KR20190030405A - Composition for immunity stimulatory activity Using an Enzyme Digest of Sargassum horneri - Google Patents

Abstract

The present invention provides a composition for improving immunity using enzyme treated Sargassum horneri, which promotes the proliferation of immunocytes in a mouse spleen and also stimulates the secretion of cytokine such as IL-1β, IL-2, and IL-10. The composition for improving immunity includes a Celluclast enzyme-treated Sargassum horneri as an active ingredient.

Description

TECHNICAL FIELD The present invention relates to a composition for enhancing immunity using an enzyme-

The present invention relates to a composition for enhancing immunity using an enzyme-treated product of Sargassum horneri .

Immunity is a series of self-protection mechanisms that occur when a substance other than its own constituents destroys or threatens the homeostasis of the body. It is divided into innate immunosurveillance and adaptive immunosurveillance (Korean J Oriental Physiol Pathol 23: 1385-1391, 2009).

Congenital immunity, also known as innate immunity, consists of leukocytes, including macrophages and natural killer cells, various cytokines such as TNF-α, mucus, and complement in blood, (Biochem Biophys Res Commun 157: 87-94, 1998; J Clin Invest.). In addition, the present invention provides a method for preventing and / or treating infectious pathogens such as microorganisms, virus infections and the like, as well as phagocytosis against aged normal cells and cancer cells 79: 319-326, 1987, Biol.Pharm.Bull. 27: 617, 2004). Acquired immunity, which plays a role in reinforcing innate immunity, is also called adaptive immunity. It is known that T-cells recognize antigen presented by predation of antigen presenting cells, and cell immunity involving T cells and antibodies produced by B cells (Korean J Food Nutr 21: 275-282, 2008).

Thus, the human body is designed to maintain homeostasis by appropriately countering innate immune and acquired immunity against invasion or infection of foreign substances such as external antigens (Food Ind Nutr 5: 21-26, 2000; Biochem Biophys Res Commun 157: 87-94, 1998).

The spleen is an important immune organ composed of various immune cells such as lymphocytes, macrophages and natural killer cells. The spleen is an immune response to blood-derived antigens and also acts to remove aged and damaged cells.

In congenital immunity, the macrophages present in the spleen play the most important role. Macrophages recognize the foreign substance invasion first and are involved in humoral immunity and cellular immunity. When activated, they increase the proliferation and spreading ability, (TNF-α, IL-1β, and IL-6) to inhibit the proliferation of various microorganisms, viruses and other infectious agents and to exhibit toxicity against cancer cells Ind Nutr 5: 21-26, 2000; Biochem Biophys Res Commun 157: 87-94, 1998; J Clin Invest 79: 319-326,1987, Biol.Pharm.Bull.27: 617, 2004. J. Immunol 144 : 1425, 1990, Proc. Soc. Exp Biol Med 211: 24, 1996, J. Exp. Med. 181: 559, 1995, J. Exp. Med. 181: 559, 1995).

In recent years, attempts have been made to search for the effect of enhancing the immunity of natural materials capable of being edible in Korea, and it has been reported that buckwheat, dolmens, ginger, red pepper, and red pepper have activities for enhancing immune cell function or promoting proliferation (J. Bacteriolo, Virol 28: 419-430, 1993; J. Kor. Diet Assoc. 11: 44-50 2005; J. Kor. Soc. Food Sci. Nutr. 31: 87-91. 2002; J. Food & Nutr. Vol. 21: 263-268, 2008).

Sargassum horneri is a perennial brown algae with hatchlings and is widely distributed in Korea, Japan and China. It is known as a major constituent of the floating moth that travels by currents to the East Sea and the Sea of Japan. It has been reported that Hornsey's mushroom has various pharmacological effects and physiological functions that contain various ingredients such as fucoidan and alginic acid as well as minerals, polyphenols and vitamin K, which are good for health and beauty. Fucoidan, a component of the sulfate polysaccharide, inhibits the growth of colon cancer cells (Shao P et al., Int J Biol Macromol 2015, 73: 124-130). It is known that LPS-stimulated macrophage cells reduce oxidative stress (Wen ZS et al., Int J Biol Macromol 2014, 68: 98-106).

The present invention discloses the immunostimulatory activity of such Houttuynia cordata enzyme treated.

It is an object of the present invention to provide a composition for enhancing immunity using a hydrogel-treated product of hoe saengmyung.

Other and further objects of the present invention will be described below.

As shown in the following Examples and Experimental Examples, the inventors of the present invention found that Houttuynia cordata cellulase treatment promotes proliferation of mouse spleen immunocytes and inhibits IL-1β, IL-2, IL-10 And promoting the secretion of cytokines such as.

