KR102292679B1 - A composition for improving immunity comprising an effective ingredient of balloon flower extract having improved safety and immunity improving ability - Google Patents

Abstract

The present invention relates to a composition for enhancing immunity containing a Platycodon grandiflorus extract having improved safety and immunity. The composition for enhancing immunity provided in one aspect of the present invention contains the Platycodon grandiflorus extract produced under specific conditions as an active component, thereby having an effect of remarkably improving safety and immunity.

Description

A composition for improving immunity comprising an effective ingredient of balloon flower extract having improved safety and immunity improving ability in a stress environment, comprising a bellflower extract having improved safety and immunity as an active ingredient

It relates to a composition for enhancing immunity comprising, as an active ingredient, a bellflower extract having improved safety and immunity improvement in a stressful environment.

Immunity is a self-defense system that exists in the body, and it is a process in which the human body recognizes and removes and metabolizes various substances or living things that invade from the outside as foreign substances against itself.

However, the immune function protects itself from damage caused by external stimuli or invasion of pathogenic microorganisms, but may also damage its own tissues, such as an inflammatory response. It regulates changes in immune function to restore normalcy or reduces the range of changes, and is classified as suppression of immune function or enhancement of immune function.

Factors that affect immune function include genetic and environmental factors, age, gender, psychological stress, nutritional status, dietary habits, and disease. Health functional food having the function of regulating the immune function is divided into the function of enhancing the reduced immune function, the function of regulating the excessive immune function, and the function of regulating the autoimmune function of regulating the immune response to self-components.

When the immune function is lowered, the morbidity of various communicable diseases may increase. An infectious disease is a disease caused by a specific pathogen or a toxic substance of a pathogen, and the infectious pathogen spreads and infects humans, resulting in a final disease. Some examples of the infectious pathogens and diseases caused by them include animal parasites (such as malaria), spirochetes (such as leptospirosis), rickettsia (such as tsutsugamushi disease), fungi (such as candidiasis), chlamydia (such as trachoma), and bacteria (such as cholera). etc.), viruses (measles, SARS, AIDS, etc.).

Infants, cancer patients or the elderly, who are prone to weakened immunity, etc., have been seeking substances that can enhance immune responses in patients with diseases. That is, the concept that disease can be eradicated by restoring or enhancing immune reactivity by applying an immune enhancer has emerged. Immune enhancers are a method of regulating one or more components of a complex immune regulatory system through an indirect method in order to improve the immune response. .

In addition, when the immunomodulatory function is lowered, it may appear in various ways such as asthma, seasonal or perennial rhinitis, allergic sinusitis, conjunctivitis, food and drug allergy, atopic dermatitis, urticaria, allergy due to bee sting due to hypersensitivity reaction. .

In order to overcome the problem of disorders in immune function, various immune enhancers and therapeutic agents are used (Korea Patent 10-1895024),

There are side effects, and what is mainly marketed is to be taken for treatment rather than prevention, and there is a need for a composition for enhancing immunity suitable for prophylaxis, which can be taken for a long time without side effects.

An object in one aspect of the present invention, bellflower ( Platycodon grandiflorum ) by providing a method for extracting, to provide a method for producing a developed bellflower extract with an increased active ingredient compared to a general bellflower extract.

It is an object of another aspect of the present invention to provide a composition for enhancing immunity containing the bellflower extract prepared by the method for preparing the bellflower extract as an active ingredient.

Another object of the present invention is to provide a functional food for enhancing immunity comprising the composition for enhancing immunity.

Another object of the present invention is to provide a pharmaceutical composition for enhancing immunity comprising the composition for enhancing immunity.

In order to achieve the above object,

One aspect of the present invention, bellflower ( Platycodon grandiflorum ) at 50 ° C. to 70 ° C. for 40 hours to 60 hours, a primary treatment step comprising the step of drying; and

A secondary treatment step comprising adding water 8 to 12 times the weight of the raw material to the raw material prepared in the first treatment step, and then leaving it at 50° C. to 70° C. for 100 to 150 minutes; It provides a method for producing a bellflower extract comprising a.

In addition, another aspect of the present invention,

It provides a composition for enhancing immunity containing the bellflower extract prepared by the method for preparing the bellflower extract as an active ingredient.

