KR20200137877A - A composition for enhancing immunity comprising the compound isolated from black ginseng extract and black ginseng as an active ingredient for companion dog - Google Patents

Abstract

The present invention provides a composition for enhancing immunity of companion dogs containing a black ginseng extract and a compound isolated from black ginseng as an active component. The black ginseng extract and the compound isolated from black ginseng of the present invention have low cytotoxicity and excellent immune suppression ability, thereby being able to be usefully used as an immunity composition, a health functional food composition for enhancing immunity, and a feed composition for enhancing immunity. In addition, the present invention provides a method for manufacturing the black ginseng extract and the compound isolated from the same, and a method for manufacturing the feed composition for enhancing immunity, thereby being able to be usefully used in related industries.

Description

A composition for enhancing immunity comprising the compound isolated from black ginseng extract and black ginseng as an active ingredient for companion dog}

The present invention is to provide a composition for enhancing immunity of dogs, comprising a black ginseng extract and a compound isolated from black ginseng as an active ingredient.

Immune is a self-defense system that exists in the body and is a process by which the human body recognizes, removes, and metabolizes various substances or living organisms that invade from the outside as foreign substances to itself.

It protects itself from damage caused by external stimuli or invasion of pathogenic microorganisms, but it can also damage its own tissues such as inflammatory reactions. It is classified into suppression of immune function or enhancement of immune function by controlling changes in immune function to restore normal or to reduce the width of change. Relief of an irritable immune response refers to suppressing an undesirably increased immune response such as an allergic reaction autoantigen or a response to a modified autoantigen resulting from adverse reactions to foreign substances.

In order to overcome the problem of such an immune response, various immunity enhancers and treatments are used, but there are side effects problems, and those that are mainly marketed are taken for treatment rather than prevention. There is also a situation in which a suitable composition for promoting immunity is required.

Accordingly, the inventors found that while researching a substance for use as a composition for improving immunity without side effects, processed ginseng was found to have a higher content of useful substances than ginseng itself, and the ginseng went through the process of proliferation and drying several times. A method of manufacturing black ginseng that can increase the content of ginseng saponin as well as not lose the ginseng saponin present was established. In addition, the present invention was completed by analyzing the additional useful ingredients from the prepared black ginseng and confirming the effect of enhancing the immunity of the dog.

(001) Korean patent publication KR 10-2019-0018175

The present invention aims to provide a method for efficiently producing black ginseng and a method for producing an extract, and more specifically, anti-inflammatory effects by extraction conditions of black ginseng and improved immunity of 7 newly generated ingredients by the black ginseng production process. It is for the purpose of functionality.

In order to achieve the above object, the present invention provides a pharmaceutical composition for enhancing immunity comprising black ginseng extract as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for enhancing immunity comprising the compound isolated from the black ginseng extract as an active ingredient.

In addition, the present invention provides a health functional food composition for enhancing immunity comprising black ginseng extract as an active ingredient.

In addition, the present invention provides a health functional food composition for enhancing immunity comprising the compound isolated from the black ginseng extract as an active ingredient.

In addition, the present invention provides a feed composition for improving immunity comprising black ginseng extract as an active ingredient.

In addition, the present invention provides a feed composition for enhancing immunity comprising the compound isolated from the black ginseng extract as an active ingredient.

In addition, the present invention provides a method for producing a black ginseng extract.

In addition, the present invention provides a method for preparing a compound isolated from black ginseng extract.

In addition, the present invention provides a method for producing a feed composition for enhancing immunity.

Since the black ginseng extract and the compound isolated from the black ginseng of the present invention have excellent immunosuppression effects, they can be usefully used as an immunity composition, a health functional food composition for improving immunity, and a feed composition for improving immunity. In addition, it provides a method for preparing a black ginseng extract and a compound isolated therefrom, a method for preparing a feed composition for enhancing immunity, and can be usefully used in related industries.

