KR101434409B1 - Anti-inflammatory composition with the extract of the xylem of Abies holophylla MAX - Google Patents

Abstract

The present invention relates to a composition for anti-inflammatory, fir (Abies holophylla The present invention also provides a composition for antiinflammation comprising an ethanol extract of woody part as an active ingredient and a dichloromethane fraction obtained by fractionating the ethanolic extract of woody part Abies holophylla MAX with dichloromethane as an active ingredient.

Description

[0001] The present invention relates to an anti-inflammatory composition containing an extract of fir wood,

The present invention relates to an anti-inflammatory composition, and more particularly, to an anti-inflammatory composition containing an extract of a fir wood part.

Inflammation is an abscess originating from a bacterium. It is an abscess caused by leukocyte death caused by leukocyte's vigorous phagocytosis by infiltrating into a tissue, and tissue or cell disruption which is disturbed by bacteria, It refers to the pathological state.

Anti-inflammatory action means that the proliferation of invading microorganisms is inhibited by an antibacterial agent or the like, or the macrophage that digests foreign substances accumulated in the abscess is activated to enhance the function of digesting and excreting them, thereby promoting the therapeutic process of the abscess.

In general, an inflammatory reaction is a defensive reaction process of a living body that attempts to repair and regenerate a damaged region when an invasion of biological changes occurs in the cell or tissue of the living body. Such a series of reactions include localized blood vessels, various tissue cells of body fluids, and immunocompromised cells.

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 recent years, efforts have been made to find the active ingredient in natural products used in the private sector.

Meanwhile, firs ( Abies holophylla MAX ) grows up to 40 m in diameter and 1 m in diameter, and the branches are spreading horizontally and conically shaped. The bark is gray or dark brown and rough. The winter snow is ovate with no hair and has some resin. Leaves are 4cm long, linear, and they are closely attached to branches. Its tip is pointed, and its back has two white and white pores, and the cross section has a syringe. The male and female flowers are cylindrical oval-shaped female flowers on the axilla of the tip of the branches at the end of each branch. The female flowers are long elliptical and 2 ~ 3 are close to each other. In the private sector, young leaves with shoots or sprouts are used for rheumatism and scurvy. In addition, the fir tree rosin was made into plaster and used as a wound remedy.

Currently, much research has been conducted on natural products or their extracts having anti-inflammatory activity, but studies investigating the anti-inflammatory function of fir extracts are currently limited. Accordingly, in the present invention, it is desired to develop and provide a material which can inhibit inflammation using a natural extract, fir extract, and which can be easily used in foods or pharmaceuticals.

Korean Patent No. 10-1150870 (a method for separating beta-sitosterol from the trunk trunk and a beta-sitosterol-containing antitumor active composition isolated by the above method) Treating the immersed product with ultrasound to obtain an ethanol extract; Sequentially obtaining a hexane fraction, a dichloromethane fraction, an ethyl acetate fraction, a butanol fraction and a distilled water fraction from the ethanol extract; Mixing the obtained hexane fraction, dichloromethane fraction, ethyl acetate fraction, butanol fraction and distilled water fraction to obtain a mixed fraction; And isolating beta-sitosterol from the mixed fraction using medium pressure liquid chromatography.

The present invention relates to firs holophylla MAX) separating the extract with anti-inflammatory and from an object to provide for the development of a anti-inflammatory compositions.

In order to achieve the above object, the present invention is characterized in that, as a first aspect, the present invention includes an extract obtained by adding ethanol to a woody part of Abies holophila MAX , Lt; / RTI > According to the experimental example of the present invention, it was confirmed that the extract of woody part had better anti-inflammatory activity than the fir leaf extract.

On the other hand, the present invention is fir (Abies to the second aspect holophylla MAX ) by adding ethanol to the woody part of the plant (a); (B) removing ethanol after the step (a), suspending it by adding water, and then adding dichloromethane and fractionating it; And a step (c) of obtaining a dichloromethane fraction layer after the step (b), wherein the dichloromethane fraction is contained as an active ingredient. According to the experimental example of the present invention, the anti-inflammatory activity was most excellent in the dichloromethane fraction layer among the various fractions of the fir wood part.

In the present invention, the term " included as an active ingredient "means that the ethanol extract of the first aspect of the present invention or the dichloromethane fraction of the second aspect is added to the composition to such an extent that it can exhibit an anti-inflammatory effect. In addition, when the composition of the present invention is a pharmaceutical composition, various components may be added as a sub ingredient for drug delivery and stabilization, and the composition may be formulated into various forms. When the composition is a food composition, Formulation bases and excipients may be added to prepare formulations.