Furthermore, another aspect of the present invention provides a functional food for enhancing immunity comprising the composition for enhancing immunity.

In addition, another aspect of the present invention provides a pharmaceutical composition for enhancing immunity comprising the composition for enhancing immunity.

The bellflower extract prepared by the method for producing a bellflower extract provided in one aspect of the present invention and a composition for enhancing immunity containing the same as an active ingredient have the effect of significantly improving safety and immunity in an environment in which immunity is lowered by stress. .

1 is a view showing the results of evaluating the toxicity of the bellflower extract prepared in the control and Example 1 (DD) and Comparative Example 1 (ND) to immune-related macrophages.
2 is a graph showing the results of evaluating the safety of the extracts prepared in Control (NC), Example 1 (DD), and Comparative Example 1 (ND) in immunocompromised mice through weight change.
3 is a graph showing the results of evaluating the effect of a bellflower extract according to a control group (NC), Example 1 (DD), and Comparative Example 1 (ND) on the level of immunoglobulin A (IgA) in blood.
4 is a graph showing the results of evaluating the effect of the bellflower extract according to the control (NC), Example 1 (DD), and Comparative Example 1 (ND) on the level of immunoglobulin G (IgG) in the blood.
5 is a graph showing the results of evaluating the effect of the bellflower extract according to the control (NC), Example 1 (DD), and Comparative Example 1 (ND) on the level of the blood cytokine TNF-α.
6 is a graph showing the results of evaluating the effect of the bellflower extract according to the control (NC), Example 1 (DD), and Comparative Example 1 (ND) on the level of the cytokine IL-6 in the blood.
7 is a view showing the results of evaluating the effect of the bellflower extract according to the control (NC), Example 1 (DD), and Comparative Example 1 (ND) on the proliferation of spleen immune cells.
8 is a graph showing the results of evaluating the effect of bellflower extract according to the control (NC), Example 1 (DD), and Comparative Example 1 (ND) on splenic NK cell activity.

Hereinafter, the present invention will be described in detail.

One aspect of the present invention is

Bellflower ( Platycodon) grandiflorum ) at 50 ° C. to 70 ° C. for 40 hours to 60 hours, a primary treatment step comprising the step of drying; and

A secondary treatment step comprising adding water 8 to 12 times the weight of the raw material to the raw material prepared in the first treatment step, and then leaving it at 50° C. to 70° C. for 100 to 150 minutes; It provides a method for producing a bellflower extract comprising a.

The bellflower extract prepared by the above method may exhibit superior safety and immunity improvement as the production process of bellflower is specified as compared to the conventional bellflower extract.

At this time, the hot air drying may be performed at a temperature condition of 40 ° C to 80 ° C, and the temperature condition may be 45 ° C to 75 ° C, 50 ° C to 70 ° C, 55 ° C to 65 ° C, Preferably, it may be dried with hot air at a temperature of 60 °C.

In addition, the hot air drying can be done for 40 hours to 60 hours, the time range may be 42 hours to 55 hours, 44 hours to 53 hours, 45 hours to 50 hours, 46 hours to It may be 50 hours, preferably hot air drying for 48 hours.

After the hot air drying step, it may be preferable to additionally go through a step of filtering with a 30 mesh mesh, and the filtering step after the hot air drying step may be a primary treatment step.

In the secondary treatment step, 8 to 12 times the weight of the raw material may be added to the raw material prepared in the first treatment step, and water or distilled water may be added. The water may be added in a mass or volume of 8 to 12 times, and may be added in 9 to 11 times, preferably 10 times. For example, 100 mL of distilled water (10 times) may be added to 10 g of the raw material prepared in the first treatment step.

Thereafter, the method further includes the step of standing at a constant temperature and time condition, in which case the temperature may be 50° C. to 70° C., and preferably, it may be left at 60° C. The time may be left to stand for 100 minutes to 150 minutes, may be 110 minutes to 140 minutes, and preferably may be left for about 120 minutes.