1 is a graph showing the content of compounds (ginsenoside F4, ginsenoside Rh4, ginsenoside Rk1, ginsenoside Rg5, compound O, ginsenoside Mc, compound Y) isolated from black ginseng prepared by the method of the present invention.
2 is a graph showing the immunosuppressive effect of black ginseng extract;
A: NO production, B: cell viability.
3 is a graph showing the results of a cytotoxicity experiment of a black ginseng extract and a compound isolated from a black ginseng extract:
A: black ginseng extract, B: compounds isolated from black ginseng extract (ginsenoside F4, ginsenoside Rh4, ginsenoside Rk1, ginsenoside Rg5, compound O, ginsenoside Mc, compound Y).
Figure 4 is a view showing the results of the immune indicator (IL-2) analysis of the black ginseng extract and the compound isolated from the black ginseng extract:
A: black ginseng extract, B: compounds isolated from black ginseng extract (ginsenoside F4, ginsenoside Rh4, ginsenoside Rk1, ginsenoside Rg5, compound O, ginsenoside Mc, compound Y).

The present invention aims to provide a method of efficiently producing black ginseng and a method of producing an extract, and more specifically, anti-inflammatory efficacy by extraction conditions of black ginseng and improved immunity of 7 newly generated ingredients by the black ginseng production process It is for the purpose of functionality.

Accordingly, the present invention provides a pharmaceutical composition for enhancing immunity comprising a black ginseng extract and a compound isolated from the black ginseng extract as an active ingredient.

The compound can be described by the following Chemical Formulas 1 to 7.

[Formula 1]

[Formula 2]

[Formula 3]

[Formula 4]

[Formula 5]

[Formula 6]

[Formula 7]

The pharmaceutical composition of the present invention may further contain an adjuvant in addition to the active ingredient. Any of the adjuvants known in the art may be used without limitation, but, for example, Freund's complete adjuvant or incomplete adjuvant may be further included to increase its immunity.

The pharmaceutical composition according to the present invention may be prepared in a form in which an active ingredient is incorporated in a pharmaceutically acceptable carrier. Here, the pharmaceutically acceptable carrier includes carriers, excipients, and diluents commonly used in the pharmaceutical field. Pharmaceutically acceptable carriers that can be used in the pharmaceutical composition of the present invention are not limited thereto, but lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, Calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oils.

The pharmaceutical compositions of the present invention can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, or sterile injectable solutions according to a conventional method. .

In the case of formulation, it can be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are commonly used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations include at least one excipient, such as starch, calcium carbonate, sucrose, lactose, gelatin, in the active ingredient. It can be prepared by mixing and the like. Further, in addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, liquid solutions, emulsions, syrups, and other excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to water and liquid paraffin, which are commonly used diluents. I can. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations, and suppositories. As the non-aqueous solvent and suspension, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like may be used. As a base for suppositories, witepsol, tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used.

The pharmaceutical composition according to the present invention can be administered to a subject by various routes. All modes of administration can be expected, for example, by oral, intravenous, intramuscular, subcutaneous, intraperitoneal injection.

The dosage of the pharmaceutical composition according to the present invention is selected in consideration of the age, weight, sex, and physical condition of the individual. It is obvious that the concentration of the active ingredient contained in the pharmaceutical composition can be selected in various ways depending on the subject, and is preferably included in the pharmaceutical composition at a concentration of 0.01 to 5,000 μg/ml. When the concentration is less than 0.01 μg/ml, pharmaceutical activity may not appear, and when the concentration exceeds 5,000 μg/ml, toxicity to humans may occur.

The pharmaceutical composition may be formulated in various oral or parenteral dosage forms.

Formulations for oral administration include, for example, tablets, pills, hard, soft capsules, solutions, suspensions, emulsifiers, syrups, and granules. These formulations include diluents (e.g. lactose, dextrose, water Krose, mannitol, sorbitol, cellulose and/or glycine), lubricants (eg, silica, talc, stearic acid and magnesium or calcium salts thereof and/or polyethylene glycol). In addition, the tablet may contain a binder such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidine, in some cases starch, agar, alginic acid Or disintegrants or boiling mixtures and/or absorbents, colorants, flavors and sweeteners such as sodium salts thereof. The formulation can be prepared by conventional mixing, granulating or coating methods.