Meanwhile, the present invention provides an anti-inflammatory food composition comprising an ethanol extract of the first aspect of the present invention and a dichloromethane fraction of the second aspect as an active ingredient, wherein the extract of the first aspect of the present invention The dichloromethane fraction of the second type is preferably added to the composition at a concentration of 1 to 20 μg / ml on a dry weight basis. Further, in the food composition, the composition for anti-inflammation may preferably be a composition for preventing inflammation. The anti-inflammatory food composition of the present invention may be, for example, meat, cereal, caffeinated beverages, ordinary beverages, chocolates, breads, snacks, confectionery, pizzas, jellies, noodles, gums, ice creams, alcoholic beverages, Or an external health supplementary food, but the present invention is not limited thereto.

On the other hand, the present invention provides an anti-inflammatory pharmaceutical composition comprising the extract of the first aspect of the present invention and the dichloromethane fraction of the second aspect as an active ingredient. In the pharmaceutical composition, the composition for anti-inflammation may preferably be a pharmaceutical composition for treating inflammation.

In addition, the pharmaceutical composition for antiinflammation 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 or therapeutic agent is a pharmaceutical agent, it may be a filler, an anticoagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent or an antiseptic agent.

On the other hand, the formulation of the pharmaceutical composition for antiinflammation of the present invention can be formulated into a desired form depending on the method of use, and can be used in the art to provide rapid, sustained or delayed release of the active ingredient, It is preferable to employ a known method to formulate it. 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 dose of the anti-inflammatory pharmaceutical composition of the present invention is preferably determined in consideration of the administration method, the age, sex, weight and severity of the disease, etc. of the recipient. 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 dosage is merely an example for illustrative purposes, and may vary according to the prescription of the physician depending on the condition of the recipient.

According to the present invention, fir (Abies holophylla ≪ / RTI > MAX ) woody ethanol extract as an active ingredient. In addition, an anti-inflammatory composition containing the dichloromethane fraction obtained by fractionating ethanol extract of woody part Abies holophylla MAX with dichloromethane can be prepared.

Figure 1 is a graph showing the effect of fir extract and fractions on NO production.
Figure 2 is a graph showing the effect of fir extract and fraction on cell proliferation.
FIG. 3 is a graph showing the effect of the dichloromethane fractionation layer of fir wood on the NO production according to the concentration.
FIG. 4 is a graph showing the effect of dichloromethane fractionation layer of fir wood on cell proliferation according to concentration. FIG.
FIG. 5 is a photograph showing the influence of the dichloromethane fraction layer in the fir wood part on iNOS and COX-2 protein expression.
FIG. 6 is a graph showing the effect of the dichloromethane fraction layer of fir wood on the expression of iNOS and COX-2 mRNA.
FIG. 7 is a graph showing the effect of dichloromethane fraction of fir wood on IL-1?, TNF-alpha and IL-6 mRNA expression.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the scope of the present invention is not limited to the following embodiments, and includes modifications of equivalent technical ideas.

Example  1: Preparation of sample

Dried fir leaf and 1 ㎏ of woody part were extracted by cooling with 80% ethanol for 3 days, respectively, and concentrated to remove ethanol to prepare an ethanol extract. The resulting ethanol extract was suspended in water and suspended in dichloromethane (DCM) and ethyl acetate (EtOAc), which are solvents having different polarities. The DCM layer, ethyl acetate layer and water layer .

On the other hand, Raw264.7 cells, which are mouse macrophages to be used for experiments, were cultured in DMEM (DMEM) supplemented with 10% FBS, penicillin 100 U / ml and streptomycin 100 U / Lt; / RTI > All cells used in the following Experimental Examples were subcultured when grown at a density of 80 to 90%.

Experimental Example  One: NO Fir extract and Fraction  Anti-inflammatory effect

In this experiment, LPS-induced NO production was measured in Raw264.7 cells, a macrophage of rats, to investigate the anti-inflammatory activity of fir extract and fractions.

Raw264.7 cells were plated at a density of 5 × 10 ⁴ cells / well in a 24-well plate using a medium supplemented with 10% FBS. After 24 hours, the medium was replaced with medium containing 1% FBS for 12 hours or more Followed by serum deprivation. After 24 hours, the control group treated with only LPS (1 mg / L) and the experimental group treated with LPS (1 mg / L) and each of the fir ethanol extracts and fractions prepared in Example 1 were separated. At this time, each fir extract and fraction was treated with 50 mg / L and cultured at 37 ° C in 5% carbon dioxide for 24 hours. The amount of NO produced from Raw264.7 cells was measured using the 'Griess reagent system' (Promega, Madison, WI, USA). (BIO-RAD, Hercules, CA, USA) at a wavelength of 540 nm, and then incubated at 37 ° C for 30 min at room temperature. The supernatant was applied to a 96-well plate and reacted with Griess reagent according to manufacturer's instructions. Was used to measure the nitrite concentration.

