KR20160079607A

KR20160079607A - Composition for anti-inflammation or immuno-stimulation containing an extract of a nut pine cone

Info

Publication number: KR20160079607A
Application number: KR1020150084425A
Authority: KR
Inventors: 김선영; 허성일; 한웅; 안원경
Original assignee: 재단법인 홍천메디칼허브연구소
Priority date: 2014-12-26
Filing date: 2015-06-15
Publication date: 2016-07-06

Abstract

The anti-inflammatory composition comprising the extract of Phellodendrons as an effective ingredient disclosed in this specification can effectively alleviate, treat or prevent inflammation by effectively inhibiting the production of NO and TNF-? Without toxicity. In addition, the immunostimulant composition comprising the extract of Phellodendrons as an effective ingredient disclosed in this specification can exhibit an effective immune enhancing effect by effectively proliferating macrophages and enhancing the expression or activity of IL-6 without toxicity.

Description

TECHNICAL FIELD [0001] The present invention relates to an anti-inflammatory or immunostimulating composition containing an extract of Phellodendron sunflower,

The present specification relates to the anti-inflammatory or immunostimulatory use of the extract of Picea magna.

Pinus koraiensis, also called a sea-son, belongs to Pinaceae. In pine nut seeds, 62.2% ~ 74.0% of fat is the most abundant, 16.4% is the protein, and other abundant inorganic minerals and vitamins such as carbohydrates and phosphorus. In addition, the leaf of the pine tree contains two alkaloid components, pichecoline and pyrene.

Many seeds of pine trees and plants come into seed pine, called pine cones, and the pine cones are called "pine nuts" in particular. Pine cones, including pine nuts, are used as a way to drink and soak up a unique flavor in a drink. In addition, although fully ripened pine nut is removed from the shell and used as a material for various foods, it is used as a feed for livestock such as pigs or fermented parts, (Korean Patent No. 10-2012-0106848), but most of them are being abandoned.

However, it is very difficult to dispose of pine nuts without using them because the pine nuts contain a considerable amount of essential oil components and other oil components that give off the pine nut aroma and contain a large amount of components that act pharmacologically, such as pine nut and pine nut. , One of the important forest resources, pine trees, is not yet fully utilized, and a method is needed that can be used more usefully.

Korean Patent Application No. 10-2012-0106848 (filed on September 26, 2012)

In one aspect, the object is to enhance immunity.

In another aspect, the object is to alleviate, treat, or prevent inflammation.

In one aspect, the present invention provides an immunostimulant composition comprising an extract of Phellodendron as an active ingredient.

In another aspect, the extract of the rice husk may be one that promotes the proliferation of immune cells.

In another aspect, the extract may be one that promotes the expression or activity of IL-6.

In one aspect, the present invention provides a anti-inflammatory composition comprising the extract of Picea magna as an active ingredient.

In another aspect, the extract of Picea japonica may inhibit the excessive production of NO and TNF-a.

In another aspect, the extract of Picea may be water or an extract of lower alcohol.

In another aspect, the extract of Pyralpine sp. May be an extract of ultrapure water or an aqueous solution of ethanol.

In another aspect, the extract of Picea may be an extract of Picea peel.

In one aspect, the composition of the present invention may be a cosmetic, a food or a pharmaceutical composition.

In one aspect, the anti-inflammatory or immunostimulating effect can be effectively obtained without toxicity.

It is possible to recycle the pellets that are discarded from the other side, thereby achieving the effect of resource saving or environmental protection.

Fig. 1 is a view showing a process for manufacturing a rice husk extract (at the time of 2L production).
FIG. 2 is a graph showing the cytotoxicity measurement results of the extract of Picea magna.
FIG. 3 is a graph showing the results of experiments for inhibiting the NO production ability of the extract of Picea magnesia.
Fig. 4 is a graph showing the results of measurement of TNF-a of the extract of Picea magnesia.
FIG. 5 is a graph showing the results of measurement of IL-6 activity of the extract of Picea magna.
FIG. 6 is a graph showing changes in the amount of TNF-.alpha.
FIG. 7 is a graph showing changes in IL-6 in mouse murine macrophages treated with the extract of Picea tulip.
FIG. 8 is a graph showing the effect of the extract of the rice husk on the proliferation of splenocytes.

In one aspect of the present invention, there is provided an immunostimulant composition comprising an extract of Picea magna as an active ingredient.

In the immunostimulant composition according to one aspect of the present invention, the extract of Picea may be an immunostimulating agent composition for promoting the proliferation of immune cells.

In one aspect of the present invention, the egg plant extract may be an immunostimulant composition that promotes the expression or activity of IL-6.

In one aspect of the present invention, there is provided a anti-inflammatory composition comprising the extract of Picea magna as an active ingredient.

In the anti-inflammatory composition according to one aspect of the present invention, the extract of Picea Capsicum may be a anti-inflammatory composition inhibiting the excessive production of NO and TNF- ?.

In the anti-inflammatory composition according to one aspect of the present invention, the extract of Zucchini spinach may be a composition which is an extract of water or a lower alcohol, specifically, a composition that is an extract of ultrapure water or an aqueous solution of ethanol.