In addition, a representative formulation for parenteral administration is a formulation for injection, and as a solvent for the formulation for injection, water, Ringer's solution, isotonic physiological saline or suspension may be mentioned. The sterile fixed oil of the injection preparation can be used as a solvent or suspension medium, and any non-irritating fixed oil including mono- and di-glycerides can be used for this purpose.

In addition, the injectable formulation may use a fatty acid such as oleic acid.

In addition, the present invention provides a health functional food composition for enhancing immunity comprising a black ginseng extract and a compound isolated from the black ginseng extract as an active ingredient.

In addition to containing the extract as an active ingredient, the food composition of the present invention may contain various flavoring agents or natural carbohydrates as an additional ingredient, like a conventional food composition.

Examples of the above-described natural carbohydrates include monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, and the like; And polysaccharides, for example, common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. The above-described flavoring agents can be advantageously used as natural flavoring agents (taumatin), stevia extracts (eg, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.). The food composition of the present invention can be formulated in the same manner as the pharmaceutical composition and used as a functional food or added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, meat, chocolate, foods, confectionery, pizza, ramen, other noodles, gums, candy, ice cream, alcoholic beverages, vitamin complexes and health supplements, etc. There is this.

In addition, the food composition includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring and natural flavoring agents, coloring agents and heavy weight agents (cheese, chocolate, etc.), pectic acid and salts thereof, Alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonates used in carbonated beverages, and the like may be contained. In addition, the food composition of the present invention may contain flesh for the production of natural fruit juice and fruit juice beverages and vegetable beverages.

The functional food composition of the present invention can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, and pills. In the present invention, the term'health functional food composition' refers to a food manufactured and processed using raw materials or ingredients having useful functions for the human body pursuant to Health Functional Food Act No.6727, and with respect to the structure and function of the human body. It refers to ingestion for the purpose of obtaining useful effects for health purposes such as controlling nutrients or physiological effects. The health functional food of the present invention may contain ordinary food additives, and whether it is suitable as a food additive is determined according to the general rules and general test methods for food additives approved by the Food and Drug Administration, unless otherwise specified. It is judged according to standards and standards. Examples of items listed in the'Food Additives Code' include chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; Natural additives such as reduced pigment, licorice extract, crystalline cellulose, high color pigment, and guar gum; Mixed preparations, such as a sodium L-glutamate preparation, an alkali additive for noodles, a preservative preparation, and a tar color preparation, etc. are mentioned. For example, in the health functional food in the form of a tablet, a mixture obtained by mixing the active ingredient of the present invention with an excipient, a binder, a disintegrant, and other additives is granulated by a conventional method, and then a lubricant, etc. The mixture can be directly compression molded. In addition, the health functional food in the form of a tablet may contain a mating agent or the like, if necessary. Among the health functional foods in the form of capsules, hard capsules can be prepared by filling a mixture of the active ingredient of the present invention with additives such as excipients in a conventional hard capsule, and the soft capsules contain the active ingredient of the present invention with additives such as excipients. The mixture mixed with can be prepared by filling a capsule base such as gelatin. The soft capsules may contain a plasticizer such as glycerin or sorbitol, a colorant, a preservative, and the like, if necessary. Ring-shaped health functional foods can be prepared by molding a mixture of the active ingredient of the present invention, excipients, binders, disintegrants, etc. by conventionally known methods, and can be coated with white sugar or other coating agents if necessary, Alternatively, the surface may be coated with a material such as starch or talc. Health functional foods in the form of granules can be prepared in granular form by a mixture of the excipients, binders, disintegrants, etc. of the active ingredients of the present invention by a known method, and if necessary, may contain flavoring agents, flavoring agents, etc. I can.

In addition, the present invention provides a feed composition for enhancing immunity comprising a black ginseng extract and a compound isolated from the black ginseng extract as an active ingredient.