_{The secondary treatment step may further include a step of extracting water, a C 1} to C ₂ lower alcohol or a mixture thereof with a solvent, once or twice, after the leaving step described above. In this case, when it further includes two times, it may further include one additional extraction step in succession after one extraction step. The description of the extraction steps below may be applied to one extraction step or to two consecutive extraction steps.

Of course, the extraction cycle may be repeated 2 to 10 times in a direction capable of enhancing the active ingredient of bellflower.

In this case, the extraction may be performed without limitation by a method of extracting a commonly known plant extract. As an example, extraction may be performed by ultrasound. As another example, the bellflower extract may extract the whole, or at least part of, the plant of bellflower using an extraction medium, for example, an inorganic or organic material in a gaseous or liquid phase, or a mixture thereof, but is not limited thereto. , as one embodiment, it can be obtained using a conventional extraction solvent, for example (a) anhydrous or hydrous lower alcohols having 1-4 carbon atoms (eg, methanol, ethanol, propanol, butanol, normal-propanol, iso-propanol) and normal-butanol), (b) a mixed solvent of the lower alcohol and water, (c) acetone, (d) ethyl acetate, (e) chloroform, (f) 1,3-butylene glycol, (g) It can be obtained by using hexane, (h) diethyl ether, (i) butyl acetate, or (j) water as an extraction solvent.

The extraction time may be 20 to 40 minutes, 25 to 35 minutes, and preferably, about 30 minutes.

The extraction solvent may be used in an amount of 5 to 25 times the volume of the raw material prepared in the first treatment step. For example, the first extraction may be used at 5 to 15 volumes, 8 to 13 volumes, and preferably about 10 volumes. For example, in the case of using 10 g of the raw material obtained in the first treatment step, it can be extracted by adding 100 mL of a solvent. Also, for example, another single extraction may be used at 15 to 25 volumes, 17 to 23 volumes, 18 to 22 volumes, preferably about 20 It can also be used as drainage volume. For example, in the case of using 10 g of the raw material obtained in the first treatment step, 200 mL of a solvent may be added to extract.

The solvent may be, for example, ethanol, and the concentration of ethanol may be 40 to 99% (v/v), for example, 50% (v/v) or 99% (v/v). . Also, for example, when the extraction is performed twice in succession, the first extraction uses ethanol at a concentration of about 95% (v/v) to 99% (v/v), and the next extraction is about 45%. Ethanol at a concentration of % (v/v) to 55% (v/v) may be used, and most preferably, the first extraction uses ethanol at a concentration of about 99% (v/v), followed by two rounds of extraction. Extraction may be performed using ethanol at a concentration of about 50% (v/v).

It would be preferable to undergo a centrifugation step after the extraction step. For example, if the extraction is performed twice in succession, centrifugation may be performed after the first extraction, and then centrifugation may be performed after the next extraction. The centrifugation may be performed under commonly adopted centrifugation conditions, for example, centrifugation may be performed at about 3000 rpm for about 10 minutes. After the final centrifugation, it is preferable to filter to obtain the desired extract.

The final step of the method for producing the developed bellflower extract with enhanced active ingredient may further include concentrating the extract under reduced pressure and freeze-drying.

Another aspect of the present invention is

It provides a composition for enhancing immunity containing the bellflower extract prepared by the method for preparing the bellflower extract as an active ingredient.

The composition for enhancing immunity, further comprising one or more of carriers, diluents, excipients, and additives, may be used in a form selected from the group consisting of tablets, pills, powders, granules, powders, capsules, and liquid formulations. .

As additives that may be further included in the composition for enhancing immunity, natural carbohydrates, flavoring agents, nutrients, vitamins, minerals (electrolytes), flavoring agents (synthetic flavoring agents, natural flavoring agents, etc.), coloring agents, fillers (cheese, chocolate, etc.) ), facic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, antioxidants, glycerin, alcohols, carbonation agents, and at least one component selected from the group consisting of pulp can

Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; disaccharides such as maltose, sucrose and the like; and polysaccharides such as conventional sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. As the flavoring agent, natural flavoring agents (taumatine, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used.

In addition to the above, the composition for enhancing immunity according to the present invention includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, coloring agents and thickening agents (cheese, chocolate, etc.), pectic acid and salts thereof , alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. In addition, the composition according to the present invention may contain the pulp for the production of natural fruit juices and vegetable beverages. These components may be used independently or in combination.