In one aspect of the present invention, the immunostimulant or anti-inflammatory composition may be a composition that is an extract of the extract of the extract of the extract of the extract of the extract of the extract of the extract of the extract of the extract of the extract of P. japonica.

In one embodiment of the present invention, the present invention is a composition comprising an extract of Peppermint roots as an active ingredient.

As used herein, the term " extract " is a broad concept that encompasses all of the substances obtained by extracting components of natural products, regardless of the type of extraction method, extraction solvent, extracted component or extract.

In one aspect of the present invention, a method for preparing the extract of Picea Capsicum is as follows, but a conventional extraction method can be used, and the scope of the present invention is not limited thereto.

A method for preparing a rice husk extract, comprising the steps of: preparing a raw material for rice husk or rice husk by-products; Drying the raw material; And extracting the dried raw material with a solvent.

In one aspect of the present invention, the pine nut of the step of preparing the raw materials of the pine nut or the pine nut byproduct may be a pine nut of the common pine tree. The by-product of the pine nut is a by-product generated in the process of producing pine nut in the pine nut, Shells and the like. The raw material may be at least one selected from the group consisting of pine nut and pellet by-products. In the case of using the pine nut by-products in one aspect, the pine nut in the process of producing pine nut to obtain pine nut in the pine nut is recycled, It is effective.

In one aspect of the present invention, the step of drying the raw material comprises a one-stage drying step and may be carried out at a temperature of 40 ° C to 500 ° C for 1 minute to 240 hours.

In one embodiment, the drying step may comprise a primary, secondary, tertiary or quaternary drying step. The drying step may proceed to an appropriate order until the moisture is sufficiently dried.

In one embodiment, the temperature of the drying step is greater than or equal to 40 ° C, greater than or equal to 45 ° C, greater than or equal to 50 ° C, greater than or equal to 60 ° C, greater than or equal to 65 ° C, greater than or equal to 70 ° C, , 95 占 폚 or higher, 100 占 폚 or higher, or 105 占 폚 or higher.

In one embodiment, the temperature of the drying step may be less than 500 ° C, less than 450 ° C, less than 400 ° C, less than 350 ° C, less than 300 ° C, less than 250 ° C, less than 200 ° C, less than 150 ° C, or less than 105 ° C.

When the temperature of the drying step is within the above-mentioned range, sufficient drying is performed to increase the anti-inflammatory or immunological activity in the production of the extract of the pressed rice husk.

In one embodiment, the drying time is at least 1 minute, at least 5 minutes, at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 40 minutes, at least 50 minutes, at least 1 hour, at least 2 hours, Hour, more than 5 hours, more than 6 hours, more than 12 hours, and more than 24 hours.

In one embodiment, the drying time may be 240 hours or less, 216 hours or less, 192 hours or less, 168 hours or less, 144 hours or less, 120 hours or less, 96 hours or less, 72 hours or 48 hours or less.

When the drying time in the drying step is within the above range, sufficient drying is performed to increase the anti-inflammatory or immunological activity in the production of the extract.

In one aspect of the present invention, the step of extracting the dried raw material may be performed by extracting the dried raw material with one or more selected from the group consisting of water, an organic solvent, and a mixture of water and an organic solvent. The organic solvent includes, but is not limited to, at least one selected from the group consisting of alcohol, acetone, ether, ethyl acetate, diethyl ether, ethyl methyl ketone and chloroform. Wherein the alcohol comprises a C ₁ to C ₅ lower alcohol and the C ₁ to C ₅ lower alcohol is selected from the group consisting of methanol, ethanol, isopropyl alcohol, n-propyl alcohol, n-butanol and isobutanol But is not limited thereto.

In one embodiment, the water may be ultra-pure water.

In one embodiment, the organic solvent may be ethanol.

In one embodiment, the mixture of water and an organic solvent may be an aqueous solution of ethanol.

In one embodiment, the concentration of the aqueous ethanol solution is at least 0.01%, at least 0.1%, at least 1%, at least 5%, at least 10%, at least 15%, at least 20% 30 wt% or more, 35 wt% or more, 40 wt% or more, 45 wt% or more, or 50 wt% or more.

In one embodiment, the concentration of the aqueous ethanol solution may be up to 100 wt%, up to 90 wt%, up to 80 wt%, up to 70 wt%, or up to 60 wt%.

When the concentration of the aqueous ethanol solution is within the above range, the anti-inflammatory or immunological activity is increased in the production of the extract.

In one aspect of the present invention, the step of extracting the dried raw material may include a step of concentration under reduced pressure and a freeze-drying step.

In one embodiment, the reduced pressure concentration can be reduced to a pressure of 5 to 50 brix at a temperature of 40 to 90 占 폚.

In one embodiment, the temperature of the reduced pressure concentration may be at least 40 ° C, at least 45 ° C, or at least 50 ° C.

In one embodiment, the temperature for the reduced pressure concentration may be 90 ° C or lower, 85 ° C or lower, 80 ° C or lower, 75 ° C or lower, 70 ° C or lower, 65 ° C or lower, 60 ° C or 55 ° C or lower.