The feed composition of the present invention can be expected to improve the health of the animal body, improve the weight gain and meat quality of livestock, and increase the milk production and immunity. The feed composition of the present invention may be prepared in the form of fermented feed, blended feed, pellet form, and silage. The fermented feed can be prepared by fermenting organic matter by adding various microorganism groups or enzymes other than the active ingredient of the present invention, and the compounded feed can be prepared by mixing various types of general feed and the active ingredient of the present invention. . The pellet-type feed may be prepared by applying heat and pressure to the blended feed in a pellet machine, and the silage may be prepared by fermenting the blue-green feed with the microorganism according to the present invention. Wet fermented feed collects and transports organic matter such as food waste, mixes excipients for sterilization and moisture control at a certain ratio, and then ferments at a temperature suitable for fermentation for more than 24 hours, so that the moisture content is about 70%. It can be manufactured by controlling. The fermented dried feed can be prepared by controlling the wet fermented feed to contain about 30% to 40% of moisture through an additional drying process.

The feed composition of the present invention may further include ingredients added to conventional feed. Examples of ingredients added to such feed may include grain powder, meat powder, and beans. In the above, the grain powder may be one or more selected from rice flour, wheat flour, barley flour, and corn flour. In the above, the meat powder may be a meat powder obtained by powdering at least one selected from chicken, beef, pork, and ostrich meat. Beans in the above may be used one or more selected from soybeans, kidney beans, peas, and black beans.

The feed composition of the present invention may add any one or more selected from nutrients and minerals in order to increase the nutritional properties of the feed in addition to grain powder, meat powder, and beans, which are ingredients added to the conventional feed mentioned above, and feed quality It may contain at least one selected from an anti-fungal agent, an antioxidant, an anticoagulant, an emulsifier, and a binder to prevent the reduction of the.

In addition, the present invention provides a method for producing a black ginseng extract.

In addition, the present invention provides a method for preparing a compound isolated from black ginseng extract.

Hereinafter, preferred embodiments of the present invention will be described in more detail. However, it should be understood that the present invention may be implemented in a number of different forms and is not limited to the described embodiments. It should be noted that the embodiments of the present invention described below are intended to sufficiently convey the spirit of the present invention to those skilled in the art.

<Example 1> Preparation of black ginseng extract

Dry the washed ginseng for 3-10 hours at 40℃ in a hot air dryer. 1st Jeungsam Black ginseng prepared by putting dried ginseng in a steamer, rapidly increasing the temperature from room temperature to 60~70℃, maintaining it for 10 minutes, raising the temperature to the target temperature (90~98℃) and repeating steamed ginseng 3-5 times for 2 to 5 hours. Was immersed in 80% MeOH aqueous solution for 24 hours, extracted three times at room temperature, and the obtained filtrates were combined and concentrated under reduced pressure to obtain a MeOH extract. The obtained MeOH extract was compared using thin layer chromatography (TLC), and the extracts were combined to obtain a total of 300 g of a concentrate. The concentrated extract was partitioned extracted three times with EtOAc (3 L)/H ₂ O (3 L), and the H ₂ O layer was partitioned extracted three times with n-BuOH (2.7 L). Each layer was concentrated under reduced pressure to obtain an EtOAc fraction, an n-BuOH fraction, and an H ₂ O fraction. Among them, the EtOAc fraction was used as a black ginseng alcohol extract.