The composition for improving immunity of the present invention is provided on the basis of the results of these experiments and is characterized in that it contains an enzyme-treated product of hoe saengmyung cellulase as an active ingredient.

As used herein, the term " hydrolyzed cellulase of hornbill seeds " means obtained by treating cellulase, a kind of cellulase, in the powder of hornblende stem, leaf, root, outpost or mixture thereof. The enzyme-treated product may be used as it is or after taking the supernatant after centrifugation. Alternatively, the enzyme-treated product or the supernatant thereof may be used in a liquid or powder form by concentration under reduced pressure and / or lyophilization, , Or the extract may be fractionated using an organic solvent having a different polarity, or the extract or fraction may be concentrated and used. The organic solvent may be selected from the group consisting of lower alcohol having 1 to 4 carbon atoms including ethanol, methylene chloride, ethylene, acetone, hexane, ether, chloroform, ethyl acetate, butyl acetate, N, N-dimethylformamide (DMF), dimethylsulfoxide ), 1,3-butylene glycol, propylene glycol, mixed solvents thereof, and the like.

In the present specification, cellulose is an enzyme separated from fungus Trichoderma reesei , which is a kind of cellulase that decomposes cellulose into glucose, cellobiose and glucose polymer.

In the present specification, the term " active ingredient " alone means an ingredient which exhibits the desired activity or which can exhibit activity together with a carrier which is not itself active.

 In the composition for enhancing immunity of the present invention, the effective ingredient may be contained in an arbitrary amount (effective amount) as long as it can exhibit an intended immunity enhancing effect, and a usual effective amount is based on the total weight of the composition To < RTI ID = 0.0 > 15% < / RTI > by weight. The term " effective amount " as used herein refers to an amount of the compound of the present invention which is capable of exhibiting an intended immunity-enhancing effect when the composition of the present invention is administered to a mammal, preferably a human, Of the active ingredient. Such effective amounts can be determined experimentally within the ordinary skill of those skilled in the art.

The composition of the present invention may further contain, in addition to the active ingredient, any compound or natural extract which has already been proven in the art for its safety and ascertained its activity for the purpose of raising or reinforcing the immunity enhancing effect.

Such compounds or extracts include compounds or extracts listed in the official pamphlet of each national pharmacopeia ("Korea Pharmacopoeia" in Korea), each country's health functional foods (in Korea, "health functional food standards and specifications" The laws of each country governing the manufacture and sale of compounds or extracts and health functional foods licensed under the laws of each country that regulate the manufacture and sale of pharmaceuticals (in Korea, the "Pharmaceutical Affairs Law") Laws and regulations "), and compounds or extracts that are individually recognized for their functionality. For example, such compounds or extracts have been recognized as having immunity enhancing function according to the Korean Health Functional Food Act, and include L-glutamine, germanium yeast, ginseng mushroom, Angelica gigantus extract, Cordyceps mellifera extract, Spirulina, Chlorella, (Polygamma glutamate potassium), mycelium of shiitake mushroom, yeast beta-glucan, and the like.

The composition of the present invention can be identified as a food composition in a specific embodiment.

The food composition of the present invention can be prepared in any form and can be used in various forms such as beverages such as tea, juice, carbonated beverage, ionic drink, processed milk such as milk and request route, gum, rice cake, Such as confectionery, cotton, etc., tablets, capsules, rings, granules, liquids, powders, flakes, pastes, syrups, gels, jellies, bars and the like. In addition, the food composition of the present invention may be classified into any product category as long as it meets the laws and regulations on the time of manufacture and distribution in the legal and functional category. For example, it is a health functional food according to the "Health Functional Food Act" in Korea or the food standard (standard and standard of food notices) of the Food Sanitation Act of Korea. Beverages, special-purpose foods, and the like.

The food composition of the present invention may contain food additives in addition to the active ingredients thereof. Food additives are generally understood to be substances that are added to foods and mixed or infiltrated into food in the manufacture, processing or preservation of food, and their safety must be ensured since they are ingested daily with food and for long periods of time. Food additives according to national laws regulating the manufacture and distribution of food (in Korea, the "Food Sanitation Act") are stipulated as safety-guaranteed food additives in terms of ingredients or function. In the Food Additives Code of Korea ("Food Additives Standards and Standards"), food additives are defined as chemical compounds, natural additives and mixed preparations in terms of ingredients. Preservatives, emulsifiers, acidulants, and thickeners.