The experimental results are shown in Table 1 below.

Inhibition rate (%) Ethanol extract Dichloromethane fraction Ethyl acetate fraction Water fraction fir xylem 34.6 100 81.7 3.7 leaf 1.4 95.8 48.5 8.5

As a result of the measurement, as shown in Table 1, the extracts and fractions of the fir leaf and the woody part all decreased the production of NO. However, the degree of inhibition was significantly higher in the woody part than in the leaf, and the dichloromethane (DCM) fraction in the woody part showed the most excellent effect by reducing the amount of NO by 100% (FIG. 1).

Experimental Example  2: MTT Assay  Fir extract and Fraction  Cytotoxicity investigation

In this experiment, we examined the cytotoxicity of MTT assay by treating LPS, fir leaf and woody part extracts and fractions at a concentration of 50 ㎎ / L for 24 hours in RAW264.7 cells, a macrophage of rats. Experimental conditions were the same as in Experimental Example 1 above.

2). When the fraction of dichloromethane (DCM) was used, the fraction of dichloromethane (DCM) of the fir leaf decreased cell proliferation by about 30%, whereas the fraction of dichloromethane (DCM) DCM) fractions did not show toxicity.

Experimental Example  3: Dichloromethane of fir wood In the fraction  Investigation of anti-inflammatory effect and cytotoxicity according to concentration

In this experiment, we compared the anti-inflammatory activity and cytotoxicity according to the concentrations of dichloromethane (DCM) fraction of fir wood.

Experimental conditions were the same as in Experimental Examples 1 and 2, and treatment concentrations of dichloromethane (DCM) fractions of fir wood were treated at 0, 5, 10 and 20 mg / L.

As a result of measuring the amount of NO, dichloromethane (DCM) fraction of fir wood inhibited the production of NO in a concentration dependent manner, and showed a suppression rate of 80% NO production at 20 mg / L (FIG. 3).

On the other hand, as a result of confirming the cytotoxicity, the dichloromethane (DCM) fraction of the fir wood did not significantly affect cell growth even when the concentration was changed (FIG. 4).

Experimental Example  4: iNOS Wow COX -2 protein expression level and mRNA  Fir wood by expression Dichloromethane Fraction  Anti-inflammatory effect

In this experiment, we investigated the expression of iNOS and COX-2, proteins involved in inflammation. As a sample, the woody dichloromethane fraction (DCM) fraction having the best NO reduction effect was used in the fir fraction.

Raw264.7 cells were plated at a density of 1 × 10 ⁶ cells / dish into a 100-mm dish. After 24 hours, the medium was replaced with a medium containing 1% FBS and serum deprivation was performed for 12 hours or more. LPS (1 mg / L) and fructose dichloromethane (DCM) fractions were added to the media containing 1% FBS for 0, 5, 10 and 20 mg / L for 24 hours.

Total protein concentration of total cell lysate was quantitated using the BCA protein assay kit (Pierce, Rockford, IL, USA). Total cell lysate (40 μg protein) was fractionated by 4 to 20% SDS-PAGE (gradient sodium dodecyl sulfate polyacrylamide gel electrophoresis) and stained with PVDF (polyvinylidene fluoride) membrane (Millipore, Bedford, MA , USA).

The membranes were stirred for 1 hour with 5% non-fat dry milk-TBST (20 mM Tris-HCl, pH 7.6, 150 mM NaCl, 0.1% Tween-20) 1000, COX-2 1: 1000, and β-actin 1: 2000) were added, and the mixture was stirred at room temperature for 1.5 hours. Thereafter, the membrane was stirred with HRP (horseradish peroxidase) -binding anti-mouse or anti-rabbit for 1 hour.

Each protein bands were visualized by using Western aunt Tourbillon Chemie Rooney four seunteu ^(TM Immobilon Western Chemiluminescent HRP Substrate). The intensity of each protein band was quantified using ImageJ (ver. 1.42, NIH Image, developed and maintained by the National Institutes of Health, Bethesda, MD, USA).

As a result, the expression of iNOS and COX-2 protein was increased by LPS treatment, and the expression of iNOS protein was decreased by treatment with dichloromethane (DCM) fraction of fir wood (FIG. 5).