When the concentration under reduced pressure is within the above range, the anti-inflammatory or immunological activity is increased in the production of the extract of the rice husk.

The extract of the rice husk produced through the above-mentioned production process has an anti-inflammatory or immunological activity effect.

In one aspect of the present invention, there is provided a composition comprising, as an active ingredient, a rice husk extract having anti-inflammatory or immunostimulatory activity.

In one embodiment, the extract of Pyranthaioptera canis is at least 10 ug / mL, at least 20 / / mL, at least 30 / / mL, at least 40 / / mL, at least 50 / / mL, at least 100 / / ML or more, 250 μg / mL or more, 300 μg / mL or more, 350 μg / mL or more, 400 μg / mL or more, 450 μg / mL or more, mL or more, 700 μg / mL or more, 800 μg / mL or more, 900 μg / mL or more, or 1000 μg / mL or more.

In one embodiment, the Yatch Rice extract may be 100,000 g / mL or less, 50,000 g / mL or less, 10,000 g / mL or less and 5000 g / mL or less based on the total composition.

When the extract of Picea zodiaca is contained within the above range, the anti-inflammatory or immunostimulating effect can be effectively obtained without toxicity.

In one embodiment, the extract is a water-extracted extract, wherein the extract is at least 10 / / mL, at least 20 / / mL, at least 30 / / mL, at least 40 / / mL, at least 50 / / mL ML, at least 150 μg / mL, at least 200 μg / mL, at least 250 μg / mL, at least 300 μg / mL, at least 350 μg / mL, at least 400 μg / ML or more, 600 μg / mL or more, 700 μg / mL or more, 800 μg / mL or more, 900 μg / mL or more or 1000 μg / mL or more.

In one embodiment, the Peppermint Water Extract may be less than 100,000 g / mL, less than 50,000 g / mL, less than 10,000 g / mL, and less than 5000 g / mL, based on the total composition.

When the water extract of Peppermint Water is contained within the above range, the anti-inflammatory or immunostimulating effect can be effectively obtained without toxicity. Specifically, when the water extract of Picea juncea is within the above-mentioned range, IL-1 is effectively inhibited by inducing NO or TNF-? Generated by induction of inflammatory reaction, and is also effective as a cytokine that activates NK cells having anti- 6 production, and the production of TNF-a which affects the activity of immune cells is increased, thereby obtaining an immune enhancing effect.

In one embodiment, the composition may be a cosmetic, a food or a pharmaceutical composition.

The composition according to one aspect of the present invention may be a pharmaceutical composition and may be various oral or parenteral formulations. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid form preparations for oral administration include tablets, pills, powders, granules, soft or hard capsules, etc. These solid preparations may contain one or more excipients such as starch, calcium carbonate, sucrose, Or lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc, and the like are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used as the non-aqueous solvent and suspension agent. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.

The pharmaceutical dosage forms of the compositions according to one aspect of the present invention may be used in the form of their pharmaceutically acceptable salts and may also be used alone or in combination with other pharmaceutically active compounds as well as in a suitable set. The salt is not particularly limited as long as it is pharmaceutically acceptable so long as it is pharmaceutically acceptable and includes, for example, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrofluoric acid, hydrobromic acid, formic acid acetic acid, tartaric acid, lactic acid, citric acid, fumaric acid, , Benzenesulfonic acid, toluenesulfonic acid, and naphthalenesulfonic acid.

The composition according to one aspect of the present invention may be administered parenterally or orally, and may be administered in an amount of 0.0001 to 10 g, preferably 0.0001 to 6 g per 1 kg of body weight per day, can do. The dosage for a particular patient may vary depending on the patient's body weight, age, sex, health condition, diet, time of administration, administration method, excretion rate, severity of disease, and the like.

The pharmaceutical composition according to one aspect of the present invention can be administered orally or parenterally in the form of powders, granules, tablets, soft or hard capsules, oral preparations such as suspensions, emulsions, syrups and aerosols, external preparations such as ointments, creams, , Injectable solutions, sterile injection solutions, and the like, and may be used in any form suitable for pharmaceutical preparations.

The composition according to one aspect of the present invention can be administered to mammals such as rats, mice, livestock, humans, and the like by various routes such as parenteral, oral, etc. All the ways of administration can be expected, Rectal, rectal or intravenous, intramuscular, subcutaneous, intrauterine or intracerebroventricular injection.

In one aspect of the invention, the food composition may be a health functional food composition.

The formulation of the food composition according to one aspect of the present invention is not particularly limited, but may be formulated into, for example, tablets, granules, powders, liquid preparations such as drinks, caramels, gels, bars and the like. The food composition of each formulation can be blended with the ingredients commonly used in the field in addition to the active ingredient without difficulty by those skilled in the art depending on the purpose of formulation or use, and synergistic effect can be obtained when the composition is applied simultaneously with other ingredients.

In the food composition according to one aspect of the present invention, the determination of the dosage of the active ingredient is within the level of those skilled in the art, and its daily dose is, for example, 0.001 g / kg / day to 10 g / kg / May be 0.1 g / kg / day to 6 g / kg / day, but is not limited thereto, and may vary depending on various factors such as the age, health condition, and complication of the subject to be administered.