The sweetener is used for imparting a sweet taste suitable for food, and both natural and synthetic sweeteners can be used in the food composition of the present invention. Preferably, natural sweeteners are used. Examples of natural sweeteners include sugar sweeteners such as corn syrup solids, honey, sucrose, fructose, lactose and maltose.

Flavors are used to improve taste and flavor, and natural and synthetic flavors can be used. Preferably, a natural one is used. When using natural ones, the purpose of nutritional fortification can be performed in addition to the flavor. Examples of natural flavoring agents include those obtained from apples, lemons, citrus fruits, grapes, strawberries, peaches, and the like, or those obtained from green tea leaves, Asiatica, Daegu, Cinnamon, Chrysanthemum leaves and Jasmine. Also, those obtained from ginseng (red ginseng), bamboo shoots, aloe vera, banks and the like can be used. The natural flavoring agent may be a liquid concentrate or a solid form of extract. If desired, a synthetic flavor agent may be used. As the synthetic flavor agent, esters, alcohols, aldehydes, terpenes and the like may be used.

As the preservative, calcium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, sodium benzoate, potassium benzoate, EDTA (ethylenediaminetetraacetic acid) and the like can be used, and as the emulsifier, acacia gum, carboxymethyl cellulose, xanthan gum, pectin And acidulant, malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartaric acid, ascorbic acid, acetic acid, phosphoric acid and the like may be used as the acidulant. The acidulant may be added so that the food composition has a proper acidity for the purpose of inhibiting the growth of microorganisms other than the purpose of enhancing the taste. Examples of the thickening agent include suspending agents, sedimentation agents, gel-forming agents, bulking agents and the like.

The food composition of the present invention may contain physiologically active substances or minerals which are known in the art and which are stable as a food additive, for the purpose of supplementing and reinforcing the functionality and nutrition, in addition to the above-mentioned food additives.

Examples of such physiologically active substances include catechins contained in green tea and the like, vitamins such as vitamin B1, vitamin C, vitamin E and vitamin B12, tocopherol, dibenzoyl thiamine, etc. Examples of minerals include calcium preparations such as calcium citrate, magnesium stearate , Iron preparations such as iron citrate, chromium chloride, potassium iodide, selenium, germanium, vanadium, zinc and the like.

The food composition of the present invention may contain an appropriate amount of the above-mentioned food additives according to the product type so as to achieve the purpose of addition thereof.

With regard to other food additives that may be included in the food composition of the present invention, reference may be made to the Food Code or the Food Additive Code of the respective country.

In another specific embodiment, the composition of the present invention can be identified as a pharmaceutical composition.

The pharmaceutical composition of the present invention may be prepared into oral formulations or parenteral formulations according to the route of administration by conventional methods known in the art, including pharmaceutically acceptable carriers in addition to the active ingredient. Where the route of administration may be any suitable route including local routes, oral routes, intravenous routes, intramuscular routes, and direct absorption through mucosal tissues, and combinations of two or more routes may be used. An example of a combination of two or more routes is a combination of two or more formulations of the drug according to the route of administration, for example, one drug is administered intravenously and another drug is administered via a local route.

Pharmaceutically acceptable carriers are well known in the art depending on the route of administration and formulation, and specific reference may be made to the pharmacopoeia of each country, including the " Korean Pharmacopoeia ".

When the pharmaceutical composition of the present invention is prepared into an oral formulation, it may be formulated into powder, granules, tablets, pills, sugar tablets, capsules, solutions, gels, syrups, suspensions, wafers And the like. Examples of suitable carriers include starches such as lactose, glucose, sucrose, dextrose, sorbitol, mannitol and xylitol, corn starch, potato starch and wheat starch, cellulose, methylcellulose, ethylcellulose, sodium carboxymethylcellulose, Hydroxypropylmethylcellulose and the like; polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate, mineral oil, malt, gelatin, talc, polyol, vegetable oil, ethanol Serol, and the like. In case of formulation, suitable binders, lubricants, disintegrants, coloring agents, diluents and the like may be included as needed. Examples of suitable binders include starch, magnesium aluminum silicate, starch pellets, gelatin, methyl cellulose, sodium carboxymethyl cellulose, polyvinyl pyrrolidone, glucose, corn sweetener, sodium alginate, polyethylene glycol, wax and the like. Examples of the disintegrating agent include starch, methylcellulose, magnesium stearate, magnesium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, silica, talcum, stearic acid, Agar, bentonite, xanthan gum, starch, alginic acid or its sodium salt, and the like. Examples of the diluent include lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, glycine and the like.