Meanwhile, as confirmed above, real-time RT-PCR was performed to investigate whether the decrease in iNOS protein expression by the dichloromethane (DCM) fraction of fir wood was controlled at the level of mRNA.

As a result, mRNA expression was increased by LPS in both iNOS and COX-2, and iNOS was decreased in a concentration-dependent manner by treatment with dichloromethane (DCM) fraction of fir wood. In particular, in the group treated with 20 mg / L, mRNA expression was reduced by 80% (FIG. 6). Thus, it was confirmed that the dichloromethane (DCM) fraction of the fir woods effectively inhibited NO production by inhibiting iNOS expression at the transcript level.

Experimental Example  5: inflammatory cytokines and mRNA  Fir wood by measurement of expression levels Dichloromethane Fraction Anti-inflammatory effect

In this experiment, mRNA expression levels of IL-6, IL-1β and TNF-α, which are inflammatory cytokines, were measured to investigate anti-inflammatory activity.

(DT) primer and SuperScript II reverse transcriptase (Invitrogen, Carlsbad, Calif., USA) were used to synthesize cDNA from total RNA using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA) Were used. Expression of mRNAs of iNOS, COX-2, TNF-α, IL-6, IL-1β and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) synthesized by cDNA was detected by real-time RT- (GeneTM 6000, Corbett Life Science, Sydney, Australia).

The mRNA to be measured was quantified using GAPDH and calculated using Rotor-gene software (ver. 6, Corbett Life Science, Sydney, Australia). Each value represents 100% of LPS control (1 mg / L LPS + 0 mg / L fir wood DCM fraction) and the same experiment was repeated three times. The required mRNA primers were prepared in Bioneer (Daejeon, Korea) and are shown in Table 2 below.

GAPDH sense 5'-GGCATGGCCTTCCGTGT-3 '
anti-sense 5'-GGTTTCTCCAGGCGGCA-3 ' iNOS sense 5'-AATGGCAACATCAGGTCGGCCATCACT-3 '
anti-sense 5'-GCTGTGTGTCACAGAAGTCTCGAACTC-3 ' COX-2 sense 5'-GGAGAGACTATCAAGATAGT-3 '
anti-sense 5'-ATGGTCAGTAGACTTTTACA-3 ' IL-6 sense 5'-CCCAATTTCCAATGCTCTCC-3 ',
anti-sense 5'-ACAGATAAGCTGGAGTCACAGAAGGAGTGGCT-3 ' IL-1? sense 5'-ACCTGTCCTGTGTAAATGAAAGACG-3 '
anti-sense 5'-TTGGTATTGCTTGGGATCC-3 ' TNF-a sense 5'-TCCAGGCGGTGCCTATGT-3 '
anti-sense 5'-CGATCACCCCGAAGTTCAGT-3 '

As a result, mRNA expression of IL-6, IL-1β and TNF-α, which were increased by LPS, was significantly decreased by treatment with dichloromethane (DCM) fraction of fir wood (FIG. 7). It was confirmed that the dichloromethane (DCM) fraction of fir wood inhibits the mRNA expression level of IL-6, IL-1β and TNF-α, which are inflammatory cytokines.

Example  3: Preparation of pharmaceutical composition for treating inflammation

In Example 3, a pharmaceutical composition for treating inflammation was prepared as follows. On the other hand, the dichloromethane fraction used in the following Example 3 was fragrant ( Abies holophylla MAX ) was extracted with ethanol, dried, suspended in water, and then fractionated by adding dichloromethane (DCM).

(1) Manufacture of powders

2 g of the dry powder of the dichloromethane fraction was mixed with 1 g of lactose and filled in an airtight container to prepare a powder.

(2) Preparation of tablets

Fir ( Abies holophylla MAX ) 100 mg of dried powder of woody dichloromethane fractions, 100 mg of corn starch, 100 mg of lactose and 2 mg of magnesium stearate were mixed and tableted by a conventional tableting method.

(3) Manufacture of capsules

Fir ( Abies holophylla MAX ) 100 mg of dried powder, 200 mg of corn starch, 100 mg of lactose and 2 mg of magnesium stearate were mixed with the dichloromethane fraction of woody part, and then filled into gelatin capsules to prepare capsules.

(4) Injection preparation

Fir ( Abies holophylla MAX ) Woody Dissolve 100 mg of dry powder of the fraction of dichloromethane by dissolving in distilled water in an appropriate amount, adjust the pH to about 7.5, and fill and sterilize 2 ml of ampoule to prepare an injection.

