KR20180128365A - Composition for immunity stimulatory activity Using a Water Extract of Orostachys japonicus - Google Patents

Abstract

The present invention relates to a composition for immunity enhancement using a water extract of Orostachys japonicus, which promotes the production of NO in a concentration-dependent manner in RAW 264.7 cells, promotes the production of cytokines such as TNF-α, IL-6, and IL-10 in a concentration-dependent manner, and also promotes the expression of iNOS in a concentration-dependent manner. The water extract of Orostachys japonicus of the present invention is obtained by boiling Orostachys japonicus in water for 18 hours or more at room temperature.

Description

TECHNICAL FIELD [0001] The present invention relates to a composition for enhancing immunity using an extract of water,

The invention wasong (Orostachys japonicus ) water extract.

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).

The innate immunity, also called natural immunity, is composed of leukocytes including macrophages, natural killer cells, and various cytokines such as TNF-α. In addition to infectious pathogens such as microorganisms and virus invasion, (Biochem Biophys Res Commun 157: 87-94, 1998; J Clin Invest 79: 319-326, 1987; Biol. Pharm. Bull, 27: 617, 2004). Acquired immunity is also known as adaptive immunity, which consists of recognizing antigen presented by antigen-presenting cells and recognizing T cells, and consists of cell-mediated immunity involving T cells and humoral immune response by antibodies produced by B cells Korean J Food Nutr 21: 275-282, 2008).

Macrophages play the most important role in innate immunity. Macrophages are the first to recognize foreign substance invasion and are involved in humoral immunity and cellular immunity. When activated, they increase proliferation and spreading ability, (Food Ind Nutr 5: 1), which is known to secrete cytokines such as nitric oxide, TNF-α, IL-1β and IL-6 to inhibit the growth of infectious agents such as various microorganisms and viruses, J Clin Imm 79: 319-326, 1987, Biol. Pharm. Bull. 27: 617, 2004, J. Immunol. 144: 1425, 1990. Biochem Biophys Res Commun 157: 87-94 Proc. Soc. Exp Biol Med 211: 24, 1996, J. Exp. Med. 181: 559, 1995, J. Exp. Med. 181: 559, 1995).

As such, the human body is designed to maintain the homeostasis against intrusion or infection of foreign substances such as external antigens by appropriately fighting innate immune and acquired immunity.

( Orostachys japonicus A. Berger) is a perennial herbaceous plant of Crassulaceae distributed in Korea, China, Japan, etc. In Korea, Orostachys japonicus A. Berger), round rocks ( Orostachys malacophyllus Fisch), Orostachys sikokianus Ohwi, and Orostachys iwarenge (Makino) H. Hara) are growing wildly (Lee, Changbok, "The Botanical Illustrated Book", Anal., 1993).

(38.2%), crude oil (15.8%), crude protein (13.2%) and reducing sugar (12.%), and aluminum (Al) , Minerals such as calcium (Ca), copper (Cu), iron (Fe), potassium (K), magnesium (Mg), manganese (Mn), sodium (Na) And the content of calcium and potassium is high. It has long been used as a cancer treatment, and pharmacologically has been known to have vasoconstriction, respiratory stimulation, and intestinal enhancement (M. It has long been used as a folk remedy for the treatment of cancer (Lee Nam Sun, Master Thesis, Chungbuk National University, Department of Oriental Medicine Resources, 2015). In addition, it has been reported that WASONG exhibits various physiological activities such as antioxidant effect [12], antibacterial effect [13], cell suicide induction effect [14] and anticancer effect [16] (J Kor Soc Food Sci Nutr 37 J Kor Soc Food Sci Nutr 43 (1): J Kor Soc Food Sci Nutr 38 (1): 14-18, 2009; Kor J Medicinal Corp Sci 12 (4): 315-320, 67-73, 2014).

The present invention discloses an immunostimulatory activity of < RTI ID = 0.0 >

It is an object of the present invention to provide a composition for enhancing immunity using a water-extract.