When the pharmaceutical composition of the present invention is prepared into a parenteral dosage form, it may be formulated in the form of an injection, transdermal drug delivery, nasal aspirate and suppository together with a suitable carrier according to methods known in the art. As the carrier suitable for injection preparation, aqueous isotonic solutions or suspensions may be used. Specifically, PBS (phosphate buffered saline) containing triethanolamine, sterile water for injection, and isotonic solution such as 5% dextrose may be used . When formulated with a transdermal preparation, it can be formulated in the form of ointments, creams, lotions, gels, external liquids, pastes, liniments, and air-lozenges. Nasal inhalers may be formulated in the form of aerosol sprays using suitable propellants such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, etc., and when formulated as a suppository, witepsol, tween 61, polyethylene glycols, cacao butter, laurin, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, and sorbitan fatty acid esters.

The formulation of pharmaceutical compositions is well known in the art and can be found, for example, in Remington ' s Pharmaceutical Sciences (19th ed., 1995). This document is considered part of this specification.

The preferred dosage of the pharmaceutical composition of the present invention is 0.001 mg / kg to 10 g / kg per day, preferably 0.001 mg / kg to 1 g / day, depending on the patient's condition, body weight, sex, age, / kg < / RTI > The administration can be carried out once or several times a day. Such dosages should in no way be construed as limiting the scope of the invention.

INDUSTRIAL APPLICABILITY As described above, according to the present invention, it is possible to provide a composition for enhancing immunity using a hydrogel-treated enzyme (Selucast).

The composition for improving immunity of the present invention can be made into a product such as food or medicine.

FIG. 1 shows the results of evaluating the effect of the hydrogel-treated enzyme on the survival rate of immune cells.
FIG. 2 shows the results of evaluating the effect of the hydrogel-treated enzyme on the proliferation rate of immune cells.
FIG. 3 shows the results of evaluating the effect of the hydrogel-treated enzyme on the number of CD4 ⁺ T cells and CD8 ⁺ cells.
FIG. 4 shows the results of evaluating the effect of the hydrogel-treated enzyme on the secretion of cytokines (IL-1β, IL-2 and IL-10) in immune cells.

Hereinafter, the present invention will be described with reference to Examples and Experimental Examples. However, the scope of the present invention is not limited to these examples and experimental examples.

< Example > Immune enhancement activity of the hydrolyzed enzyme

1-1. sample

Between March and April 2016, Sargassum horneri was collected from the coast of Jeju Island, Korea and washed with water to remove salt and other impurities. After lyophilization, the mixture was pulverized using a pulverizer, and 2 g of the sample was homogenized in 100 ml of distilled water and mixed with 20 mg of celluclast to induce the enzyme reaction at pH 4.5 and 50 ° C. After 24 hours, the samples were centrifuged at 2,774 xg for 10 minutes and filtered using a filter paper (Watman No.4, GE Healthcare, Buckinghamshire, UK) to obtain supernatant. It was lyophilized and obtained in powder form and then quantified and used in the experiment. It was named as SHC ( Sargassum horneri celluclast extract).

1-2. Prepare mouse spleen cell suspension

The spleen of C57BL / 6 mice at 8-10 weeks of age was harvested and a single cell suspension obtained through a cell filter was obtained. The cells were treated with ACK (ammonium-chloride-potassium) buffer and incubated at room temperature for 10 minutes to dissolve the red blood cells. After washing with 10% fetal bovine serum (Thermo Fisher Scientific) and 1% Penicillin / Streptomycin (Thermo Fisher Scientific), the cells were washed with phosphate buffered saline (DPBS, Thermo Fisher Scientific, MA, USA) And the cells were incubated in a cell culture incubator (Thermo Fisher Scientific) maintained at 37 ° C in 5% carbon dioxide.

1-3. Evaluation of cytotoxicity in mouse peripheral immune cells

Mouse spleen cells were seeded at 1 × 10 ⁵ cells in a 96-well plate (Thermo Fisher Scientific) and cultured for 24 or 48 hours with 0 to 500 μg / ml of SHC. After that, MTT (3- (4,5-Dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide, Sigma-Aldrich, ST, USA) was diluted to 5 mg / ml in DPBS and treated with 15 μl And then formazan crystals were formed by mitochondrial dehydrogenase reaction for 4 hours. After incubation, 100 μl of Solubilization buffer (10% sodium dodecyl sulfate in 50% dimethyl sulfoxide) was added to each well and absorbance was measured at 540 nm using an ELISA plate reader.