Example  4: Preparation of food composition for inflammation prevention

In Example 4, a food composition for preventing inflammation was prepared as follows. The dichloromethane fractions were used in the following Examples 4, fir (Abies holophylla MAX ) was extracted with ethanol, dried, suspended in water, and then fractionated by adding dichloromethane (DCM).

(1) Manufacturing of wire

Brown rice, barley, glutinous rice, and yulmu were dried by a known method and dried, and then the mixture was prepared into powder having a particle size of 60 mesh by a pulverizer. Black beans, black sesame seeds and perilla seeds were each steamed and dried by known methods, and then power distribution and pulverization were carried out to prepare powder having a particle size of 60 mesh. Then, the brown rice 30 weight%, coix 15 weight%, 20 weight%, barley, rice 9% by weight, perilla 7 weight%, 8% Black soybean, black sesame 7 weight%, fir (Abies holophylla MAX ) 3% by weight of dry powder, 0.5% by weight of Ganoderma lucidum and 0.5% by weight of ganoderate were mixed with dichloromethane fraction of woody part.

(2) Production of chewing gum

20% by weight of gum base, 76.9% by weight of sugar, 1% by weight of perfume, 2% by weight of water and Abies holophylla MAX ), and 0.1 wt% of dry powder of a dichloromethane fraction of woody part were mixed to prepare chewing gum in a usual manner.

(3) Candy manufacturing

Sugar 60% by weight, 39.8% by weight of starch syrup, 0.1% by weight perfume and fir (Abies Holophylla MAX ) 0.1% by weight of dry powder of the dichloromethane fraction of woody part were mixed to prepare a candy by a conventional method.

(4) Manufacturing of biscuits

By weight of starch, 0.77% by weight of glucose, 0.78% by weight of glucose, 11.78% by weight of palm shortening, 1.54% by weight of ammonia, 0.17% by weight of sodium bicarbonate, 0.16% by weight of sodium bisulfite 1.45 weight% of rice flour, 0.0001 weight% of vitamin B1, 0.0001 weight% of vitamin B2, 0.04 weight% of milk fractions, 20.6998 weight% of water, 1.16 weight% of whole milk powder, 0.29 weight% 0.29% by weight of spray salt and 7.27% by weight of spray oil and fir ( Abies holophylla MAX ) 1% by weight of dry powder of woody dichloromethane fraction was blended to prepare a biscuit by a conventional method.

(5) health drink manufacturing

0.0001% by weight of honey, 0.0002% by weight of chitosanic acid amide, 0.0004% by weight of nicotinic acid amide, 0.0001% by weight of sodium riboflavin hydrochloride, 0.0001% by weight of pyridoxine hydrochloride, 0.001% by weight of inositol, 0.002% by weight of orthoacids, Abies holophylla ≪ / RTI > MAX ) woody part of dried powder of dichloromethane fraction was mixed to prepare a health drink.

(6) Production of sausages

65.18 wt% of pork, 25 wt% of chicken, 3.5 wt% starch, soy protein 1.7% by weight, sodium chloride 1.62 wt%, glucose 0.5% by weight and 1.5% by weight glycerine and fir (Abies holophylla MAX ) 1% by weight of dry powder of the dichloromethane fraction of woody part were mixed and sausage was prepared in a usual manner.

(7) Health supplement manufacturing

A mixture of 55% by weight of Spirulina, 10% by weight of guar gum enzyme hydrolyzate, 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, And fir ( Abies holophylla MAX ) 10% by weight of dry powder of the dichloromethane fraction of woody part were blended to prepare a refillable health supplement food by a conventional method.

(8) Liquor production

Fir ( Abies holophylla MAX ) 0.5% by weight of dried powder of woody dichloromethane fraction was mixed with soju, beer, liquor or fruit wine to make emulsion, centrifuged in vacuum at 7,000 rpm for 15 minutes or mixed at 9,000 rpm in a high speed mixer, .

Claims (4)

delete (A) adding ethanol to the woody part of Abies holophylla MAX to extract;
(B) removing ethanol after the step (a), suspending it by adding water, and then adding dichloromethane and fractionating it; And
And (c) a step of obtaining a dichloromethane fraction layer after the step (b). The anti-inflammatory pharmaceutical composition according to claim 1, wherein the fraction of dichloromethane obtained from the step (c)
delete (A) adding ethanol to the woody part of Abies holophylla MAX to extract;
(B) removing ethanol after the step (a), suspending it by adding water, and then adding dichloromethane and fractionating it; And
And (c) a step of obtaining a dichloromethane fraction layer after the step (b). The composition for preventing or improving inflammation according to claim 1, wherein the fraction is dichloromethane.

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