<Example 2> Isolation of active ingredient from black ginseng extract

14 × 16 ㎝, CHCl₃-MeOH-H₂O=15:3:1) → CHCl₃-MeOH-H₂O=7:3:1)를 실시하여 100 ㎖씩 분취하여 분취액을 수득하였다. 상기 분취액을 TLC(CHCl₃-MeOH-H₂O=9:3:1)로 확인하여, 유사한 부분을 함께 모으고, 감압농축기를 사용하여 농축하여 10개의 분획물(BGE1 내지 BGE10)을 수득하였다.The ethyl acetate partitioned extract obtained in Example 1 was subjected to silicon dioxide column chromatography (hereinafter referred to as'SiO ₂ cc') (

14×16 cm, CHCl ₃ -MeOH-H ₂ O=15:3:1) → CHCl ₃ -MeOH-H ₂ O=7:3:1) was carried out, and 100 ml of aliquots were aliquoted to obtain aliquots. The aliquot was confirmed by TLC (CHCl ₃ -MeOH-H ₂ O=9:3:1), similar portions were collected together, and concentrated using a vacuum concentrator to obtain 10 fractions (BGE1 to BGE10).

Then, the compound was separated and purified using the 10 fractions.

(1) Octadecyl Silicas column chromatography on the first fraction (BGE, 1 g) of the 10 fractions; ODS cc, manufactured by Waters; Φ 4×9 cm, MeOH- H ₂ O=3:1) was carried out to obtain a total of 5 fractions (BGE1-1 to BGE1-5).

ODS column chromatography (Φ 2.5×7 cm, MeOH-H2O=3:1) was performed on the first fraction (BGE1-1-1, 300 mg) of the five fractions, and a total of eight fractions (BGE1-1 -1-1 to BGE1-1-1-8) were obtained, and finally compound O (8 mg) was isolated.

(2) Then, ODS column chromatography (Φ 3×8 cm, MeOH-H ₂ O=3:1) was performed on the third fraction (BGE3, 1.5 g) of the 10 fractions, and a total of 5 fractions (BGE3-1 to BGE3-5) was obtained, of which the 5th fraction (BGE3-5, 500 mg) was subjected to ODS column chromatography (Φ 3×7 cm, MeOH-H ₂ O=2.5:1). Thus, a total of 10 fractions (BGE3-5-1 to BGE3-5-10) were obtained, and ginsenoside F4 (8 mg) was finally isolated.

(3) ODS column chromatography (Φ 3×10 cm, MeOH-H2O=4:1) was performed on the 7th fraction (BGE7, 3.30 g) of the first 10 fractions, and a total of 8 fractions (BGE7-1 to BGE7-8) was obtained, and for the BGE7-5 fraction (400 mg), about 100 mg each was dissolved in 1 ml of solvent and injected using prep LC, and CH ₂ Cl ₂ -MeOH (5:1) It was flowed at a rate of 1.5 ml per minute. At this time, the detector used was a refractive index detector, and the signal was recovered while checking the signal coming out of the detector, and the same fractions were collected by repeating the above process and confirmed by TLC. ginsenoside MC (5 mg), compound Y (6 mg), ginsenoside Rk1 (18 mg), Rg 5 (12 mg), and ginsenoside Rk4 were isolated.

<Example 3> Comparative analysis of the content of the compound isolated from the black ginseng extract

The contents of compounds ginsenoside F4, ginsenoside Rh4, ginsenoside Rk1, ginsenoside Rg5, compound O, ginsenoside Mc, and compound Y were analyzed from the black ginseng extract prepared in Example 1 by the method described in Example 2.

As a comparative example, commercially available black ginseng Cheonil Goryeo ginseng (C company black ginseng) and Daedong ginseng (D company black ginseng) were used.

As a result, it was confirmed that the content of 7 kinds of compounds isolated from black ginseng prepared by the method described in Example 1 of the present invention was higher than that of commercial black ginseng (FIG. 1).

<Example 4> Analysis of the immune enhancing effect of black ginseng extract

<4-1> cell culture

BV2 cells used in this experiment were purchased from Korea Cell Line Bank and used. 10% FBS and 1% penicillin-streptomycin were added to DMEM medium and cultured at 37° C. and 5% CO ₂ . In all experiments, BV2 cells were pre-treated with each extract for each black ginseng extraction solvent for 1 hour and then treated with LPS (500 ng/ml) and cultured for 24 hours.