Specific examples of the carrier, excipient, diluent and additive include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, erythritol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium phosphate, calcium Silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, polyvinylpyrrolidone, methylcellulose, water, sugar syrup, methylcellulose, methyl hydroxy benzoate, propyl hydroxy benzoate, talc, magnesium stearate and at least one selected from the group consisting of mineral oil is preferably used.

When formulating the composition according to the present invention, it is prepared using a diluent or excipient such as a filler, extender, binder, wetting agent, disintegrant, surfactant, etc. commonly used.

The composition for enhancing immunity may enhance immunity by regulating cytokines in the intestine of animals, wherein the cytokine may be interleukin-6 (IL-6). In addition, the composition for enhancing immunity may enhance immunity by regulating the level of TNF-α. Furthermore, the composition for enhancing immunity may enhance immunity by improving the level of immunoglobulin (IgG or IgA) in the blood. In addition, the composition for enhancing immunity may enhance immunity by improving the proliferation of spleen immune cells. This effect is supported by the experimental examples to be described later.

Another aspect of the present invention provides a functional food for enhancing immunity comprising the composition for enhancing immunity and a pharmaceutical composition for enhancing immunity.

When the composition for enhancing immunity is used as a functional food for enhancing immunity,

The immune enhancing composition may be included in an amount of 0.0001 to 90% by weight, preferably 1 to 60% by weight, and even more preferably 30 to 50% by weight based on the total weight of the functional food composition for enhancing immunity. Foods to which the composition for enhancing immunity of the present invention can be added include various foods, powders, granules, tablets, capsules, syrups, beverages, gums, teas, vitamin complexes, and health functional foods.

When the composition for enhancing immunity is used as a pharmaceutical composition for enhancing immunity,

The content of the composition for enhancing immunity according to the present invention as an active ingredient in the pharmaceutical composition can be appropriately adjusted depending on the type and purpose of use, the patient's condition, the type and severity of symptoms, and the like, and according to an embodiment of the present invention, The pharmaceutical composition may include, but is not limited to, 1 to 100 mg/kg of body weight in an effective amount on a daily basis of the composition for enhancing immunity.

The composition according to the present invention relates to the enhancement of immune function, refers to a composition that contributes to enhancing the immune function of the human body that has been lowered, and may also include the case of enhancing normal immune function.

Biomarkers for confirming the enhancement function of these immune functions are,

In the case of innate immunity, there are NK cells, cytokines (IFN-γ, IL-1β, TNF-α, IL-6, IL-12), phagocytic ability, NO, iNOS, COX-2, complement system, etc. In the case of adaptive immunity, Cytokines (IFN-γ, TNF-α, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13), lymphocyte subpopulation ratio, immunoglobulin, etc. . In addition, in innate immunity and adaptive immunity, there are common cell viability (increase in the number of immune cells), splenocyte proliferation, and tissue weight (spleen, thymus). The details of each biomarker are as follows.

The cell viability refers to the enhancement of immune function as the number of immune cells increases.

NO (nitric oxide) is produced by the action of iNOS (inducible nitric oxide synthase) enzymes in macrophages, and the generated NO inhibits the transformation of the preyed microorganism (pathogen) components and the action of enzymes containing Fe-S to exhibit antimicrobial action. In addition, NO is secreted from activated macrophages or neutrophils to kill extracellular pathogens. Inducible nitric oxide synthase (iNOS) oxidizes L-arginine to produce L-citrulline and NO, and iNOS is expressed in almost all cells by stimuli such as cytokines. COX-2 (Cyclooxygenase-2) is an enzyme that converts arachidonic acid to prostaglandin E2 (PGE2). It is expressed by inflammation or immune stimulation, and is induced in response to various cytokine growth factors. Phagocytosis, also called phagocytosis, is an action in which macrophages such as white blood cells phagocytose to remove bacteria or foreign substances. Macrophages have receptors for various molecular substances, and these receptors interact with ligands and microorganisms when they meet It increases the phagocytic ability of the pathogen by causing a reaction.

