KR101284277B1 - The extract of the bark of Acer barbinerve Max with anti-inflammatory effect and composition thereof - Google Patents

Abstract

The present invention relates to a blue bark extract bark extract exhibiting anti-inflammatory activity and a composition containing the same, the blue bark ( Acer barbinerve The Max), while the xylem extract as negligible anti-inflammatory activity, cheongsi of the invention Doc tree (Acer barbinerve Max ) bark extract showed significant anti-inflammatory activity below the cell death concentration.

Description

Extract of the bark of tree bark showing anti-inflammatory activity and composition containing same {The extract of the bark of Acer barbinerve Max with anti-inflammatory effect and composition

The present invention relates to the extract of the bark of the tree bark showing anti-inflammatory activity and a composition containing the same.

Inflammation refers to a pathological condition in which an abscess derived from bacteria is a dead body of leukocytes, tissues destroyed by bacteria, cell debris, etc. in which tissues are melted and accumulated. When an invasion that causes some organic changes in cells or tissues is applied, it may be interpreted as a biological defense process that attempts to repair and repair the damaged area.

Currently, anti-inflammatory drugs are steroidal and non-steroidal anti-inflammatory drugs, which are difficult to use for long term due to various side effects. In connection with the problem of overcoming these side effects, efforts have recently been made to find the active ingredient in natural products that are used in the private sector.

Blue Tak Tree ( Acer barbinerve Max ) is a deciduous broad-leafed arborescent with maple leaves, about 10 m high. The leaves are opposite, broad egg-shaped or round, divided into 3 ~ 5 branches, and have a lot of fine hair on the back side. Stems and branches grow in medicinal and deep mountains, and are distributed in Korea, Japan, and Manchuria. Bark is green or greyish brown with yellow branches, but sometimes reddish, with hairs.

Many studies have been conducted on natural products having anti-inflammatory activity or extracts thereof, and studies on the anti-inflammatory function of the tree seedling extracts are currently insignificant.

The present invention is a blue-green tree ( Acer barbinerve Max ) is to isolate and extract the anti-inflammatory extract to provide an anti-inflammatory composition for the purpose.

The present invention is the first form of green pine ( Acer barbinerve After the hydrothermal extraction of the bark of Max ), an extract having anti-inflammatory activity obtained by separating the extract is provided. According to experiments of the present invention to shut cheongsi tree (Acer barbinerve Max ) It was confirmed that the anti-inflammatory effect of the hydrothermal extract of the bark skin was remarkable. Whereas the bark had a significant anti-inflammatory effect, the wood did not show a significant anti-inflammatory effect.

On the other hand, the present invention in the second form Cheongdak wood ( Acer barbinerve Max ) bark skin was extracted by hot water extraction, the extracts were separated, fractionated with ethyl acetate, and the extracts having anti-inflammatory activity obtained by separating the fractions were provided. According to the experiments of the present invention, the most effective anti-inflammatory activity in the ethyl acetate fraction of the various fractions.

On the other hand, the present invention provides an anti-inflammatory composition comprising an extract of the first or second form of the present invention as an active ingredient in a third form. In the present invention, "contained as an active ingredient" means that the extract of the first form or the second form of the present invention is added to the composition to an extent capable of exhibiting an anti-inflammatory effect. That is, when the composition is a pharmaceutical composition, it can be formulated in various forms by adding a variety of ingredients as a sub-component for drug delivery and stabilization, etc., if the form of the composition is a food or cosmetic composition, various formulations It means that the formulation base and excipient can be added to prepare the.

The term 'extract' in the present invention is used in the sense including both the liquid form containing the extraction solvent and the form of the remaining solid powder volatilize the extraction solvent.

On the other hand, the present invention provides an anti-inflammatory food composition comprising an extract of the first or second form of the present invention or a dry powder thereof as an active ingredient, the extract of the first or second form of the present invention Is preferably added in the composition at a concentration of 1-20 μg / mL relative to dry weight.

