, 5mg / mL MTT reagent (3- (4,5-dimethylthiaxo-2-yl) -2,5-diphenyl tetrazolium bromide) was added 10 uL. After that, it was left for 1 to 2 hours and the resulting formazan was dissolved in dimethyl sulfoxide (DMSO) to measure absorbance at 540 nm. The negative control group, which was not treated with the thawed blood hot water extract or the thawed blood 80% ethanol extract, was converted to 100% to express the cell viability of each group, and when it was 80% or less, it was judged to be toxic.

1 is a graph showing the effect on the cytotoxicity of the extract of Haedongpi hot water. As in FIG. 1, the thawed blood hot water extract did not show toxicity at all concentrations treated.

Figure 2 is a graph showing the effect on the cytotoxicity of 80% ethanol extract of haedongpi. As shown in Figure 2, the 80% ethanol extract of thawed blood did not show cytotoxicity at the corresponding concentration of 20 or 100 ug / mL.

Experimental Example 2. Nitric oxide (NO) measurement test

Using the extract of thawed skin prepared in Example 1 and Comparative Example 1, a test was performed to measure the amount of nitric oxide produced in the medium using the Greries method. 2 x 10 ⁵ cells were dispensed into each well with 100 uL medium and attached to a 96 well plate for 20 to 24 hours. After that, the medium was exchanged with no fetal bovine serum (FBS) (starvation process). After 2 hours, the thawed blood hot water extract or the thawed blood 80% ethanol extract was treated to the final concentration, and after 20 to 22 hours, 50 uL of the medium was taken and reacted with the same amount of Griess reagent. After 10-15 minutes, absorbance was measured at 540 nm. Lipid polysaccharide (LPS) of 1 ug / mL was used as a positive control, and the negative control treated with DMSO without treating the defrosted hot water extract or 80% ethanol extract of defrosted blood was converted to 100% to show nitric oxide generation ability. .

Figure 3 is a graph showing the effect on the production of nitrogen oxides of Haedongpi hot water extract. 3, it was confirmed that the production of nitrogen oxides was increased at all concentrations of the thawed skin hot water extract.

4 is a graph showing the effect of 80% ethanol extract of hadongpi on nitric oxide production. As shown in FIG. 7, it was confirmed that the 80% ethanol extract of defrosted blood did not increase the production of nitrogen oxide at all concentrations treated, and thus did not affect the amount of nitrogen oxide produced in the medium.

Experimental Example 3. Measurement of iNOS and COX-2 expression levels related to the immune response

Using the extract of thawed blood prepared in Example 1 and Comparative Example 1, Western blot was used to measure the expression level of enzymes that produce immune-related substances. Representative enzymes, iNOS and COX-2, were selected for the immune response. Cells of 2 x 10 ⁶ were dispensed into each well with 2 mL medium and attached to 6 well plates for 20 to 24 hours. Thereafter, the medium was exchanged for serum-free (starvation process). After 2 hours, the thawed skin hydrothermal extract was treated to a final concentration, and after 20 to 22 hours, the attached cells were scraped using Laemmli buffer solution, and then the expression level of the enzymes was measured using a Western blot method. Lipid polysaccharide (LPS) of 1 ug / mL was used as a positive control, and the negative control that was not treated with the thawed blood hot water extract was converted to 100% to show the expression level of enzymes.

5 is a graph showing the effect on the expression level of the immune-reactive enzymes of Haedongpi hot water extract. As shown in FIG. 5, the thawed blood hot water extract increased the expression level of iNOS and COX-2, which are immunoreactive enzymes, in a concentration gradient.