Cytokine is a protein that mediates signal transduction between cells and includes interleukin (IL), interferon (IFN), and tumor necrosis factors (TNF). Mainly produced by activated macrophages or lymphocytes, the types of cytokines secreted by TH1-helper T lymphocytes are IL-1, IL-2, IL-12, IL-15, IL-18, IFN-ν, TNF- α and the like, which exhibit cytotoxicity as inflammatory cytokines and are involved in cell-mediated immune responses.

Lymphocyte subpopulation ratio is a white blood cell produced by the differentiation and maturation of hematopoietic stem cells into lymphoid hematopoietic stem cells, and is largely divided into B lymphocytes, T lymphocytes, and NK cells. It is morphologically difficult, including observation by Their differentiation is possible by using an antibody of a cell surface marker (CD; Cluster of Differentiation) characteristic of each cell. These markers are characteristically different depending on the lymphocytes. NK cells are CD3, CD56, CD16, CD94, CD161, etc., B lymphocytes are CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD40, etc., T lymphocytes are CD3 , Helper T lymphocytes express CD4 and cytotoxic T lymphocyte CD8 as markers on the cell surface.

The spleen and thymus are tissues involved in immune function, the spleen is involved in lymphocyte production, and the thymus is the tissue involved in T lymphocyte differentiation and maturation. Therefore, the spleen and thymus tissues are weighed to see the systemic effects of immune-related tissues.

The spleen is a tissue that makes lymphocytes and destroys old red blood cells. Therefore, as the number of splenocytes isolated from the experimental animals ingesting the test substance increases, it means that the immune function is improved.

Immunoglobulin (Ig) is a serum protein that plays a major role in humoral immunity and is classified into IgA, IgD, IgE, IgG, and IgM. IgA accounts for about 20% of serum immunoglobulins and is present in mucus such as saliva and upper respiratory tract secretions and is involved in the defense against infection of the mucous membrane. IgG is divided into four subgroups, IgG1, IgG2, IgG3, and IgG4, and is involved in neutralization, sedimentation, and aggregation of toxins or viruses. IgM is complement-binding and has high aggregation reactivity. An increase in immunoglobulin (IgA, IgG, IgM) means enhancement of immune function.

Complement is a protein contained in blood and lymph. Complement has no immunological specificity and binds to the antigen-antibody complex and performs hemolysis and lysis to remove bacteria and antigens that have passed through the physical barrier. Complement consists of more than 20 proteins, mainly measuring C3, C4, and C5. Activation of complement is related to various immune responses such as activation of macrophages and lymphocytes and enhancement of immune function.

NK cells are leukocytes in the blood that are responsible for innate immunity, and are cells that either recognize virus-infected cells or stressed cells and either directly kill them or secrete inflammatory cytokines to kill them. It can be determined whether the immune function is enhanced through the change in NK cell activity.

The composition for enhancing immunity provided in one aspect of the present invention has the effect of remarkably improving safety and immunity by containing the extract of bellflower produced under specific conditions as an active ingredient.

Hereinafter, Examples and Experimental Examples of the present invention will be specifically illustrated and described below. However, the Examples and Experimental Examples to be described below are merely illustrative of a part of the present invention, and the present invention is not limited thereto.

< Example 1> Manufacture of developed bellflower extract

The bellflower used in this experiment was purchased from Yeongdong, Chungbuk, and a developed bellflower (DD) extract with improved active ingredient by the extraction method of the present application was prepared in the following manner.

1-1. primary treatment

Bellflower (Scientific name: Platycodon) grandiflorum ) was washed with water to clean the soil or dirt, and then the bellflower washed at a temperature of about 60 ℃ or lower for 48 hours was dried with hot air and filtered through 30 mesh.

1-2. secondary processing

100 mL of distilled water about 10 times the weight of the bellflower raw material was added to 10 g of the bellflower raw material on which the primary treatment was completed, and left at 60° C. for 120 minutes. Then, 100 mL of 99% ethanol was added, followed by ultrasonic extraction for 30 minutes. After centrifugation at 3,000 rpm for 10 minutes, filter press filtration, 200 mL of 50% ethanol was added, and ultrasonic extraction was performed for 30 minutes. This was centrifuged at 3,000 rpm for 10 minutes, filtered, concentrated under reduced pressure, and freeze-dried to prepare a developed bellflower (DD) with improved active ingredient.