Food composition for preventing inflammation of the present invention is preferably meat, cereals, caffeine beverages, general beverages, chocolate, breads, snacks, confectionery, pizza, jelly, noodles, gums, ice creams, alcoholic beverages, alcohol, vitamin complexes and other It may be any one selected from health supplements, but is not necessarily limited thereto.

On the other hand, the present invention provides a pharmaceutical composition for treating inflammation, characterized in that it contains an active ingredient of the first or second form of the present invention or a dry powder thereof. The content of the extract of the first or second form of the present invention or the dry powder of the present invention contained in the pharmaceutical composition for treating inflammation according to the present invention depends on the method of using prophylactic and therapeutic agents, the condition of the user, the type of disease, and the severity of the disease. Preferably controlled, preferably added in a composition at a concentration of 1-20 μg / mL relative to dry weight.

On the other hand, the pharmaceutical composition for treating inflammation of the present invention may further comprise a pharmaceutically acceptable carrier, diluent or excipient in addition to the active ingredient. Examples of usable carriers, excipients or diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. These may be used singly or in combination. When the preventive and therapeutic agent is a pharmaceutical agent, a filler, an anticoagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent or an antiseptic agent may be additionally included.

On the other hand, the dosage form of the pharmaceutical composition for treating inflammation of the present invention may be formulated in a preferred form according to the method of use, and in particular in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. It is desirable to formulate by employing known methods. Examples of specific formulations include PLASTERS, GRANULES, LPTIONS, LINIMENTS, LEMONADES, AROMATIC WATERS, POWDERS, Syrups SYRUPS, OPHTALMIC OINTMENTS, LIQUIDS AND SOLUTIONS, AEROSOLS, EXTRACTS, ELIXIRS, OINTMENTS, FLUIDEXTRACTS, EMULSIONS, ), Suspensions, DECOCTIONS, INFUSIONS, OPHTHALMIC SOLUTIONS, TABLETS, SUPPOSITIORIES, INJECTIONS, SPIRITS, CATAPLSMA, ), Capsules, CREAMS, TROCHES, TINCTURES, PASTES, PILLS, soft or hard gelatin capsules.

On the other hand, the dosage of the pharmaceutical composition for treating inflammation of the present invention may be determined in consideration of the administration method, the age, sex and weight of the recipient and the severity of the disease. For example, the inflammatory therapeutic agent of the present invention can be administered at least once at a dose of 0.1 to 100 mg / kg (body weight) per day based on the active ingredient. However, the above dosage is only one example to illustrate, and may vary depending on the doctor's prescription depending on the condition of the patient.

As confirmed by the present invention, the green tree ( Acer barbinerve The Max), while extracts of xylem as negligible anti-inflammatory activity, cheongsi Doc trees (Acer barbinerve Max ) bark extract showed significant anti-inflammatory activity below the cell death concentration.

1 is an MTT assay result for examining cell proliferation ability (killing ability).
2 is a result of the anti-inflammatory efficacy investigation by the measurement of NO amount.
Figure 3 shows the results of anti-inflammatory efficacy by measuring the amount of protein expression of iNOS and COX-2.
Figure 4 shows the results of anti-inflammatory efficacy by measuring the mRNA expression level of iNOS, COX-2, IL-6, IL-1β and TNF-α.

Hereinafter, the content of the present invention will be described in detail in the following Examples and Experimental Examples. However, the scope of the present invention is not limited to the following embodiments, and includes modifications of equivalent technical ideas.

Experimental Example  One: MTT Assay  Cell through Proliferative ability ( Death ) Research

In the present experimental example, RAW264.7 cells, which are mouse macrophages, were treated with a fraction of LPS and blue bark bark at a concentration of 20 μg / mL for 24 hours to investigate cell death ability (proliferative capacity) by MTT assay.