<4-2> Cytotoxicity investigation by MTT assay

BV2 cells were dispensed into 6 well plates for cell culture at 6 × 10 ⁵ cells/well and stabilized for 24 hours. After pre-treatment of each extract for each concentration of black ginseng extract for 1 hour, LPS was post-treated and cultured for 24 hours. Thereafter, all the medium was removed, tetrazolium bromide salt (MTT) was diluted to a concentration of 0.5 mg/ml, and 1 ml per well was dispensed. After incubation for 2 hours, the supernatant was removed, all formazin was dissolved with dimethylsulfoxide (DMSO), transferred 200 μl to a 96 well plate, and the absorbance was measured with an ELISA reader (Molecular Devices, Sunnyvale, CA, USA).

<4-3> Measurement of NO

NO production was measured through Griess assay. To this end, after culturing the cells in a 6 well plate for cell culture in the manner described above, 100 μl of the supernatant was collected from each well and each Griess reagent[1% sulfanilamide, 0.1% N-(1-naphthyl)-ethylenediamine dihydrochloride , 2.5% H ₃ PO ₄ ] was mixed with 100 μl and dispensed into a 96 well plate. After measuring the absorbance using an ELISA reader (540nm), the NO concentration was calculated based on the standard curve of sodium nitrite (NaNO ₂ ).

<4-4> Analysis result of NO production of black ginseng extract

When the inflammatory reaction is initiated by stimulation such as LPS in the body or in the cell system, the inflammatory gene iNOS is expressed, and excessive NO is produced by the inflammatory reaction involving iNOS. (Chen et al., 1998) Therefore, the disappearance of NO in the body means that the inflammatory response is inhibited. As a result of measuring the NO inhibitory ability of the black ginseng extract (40%, 70% EtOH), it was confirmed that NO production was reduced in the group treated with 0.1 μg/ml of LPS (FIG. 2A).

<4-5> Analysis results of the effect of black ginseng extract on cell viability

As a result of confirming the cytotoxicity of the black ginseng extract to BV2 cells, there was no significant difference that inhibited cell survival in all treatment groups of the black ginseng extract, and it is believed that the black ginseng extract had no effect on the survival of BV2 cells (Fig. 2B). .

<Example 5> Analysis of the immune enhancing effect of the black ginseng extract and the compound isolated from the black ginseng extract

<5-1> cell culture

Immune cells Jurkat T cells are in CO ₂ cell incubator (37°C, 5%, CO ₂ ) in fetal bovine serum (10%), penicillin G (100 IU/mL), streptomycin (100 ug/mL), and L- It was cultured in RPMI640 medium to which glutamine (2 mM) was added. As a stimulant in Jurkat T cells, phorbol 12-myristate 13-acetate (PMA) and A23187 were used as reagents Sigma (St. Louis, MO). Cells corresponding to log-phase were dispensed into 12-well, 6-well, or 60-mm dishes according to each experiment and used.

<5-2> MTT assay

Human Jurkat Tcell (5 X 10 ⁵ Cell/ml) was dispensed into a 48 well plate and cultured in a 5% carbon dioxide incubator at 37°C for one day. Each well was cultured in DMEM culture solution containing 10% fetal bovine serum and 1% antibiotic, and then treated with reagents. As a negative control, dimethyl sulfoxide (DMSO) was added as a solvent. After culturing in a 5% carbon dioxide incubator at 37°C for one day, the degree of growth of the cells was confirmed by the MTT method. Add 100 ul of 0.1% MTT (Sigma M2128) solution to each well of a 48 well culture vessel and react in a 5% carbon dioxide incubator at 37°C for 3 hours. After removing the culture solution and MTT solution, 100 ul of dimethyl sulfoxide is added to dissolve the formed crystals. Absorbance was measured at 540 nm using a microplate reader.