< comparative example 1> Manufacture of common bellflower extract

Common bellflower (ND) extract was prepared as follows. After washing and drying in hot air (60 degrees or less, 48 hours), the bellflower was pulverized with a grinder (FM909T, Hanil, Wonju, Korea) and passed through a 30 mesh sieve. The prepared bellflower powder was extracted twice for 24 hours at room temperature with 70% alcohol, filtered, concentrated and freeze-dried (PVTFD 10R, Ilsin Lab, Yangju, Korea) and used as a general bellflower (ND) sample.

< Experimental example 1> Cytotoxicity assessment

The toxicity of the bellflower extract prepared in Example 1 and Comparative Example 1 to immune-related macrophages was evaluated as follows.

Macrophages (Raw 264.7) were purchased from the Korea Cell Line Bank (Seoul, Korea) and used in the experiment. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco, Grand Island, NY) medium containing 10% Fetal Bobin Serum (FBS; Invitrogen, Carlsbad, USA) and 100 units/mL penicillin streptomycin (HyClone, Logan, UT). using a CO ₂ incubator (CO ₂ 5%, 37 °C). 100 μL of healthy cells were dispensed in a 96-well plate to 1×10 ⁶ cells/mL, and 100 μL of the bellflower extract prepared in Example 1 and Comparative Example 1 at each concentration (125, 250, 500 μg/mL) each and incubated for 46 hours. In order to evaluate cytotoxicity, 30 μL of MTS assay reagent (Promega Corporation, Madison, WI)) was dispensed, cultured under the same conditions, and absorbance (490 nm) was measured using a microplate reader (Molecular Devies, CA, USA). . Cell viability was expressed as a percentage compared to the control group that did not contain the bellflower extract.

The results are shown in FIG. 1 .

1 is a view showing the results of evaluation of toxicity to immune-related macrophages of a bellflower extract prepared in a control group (NC), Example 1 (DD), and Comparative Example 1 (ND).

As shown in Figure 1,

The developed bellflower (DD) extract with an increased active ingredient according to the bellflower extract of the present application according to Example 1, compared to the general bellflower (ND) extract according to Comparative Example 1, showed a 31% improvement in toxicity and safety to macrophages. . Therefore, it can be seen that the developed bellflower extract extracted by the extraction method according to Example 1 has an excellent effect of improving toxicity on immune-related cells.

< Experimental example 2> Safety evaluation in immunocompromised mice

In order to evaluate the safety in mice with reduced immunity due to induction of stress, the following experiments were conducted. CPA (cyclophosphamide) was administered to experimental animals (C57BL/6 mice, 6 weeks old, male) to induce immunosuppression, and developed bellflower (DD) extract (Example 1) and general bellflower (ND) extract (Comparative Example 1) was orally administered at a level of 75 mg/kg for 7 days to evaluate how much the body weight of mice with reduced immunity was improved by the bellflower extract. The control group (NC) was administered only distilled water in which the bellflower extract was dissolved.

The results are shown in FIG. 2 .

2 is a graph showing the results of evaluating the safety of the extracts prepared in Control (NC), Example 1 (DD), and Comparative Example 1 (ND) in immunocompromised mice through weight change.

As shown in Figure 2,

In the general bellflower extract intake group and the developed bellflower extract intake group, body weight increased compared to the control group, but in particular, the group administered with the developed bellflower extract (Example 1) showed a significant improvement of about 10.8% compared to the control group. Through this, it can be seen that the developed bellflower extract extracted by the extraction method according to Example 1 can effectively restore the weight reduced by the stress of suppressing immunity.

< Experimental example 3> Assessment of effects on blood immunoglobulins and related cytokines

In order to evaluate the effect of the bellflower extract prepared in Example 1 and Comparative Example 1 on blood immunoglobulins and related cytokines, the experiment was performed as follows.

In order to evaluate the effect on the blood immune-related indicators of bellflower, blood was obtained from the mice fed the extract in Experimental Example 2, and immunoglobulin A (IgA), immunoglobulin G (IgG), cytokines (IL-6, TNF) -α) content was measured.