RAW264.7 cells were cultured in a humidified CO ₂ incubator at 37 ° C. using a culture medium in which 10% FBS, 1% penicillin streptomycin was added to DMEM medium. When the cells were about 70-80% of the culture dish, the monolayers of cells were washed with PBS (phosphate-buffered saline, pH 7.4), and then the cells were passaged and the medium was changed every two days.

RAW264.7 cells incubated as above were diluted in DMEM with 10% FBS and dispensed into 24 well-plates at a density of 50,000 cells / well. After 24 hours of incubation, the cells were further incubated for 24 hours with a serum-deprivation medium (SDM) containing 1% FBS. Thereafter, the cells were exchanged with SDM containing the bark fraction of 20 μg / mL concentration, and after 24 hours, the number of living cells was measured by MTT assay.

As a result of confirming the proliferative capacity of the cells, it was confirmed that they did not affect the proliferation of the cells. (FIG. 1).

Experimental Example  2: NO Investigation of anti-inflammatory effect by amount measurement

Blue Tak Tree ( Acer barbinerve Max ) extract did not affect the proliferation of macrophages at a concentration of 20 μg / mL, this experiment was to explore the anti-inflammatory efficacy within this range.

Raw264.7 cells were incubated for 24 hours in a cultured blue bark fraction and LPS added as in Experimental Example 1, the medium was collected and the amount of NO secretion was measured using the 'Griess reagent system' (Promega).

As a result, it was confirmed that NO was increased during LPS treatment and that NO increased by treatment of bark fraction. (Fig. 2). Among the fractions, the Etoac (ethyl acetate) fraction was the most effective, using the Etoac fraction was further carried out the following experiment.

Experimental Example  3: iNOS Wow COX -Investigation of anti-inflammatory efficacy by measuring protein expression level of -2

Since it was confirmed that the NO decrease in Experimental Example 2, this experiment was to investigate the expression of iNOS and COX-2 proteins related to the inflammatory response. As a sample, an ethyl acetate fraction having the highest NO reduction effect among the Chengsidac extracts was used.

RAW264.7 cells were dispensed in 100 mm dishes at a density of 1 × 10 ⁶ cells / dish. After 24 hours of incubation, the cells were incubated for 24 hours with SDM. After incubation, the ethyl acetate fractions of the bark of the green bark were added at various concentrations to culture the cells. After 24 hours, the cells were rinsed with cold PBS (including 1 mmol / L iodoacetic acid and 1 mmol / L phenylmethylsulfonylfluoride (PMSF)), and the cells were collected with a scraper and centrifuged (2,000 rpm, 2 min, 4 ° C.). Lysis buffer in pellet (20 mmol / L Hepes, pH 7.5, 1% Triton X-100, 150 mmol / L NaCl, 1 mmol / L EDTA, 1 mmol / L EGTA, 100 mmol / L NaF, 10 mmol. L iodoacetic acid, 0.2 mmol / L PMSF, 20 mg / L aprotinin, 10 mg / L antipain, 10 mg / L lepeptin, 80 mg / L benzamidine HCl) was added and stirred at 4 ° C. for 40 minutes to lyse the cells. The supernatant was separated and stored at -70 ° C. Lysates (Lysate, 50 μg protein) were separated by 4-20% gradient SDS-PAGE and transferred to polyvinylodene fluoride membrane (PVDF). The membrane obtained was brazed for 5 hours in 5% 'non-fat milk-TBST' (20 mmol / L Tris-HCl, pH 7.6, 150 mmol / L NaCl, 0.1% Tween 20), followed by 3 minutes in TBST. Rinse times. COX-2 and iNOS antibodies were added to the membrane and incubated at 4 ° C. for 24 hours, followed by rinsing three times with TBST for 10 minutes. The membrane was then incubated for 1 hour with horseradish peroxidase (HRP) -binding anti-rabbit, mouse or goat IgG and then rinsed three times with TBST for 10 minutes. Signals of the proteins bound to the antibody were visualized by using an Immobilon Western Chemiluminescent HRP substrate.