The food composition according to one aspect of the present invention can be used in various foods such as chewing gum, caramel product, candy, ice cream, confectionery, beverage such as soft drink, mineral water, alcoholic beverage, health including vitamins and minerals It may be a functional food.

In addition to the above, the food composition which is one aspect of the present invention can be used as a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and enhancers (cheese, chocolate etc.), pectic acid and its salts, Alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages and the like. In addition, the functional food compositions of the present invention may comprise natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The proportion of such additives is not so critical, but is generally included in the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

The formulation of the cosmetic composition according to one aspect of the present invention is not particularly limited and can be appropriately selected according to the purpose. For example, skin lotion, skin softener, skin toner, astringent, lotion, milky lotion, moisturizing lotion, nutrition lotion, massage cream, nutritional cream, moisturizing cream, hand cream, foundation, essence, nutrition essence, pack, soap, cleansing But the present invention is not limited thereto, and may be manufactured by any one or more formulations selected from the group consisting of a foam, a cleansing lotion, a cleansing cream, a body lotion and a body cleanser.

When the formulation of the cosmetic composition according to one aspect of the present invention is a paste, cream or gel, the carrier component may include animal fibers, plant fibers, waxes, paraffins, starch, trachants, cellulose derivatives, polyethylene glycols, silicones, bentonites, Or zinc oxide may be used.

If the formulation of the cosmetic composition according to one aspect of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as the carrier component, Propellants such as chlorofluorohydrocarbons, propane / butane or dimethyl ether.

Solvents, solubilizing agents or emulsifying agents are used as carrier components in the case of solutions or emulsions in the form of cosmetic compositions according to one aspect of the present invention, and examples thereof include water, ethanol, isopropanol, ethylcarbonate, ethyl acetate, benzyl alcohol, Benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid esters of sorbitan.

When the formulation of the cosmetic composition according to one aspect of the present invention is a suspension, the carrier component may include water, a liquid diluent such as ethanol or propylene glycol, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester The same suspending agent, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tracant, etc. may be used.

When the formulation of the cosmetic composition according to one aspect of the present invention is an interface-active agent-containing cleansing, the carrier component is selected from aliphatic alcohol sulfates, aliphatic alcohol ether sulfates, sulfosuccinic acid monoesters, isethionates, imidazolinium derivatives, , Fatty acid amide ether sulfate, alkylamido betaine, aliphatic alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, linolenic derivative, or ethoxylated glycerol fatty acid ester can be used.

The content of the active ingredient is not particularly limited, but may be in the range of 0.001 to 100% by weight based on the total weight of the cosmetic composition according to one aspect of the present invention. When the active ingredient satisfies the above content, it can exhibit excellent efficacy without side effects.

The cosmetic composition may further contain ingredients included in a functional additive and a general cosmetic composition. The functional additives may include water-soluble vitamins, oil-soluble vitamins, polymer peptides, polymeric polysaccharides, sphingolipids and seaweed extracts.

The cosmetic composition according to one aspect of the present invention may further comprise, in addition to the above-described functional additive, components contained in a general cosmetic composition as required. Examples of the other ingredients that can be included in the composition include humectants, emollients, surfactants, organic and inorganic pigments, organic powders, ultraviolet absorbents, preservatives, bactericides, antioxidants, plant extracts, pH adjusters, alcohols, Accelerators, coolants, antiperspirants, purified water, and the like.

Hereinafter, the constitution and effects of the present invention will be described in more detail with reference to Examples and Experimental Examples. However, these examples and experimental examples are provided for illustrative purposes only in order to facilitate understanding of the present invention, and the scope and scope of the present invention are not limited thereto.

EXAMPLES Preparation of the extract of rice husk

Preparation

The extracts were collected from Hongcheon County, and the extracts were removed from the pine nuts. The extracts from the forests of Hongcheon County, Gangwon Province were used for the extraction. For the preparation of the extracts, fermented syrup (95%, manufactured by Kohung Life) and ultrapure water (Millipore, MilliQ) were used.

How to make Extract

1. Moisture content measurement of Phellodendron husk

The samples were placed in a dryer for agricultural products, and the samples were dried at 50 ° C for 24 hours, 24 hours for moisture discharge, and 24 hours for primary drying for 24 hours.

The dried sample is dried in a drying oven at 105 ° C for 3 hours, left in a desiccator for 1 hour, and then weighed. After leaving for 1 hour in the desiccator again, calculate the water content when there is no weight change, and it repeats if the weight continues to decrease. The water content (%) is expressed as a percentage of the weight before drying to the weight before drying, minus the weight after drying.

The drying results are shown in Table 1. The average moisture content of the first dried sample was 12.83%. In the drying process for water content measurement, some of the rosin component melts and some of it flows to the bottom of the container and sticky sticky.

sample Before drying (g) After drying (g) Moisture content (%) Average Standard Deviation One 38.8181 33.9071 12.65
12.83%
0.003366 2 48.4272 42.3134 12.62 3 61.2649 53.1653 13.22

2. Manufacture of Phellodendron bark extract

Each of the by-products of dried pomace peel, which had been dried in the agricultural product dryer, was ground in a blender and put into a 2 L round-shaped flask (inlet size 24/40) in an amount of 200 g each. Then, 1 L of tertiary distilled water and 50% Reflux extraction was carried out in a constant-temperature water bath at 90 ° C and a cooler at -10 ° C for 4 hours, then the extract was transferred to another vessel, and then 1 liter of the same solvent was added thereto. 2 L of the extract was filtered using a funnel funnel and used for concentration.