<5-3> RNA isolation

For Jurkat T cells, cells (2×10 ⁶ ) grown in a 6-well plate were cultured by treatment with extracts and compounds, and chlorform was added to extract RNA to make a voltex of 20-30 seconds at 4℃ at 15,000 rpm. Centrifuged for 15 minutes. At this time, the solution was divided into three parts. After the supernatant was transferred to a new tube, an equal amount of isopropanol was added and placed at room temperature for 10 minutes, followed by centrifugation at 15,000 rpm for 20 minutes at 4°C. The supernatant was discarded, washed with 75% EtOH (in DEPC water), centrifuged at 13,000 rpm for 5 minutes at 4° C., the supernatant was discarded, and the pellet was dissolved using DEPC water. RNA thus extracted was stored at -80 ℃. Total RNA concentration was measured by dissolving in 0.1% DEPC water treated with RNase, and absorbance was measured at 260 nm/280 nm with a UV spectrophotometer.

<5-4> Reverse Transcription-Polymerase Chain Reaction (RT-PCR)

In Jurkat T cells, PCR products for mRNA were quantified using a semiquantitative RT-PCR method. Reverse transcription reaction is performed by accupowering the extracted total RNA (1 μg) and oligonucleotide dT primer (100 p mol).After mixing in RT PreMix (Bioneer Co, Korea) tube, the total volume of the reaction was 20 μl and pretreated at 70° C. for 5 minutes. cDNA synthesis was reacted at 42° C. for 1 hour, and heat treated at 80° C. for 15 minutes to inactivate the reverse transcriptase. PCR (Polymerase Chain Reaction) is Accupower Polymerization was carried out using 1 μg of cDNA reverse transcribed to PCR PreMix (Bioneer Co., Korea) tube and 10 p mol of Forward and Reverse Primer, respectively. The polymerization reaction conditions were initially heat-treated at 94°C for 5 minutes to denature cDNA, and then carried out as follows. Human primer IL-2 (s 5′-CCG GAG AGG AGA CTT CAC AG-3′ (SEQ ID NO: 1) as 5′-GGA AAT TGG GGT AGGAAGGA-3′ (SEQ ID NO: 2)) and GAPDH expression were 5 It was confirmed using'-CGG AG TCA ACG GAT TTG GTC GTA T-3' (sense) (SEQ ID NO: 3) and 5-AGC TTC TCC ATG GT GGT GAA GAC-3' (antisense) (SEQ ID NO: 4). The amplified PCR products were electrophoresed on 1% agarose gel to quantify the band using ImageJ 1.44d (Image J is a public domain Java image processing program inspired by NIH Image).

<5-5> Analysis of the effect of black ginseng extract and black ginseng-derived compounds on cytotoxicity in Jurkat T cells

Jurkat Tcell (5 X 10 ⁵ Cell/ml) was dispensed into a 48 well plate and cultured in a 5% carbon dioxide incubator at 37°C for one day. Each well was cultured in DMEM culture solution containing 10% fetal bovine serum and 1% antibiotic, and then treated with reagents. As a negative control, dimethyl sulfoxide (DMSO) was added as a solvent. After culturing in a 5% carbon dioxide incubator at 37°C for one day, the degree of growth of the cells was confirmed by the MTT method. Add 100 ul of 0.1% MTT (Sigma M2128) solution to each well of a 48 well culture vessel and react in a 5% carbon dioxide incubator at 37°C for 3 hours. After removing the culture solution and MTT solution, 100 ul of dimethyl sulfoxide is added to dissolve the formed crystals. Absorbance was measured at 540 nm using a microplate reader. Jurkat Tcell was incubated for 12 hours in a culture medium containing RPMI + 10% FBS. The extract was treated at a concentration of 200 ug/ml and 7 kinds of compounds at a concentration of 50 ug/ml. After 24 hours, 1 mg/ml of MTT reagent (Sigma M2128) was added, and absorbance was measured at 540 nm using a microplate reader after 4 hours. The results of Jurkat T cells are as follows, and the results of cell viability in extracts and compounds are as follows, and the best inhibitory activity was shown in 70% black ginseng extract (FIG. When the compound was treated at a concentration of 50 ug/ml, significant effects were confirmed in all compounds except MC, and particularly good activity was confirmed in Rh4, Rk1, Rg5, and compound O (Fig. 3B).