As a result, both iNOS and COX-2 were increased by LPS treatment, but iNOS was decreased in concentration-dependent manner by Etoac fractionation of bark of blue pine. (FIG. 3).

Experimental Example  4: iNOS , COX -2, IL -6, IL -1β and TNF of -α mRNA  Investigation of anti-inflammatory efficacy by measuring expression level

In this experimental example, the anti-inflammatory efficacy was investigated by measuring the mRNA expression levels of iNOS, COX-2, IL-6, IL-1β and TNF-α.

After the cells were treated with various concentrations of bark fractions, total RNA was isolated using the 'Qiagen RNase mini kit' (Qiagen, Hilden, Germany). RNA (3 μg) was incubated with 'Superscript II reverse transcriptase' (Invitrogen, Carlsabd, Calif., USA) for 1 hour 45 minutes at 42 ° C. and 15 minutes at 70 ° C. to obtain cDNA, followed by real-time Quantification by PCR. The real-time PCR results were expressed by converting the amount of mRNA compared to GAPDH using 'ROTOR-GENE 6000 Series software 1.7' and then displaying the content of the control. Primers and experimental conditions are listed in Table 1.

First, as a result of observing the mRNA levels of iNOS and COX-2, it was observed that the transcript level was increased by LPS but then decreased by treatment of the bark Etoac fraction. (Fig. 4).

In addition, mRNA levels of cytokines IL-6, IL-1β, and TNF-α related to the inflammatory response were measured, and the mRNA levels of cytokines were increased by LPS treatment. It was confirmed that the mRNA level of cytokines decreased by. (Figure 4)

Example  1: Preparation of pharmaceutical composition for treating inflammation

In Example 1, a pharmaceutical composition for treating inflammation was prepared as follows.

(1) Manufacture of powders

Blue Tak Tree ( Acer barbinerve Max ) bark skin was extracted with hot water, and then 1 g of lactose was mixed with 2 g of dry powder of the fraction obtained by fractionation with ethyl acetate, and filled with an airtight cloth to prepare a powder.

(2) Preparation of tablets

Blue Tak Tree ( Acer barbinerve Max ) bark skin was extracted with hot water, and then 100 mg of dry powder, 100 mg of corn starch, 100 mg of lactose and 2 mg of magnesium stearate were mixed with ethyl acetate. The tablets were prepared by tableting according.

(3) Manufacture of capsules

Blue Tak Tree ( Acer barbinerve Max ) bark skin was extracted with hot water, and then 100 mg of dried powder, 100 mg of corn starch, 100 mg of lactose and 2 mg of magnesium stearate were mixed with ethyl acetate, and then filled into gelatin capsules. Capsules were prepared.

(4) Injection preparation

Blue Tak Tree ( Acer barbinerve Max ) bark skin was extracted with hot water, and then 100 ml of dry powder of the fraction obtained by fractionation with ethyl acetate was added for dissolution of distilled water for injection. The pH was adjusted to about 7.5, followed by a 2 ml ampoule. Injections were made by filling and sterilizing.

Example  2: Preparation of food composition for preventing inflammation

In Example 2 was prepared a food composition for preventing inflammation as follows.

(1) Manufacturing of wire

Brown rice, barley, glutinous rice, and yulmu were alphad by a known method, and then dried and roasted to prepare a powder having a particle size of 60 mesh. Black beans, black sesame seeds and perilla were each steamed and dried in a known manner, then roasted and ground to prepare a powder having a particle size of 60 mesh. Then, the brown rice 30 weight%, coix 15 weight%, barley 20 weight%, and 9% by weight of glutinous rice, perilla 7 weight%, 8% Black soybean, black sesame 7 weight%, cheongsi shut tree (Acer barbinerve Max ) bark skin was extracted with hot water, and then, 3% by weight of the dry powder, 0.5% by weight of Ganoderma lucidum and 0.5% by weight of sulfuric acid were obtained by fractionation with ethyl acetate.