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

The inventors of the present invention found that when water extract, 30% ethanol extract, 50% ethanol extract, 70% ethanol extract and 100% ethanol extract of watsong were treated with RAW 264.7 cells at a concentration of 50, 100 and 200 ㎍ / , Water extract alone promotes the production of NO (nitric oxide), and the water extract obtained by setting the extraction temperature at room temperature, 30 ° C, 60 ° C, and 90 ° C and the extraction time of 24 hours, 12.5, 25, and 50 ㎍ / mL, RAW 264.7 cells were treated with room temperature extract, and the extraction temperature was maintained at room temperature. The extraction time was 6 hours, 12 hours, 18 hours, When water extracts obtained from 24 hours were also treated with RAW 264.7 cells at the lower concentrations of 12.5, 25 and 50 ㎍ / mL, only water extracts of 18 hours and 24 hours were found to be effective. In addition, the 24-h water extract promoted cytokine production such as TNF-α, IL-6 and IL-10 in RAW 264.7 cells in a concentration-dependent manner and promoted the expression of iNOS in a concentration-dependent manner.

The immunoconjugation composition of the present invention is provided on the basis of the results of the experiment and is characterized in that it comprises an extract of water as an active ingredient and the extract of the extract is obtained by leaching water at room temperature for 18 hours or more .

In the present specification, the term " extract " means a crude extract immediately after extraction, a filtrate to extract the extracted residue, a crude extract or an extract filtrate to concentrate the extracted solvent by freeze drying, vacuum drying, hot air drying, spray drying, Or a solid form of the extract.

In the present specification, the term " room temperature " refers to the temperature at which the artificial heat is not applied to the extraction site at the time of extraction, that is, the natural state of the extraction site.

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 active ingredient may be contained in an arbitrary amount (effective amount) as long as it can exhibit an immunostimulating effect, and a typical effective amount is 0.0001 To 15% 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 immunological 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 increasing or enhancing the immune 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. Examples of such compounds or extracts include L-glutamine, germanium yeast, ginseng mushroom, Angelica japonica extract, Cordyceps mellifera extract, Spirulina, Chlorella, etc. as individual approved raw materials according to the Korean Health Functional Food Code or the Korean Health Functional Food Act , A purified product of cultivating a soybean curd fungus (potassium polygamargatate), 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 an extract of dried fish.

The composition for enhancing immunity of the present invention can be made into a food or medicine.

FIG. 1 shows the results of the NO production promoting activity of each extract solvent (extraction temperature at room temperature, extraction time 24 hours).
Fig. 2 shows the results of the NO production promoting activity of the water extract (extraction time: 24 hours) by the extraction temperature.
Fig. 3 shows the results of the NO production promoting activity of the water extract (extraction temperature at room temperature) by extraction time.
Figs. 4 to 6 are the results of the water extract for 24 hours at room temperature showing cytokine production promoting activity such as TNF-a, IL-6 and IL-10.
FIG. 7 shows the measurement results of the effect of water extract for 24 hours at room temperature on the expression of iNOS and COX-2.

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 > Welcome  Immune enhancing activity of water extract

1. Samples and Experimental Methods

1-1. Sample production

The samples were washed 2 ~ 3 times with distilled water, and after removing the water, they were dried with hot air, and then transferred to a grinding machine. And used as a sample. Extracts were extracted with 100% water and 30%, 50%, 70% and 100% of ethanol (EtOH). That is, the dried powder was repeatedly extracted once per solvent condition and filtered. The extract was concentrated under reduced pressure and completely dried with a freeze dryer to prepare an extract for each solvent condition.

1-2. Cell culture

Receive pre-sale a Murine macrophage cell line RAW 264.7 cells from KCLB (Korean Cell Line Bank) 100 units / mL penicilin-streptomycin and 10% fetal bovine serum (FBS) is by using a DMEM medium containing 37 ℃, 5% CO ₂ thermostat , And subculture was performed once every three days. Lipopolysaccharide (LPS, E, coli serotype 0111: B4) was purchased from Sigma.

1-3. Nitric oxide (NO) assay

RAW 264.7 cells (1.5 × 10 ⁵ cells / mL) were inoculated into a 24-well plate using DMEM medium and cultured for 24 h at the same time with fresh medium containing LPS (1 μg / mL). The amount of NO produced was measured in the form of NO ₂ ^- present in the cell culture medium using Griess reagent. 100 μl of cell culture supernatant and 100 μl of Griess reagent [1% (w / v) sulfani-lamide, 0.1% (w / v) naphylethylenediamine in 2.5% (v / v) phosphoric acid] The absorbance was measured at 540 nm using an ELISA reader. Standard concentration curves were obtained by serial dilution of sodium nitrite (NaNO ₂ ).