<5-6> Results of inhibition of IL-2 mRNA expression of extracts and compounds by PMA/A23187 treatment in Jurkat cells

In Jurkat T cells, PCR products for mRNA were quantified using a semiquantitative RT-PCR method. Reverse transcription reaction is performed by accupowering the extracted total RNA (1 μg) and oligonucleotide dT primer (100 p mol).After mixing in RT PreMix (Bioneer Co, Korea) tube, the total volume of the reaction was 20 μl and pretreated at 70° C. for 5 minutes. The cDNA synthesis was reacted at 42° C. for 1 hour, and heat treatment was performed at 80° C. for 15 minutes to inactivate the reverse transcriptase. PCR (Polymerase Chain Reaction) is Accupower  Polymerization was carried out using 1 μg of cDNA reverse transcribed to PCR PreMix (Bioneer Co., Korea) tube and 10 p mol of Forward and Reverse Primer, respectively.

In addition, after stimulation with PMA (200 nM) A23187 (1 uM), the concentration of extract 200 ug/ml and 7 types of compounds were pretreated with 50 ug/ml and harvested 6 hours later to extract RNA, and the concentration was measured. After the analysis, IL-2 was analyzed by RT-PCR.

As a result, it was confirmed that interleukin 2 (IL-2) elevated after treatment with PMA (200 nM) A23187 (1 uM) was inhibited in 70% black ginseng extract (FIG. 4A).

Further, it was confirmed that IL-2 was inhibited by treatment with Rh4, Rk1, Rg5, and compound O (Fig. 4B).

Claims (11)

A pharmaceutical composition for enhancing immunity comprising black ginseng extract as an active ingredient. A pharmaceutical composition for enhancing immunity comprising a compound isolated from black ginseng extract as an active ingredient. The method of claim 2, wherein the compound is:
[Formula 1]

,
[Formula 2]

,
[Formula 3]

,
[Formula 4]

,
[Formula 5]

,
[Formula 6]

or
[Formula 7]

A pharmaceutical composition for enhancing immunity comprising a. A health functional food composition for improving immunity comprising black ginseng extract as an active ingredient. A health functional food composition for enhancing immunity comprising a compound isolated from black ginseng extract as an active ingredient. A feed composition for enhancing immunity containing black ginseng extract as an active ingredient. A feed composition for enhancing immunity comprising a compound isolated from black ginseng extract as an active ingredient. a) drying ginseng;
b) increasing the temperature of the dried ginseng from room temperature to 50°C to 80°C to increase ginseng;
c) re-evaporating the ginseng steamed at 50 to 80°C by raising the temperature to 80 to 110°C;
d) repeating steps b) and c) 2 to 6 times to prepare black ginseng; And
e) extracting the black ginseng extract from the black ginseng using an extraction solvent;
Method for producing a black ginseng extract comprising a. a) drying ginseng;
b) increasing the temperature of the dried ginseng from room temperature to 50°C to 80°C to increase ginseng;
c) re-evaporating the ginseng steamed at 50 to 80°C by raising the temperature to 80 to 110°C;
d) repeating steps b) and c) 2 to 6 times to prepare black ginseng;
e) extracting the black ginseng extract from the black ginseng using an extraction solvent; And
f) Separating and purifying the compound using an extraction solvent in the black ginseng extract; method for producing a compound isolated from the black ginseng extract comprising. a) drying ginseng;
b) increasing the temperature of the dried ginseng from room temperature to 50°C to 80°C to increase ginseng;
c) re-evaporating the ginseng steamed at 50 to 80°C by raising the temperature to 80 to 110°C;
d) repeating steps b) and c) 2 to 6 times to prepare black ginseng; And
e) extracting the black ginseng extract from the black ginseng using an extraction solvent;
A method for producing a feed composition for enhancing immunity comprising a. The method of claim 10,
The step e) is a step of separating and purifying the compound using an extraction solvent in the black ginseng extract; further comprising, a method for producing a feed composition for enhancing immunity.

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