(2) Production of chewing gum

Gum base 20%, 76.9% sugar by weight and 1% by weight of spices, 2% water and cheongsi Doc trees (Acer barbinerve Max ) bark skin was extracted with hot water, and then 0.1% by weight of the dry powder of the fraction obtained by fractionation with ethyl acetate was prepared to prepare chewing gum in a conventional manner.

(3) Candy manufacturing

60 wt% of sugar, 39.8 wt% of starch syrup, 0.1 wt% of fragrance, and 0.1 wt% of dry powder of the fraction obtained by fractionation with ethyl acetate after hydrothermal extraction of the bark of Acer barbinerve Max The candy was prepared by the conventional method by combining.

(4) Manufacturing of biscuits

Force 1st class 25.59 wt%, 1st class gravity 22.22 wt%, white sugar 4.80 wt%, salt 0.73 wt%, glucose 0.78 wt%, palm shortening 11.78 wt%, ammonium 1.54 wt%, sodium bicarbonate 0.17 wt%, sodium bisulfite 0.16 wt %, Rice flour 1.45 wt%, Vitamin B₁0.0001 wt%, Vitamin B20.0001 wt%, Milk flavor 0.04 wt%, Water 20.6998 wt%, Whole milk powder 1.16 wt%, Substitute milk powder 0.29 wt%, Calcium phosphate 0.03 wt% , 0.29 wt% salt spray and spray oil 7.27% by weight and cheongsi shut tree (Acer barbinerve Max ) bark skin was extracted with hot water, and then 1% by weight of dry powder of the fraction obtained by fractionation with ethyl acetate was prepared to prepare a biscuit in a conventional manner.

(5) health drink manufacturing

0.26% by weight of honey, 0.0002% by weight of thioctoamide, 0.0004% by weight of nicotinic acid, 0.0001% by weight of riboflavin hydrochloride, 0.0001% by weight of pyridoxine hydrochloride, 0.001% by weight of inositol, 0.002% by weight of ortic acid, 98.7362% by weight of water and cyansid Hot water was extracted from the bark of the trees ( Acer barbinerve Max ), and then 1% by weight of the dry powder of the fraction obtained by fractionation with ethyl acetate was prepared to prepare a health beverage in a conventional manner.

(6) Production of sausages

Pork 65.18%, poultry 25%, starch 3.5%, soy protein 1.7%, salt 1.62%, glucose 0.5% and glycerin 1.5% and bark of Acer barbinerve Max ) Was extracted with hot water, and then 1% by weight of the dry powder of the fraction obtained by fractionation with ethyl acetate, sausage was prepared in a conventional manner.

(7) Health supplement manufacturing

55% by weight of spirulina, 10% by weight of guar gum enzyme digestion, 0.01% by weight of vitamin B₁ hydrochloride, 0.01% by weight of vitamin B6 hydrochloride, 0.23% by weight of DL-methionine, 0.7% by weight of magnesium stearate, 22.2% by weight of lactose and 1.85% by weight of corn starch Hot water was extracted from the bark of Acer barbinerve Max , and 10% by weight of the dry powder of the fraction obtained by fractionation with ethyl acetate to prepare a tablet health supplement in a conventional manner. .

(8) Liquor production

Blue Tak Tree ( Acer barbinerve Max ) bark skin was extracted with hot water, and then 0.5% by weight of the dry powder of the fraction obtained by fractionation with ethyl acetate was mixed with soju, beer, liquor or fruit wine to make an emulsion, and then 7,000 rpm under vacuum. Centrifuged for 15 minutes or mixed at 9,000rpm with a high speed mixer to prepare a liquor.

Claims (3)

An anti-inflammatory composition comprising an extract having anti-inflammatory activity obtained by separating the extract after hydrothermal extraction of the bark of Acer barbinerve Max as an active ingredient
Anti-inflammatory activity, characterized in that it comprises an extract having anti-inflammatory activity obtained by separating the fractions fractionated with ethyl acetate after hydrothermal extraction of the bark of Acer barbinerve Max Composition
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