The collected extract was concentrated under reduced pressure to a temperature of 55 캜 at 15 brix. The concentrate was frozen at -80 DEG C for 2 hours or more and then lyophilized in a freeze dryer. The lyophilized state was checked every 24 hours, and when the lyophilization was not completed, the ice of the trap was removed and the lyophilized was again dried. After freeze-drying, the weight of the extract including the container was measured, and the weight of the extract was determined by subtracting the weight of the container previously measured, and the extraction yield was calculated. The experimental results are shown in Table 2 below.

menstruum Tare Weight
(g) Weight after drying (g) Sample weight
(g) Extract dry weight (g) yield Ultrapure water 16.856 31.949 200 15.093 7.55% 50% Ethanol 16.86 52.408 200 35.548 17.77%

The yield of hydrothermal extract was 7.55% and the yield of 50% ethanol extract was 17.77%. It was judged that the yield of 50% ethanol extract was high due to the large amount of oil containing the rosin.

When the 50% ethanol extract was condensed in a waste flask under reduced pressure concentration, it was divided into three layers and it was considered to be a water - essential oil - ethanol layer from below.

The part considered as an essential oil layer was about 150 ~ 200ml in volume.

[Experimental Example 1] Cytotoxicity test of extract of rice husk

In order to evaluate the anti - inflammatory and immunological activity of the extracts of Phellodendron chestnut, we examined the cytotoxicity of extracts in the cells used in the experiments.

Raw 264.7 cells (KCLB No. 40071, Korea Cell Line Bank) were subcultured in a 96-well plate at a density of 1 × 10 ⁴ cells / well in an amount of 100 μL. On the next day, the medium was removed, and the water extract and 50% ethanol extract (hereinafter referred to as "sample") of the zucchini by-product prepared in the examples were treated for 72 hours at 37 ° C in a 5% CO ₂ incubator. The concentration of each sample was 50, 100, 200, 500, 1000 ㎍ / mL in final concentration. After incubation for 72 hours, the medium was removed and 100 μL of MTT (1 mg / mL) reagent was added. After incubation for 4 to 6 hours in a CO ₂ incubator, MTT reagent was removed and 100 μL of DMSO reagent was added After incubation at room temperature (RT), the absorbance (OD) was measured at 570 nm with a microplate reader.

Cytotoxicity test results showed no signs of toxicity by the samples up to the concentration of 1000 ㎍ / ㎖ in the water extract of Picea juncea and 50% ethanol extract, and the cells were increased with increasing concentration. The results are shown in Fig. Fig. 2 shows the results of cytotoxicity measurement of the extract of Yukisugi.

[Experimental Example 2] Evaluation of anti-inflammatory (NO, TNF-α)

NO is known as a very unstable molecule as one of the free radicals and NO is a molecule which is formed by NO ₂ , N ₂ O ₃ , N ₂ O ₄ , NO ₂ - and NO ₃ - by superoxide, (L-arginine), nitric oxide synthase (NOS), and NOS are endothelial NOS (eNOS) and neuronal , And inducible NOS (iNOS) induced by NOS (nNOS) and inflammatory factors.

TNF-α is an inflammation-mediated cytokine that acts in vivo alone or with cytokines such as IL-1 and IFN-γ to damage tumor vessels, resulting in tumor necrosis or infection by microorganisms Leads to resistance of the host, and abrupt overproduction has a deleterious effect on the living body and can induce iNOS expression in a wide variety of tissues or organs, tumors, and cell lines

The macrophage acts as a phagocyte that eats the antigen that enters the immune system when it enters the immune system. In this case, the secretion of NO, which is a kind of peroxide secreted when the macrophage is activated, is increased and inflammation is caused by macrophages TNF-α, which causes symptoms and worsens, is overproduced.

In order to measure NO and TNF-alpha, which are the criteria for evaluation of inflammatory activity, the inhibitory effect of LPS (lipopolysaccharide) on the production of NO, which is an inflammatory reaction product, in Raw 264.7 cells, And the amount of TNF-? Produced was measured according to the following steps.

(1) Cell culture

1) Raw 264.7 cells were subcultured in a 96-well plate at 1 × 10 ⁴ cells / well.

2) Overnight After removing media, water extracts and 50% ethanol extracts (hereinafter referred to as "samples") of the zucchini by-products prepared in the Examples were treated for 1 hour at 37 ° C in 5% CO ₂ And cultured in an incubator (Incubator). The concentrations of the samples were 50, 100, 200, 500, and 1000 ㎍ / mL in the final concentration.

3) One hour later, LPS was adjusted to 0.5 ug / mL and placed in a group with LPS treatment.

4) After 24 hours, measurements of NO and TNF-a were performed using the supernatant using griess reagent and ELISA kit, respectively.