An ELISA kit (Abcam, London, UK) was used for the analysis, and 50 μL of a serum sample was added to each well, and 50 μL of an antibody cocktail was added. Sealed and incubated for 1 hour at room temperature, washing each well with 350 μL 1X Wash Buffer was repeated 3 times. After the last washing process, all excess liquid was removed and , 100 μL of TMB Substrate was added to each well and incubated for 15 minutes, and 100 μL of Stop Solution was added to each well. After reacting for 1 minute, absorbance was measured at 450 nm using a microplate reader (Microplate reader, Molecular Devices, CA, USA).

The results are shown in FIGS. 3 to 6 .

3 is a graph showing the results of evaluating the effect of a bellflower extract according to a control group (NC), Example 1 (DD), and Comparative Example 1 (ND) on the level of immunoglobulin A (IgA) in blood.

4 is a graph showing the results of evaluating the effect of the bellflower extract according to the control (NC), Example 1 (DD), and Comparative Example 1 (ND) on the level of immunoglobulin G (IgG) in blood.

5 is a graph showing the results of evaluating the effect of the bellflower extract according to the control (NC), Example 1 (DD), and Comparative Example 1 (ND) on the level of the blood cytokine TNF-α.

6 is a graph showing the results of evaluating the effect of the bellflower extract according to the control (NC), Example 1 (DD), and Comparative Example 1 (ND) on the level of the cytokine IL-6 in the blood.

As shown in Figure 3,

The general bellflower extract according to Comparative Example 1 did not have a significant effect on the blood immunoglobulin IgA level, but the developed bellflower extract according to Example 1 was found to improve blood immunoglobulin by about 19.5% or more higher than that of the control group. .

As shown in Figure 4,

The general bellflower extract according to Comparative Example 1 did not have a significant effect on the blood immunoglobulin IgG level, but the developed bellflower extract according to Example 1 showed a significant improvement of about 2.1% compared to the control group.

As shown in Figure 5,

The general bellflower extract according to Comparative Example 1 showed a tendency to rather increase the TNF-α level increased in a mouse model with reduced immunity due to stress, but the developed bellflower extract according to Example 1 was superior to the control by about 53.1%. showed the effect of reducing it.

As shown in Figure 6,

In a mouse model with reduced immunity due to stress, the general bellflower extract according to Comparative Example 1 showed a decrease of about 11.2%, whereas the developed bellflower extract according to Example 1 showed a decrease of about 35.3%, the general Compared to the bellflower extract, it showed about 3.2 times superior reduction effect.

Therefore, the developed bellflower extract prepared according to Example 1 has the effect of improving both IgA and IgG in blood to a significant level in a stressed mouse model with reduced immunity, compared to the control, and increases blood cytokines due to stress. Since it has an effect of regulating TNF-α and IL-6 levels, it has an excellent effect on enhancing immunity.

< Experimental example 4> Evaluation of spleen immune cell proliferation effect

In order to evaluate the effect of the bellflower extract prepared in Example 1 and Comparative Example 1 on the proliferation of spleen immune cells, an experiment was performed as follows.

The spleen was extracted from the mice fed the extract in Experimental Example 2, washed twice with a culture medium (RPMI 1640, 10% FBS, 100 units/mL penicillin streptomycin), and after adding 10 mL of the culture medium in a cell culture dish, 40 Homogenization was performed using a μm nylon cell strainer (BD Biosciences, San Jose, CA, USA). The splenocytes thus obtained were treated on Histopaque (Sigma, USA) to separate lymphocytes, washed by repeated centrifugation, ^{and dispersed at 3.0 × 10 6} cells/mL in the culture medium in a 96 well plate (Nunc, Rosklide, Denmark) 100 μL of each was dispensed and used for cell proliferation measurement. LPS (Sigma-Aldrich Co, USA) was added to a final concentration of 1 μL/mL, and an equal amount of the culture medium was dispensed to the control group (NC), and cultured at 37 ° C., 5% CO₂ incubator. After 46 hours of incubation, each well was reacted for 2 hours using 30 μL of MTS (Promega Corporation, Madison, WI, USA), and a microplate reader (microplate reader, Molecular Devices, CA, USA) was used to measure the absorbance (OD) at 490 nm to compare the activity.