1-4. Evaluation of cell proliferation in mouse peripheral immune cells

Mouse splenocytes were seeded at 4 × 10 ⁵ cells in a 96-well plate (Thermo Fisher Scientific) and cultured for 54 hours at a concentration of SHC. Each well was treated with 1 μCi of ³ H-thymidine (42 Ci / mmol; Amersham, Little Chalfont, UK) and incubated for 18 hours. After incubation for 72 hours, the cells were captured on glass fiber, dried and the amount of radioactive isotopes ( ³ H) was measured using a radiation meter (Microbeta TriLux, PerkinElmer, MA, USA).

1-5. Evaluation of the effect on number variation in mouse peripheral immune cells

Mouse splenocytes were seeded at 4 × 10 ⁶ cells in a 24-well plate (Thermo Fisher Scientific) and cultured at 0 or 125 μg / ml for 48 hours. After subsequent out to harvest the cells washed with DPBS, purified anti-mouse CD16 / 32 mAb (5 ㎍ / ml) for 15 minutes at 4 ℃ reaction the process to reduce the non-specific reaction, 1 × 10 ⁶ of each tube (FITC labeled anti mouse CD3e (145-2C11), APC-Cy7 labeled anti mouse CD4 (H129.19), V500 labeled anti mouse CD8a (53-6.7), APC labeled CD25 PC61.5), BD Biosciences, CA, USA) and reacted at 4 ° C for 15 minutes. The cells were then washed with phosphate buffered saline (DPBS, Thermo Fisher Scientific), fixed in phosphate-buffered saline (DPBS, Thermo Fisher Scientific) containing 1% prafaformaldehyde and collected using a flow cytometer (BD LSRFortessa, BD Biosciences) And analyzed using BD FACSDiva Software (BD Biosciences).

1-6. Evaluation of cytokine (IL-1, IL-2, IL-10) secretion in mouse peripheral immune cells

ELISA (enzyme-linked immunosorbent assay) was performed to determine the effect of SHC on IL-1β, IL-2, or IL-10 production in mouse peripheral immune cells. Mouse splenocytes were seeded at 4 × 10 ⁶ cells in a 24-well plate (Thermo Fisher Scientific) and cultured for 48 hours at a SHC concentration of 0, 62.5, or 125 μg / ml. After 48 hours, the supernatant was harvested and tested according to the indicated method using an ELISA kit (BioLegend, CA, USA) capable of measuring the amount of IL-1β, IL-2, or IL-10 produced. The absorbance of the final product was measured at 450 nm using an ELISA plate reader.

2. Experimental results

2-1. Cytotoxicity Assessment

The results are shown in Fig. When SHC was treated with SPC for 24 or 48 hours, the cell viability was increased in a dose - dependent manner and increased to a maximum of 129%.

2-2. Assessment of cell proliferation

The results are shown in Fig. SHC was increased at the concentration of 125 g / ml compared to the control without any treatment. In particular, when the concentration of SHC was treated at a concentration of 125 g / ml, the cell proliferative capacity was the highest at about 8.3 Shipments.

2-3. Evaluation of effects on number change of peripheral immune cells

The results are shown in Fig. When SHC was treated at the concentration of 125 ㎍ / ml, which showed the highest proliferative capacity, there was no significant difference in total number of CD4 ⁺ T cells, but CD8 ⁺ T cells were significantly increased in comparison with the control group (25.8 ± 2.33% vs. 35.9 ± 3.04%, p <0.05).

2.4 Assessment of the effects on the secretion of cytokines (IL-1, IL-2, IL-10)

The results are shown in Fig. SHC increased the expression of cytokines such as IL-1, IL-2, and IL-10 in response to immune cell responses in a concentration-dependent manner.

Among cytokines, IL-2 is a growth, survival and differentiation factor in T cells and plays an important role in T cell response regulation. In addition, IL-1 is a biologically active cytokine, mainly known to be produced in activated macrophages. IL-10 is one of Th2-type cytokines secreted mainly by macrophages and T cells, especially regulatory T cells, and is known to be involved in the regulation of the immune response and the inflammatory response (Abbas AK et al. , Cellular and Molecular Immunology 8 ^th edition).

Claims (4)

A composition for enhancing immunity comprising an active ingredient of a hydrogel-treated cellulase of hornbill.
The method according to claim 1,
The cellulase enzyme-treated product is an enzyme-treated product, an extract obtained by centrifuging the enzyme-treated product, an extract obtained by concentrating the supernatant, an enzyme-treated product thereof, ethanol, or a mixed solvent thereof / RTI &gt;
The method according to claim 1,
Wherein the composition is a food composition.
The method according to claim 1,
Wherein the composition is a pharmaceutical composition.

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