(2) NO assay: Measurement using a grease reagent

1) A plate was taken out from the incubator for the NO assay.

2) The culture supernatant of the above (1) was transferred into a new 96-well plate in an amount of 70 μl.

3) Griess reagent was taken out from the refrigerator and allowed to stand at room temperature 30 minutes before the assay.

4) 70 μl of sulfanilamide solution was added to a 96-well plate containing 30 μl of the solution.

5) After 30 minutes, 70 μl of NED (N-1-naphthylethylenediamine dihydrochloride) solution was added and left in a dark place for 30 minutes.

6) At the end of the color reaction, the absorbance value (OD) was measured at 520 nm with a microplate spectrophotometer.

As a result, NO production was slightly increased as the treatment concentration was increased when the extract was treated with Raw 264.7 cells alone (green solid line in FIG. 3), but it was not significantly increased compared with LPS treatment, And 50% ethanol extract showed lower NO production than water extract.

The inhibitory effect of NO on LPS-induced NO production (purple solid line 'with LPA' in Fig. 3) inhibited the production of NO at a concentration of 500 and 1000 ㎍ / ㎖ in water extract of PJ % Ethanol (ETOH) extract showed a tendency to inhibit the production of NO but the effect was lower than that of the water extract and the inhibition rate was about 10% lower.

The results are shown in Fig. 3 (results of inhibition of the NO production ability of the extract of Picea magnesia).

(3) Measurement of TNF-a: ELISA assay

1) A capture antibody (capture anti-body (ab)) of ELISA kit (R & D Systems, USA) was diluted with a dilution factor according to the instruction manual and coated on a 96 well nunc plate for ELISA (Nunc cat No. 439454 ) Was dispensed in 100 쨉 l portions at 4 째 C overnight.

2) 200 [mu] l of 0.05% Tween 20 was added to each well and washed twice.

3) 200 μl of 2% skim milk solution was added and blocking was performed at room temperature (RT) for 1 hour.

4) Washing solution was washed twice with 200 ㎕ each.

5) 100 ㎕ of the supernatant of the culture completed in (1) and the standard solution diluted in steps 1) to 4) was dispensed into each well and reacted for 2 hours in a 37 ° C CO ₂ incubator .

6) Washing solution was washed 5 times with 200 ㎕ each.

7) detection anti-body (ab) and streptavidin-HRP were diluted with a dilution factor of the protocol in a kit and 100 ㎕ each was dispensed. After incubation at 37 ° C in a CO ₂ incubator, For 1 hour.

8) Washing solution was washed by 7 times with 200 ㎕ each.

9) 100 μl of TMB (3,3 ', 5,5'-Tetramethylbenzidine) substrate solution was dispensed and dispensed.

10) 2N H ₂ SO ₄ as a stop solution was dispensed in an amount of 100 μl, and color development was stopped.

11) The absorbance value (OD) was measured using a microplate spectrophotometer at a wavelength of 450 nm within 40 minutes after stopping the color development.

As a result, TNF-α, which is an inflammatory cytokine, was measured in culture supernatant, and it was confirmed that the amount of TNF-α produced by LPS in a concentration-dependent manner was lowered in the extract of Phellodendra spinach. Similar trend was observed in the 50% ETOH extract, but less than that of the water extract. The results are shown in Fig. 4 (TNF-alpha measurement results of the extract of Phellodendron husk).

The results of (2) and (3) above showed that the water extract of P. junosum inhibited excessive production of NO and TNF-a due to inflammation induced by LPS treatment at a concentration of 500 and 1000 μg / ml, .

Although 50% ethanol extract showed anti - inflammatory activity, it showed less activity than water extract.

[Experimental Example 3] Evaluation of the immunological activity of the extract of the rice husk

IL-6 cytokine production and innate immune responses in Raw 264.7 cell (murine leukemia tumor, macrophage) and murine macrophage induced by extract of Phellodendron chinensis extract and TNF -α was confirmed by the following steps.

(1) Cell culture

2) After removal of media after Overnight, water extracts and 50% ethanol extracts (hereinafter referred to as "samples") of zucchini by-products prepared in the Examples were treated for 1 hour at 37 ° C in a 5% CO ₂ incubator ). The concentrations of the samples were 50, 100, 200, 500, and 1000 ㎍ / mL in the final concentration.

3) One hour later, for the measurement of IL-6, LPS was adjusted to 5 ug / mL and placed in a group with LPS treatment. (In the case of TNF-α measurement, only the extract is treated alone)

4) After 24 hours, the activity of IL-6 and TNF-α was measured using supernatant using an ELISA kit.

(2) Measurement of IL-6 and TNF-α activity in RAW 264.7 cells: ELIA assay

1) Capture antibody (capture anti-body (ab)) of ELISA kit (BD, IL-6, TNF-α) was diluted in the coating buffer with a dilution factor according to the instruction manual and diluted with 96 well nunc plate for ELISA cat no. 439454) were dispensed at 100 uL and placed overnight at 4 ° C. (Overnight).

2) 200 uL each with washing solution containing 0.05% Tween 20 and washed twice.