The results are shown in FIG. 7 .

7 is a view showing the results of evaluating the effect of the bellflower extract according to the control (NC), Example 1 (DD), and Comparative Example 1 (ND) on the proliferation of spleen immune cells.

As shown in Figure 7,

The general bellflower extract according to Comparative Example 1 did not affect the proliferation of spleen immune cells against stress caused by LPS (lipopolysaccharide), whereas in the case of the group administered with the developed bellflower extract according to Example 1, about 9.4% concentration-dependent effect appeared to show

< Experimental example 5> spleen NK cells Assessing the impact on activity

In order to evaluate the effect of the bellflower extract prepared in Example 1 and Comparative Example 1 on splenic NK cell activity, the experiment was performed as follows.

Splenocytes obtained from mice fed the extract in Experimental Example 2 were each dispensed into a 96-well plate at 1×10 ⁶ cells/mL per well, and the target cells were Yac-1 cells at 1×10 ⁴ cells/well. It was aliquoted (effector-to-target cell ratio 100:1), _{and incubated at 37° C., 5% CO 2} for 24 hours. After incubation, the absorbance was measured at 490 nm using a microplate reader using an MTS assay kit (Promega Corporation, Madison, WI, USA), and the activity was calculated according to the following formula.

NK cell activity (%) = [1 - (B-A) / A] × 100

A (OD): NK cells only

B (OD): NK cells + Yac-1

The results are shown in FIG. 8 .

8 is a graph showing the results of evaluating the effect of bellflower extract according to the control (NC), Example 1 (DD), and Comparative Example 1 (ND) on splenic NK cell activity.

As shown in Figure 8,

The NK cell activity of the spleen was improved by 8% in the developed bellflower extract according to Example 1 compared to the control, and showed a significant improvement of about 32.4% compared to the general bellflower extract according to Comparative Example 1.

As mentioned above, although the present invention has been described in detail through preferred examples and experimental examples, the scope of the present invention is not limited to specific examples, and should be interpreted by the appended claims. In addition, those skilled in the art should understand that many modifications and variations are possible without departing from the scope of the present invention.

Claims (15)

Bellflower ( Platycodon grandiflorum ) A primary treatment step comprising the step of hot air drying at 60 ℃ or less for 48 hours; and
After adding water 8 to 12 times the weight of the raw material to the raw material prepared in the first treatment step, and leaving it at 50 to 70 ° C for 100 to 150 minutes, 99% (v/v) A secondary treatment step comprising the step of primary extraction with ethanol and secondary extraction with 50% (v/v) ethanol; containing a bellflower extract prepared as an active ingredient;
delete delete According to claim 1,
The bellflower extract is a composition for enhancing reduced immune function, characterized in that it is prepared by treating ethanol in a volume of 5 to 25 times the weight of the raw material prepared in the primary treatment step.
According to claim 1,
The bellflower extract is characterized in that it is prepared by extracting for 20 to 40 minutes, a composition for enhancing reduced immune function.
delete delete According to claim 1,
The composition for enhancing the reduced immune function, characterized in that for regulating the cytokines in the blood of animals, the composition for enhancing the reduced immune function.
9. The method of claim 8,
The cytokine is IL-6 or TNF-α, characterized in that the reduced immune function enhancement composition.
According to claim 1,
The composition for enhancing the reduced immune function, characterized in that to improve the level of immunoglobulin in the blood of an animal, the composition for enhancing the reduced immune function.
11. The method of claim 10,
The immunoglobulin is IgA or IgG, characterized in that the reduced immune function enhancement composition.
According to claim 1,
The composition for enhancing the reduced immune function, characterized in that it improves the proliferation of stress-induced spleen cells, the composition for enhancing the reduced immune function.
According to claim 1,
The composition for enhancing the reduced immune function, characterized in that it enhances the activity of the stress-induced splenic NK cells, the composition for enhancing the reduced immune function.
A functional food for enhancing reduced immune function comprising the composition for enhancing reduced immune function according to claim 1 .
A pharmaceutical composition for enhancing reduced immune function, comprising the composition for enhancing reduced immune function according to claim 1.

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