1-4. Immune regulatory bio Marker Inn  Assessment of inhibitory effect on cytokine production

RAW 264.7 cells were adjusted to 1.5 × 10 ⁵ cells / mL using DMEM medium, inoculated into 24-well plates, and incubated 18 hours before in a 5% CO ₂ incubator. After the medium was removed, RAW 264.7 cells were treated at the same time with 50 μl of extract samples prepared at 10 times concentration and 450 μl of LPS (1 μg / ml) at the same time. After 24 hours, the cytokine production of the supernatant obtained by centrifuging the culture medium (12,000 rpm, 3 min) was measured. All samples were stored at -20 ° C or lower before quantification. Quantitation of cytokines was quantitated using a mouse enzyme-linked immunosorbent assay (ELISA) kit (R & D System Inc., Minneapolis, MN, USA). The r ² value of the standard curve for the standard was 0.99 or more.

1-5. Immuno  blot analysis

RAW 264.7 cells (1.0 × 10 ⁶ cells / mL) were cultured for 18 hours before stimulation with LPS (1 μg / mL), and the samples were treated at the same time and cultured for 24 hours under the same conditions as the pre-culture. After incubation, the cells were washed 2-3 times with PBS (phosphate buffered saline), and lysed in 300 μl of lysis buffer for 30 minutes to 1 hour. After centrifugation (15,000 rpm, 15 min) . Protein concentration was quantified by standardizing bovine serum albumin (BSA) using the Bio-Rad Protein assay kit. 20 ~ 30 ㎍ of lysate was denatured by 8% mini gell SDS-PAGE (polyacrylamide gel electrophoresis) and transferred to PVDF membrane (BIORAD) at 200 mA for 2 hours. The blocking of memvrane was performed in TTBS (TBS + 0.1% Tween 20) solution containing 5% skin milk at room temperature for 2 hours. (BD Biosciences, San Jose, Calif., USA) as anti-mouse iNOS (Calbiochem, La Jolla, CA, USA) and anti-goat COX-2 as a primary antibody were used to measure the expression levels of iNOS and COX- (1: 1000), reacted at room temperature for 2 hours, and washed three times with TTBS. Anti-mouse IgG conjugated with horse radish peroxidase (HRP) and anti-rabbit IgG (Cell Signaling Technology, Beverly, Mass., USA) were diluted 1: 5000 and reacted at room temperature for 30 min. (Amersham Biosciences, Piscataway, NJ, USA) for 1 min and then exposed to X-ray film.

1-6. Statistical analysis

All experiments were repeated 3 times or more. The mean value SD (SD) was calculated according to each item and the statistical significance was evaluated at 95% confidence level (p <0.05).

2. Experimental results

2-1. NO production promoting activity

The results of the NO production promoting activity of each extractant extract (extraction temperature at room temperature, extraction time 24 hours) are shown in FIG. 1, and the results of the NO production promoting activity of water extract (extraction time 24 hours) FIG. 2 shows the results of the water extract (extracting temperature at room temperature) for promoting NO production by extraction time.

Referring to FIG. 1, the extract of the extracting solvent such as 30% ethanol showed almost no NO production promoting activity, but the water extract showed higher NO production promoting activity than LPS.

Also, referring to FIG. 2, the room temperature water extract showed high NO production promoting activity and did not further promote NO production when treated with LPS.

Also, referring to FIG. 3, the water extract showed marked NO production promoting activity from the time when the extraction time was 18 hours or more, and the NO production was not further promoted by the simultaneous treatment with LPS, as in the result shown in FIG.

2-2. Cytokine production-promoting activity

The results of water extract for 24 hours at room temperature on cytokine production promoting activity such as TNF-a, IL-6 and IL-10 are shown in Fig. 4 to Fig.

4 to 6, water extracts at room temperature for 24 hours showed TNF-?, IL-6 and IL-10 production-promoting activities in a concentration-dependent manner, and when co-treated with LPS, TNF- ?, IL -6 and &lt; RTI ID = 0.0 &gt; IL-10. &Lt; / RTI &gt;

2-3. iNOS  And COX-2 production promoting activity

The results of measurement of the effect of water extract at room temperature for 24 hours on the expression of iNOS and COX-2 are shown in Fig. Water extracts at room temperature for 24 hours did not affect the production of COX-2 but stimulated iNOS production in a concentration-dependent manner.

Claims (3)

Wherein the extract is an extract obtained by extracting water with water for at least 18 hours at room temperature.
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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