3) 200 μl of 2% skim milk solution was added and the mixture was blocked at room temperature (RT) for 1 hour.

4) 200 uL each with washing solution.

5) 100 L of the supernatant of step (1) and step diluted standard solutions (1) to 4) were dispensed into each well and incubated in a 37 ° C CO ₂ incubator for 2 hours .

6) Washing solution was washed 5 times with 200 uL each.

7) detection anti-body (ab) and streptavidin-HRP were diluted with a dilution factor of the protocol in the kit and 100 uL each was dispensed. After incubation at 37 ° C in a CO ₂ incubator, For 1 hour.

8) 200 uL aliquots were washed with washing solution 7 times.

9) TMB substrate solution was dispensed in 100 μl aliquots, and the color development was observed in cows.

10) 2N H ₂ SO ₄ as a stop solution was dispensed in 100 μl aliquots to stop color development.

In the supernatant of Raw 264.7 cells, cytokine IL-6 was found to increase with increasing concentrations of water extracts and 50% ethanol extracts. 500 and 1000 ug / mL were similar to positive control (positive control (treated with LPS alone)). In the case of 50% ethanol extract, IL-6 production was increased in a dose dependent manner, And about 40% of positive control group. The results are shown in Fig. Fig. 5 shows the results of measurement of IL-6 activity of the extract of Phellodendrons sp.

When TNF-α was cultured in culture supernatant of Raw 264.7 cells, TNF-α production was increased with increasing concentrations of extracts of Peppermint water and 50% ethanol (ETOH) The extracts showed higher yields than the 50% ethanol extract at the concentrations of 50 and 100 ug / mL. 6 is a graph showing changes in the amount of TNF-?

(3) Analysis of IL-6 activity in mouse murine macrophages: ELISA assay

RAW 264.7 cells are stable in the stable cell line of murine leukemia virus-induced tumors and are thus widely used in basic experiments. However, The murine macrophage, a primer cell, was extracted from the mouse and the reaction in the macrophage was confirmed by the following steps.

1) A 3% TG (thioglycolate medium) aged for 2 days was intraperitoneally administered in 1 mL of a mouse (ICR mouse, Orient Biotechnology, Korea) and left for 4 days.

2) Fill a 10 mL syringe with 10 mL of sterile PBS (Phosphate Buffered Saline).

3) Mice were dislocated by cervical dislocation, and the abdominal cavity was spray-sterilized with 70% ETOH.

4) In the clean hood, the foam and scissors were sterilized by flame to incise the abdominal skin.

5) After the incision, when the dermis layer is removed, the abdominal cavity is washed with PBS in a syringe and the cells are harvested.

6) Centrifugation was performed at 1500 rpm for 5 minutes.

After removing the supernatant, cells were counted and 100 μL of macrophage was added to 96-well plate at 1 × 10 ⁶ cells / well.

8) After Overnight, the media was removed and the samples were treated for 24 hours at 37 ° C in a 5% CO ₂ incubator.

9) Final concentration of each sample was 50, 100, 200, 500, and 1000 ㎍ / mL, respectively.

10) After 24 hours, IL-6 activity was measured by the ELISA assay of (2) above using the culture supernatant as a sample for ELISA assay.

As a result of measuring cytokine IL-6 in order to confirm the immunological activity of the extract of Phellodendra spinach on murine macrophage cells, RAW 264.7 cell similar to the above (2) 6, and water extracts showed higher activity. The results are shown in Fig. FIG. 7 shows IL-6 changes in mouse murine macrophages treated with the extract of Picea tulip.

In addition, as in Experimental Example 1, the cytotoxicity test showed no signs of toxicity by the samples up to the concentration of 1000 μg / ml in the water extract of Picea japonica and 50% ethanol, and it was confirmed that the cells increased with increasing concentration .

[Experimental Example 4] Evaluation of proliferation activity of the extract of Picea magna

Spleen is an organs with a lot of lymphocytes. It plays a role of destroying aged red blood cells, eliminating immune complexes and blood cells, and plays a role as a storage depot of lymphocytes. In response to the immune activity, APCs cells such as macrophages secrete various cytokines by stimulating foreign substances or active ingredients, and help T-cells (helper t-cells) Activation leads to the proliferation of IL-2 cytokines and secretory lymphocytes. Whether the extract of Picea junea stimulates the immune cells of splenocytes to increase the proliferation was confirmed by the following steps.

1) The mice were euthanized by dislocation of the cervical vertebrae and then sprayed with 70% ethanol (ETOH) on the left side of the back of the mouse.

2) In the clean hood, the foils and scissors were sterilized by flame, and the skin of the left side of the dorsal portion was incised.

3) After the incision, the spleen was observed through the naked eye and the dermis was incised, and the spleen was stored in the prepared PBS for a short time.

4) The spleen was pulverized using steel 100 mesh which was sterilized by flame.

5) After the pulverization, centrifugation was performed at 1500 rpm for 5 minutes.

6) After removing the supernatant, add 1 mL of sterilized 0.2% NaCl solution and shake well for hemolysis.

7) 9 mL PBS was added and centrifuged at 1500 rpm for 5 minutes.