Experimental Example 4. Immunity-related cytokines IL-1β, IL-6, IL-12, and TNF-α expression level measurement test

The expression level of cytokines related to the immune response was measured using the enzyme-linked immunosorbent assay (ELISA) method using the thawed blood extracts prepared in Example 1 and Comparative Example 1. As cytokines to be measured, representative cytokines IL-1β, IL-6, IL-12, and TNF-α were selected in relation to the immune response. Cells of 2 x 10 ⁶ were dispensed into each well with 2 mL medium and attached to 6 well plates for 20 to 24 hours. Thereafter, the medium was exchanged for serum-free (starvation process). After 2 hours, the thawed skin hydrothermal extract was treated to the final concentration, and after 20 to 22 hours, the medium was taken, and the expression levels of the selected cytokines IL-1β, IL-6, IL-12, and TNF-α were used by ELISA. Was measured. Lipid polysaccharide (LPS) of 1 ug / mL was used as a positive control, and the negative control not treated with the thawed blood hot water extract was converted to 100% to represent the expression level of cytokines.

6 is a graph showing the effect of the hot water extract of Haedongpi on the production of cytokines. As shown in FIG. 6, the thawed blood hot water extract increased the expression level of the immunoreactive cytokines IL-1β, IL-6, IL-12, and TNF-α in a gradient.

Experimental Example 5. Macrophage (macrophage) phagocytosis test

Macrophage phagocytosis was measured using the thawed blood extract prepared in Example 1 and Comparative Example 1. Specifically, the effect of thawed blood extract on macrophage phagocytosis was measured using a kit (CytoSlelect ^TM 96-well phagocytosis assay kit, Cell Biolabs Inc.) using zymosan.Zymosan is a polysaccharide extracted from yeast. In the body, it acts as an antigen to induce phagocytosis of macrophages: 2 x 10 ⁵ cells in a 96 well plate are dispensed into each well with 100 uL medium and attached for 20 to 24 hours. ) Was added and left for 2 hours, and then experiments were performed according to the protocol provided by the kit.The degree of macrophage phagocytosis was confirmed by measuring the amount of phagocytosed zymosan and absorbance was performed at 405 nm.

7 is a graph showing the effect on the phagocytosis of Haedongpi hot water extract. As shown in FIG. 7, the extract of hadongpi increased the phagocytosis in a concentration gradient.

This study is the result of the research conducted by the Ministry of Trade, Industry and Energy and the Korea Institute of Industrial Technology, "Support Project for Regional Industrial Bases" (Task No .: R0004851).

Those skilled in the art to which the present invention pertains will understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an explanatory point of view rather than a restrictive point of view. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the equivalent range should be interpreted as being included in the present invention.

Claims (9)

Haedongpi (Kalopanacis Cortex) A pharmaceutical composition for enhancing congenital immunity comprising a hot water extract as an active ingredient. The method according to claim 1, wherein the hot water is a pharmaceutical composition comprising 80 to 120 ℃ water. The method according to claim 1, Pharmaceutical composition comprising 1 to 80% by weight of defrosted hot water extract based on the total weight of the composition. Haedongpi (Kalopanacis Cortex) A food composition for enhancing congenital immunity comprising a hot water extract as an active ingredient. The food composition according to claim 4, wherein the hot water includes water at 80 to 120 ° C. The method according to claim 4, Food composition comprising 1 to 80% by weight of defrosted hot water extract based on the total weight of the composition. Haedongpi (Kalopanacis Cortex) A feed composition for enhancing congenital immunity comprising a hot water extract as an active ingredient. Haedongpi (Kalopanacis Cortex) method of enhancing the innate immunity of an animal, characterized by administering a hot water extract to an animal in need of enhancement of innate immunity. A step of obtaining a solution of thawed blood by adding distilled water to the powder (Kalopanacis Cortex);
Heating the solution at a temperature of 80 to 120 ° C;
A first extraction step of concentrating the heated solution under reduced pressure to obtain a first extract; And
Comprising a second extraction step of obtaining a second extract by freeze-drying the first extract, a method for preparing a pharmaceutical composition for enhancing innate immunity, comprising a hot water extract of Kadonganacis Cortex of claim 1.

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