8) After removal of the supernatant, 1 mL of PBS was added and the debris was removed using an auto pipette.

9) Cells were counted, and then 100 μL of each was dispensed into 96-well plates at 2 × 10 ⁶ cells / well.

10) The concentration of the water extract and 50% ethanol extract (hereinafter referred to as "sample") of the zucchini by-product produced in the examples after maintaining in an incubator at 7 ° C in a 5% CO ₂ for 2 hours was 50, 100, 200, 500, and 1000 ㎍ / mL, respectively.

11) After culturing for 3 days, 10 uL of CCK (cell count kit) using WST-1 assay was added, and the OD value was measured at 450 nm after confirming the coloration for 30 to 60 minutes.

The proliferation activity of the extract of Phellinus linteus on the splenocyte obtained from mouse was confirmed to be 10% at the concentration of 1000 ug / mL in the water extract and 50% It was confirmed to happen. This was the same as the result of cytotoxicity test of macrophage RAW 264.7 cell.

The results are shown in Fig. 8 is a graph showing the effect of the extract of Picea magna on the proliferation of splenocytes.

Compared to Experiments 1 to 4, the extracts of water and 50% ethanol of Zizuria japonica showed anti-inflammatory effects by inhibiting excessive production of NO and TNF-α by induction of inflammatory reaction by LPS treatment, IL-6 production, a cytokine that activates NK cells with innate immune system, particularly anti-cancer effect, by reacting with Raw 264.7 cell, which is a stable cell line, and murine macrophage, which is a primer cell, And increased the production of TNF- [alpha], which influences immune cell activity, and thus has an immune activity. It increases the proliferation activity of Raw 264.7 cell and splenocyte, And increased cell proliferation.

Therefore, the extract of Phellodendron japonica has been shown to be effective for antiinflammation, immunological activity and immune cell proliferation.

Hereinafter, a formulation example of a composition having anti-inflammatory or immunostimulating effect according to an aspect of the present invention will be described below, but it may be applied to various other formulations, .

[Formulation Example 1] Health food

Peppermint extract ................... 1000 mg

Vitamin mixture

Vitamin A Acetate ............... 70 ㎍

Vitamin E ....................... 1.0 mg

Vitamin B1 ...................... 0.13 mg

Vitamin B2 ...................... 0.15 mg

Vitamin B6 ........................ 0.5 mg

Vitamin B12 ....................... 0.2 g

Vitamin C .......................... 10 mg

Biotin ............................. 10 μg

Nicotinic acid amide ... 1.7 mg

Folic acid ............................... 50 ㎍

Calcium pantothenate ..................... 0.5 mg

Mineral mixture

Ferrous sulfate ........................ 1.75 mg

Zinc oxide .......................... 0.82 mg

Magnesium carbonate ...................... 25.3 mg

Potassium Phosphate ......................... 15 mg

Secondary Calcium Phosphate ..................... 55 mg

Potassium citrate .......................... 90 mg

Calcium carbonate ........................... 100 mg

Magnesium chloride ...................... 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with the ingredient suitable for health food as a preferred embodiment, the compounding ratio thereof may be arbitrarily modified.

[Formulation Example 2] Health drinks

Peppermint extract .......................... 1000 mg

Citric acid ................................ 1000 mg

Oligosaccharides ................................ 100 g

Taurine .................................... 1 g

Purified water .................................. Remaining amount

The above components are mixed according to a conventional health drink manufacturing method, and the mixture is stirred and heated at 85 DEG C for about 1 hour, and then the solution is sterilized by filtration.

[Formulation Example 3] Tablets

The granules were formed by mixing 100 mg of Extract of Phellinus crassa, 50 mg of soybean extract, 100 mg of glucose, 50 mg of red ginseng extract, 96 mg of starch and 4 mg of magnesium stearate and 40 mg of ethanol at 30% Tablets are tableted using a tablet machine.

[Formulation Example 4]

The granules were prepared by mixing 100 mg of Extract of Yuzunkoe, 50 mg of Soybean Extract, 100 mg of Glucose and 600 mg of Starch and 100 mg of 30% Ethanol, and dried at 60 ° C to form granules.

Claims

The present invention relates to an immunostimulant composition comprising an extract of Phellodendron japonica as an active ingredient.

The method according to claim 1,
The extract of Picea japonica promotes the proliferation of immune cells.

3. The method of claim 2,
The extract of Picea japonica promotes the expression or activity of IL-6.

An antiinflammatory composition comprising an extract of Picea magna as an active ingredient.

5. The method of claim 4,
The extract of Phellodendra japonica inhibits the excessive production of NO and TNF-α.

6. The method according to any one of claims 1 to 5,
Wherein said extract is an extract of an aqueous ethanol solution.

The method according to claim 6,
Wherein the Picea jute extract is an extract of water.

6. The method according to any one of claims 1 to 5,
Wherein the extract of Picea zodiacus is an extract of Picea zooplankton.

6. The method according to any one of claims 1 to 5,
The extract of Picea japonica has a composition of 10 / / mL to 100,000 / / mL

6. The method according to any one of claims 1 to 5,
Wherein said composition is a cosmetic, food or pharmaceutical composition.